CN105613568B - It is a kind of using siRNA using plant as the biological pesticide of generator - Google Patents

Abstract

The invention belongs to the field of genetic engineering and relates to a biopesticide using siRNA with plants as generators. The biological pesticide uses plants expressing siRNA targeting key genes of life metabolism of pests as raw materials. The siRNA described in the present invention is expressed by transgenic plants, and after being contacted or eaten by pests, the key genes of life metabolism of the targeted pests will be silenced through the RNAi mechanism. The invention aims at designing siRNA specifically targeting key genes of life metabolism of pests, and creatively uses plants as siRNA carriers to produce biological pesticides. By transferring the siRNA template sequence of highly targeted pests into the plant, the plant expresses the corresponding target siRNA, and the transgenic plant is planted on a large scale, and its extract is used to manufacture biopesticides, which are harmless to the environment and are Kills pests at the genetic level, hinders the development of pests, has high killing efficiency, and is not easy to cause pest resistance.

Description

A biopesticide using siRNA to use plants as generators

technical field

The invention belongs to the field of genetic engineering and relates to a biopesticide using siRNA with plants as generators.

Background technique

Small interfering ribonucleic acid (siRNA) is a type of double-stranded RNA molecule composed of more than 20 nucleotides, which can silence gene expression by specifically degrading the messenger RNA (mRNA) of the target gene. One process is called RNA interference (RNA interference, RNAi), which plays an important role in gene regulation and growth and development.

RNA interference (RNAi) is an antiviral mechanism. It is the post-transcriptional gene silencing of sequence-specific homologous genes mediated by dsRNA, and the effective mode of action in cells is siRNA. RNA interference can use siRNA or siRNA expression vector to specifically silence the target gene. This method is fast, economical, simple, and has a high degree of sequence specificity. It can obtain the loss of function or reduce the expression of the target gene in a specific way to reduce the mutation sequence, and become an exploration gene. An important research tool for functions. A large number of experiments have proved that the phenomenon of RNAi widely exists in the biological world, and it is one of the ancient defense mechanisms for organisms to resist virus infection and block transposons. With the deepening of the research, the mechanism of RNAi is being gradually elucidated, and at the same time, as an effective tool in functional genomics and gene therapy research, RNAi has attracted more and more attention. Studies have shown that siRNA can successfully knock out some drug resistance genes of cotton bollworm, diamondback moth and pea sprouts, and can prevent insect pests by knocking out acetylcholinesterase of insects, thus playing a role in pest control that is comparable to that of pesticides. Quite effective.

At present, pesticides on the market can be divided into organic pesticides, inorganic pesticides, botanical pesticides, and microbial pesticides according to the source of raw materials. In addition, there are insect hormones. According to the processing dosage form, it can be divided into powder, wettable powder, soluble powder, emulsion, emulsifiable concentrate, concentrated emulsion, cream, paste, colloid, fumigant, fumigant, aerosol, oil, granule and microgranule, etc. . Most are liquids or solids, and a few are gases. The loss of pesticides into the environment will cause serious environmental pollution, and sometimes even cause extremely dangerous consequences.

Traditional pesticides have the following disadvantages: 1. Pollution of the air and water environment, causing soil compaction. 2. Enhance the resistance of germs and pests to pesticides 3. Kill beneficial organisms 4. Wild life and livestock poisoning 5. Harm to human health. In order to overcome the above shortcomings, high efficiency, low toxicity and low residue are the development direction of the pesticide industry.

Although researchers have made preliminary studies on the use of RNA interference to control insect pests, there are still some insoluble problems that limit its commercial development. Among them, how to deliver siRNA and how to cull pests on a large scale are the main reasons hindering its application.

Contents of the invention

The object of the present invention is to address the above-mentioned defects in the prior art, and provide an siRNA expression vector for preparing biopesticides and cells containing the vector.

Another object of the present invention is to provide a biological pesticide.

Another object of the present invention is to provide a preparation method of the biopesticide.

The purpose of the present invention can be achieved through the following technical solutions:

The invention relates to a biological pesticide, which uses plants expressing siRNA targeting key genes of life metabolism of pests as raw materials. The siRNA described in the present invention is expressed by transgenic plants, and after being contacted or eaten by pests, the key genes of life metabolism of the targeted pests will be silenced through the RNAi mechanism.

Among them, the "key gene of life metabolism" refers to a gene that plays an important role in the life metabolism of pests, and the biopesticide of the present invention uses plants expressing siRNA targeting key genes of life metabolism of pests as raw materials. Key genes for life metabolism of pests that can be used in the present invention are not particularly limited, and generally include digestive system and nervous system-specific toxic genes, genes that affect insect growth and development through phenotypic screening, such as insect chitin synthase gene, ecdysone receptor gene, glutathione-S-transferase gene, etc.

Key genes for life metabolism of pests include acetylcholinesterase gene, AV2 gene, GPCR gene, V-ATPase gene, Cathespin gene, IAP gene, APN gene of Lepidoptera, Diptera or Hemiptera pests, more preferably, in One embodiment includes key genes for life metabolism of pests, including preferably acetylcholinesterase genes of pests of Lepidoptera, Diptera or Hemiptera.

For the biopesticide, it is preferable to construct a transgenic plant expressing siRNA that highly targets key genes of life metabolism of pests by means of genetic engineering, plant the transgenic plant, and use the transgenic plant or its extract for the production of biopesticide.

For the biopesticide, it is further preferred to prepare the leaves or other organs of the transgenic plant into powder as the biopesticide; or extract the siRNA expression product in the transgenic plant as the biopesticide.

In the present invention, exogenous siRNA can be introduced into the vector by conventional technical means in the art, and expressed in transgenic plants. The species of plants that can be used in the present invention are not particularly limited, and generally include Poaceae, Typhaceae, Compositae, Labiatae, Liliaceae, Amaryllidaceae, Boraginaceae, Araceae, Umbelliferae, Cruciferae, Primula Polygonaceae, Chenopodiaceae, Caryophyllaceae, Willowaceae, Urticaceae, Plantainaceae, Myricaceae, Moraceae, Cannabisaceae, Saxifragaceae, Fabaceae, Fernaceae, Violaceae, Silver Lactucaceae, Amaranthaceae, Fagaceae, Chlorellaceae, Theaceae, Rubiaceae, Sycamore, Pinaceae, Cucurbitaceae, Cyperaceae, Podocarpus, Betulaceae, Juglandaceae, Pepperaceae, Magnoliaceae , Dendtaceae, Agaricaceae, Tricholomaceae, Agaricaceae, Russulaceae, Rhododendronaceae, Rosaceae, Actinidiaceae, Apricotaceae, Grapeaceae, Anemoneaceae, Begoniaceae, Bromeliaceae, Lilyaceae, Ginkgoceae, Star Aniseceae, Gingeraceae, Pomegranateceae, Ranunculaceae, Apocynaceae, Berberidaceae, Rutaceae, Solanaceae, Poppyaceae, Verbenaceae, Wintergreenaceae, Commelinaceae, Daphneaceae , Mulberry family, Radixaceae, Sanbaiaceae, Crassulaceae, Lepidopteridaceae, Purslaneceae, Alismaceae, Malvaceae, Scrophulariaceae, Bignoniaceae, Fangzhike, Haijinsha Branches, Acanthaceae, Convolvulaceae, Polyporaceae, Sapindaceae, Cupressaceae, Bitterwoodaceae, Euphorbiaceae, Meliaceae, Gentlemenaceae, Lonicera, Tamaraceae, Liedangaceae, Rushaceae, Hamameliaceae, Gentianaceae, Tiliaceae, Calamusaceae, Ilexaceae, Plantaceae, Anacardiaceae, Schisandraceae, Araliaceae, Kapokaceae, Aristolochiaceae, Oleaceae, Palmaceae, Nymphaeaceae, Sandalaceae One or a combination of plants.

Preferably, the transgenic plants include lettuce, rice, wheat, corn, peanut, sorghum, soybean, potato, buckwheat, quinoa, thistle, pepper, star anise, fennel, peach, apricot, pear, apple, banana, Hericium erinaceus, fungus, Yam, hawthorn, ginseng, angelica, tomato, pepper, eggplant, carrot, cabbage, cauliflower, Chinese cabbage, pak choy, rape, spinach, mustard greens, peas, pumpkin, cucumber, watermelon, melon, shrub, onion, or combination.

More preferably, the transgenic plant comprises lettuce, alfalfa, ryegrass, soybean or rice.

For the biopesticide, it is also preferable to express the siRNA in the same plant together with at least one second insecticidal protein that is different from the expression product of the siRNA target gene or express it in different plants and then mix it as Raw materials for biopesticides.

Wherein, the second insecticidal protein is Vip insecticidal protein, protease inhibitor, lectin, α-amylase or peroxidase.

The expression of the siRNA in the same plant together with at least one second insecticidal protein different from the expression product of the siRNA target gene is achieved by genetically engineering the plant to contain and express the siRNA and the second insecticidal protein; or by Use genetic engineering to make the first parent plant express siRNA and the second parent plant express the second insecticidal protein, and then cross the first parent and the second parent to obtain offspring plants expressing all the genes introduced into the first parent and the second parent .

An siRNA expression vector for preparing biological pesticides, which is obtained by inserting the template sequence of siRNA for key genes of pest life metabolism into a binary expression vector; the key genes for life metabolism of pests are selected from Specific poisonous genes, or genes that affect the growth and development of insects after phenotypic screening; preferably the acetylcholinesterase gene, AV2 gene, GPCR gene, V-ATPase gene, Cathespin of Lepidoptera, Diptera or Hemiptera pests gene, IAP gene, APN gene, preferably the acetylcholinesterase gene of Lepidoptera, Diptera or Hemiptera pests; the binary expression vector is selected from pcambia1301, pcambia1300, pcambia2300 or other vectors selected from the pcambia series. The binary expression vector used in the present invention is preferably pCAMBIA2301 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.)

The siRNA expression vector for preparing biopesticides is preferably obtained by inserting the siRNA template sequence shown in SEQ ID NO.19 designed for the acetylcholinesterase target gene shown in SEQ ID NO.1 into the binary expression vector pCAMBIA2301.

A cell containing the siRNA expression vector of the present invention.

Application of the siRNA expression vector and the cell containing the siRNA expression vector in the preparation of biological pesticides.

A method for controlling pests, which comprises contacting the pests attacking the plant with the biopesticide or the composition containing the biopesticide, thereby controlling the attack of the pests on the plant.

The term "RNA interference (RNAi)" refers to some RNAs that can efficiently and specifically block the expression of specific genes in the body, promote mRNA degradation, and induce cells to show a phenotype of specific gene deletion, which is also called RNA interference Or interfere. RNA interference is a highly specific gene silencing mechanism at the mRNA level.

The term "interfering RNA" or "dsRNA" refers to an RNA molecule that can degrade a specific mRNA with a homologous complementary sequence mRNA as its target. This process is the RNA interference pathway (RNAinterference pathway).

Beneficial effect:

The invention aims at the design of siRNA specially targeting key genes of life metabolism of pests, and creatively uses plants as siRNA carriers to produce biological pesticides. By transferring the siRNA template sequence of highly targeted pests into the plant, the plant expresses the corresponding target siRNA, and the transgenic plant is planted on a large scale, and its extract is used to manufacture biopesticides, which are harmless to the environment and are Kills pests at the genetic level, hinders the development of pests, has high killing efficiency, and is not easy to cause pest resistance.

As a preferred mode of the present invention, multiple siRNA sequences that can kill pests with targeted properties were designed and screened for the acetylcholinesterase gene, introduced into plants for expression, crushed and used as biopesticides. Has an ideal lethal effect.

Description of drawings

Figure 1 Vector construction flow chart.

Detailed ways

Embodiment 1: construct the method for the plasmid vector of stably expressing exogenous siRNA in plant

This example provides specific methods for screening, obtaining and constructing a nucleic acid construct vector containing the target gene. Through screening, multiple siRNA sequences that can target and kill pests are obtained.

The following are the target genes and corresponding siRNA sequences:

Plutella xylostella target gene: CATGCCTGGTGATGTAATA (SEQ ID NO.1)

Sense strand: CAUGCCUGGUGAUGUAAUA (SEQ ID NO.2)

Antisense strand: UAUUACAUCACCAGGCAUG (SEQ ID NO.3)

Aphid target gene: TGGTTGGTATTAGAAACAATGTT (SEQ ID NO.4)

Sense strand: UGGUUGGUAUUAGAAACAAUGUU (SEQ ID NO.5)

Antisense strand: AACAUUGUUUCUAAUACCAACCA (SEQ ID NO.6)

Drosophila melanogaster target gene: AACTATAAAGTTGATGATATTAT (SEQ ID NO.7)

Sense strand: AACUAUAAAGUUGAUGAUAUUAU (SEQ ID NO.8)

Antisense strand: AUAAUAUCAUCAACUUUAUAGUU (SEQ ID NO.9)

Target gene of Lygus graminis: TATACGCTATCTGATACCATGTA (SEQ ID NO.10)

Sense strand: UAUACGCUAUCUGAUACCAUGUA (SEQ ID NO.11)

Antisense strand: UAGAUGGUAUCAGAUAGCGUAUA (SEQ ID NO.12)

Asian corn borer target gene: TCGTACCATCGAGTACCTACG (SEQ ID NO.13)

Sense strand: UCGUACCAUCGAGUACCUACG (SEQ ID NO.14)

Antisense strand: CGUAGGUACUCGAUGGUACGU (SEQ ID NO.15)

Target gene of Spodoptera spp.: GTATCGGTATAGCCGATCGTA (SEQ ID NO.16)

Sense strand: GUAUCGGUAUAGCCGAUCGUA (SEQ ID NO.17)

Antisense strand: UACGAUCGGCUAUACCGAUAC (SEQ ID NO.18)

The principle of each small interfering RNA insecticide:

Plutella xylostella acetylcholinesterase gene: The target sequence used to produce dsRNA is Plutella xylostella acetylcholinesterase gene, siRNA can silence acetylcholinesterase genes 1 and 2, thereby affecting the normal nerve impulse transmission of insects, making insects continue to be in a state of excitement Death, and then realized the management of insects.

Aphid AV2 gene: The target sequence used to generate the dsRNA was a homologous fragment of the A subunit gene of the V-type proton pump ATPase isolated from cotton aphid. But the target sequence may not be limited to this gene and cotton aphid. In the carrier of the present invention, the transcription of the dsRNA is driven by a specific promoter, so that the dsRNA is mainly accumulated in the sieve tube. Its potential pest control targets are aphids, whiteflies, and scale insects that feed on piercing-sucking mouthparts.

Drosophila melanogaster GPCR gene: Drosophila melanogaster octopamine receptor activity G protein-coupled receptor (GPCR) as an insecticide target, affects the digestive system of Drosophila melanogaster through siRNA, thereby killing pests.

Lygus pods, Asian corn borer, Spodoptera exigua: affect the digestive system of pests through siRNA, so as to kill pests.

To sum up, this example provides multiple sequences covering various pests of Lepidoptera, Diptera, Hemiptera, etc., and can effectively kill pests whose toxicology is similar or similar to the pests of the above orders.

According to Plutella xylostella target gene siRNA, aphid target gene siRNA, Asian corn borer target gene siRNA, and Drosophila melanogaster target gene siRNA, respectively construct the required nucleic acid constructs, transform the prokaryotic expression elements, and design the primers used in the experiment, specific steps include:

(1) Acquisition of siRNA recombinant expression vector of Plutella xylostella

Using the backbone vector pAd/PL-DEST (purchased from invitrogen) as a template to obtain three fragments SCmx 1, SCmx2, and SCmx 3 through PCR reaction, the primer sequences of the three fragments are respectively:

SCmx 1:FORWARD:5-ATATCGTTACGTACGG-3

REVERSE: 5-GGCCGTATCGTAATAT-3

SCmx2:FORWARD:5-GAGCCACACGTGACG-3

REVERSE: 5-TACGGTACTTGTCATTT-3

SCmx2:FORWARD:5-ATTTGTCGAGCAAGACC-3

REVERSE: 5-CCACTACGATGCGATCG-3

Separation by agar 1.5% sugar gel electrophoresis and recovery by tapping the gel.

Using the recovered three fragments as templates, and then using high-fidelity DNA polymerase (Takara), after overlapping PCR reaction, the overlapping PCR reaction system is as follows: 100 μl PCR reaction solution includes 30 pmol of forward primer and reverse primer, forward primer F1: 5-TTGATCGATGCAACGTAC-3 (SEQ ID NO.25); reverse primer R1: 5-GAGACATGGCCCATGGATA-3 (SEQ ID NO.26), 0.2 μg of template DNA, 0.25 units of Taq enzyme; the reaction procedure is first After pre-denaturation at 95°C for 3 minutes, the reaction cycle was carried out 30 times at 94°C for 30 s, 55°C for 30 s, and 72°C for 1 min, and finally at 72°C for 10 min. The desired full-length fragment was obtained as shown in SEQ ID NO.19.

The nucleotide sequence was ligated into a T vector, and the T vector was pGEM-T easy (purchased from Shanghai Shrek Biotechnology Co., Ltd.). The cloning vector pEASY01-T obtained by recombination was transformed into Escherichia coli T1 competent cells by heat shock method. After the cells were cultured, the plasmid was extracted and identified by EcoRV and Smal digestion, and the positive clones were sequenced and verified. The results showed that the recombinant cloning vector pEASY01-T Insert the desired sequence.

The recombinant cloning vector pEASY01-T and the expression vector pCAMBIA2301 (purchased from Beijing Dingguo Changsheng Biotechnology Co., Ltd.) were digested with restriction endonucleases Spel and Pvul, respectively. The vector construction process is shown in Figure 1. Insert the excised target fragment into the expression vector pCAMBIA2301, the insertion site is between the Spel and Pvul sites, and the construction of the vector by conventional enzyme digestion method is well known to those skilled in the art, and the recombinant expression vector pEASY-3M is constructed -01.

(2) Acquisition of the siRNA recombinant expression vector for the target gene of Ostrinia sativa

Using the backbone vector pAd/PL-DEST (purchased from invitrogen) as a template to obtain three fragments OFmx1, OFmx2, and OFmx3 through PCR reaction, the primer sequences of the three fragments are respectively:

OFmx1:FORWARD:5-AATATCACGTACGTGTG-3

REVERSE: 5-CAGTACCGACTCAATGT-3

OFmx2:FORWARD:5-TCAATCGCATACCAATCT-3

REVERSE: 5-CACCATATCAGATCGATC-3

OFmx3:FORWARD:5-ATTCTACTAGGAATACT-3

REVERSE: 5-GTACATACTACCATCG-3

Separation by agar 1.5% sugar gel electrophoresis and recovery by tapping the gel.

Obtain the siRNA template sequence by overlapping PCR, the experimental procedure is as described in (1), and the difference is the primer sequence, and the primer sequence is as follows:

Forward primer F2: 5-ACTAACCAGACTGTATGACAA-3

Reverse primer R2: 5-TTAGATCAGTCCACTTCACC-3

The specific experimental operations for obtaining the template sequence and obtaining the recombinant expression vector pEASY-3M-02 are the same as those described in (1) for the starting vector, and the template sequence is shown in SEQ ID NO.20. According to the method in (1), the template sequence was cloned into the expression vector pCAMBIA2301 to obtain the recombinant expression vector pEASY-3M-02.

(3) Acquisition of aphid target gene siRNA recombinant expression vector

Using the backbone vector pAd/PL-DEST (purchased from invitrogen) as a template to obtain three fragments APmx1, APmx2, and APmx3 through PCR reaction, the primer sequences of the three fragments are respectively:

APmx1:FORWARD:5-TCCGACGACCAGTCCGT-3

REVERSE: 5-TAATTCGGCAACTACGT-3

APmx2:FORWARD:5-ACCGTTGCAAACACGTCT-3

REVERSE: 5-AATCGTAACACAGACTTC-3

APmx3:FORWARD:5-ATACGTCTAGCCATACT-3

REVERSE: 5-GCACATCCTTACAGTCG-3

Separation by agar 1.5% sugar gel electrophoresis and recovery by tapping the gel.

Obtain the siRNA template sequence by overlapping PCR, the experimental procedure is as described in (1), and the difference is the primer sequence, and the primer sequence is as follows:

Forward primer F3: 5-ACCAACCACAATCTTTGTTACAA-3

Reverse primer R3: 5-TTACGTCACTCGATCACCCT-3

The specific experimental operations for obtaining the template sequence and obtaining the recombinant expression vector pEASY-3M-03 are the same as those described in (1) for the starting vector, and the template sequence is shown in SEQ ID NO.21. According to the method in (1), the template sequence was cloned into the expression vector pCAMBIA2301 to obtain the recombinant expression vector pEASY-3M-03.

(4) Acquisition of Drosophila melanogaster target gene siRNA

Using the backbone vector pAd/PL-DEST (purchased from invitrogen) as a template to obtain three fragments DMmx1, DMmx2, and DMmx3 through PCR reaction, the primer sequences of the three fragments are respectively:

DMmx1:FORWARD:5-ATCGACGAACTCAACT-3

REVERSE: 5-ATTCACGTACAACGGT-3

DMmx2:FORWARD:5-GGCTAGCTACAATCGAAT-3

REVERSE: 5-TAATAAGCTAAGCCTCC-3

DMmx3:FORWARD:5-GGATACTTACCATCAAT-3

REVERSE: 5-GCCTAATACATGCTACTG-3

Separation by agar 1.5% sugar gel electrophoresis and recovery by tapping the gel.

Obtain the siRNA template sequence by overlapping PCR, the experimental procedure is as described in (1), and the difference is the primer sequence, and the primer sequence is as follows:

Forward primer F4: 5-GGCTCTAGCTCTAATCGATC-3

Reverse primer R4: 5-CAACTAGCTTACCATTCCTA-3

The specific experimental operations for obtaining the template sequence and the recombinant expression vector pEASY-3M-04 are the same as those described in (1) for the starting vector, and the template sequence is shown in SEQ ID NO.22. According to the method in (1), the template sequence was cloned into the expression vector pCAMBIA2301 to obtain the recombinant expression vector pEASY-3M-04.

(5) Acquisition of target gene siRNA of Lygus japonica

Using the backbone vector pAd/PL-DEST (purchased from invitrogen) as a template to obtain three fragments DMmx1, DMmx2, and DMmx3 through PCR reaction, the primer sequences of the three fragments are respectively:

DMmx1:FORWARD:5-ATCGACGAACTCAACT-3

REVERSE: 5-ATTCACGTACAACGGT-3

DMmx2:FORWARD:5-GGCTAGCTACAATCGAAT-3

REVERSE: 5-TAATAAGCTAAGCCTCC-3

DMmx3:FORWARD:5-GGATACTTACCATCAAT-3

REVERSE: 5-GCCTAATACATGCTACTG-3

Separation by agar 1.5% sugar gel electrophoresis and recovery by tapping the gel.

Obtain the siRNA template sequence by overlapping PCR, the experimental procedure is as described in (1), and the difference is the primer sequence, and the primer sequence is as follows:

Forward primer F5: 5-GGCTCTAGCTCTAATCGATC-3

Reverse primer R5: 5-CAACTAGCTTACCATTCCTA-3

The specific experimental operations for obtaining the template sequence and obtaining the recombinant expression vector pEASY-3M-05 are the same as those described in (1) for the starting vector, and the template sequence is shown in SEQ ID NO.23. According to the method in (1), the template sequence was cloned into the expression vector pCAMBIA2301 to obtain the recombinant expression vector pEASY-3M-05.

(6) Acquisition of gene siRNA of the two-pointed worm

Using the backbone vector pAd/PL-DEST (purchased from invitrogen) as a template to obtain three fragments DMmx1, DMmx2, and DMmx3 through PCR reaction, the primer sequences of the three fragments are respectively:

DMmx1:FORWARD:5-ATCGACGAACTCAACT-3

REVERSE: 5-ATTCACGTACAACGGT-3

DMmx2:FORWARD:5-GGCTAGCTACAATCGAAT-3

REVERSE: 5-TAATAAGCTAAGCCTCC-3

DMmx3:FORWARD:5-GGATACTTACCATCAAT-3

REVERSE: 5-GCCTAATACATGCTACTG-3

Separation by agar 1.5% sugar gel electrophoresis and recovery by tapping the gel.

Obtain the siRNA template sequence by overlapping PCR, the experimental procedure is as described in (1), and the difference is the primer sequence, and the primer sequence is as follows:

Forward primer F6: 5-GGCTCTAGCTCTAATCGATC-3

Reverse primer R6: 5-CAACTAGCTTACCATTCCTA-3

The specific experimental operations for obtaining the template sequence and obtaining the recombinant expression vector pEASY-3M-06 are the same as those described in (1) for the starting vector, and the template sequence is shown in SEQ ID NO.24. According to the method in (1), the template sequence was cloned into the expression vector pCAMBIA2301 to obtain the recombinant expression vector pEASY-3M-06.

Embodiment 2: exogenous siRNA expression vector transforms cruciferous lettuce

Target siRNA sense strand nucleotide sequence (sequence is as follows):

5'-CAUGCCUGGUGAUGUAAUA-3' (SEQ ID NO.2)

5'-UGGUUGGUAUUAGAAACAAUGUU-3' (SEQ ID NO.5)

5'-AACUAUAAAGUUGAUGAUAUUAU-3' (SEQ ID NO.8)

5'-UAUACGCUAUCUGAUACCAUGUA-3' (SEQ ID NO.11)

5'-UCGUACCAUCGAGUACCUACG-3' (SEQ ID NO.14)

5'-GUAUCGGUAUAGCCGAUCGUA-3' (SEQ ID NO.17)

5'-CUAACGAACUAUGCCGCGUAUA-3' (SEQ ID NO.18)

The experiments of transforming plants all included culture of sterile seedlings, infection of Agrobacterium EHA105 with the recombinant expression vector constructed in Example 1, culture of plant callus bud induction, and seedling root induction culture. Specifically include the following steps:

The transformed plants were respectively adopted the leaf disc method mediated by Agrobacterium.

The surface of the lettuce seeds is sterilized by shaking with 10% NaClO for about 10 minutes; rinsed with sterile water 4-5 times; dried and inoculated on the medium, and cultivated at 25°C for 5 days. Cut off the fresh leaf buds for the next step of plasmid transformation.

The exogenous siRNA expression plasmids pEASY-3M-01 (containing Plutella xylostella siRNA), pEASY-3M-02 (containing the siRNA of the target gene of O. 3M-04 (containing the siRNA of the target gene of Drosophila melanogaster), pEASY-3M-05 (containing the siRNA of the target gene of Lygus graminis), and pEASY-3M-06 (containing the siRNA of the target gene of S. Culture on a shaker at 28°C, collect cells by centrifugation, resuspend in sucrose solution, and this mixture is the infection solution.

Place the freshly cut weed leaf buds in the above infection solution for suspension culture for 10 minutes, then place them on sterile filter paper to absorb excess liquid, and inoculate the cotyledons on 1/2 MS medium covered with a layer of sterile filter paper in advance superior. After culturing for 2 days, the leaf buds were rinsed 4-5 times with sterile water containing 4 mg/ml carbenicillin, and then inoculated on the bud-inducing medium.

After the induced bud grows, it is cut off from the explant, and transferred to the root-inducing medium, and the generation of young roots can be seen in about 7-10 days. After the seedlings take root, they are transferred to the soil for cultivation, and the test example Expression of siRNA sense strands (SEQ ID NO.2, SEQ ID NO.5, SEQ ID NO.8, SEQ ID NO.11, SEQ ID NO.14, SEQ ID NO.17) described in 1 in plants. Finally, six kinds of transgenic lettuce were obtained, and the following six recombinant plasmids were integrated respectively: pEASY-3M-01 (containing Plutella xylostella siRNA), pEASY-3M-02 (containing siRNA of the target gene of O. Aphid target gene siRNA), pEASY-3M-04 (containing Drosophila melanogaster target gene siRNA), pEASY-3M-05 (containing Lygus pod gene siRNA).

The present invention also provides the following transfection methods:

1. Calcium Phosphate Co-precipitation

Calcium chloride, RNA (or DNA) and phosphate buffer are mixed and precipitated to form very small insoluble calcium phosphate particles containing DNA. The calcium phosphate-DNA complex adheres to the cell membrane and enters the cytoplasm of the target cell by pinocytosis. The size and quality of the pellet are critical to the success of calcium phosphate transfection.

2. Electroporation

Electroporation transduces molecules by exposing cells to brief pulses of high field strength electricity. Placing a cell suspension in an electric field induces a voltage difference along the cell membrane that is thought to cause the cell membrane to temporarily perforate. The optimization of electric pulse and field strength is very important for successful transfection, because too high field strength and too long electric pulse time can irreversibly damage the cell membrane and lyse the cells. Typically, successful electroporation procedures are accompanied by high levels (50% or higher) of toxicity.

3. DEAE-dextran and polybrene multimeric complex

The positively charged DEAE-dextran or polybrene multimer complexes and negatively charged DNA molecules allow DNA to bind to the cell surface. The DNA complexes were introduced by osmotic shock obtained with DMSO or glycerol. Both reagents have been used successfully for transfection.

4. Cationic Liposome Reagent

When added to water under optimized conditions, cationic liposome reagents can form tiny unilamellar liposomes. These liposomes are positively charged and can bind electrostatically to the phosphate backbone of DNA and to the negatively charged surface of cell membranes. Using cationic liposome reagents, DNA is not pre-embedded in liposomes, but negatively charged DNA automatically binds to positively charged liposomes to form DNA-cationic liposome complexes.

After the completion of the construction, in order to confirm whether the transformation plant was successfully constructed, the following work was carried out, see Example 4.

Example 3 Transformation of Poaceae Lolium with Exogenous siRNA Expression Vector

The transformed plants were induced by Agrobacterium.

According to the routinely used Agrobacterium dipping method, the aseptically cultured young ryegrass embryos were co-cultured with the recombinant Agrobacterium described in Example 2, so as to transfer the T-DNA in the recombinant expression vector constructed in Example 1 into Into the ryegrass chromosome, a ryegrass plant (positive control) transformed with exogenous siRNA was obtained; at the same time, wild-type ryegrass was used as a negative control.

For Agrobacterium-mediated transformation of ryegrass, briefly, immature embryos are isolated from ryegrass, and the embryos are contacted with a suspension of Agrobacterium capable of delivering a nucleotide sequence to at least one of the embryos. A cell with the nucleic acid construct operably linked to the ryegrass genome. In this step, the immature embryos are preferably immersed in an Agrobacterium suspension (OD600=0.4-0.6, infiltration medium). Preferably, immature embryos are cultured on solid medium with antibiotics, but without selection, to eliminate Agrobacterium and to provide a recovery period for infected cells. Afterwards, the transgenic ryegrass callus was screened out by selection medium. The callus then re-forms the plant.

Finally, six kinds of transgenic ryegrass were obtained, and the following six recombinant plasmids were integrated respectively: pEASY-3M-01 (containing Plutella xylostella siRNA), pEASY-3M-02 (containing siRNA of the target gene of O. Aphid target gene siRNA), pEASY-3M-04 (Drosophila melanogaster target gene siRNA), pEASY-3M-05 (L. moth gene siRNA).

Example 4 Detection experiment of exogenous siRNA in transformed plants

Verification of transgenic ryegrass by Taqman method. The specific operation steps are as follows:

Take 100 mg of leaves of ryegrass and wild-type ryegrass with the target siRNA sequence, grind them into a homogenate with liquid nitrogen in a mortar, and take 3 replicates for each sample;

The genomic DNA of the above samples was extracted with Qiagen DNeasy Plant Mini Kit, and the concentration was adjusted to 80-100ng/ul;

The copy number of the sample was identified by the Taqman probe fluorescent quantitative PCR method, and the probe primers were as follows:

Plutella xylostella target gene probe 5'-UAUUACAUCACCAGGCAUG-3'

Asian corn borer target gene probe 5'-AACAUUGUUUCUAAUACCAACCA-3'

Aphid target gene probe 5'-AUAAUAUCAUCAACUUUAUAGUU-3'

Drosophila melanogaster target gene probe 5'-UAGAUGGUAUCAGAUAGCGUAUA-3'

Target gene probe 5'-CGUAGGUACUCGAUGGUACGU-3'

Probe 5'-UACGAUCGGCUAUACCGAUAC-3'

The identified sample with known copy number was used as a standard, and the wild type was used as a negative control. Each sample was replicated three times, and finally a single copy of siRNA-transferred ryegrass was obtained.

This embodiment provides another implementation:

1. Extract the genomic DNA of the plant to be tested, using conventional methods such as CTAB;

2. Using genomic DNA as a template for multiplex PCR amplification;

3. Denaturation and renaturation treatment of PCR products;

4. Detection of PCR products by agarose gel electrophoresis after denatured and refolded treatment;

If the electrophoretic band migrates farther than the untreated PCR product after renaturation treatment, it indicates that after renaturation treatment, the PCR product forms a hairpin-like folded structure, and the hairpin-like structure is the siRNA expression vector. The unique structure designed for gene silencing, based on which it can be inferred that the sample belongs to siRNA transgenic plants; if the migration distance of the electrophoretic bands does not change after renaturation treatment, it indicates that no new folding structure has been formed, and it can be judged that the sample is not Belongs to transgenic plants. It was tested that the transgenic lettuce and transgenic ryegrass constructed in Examples 2 and 3 both expressed the target exogenous siRNA.

Example 5 Changes in Physiological Conditions of Lepidoptera Insects After Ingesting Genetically Engineered Plants

Lepidoptera (scientific name Lepidoptera) belongs to the winged subclass, holometabolism. About 200,000 species are known in the world, and more than 8,000 species are known in China. This order is the second largest order in the class Insecta after Coleoptera. Among them, there are 6,000 species of moths and 2,000 species of butterflies. It is also an order with the most agricultural and forestry pests, such as corn borer, rice leaf roller, diamondback moth, etc. In this example, a bioassay test was carried out on Lepidoptera insects Plutella xylostella and Ostrinia sativa to determine the killing effect of genetically engineered plants on target insects. The transgenic plants used in the Plutella xylostella experiment were transgenic lettuces carrying the exogenous siRNA expression plasmid pEASY-3M-01 constructed in Example 2, and the transgenic plants used in the Asian corn borer experiment were the exogenous siRNA expression plasmid pEASY-3M constructed in Example 2 -02 Genetically modified lettuce.

The specific implementation steps are as follows: take the lettuce leaves that have been transferred to the siRNA recombinant expression vector designed for insects, the wild-type lettuce leaves, and the lettuce leaves identified as non-transgenic by Taqman, rinse them with sterile water, and wipe the water on the leaves with gauze. Blot dry, remove leaf veins, grind into powder, stir evenly with insect bait, and feed target insects regularly. Put 10 artificially raised target insects in each petri dish, after the insect test petri dish is covered, under the conditions of temperature 26-28 ℃, relative humidity 70%-80%, photoperiod (light/dark) 16:8 After standing for 3 days, the larval mortality was counted, and the average mortality rate of pests in each sample was calculated. A total of 3 strains (S1, S2 and S3) transferred to the siRNA recombinant expression vector, a total of 2 strains (S4 and S5) identified as non-transgenic by Taqman, and 1 wild-type strain (CK); Five plants were selected from each strain for testing, and each plant was repeated 5 times. The specific results are shown in Table 1.

Table 1. Bioassay results of lettuce leaves transfected with siRNA against Plutella xylostella

It can be seen from Table 1 that after the transgenic lettuce leaves (S1, S2, S3) fed the target pest Plutella xylostella, the lethality rate of Plutella xylostella significantly increased compared with the non-transgenic group (S4, S5) and the wild-type lettuce group (CK). The average lethality rate of the transgenic group was 79.67%, the average lethality rate of the non-transgenic plant group was 20.5%, and the lethality rate of the CK group was 8.6%. It can be seen from the data that the average lethality rate of the transgenic group increased by 59.17% compared with the non-transgenic plant group Compared with the CK group, it has increased by 71.09%. The above data prove that the transgenic lettuce obtained in Example 2 can successfully express the siRNA described for Plutella xylostella acetylcholinesterase; the genetic engineering plant provided by the invention can have obvious killing effect on Plutella xylostella. Deadly effect.

Table 2. Bioassay results of lettuce leaves transfected with siRNA against O. corn borer

It can be seen from Table 2 that after feeding the target pests, the transgenic genetically engineered plant group (S1, S2, S3) had a significantly higher lethality rate of O. The average lethality rate of the transgenic group was 79.53%, the average lethality rate of the non-transgenic plant group was 20.3%, and the lethality rate of the CK group was 9.0%. It can be seen from the data that the average lethality rate of the transgenic group increased by 59.23% compared with the non-transgenic plant group , increased by 70.53% compared with the CK group, the above data prove that the transgenic lettuce obtained in Example 2 can successfully express the siRNA of the target gene of the Asian corn borer target gene; the genetic engineering plant provided by the invention can be effective against the Asian corn borer It has obvious killing effect.

Embodiment 6: The lethal effect of powder genetically engineered plants on Lepidoptera insects

The transgenic ryegrass obtained in Example 3 containing pEASY-3M-01 (containing Plutella xylostella siRNA) and pEASY-3M-06 (containing the siRNA of Plutella xylostella gene) were ground into powder respectively; containing pEASY-3M- The ryegrass powder of 01 was fed to Plutella xylostella, and the ryegrass powder containing pEASY-3M-06 was fed to Spodoptera spp. 30 of each kind of insects were divided into two groups: each 15 of the experimental group and the control group, 15 of each group were divided into three groups of contrasts, the experimental groups (S1, S2, S3) were regularly fed with transgenic plant powder, and were continuously fed for two weeks, and the control group ( CK1, CK2, CK3) were fed non-transgenic ryegrass powder regularly for two consecutive weeks, recorded the physiological and biochemical changes of insects and calculated the average mortality rate of pests in the experimental group and the control group. The specific results are shown in Table 3.

Table 3. Mortality of powdered genetically engineered plants to diamondback moth

plant S1 S2 S3 CK1 CK2 CK3 average fatality rate 80% 60% 100% 0 20% 0

As can be seen from Table 3, the genetic engineering plant group (S1, S2, S3) powder has a higher killing rate to the target insect diamondback moth, and the average killing rate can reach 80%; the control group (CK1, CK2, CK3) diamondback moth average Mortality rate is 6.7%. Compared with the control group, transgenic engineering plant powder has increased the mortality rate of diamondback moth by 73.3%. Through this example, it can be seen that grinding transgenic engineering plants into powder to make biological pesticides will not affect its target The lethal effect of the insect diamondback moth illustrates the feasibility of using genetically engineered plants as biopesticides from another angle.

Table 4. The lethality rate of powdered genetically engineered plants to Spodoptera spp.

plant S1 S2 S3 CK1 CK2 CK3 average fatality rate 100% 80% 100% 20% 20% 0

As can be seen from Table 4, the powder of the genetically engineered plant group (S1, S2, S3) has a higher killing rate to the target insect Spodoptera spp., and the average killing rate can reach 93.3%; the control group (CK1, CK2, CK3) The average death rate of Spodoptera spp. is 16.7%. Compared with the control group, the transgenic engineering plant powder has increased the mortality rate of Spodoptera 2. It can be seen that the transgenic engineering plants are ground into powder Biopesticides will not affect their lethal effect on target insects, which illustrates the feasibility of using genetically engineered plants as biopesticides from another perspective.

Example 7: Changes in Physiological Conditions of Aphidoidea Insects After Ingesting Genetically Engineered Plants

Aphidoidea insects belong to the Homoptera aphid class Aphidinea, which is a larger group in the Homoptera. It has a variety of feeding habits and a wide range of hosts. It is a very important pest in vegetable production and occurs all year round. Although its body is small, it is very harmful to plants. It not only sucks the juice directly, affects the yield and reduces the quality, but also induces mildew and spreads viral diseases. Among them, gallaphid is a beneficial insect, and the rest can be said to be destructive pests. In this embodiment, the experimental design and verification are carried out for aphids.

The specific implementation steps are as follows: take the fresh leaves obtained in Example 3 containing pEASY-3M-03 transgenic ryegrass, wild-type weed plants and non-transgenic weed plants identified by Taqman, rinse them with sterile water and use Gauze absorbs the water on the leaves, removes the veins, grinds them into powder, mixes them evenly with insect bait, and feeds the target insects regularly. Put 10 artificially raised target insects in each petri dish, after the insect test petri dish is covered, under the conditions of temperature 26-28 ℃, relative humidity 70%-80%, photoperiod (light/dark) 16:8 After 3 days of standing, the larval mortality was counted, and the average mortality of pests in each sample was calculated. A total of 3 strains (S1, S2 and S3) transferred to the siRNA recombinant expression vector, a total of 2 strains (S4 and S5) identified as non-transgenic by Taqman, and 1 wild-type strain (CK); Five plants were selected from each strain for testing, and each plant was repeated 5 times. The specific results are shown in Table 5.

Table 5. Bioassay results of trans siRNA weeds on aphids

It can be seen from Table 5 that the aphid lethality rate of the transgenic genetically engineered plant group (S1, S2, S3) was significantly higher than that of the non-transgenic group (S4, S5) and wild-type plant group (CK) after feeding the target pests. The average lethality rate of the transgenic group was 73.86%, the average lethality rate of the non-transgenic plant group was 16.88%, and the lethality rate of the CK group was 9%. It can be seen from the data that the average lethality rate of the transgenic group increased by 56.98% compared with the non-transgenic plant group , increased by 67.86% compared with the CK group. The above data prove that the siRNA recombinant expression vector of the present invention can be successfully transferred into plants to form genetically engineered plants, and express siRNA targeting the target sequence to silence the target gene; the genetically engineered plants provided by the invention can be It has obvious killing effect on aphids.

Example 8: Lethal effect of powdered genetically engineered plants on Aphidoidea insects

The transgenic ryegrass containing pEASY-3M-03 obtained in Example 3 will be ground into powder; 30 aphids are divided into two groups: each 15 of the experimental group and the control group, 15 of each group are divided into three groups of controls, the experimental group (S1, S2, S3) regularly fed transgenic plant powder for two consecutive weeks, and the control group (CK1, CK2, CK3) regularly fed non-transgenic weed powder for two consecutive weeks, recorded the physiological and biochemical changes of aphids and calculated the experimental group, Average mortality of aphids in the control group. The specific results are shown in Table 6.

Table 6. Mortality rate of powdered genetically engineered plants to aphids

plant S1 S2 S3 CK1 CK2 CK3 average fatality rate 60% 60% 80% 20% 0 0

As can be seen from Table 6, the genetically engineered plant group (S1, S2, S3) powder has a higher killing rate to target insects, and the average killing rate can reach 76.7%; the control group (CK1, CK2, CK3) insect average death rate is 16.7%, compared with the control group, the death rate of transgenic engineering plant powder to insects has increased by 50%. It can be seen from this example that grinding transgenic engineering plants into powder to make biopesticides will not affect its lethal effect on aphids. From another perspective, the feasibility of using transgenic engineering plants as biological pesticides is illustrated.

Example 9: Changes in Physiological Conditions of Diptera Insects After Ingesting Genetically Engineered Plants

Diptera (Diptera) is the fourth class of insects, currently there are more than 4000 species. The larvae of certain groups of Diptera, such as seed flies, leaf miners, fruit flies, and wheat gall midges, are important agricultural pests. The larvae of the genus Anthropoda in the family Anthropidae damage coniferous cones, seriously affecting the afforestation work in northern China; It damages a variety of leguminous plants; many species of Tephritidae damage citrus, pears, peaches, etc. In this example, the experimental design and verification are carried out for Drosophila melanogaster of Diptera.

The specific implementation steps are as follows: take the fresh leaves of the transgenic ryegrass containing pEASY-3M-04 obtained in Example 3, wild-type weed plants, and non-transgenic weed plants identified by Taqman, and wash them with sterile water Clean and dry the water on the leaves with gauze, remove the veins, grind them into powder, stir them evenly with insect bait, and feed the target insects regularly. Put 10 artificially raised target insects in each petri dish, after the insect test petri dish is covered, under the conditions of temperature 26-28 ℃, relative humidity 70%-80%, photoperiod (light/dark) 16:8 After standing for 3 days, the larval mortality was counted, and the average mortality rate of pests in each sample was calculated. A total of 3 strains (S1, S2 and S3) transferred to the siRNA recombinant expression vector, a total of 2 strains (S4 and S5) identified as non-transgenic by Taqman, and 1 wild-type strain (CK); Five plants were selected from each strain for testing, and each plant was repeated 5 times. The specific results are shown in Table 7.

Table 7. Bioassay results of trans siRNA weeds on Drosophila melanogaster

It can be seen from Table 7 that after the transgenic genetically engineered plant groups (S1, S2, S3) were fed with target pests, the lethality of D. . The average lethality rate of the transgenic group was 82.8%, the average lethality rate of the non-transgenic plant group was 21.5%, and the lethality rate of the CK group was 9.4%. It can be seen from the data that the average lethality rate of the transgenic group increased by 61.3% compared with the non-transgenic plant group , increased by 73.4% compared with the CK group. The above data prove that the transgenic ryegrass obtained by the present invention can successfully express the siRNA sequence to silence the target gene; killing effect.

Example 10: Lethal effect of powdered genetically engineered plants on Diptera insects

The transgenic ryegrass containing pEASY-3M-04 obtained in Example 3 was ground into powder; 30 heads of Drosophila melanogaster were divided into two groups: each 15 heads of the experimental group and the control group, and 15 heads of each group were divided into three groups of contrasts, The experimental groups (S1, S2, S3) were regularly fed with transgenic plant powder for two consecutive weeks, and the control group (CK1, CK2, CK3) were regularly fed with non-transgenic weed powder for two consecutive weeks, and the physiological and biochemical changes of Drosophila melanogaster were recorded And calculate the average death rate of Drosophila melanogaster in the experimental group and the control group. The specific results are shown in Table 8.

Table 8. Mortality of powdered genetically engineered plants to Drosophila melanogaster

plant S1 S2 S3 CK1 CK2 CK3 average fatality rate 80% 60% 100% 20% 0 20%

As can be seen from Table 8, the genetically engineered plant group (S1, S2, S3) powder has a higher killing rate to target insects, and the average killing rate can reach 80%; the average killing rate of the control group (CK1, CK2, CK3) insects is 16.7%, compared with the control group, the transgenic engineering plant powder has increased the mortality rate of insects by 63.3%. Through this example, it can be seen that grinding transgenic engineering plants into powder to make biopesticides will not affect its effect on Drosophila melanogaster. The lethal effect illustrates the feasibility of using genetically engineered plants as biopesticides from another angle.

Claims (3)

1. a kind of SiRNA expression vector being used to prepare biological pesticide, it is characterised in that will be directed to shown in SEQ ID NO.13 The template sequence of siRNA shown in the SEQ ID NO.20 of Ostrinia furnacalis IAP target genes design is inserted into binary expression vector institute .

2. application of the SiRNA expression vector described in claim 1 in preparing biological pesticide.

3. application of the cell containing SiRNA expression vector described in claim 1 in preparing biological pesticide.