CN1366045A - Inducible promoter of fungus - Google Patents

Abstract

It has a fungal-inducible promoter sequence, and the promoter is derived from the antifungal protein gene of Gastrodia elata, which can enable foreign genes to be expressed in transgenic plants under the induction of fungi.

Description

fungal inducible promoter

The present invention relates to a fungal inducible promoter sequence and a construct comprising the promoter.

Specifically, the present invention relates to the promoter sequence of the fungus-induced expression activity of the antifungal protein gene derived from Gastrodia elata, as well as the gene construct, expression system and transgenic plant comprising the promoter sequence.

Gastrodia elata Bl. is an important traditional Chinese medicine in my country and belongs to the Orchidaceae plant. Gastrodia Antifungal Protein (GAFP) is the first protein that Hu Zhong isolated and purified from Gastrodia elata, which has a strong inhibitory effect on fungi (Hu Zhong et al., Yunnan Plant Research, 10:373-380, 1988). In vitro antibacterial experiments have proved that it has obvious antibacterial effects on pathogenic bacteria of many important crops, such as cotton wilt pathogen, gibberella wheat, Phytophthora melon, gibberella sugarcane and black shank of tobacco. GAFP is specifically distributed in the epidermis and cortex of Gastrodia elata secondary tubers (Jiang Liu et al., Acta Botanica Sinica, 35:593-599, 1993). Fungal infection has a strong induction effect on the expression of GAFP. Fungal infection Afterwards, the expression level of GAFP can be increased dozens of times (Hu Zhong et al., Yunnan Plant Research, 16: 169-177, 1994). Recently, three domestic research groups have cloned the cDNA of GAFP (Hu Zhong et al., Yunnan Plant Research, 21:131-138, 1999; Wang Xiaochen et al., Acta Botany, 41:1041-1045, 1999; Wang Yiqin et al., High Technology Communications , 10:10-14, 2000). Judging from the cDNA sequence, the Gastrodia elata antifungal protein gene may be a gene family, and its members have high homology (more than 80%) in protein sequence and nucleic acid sequence, and have high homology with lectin sex. So far, at least six GAFP cDNA sequences have been registered in Gene Bank.

In recent years, with the development of molecular biology techniques, some fungal-induced expression promoters in plants have been cloned. Their structure and function are well understood. Most of the genes containing these promoters are genes encoding key enzymes in the phytoalexin synthesis pathway, or genes related to proteins related to plant defense responses. These promoters include: Chalcone synthase Chs (Dron et al., Proc Natl Acad Sci USA, 85:6738-6742, 1988), phenylalanine lyase (Pal) (Rodrigo et al., EMBO J, 8:1641-1648,1989), isoflavone reductase (IFR) (Oommen et al., PlantCell, 6:1789-1803,1994), pine 1,2-stilbene synthase (PST) ( Regina et al., Plant Mol Biol, 39:221-229, 1999), the promoter of Arabidopsis plant defensin PDF gene (Manners et al., Plant Mol Biol, 38:1071-1080, 1998).

The purpose of the present invention is to provide a novel fungus-induced expression promoter, which can induce gene expression under the action of fungi. Another object of the present invention is to provide a method for expressing effective genes in plants using the promoter.

For this reason, the inventors of the present invention have successfully discovered a novel fungus-induced expression promoter through intensive research, and determined the base sequence of the promoter DNA. Then, link the promoter DNA with the reporter gene GUS, and transform tobacco with the carrier of the fusion gene, which proves that the promoter can promote the expression of the GUS gene in transgenic tobacco under the induction of fungi, and its activity is much higher than that expressed by plants. CaMV35S promoter commonly used in the system.

That is, the present invention provides novel DNA sequences having fungal-induced expression activity. The DNA sequence is a sequence composed of bases 1-1310 in the base sequences shown in the sequence table, or a partial fragment thereof having fungal-induced expression promoter activity. Also included are sequences having one or more nucleotide deletions, substitutions or insertions in these sequences.

According to the present invention, a novel fungal inducible promoter sequence is provided, and the promoter can induce gene expression under fungal induction in transgenic plants.

According to another aspect of the present invention, the above-mentioned isolated DNA and the recombinant DNA of the effective gene are provided, and relate to a vector containing the recombinant DNA and a plant culture cell, protoplast or plant containing the recombinant DNA.

The method for implementing the present invention will be described in detail below with reference to the accompanying drawings.

In the sequence listing, ATG Nos. 1306-1309 are initiation codons, TAG Nos. 1820-1822 are stop codons, Nos. 1306-1822 are coding region sequences, and Nos. 1-1305 are promoter sequences.

Figure 1 shows the restriction map (linear) of the plant expression vector pGGUS plasmid containing the GAFP promoter/GUS fusion gene.

The term "fungi-inducible" as used herein refers to the expression of a gene at a significantly higher level in the presence of fungi than in the background (ie, in the absence of fungus) levels.

The term "effective gene" refers to a gene encoding a protein that can be expressed in plant cells.

The term "construct" refers to a recombinant DNA or vector comprising the promoter sequence or partial sequence.

The term "transgenic plant" refers to plant seedlings or plants carrying the isolated DNA or partial fragments thereof.

As described in the examples, the discovery process of the present invention is as follows:

The GAFP cDNA (Wang Yiqin et al., High Technology Communication, 10: 10-14, 2000) isolated from Gastrodia elata cDNA library by the research group Wang Yiqin et al. can be used as a probe in the present invention. The Gastrodia elata genome library was constructed, and GAFP cDNA was used as a probe to screen the genome library, and the genes described in the sequence list were obtained. The DNA sequence of the gene was determined, and it was found that there was no intron in the gene, but only one coding frame. The sequence before ATG was connected with the reporter gene GUS and introduced into tobacco. As a result, under the induction of the fungus, the GUS gene was expressed at a high level in tobacco. Thus, the fungal inducibility of the promoter was confirmed. The process of obtaining the DNA fragment of the present invention will be described in detail below. (1) Construction of Gastrodia elata genome library

Gastrodia elata total DNA was extracted, genomic DNA was partially digested with Sau3AI, and fragments of 12-23Kb were recovered, connected to the λDASHII phage vector (Stratagene Company) digested by BamHI, and packaged with packaging protein Packagene Extracts (Promega Company), Preparation of phage library of Gastrodia elata genome. (2) Screening of Gastrodia elata genome library

The library was cultured on a plate, and the obtained phage plaques were transferred to a nylon membrane to denature the DNA, and fixed to the nylon membrane by ultraviolet light fixation. Then the membrane is hybridized with the isotope-labeled probe in the hybridization solution, so that the DNA on the membrane and the denatured probe form a hybrid. After the hybridization is completed, the non-specifically adsorbed probes on the membrane are washed away, and autoradiography is performed to identify clones that form hybrids with the probes. The second and third rounds of hybridization were performed until a single clone was obtained (Molecular Cloning, Second Edition, Cold Spring Harbor Laboratory, 1989). (3) Determination of DNA sequence

Extract the λDNA of the phage monoclonal, digest it with restriction endonuclease, perform agarose electrophoresis, and transfer it to a nylon membrane. After the same steps of DNA denaturation and fixation, hybridize with an isotope-labeled denatured cDNA probe, to identify specific bands. Find that XbaI can cut out a 2.2Kb band, reclaim this band, connect to the Bluescript SK carrier (Stratagene Company) through the same digestion, carry out the mensuration of DNA sequence (Liuhetong, Boya Company), obtained such as DNA sequences shown in Sequence Table 1. The search results in Genebank using NCBI's Blast software showed that the promoter sequence had no identity with known sequences, so it was a novel DNA sequence. (4) Identification of promoter activity

In the DNA sequence shown in the sequence table, there is an NcoI restriction site at the position of the 1305th base, and the vector described in (3) with the DNA fragment of the present invention is digested with SalI and NcoI to obtain the promoter region, The promoter region is connected with the GUS gene, and the carrier with the fusion gene is transformed with methods such as Agrobacterium-mediated genetic transformation or gene gun transformation (Handbook of Plant Genetic Transformation Technology, Fu Rongzhao, etc., China Science and Technology Press, 1994), introduced into tobacco to obtain transgenic tobacco containing the fusion gene carrier. Respectively in the two cases of fungal induction and no fungal induction, the expression of GUS gene was detected by methods such as histochemistry or fluorescent staining (Handbook of Plant Genetic Transformation Technology, Fu Rongzhao, etc., China Science and Technology Press, 1994), to prove that the activation The promoter has fungi-inducing activity, and under fungal-inducing conditions, the expression level of the GUS gene is 3-5 times that of the CaMV35S promoter as a control.

The fungi-inducible promoter of the present invention can express its gene under the induction of fungi.

Another feature of the present invention is that the effective gene can be linked to the downstream of the promoter of the present invention in an expressible manner to construct a plant expression vector and transform plant cells, protoplasts or plants. For this reason, the plasmid containing the promoter fragment of the present invention and the plasmid used to construct the expression vector can be digested with a suitable restriction enzyme, and then connected with a ligase to obtain a plant containing the promoter fragment of the present invention and an effective gene. Expression vector. If you can’t find a suitable endonuclease, you can use T4 DNA polymerase to fill in the blunt ends, and then use the above-mentioned DNA ligase to connect the blunt ends.

Effective genes used in the present invention include, but are not limited to Gastrodia elata antifungal protein gene, PR-1 gene, chitinase gene, dextranase gene, xanthanin-like protein gene (PR-5), osmotic protein gene , radish antifungal protein gene (As-AFP).

The DNA fragment obtained by linking the promoter region of the present invention with an effective gene is inserted into a plasmid to obtain a plant expression vector.

Agrobacterium-mediated genetic transformation, gene gun transformation, electroporation and other methods can be used to introduce the plasmid containing the DNA fragment and effective gene of the present invention into plant culture cells, protoplasts or plants.

The present invention can be used for any plant that can utilize the transformation method, and these plants include crops, such as wheat, barley, rice, corn, soybean, soybean, peanut, sorghum, cotton; fruits, such as apple, peach, pear, banana, watermelon, ; Vegetables such as cabbage, radish, lettuce, onion, cabbage, potato; and forest trees such as poplar and willow. However, it is not limited to the above-mentioned plant species.

The present invention will be further described below through embodiment.

Cloning of the gastrodia elata antifungal protein gene promoter of embodiment 1, the construction of the gastrodia elata genome library: 1, the extraction of the gastrodia elata total DNA: claim the gastrodia elata tuber that 1-3 gram-70 ℃ preserves, grind into powder in liquid nitrogen, add 20ml 65°C preheated extraction buffer (100mM Tris-Hcl, 500mM Nacl, 50mM EDTA, add 0.2% β-mercaptoethanol before use), shake; add 20% SDS 2ml, mix well, heat at 65°C for 10-20 minutes; Add 5M o 5ml, mix well, place on ice for 30 minutes; centrifuge at 20000g for 20 minutes; supernatant is precipitated with 2 times the volume of absolute ethanol; ). 2. Construction of Gastrodia elata genomic library: Take 20 μg of Gastrodia elata total DNA, perform partial enzyme digestion with Sau3AI, recover 12-23Kb fragments, connect with λDASHII (Stratagene) vector, and package the ligated product with packaging protein (Promega Company), and measure phage library drop degree, it can be used for library screening (Plant Molecular Biology, a Laboratory Manual, Springer, 1997). 2. Screening of Gastrodia elata genome library:

The specific operation steps of phage library plating, transmembrane transfer and immobilization were in accordance with "Molecular Cloning" (Sambrook et al., Molecular Cloning, cold spring harbor laboratory, 1989). The probe was GAFP cDNA, and the probe was labeled with Rche’s random primer labeling kit, and hybridized overnight at 65°C. Membrane washing: 4×SSC, 0.1% SDS, 60°C for 20 minutes; 2×SSC, 0.1% SDS, 60°C for 20 minutes; 1×SSC, 0.1% SDS, 60°C for 20 minutes. After washing the membrane, the membrane was dried and pressed against an X-ray film for autoradiography. 3. Sequence determination:

After the second and third rounds of screening, isolate and purify candidate positive clones, extract λDNA, digest with different restriction endonucleases, perform agarose electrophoresis, transfer to membrane, and hybridize with labeled probes. Then, the hybridization-positive bands with appropriate size were recovered, cloned into the Bluescript SK (Stratagene) vector, and sequenced.

Example IIGAFP gene promoter-GUS reporter gene fusion expression vector construction

The Bluescript SK vector with the promoter was digested with NcoI (New England Biolab), filled with T4 DNA polymerase (New England Biolab), after that, digested with SalI (New England Biolab), performed agarose electrophoresis, and recovered 1.3kb The specific fragment was connected to the pBI101.2 vector digested by SmaI and SalI with T4 DNA ligase, transformed into Escherichia coli, and the plasmid was extracted for identification, and the expression vector pGGUS for plant transformation was obtained.

Example III Genetic Transformation of Tobacco 1. Transformation of Recombinant Plasmids to Agrobacterium 1. Preparation of Competent Agrobacterium: Inoculate a single colony of Agrobacterium (LBA4404) in 50 ml of YEP medium, culture with shaking at 28° C. until OD ₆₀₀ is 0.5 , ice bath for 30 minutes, centrifuge at 5000rpm for 5 minutes, collect the bacteria, resuspend in 10ml 0.5M NaCl; centrifuge again, resuspend the bacteria in 1ml 20mM CaCl ₂ solution; take 50μl of the bacteria solution and put it in a 1.5ml centrifuge tube , stored at -70°C (Handbook of Plant Genetic Transformation Technology, Fu Rongzhao et al., China Science and Technology Press, 1994). 2. Transformation of plasmids to Agrobacterium: Take 0.5-1 μg of the above-mentioned plasmid DNA, add it to 50 μl of the above-mentioned competent bacteria, mix well, ice-bath for 30 minutes, freeze in liquid nitrogen for 1 minute, after melting at 37°C, add 1ml of YEP Cultivate the culture solution at 28°C for 2-4 hours; centrifuge at 6000rpm, discard most of the supernatant, spread on a plate containing 50 μg/ml streptomycin and 50 μ/ml kanamycin, and cultivate at 28°C. After the colony grows, the plasmid is extracted and identified by enzyme digestion (Handbook of Plant Genetic Transformation Technology, Fu Rongzhao et al., China Science and Technology Press, 1994). 2. Tobacco Transformation

Cut the true leaves of sterile seedlings into small pieces of 0.5cm square; centrifuge the overnight cultivated Agrobacterium at 4000rpm, suspend in callus or differentiation medium, and co-infect with exosomes for 5-10 minutes; suck the explants Dry, place on MS medium covered with filter paper, and culture in the dark at 28°C for 2 days; wash off the Agrobacterium on the surface of the explants, blot dry, and place on a medium containing 50 μg/ml kanamycin and 250 μg/ml carbenicillin After the buds have differentiated, they are cut and inserted on the rooting medium. After growing into plants, total DNA was extracted and Southern hybridization was performed to identify transgenic plants.

Identification of promoter activity in Example IV 1. Detection of GUS activity in transgenic tobacco 1. Histochemical analysis method: in the transformed tobacco stem, add an appropriate amount of fixative, and shake gently at room temperature for 30-60 minutes; use 50mM phosphate buffer (PH7 .0) Wash for 10-15 minutes, repeat several times; vacuum filter the x-Gluc staining solution for 1 minute; put the plant material in x-Gluc, keep warm at 37°C for 30 minutes; soak the material in 70% ethanol 5 minutes; observe under a microscope. 2. Fluorescence analysis method: add 100 μl extract to 100 mg transformed tobacco stem, grind in liquid nitrogen, thaw in ice bath; centrifuge at 4000 rpm for 2 minutes, take 0.5ml MUG analysis buffer, preheat at 37°C, add 1-50 μl enzyme Extract the solution, mix well, take out 100μl after 1-2 minutes, add to 0.9ml stop solution, after 5 minutes, 30 minutes, 60 minutes, take 100μl each, add to 0.9μl stop solution; measure the fluorescence respectively plant. For the preparation method of the solution, please refer to "Handbook of Plant Genetic Transformation Technology" (Fu Rongzhao et al., China Science and Technology Press, 1994). 2. Fungal induced expression

Inject Trichoderma viride spore suspension (5×10 ⁵ spores/ml water) on the leaves of transformed tobacco with a syringe, 4 drops per leaf, 5 μl per drop. GUS activity detection was performed every 2, 4, and 6 days (Manners et al., Plant Molecular Biology, 38: 1071-1080, 1998).

Sequence Listing Information (1) Sequence Features (A) Length: 2306kb (B) Type: Nucleic Acid (C) Strand Type: Double Strand (D) Geometry: Linear (2) Molecular Type: Genomic DNA (3) Deduction : not (4) antisense: not (5) sequence description:

Claims (10)

1, the DNA that exsomatizes is characterized in that, this stripped DNA by in the represented base sequence of sequence table, the sequence formed of No. 1-No. 1822 base sequence.

2, have the stripped DNA of fungal induction, it is characterized in that, this stripped DNA by in the represented base sequence of sequence table 1, No. 1-No. 1305 base sequence form or its have the active partial sequence of inducible promoter of fungus; Disappearance, displacement, the insertion of one or more bases are perhaps arranged in these sequences.

3, recombinant DNA, it contains the stripped dna fragmentation of claim 1 or claim 2 and carries out the effective gene that operability connects with it.

4, the carrier that contains the recombinant DNA of claim 2 or 3.

5, according to the carrier of claim 4, wherein carrier is plasmid vector or virus vector.

6, vegetable cell or protoplastis have wherein comprised the recombinant DNA of claim 2 or 3.

7, the plant that contains the vegetable cell of claim 6.

8, the vegetable cell or the protoplastis that contain the carrier of claim 4 or 5.

9, the plant that contains the vegetable cell of claim 8.

10, express the method for effective gene with the described stripped DNA of claim 1, this method comprises the following steps:

(1) this effective gene is connected to the downstream of the stripped DNA of claim 1 in effable mode;

(2) this recombinant dna fragment is inserted in the carrier;

(3) with method for transformation such as agriculture bacillus mediated genetic transformation, particle gun conversion, electroporation or pollen tube importings this carrier is imported in vegetable cell, protoplastis or the plant;

(4) culturing plants cell, protoplastis or plant.

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