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WO2017128791A1 - 基因组合及其用途 - Google Patents

基因组合及其用途 Download PDF

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Publication number
WO2017128791A1
WO2017128791A1 PCT/CN2016/103934 CN2016103934W WO2017128791A1 WO 2017128791 A1 WO2017128791 A1 WO 2017128791A1 CN 2016103934 W CN2016103934 W CN 2016103934W WO 2017128791 A1 WO2017128791 A1 WO 2017128791A1
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Prior art keywords
gene
promoter
nucleotide sequence
rice
seq
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PCT/CN2016/103934
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English (en)
French (fr)
Inventor
凃巨民
陈浩
胡培松
罗炬
张晓波
刘玉君
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Zhejiang University ZJU
China National Rice Research Institute
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Zhejiang University ZJU
China National Rice Research Institute
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Priority to US16/073,177 priority Critical patent/US11674146B2/en
Priority to CN202311567386.XA priority patent/CN118028290A/zh
Priority to CN201680080151.XA priority patent/CN109312334B/zh
Publication of WO2017128791A1 publication Critical patent/WO2017128791A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8285—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for nematode resistance
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    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to the field of transgenic plants.
  • the present invention relates to a combination of genes for controlling expression of a foreign gene of interest in a plant, and a method of cultivating a transgenic plant using the combination of genes.
  • GM crops have been grown in 28 countries around the world, with a production area of 181.5 million hectares in 2014 (James, et al., 2014).
  • GM crops increased crop production value by $133 billion and saved pesticides 500 million kg in the 17 years from 1996 to 2013, only in 2013.
  • GM crops continue to have a significant positive impact on food safety, sustainable development and climate change.
  • Rice is the staple food crop of 50% of the world's population. Although it was announced in 2000 that the transgenic rice line was successfully developed (Tu, et al., 2000), for example, the excellent transgenic restorer line Minghui 63/Bt (Huahui No. 1) and its hybrid rice cultivar Yujing 63/Bt, and In November 2009, after repeated verification by the developer and third parties that there was no biosafety risk, the biosafety certificate issued by the Ministry of Agriculture of China was obtained.
  • these traditional transgenic Bt-resistant rice are endosperm-expressing, that is, the expression product of Bt gene is accumulated in the transgenic rice produced, it is still difficult for the public to recognize its food safety, and its commercial planting is also Therefore, it has not been approved. This situation is not only true in China, but also in other parts of the world and countries such as Europe and Japan. 58% of the people surveyed in Europe in 2006 thought that GM foods should not be encouraged.
  • the technology automatically removes foreign genes from pollen and seeds before and after transgenic flowering, as the computer “unloads” the application software, so that the foreign genes in the transgenic plants are automatically cleared before diffusion and before use (Zhao Degang et al., 2008). ).
  • This technology can be applied directly to asexually propagated crops, but improvements are needed to apply to sexually propagated crops.
  • the main problem is that the exogenous gene of interest in the seeds of sexually propagated crops is no longer retained once removed, thereby rendering the next generation that is propagated through the seed lose the transgenic trait.
  • the inventors have developed a combination of genes capable of shutting down expression of a gene of interest in a particular tissue/organ (eg, endosperm).
  • the technique consists essentially of a locking element that locks the expression of an exogenous gene of interest and a key gene that unlocks the expression of the locked foreign gene of interest.
  • the exogenous gene of interest carried by the former is permanently locked, and the key gene carried by the latter that can open the locked expression of the exogenous gene of interest is expressed only in a specific tissue/organ.
  • the two components are separately transformed and integrated into the parental genome such as rice, and then they can be integrated by hybrid production, so that the expression of the exogenous gene can be opened in the specific tissue/organ.
  • the desired phenotype occurs, and the expression of the exogenous gene of interest is always turned off in a particular tissue/organ (eg, an edible organ, such as a rice endosperm), resulting in a transgenic plant that does not contain any transgenic components in the portion for consumption.
  • tissue/organ eg, an edible organ, such as a rice endosperm
  • the same effect can be obtained by introducing and integrating these two components into the genome of the same recipient plant.
  • the present invention provides a gene combination for controlling expression of an exogenous gene of interest in a plant, which consists of a locking element and a key gene, wherein
  • the locking element comprises the nucleotide sequence set forth in SEQ ID NO: 5 or 8, or comprises at least 80% homology to the nucleotide sequence set forth in SEQ ID NO: 5 or 8, and is capable of locking A nucleotide sequence operably linked to the expression of the foreign gene of interest;
  • the key gene comprising the nucleotide sequence set forth in SEQ ID NO: 3 or 6, or comprising the core set forth in SEQ ID NO: 3 or
  • the nucleotide sequence has at least 40% homology and is capable of releasing the nucleotide sequence locked by the locking element for expression of the foreign gene of interest.
  • the invention provides a locking element for locking expression of a foreign gene of interest linked thereto in a plant, comprising the nucleotide sequence set forth in SEQ ID NO: 5 or 8, or comprising and SEQ ID A nucleotide sequence represented by NO: 5 or 8 has at least 80% homology and is capable of locking a nucleotide sequence of expression of an exogenous gene of interest operably linked thereto.
  • the present invention provides a key gene for releasing expression locking of an exogenous gene of interest in a plant-specific tissue, comprising the nucleoside represented by SEQ ID NO: 3 or 6.
  • the invention provides a method for controlling expression of a foreign gene of interest in a plant, the method comprising introducing a locking element and a key gene into a recipient plant, wherein the locking element comprises SEQ ID NO: a nucleotide sequence represented by 5 or 8, or comprising at least 80% homology to the nucleotide sequence shown in SEQ ID NO: 5 or 8, and capable of locking an exogenous purpose to which it is operably linked a nucleotide sequence for expression of the gene; the key gene comprising the nucleotide sequence set forth in SEQ ID NO: 3 or 6, or comprising at least 40% of the nucleotide sequence set forth in SEQ ID NO: 3 or The homology is and the nucleotide sequence capable of unlocking the expression of the foreign gene of interest by the locking element can be released.
  • the locking element comprises SEQ ID NO: a nucleotide sequence represented by 5 or 8, or comprising at least 80% homology to the nucleotide sequence shown in SEQ ID NO: 5 or 8, and
  • the invention provides a method for controlling expression of a foreign gene of interest in a plant, the method comprising operatively linking the exogenous gene of interest to a locking element, the locking element comprising SEQ ID NO: a nucleotide sequence represented by 5 or 8, or comprising at least 80% homology to the nucleotide sequence set forth in SEQ ID NO: 5 or 8, and capable of locking an exogenous gene of interest operably linked thereto The expressed nucleotide sequence.
  • the present invention provides a method for releasing expression locking of an exogenous gene of interest in a plant, the method comprising introducing a key gene into the plant, the key gene comprising Nucleotide sequence shown in SEQ ID NO: 3 or 6 The column, or a nucleotide sequence comprising at least 40% homology to the nucleotide sequence set forth in SEQ ID NO: 3 or 6, and capable of releasing the locked expression of the foreign element of interest by the locking element.
  • the present invention provides a method of breeding a transgenic plant, the method comprising: hybridizing a first parent comprising a locking element to a second parent comprising a key gene, thereby obtaining the transgenic plant, wherein
  • the locking element comprises the nucleotide sequence set forth in SEQ ID NO: 5 or 8, or comprises at least 80% homology to the nucleotide sequence set forth in SEQ ID NO: 5 or 8, and is capable of being locked and operable a nucleotide sequence for expression of the ligated exogenous gene of interest;
  • the key gene comprising the nucleotide sequence set forth in SEQ ID NO: 3 or 6, or comprising the nucleotide set forth in SEQ ID NO: 3 or
  • a sequence has at least 40% homology and is capable of releasing a nucleotide sequence that the locking element locks for expression of an exogenous gene of interest.
  • the method further comprises introducing and/or integrating the locking element into the genome of the first parent prior to the hybridizing step, and introducing and/or integrating the key gene into the genome In the genome of the second parent.
  • the invention provides a method of breeding a transgenic plant, the method comprising introducing and/or integrating a locking element and a key gene into the genome of the same plant, thereby obtaining the comprising the locking element And a transgenic plant of a key gene, wherein the locking element comprises the nucleotide sequence set forth in SEQ ID NO: 5 or 8, or comprises at least 80% of the nucleotide sequence set forth in SEQ ID NO: 5 or 8.
  • nucleotide sequence which is homologous and capable of locking expression of an exogenous gene of interest operably linked thereto; the key gene comprising the nucleotide sequence set forth in SEQ ID NO: 3 or 6, or comprising SEQ ID NO
  • the nucleotide sequence shown by 3 or 6 has at least 40% homology and is capable of releasing the nucleotide sequence in which the locking element is locked for expression of the exogenous gene of interest.
  • the invention provides the use of a combination of a locking element and a key gene for regulating expression of a foreign gene of interest in a plant, wherein the locking element is capable of locking expression of an exogenous gene of interest operably linked thereto;
  • the key gene is capable of releasing the expression lock of the locking element for an exogenous gene of interest.
  • the locking element comprises the nucleotide sequence set forth in SEQ ID NO: 5 or 8, or comprises at least 80% of the nucleotide sequence set forth in SEQ ID NO: 5 or 8. And a nucleotide sequence capable of locking the expression of an exogenous gene of interest operably linked thereto; the key gene comprising the SEQ ID NO: 3 or 6 a nucleotide sequence, or a nucleotide comprising at least 40% homology to the nucleotide sequence set forth in SEQ ID NO: 3 or 6, and capable of releasing the locked expression of the foreign element of interest by the locking element sequence.
  • the gene combination is a locking element comprising a nucleotide sequence of SEQ ID NO: 5 or at least 80% homology to SEQ ID NO: 5, and comprising SEQ ID NO : 3 or a key gene of a nucleotide sequence having at least 40% homology to SEQ ID NO: 3.
  • the gene combination is a locking element comprising a nucleotide sequence of SEQ ID NO: 8 or at least 80% homology to SEQ ID NO: 8, and comprising SEQ ID NO: 6 or a key gene of a nucleotide sequence having at least 40% homology to SEQ ID NO: 6.
  • the locking element is located between the constitutive promoter and the exogenous gene of interest and is operably linked to the constitutive promoter and the exogenous gene of interest.
  • the key gene is operably linked to a tissue-specific promoter.
  • the constitutive promoter is selected from the group consisting of the cauliflower mosaic virus (CaMV) 35S promoter, the nopaline synthase gene Ocs promoter from the T-DNA region of the Agrobacterium tumefaciens Ti plasmid, rice muscle movement
  • the tissue-specific promoter is selected from the group consisting of a rice green tissue-specific promoter ribulose-1,5-bisphosphate carboxylase small subunit rbcS promoter (Kyozuka et al, 1993; Nomura) Et al, 2000), maize phosphoenolpyruvate carboxylase PEPC promoter (Yanagisawa et al, 1989), rice green tissue-specific expression of DX1 promoter (Ye et al, 2012), rice photosystem II10kDa peptide D540 initiation (Cai et al, 2007), rice leucine-rich leucine repeat-like receptor protein kinase LP2 promoter (Thilmony et al, 2009), and maize chloroplast C4Pdk promoter (Jang et al, 1999); preferably,
  • the tissue-specific promoter is the rice green tissue-specific promoter rbcS promoter.
  • the exogenous gene of interest may be an insect resistance gene or a herbicide resistance gene.
  • the insect resistance gene includes Bacillus thuringiensis (Bacillus) Thryiensis) cry1Ab, cry1Ac, cry1Ab/1Ac, cry1C, cry2A and Vip3 insect-resistant genes, anf, sep gene from Serratia entomophila, cmb gene from Clostridium bifermentans, The mtx gene from Bacillus sphaericus, the insecticidal protein gene from Xenorhabdus nematophilus, the tca, tcb gene from Photorhabdus luminescens, and the Metarhizium anisopliae Metarliziz anisopliae) prl gene, but is not limited to this.
  • nucleotide coding sequence and amino acid sequence of the Bt insect-resistant gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • the herbicide resistance gene comprises an EPSP synthase gene resistant to glyphosate, an EPSA mutant gene aroA of Salmonella typhimurium, a bar gene resistant to glufosinate, an ALS mutant gene Ilv against moxazolinone G, the anti-Spirulina AccL-s2 gene, the anti-bromobenzonitrile bxn gene, and the chlorsulfuron-resistant csrl gene, but are not limited thereto.
  • the plant is selected from the group consisting of rice, wheat, barley, oats, corn, corn, sorghum, glutinous rice, sweet potatoes, potatoes, lotus seeds, soybeans, and peanuts.
  • the plant is rice.
  • Locking element Locking element (LOCK) and key gene (KEY)
  • the present inventors invented a set of regulatory elements and gene sequences having a function of regulating gene expression by DNA recombination, and named them as a locking element (LOCK) and a key gene (KEY) according to their respective functions.
  • LOCK locking element
  • KEY key gene
  • the locking element is capable of locking the expression of an exogenous gene of interest to which it is linked.
  • the locking element comprises the nucleotide sequence shown in SEQ ID NO: 5 or 8, or comprises at least 80% homology to the nucleotide sequence shown in SEQ ID NO: 5 or 8, and A nucleotide sequence capable of locking the expression of an exogenous gene of interest operably linked thereto.
  • the locked gene comprises the nucleotide sequence shown in SEQ ID NO: 5 or 8, or consists of the nucleotide sequence shown in SEQ ID NO: 5 or 8.
  • the key gene is capable of releasing the expression lock of the locking element for an exogenous gene of interest.
  • the key gene comprises the nucleotide sequence shown in SEQ ID NO: 3 or 6, or comprises at least 40% homology to the nucleotide sequence shown in SEQ ID NO: 3 or 6, and A nucleotide sequence capable of unlocking the expression of an exogenous gene of interest by the locking element.
  • the locked gene comprises the nucleotide sequence shown in SEQ ID NO: 3 or consists of the nucleotide sequence shown in SEQ ID NO: 3.
  • the degree of homology between two nucleotide sequences can be determined by algorithms known in the art.
  • the optimal alignment of the sequences used for comparison can be performed by: local homology algorithm [Smith and Waterman Add. APL. Math. 2: 482 (1981)]; homology alignment algorithm [Needleman and Wunsch J. Mol. Biol. 48: 443 (1970); Retrieval similarity algorithm [Pearson and Lipman Proc. Natl. Acad Sci. (USA) 85: 2444 (1988)]; computer programs of these algorithms and their default parameters [Wisconsin Genetics software] GAP, BESTFIT, BLAST, PASTA, and TFASTA, Genetics Computer Group (GCG), 575 Science Dr., Madison, WI in the package; or by visual inspection.
  • the locking element may comprise at least 80%, such as at least 91%, 92%, 93%, 94%, 95%, 96%, of the nucleotide sequence set forth in SEQ ID NO: 5 or 8. 97%, 98%, 99%, 99.5%, 99.8% or 100% homology, and is capable of locking the nucleotide sequence of expression of an exogenous gene of interest operably linked thereto.
  • the homology can be calculated using BLAST and its default parameters.
  • the key gene may comprise at least 40%, such as at least 40%, 41%, 42%, 43%, 44%, 45%, of the nucleotide sequence set forth in SEQ ID NO: 3 or 6. 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62% , 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79 %, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.8% or 100% homology, and a nucleotide sequence capable of releasing the locked expression of the foreign element of interest by the locking element.
  • the gene combination of the invention is a locked element comprising a nucleotide sequence of SEQ ID NO: 5 or at least 80% homology to SEQ ID NO: And a key gene comprising a nucleotide sequence having at least 40% homology to SEQ ID NO: 3 or SEQ ID NO: 3.
  • the gene combination of the invention is a locking element comprising a nucleotide sequence of SEQ ID NO: 8 or at least 80% homology to SEQ ID NO: 8, and comprising SEQ ID NO : 6 or a key gene of a nucleotide sequence having at least 40% homology to SEQ ID NO: 6.
  • the locking element may be: 1) comprising the nucleotide sequence set forth in SEQ ID NO: 5 or 8, or 2) comprising a substitution by the nucleotide sequence set forth in SEQ ID NO: 5 or 8, A nucleotide sequence derived from a deletion or addition of one or more nucleotides and capable of locking the expression of an exogenous gene of interest operably linked thereto.
  • the key gene may be: 1) comprising the nucleotide sequence shown in SEQ ID NO: 3 or 6, or 2) comprising a nucleotide sequence represented by SEQ ID NO: 3 or 6, A nucleotide sequence derived by deleting or adding one or more nucleotides and capable of releasing the locked expression of the foreign element of interest by the locking element.
  • the key gene may: 1) encode the amino acid sequence shown in SEQ ID NO: 4 or 7, or 2) encode, replace or delete in the amino acid sequence shown in SEQ ID NO: 4 or 7.
  • the locking element may: 1) comprise the nucleotide sequence set forth in SEQ ID NO: 5 or 8, or 2) under stringent conditions and the nucleotide sequence set forth in SEQ ID NO: 5 or 8.
  • the complementary sequence hybridizes and is capable of locking the nucleotide sequence of expression of the exogenous gene of interest to which it is operably linked.
  • the key gene may be: 1) comprising the nucleotide sequence shown in SEQ ID NO: 3 or 6, or 2) under stringent conditions and the nucleotide sequence shown in SEQ ID NO: 3 or 6.
  • the complementary sequence hybridizes and is capable of releasing the nucleotide sequence locked by the locking element for expression of the foreign gene of interest.
  • Stringent conditions are used herein to describe hybridization to a nucleotide sequence in 6X sodium chloride/sodium citrate (SSC) at about 45 °C followed by 0.2 x SSC at about 50-65 °C. One or more washes in /0.1% SDS.
  • the "stringent condition” is “highly stringent condition”.
  • the term “highly stringent conditions” refers to, for example, hybridization to a nucleotide sequence in 6x SSC at about 45 °C followed by one or more washes in 0.1 x SSC/0.2% SDS at about 68 °C.
  • the combination of the regulatory gene composed of the locking element and the key gene can control the expression of the exogenous gene of interest in the plant, thereby locking the exogenous gene of interest in the endosperm while realizing the function of the exogenous gene of interest.
  • the locking element is placed between the constitutive promoter and the exogenous gene of interest to thereby lock expression of the exogenous gene of interest in the whole transgenic plant body comprising the locking element;
  • the key gene is operably linked to a tissue-specific promoter, thereby unlocking expression of the locked gene for the foreign gene of interest in a specific tissue, and realizing the function of the exogenous gene of interest.
  • promoter refers to a genetic element that initiates transcription. Promoters can be classified into constitutive promoters, tissue-specific promoters, and inducible promoters according to their mode of action.
  • the "constitutive promoter” refers to a promoter that maintains sustained activity in most or all tissues. Under the control of constitutive promoters, there was no significant difference in gene expression between different tissues and developmental stages.
  • the constitutive promoter for use in the present invention is a promoter derived from a plant or having constitutive activity in a plant.
  • constitutive promoters useful in the present invention include, but are not limited to, the cauliflower mosaic virus (CaMV) 35S promoter, the nopaline synthase gene Ocs promoter from the T-DNA region of the Agrobacterium tumefaciens Ti plasmid, rice muscle The kinesin promoter Actin I, the ubiquitin gene promoter Ubi, and the like.
  • the constitutive promoter of the invention is the rice actin promoter Actin I.
  • the tissue-specific promoter refers to a promoter that is active in a particular type of cell or tissue. Under the control of tissue-specific promoters, genes are expressed only in certain specific organs or tissues.
  • the tissue-specific promoter used in the present invention is a promoter derived from a plant or having tissue-specific activity in a plant.
  • a person skilled in the art can express an organ according to a plant species, a desired/undesired exogenous gene of interest/ Tissues (eg, plant tissues/organs for consumption) are selected to select tissue-specific promoters.
  • Tissue-specific promoters useful in the present invention include: a root-specific promoter, a stem-specific promoter, and a leaf-specific promoter.
  • the tissue-specific promoter of the present invention is A green tissue-specific promoter of a plant, such as a stem and/or leaf-specific promoter.
  • tissue-specific promoter examples include a rice green tissue-specific promoter ribulose-1,5-bisphosphate carboxylase small subunit rbcS promoter, corn phosphoenolpyruvate carboxylate
  • the enzyme PEPC promoter rice green tissue-specific expression of DX1 promoter, rice photosystem II10kDa polypeptide D540 promoter, rice leucine-rich repeat-like receptor protein kinase LP2 promoter, and maize chloroplast C4Pdk promoter, but not limited to this.
  • the tissue-specific promoter of the invention is a rice green tissue-specific promoter rbcS promoter.
  • the promoter is "operably linked" to the locked gene or key gene.
  • the "operably connected” refers to an arrangement of elements that are configured to perform their normal function. For example, if a promoter affects the transcription of a coding sequence, it is operably linked to the coding sequence.
  • the "exogenous gene of interest” refers to a foreign gene which is not found in a recipient plant in a natural state.
  • a variety of exogenous gene of interest has been introduced into recipient plants for various purposes to increase plant resistance to pests, herbicides, and the like, and to increase and stabilize crop yields and the like.
  • the exogenous gene of interest may be an insect resistance gene or a herbicide resistance gene.
  • the insect-resistant genes include cry1Ab, cry1Ac, cry1Ab/1Ac, cry1C, cry2A and Vip3 insect-resistant genes from Bacillus thuringiensis, anf, sep gene from Serratia entomophila, The cmb gene from Clostridium bifermentans, the mtx gene from Bacillus sphaericus, the insecticidal protein gene from Xenorhabdus nematophilus, and Photorhabdus luminescens The tca, tcb gene, and the prl gene from Metarhizium anisopliae are not limited thereto.
  • nucleotide coding sequence and amino acid sequence of the Bt insect-resistant gene are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • the herbicide resistance gene comprises an EPSP synthase gene resistant to glyphosate, an EPSA mutant gene aroA of Salmonella typhimurium, a bar gene resistant to glufosinate, an ALS mutant gene Ilv G against moxazolinone, and an antibiotic.
  • Scleroside of AccL-s2 gene, anti-bromobenzonitrile The bxn gene and the csrl gene against chlorsulfuron are not limited thereto.
  • expression of the exogenous gene of interest is controlled by the locking element and the key gene of the present invention.
  • the foreign gene is operably linked to a locking element of the invention.
  • sequence encoding the exogenous gene of interest is also operably linked to appropriate regulatory factors, including promoters, enhancers, terminators, and signal peptides.
  • the invention provides a method of breeding a transgenic plant, the method comprising crossing a first parent comprising a locking element of the invention with a second parent comprising a key gene of the invention, thereby obtaining the transgenic plant.
  • the method for breeding a transgenic plant of the present invention further comprises introducing and integrating the locking element and a gene of interest linked thereto with the target gene into the first parental genome, and The key gene is introduced and integrated into the second parental genome.
  • the invention provides a method of breeding a transgenic plant, the method comprising introducing and integrating a locking element and a key gene of the invention into the genome of the same plant, thereby obtaining the transgenic plant .
  • Methods for introducing genes into recipient plants include, for example, Agrobacterium-mediated gene transformation, gene gun transformation, pollen tube pathway methods, etc., wherein Agrobacterium-mediated gene transformation is obtained in plant transformation.
  • Agrobacterium-mediated gene transformation is obtained in plant transformation.
  • the specific steps can be found in the attached embodiments.
  • specific exogenous genes such as marker genes for screening, can be isolated and eliminated from the transgenic plants by integration.
  • these transgenic plants after knocking out the marker gene are still included in the scope of the transgenic plants of the present invention.
  • the present invention provides a combination of genes for controlling expression of a foreign gene in a plant that is not expressed in a specific tissue, and a method of cultivating the transgenic plant using the combination of the genes.
  • the method can be used to cultivate endosperm 0-expressing transgenic anti-aphid rice, that is, there is no transgenic product egg in the rice endosperm produced by the aphid-resistant rice. White synthesis and accumulation. Therefore, public concerns about the safety of food for GM food crops can be ruled out.
  • the target Bt insect resistance gene was not expressed in the Japanese hyalinated rice endosperm of the obtained locking element and key gene, and the Bt protein detection result was not significantly different from the Nipponbare wild type control, that is, 0. However, it is highly expressed in the stem and leaf tissues of the designed expression. Since the target gene expression product was not detected in the rice endosperm, the inventors referred to it as the endosperm 0 expression type Bt transgenic rice, which was distinguished from the traditional endosperm expression type Bt transgenic rice.
  • the invention has broad applicability, and is suitable for a wide range of crops, such as rice, wheat, barley, oats, corn, corn, sorghum, glutinous rice, sweet potato, potato, lotus seed, soybean, and the like for the purpose of production of fruits, seeds, roots and tubers. Peanuts and so on.
  • FIG. 1 Map of pSB130 plasmid vector.
  • the plasmid vector has two T-DNA regions, one of which carries a hygromycin B-resistant marker gene (hpt) and the other carries a multiple cloning site (MCS) for a key gene or a gene of interest. The loading of the locked component sequence.
  • hpt hygromycin B-resistant marker gene
  • MCS multiple cloning site
  • Figure 2 is a flow diagram showing the construction of the expression vector pKey1 of the gene key (Key1; SEQ ID NO: 5) according to one embodiment of the present invention, wherein Figure 2A shows: the first step, the specificity of the recognition site by introducing KpnI and EcoRI The primers were amplified to obtain the NosT fragment and ligated into the corresponding multiple cloning site of pSB130; and in the second step, the Key1 3' fragment was amplified by specific primers introducing the XbaI/SmaI and KpnI recognition sites, and Linked to the corresponding multiple cloning site of pSB130; Figure 2B shows: In the third step, the Key1 5' fragment was amplified by specific primers introduced into the SalI and SmaI recognition sites, and ligated into pSB130 corresponding polyclonal At the locus; and the last step, the rbcS fragment was amplified by specific primers introducing the HindIII and SalI recognition
  • Figure 3 is a flow chart showing the construction of the expression vector pLY1 of the locking element (Lock1; SEQ ID NO: 8) and its linked reporter gene (eYFP) according to one embodiment of the present invention, wherein Figure 3A shows: The NosT fragment was amplified and cloned with pKey1; and in the second step, the Lock1 fragment was amplified by specific primers introduced into the PstI/XbaI recognition site and ligated into the corresponding multiple cloning site of pSB130; Figure 3B shows: In three steps, the ActinI fragment was amplified by specific primers introduced into the HindIII and PstI recognition sites, and ligated into the corresponding multiple cloning site of pSB130; and the last step was amplified with specific primers introducing XbaI and KpnI recognition sites. The eYFP fragment was obtained and ligated into the corresponding multiple cloning site of pSB130 to construct a pLY1
  • Figure 4 is a flow chart showing the construction of the expression vector pKey2 of the key gene (Key2; SEQ ID NO: 3) according to another embodiment of the present invention, wherein Figure 4A shows the first step of identifying a site by introducing SacI and EcoRI. The specific primers were amplified to obtain the NosT fragment and ligated into the corresponding multiple cloning site of pSB130. In the second step, the rbcS fragment was digested with the SalI and HindIII from the pKey1 expression vector and ligated into pSB130.
  • Figure 4B shows:
  • the Key2 3' fragment was amplified by specific primers introducing the SacI and XbaI recognition sites, and ligated into the corresponding multiple cloning site of pSB130;
  • the Key2 5' fragment was amplified by specific primers introducing the SalI and XbaI recognition sites, and ligated into the corresponding multiple cloning site of pSB130 to construct a pKey2 expression vector.
  • Figure 5 is a flow diagram showing the construction of the expression vector pLY2 of a locking element (Lock2; SEQ ID NO: 6) linked to a reporter gene (eYFP) according to another embodiment of the present invention, wherein Figure 5A shows the first step, using KpnI and EcoRI was digested with pLY1 to obtain a Nos fragment and ligated into the corresponding multiple cloning site of pSB130; and in the second step, the Actin I fragment was amplified by specific primers introducing the PstI and HindIII recognition sites, and was ligated.
  • Figure 5A shows the first step, using KpnI and EcoRI was digested with pLY1 to obtain a Nos fragment and ligated into the corresponding multiple cloning site of pSB130; and in the second step, the Actin I fragment was amplified by specific primers introducing the PstI and HindIII recognition sites, and was ligated.
  • FIG. 5B shows:
  • the Lock2 fragment was amplified by introducing SalI and PstI specific primers and ligated into the corresponding multiple cloning site of pSB130; and the last step was used.
  • KpnI and SalI were digested from pLY1 to obtain an eYFP fragment, which was ligated into the corresponding multiple cloning site of pSB130 to construct a pLY2 expression vector.
  • Figure 6 Locking element (Lock1; SEQ ID NO: according to one embodiment of the present invention) 8) A flow chart of the construction of the expression vector pLB of the addition gene (cry1Ab/1Ac), wherein Figure 6A shows: in the first step, a Nos fragment is obtained by amplification with a specific primer introducing a KpnI/SalI and EcoRI recognition site, and Linked to the corresponding multiple cloning site of pSB130; and in the second step, the specific fragment was introduced with the PstI and XbaI recognition sites to obtain a Lock fragment, which was ligated into the corresponding multiple cloning site of pSB130; Figure 6B shows In the third step, the ActinI fragment was amplified by specific primers introducing the HindIII and PstI recognition sites, and ligated into the corresponding multiple cloning site of pSB130; and the final step was to identify the specificity of the site by introducing KpnI and SalI. Prime
  • Figure 7 is a graphical representation of functional verification of a set of locking elements and key genes in accordance with one embodiment of the present invention, wherein pLY1-C+pKey1 is an undifferentiated pLY1 positive callus with no green dots and is re-keyed by the key gene pKey1
  • pLY1-C+pKey1 is an undifferentiated pLY1 positive callus with no green dots and is re-keyed by the key gene pKey1
  • pLY1-S+pKey1 was a differentiated pLY1 positive callus with green dots and then re-transformed and transiently expressed by key gene pKey1.
  • Figure 9 Copy number analysis of Nipponbare pKey1 and pLB positive transformed lines, wherein: Figure 9A shows the copy number analysis of 9 Nipponbare pKey1 positive transformed lines, and the results show that 6 of them are single copies; Figure 9B shows 10 selected The copy number analysis of Nipponbare pLB positive transformation lines showed that 6 of them were single copies. Combining the agronomic traits such as seed setting rate, plant height and growth period and other yield traits, two single-copy transformation lines were selected from pKey1 and pLB independent transformants (referred to as KEY and LB, respectively) (the red line in the figure shows For the subsequent production of LB/KEY and KEY/LB heterozygous parents, the selected material number is directly marked above the rectangular frame.
  • M is DNA Molecular weight Marker II (DIG-labeled)
  • P is a plasmid positive control
  • N is a non-transgenic wild type Nipponbare negative control.
  • Figure 10 The homozygous situation of a T 2 generation strain of Nipponbare pLB positive single copy transformation line and its PCR detection results, wherein: M-DL2000 stands for DL2000 DNA Marker; "+” stands for positive plasmid control, “-” stands for Non-transgenic negative control; 1-24 represents 24 independent individuals in one T 2 generation line of Nipponbare pLB positive single copy transformation line.
  • Figure 11 ELISA quantitative analysis of Bt protein content in stem and leaf tissues of 6 reciprocal crosses of Nipponbare and 2 pLB single copy transformation lines in the tillering, heading and filling stages, :KEY1, KEY2, LB3, and LB7 are single-copy pure parentes selected from pKey1 and pLB transformants; LB3/KEY1, LB7/KEY1, LB3/KEY2, LB7/KEY2, KEY1/LB3, and KEY2/LB3 are the above-mentioned singles. Copy the heterozygous species from the pure parent. T51-1 and Zheyou 3 were positive controls.
  • Figure 12 Locking element and key gene heterozygous LB3/KEY2 (left) and wild-type Nipponbare control (right) on artificially inoculated larvae of the first infested larvae under field conditions (about 100 worms per egg) Resistance performance of strain).
  • LB3/KEY2 left
  • wild-type Nipponbare control right
  • a high ratio of white spikes appears on the wild type control, and almost no white spikes appear on the transgenic outcrosses.
  • Figure 13 Resistance of in vitro leaves of LB3/KEY2 outcrossed leaves of locking element and key gene to indoor inoculated larvae of the first instar larvae, A: positive control TT51-1; B: wild type Minghui 63 Control; C: LB3/KEY2 outcrossed species; D: wild type Nipponbare negative control; E: Nipponbare KEY2 parental transformation line; F: Nipponbare LB3 parental transformation line.
  • the picture shows that the resistance of transgenic heterozygous LB3/KEY2 to Chilo suppressalis is consistent with the positive control T51-1.
  • Figure 14 Resistance performance of isolated stalks of locking elements and key genes LB3/KEY2 outcrosses and wild-type Nipponbare negative control (Nip-CK) to indoor artificial inoculation of the first instar larvae of Chilo suppressalis.
  • the picture shows that no signs of harm to the stem borer were observed on the stems of the transgenic LB3/KEY2 outcrossed stalks, and almost all of the inoculated mites were killed; while the wormholes and phlegm that were harmed by the stem borer were observed on the control stems. Slots and their excreta, and there are surviving larvae.
  • Example 1 Design of the gene combination of the present invention and its feasibility study and verification
  • the locking element (Lock) and the key gene (Key) that can specifically open the locking element are composed of two components.
  • two sets of such components have been designed, wherein the DNA sequences of the first set of components are SEQ ID NO: 5 (Lock1) and SEQ ID NO: 3 (Key1), respectively, and the DNA sequence of the second set of components Is SEQ ID NO: 8 (Lock 2) and SEQ ID NO: 6 (Key 2).
  • the locking element is designed to be placed between the constitutive promoter of Actin I and the start codon of the gene of interest, and its function is to prevent the expression of the gene of interest: while the key gene is designed to be placed in rbcS. Under the control of a green tissue-specific expression promoter, its function is to open the expression of the gene of interest locked by the locked element in a specific tissue (such as green tissue).
  • a second set of expression vectors was constructed, in which some functional fragments were obtained from the first set of expression vectors by restriction enzyme digestion, and the other functional fragments still passed specific primers (Table 1). And high-fidelity PCR amplification technology was amplified, and then these functional fragments were ligated into the pSB130 plasmid vector (Fig.
  • the application of the applied expression vector is mainly directed to the locking element and the target gene.
  • the fragment is carried out.
  • a functional fragment which is partially versatile with a validated expression vector is obtained by enzymatic cleavage, and on the other hand, the first set of expression vectors selected in the examples of the present application are respectively obtained by specific primers (Table 1) and high-fidelity PCR amplification techniques.
  • the locking element and the two functional fragments of the insect-resistant gene to be ligated are then ligated into the pSB130 plasmid vector (Fig.
  • the pSB130 plasmid vector used in the examples of the present application comprises two "T-DNA” regions, wherein one "T-DNA” region is used to load a locking element or a key gene expression unit which is linked to a gene of interest, and the other is used for loading a moisture-proof mold.
  • Prime marker gene Hpt expression unit The purpose of constructing the double T-DNA expression vector is to focus on the practical application in the future, because the double T-DNA plasmid vector can transform the rice to enable the target gene and the marker gene to have independent integration in the receptor genome, so as to facilitate the marker gene. Separation and rejection by self-crossing in subsequent segregation generations.
  • the verification of the gene combination locking and opening function of the present invention is carried out using a two-step transformation method.
  • the two locking elements pLY1 and pLY2 in the validated expression vector prepared in Example 1 were introduced into rice embryogenic callus for permanent expression by Agrobacterium-mediated method (Liu et al., 1998). After that, after several consecutive rounds (3-5 rounds, 12-14 days/round) of antibiotic screening, resistant callus pre-differentiation (7-9 days) and differentiation culture until green spots are formed (7-9 days);
  • the second step using the gene gun-mediated method (Tu et al., 2000), the two key genes pKey1 and pKey2 in the validated expression vector were bombarded into a differentiated positive callus with green spots and no green spots.
  • the undifferentiated positive callus was transiently expressed, and the luminescence of the yellow fluorescent protein was observed under confocal microscopy after 24-36 h. If the designed gene combination has complete locking and unlocking functions, the green dots transformed by pKey1 and pKey2 Undifferentiated positive callus does not fluoresce at all, and they are fluorescing when they are transformed with green dot differentiation positive callus. The detailed test procedures are described below:
  • the receptor variety used for genetic transformation of the above-described verification type locking elements pLY1 and pLY2 is indica Nipponbare. This variety is a model of rice genetic transformation, and its mature embryo callus is easy to induce and has high genetic transformation efficiency.
  • Induction/subculture medium (per liter): 4.1 g/L N6 (Chu et al, 1975) base salt component + N6 organic component (Table 2) + 2 mg/L 2,4-dichlorophenoxy Acetic acid (2,4-D)+2.0g/L hydrolyzed casein+30g/L sucrose+3g/L agar, pH 5.9
  • Infectious medium per liter: AA basal medium (Table 3) (Toriyama & Hinata, 1985) + 200 ⁇ M acetosyringone, pH 5.9.
  • Co-cultivation medium per liter: CC basal medium (Table 4) (Hiei et al, 1994) + 200 ⁇ M acetosyringone, pH 5.9.
  • Antibacterial medium per liter: induction/subculture medium + 500 mg/L cephalosporin, pH 5.9.
  • Screening medium (per liter): induction/subculture medium + 50 mg/L hygromycin + 500 mg/L cephalosporin, pH 5.9.
  • Regeneration medium (per liter): 4.1g/L N6 base salt component + N6 organic component (Table 2) + 2.0g / L hydrolyzed casein + 30g / L sucrose + 6g / L agar + 2mg / L Kinetin +1 mg/L ⁇ -naphthylacetic acid, pH 5.9.
  • N6 basic salt component (Shanghai Shenggong) and organic components
  • the genetic transformation of the proof-type locking element expression vectors pLY1 and pLY2 was carried out using Agrobacterium-mediated methods.
  • the Agrobacterium strain used was EHA105 (Bio Vector NTCC) Inc.), the recipient cell used is an embryogenic callus induced by Nipponbare mature embryos.
  • the specific steps of Agrobacterium transformation are as follows:
  • the nascent callus was cut and transferred to a new callus induction and subculture medium, subcultured under the same conditions, subcultured every 2 weeks until the cytoplasm was thick, the color was bright yellow, the texture was hard and the cell mass was present. Granular embryogenic callus formation.
  • 0.5 ⁇ L of the expression vector plasmid was added to a 1.5 mL centrifuge tube containing 60 ⁇ L of Agrobacterium electroporation EHA105. After the gun was sucked and mixed, it was transferred into the electrode cup. After the electric shock, 1 mL of LB liquid medium was quickly added and sucked. After mixing, transfer to the previous 1.5mL centrifuge tube and shake it for 1h at 28 °C. After the bacterial solution is resuscitated, draw 100 ⁇ L of the bacterial solution and apply it evenly to the LB solid screening (containing 50 mg/L kanamycin, 25 mg).
  • Agrobacterium infection was carried out according to the method steps reported by Yang et al (2011). The specific procedure is as follows: the Agrobacterium liquid stored at -80 ° C is taken out, and 200 ⁇ L of the solution is uniformly applied to the surface of the LB solid medium containing 25 mg/l of rifampicin and 50 mg/L of kanamycin at 28 ° C. The culture was carried out overnight; then the single colony was selected and expanded with LB liquid medium; then, 200-300 ⁇ L of fresh bacterial solution was taken from the solution and added to 20 mL of LB liquid culture containing 25 mg/L rifampicin and 50 mg/L kanamycin.
  • the medium was incubated at 28 ° C with shaking (220 rpm) for 16-18 h. After taking enough bacteria to centrifuge at 4000 rpm for 15 min, discard the supernatant of LB medium; add 20 mL of 0.1 M MgSO 4 solution to resuspend Agrobacterium (lightly pipetted with a pipette), and centrifuge at 4000 rpm for 10-15 min. The MgSO 4 supernatant containing antibiotics was discarded; 5 mL of AA infecting medium containing 200 ⁇ M acetosyringone (AS) was added (Table 3) to resuspend Agrobacterium, and then added with appropriate amount of AA-AS infection culture. Base, the OD 600 value of the bacterial liquid is finally adjusted between 0.8-1.0; after the concentration adjustment, the bacterial liquid is dispensed in a sterile 50 mL centrifuge tube, 20-25 mL / tube, and used.
  • AS acetosyringone
  • the embryogenic callus was pre-cultured for about 7 days, and then transferred from the subculture dish to the empty culture dish covered with sterile filter paper, and air-dried on the ultra-clean workbench 10- After about 15 minutes, slowly tumbling the callus with a sterilized teaspoon to make it fully dry; after drying, transfer it to a centrifuge tube containing the bacterial liquid, gently shake it at room temperature for 40 minutes, and centrifuge the tube for super Leave on the net work bench for 10 min; remove the bacterial solution, place the embryogenic callus on sterile filter paper for about 15 minutes, then transfer it to the surface with a sterile filter paper containing AS (200 ⁇ M) CC.
  • the culture medium (Table 4) was cultured at 28 ° C for 50-55 h; the embryogenic callus which was not grown or contaminated by Agrobacterium was selected and transferred to 2,4-D, 500 mg containing 2.0 mg/L. /L cefotaxime (Cefortaxim) on the N6 bacteriostatic medium, bacteriostatic culture in the dark room at 28 °C for 3-4 days; then the callus after bacteriostatic culture was transferred to 500mg/L cephalosporin and 50mg/L On the screening medium of Hygromycin, culture in a dark room at 28 ° C; picking up well-grown callus every half month for subculture, The concentration of cephalosporin in the culture is adjusted according to the degree of pollution. In general, the concentration can be reduced by half in the third or fourth round. This was subcultured until a rapidly growing, large, and vivid resistant callus was obtained (4-6 rounds of screening and subculture).
  • the resistant callus obtained in the previous step was transferred to N6 regeneration medium, pre-differentiated for one week at 28 °C, then transferred to fresh N6 regeneration medium, and cultured in a light chamber at 25 ° C until green dot formation ( It takes about two weeks).
  • the green callus was formed into a receptor in the above step, and the green-type differentiation of the key gene expression vectors pKey1 and pKey2 prepared according to Example 1 was introduced by the gene gun-mediated method described in Tu et al (2000). The callus and the undifferentiated callus without green spots were transiently expressed. After 24 to 36 h of recovery, the fluorescence signal of yellow fluorescent protein was observed under a confocal microscope and photographed and stored.
  • the genetic transformation of the application-type locking element and the key gene was carried out by Agrobacterium-mediated method.
  • the selection of the acceptor material, the various media used, the genetic transformation of the vector and the positive resistance callus Both the differentiation and differentiation cultures were identical to the corresponding test procedures described in Example 2 until the positive resistant callus differentiated into green shoots.
  • the obtained green seedlings after washing the medium on the root system, or directly (the simultaneous differentiation of root buds), or after the rooting of the N6 rooting medium (the bud-differentiated type) is transferred into the Yoshida culture solution (Table 5) After cultivation, after the growth state is good and stable, transplant it to the greenhouse until it matures.
  • the supernatant was centrifuged at 4000 rpm for 20 minutes at room temperature (temperature > 18 ° C), shaken by adding 1/10 volume of pre-heated 10 X CTAB at 56 ° C, and once again added a volume of 24:1 (chloroform: isoamyl alcohol) and gently swirled. Shake for 30 minutes. Centrifuge at 4000 rpm for 20 min at room temperature (temperature > 18 ° C). After that, the supernatant was aspirated with a 5 mL pipette tip, and then a volume of 1 X CTAB was added. After the shaking, flocculation of DNA was observed, and the tip was used to pick it out at 1.5.
  • the bottom of the tube was centrifuged and poured on absorbent paper for 10 min. Then 10 ⁇ L of RNase and 0.5 ml of sterilized 1 M NaCl were added and dissolved in a water bath at room temperature or 56 °C. The dissolved DNA was added to a 2-fold (-20 ° C) volume of -20 ° C pre-cooled 95% ethanol, and the DNA was flocculated and precipitated by shaking. Use the tip of the gun to pick up the flocculent DNA and put it into a 1.5 mL centrifuge tube, add 1.5 mL of 75% ethanol to wash the DNA, then pour the ethanol, add ultrapure water and store at 4 ° C for later use.
  • Detection and pure line identification of positive transgenic plants were performed using PCR techniques. Due to Hpt The marker gene and the locking element or key gene are co-transformed by double T-DNA, and the identification of the T0 generation positive transformation line is based only on the gene of interest. Therefore, only the plants whose target genes are positive after the test are continued to be cultivated to maturity, and the non-positive plants are all eliminated to reduce the workload of subsequent generations.
  • the anti-hygromycin gene amplification primers used were: Hpt-F, 5'-GCTGTTATGCGGCCATTGTC-3' (SEQ ID NO: 9) and Hpt-R, 5'-GACGTCTGTCGAGAAGTTTC-3' (SEQ ID NO: 10);
  • the PCR reaction system 25 ⁇ L system was: 1 ul of DNA template to be tested, 2.5 ⁇ L of 10 ⁇ PCR buffer, 2 ⁇ L of 10 mM dNTP, 0.25 ⁇ L of 20 uM primer, 0.5 ⁇ L of 2 U/ ⁇ L Tagase, and ddH 2 O to 25 ⁇ L.
  • the PCR reaction procedure was: denaturation at 94 ° C for 3 min; followed by denaturation at 94 ° C for 30 ec, annealing at 55 ° C for 30 sec, and extension at 72 ° C for 40 sec for 35 cycles; thereafter, extension at 72 ° C for 5 min at 10 ° C.
  • the amplified products were identified by 1% agarose gel electrophoresis and stored after photographing.
  • the Bt gene amplification primer used was: BtF, 5'-GGCCATACAACTGCTTGAGT-3' (SEQ ID NO: 11); BtR, 5'-GCGTTTCCCATAGTTCCATA-3' (SEQ ID NO: 12), and the amplified fragment was 1 Kb in length.
  • the PCR reaction procedure was: denaturation at 94 ° C for 3 min; subsequent denaturation at 94 ° C for 30 sec, annealing at 55 ° C for 30 sec, extension at 72 ° C for 1 min for 35 cycles; thereafter, extension at 72 ° C for 5 min at 10 ° C.
  • the amplified products were identified by 1% agarose gel electrophoresis and stored after photographing.
  • the PCR reaction system is shown in Table 6.
  • the key gene amplification primer used was: KeyF, 5'-AACGAGTGATGAGGTTCGCA-3' (SEQ ID NO: 13); KeyR, 5'-ACCCGGCAAAACAGGTAGTT-3' (SEQ ID NO: 14), and the amplified fragment was 672 bp in length.
  • the PCR reaction procedure was: denaturation at 94 ° C for 3 min; subsequent denaturation at 94 ° C for 30 sec, annealing at 55 ° C for 30 sec, and extension at 72 ° C for 1 min for a total of 35 cycles; thereafter, extension at 72 ° C for 7 min at 4 ° C.
  • the amplified products were identified by 1% agarose gel electrophoresis and stored after photographing.
  • the PCR reaction system is shown in Table 6.
  • 21 pKey1 and 22 pLB positive transformed lines were identified from 41 pKey1 and 50 pLB independent transformed lines of Nipponbare, respectively.
  • Transgenic copy number detection was performed using Southern hybridization techniques. First, the total rice DNA was digested with restriction endonuclease HindIII overnight at 37 °C (8-12 h); the digested DNA was separated by 0.8% (w/v) agarose gel electrophoresis (40V). , 12h), after which the electrophoreticly separated DNA blot was transferred to a nylon membrane (GE Healthcare, UK). Preparation of hybridization probes and Southern hybridization and hybridization signal detection were carried out according to the instructions provided by Roche DIG-High Prime DNA Labeling and Detection Starter Kit II (Switzerland), wherein the hybrid probe preparation primers were respectively :
  • pKey1 Key-1U20, 5'-ATGTCCAATTTACTGACCGT-3' (SEQ ID NO: 15), and
  • pLB Bt-577U24, 5'-AGGCTGATTGGAAACTACACCGAC-3' (SEQ ID NO: 17), and Bt-1161L24, 5'-ACAGCGGATGGCAAGTTAGAAGAG-3' (SEQ ID NO: 18).
  • Fig. 9 The results of copy number analysis of the pKey1 and pLB positive transformed lines of Nipponbare are shown in Fig. 9. As can be seen from Fig. 9, in the 9 parts of the Nipponbare pKey1 positive transformation line (Fig. 9A) detected, 6 copies were single copies, and in the 10 pLB positive transformation lines (Fig. 9B) detected, there were also 6 copies. It is a single copy.
  • Fig. 10 shows the results of PCR detection of one T 2 generation homozygous strain of Nipponbare pLB single copy transformation line. As shown in Fig. 10, 24 individuals of the T 2 generation strain showed a positive LB gene amplified fragment, indicating that the T 2 generation strain was an LB transgenic pure line. PCR amplification assays of other pLB and pKey1 transgenic lines were performed as follows (data not shown).
  • Example 5 Determination of Bt protein content and identification of insect resistance of stem and leaf of heterologous species with applied locking elements and key genes
  • all tested materials included wild-type Nipponbare negative control, 1 KEY (KEY1) and 2 LB (LB3 and LB7) transformed line parents and were configured by the above pKey1 single copy transgenic line and pLB single copy transgenic line.
  • test materials were the same as in 2014. All the tested materials were sown on May 25, and after 15 days, Bt Cry1Ab/1Ac transgenic rapid test strips (Cat. No.: AA0232, Youlong, Shanghai) were tested and confirmed as positive cross-species seedlings and wild-type control and parental materials. The seedlings were planted in 2 rows/cells, 12 plants/row, and repeated 3 times. On June 25, they were transplanted into the mud pool of the special genetic isolation grid of the transgene, planted in wide and narrow rows, 40 cm wide and 15 cm narrow. The plant spacing is 20cm, and the rest of the cultivation management is the same as in 2014.
  • the Bt protein content in the stalks and leaves of Nipponbare KEY/LB and LB/KEY was detected by Enzyme Co. (EnviroLogixTM Inc., Portland, USA) enzyme-linked immunosorbent assay (ELISA) kit. Rice stem and leaf samples were sampled in three stages: tillering stage, heading stage and filling stage.
  • Enzyme Co. EnviroLogixTM Inc., Portland, USA
  • ELISA enzyme-linked immunosorbent assay
  • the working fluid is determined to include an extract/diluent and an eluent.
  • the eluent is prepared by adding the phosphate attached to the kit to 1 L of double distilled water and then stirring to completely dissolve it.
  • the extract/diluent was prepared by taking 200 ⁇ L of the eluate and adding 1 mL of Tween-20 to dissolve completely. Both working solutions were stored at 4 ° C and preheated to room temperature before use.
  • Each replicate was set up with 3 replicates, 1 strain/repeat, and the fresh sample retrieved with liquid nitrogen was ground into a powder. Each material took about 20 mg of powder, and the extract/diluent was added to the mixture at a ratio of 20 mg/500 ⁇ L.
  • the dilution ratios of stem and leaf samples at various times are shown in Table 7.
  • the ELISA reaction process is carried out according to the instructions of the kit. The specific steps are as follows:
  • the absorbance values were measured with a microplate reader (Synergy H1, USA) and read at a wavelength of 450 nm.
  • a standard curve is drawn using the concentration and absorbance of the standard sample, the concentration of the test sample is read on the standard curve, and the protein content in the rice tissue is calculated based on the dilution factor.
  • Figure 11 The results of the two-year analysis are shown in Figure 11: In the three growth stages, the leaves of the six positive and negative crosses contained roughly the same as the positive control T51-1 and its two-line hybrid rice Zheyou 3 (Zheyou 3). Or higher Bt protein content ( Figures 11A and 11C); and Bt consistent with Zheyou 3 positive control in 3 outcrossed species LB3/KEY2, LB7/KEY2 and KEY2/LB3 stems The protein content, the other three outcrossed species LB3/KEY1, LB7/KEY1 and KEY1/LB3 stems contained lower Bt protein content than the above two positive controls ( Figures 11B and 11D).
  • the KEY/LB and LB/KEY positive and negative crosses were used to evaluate the field insect resistance of the stem borer.
  • the natural insect method and the artificial insect method were used.
  • the natural insecticide method is carried out 5-7 days after the peak of the damage of the stem borer.
  • Survey indicators include the number of dead hearts and the number of white spikes.
  • the artificial insects were divided twice in the highest tillering period and one week before heading.
  • the eggs were purchased from Jiangxi Shennong Technology Co., Ltd., and the purchased eggs were placed in a glass test tube with a length of 12 cm and a diameter of 2.0 cm and a wet cotton ball on the bottom, an egg block/tube, a cotton plug, and a nozzle. Put a black cloth on the outside.
  • Egg hatching was carried out at a temperature of 26-28 ° C, 80% humidity and 16 hours light / 8 hours dark.
  • the hatched first-instar larvae were inoculated with the test tube to the base of the rice blast within 6 hours after hatching.
  • the body operation is as follows: open the black cloth and remove the tampon, insert the base of the test tube into the paddy soil, and make the tube mouth close to the rice straw, so that the larva can climb from the test tube to the rice straw for damage.
  • the amount of insects was 1 egg block/strain, about 80 first-instar larvae.
  • Two weeks later, the number of dead hearts or white spikes of the damage of the stem borer was investigated, and at the same time, the total number of tillers or the total number of effective spikes of the test plants were investigated.
  • the field insect resistance of LB/KEY and KEY/LB reciprocal crosses was evaluated by the natural insect inoculation method.
  • the number of damaged leaves and the degree of damage of leaves per plant damaged by Cnaphalocrocis medinalis were carried out 5-7 days after the occurrence of the hazard peak.
  • Table 8 6 LB / KEY and hybrid combinations KEY / LB, 3 and KEY th LB of the parents and the wild type Nipponbare, and controls the resistance of Chilo suppressalis and Cnaphalocrocis medinalis performance under field conditions a
  • Table 9 Resistance performance of LB3/KEY2 hybrid combinations to inoculated sorghum in the tillering stage and 7 days before heading in field conditions
  • the indoor insect resistance evaluation was carried out for the stem borer, and the eggs were also purchased from Jiangxi Shennong Technology Co., Ltd., and the egg hatching method was the same as before.
  • the methods used for the evaluation of insect resistance were isolated stems and leaf worms.
  • the method of in vitro stem stalk is to take the stem of rice after jointing, cut into 12cm long section and stem section of leaf sheath. Each insect tube is placed in two stem segments of the same plant and 20 heads are in the same age. ⁇ larvae. The insect tube was sealed with a cotton plug, placed at 25-27 ° C and 80% relative humidity, and the larval mortality was counted after 6 days.
  • the in vitro leaf worm method is to take the blade leaves and the second leaf leaves at the booting stage, cut into 8 cm long leaf segments, and place them in a petri dish with filter paper moistened with 2 mL of distilled water, and place 4 segments of each culture dish. And 15 first-instar larvae of the mites, and wrapped with PARAFILM four times to prevent larvae from drilling out. Thereafter, they were housed at room temperature (25 ° C), and the mortality of the larvae was counted after 6 days.
  • Example 6 Determination of Cry1Ab/1Ac protein content in endosperm of application-type locking element/key gene heterologous seed
  • the present invention further determined the content of Cry1Ab/1Ac protein in the endosperm of the six locking element/key gene outcrosses described in Example 5.
  • the Cry1Ab/1Ac protein content in Nipponbare pKey1/pLB heterologous endosperm was also determined using an enzyme-linked immunosorbent assay (ELISA) kit from EnronoLogixTM Inc. (Portland, USA).
  • ELISA enzyme-linked immunosorbent assay
  • the working fluid is determined to include an extract/diluent and an eluent.
  • the eluent is prepared by adding the phosphate attached to the kit to 1 L of double distilled water and then stirring to completely dissolve it.
  • the extract/diluent was prepared by taking 200 ⁇ L of the eluate and adding 1 mL of Tween-20 to dissolve completely. Both working solutions were stored at 4 ° C and preheated to room temperature before use.
  • Each hybrid combination consists of 3 replicates, 1 strain/repeated, 50 seeds/strain.
  • the harvested seeds are removed from the hulls and prepared into brown rice for use.
  • some brown rice is ground into polished rice by using a rice mill, and then 50 grains of brown rice are separately selected.
  • the polished rice sample was ground into a powder, and about 20 mg of powder was taken from each material, and the extract/diluted solution was added in a ratio of 20 mg/500 ⁇ L to sufficiently oscillate to extract a protein sample.
  • the turbulent homogenate was placed in a 1.5 mL centrifuge tube, allowed to stand on ice for 30 min, centrifuged at 4 ° C for 3 min at 4000 rpm, and the supernatant was aspirated for use.
  • the protein sample extracted from the polished rice and brown rice of the positive control T51-1 and its two-line hybrid rice hybrid Zheyou 3 was diluted 50 times with extraction/diluent, and then the Bt protein concentration was measured.
  • the heterozygous and negative controls do not need to be diluted.
  • the ELISA reaction process was carried out according to the instructions of the kit, and the specific steps were the same as those of the aforementioned stem and leaf tissue.
  • the content of Cry1Ab/1Ac protein determined in some outcrossed brown rice was significantly higher than that of the wild type Nipponbare negative control brown rice (less than 1 ng/g powder) (Table 11), possibly with
  • the peel in the brown rice cortex is related to the chloroplast at the beginning of development.
  • the cortex of the brown rice was crushed as a layer of ruthenium, and therefore, a very small amount of Cry1Ab/1Ac protein accumulated therein was also removed.
  • a. Indicates that the data in the table is measured from 50 grains/test material/repeated random sampled kernels (three repetitions in total);
  • WT is a wild-type Nipponbare negative control, and the measured data of all other strains are compared. Data were analyzed using the Dunnett t test of the SPSS 22.0 software package.
  • c. Values represent mean ⁇ mean standard deviation. * indicates a difference of P ⁇ 0.05 from the wild type Nipponbare negative control. ** indicates a difference of P ⁇ 0.01 with the wild type Nipponbare negative control.
  • the present invention provides a nucleotide sequence of a foreign gene Bt, a gene lock Lock1/Lock2 and a gene key Key1/Key2 for constructing a "gene combination" and a deduced amino acid sequence thereof, wherein the Bt gene cry1Ab/1Ac is from China Academician Fan Yunliu of the Academy of Agricultural Sciences was artificially synthesized and provided for use; the locking element Lock1/Lock2 and the key gene Key1/Key2 were artificially synthesized.
  • the sequences listed in the sequence listing are as follows.

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Abstract

提供了一种用于控制外源基因不在植物特定组织中表达的新的基因组合，以及应用该基因组合培育转基因植物的方法，该方法可用于培育例如胚乳0表达型转基因水稻，即由所述水稻所产生的稻米胚乳中没有任何转基因产物蛋白的合成和积累。

Description

基因组合及其用途 技术领域
本发明涉及转基因植物领域。具体地，本发明涉及一种用于控制植物中的外源目的基因的表达的基因组合，以及应用该基因组合培育转基因植物的方法。
背景技术
转基因技术自上世纪八十年代发明以来取得了举世瞩目的成就。培育的转基因作物已在全球28个国家种植，2014年生产面积已达1.815亿公顷(James，et al.，2014)。据英国PG经济公司的布鲁克斯和巴菲特提供的最新即时信息表明，自1996至2013年的17年间，转基因作物提高了作物生产价值1330亿美元，节省农药活性成分5亿kg，仅在2013年就减少二氧化碳排放量28亿kg，相当于1240万辆汽车一年的尾气排放量，帮助缓解了一些在世界上最贫穷的国家超过165万小农场和他们的家庭共计6500万人的贫困(James，et al.，2014)。因此他们得出的结论是转基因作物继续对食品安全、可持续发展和气候变化产生显著的正影响。目前，已有超过10个粮食和纤维作物被批准商业化种植，范围从大宗的粮食、经济作物如玉米、大豆和棉花等到小宗的水果、蔬菜作物如木瓜、茄子和南瓜等。
水稻是全球50％人口的主粮作物。尽管已于2000年宣告成功研制出转基因水稻系(Tu，et al.，2000)，例如优良的转基因恢复系明恢63/Bt(华恢1号)及其杂交稻汕优63/Bt，并于2009年11月在经过研制方和第三方反复验证其无生物安全性风险的情况下，获得我国农业部颁发的生物安全证书。然而，由于这些传统的转Bt基因抗虫水稻是胚乳表达型的，即所生产的转基因大米中有Bt基因的表达产物积累，依然很难使公众认可它的食用安全性，其商业化种植也因此一直未获批准。这种状况不仅在中国是如此，在世界其它一些地区和国家如欧洲和日本也是如此。2006年在欧洲调查的人群中有58％的人认为转基因食品“不应该被鼓励”。
为了解决人们对转基因植物生态安全性和食品安全性的恐惧和担 忧，许多安全生物技术先后被研制出来，如转基因花粉不育技术(Paoletti and Pimentel，1996)、种子不育或无籽技术(Daniell，2002；Li，1998)、叶绿体转基因技术(Bock，2001)、“终结者”种子技术(Oliver，et al.，1998)和外源基因删除技术(Luo，et al.，2007)等。在这些技术中，影响较大的是美国康涅狄格大学李义教授及其学生罗克明等人研发的外源基因删除技术。该技术是在转基因开花前后自动除去花粉和种子中的外源基因，如同计算机“卸载”应用软件一样，使转基因植物中的外源基因在扩散前和人们使用前被自动清除(赵德刚等，2008)。该技术可直接应用于无性繁殖作物，但还需改进才能应用于有性繁殖作物。主要的问题是有性繁殖作物种子中的外源目的基因一旦被删除就不再保有，从而使通过种子繁殖的下一代丢失转基因性状。
因此，在转基因粮食、蔬菜和水果等作物上，迫切需要能够在可食用的组织或器官中关闭目的基因表达，但又不至于在通过种子繁殖的下一代中丢失转基因性状的新型生物技术体系。
发明内容
出于以上目的，本发明人研发了能够在特定组织/器官(例如，胚乳中)关闭目的基因表达的基因组合。该技术主要由能锁定外源目的基因表达的锁定元件和能开启被锁定的外源目的基因表达的钥匙基因两个组件构成。在独立存在条件下，前者携带的外源目的基因是永久锁定的，而后者携带的能开启被锁定的外源目的基因表达的钥匙基因仅在特定组织/器官中是表达的。将这两个组件分别转化和整合到诸如水稻的父母本基因组中，然后通过杂交生产就可以将其综合到一起，也就可以获得能在所述特定组织/器官中开启外源目的基因表达，以致出现期望的表型，而在特定组织/器官(例如食用器官，如水稻胚乳)中则始终关闭该外源目的基因表达，从而产生在供食用的部分中不含任何转基因成分的转基因植物。同样地，将这两个组件分别导入和整合到同一个受体植株的基因组中，也能获得有同样的效果。同时，不存在由于目的基因DNA在繁殖过程中被删除而不能遗传的问题，因此，拥有广泛的适用性。
具体地，在第一个方面，本发明提供了一种用于控制植物中的外源目的基因的表达的基因组合，其由锁定元件和钥匙基因组成，其中 所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
一方面，本发明提供了一种用于在植物中锁定与其连接的外源目的基因的表达的锁定元件，其包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列。
相应地，一方面，本发明提供了一种用于在植物特定组织中解除所述锁定元件对于外源目的基因的表达锁定的钥匙基因，其包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
在第二方面，本发明提供了一种用于控制植物中的外源目的基因的表达的方法，所述方法包括将锁定元件和钥匙基因导入受体植物中，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
一方面，本发明提供了一种用于控制外源目的基因在植物中表达的方法，所述方法包括将所述外源目的基因可操作连接至锁定元件，所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列。
相应地，一方面，本发明提供了一种用于解除所述锁定元件对外源目的基因在植物中的表达锁定的方法，所述方法包括将钥匙基因导入所述植物中，所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序 列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
在第三方面，本发明提供了一种培育转基因植物的方法，所述方法包括将包含锁定元件的第一亲本与包含钥匙基因的第二亲本杂交，从而获得所述的转基因植物，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
在具体实施方式中，所述方法还包括在所述杂交步骤之前，将所述锁定元件导入和/或整合到所述第一亲本的基因组中，以及将所述钥匙基因导入和/或整合到所述第二亲本的基因组中。
在可选的实施方式中，本发明提供了一种培育转基因植物的方法，所述方法包括将将锁定元件和钥匙基因导入和/或整合到同一植株的基因组中，从而获得包含所述锁定元件和钥匙基因的转基因植物，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
在第四方面，本发明提供了锁定元件和钥匙基因的组合用于调控植物中的外源目的基因的表达的用途，其中所述锁定元件能够锁定与其可操作连接的外源目的基因的表达；所述钥匙基因能够解除所述锁定元件对于外源目的基因的表达锁定。
在该方面的具体实施方式中，所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的 核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
根据本发明的一些具体实施方式，所述基因组合为包含SEQ ID NO：5或与SEQ ID NO：5所具有至少80％的同源性的核苷酸序列的锁定元件，和包含SEQ ID NO：3或与SEQ ID NO：3所具有至少40％的同源性的核苷酸序列的钥匙基因。
根据本发明的另一些具体实施方式，所述基因组合为包含SEQ ID NO：8或与SEQ ID NO：8所具有至少80％的同源性的核苷酸序列的锁定元件，和包含SEQ ID NO：6或与SEQ ID NO：6所具有至少40％的同源性的核苷酸序列的钥匙基因。
根据本发明的各个方面，所述锁定元件位于组成型启动子和所述外源目的基因之间，并与所述组成型启动子和所述外源目的基因可操作连接。
根据本发明的各个方面，所述钥匙基因与组织特异性启动子可操作连接。
根据本发明的各个方面，所述组成型启动子选自花椰菜花叶病毒(CaMV)35S启动子、来自根癌农杆菌Ti质粒T-DNA区域的胭脂碱合成酶基因Ocs启动子、水稻肌动蛋白启动子Actin I、玉米泛素(ubiquitin)基因启动子Ubi；优选地，所述组成型启动子是水稻肌动蛋白启动子Actin I。
根据本发明的各个方面，所述组织特异性启动子选自水稻绿色组织特异性启动子核酮糖-1，5-二磷酸羧化酶小亚基rbcS启动子(Kyozuka et al，1993；Nomura et al，2000)、玉米磷酸烯醇式丙酮酸羧化酶PEPC启动子(Yanagisawa et al，1989)、水稻绿色组织特异表达DX1启动子(Ye et al，2012)、水稻光系统II10kDa多肽D540启动子(Cai et al，2007)、水稻富亮氨酸重复序列类受体蛋白激酶LP2启动子(Thilmony et al，2009)，以及玉米叶绿体C4Pdk启动子(Jang et al，1999)；优选地，所述组织特异性启动子是水稻绿色组织特异性启动子rbcS启动子。
根据本发明的各个方面，所述外源目的基因可以是抗虫基因或抗除草剂基因。所述抗虫基因包括来自苏云金芽胞杆菌(Bacillus  thuringiensis)的cry1Ab、cry1Ac、cry1Ab/1Ac、cry1C、cry2A和Vip3抗虫基因、来自嗜虫沙雷氏菌(Serratia entomophila)的anf、sep基因，来自双酶梭菌(Clostridium bifermentans)的cmb基因、来自球形芽孢杆菌(Bacillus sphaericus)的mtx基因、来自嗜线虫致病杆菌(Xenorhabdus nematophilus)的杀虫蛋白基因、来自发光光杆状菌(Photorhabdus luminescens)的tca、tcb基因，以及来自金龟子绿僵菌(Metarhizium anisopliae)的prl基因，但不限于此。
根据本发明，所述Bt抗虫基因的核苷酸编码序列和氨基酸序列分别如SEQ ID NO：1和SEQ ID NO：2中所示。
根据本发明的各个方面，所述抗除草剂基因包括抗草甘膦的EPSP合成酶基因、鼠伤寒沙门氏菌EPSP突变基因aroA、抗草丁膦的bar基因、抗米唑啉酮的ALS突变基因Ilv G、抗稀禾定的AccL-s2基因、抗溴苯腈的bxn基因和抗绿磺隆的csrl基因，但不限于此。
根据本发明的各个方面，所述植物选自水稻、小麦、大麦、燕麦、玉米、粟米、高粱、薏米、红薯、马铃薯、莲子、大豆和花生。优选地，所述植物为水稻。
本文中使用以下定义来进一步定义和描述本公开。除非在特定情况下另有限定，否则这些定义适用于本说明书中通篇所用的术语。
除非另有定义，本文所用的所有技术和科学术语的含义均与本发明所属领域的普通技术人员通常理解的一样。如发生矛盾，则以本说明书，包括本文给出的定义为准。
锁定元件(LOCK)和钥匙基因(KEY)
本发明人经DNA重组发明了一组具有调控基因表达功能的调控元件和基因序列，并且根据其各自的功能分别命名为锁定元件(LOCK)和钥匙基因(KEY)。
所述锁定元件能够锁定与其连接的外源目的基因的表达。具体地，所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列。优选地，所述锁定基因包含SEQ ID NO：5或8所示的核苷酸序列，或由SEQ ID NO：5或8所示的核苷酸序列组成。
所述钥匙基因能够解除所述锁定元件对于外源目的基因的表达锁定。具体地，所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。优选地，所述锁定基因包含SEQ ID NO：3所示的核苷酸序列，或由SEQ ID NO：3所示的核苷酸序列组成。
可通过本领域中已知的算法测定两条核苷酸序列之间同源性的程度。用于比较的序列的最佳比对可通过以下进行：局部同源性算法【Smith和Waterman Add.APL.Math.2：482(1981)】；同源性比对算法【Needleman和Wunsch J.Mol.Biol.48：443(1970)；检索类似性算法【Pearson和Lipman Proc.Natl.Acad Sci.(USA)85：2444(1988)】；这些算法的计算机程序及其默认参数【Wisconsin Genetics软件包中的GAP、BESTFIT、BLAST、PASTA，和TFASTA，Genetics Computer Group(GCG)，575 Science Dr.，Madison，WI】；或通过目视检查。
根据本发明，所述锁定元件可以包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％，例如至少91％，92％，93％，94％，95％，96％，97％，98％，99％，99.5％，99.8％或100％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列。所述同源性可以使用BLAST及其默认参数计算。
根据本发明，所述钥匙基因可以包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％，例如至少40％，41％，42％，43％，44％，45％，46％，47％，48％，49％，50％，51％，52％，53％，54％，55％，56％，57％，58％，59％，60％，61％，62％，63％，64％，65％，66％，67％，68％，69％，70％，71％，72％，73％，74％，75％，76％，77％，78％，79％，80％，81％，82％，83％，84％，85％，86％，87％，88％，89％，90％，91％，92％，93％，94％，95％，96％，97％，98％，99％，99.5％，99.8％或100％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。所述同源性可以使用BLAST及其默认参数计算。
在一个具体实施方式中，本发明的基因组合为包含SEQ ID NO：5或与SEQ ID NO：5所具有至少80％的同源性的核苷酸序列的锁定元 件，和包含SEQ ID NO：3或与SEQ ID NO：3所具有至少40％的同源性的核苷酸序列的钥匙基因。
在另一个具体实施方式中，本发明的基因组合为包含SEQ ID NO：8或与SEQ ID NO：8所具有至少80％的同源性的核苷酸序列的锁定元件，和包含SEQ ID NO：6或与SEQ ID NO：6所具有至少40％的同源性的核苷酸序列的钥匙基因。
根据本发明，所述锁定元件可以：1)包含SEQ ID NO：5或8所示的核苷酸序列，或者2)包含由SEQ ID NO：5或8所示的核苷酸序列经过取代、缺失或添加一个或多个核苷酸而衍生的，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列。
根据本发明，所述钥匙基因可以：1)包含SEQ ID NO：3或6所示的核苷酸序列，或者2)包含由SEQ ID NO：3或6所示的核苷酸序列经过取代、缺失或添加一个或多个核苷酸而衍生的，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
根据本发明，所述钥匙基因可以：1)编码SEQ ID NO：4或7所示的氨基酸序列，或者2)编码在SEQ ID NO：4或7所示的氨基酸序列中经过取代、缺失或添加一个或几个氨基酸而衍生的，并且能够解除所述锁定元件对于外源目的基因的表达锁定的氨基酸序列。
根据本发明，所述锁定元件可以：1)包含SEQ ID NO：5或8所示的核苷酸序列，或者2)在严格条件下与SEQ ID NO：5或8所示的核苷酸序列的互补序列杂交，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列。
根据本发明，所述钥匙基因可以：1)包含SEQ ID NO：3或6所示的核苷酸序列，或者2)在严格条件下与SEQ ID NO：3或6所示的核苷酸序列的互补序列杂交，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
“严格条件”在本文中用于描述在约45℃下在6×氯化钠/柠檬酸钠(SSC)中与核苷酸序列的杂交，随后为在约50-65℃下在0.2×SSC/0.1％SDS中的一次或多次洗涤。优选地，所述“严格条件”为“高度严格条件”。术语“高度严格条件”指例如在约45℃下在6xSSC中与核苷酸序列的杂交，随后为在约68℃下在0.1×SSC/0.2％SDS中的一次或多次洗涤。
根据本发明，由所述锁定元件和所述钥匙基因构成的调控基因组合可以控制外源目的基因在植物体中的表达，从而在实现外源目的基因功能的同时，锁定外源目的基因在胚乳、块根、块茎和果肉等储藏组织或器官中的表达，以便获得无转基因表达产物的胚乳、块根、块茎和果肉等，但又不影响其通过有性繁殖过程遗传给下一代的能力。
在具体的实施方式中，将所述锁定元件置于组成型启动子和外源目的基因之间，从而在包含所述锁定元件的整株转基因植物体中锁定外源目的基因的表达；同时，将所述钥匙基因与组织特异地启动子可操作地连接，从而在特定组织中解除所述锁定基因对于外源目的基因的表达锁定，实现外源目的基因功能。
启动子
根据本发明，所述“启动子”是指起始转录作用的基因元件。启动子按照其作用方式可以分为组成型启动子、组织特异性启动子和诱导型启动子等。
根据本发明，所述“组成型启动子”是指在多数或全部组织中保持持续的活性的启动子。在组成型启动子调控下，不同组织器官和发育阶段的基因表达没有明显差异。优选地，用于本发明的组成型启动子是源自植物或在植物中具有组成型活性的启动子。
可用于本发明的组成型启动子的实例包括但不限于：花椰菜花叶病毒(CaMV)35S启动子、来自根癌农杆菌Ti质粒T-DNA区域的胭脂碱合成酶基因Ocs启动子、水稻肌动蛋白启动子Actin I、玉米泛素(ubiquitin)基因启动子Ubi等。优选地，本发明的组成型启动子是水稻肌动蛋白启动子Actin I。
根据本发明，所述组织特异性启动子是指在特定类型的细胞或组织中具有活性的启动子。在组织特异性启动子调控下，基因只在某些特定的器官或组织部位表达。优选地，用于本发明的组织特异性启动子是源自植物或在植物中具有组织特异性活性的启动子本领域技术人员可以根据植物种类、期望/不期望外源目的基因表达的器官/组织(例如供食用的植物组织/器官)来选择组织特异性启动子。
可用于本发明的组织特异性启动子包括：根特异启动子、茎特异启动子、叶特异启动子。优选地，本发明所述的组织特异性启动子是 植物的绿色组织特异性启动子，例如茎和/或叶特异性启动子。具体地，根据本发明的组织特异性启动子的实例包括水稻绿色组织特异性启动子核酮糖-1，5-二磷酸羧化酶小亚基rbcS启动子、玉米磷酸烯醇式丙酮酸羧化酶PEPC启动子、水稻绿色组织特异表达DX1启动子、水稻光系统II10kDa多肽D540启动子、水稻富亮氨酸重复序列类受体蛋白激酶LP2启动子，以及玉米叶绿体C4Pdk启动子，但不限于此。优选地，本发明的组织特异性启动子是水稻绿色组织特异性启动子rbcS启动子。
根据本发明，所述启动子与所述锁定基因或钥匙基因是“可操作连接的”。所述“可操作连接”是指元件的排列，其中所述元件经配置以发挥其正常功能。例如，如果启动子影响编码序列的转录，那么它是与该编码序列可操作连接的。
外源目的基因
根据本发明，所述“外源目的基因”是指在天然状态下不存在于受体植物中的外来基因。现已出于各种目的，将多种外源目的基因导入到受体植物中，以提高植物对害虫、除草剂等的抗性，并提高和稳定作物产量等。
根据本发明，所述外源目的基因可以是抗虫基因或抗除草剂基因。所述抗虫基因包括来自苏云金芽胞杆菌(Bacillus thuringiensis)的cry1Ab、cry1Ac、cry1Ab/1Ac、cry1C、cry2A和Vip3类抗虫基因、来自嗜虫沙雷氏菌(Serratia entomophila)的anf、sep基因，来自双酶梭菌(Clostridium bifermentans)的cmb基因、来自球形芽孢杆菌(Bacillus sphaericus)的mtx基因、来自嗜线虫致病杆菌(Xenorhabdus nematophilus)的杀虫蛋白基因、来自发光光杆状菌(Photorhabdus luminescens)的tca、tcb基因，以及来自金龟子绿僵菌(Metarhizium anisopliae)的prl基因，但不限于此。
根据本发明，所述Bt抗虫基因的核苷酸编码序列和氨基酸序列分别如SEQ ID NO：1和SEQ ID NO：2中所示。
根据本发明，所述抗除草剂基因包括抗草甘膦的EPSP合成酶基因、鼠伤寒沙门氏菌EPSP突变基因aroA、抗草丁膦的bar基因、抗米唑啉酮的ALS突变基因Ilv G、抗稀禾定的AccL-s2基因、抗溴苯腈的 bxn基因和抗绿磺隆的csrl基因，但不限于此。
根据本发明，所述外源目的基因的表达受到本发明的锁定元件和钥匙基因的控制。在具体的实施方式中，所述外源基因与本发明的锁定元件可操作地连接。
如果需要，编码外源目的基因的序列也可操作地连接至合适的调控因子，包括启动子、增强子、终止子和信号肽等。
转基因植物
本发明提供了一种培育转基因植物的方法，所述方法包括将包含本发明锁定元件的第一亲本与包含本发明钥匙基因的第二亲本杂交，从而获得所述转基因植物。
在具体实施方式中，本发明所述的培育转基因植物方法还包括在所述杂交步骤之前，将所述锁定元件和与其操作链接的目的基因导入和整合到所述第一亲本基因组中，以及将所述钥匙基因导入和整合到所述第二亲本基因组中。
在可选的实施方式中，本发明提供了一种培育转基因植物的方法，所述方法包括将将本发明的锁定元件和钥匙基因导入和整合到同一植株的基因组中，从而获得所述转基因植物。
将基因导入受体植物的方法是本领域中已知的，包括例如农杆菌介导基因转化、基因枪转化、花粉管通道法等，其中，由农杆菌介导的基因转化在植物转化中得到了广泛的使用，具体步骤可参见后附的实施例。
在本发明的进一步考虑的实施方式中，可以通过整合的方式，将特定的外源基因，例如用于筛选的标记基因从转基因植物中分离和剔除。然而，这些剔除标记基因后的转基因植物仍包含在本发明所述的转基因植物的范畴之内。
本发明的优点
本发明提供了一种用于控制植物中的外源基因不在特定组织中表达的基因组合，以及应用该基因组合培育转基因植物的方法。以水稻和Bt抗虫基因为例，该方法可用于培育胚乳0表达型转基因抗螟虫的水稻，即这种抗螟虫水稻所生产的稻米胚乳中没有任何转基因产物蛋 白的合成和积累。因此，可排除公众对转基因粮食作物食用安全性的担忧。
已有的试验结果显示，目的Bt抗虫基因在所获得的锁定元件和钥匙基因的日本晴异交种稻米胚乳中不表达，其Bt蛋白检测结果与日本晴野生型对照无显著差异，也即为0，但在设计表达的茎、叶组织中则高效表达。由于稻米胚乳中检测不到目的基因表达产物，本发明人将其称之为胚乳0表达型转Bt基因抗虫水稻，以与传统的胚乳表达型转Bt基因抗虫水稻相区别。进一步的抗虫性鉴定结果表明，携带本发明的锁定元件和钥匙基因的日本晴异交种(30株)对自然爆发的稻纵卷叶螟的抗虫效果接近0危害，与非转基因对照(30株)的100％受害株率相比呈极显著差异；其对人工接种的二化螟一龄幼虫(1个卵块的虫量/株)的抗虫效率也都达到抗至高抗水平。因此，本发明的基因组合和胚乳0表达型转基因抗虫水稻的成功选育将不仅有望突破传统的胚乳表达型转Bt基因水稻不被公众接受的僵局，同时，也将有力地推进转基因粮食作物的产业化进程。
本发明具有广适性，即适用于以果实、种子、块根、块茎为生产目的的广大作物，如水稻、小麦、大麦、燕麦、玉米、粟米、高粱、薏米、红薯、马铃薯、莲子、大豆和花生等等。本发明的这些和其他方面将参照以下附图和详细说明而得到更好的理解。
附图说明
图1：pSB130质粒载体图谱。该质粒载体有两个T-DNA区，其中，一个携带抗潮霉素(Hgromycin B)的标记基因(hpt)；另一个携带多克隆位点(MCS)，用于钥匙基因或连接了目的基因的锁定元件序列的装载。
图2：根据本发明的一个实施方式的基因钥匙(Key1；SEQ ID NO：5)的表达载体pKey1构建的流程图，其中图2A显示：第一步，用引入KpnI和EcoRI识别位点的特异引物扩增获得NosT片段，并将其连入pSB130相应的多克隆位点上；和第二步，用引入XbaI/SmaI和KpnI识别位点的特异引物扩增获得Key1 3′片段，并将其连入pSB130相应的多克隆位点上；图2B显示：第三步，用引入SalI和SmaI识别位点的特异引物扩增获得Key1 5′片段，并将其连入pSB130相应的多克隆 位点上；和最后一步，用引入HindIII和SalI识别位点的特异引物扩增获得rbcS片段，并将其连入pSB130相应的多克隆位点上，从而构建出pKey1表达载体。
图3：根据本发明的一个实施方式的锁定元件(Lock1；SEQ ID NO：8)及其相连接的报告基因(eYFP)的表达载体pLY1构建的流程图，其中图3A显示：第一步，NosT片段扩增和克隆同pKey1；和第二步，用引入PstI/XbaI识别位点的特异引物扩增获得Lock1片段，并将其连入pSB130相应的多克隆位点上；图3B显示：第三步，用引入HindIII和PstI识别位点的特异引物扩增获得ActinI片段，并将其连入pSB130相应的多克隆位点上；和最后一步，用引入XbaI和KpnI识别位点的特异引物扩增获得eYFP片段，并将其连入pSB130相应的多克隆位点上，从而构建出pLY1表达载体。
图4：根据本发明的另一个实施方式的钥匙基因(Key2；SEQ ID NO：3)的表达载体pKey2构建的流程图，其中图4A显示：第一步，用引入SacI和EcoRI识别位点的特异引物扩增获得NosT片段，并将其连入pSB130相应的多克隆位点上；和第二步，用SalI和HindIII从pKey1表达载体上酶切分离获得rbcS片段，并将其连入pSB130相应的多克隆位点上；图4B显示：第三步，用引入SacI和XbaI识别位点的特异引物扩增获得Key2 3′片段，并将其连入pSB130相应的多克隆位点上；和最后一步，用引入SalI和XbaI识别位点的特异引物扩增获得Key2 5′片段，并将其连入pSB130相应的多克隆位点上，从而构建出pKey2表达载体。
图5：根据本发明的另一个实施方式的锁定元件(Lock2；SEQ ID NO：6)连接报告基因(eYFP)的表达载体pLY2构建的流程图，其中图5A显示：第一步，用KpnI和EcoRI从pLY1酶切分离获得Nos片段，并将其连入pSB130相应的多克隆位点上；和第二步，用引入PstI和HindIII识别位点的特异引物扩增获得ActinI片段，并将其连入pSB130相应的多克隆位点上；图5B显示：第三步，用引入SalI和PstI特异引物扩增获得Lock2片段，并将其连入pSB130相应的多克隆位点上；和最后一步，用KpnI和SalI从pLY1酶切分离获得eYFP片段，并将其连入pSB130相应的多克隆位点上，从而构建出pLY2表达载体。
图6：根据本发明的一个实施方式的锁定元件(Lock1；SEQ ID NO： 8)加目的基因(cry1Ab/1Ac)的表达载体pLB构建的流程图，其中图6A显示：第一步，用引入KpnI/SalI和EcoRI识别位点的特异引物扩增获得Nos片段，并将其连入pSB130相应的多克隆位点上；和第二步，用引入PstI和XbaI识别位点的特异引物扩增获得Lock片段，并将其连入pSB130相应的多克隆位点上；图6B显示：第三步，用引入HindIII和PstI识别位点的特异引物扩增获得ActinI片段，并将其连入pSB130相应的多克隆位点上；和最后一步，用引入KpnI和SalI识别位点的特异引物扩增获得cry1Ab/1Ac片段，并将其连入pSB130相应的多克隆位点上，从而构建出pLB表达载体。
图7：根据本发明的一个实施方式的一组锁定元件和钥匙基因的功能验证的图示，其中，pLY1-C+pKey1为无绿点的未分化型pLY1阳性愈伤再经钥匙基因pKey1复转化和瞬时表达后在共聚焦显微镜下的观察结果，暗场条件下未呈现荧光信号；pLY1-S+pKey1为有绿点的分化型pLY1阳性愈伤再经钥匙基因pKey1复转化和瞬时表达后在共聚焦显微镜下的观察结果，暗场条件下呈现荧光信号，图中，线段＝20μM。
图8：根据本发明的另一个实施方式的一组锁定元件和钥匙基因功能验证的图示，其中，pLY2C+pKey2为无绿点的未分化型pLY2阳性愈伤再经钥匙基因pKey2复转化和瞬时表达后在共聚焦显微镜下的观察结果，暗场条件下未呈现荧光信号；pLY2-S+pKey2为为有绿点的分化型pLY2阳性愈伤再经钥匙基因pKey2复转化和瞬时表达后在共聚焦显微镜下的观察结果，暗场条件下呈现荧光信号，图中，线段＝20μM。
图9：日本晴pKey1和pLB阳性转化系的拷贝数分析，其中：图9A为选取的9个日本晴pKey1阳性转化系拷贝数分析，结果显示其中的6个为单拷贝；图9B为选取的10个日本晴pLB阳性转化系拷贝数分析，结果显示其中也有6个为单拷贝。结合结实率、株高和生育期及其他产量性状等重要农艺性状表现从pKey1和pLB独立转化体(分别简称KEY和LB)中各选择出2个单拷贝转化系(图中红色线框示出)用于后续生产LB/KEY和KEY/LB异交种的亲本，所选材料编号直接标注于矩形框上方。图中：M为DNA Molecular weight Marker II(DIG-labeled)，P为质粒阳性对照，N为非转基因野生型日本晴阴性对照。
图10：日本晴pLB阳性单拷贝转化系的1个T2代株系的纯合情况 及其PCR检测结果，其中：M-DL2000代表DL2000 DNA Marker；“+”代表阳性质粒对照，“-”代表非转基因阴性对照；1-24代表日本晴pLB阳性单拷贝转化系的1个T2代株系中24个独立的单株。
图11：日本晴2个pKey1和2个pLB单拷贝转化系组配的6个正反异交种在分蘖期、抽穗期和灌浆期的茎、叶组织中Bt蛋白含量的ELISA定量分析结果，其中：KEY1、KEY2和LB3、LB7分别为pKey1和pLB转化体中筛选的单拷贝纯系亲本；LB3/KEY1、LB7/KEY1、LB3/KEY2、LB7/KEY2、KEY1/LB3和KEY2/LB3为上述单拷贝纯系亲本所组配的异交种。T51-1和Zheyou 3为阳性对照。
图12：锁定元件和钥匙基因异交种LB3/KEY2(左侧)和野生型日本晴对照(右侧)在大田条件下对人工接种的二化螟一龄幼虫(1个卵块约80头虫/株)的抗性表现。图中，可以看到野生型对照上出现高比率的白穗，而转基因异交种上几乎没有出现白穗。
图13：锁定元件和钥匙基因LB3/KEY2异交种离体叶片对室内人工接种的二化螟一龄幼虫的抗性表现，图中A：阳性对照TT51-1；B：野生型明恢63对照；C：LB3/KEY2异交种；D：野生型日本晴阴性对照；E：日本晴KEY2亲本转化系；F：日本晴LB3亲本转化系。图片显示转基因异交种LB3/KEY2对二化螟的抗性表现与阳性对照T51-1一致。
图14：锁定元件和钥匙基因LB3/KEY2异交种和野生型日本晴阴性对照(Nip-CK)的离体茎秆对室内人工接种二化螟一龄幼虫的抗性表现。图片显示转基因LB3/KEY2异交种离体茎秆上没有观察到二化螟危害的迹象，接种的二化螟几乎全部死亡；而对照茎秆上观察到二化螟危害的虫孔、啃噬槽及其排泄物，并有存活的幼虫。
通过参考以下非限定性实施例可更好地理解本发明，提供所述非限定性实施例作为本发明的示例。介绍以下实施例以更充分地说明本发明的实施方式，且决不应解释为对本发明广泛范围的限制。
具体实施方式
实施例1：本发明的基因组合的设计及其可行性研究与验证
1.基因组合的设计
如前所述，完整的基因组合在设计上是由能锁定目的基因表达的 锁定元件(Lock)和能组织特异开启锁定元件的钥匙基因(Key)两个组件组成。为了使用的方便，共设计了两套这样的组件，其中，第一套组件的DNA序列分别是SEQ ID NO：5(Lock1)和SEQ ID NO：3(Key1)，第二套组件的DNA序列是SEQ ID NO：8(Lock2)和SEQ ID NO：6(Key2)。作为锁定元件在设计上是被设置在Actin I等组成型表达启动子和目的基因起始密码子之间，其功能是阻止目的基因的表达：而作为钥匙基因在设计上则是被置于rbcS等绿色组织特异型表达启动子的控制之下，其功能是在特定组织(如绿色组织)里开启被锁定元件锁定的目的基因的表达。
为了验证上述基因组合设计是否具备预期的锁定和开启功能，我们先利用增强型黄色荧光蛋白基因eYFP对各个组件进行了测试。然后在获得肯定结果的前提下，我们再利用抗虫的Bt杂种蛋白基因cry1Ab/1Ac进一步对其开展了应用性研究。
2.用于锁定和开启功能验证的验证型表达载体构建
为了构建第一套锁定元件和钥匙基因的表达载体，利用引入了限制性核酸内切酶识别位点的特异引物(表1)和高保真PCR技术扩增所有功能片段，然后依设计顺序将这些功能片段分别连入pSB130质粒载体(图1)，从而获得相应的基因钥匙表达载体pSB130：：rbcS：：Key1：：NosT(简称pKey1)和锁定元件表达载体pSB130：：ActinI：：Lock1：：eYFP：：NosT(简称pLY1)，其具体构建流程分别示于图2和图3。
以构建成功的第一套表达载体作为基础，构建第二套表达载体，其中部分功能片段可以通过酶切方法从第一套表达载体中获得，另一部分功能片段则依然通过特异引物(表1)和高保真PCR扩增技术扩增出来，然后依设计顺序将这些功能片段连入pSB130质粒载体(图1)，从而获得相应的基因钥匙表达载体pSB130：：rbcS：：Key2：：NosT(简称pKey2)和锁定元件表达载体pSB130：：ActinI：：Lock2：：eYFP：：NosT(简称pLY2)，这两个表达载体的具体构建流程图分别示于图4和图5。
3.用于抗虫目的应用型表达载体构建
由于验证型和应用型表达载体在基因钥匙部分完全相同，而主要差别仅在于与锁定元件所连接的目的基因片段的不同，因此，这套应用型表达载体的构建主要是针对锁定元件和目的基因片段进行。构建 时，一方面通过酶切获得部分与验证型表达载体通用的功能片段，另一方面通过特异引物(表1)和高保真PCR扩增技术分别获得本申请实施例选用的第一套表达载体中的锁定元件以及待连接的抗虫基因两个功能片段，然后依设计顺序将这些功能片段连入pSB130质粒载体(图1)，便可获得相应的应用型锁定元件表达载体pSB130：：ActinI：：LB1：：NosT(简称pLB1)，该表达载体的具体构建流程图示于图6。
本申请实施例所用的pSB130质粒载体包含两个“T-DNA”区，其中一个“T-DNA”区用于装载连接目的基因的锁定元件或钥匙基因表达单位，另一个用于装载抗潮霉素标记基因Hpt表达单位。构建双T-DNA表达载体的目的在于着眼于今后的实际应用，因为双T-DNA质粒载体转化水稻后能使目的基因和标记基因在受体基因组中有机会发生独立整合，以便于标记基因能在随后的分离世代中通过自交方式分离和剔除。
Figure PCTCN2016103934-appb-000001
Figure PCTCN2016103934-appb-000002
实施例2：外源目的基因表达锁定与开启功能的验证
本发明的基因组合锁定与开启功能的验证采用两步转化法进行。第一步，先利用农杆菌介导法(Liu et al.，1998)将按照实施例1中制备的验证型表达载体中的两个锁定元件pLY1和pLY2导入水稻胚性愈伤组织进行永久表达，之后，经过连续多轮(3-5轮，12-14天/轮)抗生素筛选，抗性愈伤预分化(7-9天)和分化培养，直到形成绿点(7-9天)；第二步，再利用基因枪介导法(Tu et al.，2000)将验证型表达载体中的两个钥匙基因pKey1和pKey2分别轰入有绿点的分化型阳性愈伤和没有绿点的未分化型阳性愈伤进行瞬时表达，24-36h后在共聚焦显微镜下观察黄色荧光蛋白的发光情况，如果所设计的基因组合具有完备的锁定和开锁功能，则pKey1和pKey2转化的无绿点未分化型阳性愈伤完全不发荧光，而它们转化的有绿点分化型阳性愈伤则能够发荧光。详细试验步骤叙述如下：
1.受体材料
用于上述验证型锁定元件pLY1和pLY2遗传转化的受体品种为粳稻日本晴。该品种是水稻遗传转化的模式品种，其成熟胚愈伤组织诱导容易，遗传转化效率高。
2.水稻转化及生长所使用的培养基及组成成分
2.1.诱导/继代培养基(每升)：4.1g/L N6(Chu et al，1975)基础盐组分+N6有机组分(表2)+2mg/L 2，4-二氯苯氧乙酸(2，4-D)+2.0g/L水解酪蛋白+30g/L蔗糖+3g/L琼脂，pH 5.9
2.2.侵染培养基(每升)：AA基础培养基(表3)(Toriyama&Hinata，1985)+200μM乙酰丁香酮，pH 5.9。
2.3.共培养培养基(每升)：CC基础培养基(表4)(Hiei et al，1994)+200μM乙酰丁香酮，pH 5.9。
2.4.抑菌培养基(每升)：诱导/继代培养基+500mg/L头孢霉素，pH 5.9。
2.5.筛选培养基(每升)：诱导/继代培养基+50mg/L潮霉素+500mg/L头孢霉素，pH 5.9。
2.6.再生培养基(每升)：4.1g/L N6基础盐组分+N6有机组分(表2)+2.0g/L水解酪蛋白+30g/L蔗糖+6g/L琼脂+2mg/L激动素+1mg/Lα-萘乙酸，pH 5.9。
2.7.水稻生根培养基(每升)：2.05g/L N6基础盐组分+1/2的N6有机组分(表2)+1.0g/L水解酪蛋白+15g/L蔗糖+3g/L琼脂，pH 5.9。
表2：N6基础盐组分(上海生工)和有机组分
Figure PCTCN2016103934-appb-000003
表3：AA培养基配方
Figure PCTCN2016103934-appb-000004
表4：CC培养基配方
Figure PCTCN2016103934-appb-000005
3.锁定元件转化
如前所述，验证型锁定元件表达载体pLY1和pLY2的遗传转化采用农杆菌介导法进行。所用的农杆菌菌株为EHA105(Bio Vector NTCC  Inc.)，所用的受体细胞为日本晴成熟胚诱导的胚性愈伤组织。农杆菌转化的具体步骤如下：
3.1水稻胚性愈伤诱导
取成熟的日本晴种子若干，去壳后，挑选粒型饱满、胚完好的糙米放入预先高压灭菌的烧杯中，先用70％酒精浸泡1min，并不间断地振荡，以除去表面杂质；再用20％的次氯酸钠浸泡灭菌20min(可置于摇床上振荡)；再用无菌蒸馏水冲洗4～5次，以便稀释并除去糙米表面残留的次氯酸钠。将无菌处理过的糙米接种于含有2.0mg/L 2，4-D的MS愈伤诱导和继代培养基(表2)上，28℃暗培养2周左右，直至成熟胚的盾片处产生合适大小的初生愈伤。之后，切下初生愈伤，并转移到新的愈伤诱导和继代培养基上，在相同条件下继代培养，每2周继代一次，直至细胞质浓、颜色鲜黄、质地硬实和细胞团呈颗粒状的胚性愈伤形成为止。
3.2农杆菌EHA105菌株的遗传转化和摇菌培养
取0.5μL表达载体质粒加入到含有60μL农杆菌电击感受态EHA105的1.5mL离心管中，待枪头吸打混匀后移入电极杯中；电击后，迅速加入1mL的LB液体培养基，吸打混匀后移入先前的1.5mL离心管中，于28℃振荡仪摇上1h；菌液复苏后，吸取100μL菌液，均匀涂布于LB固体筛选(含50mg/L的卡那霉素、25mg/L的利福平)培养基(上海生工)表面，28℃培养2天；菌落PCR验证阳性克隆后，对阳性克隆进行摇菌培养，所得菌液于50％的甘油浓度和-80℃条件下保存备用。
3.3水稻愈伤的农杆菌侵染和筛选继代
农杆菌侵染按Yang et al(2011)报道的方法步骤进行。其具体程序是：取出保存于-80℃的农杆菌菌液，从中吸取200μL均匀涂布于到含有25mg/l利福平和50mg/L卡那霉素的LB固体培养基表面，于28℃条件下培养过夜；再从中挑单菌落用LB液体培养基扩大培养；之后，从中吸取200-300μL的新鲜菌液接入到20mL含有25mg/L利福平和50mg/L卡那霉素的LB液体培养基中，28℃振荡(220rpm)培养16-18h。取足量的菌液于4000rpm下离心15min之后，弃去LB培养基上清液；加入20mL 0.1M MgSO4溶液重新悬浮农杆菌(用移液枪轻轻吹打)，于4000rpm下离心10-15min，弃去含有抗生素的MgSO4上清 液；再加入5mL含有200μM乙酰丁香酮(Acetosyringone，AS)的AA侵染培养基(表3)重新悬浮农杆菌，再加入适量的AA-AS侵染培养基，使菌液的OD600值最终调整在0.8-1.0之间；浓度调整后，用无菌的50mL离心管分装菌液，20-25mL/管，待用。
农杆菌侵染前，先将胚性愈伤组织预培养7天左右，再将其从继代培养皿中转移至覆有无菌滤纸的空培养皿中，在超净工作台上风干10-15min左右，期间用灭菌过的小勺缓缓翻滚愈伤组织使之充分干燥；待其干燥后，移入盛有菌液的离心管，在室温下轻轻摇晃40min，将该离心管于超净工作台上静置10min；倒除菌液，将胚性愈伤组织置于无菌滤纸上干燥15min左右，然后，将其转移至表面以无菌滤纸覆盖的含有AS(200μM)的CC共培养培养基(表4)上，于28℃暗培养50-55h；挑选表面农杆菌未大量生长或未污染的胚性愈伤组织，移至含有2.0mg/L的2，4-D，500mg/L头孢霉素(Cefortaxim)的N6抑菌培养基上，28℃暗室中抑菌培养3-4d；再将抑菌培养后的愈伤组织移至含有500mg/L头孢霉素和50mg/L潮霉素(Hygromycin)的筛选培养基上，28℃暗室培养；每隔半月挑选生长状态良好的愈伤组织作继代，并根据污染的程度来调整培养中头孢霉素的浓度，一般情况下第三或第四轮继代的时候可考虑浓度减半。这样继代培养直至获得生长迅速、量大和颜色鲜活的抗性愈伤组织为止(4-6轮筛选和继代培养)。
3.4抗性愈伤组织分化培养
将上一步获得的抗性愈伤组织转移至N6再生培养基上，28℃暗室预分化一周，再移至新鲜的N6再生培养基上，于25℃光室进行分化培养，直至绿点形成(约需两周时间)为止。
4.基因钥匙转化
以上一步形成绿点的愈伤组织为受体，利用Tu et al(2000)描述的基因枪介导法将按照实施例1制备的验证型用钥匙基因表达载体pKey1和pKey2导入有绿点的分化型愈伤组织和无绿点的未分化型愈伤组织进行瞬时表达，经24-36h小时恢复培养后，在共聚焦显微镜下观察黄色荧光蛋白的荧光信号，并拍照记录和保存。
5.实验结果
试验结果示于图7和图8。从图中可以看出，在共聚焦显微镜下，无绿点的未分化型pLY1和pLY2阳性愈伤再经过钥匙基因表达载体 pKey1和pKey2复转化和瞬时表达后未能观察到荧光信号，而只有当有绿点的分化型pLY1和pLY2阳性愈伤再经过钥匙基因表达载体pKey1和pKey2复转化和瞬时表达后才能观察到荧光信号。这些试验结果因此证实所设计的两套基因组合都能正常和有效地行驶锁定和开启功能。
实施例3：应用型锁定元件和钥匙基因转化系的获得及其异交种生产
应用型锁定元件和钥匙基因的遗传转化采用农杆菌介导法进行。除了使用按照实施例1制备的应用型锁定元件表达载体pLB1和基因钥匙表达载体pKey1之外，受体材料的选用、所使用的各种培养基、载体的遗传转化和阳性抗性愈伤的预分化和分化培养均与实施例2中所述相应的试验步骤相同，直至阳性抗性愈伤分化成绿苗。所获得的绿苗，洗净根系上的培养基后，或直接(根芽同时分化型)，或经前述N6生根培养基壮根后(芽先分化型)移入Yoshida培养液(表5)中过渡培养，待其生长状态良好与稳定后，再移栽到温室，直至成熟。
表5：Yoshida营养液配方(Yoshida，1976)
Figure PCTCN2016103934-appb-000006
经过上述步骤的遗传转化，先后获得pKey1独立转化体42份和pLB1独立转化体59份。之后，分别针对这两种独立转化体进行了纯系选育及其它们之间的异交种生产和抗虫性鉴定。具体如下文所述。
实施例4：应用型锁定元件和钥匙基因转化系的分子分析
1.DNA抽提
1.1 DNA小样抽提
从按照实施例3获得的转基因植株上取下2-3cm长的新鲜叶片置于碾钵中，加入500μL的1.5×CTAB提取液，并碾磨至匀浆，转移匀浆至1.5mL离心管中，置于56℃水浴保温20min，之后加入500μL的氯仿∶异戊醇(24∶1)，并充分混匀(上下颠倒数次)，室温下离心5min(14,000r/min)后取300ul上清液，加入到600μL的的无水乙醇(-20℃预冷)中，混匀后于-20℃中放置30min以上，室温离心5min(14,000r/min)后弃去上清，用75％乙醇浸洗DNA沉淀，之后，弃去乙醇，室温风干DNA沉淀，最后用200μL无菌水溶解，待用。
1.2 DNA大样抽提
取3-5片(3-5g)成株期水稻叶片，加入液氮迅速研磨至粉末状，转移至经预先冷冻的50mL离心管中，立即加入15-20mL煮沸预热的1.5 X CTAB，摇匀后将其置入56℃水浴30min。然后加入一倍体积24∶1(三氯甲烷∶异戊醇)，轻柔旋摇30min至溶液分三层，上中下依次为黄色、绿色、黑色。常温(温度＞18℃)4000rpm离心20min取上清，加入1/10体积56℃预热的10 X CTAB摇匀，再次加入一倍体积24∶1(三氯甲烷∶异戊醇)并轻柔旋摇30min。常温(温度＞18℃)4000rpm离心20min，之后，用5mL枪头吸出上清，再加入一倍体积1 X CTAB，旋摇后会出现絮状DNA沉淀，利用枪头将其挑出置于1.5mL离心管管底，倒转于吸水纸上沥干10min。然后加入10μL RNase及0.5ml已灭菌的1M NaCl，室温或56℃水浴溶解。将溶解后的DNA加入2倍(-20℃)体积-20℃预冷的95％乙醇，旋摇使DNA絮状沉淀析出。利用枪头挑取絮状DNA放进1.5mL离心管中，加1.5mL75％乙醇清洗DNA，然后倒去乙醇，加入超纯水于4℃保存备用。
2.阳性转基因植株的检测和纯系鉴定
阳性转基因植株的检测和纯系鉴定利用PCR技术进行。由于Hpt 标记基因和锁定元件或钥匙基因是通过双T-DNA共转化的，T0代阳性转化系的鉴定仅基于目的基因进行。因此检测后仅保留目的基因为阳性的植株继续栽培至成熟，非阳性的植株则全部清除，以减少后续世代的工作量。
所用的抗潮霉素基因扩增引物为：Hpt-F，5′-GCTGTTATGCGGCCATTGTC-3′(SEQ ID NO：9)和Hpt-R，5′-GACGTCTGTCGAGAAGTTTC-3′(SEQ ID NO：10)；PCR反应体系(25μL体系)为：待测样品DNA模板1ul，10×PCR buffer 2.5μL，10mM dNTP 2μL，20uM引物0.25μL，2U/μL Tag酶0.5μL，加ddH2O至25μL。PCR反应程序为：94℃变性3min；接着94℃变性30ec，55℃退火30sec，72℃延伸40sec，共35个循环；之后，经72℃延伸5min于10℃保持。扩增产物经1％琼脂糖凝胶电泳鉴定，拍照后保存。
所用的Bt基因扩增引物为：BtF，5′-GGCCATACAACTGCTTGAGT-3′(SEQ ID NO：11)；BtR，5′-GCGTTTCCCATAGTTCCATA-3′(SEQ ID NO：12)，扩增片段长度为1Kb。PCR反应程序为：94℃变性3min；接着94℃变性30sec，55℃退火30sec，72℃延伸1min，共35个循环；之后，经72℃延伸5min于10℃保持。扩增产物经1％琼脂糖凝胶电泳鉴定，拍照后保存。PCR反应体系见表6。
所用的钥匙基因扩增引物为：KeyF，5′-AACGAGTGATGAGGTTCGCA-3′(SEQ ID NO：13)；KeyR，5′-ACCCGGCAAAACAGGTAGTT-3′(SEQ ID NO：14)，扩增片段长度为672bp。PCR反应程序为：94℃变性3min；接着94℃变性30sec，55℃退火30sec，72℃延伸1min，共35个循环；之后，经72℃延伸7min于4℃保持。扩增产物经1％琼脂糖凝胶电泳鉴定，拍照后保存。PCR反应体系见表6。
表6：PCR反应体系
Figure PCTCN2016103934-appb-000007
这样，从日本晴的41份pKey1和50份pLB独立转化系中分别鉴定出21份pKey1和22份pLB阳性转化系。
3.转基因拷贝数检测
转基因拷贝数检测利用Southern杂交技术进行。首先，用限制性内切酶HindIII对水稻总DNA在37℃条件下酶切过夜(8-12h)；将酶切后的DNA通过0.8％(w/v)的琼脂糖凝胶电泳分离(40V，12h)，之后，将电泳分离的DNA印迹转移到尼龙膜(GE Healthcare，UK)上。杂交探针的制备和Southern杂交及杂交信号检测均按罗氏地高辛试剂盒[Roche DIG-High Prime DNA Labeling and Detection Starter Kit II(Switzerland)]提供的说明书进行，其中杂交探针制备引物分别为：
pKey1：Key-1U20，5′-ATGTCCAATTTACTGACCGT-3′(SEQ ID NO：15)，和
Key-800L20，5′-GCTTCAAAAATCCCTTCCAG-3′(SEQ ID NO：16)；
pLB：Bt-577U24，5′-AGGCTGATTGGAAACTACACCGAC-3′(SEQ ID NO：17)，和Bt-1161L24，5′-ACAGCGGATGGCAAGTTAGAAGAG-3′(SEQ ID NO：18)。
日本晴的pKey1和pLB阳性转化系的拷贝数分析结果示于图9。从图9可见，在所检测的9份日本晴pKey1阳性转化系(图9A)中，有6份是单拷贝的，而在所检测的10份pLB阳性转化系(图9B)中，也有6份是单拷贝的。
4.单拷贝转基因纯系的PCR扩增检测与筛选
单拷贝转基因纯系的PCR扩增检测与筛选在T2代株系中进行。分别将KEY1和LB单拷贝转化系自交两代后获取T2代种子和植株，之后，经PCR扩增检测筛选出分别带有KEY1和LB的纯系。图10是日本晴pLB单拷贝转化系的1个T2代纯合株系的PCR检测结果。如图10所示，该T2代株系的24个单株都显示阳性LB基因扩增片段，表明该T2代株系为LB转基因纯系。其它pLB和pKey1转基因纯系的PCR扩增检测均依此进行(数据未显示)。
实施例5：应用型锁定元件和钥匙基因所配异交种的茎叶Bt蛋白含量测定和抗虫性鉴定
1、材料种植
将经过实施例4中所述的PCR扩增检测筛选的、且结实率和其它农艺性状优良的日本晴pKey1单拷贝转基因纯系KEY1、KEY2分别与pLB单拷贝转基因纯系LB3、LB7进行正的和反的异交，共获得6个异交种。
2014年，所有供试材料包括野生型日本晴阴性对照、1个KEY(KEY1)和2个LB(LB3和LB7)转化系亲本以及由上述pKey1单拷贝转基因纯系与pLB单拷贝转基因纯系配置的6个正反异交种共10份于6月25日播种，15天后，利用Bt Cry1Ab/1Ac转基因快速检测试纸(货号：AA0232，佑隆，上海)，按说产品明书中描述的步骤，对6个KEY/LB和LB/KEY正反异交种供试幼苗进行阳性检测，经检测确认为阳性的异交种幼苗和野生型阴性对照及亲本材料的幼苗一起，按1行/小区，10株/行，共3次重复，于7月28日移栽到转基因专用隔离网室泥池中，株距20cm，行距40cm。整个试验区周围种植2行非转基因水稻植株作为保护行。全生育期不施用杀虫剂，水肥管理同当地大田。
2015年，供试材料与2014年相同。全部供试材料于5月25日播种，15天后，经Bt Cry1Ab/1Ac转基因快速检测试纸(货号：AA0232，佑隆，上海)检测，确认为阳性的异交种幼苗和野生型对照及亲本材料的幼苗一起按2行/小区，12株/行，共3次重复，于6月25日移栽到转基因专用隔离网室泥池中，宽窄行种植，宽行40cm，窄行15cm， 株距20cm，其余栽培管理同2014年。
2、茎叶Bt蛋白含量测定
(1)、ELISA试剂盒和水稻样品
日本晴KEY/LB和LB/KEY正反异交种茎秆和叶片中的Bt蛋白含量用一龙公司(EnviroLogixTM Inc.，Portland，USA)酶联免疫吸附(ELISA)试剂盒测定。水稻茎、叶样品分别于分蘖期、抽穗期和灌浆期三个时期取样。
(2)、Bt蛋白含量测定工作液和样品的制备
测定工作液包括抽提液/稀释液和洗脱液。洗脱液是将试剂盒附带的磷酸盐加入1L双蒸水，然后搅拌使之完全溶解制成。抽提液/稀释液是将该洗脱液取出200μL加入1mL Tween-20搅拌完全溶解配制而成。两种工作液都在4℃保存，使用前预热至室温。
每个异交种设3个重复，1株/重复，用液氮将取回的新鲜样品研磨成粉末，每个材料取约20mg粉末，按照20mg/500μL的比例加入抽提/稀释液充分震荡以提取蛋白样品。将匀浆装入1.5mL的离心管中冰上静置30min，然后吸取上清夜，4℃低温4000rpm离心3min，弃去上清后，用抽提/稀释液稀释一定的倍数后测定其中的Bt蛋白浓度，阴性对照无需稀释。各个时期茎、叶样品的稀释倍数见表7。
表7 Bt蛋白样品的稀释倍数_
Figure PCTCN2016103934-appb-000008
3.ELISA反应和蛋白含量计算
ELISA反应过程参照试剂盒的说明书进行，具体步骤如下：
(1)加入50μL Cry1Ab/1Ac酶标抗体至酶标板(96孔)的每一孔中；
(2)分别加入不同浓度梯度(1、0.5、0.25、0.1ppb)的Cry1Ab/1Ac标准样品、50μL空白样品(抽提/稀释液)和待测样品到上述96孔板的孔中，1个样品/孔，1个样品设置两个技术重复，然后用封口胶将 96孔板密封后低速(200rpm)振荡1-2h；
(3)倒去上清夜，向96孔板的每个孔中加入300μL洗脱液，轻微振荡1min，倒去洗脱液，倒扣在吸水纸上将残余液体沥干；
(4)将步骤3重复4次；
(5)向96孔板的每个孔中加入100μL底物，含有Bt蛋白的样品对应的孔中将呈现蓝色，将各个孔密封后避光，低速振荡(200rpm)30min左右；
(6)向96孔板的每个孔中加入100μL反应终止液，将蓝色转换成黄色，立即读取吸光度值。
吸光度值用酶标仪(Synergy H1，USA)测定，在450nm波长下读取。用标准样品的浓度和吸光度绘制标准曲线，在标准曲线上读出测试样品的浓度，再根据稀释倍数计算出水稻组织中的蛋白含量。蛋白含量的计算公式为：Bt蛋白含量(μg/g鲜重)＝测试样品浓度(ng/mL)×稀释倍数×样品上样体积(μL)/组织鲜重(mg)。蛋白浓度在不同杂交组合、不同时期和组织之间的差异用成组数据的多重t测验进行比较。
两年的分析结果如图11所示：在三个生长时期，6个正反异交种叶片中均含有与阳性对照T51-1及其两系杂交稻浙优3号(Zheyou 3)大体一致或更高的Bt蛋白含量(图11A和11C)；而在3个异交种LB3/KEY2，LB7/KEY2和KEY2/LB3茎秆中含有与浙优3号(Zheyou 3)阳性对照一致的Bt蛋白含量，另外3个异交种LB3/KEY1、LB7/KEY1和KEY1/LB3茎秆中则含有比上述2个阳性对照都低的Bt蛋白含量(图11B和11D)。
4、田间抗虫性评价
KEY/LB和LB/KEY正反异交种对二化螟的田间抗虫性评价利用自然接虫法和人工接虫法两种方法进行。自然接虫法是在二化螟爆发危害高峰出现后的5-7天进行。调查指标包括枯心数和白穗数。人工接虫分两次在最高分蘖期和抽穗前1星期进行。虫卵购自江西神农科技公司，购回的虫卵置入长12cm和直径2.0cm、且底部置有湿润棉球的玻璃试管中，1个卵块/管，塞上棉塞，并在管口外面扎上黑布。虫卵孵化在26-28℃温度、80％湿度和16小时光照/8小时黑暗条件下进行。孵化的一龄幼虫于孵化后6小时内连同试管接种到稻蔸基部。具 体操作如下：打开黑布并拔除棉塞，将试管基部插入稻田土中，使管口紧靠稻杆，方便幼虫从试管爬到稻杆上进行危害。接虫量为1个卵块/株，约80头一龄幼虫。两周后，调查二化螟危害的枯心数或白穗数，同时，调查供试单株的总分蘖数或总有效穗数。
LB/KEY和KEY/LB正反异交种对稻纵卷叶螟的田间抗虫性评价利用自然接虫法进行。稻纵卷叶螟危害的单株受害叶片数和叶片受害程度调查在危害高峰出现后的5-7天进行。
LB/KEY和KEY/LB正反异交种对二化螟和稻纵卷叶螟的田间抗性评价指标的统计分析用SPSS22.0(SPSS，Chicago，USA)软件完成，其二化螟危害率与野生型日本晴及带锁定元件和Bt基因的亲本对照差异的显著性用Dunnett t检验完成。
2014年田间自然接虫鉴定结果表明：6个正反异交种对稻纵卷叶螟具有高效的抗性，在野生型日本晴及其带锁定元件和Bt基因的亲本品系单株受害分蘖数和单株受害叶片数分别达到54.81-65.27％和9.17-14.60％的情况下，6个正反异交种的这2个指标仅为0.00-1.33％和0.00-0.13％(表8)，两者的差异达到P＝0.01极显著水平；另外，所检测的4个异交种LB3/KEY2、LB7/KEY2、KEY1/LB3和KEY2/LB3对自然发生的二化螟也具有较好的抗性，与野生型日本晴及带锁定元件和Bt基因的亲本品系6.96-8.02％的二化螟危害率相比(表8)，这些异交种的二化螟危害率仅为0.00-0.63％，两者的差异也达到P＝0.01极显著水平。
2015年田间自然接虫鉴定结果与2014年基本一致，6个正反异交种都表现出了对稻纵卷叶螟和二化螟的良好田间抗性，它们与野生型日本晴及带锁定元件和Bt基因的亲本品系的差异都达到了P＝0.01的极显著水平(表8)。
至于人工接虫鉴定，2014年选择了2个茎叶组织中Bt蛋白含量低的异交种LB3/KEY1和LB7/KEY1作为待测材料，并以野生型日本晴和带锁定元件及Bt基因的LB3及LB7两个亲本为感虫对照。结果显示：4个对照品系的二化螟危害率达到23％以上，而2个异交种的二化螟危害率则不超过8％(表9)，因此，显示了良好的二化螟抗性。
2015年则选用了茎杆中Bt蛋白含量相对较高的一个异交种LB3/KEY2重复进行了大田人工接虫鉴定，结果显示该异交种受二化螟 危害程度极低，平均只有0.78％的分蘖或稻穗受到危害，极显著的低于野生型日本晴阴性对照的56.26％(表9和图12)。
表8：6个LB/KEY和KEY/LB杂交组合、3个KEY及LB亲本及和野生型日本晴对照在大田条件下对二化螟和稻纵卷叶螟的抗性表现a
Figure PCTCN2016103934-appb-000009
a.每品种均基于三次重复获得的数据，每品系每重复随机调查10个单株。b.WT为野生型日本晴对照，所有其它9个待测品系的鉴定数据均与其对比，数据采用SPSS 22.0软件包的Dunnett t检验进行分析。c.数值表示平均值±平均标准差。ND.表示没有数据。**表示极显著差异P＜0.01。
表9：LB3/KEY2杂交组合在大田条件下对分蘖期和抽穗前7天人工接种的二化螟的抗性表现
Figure PCTCN2016103934-appb-000010
*表示与对照的差异达P＜0.05显著水平，**表示与对照的差异达P＜0.01 极显著水平.
3、室内抗虫性评价
室内抗虫性评价是针对二化螟进行，虫卵同样购买自江西神农科技公司，虫卵孵化方法同前。抗虫性评价采用的方法有离体茎杆和叶片接虫法。
离体茎杆接虫法是取拔节后的水稻茎杆，切成12cm长带节和叶鞘的茎段，每个养虫管放入同一单株的2个茎段和20头一龄三化螟幼虫。养虫管用棉塞封口后，置于25-27℃和80％的相对湿度下饲养，6天后统计幼虫的死亡率。
离体叶片接虫法是取孕穗期的剑叶和倒二叶叶片，切成8cm长的叶段，置入垫有用2mL蒸馏水湿润的滤纸的培养皿中，每个培养皿放4段叶段和15头一龄二化螟幼虫，并用PARAFILM缠4次封口，以防幼虫钻出。之后，置于室温下(25℃)饲养，6天后统计幼虫的死亡率。
每个养虫管或培养皿统计为1次重复，每样本设置3次重复。不同LB/Key1及Key1/LB转化系异交种对二化螟的抗性用成组茎秆和叶片数据的t测验进行比较。结果表明：在室内人工接种条件下，LB3/KEY2异交种的离体叶片和茎秆对二化螟的抗虫表现也十分优异(图13和14)，其中，LB3/KEY2异交种离体叶片喂养的二化螟幼虫死亡率达100％，其离体茎秆喂养的二化螟幼虫死亡率也达到95％以上，而对照的离体叶片和茎秆喂养的二化螟幼虫死亡率则都不超过20％(表10)。
表10：LB3/KEY2异交种离体叶片和茎秆对人工接种二化螟的抗性表现
Figure PCTCN2016103934-appb-000011
实施例6：应用型锁定元件/钥匙基因异交种胚乳中Cry1Ab/1Ac蛋白含量的测定
为了验证基因组合是否具有胚乳特异性关闭目的基因的功能，本发明对实施例5中所述的6个锁定元件/钥匙基因异交种的胚乳进一步进行了Cry1Ab/1Ac蛋白含量测定.
1.ELISA试剂盒和水稻样品
日本晴pKey1/pLB异交种胚乳中Cry1Ab/1Ac蛋白含量同样用一龙公司(EnviroLogixTM Inc.，Portland，USA)酶联免疫吸附(ELISA)试剂盒进行测定。水稻检测的时期分别是灌浆完熟期。
2.Cry1Ab/1Ac蛋白含量测定工作液和样品的制备
测定工作液包括抽提液/稀释液和洗脱液。洗脱液是将试剂盒附带的磷酸盐加入1L双蒸水，然后搅拌使之完全溶解制成。抽提液/稀释液是将该洗脱液取出200μL加入1mL Tween-20搅拌完全溶解配制而成。两种工作液都在4℃保存，使用前预热至室温。
每个杂交组合设3个重复，1株/重复，50粒种子/株，将收获的种子去除颖壳后制备成糙米备用，然后利用精米机将部分糙米研磨成精米，再分别选取50粒糙米和精米样品研磨成粉末，每个材料取约20mg粉末，按照20mg/500μL的比例加入抽提/稀释液充分震荡以提取蛋白样品。将震荡后的匀浆装入1.5mL的离心管中，冰上静置30min，4℃低温、4000rpm离心3min，吸取上清备用。之后，阳性对照T51-1及其两系杂交稻杂种浙优3号(Zheyou 3)的精米和糙米中抽提的蛋白样品用抽提/稀释液稀释50倍数后，再测定其中的Bt蛋白浓度，而异交种和阴性对照则无需稀释。
3.ELISA反应和蛋白含量计算
ELISA反应过程参照试剂盒的说明书进行，具体步骤同前述茎叶组织中Bt蛋白含量测定。
4. 6个异交种精米胚乳和糙米米粉中Cry1Ab/1Ac蛋白含量的测定结果
2014年和2015年两年重复测定的结果均显示：6个异交种精米胚乳的Cry1Ab/1Ac蛋白含量检测值都与野生型日本晴阴性对照以及携带锁定元件及Bt基因的LB3亲本品系的检测值无明显差异，它们的实测值都小于1ng/g粉末，极显著地低于阳性对照T51-1及其两系杂交稻 浙优3号(Zheyou 3)在两年中的平均测定值338.45ng/g粉末和184.75ng/g粉末(表11)。该结果因此证实本发明设计的“基因组合”确实能完全关闭外源目的基因在稻米胚乳中的表达。
至于部分异交种糙米中所测定的Cry1Ab/1Ac蛋白含量达到1-3ng/g粉末，显著地高于野生型日本晴阴性对照糙米的测定值(小于1ng/g粉末)(表11)，可能与糙米皮层(由果皮、种皮、外胚乳和糊粉层等几部分组成)中的果皮在发育之初含有叶绿体有关。但在碾精时，糙米的皮层作为糠层被碾掉，因此，其中所积累的极微量的Cry1Ab/1Ac蛋白也被一同去掉。
表11.亲本KEY及LB品系、杂种LB/KEY和KEY/LB品系、阳性对照T51-1和浙优3号(Zheyou 3)和野生型阴性对照日本晴糙米和精米Cry1Ab/1Ac蛋白的ELISA定量分析
Figure PCTCN2016103934-appb-000012
a.表示表中数据测量自50粒/待测材料/重复随机取样的籽粒(共三次重复)；b.WT为野生型日本晴阴性对照，所有其它品系的测量数据均与其对比。数据采用SPSS 22.0软件包的Dunnett t检验分析。c.数值表示平均值±平均标准差。*表示与野生型日本晴阴性对照的差异达P＜0.05显著水平。**表示与野生型日本晴阴性对照的差异达P＜0.01极显著水平。
序列表和注释
本发明提供了用于构建“基因组合”的外源目的基因Bt、基因锁Lock1/Lock2和基因钥匙Key1/Key2的核苷酸序列及其推导的氨基酸序列，其中，Bt基因cry1Ab/1Ac由中国农科院范云六院士人工合成并提供使用；锁定元件Lock1/Lock2和钥匙基因Key1/Key2为人工合成。序列表中所列的各序列具体如下。
Figure PCTCN2016103934-appb-000013
Figure PCTCN2016103934-appb-000014
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21.Zhao D.，Lv L.，He A.，Luo K.，Duan H.，Zheng X.，Deng W.，Chen Y.，An X.，He M.，2008，The gene-deletor technology：principle and potential application in genetically engineered agriculture，Molecular Plant Breeding，2008，6(3)：413-418.
本发明不限于本文所述的具体实施方式的范围。实际上，除本文所述那些之外的本发明的各种变式对于参阅上述说明书的本领域技术人员将是显而易见的。这些变式应落入后附权利要求书的范围之内。
还应理解，对于核酸或多肽所给出的所有的碱基大小或氨基酸大小，和所有的分子量或分子质量数值都是近似值，并且提供以用于说明。

Claims (25)

  1. 一种用于控制植物中的外源目的基因的表达的基因组合，其由锁定元件和钥匙基因组成，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
  2. 权利要求1的基因组合，其中所述锁定元件位于组成型启动子和所述外源目的基因之间，并与所述组成型启动子和所述外源目的基因可操作连接。
  3. 权利要求2的基因组合，其中所述组成型启动子选自花椰菜花叶病毒35S启动子、来自根癌农杆菌Ti质粒T-DNA区域的胭脂碱合成酶基因Ocs启动子、水稻肌动蛋白启动子Actin I和玉米泛素基因启动子Ubi；优选地，所述组成型启动子是水稻肌动蛋白启动子Actin I。
  4. 权利要求1或2的基因组合，其中所述钥匙基因与组织特异性启动子可操作连接。
  5. 权利要求4的基因组合，其中所述组织特异性启动子选自水稻绿色组织特异性启动子核酮糖-1，5-二磷酸羧化酶小亚基rbcS启动子、玉米磷酸烯醇式丙酮酸羧化酶PEPC启动子、水稻绿色组织特异表达DX1启动子、水稻光系统II10kDa多肽D540启动子、水稻富亮氨酸重复序列类受体蛋白激酶LP2启动子，以及玉米叶绿体C4Pdk启动子；优选地，所述组织特异性启动子是水稻绿色组织特异性启动子rbcS启动子。
  6. 权利要求1-5中任一项所述的基因组合，其中所述外源目的基因是抗虫基因或抗除草剂基因，其中所述抗虫基因选自来自自苏云金芽胞杆菌(Bacillus thuringiensis)的cry1Ab、cry1Ac、cry1Ab/Ac、cry1C、cry2A和Vip3抗虫基因、来自嗜虫沙雷氏菌(Serratia entomophila)的anf、sep基因，来自双酶梭菌(Clostridium bifermentans)的cmb基因、来自球形芽孢杆菌(Bacillus sphaericus)的mtx基因、来自嗜线虫致 病杆菌(Xenorhabdus nematophilus)的杀虫蛋白基因、来自发光光杆状菌(Photorhabdus luminescens)的tca、tcb基因，以及来自金龟子绿僵菌(Metarhizium anisopliae)的prl基因；和/或所述抗除草剂基因选自抗草甘膦的EPSP合成酶基因、鼠伤寒沙门氏菌EPSP突变基因aroA、抗草丁膦的bar基因、抗米唑啉酮的ALS突变基因Ilv G、抗稀禾定的AccL-s2基因、抗溴苯腈的bxn基因和抗绿磺隆的csrl基因。
  7. 权利要求1-6中任一项所述的基因组合，其中所述植物选自水稻、小麦、大麦、燕麦、玉米、粟米、高粱、薏米、红薯、马铃薯、莲子、大豆和花生；优选地，所述植物是水稻。
  8. 一种用于控制植物中的外源目的基因的表达的方法，所述方法包括将锁定元件和钥匙基因导入受体植物中，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
  9. 一种培育转基因植物的方法，所述方法包括将包含锁定元件的第一亲本与包含钥匙基因的第二亲本进行杂交，从而获得包含所述锁定元件和钥匙基因的转基因植物，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
  10. 权利要求9的方法，其还包括在所述杂交步骤之前，将所述锁定元件导入和整合到所述第一亲本的基因组中，以及将所述钥匙基因导入和整合到所述第二亲本的基因组中。
  11. 一种培育转基因植物的方法，所述方法包括将将锁定元件和钥匙基因导入和整合到同一植株的基因组中，从而获得所述转基因植物，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含 与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
  12. 权利要求8-11中任一项的方法，其中使所述锁定元件位于组成型启动子和所述外源目的基因之间，并与所述组成型启动子和所述外源目的基因可操作连接。
  13. 权利要求12的方法，其中所述组成型启动子选自花椰菜花叶病毒35S启动子、来自根癌农杆菌Ti质粒T-DNA区域的胭脂碱合成酶基因Ocs启动子、水稻肌动蛋白启动子Actin I、玉米泛素基因启动子Ubi；优选地，所述组成型启动子是水稻肌动蛋白启动子Actin I。
  14. 权利要求8-12中任一项的方法，其中使所述钥匙基因与组织特异性启动子可操作连接。
  15. 权利要求14的方法，其中所述组织特异性启动子选自水稻绿色组织特异性启动子核酮糖-1，5-二磷酸羧化酶小亚基rbcS启动子、玉米磷酸烯醇式丙酮酸羧化酶PEPC启动子、水稻绿色组织特异表达DX1启动子、水稻光系统II10kDa多肽D540启动子、水稻富亮氨酸重复序列类受体蛋白激酶LP2启动子，以及玉米叶绿体C4Pdk启动子；优选地，所述组织特异性启动子是水稻绿色组织特异性启动子rbcS启动子。
  16. 权利要求8-15中任一项所述的方法，其中所述外源目的基因是抗虫基因或抗除草剂基因，其中所述抗虫基因选自来自苏云金芽胞杆菌(Bacillus thuringiensis)的cry1Ab、cry1Ac、cry1Ab/Ac、cry1C、cry2A和Vip3抗虫基因、来自嗜虫沙雷氏菌(Serratia entomophila)的anf、sep基因，来自双酶梭菌(Clostridium bifermentans)的cmb基因、来自球形芽孢杆菌(Bacillus sphaericus)的mtx基因、来自嗜线虫致病杆菌(Xenorhabdus nematophilus)的杀虫蛋白基因、来自发光光杆状菌(Photorhabdus luminescens)的tca、tcb基因，以及来自金龟子绿僵菌(Metarhizium anisopliae)的prl基因；和/或所述抗除草剂基因选自抗草甘膦的EPSP合成酶基因、鼠伤寒沙门氏菌EPSP突变基因aroA、抗草丁膦的bar基因、抗米唑啉酮的ALS突变基因Ilv G、抗稀禾定的AccL-s2基因、抗溴苯腈的bxn基因和抗绿磺隆的csrl基因。
  17. 权利要求8-16中任一项所述的方法，其中所述植物选自水稻、小麦、大麦、燕麦、玉米、粟米、高粱、薏米、红薯、马铃薯、莲子、大豆和花生；优选地，所述植物是水稻。
  18. 锁定元件和钥匙基因的基因组合用于调控植物中的外源目的基因的表达的用途，其中所述锁定元件能够锁定与其可操作连接的外源目的基因的表达；所述钥匙基因能够解除所述锁定元件对于外源目的基因的表达锁定。
  19. 如权利要求18所述的用途，其中所述锁定元件包含SEQ ID NO：5或8所示的核苷酸序列，或包含与SEQ ID NO：5或8所示的核苷酸序列具有至少80％的同源性，并且能够锁定与其可操作连接的外源目的基因的表达的核苷酸序列；所述钥匙基因包含SEQ ID NO：3或6所示的核苷酸序列，或包含与SEQ ID NO：3或6所示的核苷酸序列具有至少40％的同源性，并且能够解除所述锁定元件对于外源目的基因的表达锁定的核苷酸序列。
  20. 如权利要求18或19所述的用途，其中使所述锁定元件位于组成型启动子和所述外源目的基因之间，并与所述组成型启动子和所述外源目的基因可操作连接。
  21. 权利要求20的用途，其中所述组成型启动子选自花椰菜花叶病毒35S启动子、来自根癌农杆菌Ti质粒T-DNA区域的胭脂碱合成酶基因Ocs启动子、水稻肌动蛋白启动子Actin I、玉米泛素基因启动子Ubi；优选地，所述组成型启动子是水稻肌动蛋白启动子Actin I。
  22. 权利要求18-21中任一项的用途，其中使所述钥匙基因与组织特异性启动子可操作连接。
  23. 权利要求22的用途，其中所述组织特异性启动子选自水稻绿色组织特异性启动子核酮糖-1，5-二磷酸羧化酶小亚基rbcS启动子、玉米磷酸烯醇式丙酮酸羧化酶PEPC启动子、水稻绿色组织特异表达DX1启动子、水稻光系统II10kDa多肽D540启动子、水稻富亮氨酸重复序列类受体蛋白激酶LP2启动子，以及玉米叶绿体C4Pdk启动子；优选地，所述组织特异性启动子是水稻绿色组织特异性启动子rbcS启动子。
  24. 权利要求18-23中任一项所述的用途，其中所述外源目的基因是抗虫基因或抗除草剂基因，其中所述抗虫基因选自来自苏云金芽胞杆菌(Bacillus thuringiensis)的cry1Ab、cry1Ac、cry1Ab/Ac、cry1C、 cry2A和Vip3抗虫基因、来自嗜虫沙雷氏菌(Serratia entomophila)的anf、sep基因，来自双酶梭菌(Clostridium bifermentans)的cmb基因、来自球形芽孢杆菌(Bacillus sphaericus)的mtx基因、来自嗜线虫致病杆菌(Xenorhabdus nematophilus)的杀虫蛋白基因、来自发光光杆状菌(Photorhabdus luminescens)的tca、tcb基因，以及来自金龟子绿僵菌(Metarhizium anisopliae)的prl基因；和/或所述抗除草剂基因选自抗草甘膦的EPSP合成酶基因、鼠伤寒沙门氏菌EPSP突变基因aroA、抗草丁膦的bar基因、抗米唑啉酮的ALS突变基因Ilv G、抗稀禾定的AccL-s2基因、抗溴苯腈的bxn基因和抗绿磺隆的csrl基因。
  25. 权利要求18-24中任一项所述的用途，其中所述植物选自水稻、小麦、大麦、燕麦、玉米、粟米、高粱、薏米、红薯、马铃薯、莲子、大豆和花生；优选地，所述植物是水稻。
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