CN113801886A - Application of BZR1 gene in regulation and control of insect pest stress resistance of plants - Google Patents

Abstract

The invention discloses an application of BZR1 gene in regulation and control of insect pest stress resistance of plants, belonging to the technical field of biology. The application is as follows: the BZR1 gene function is deleted by using a biotechnology means, so that the resistance of the plant to insect pest stress is improved; the coding sequence of the BZR1 gene is shown as SEQ ID NO.1 or has at least 70% homology with the sequence shown as SEQ ID NO.1, and the coded proteins are functionally equivalent. The invention discloses the application of the BZR1 gene in regulating and controlling insect pest stress, especially cotton bollworm resistance for the first time, the BZR1 gene function is deleted by a gene editing technology, the cotton bollworm resistance of a plant can be obviously enhanced, and the BZR1 gene can be applied to cultivation of insect pest stress resistant plant varieties.

Description

Application of BZR1 gene in regulation and control of insect pest stress resistance of plants

Technical Field

The invention relates to the technical field of biology, in particular to a method for improving the resistance of plants to insect pests, especially cotton bollworms by deleting the function of BZR1 gene.

Background

Tomato (Solanum lycopersicum L.) is one of the most widely consumed and grown vegetables in the world. However, during the whole growth cycle, tomatoes are subject to attack by various pests, resulting in huge economic losses. If a large amount of insecticide is applied for prevention and treatment, the problems of pesticide residue, environmental pollution, drug resistance and the like are caused. In view of the advantages of relatively short life cycle, small genome, high genetic diversity, easy transgenosis and cultivation, and the like of the tomato, and the complete genome sequencing and the development of molecular genetics research, the tomato becomes a classical model plant for researching the interaction between plants and plant diseases and insect pests. Therefore, the research on the molecular mechanism of the plant for resisting insect pests by taking the tomatoes as objects has positive ecological and economic significance for insect pest control and agricultural product safety production.

Helicoverpa armigera (Helicoverpa armigera) is an important crop pest occurring in both cotton and vegetable growing areas of China. The cotton bollworm has wide feeding range, and the host plants can be more than 30 families and more than 200 species, including important horticultural crop-tomato. The cotton bollworm can occur in the whole tomato planting period, the biggest harm is to specially eat the fruits, the fruits are rotten, the yield loss reaches more than 30 percent, and the cotton bollworm becomes a main obstacle factor for developing the production of the later-cropping tomatoes. Therefore, the research on the molecular mechanism of tomato internal defense against cotton bollworms has certain guiding significance for breeding new insect-resistant varieties.

As sessile organisms, plants have evolved a number of defense strategies against herbivores to successfully survive and reproduce. The Defense response of plants to herbivore attack is mediated by Plant hormones (Pietese, C.M.J., et al, "Signalling Networks invent In Induced Resistance." In Induced Resistance for Plant Resistance, 2014, 58-80), where the Jasmonic Acid (JA) signaling pathway has been recognized as the core pathway regulating the Plant's Defense response to herbivores (Howe GA et al, "modulation In jasmate signaling for multiple Resistance." Annu. Rev. Plant biol., 2018, 69, 387 415). When herbivores attack, the JA signal is activated, modifying the physical and chemical phenotype of the plant, thereby increasing the plant's resistance. Intensive studies over the past 20 years have revealed an important molecular composition of the JA pathway (Howe GA et al, "modulation in jasmonate signaling for multistress resilience." Annu. Rev. plant biol., 2018, 69, 387-415). Upon injury to the plant, the acyl hydrolase releases α -linolenic acid (18:3) from the galactolipids in the plastid membrane, which is oxidized by the 13-lipoxygenase enzyme, followed by epoxidation and cyclization reactions to 12-oxo-phytodienoic acid (OPDA). OPDA is transported to peroxisomes, forms (+) -7-iso-JA through three beta-oxidation cycles mediated by reduction and acetyl-CoA oxidase (Acx1), etc., and is converted into a more stable trans-configuration, namely JA, through epimerization. Further, JA binds with isoleucine to form jasmonic acid-isoleucine (JA-Ile), an active derivative. JA-Ile binds to a receptor consisting of COI1, JAZ and inositol polyphosphate cofactor, so that the inhibitor JAZ is degraded by 26S proteasome, thereby releasing MYC transcription factor and activating the expression of downstream defense genes. Although the JA signal transduction pathway has been extensively studied, further investigation of the regulatory mechanism of the JA synthesis pathway is urgently needed.

Transcriptional regulation plays a key role in the formation of a regulatory network of plant resistance and is the key point for studying the generation of defense response of plants. Transcription factor is combined with cis-acting element in promoter region to regulate the expression of serial genes to resist pest attack. BZR1(BRASSINAZOLE RESISTANT 1) is a key transcription factor in brassinolide signaling pathway, is involved in multiple growth and development processes such as Plant cell elongation, cell division, flowering and seed development, and is also found to play an important role in resisting abiotic stresses such as high and low temperature and salt stress (Qianfeng Li et al, "The branched-regulated transformation factors BZR1/BES1 function a cordinator in multiple-regulated Plant growth," BBA-Gene regulation Mechanisms, 2018, 861, 561-. However, the role and mechanism of BZR1 in pest stress is still unclear. Therefore, the research on the resistance mechanism of the tomato SlBZR1 gene to cotton bollworm has certain guiding significance for the breeding of the cotton bollworm-resistant tomato variety.

Disclosure of Invention

The invention aims to provide a gene capable of regulating and controlling plant resistance to insect pest stress, particularly to cotton bollworm, and the gene can be used for improving the resistance of the plant to the cotton bollworm through gene modification, so that a basis is provided for cultivating plant varieties resistant to insect pests.

In order to achieve the purpose, the invention adopts the following technical scheme:

the invention provides an application of BZR1 gene in regulation and control of insect pest stress resistance of plants, which comprises the following steps: the BZR1 gene function is deleted by using a biotechnology means, so that the resistance of the plant to insect pest stress is improved;

the coding sequence of the BZR1 gene is shown as SEQ ID NO.1 or has at least 70% homology with the sequence shown as SEQ ID NO.1, and the coded proteins are functionally equivalent.

Further, the protein encoded by the BZR1 gene negatively regulates the synthesis of jasmonic acid.

Furthermore, the protein coded by the BZR1 gene is combined with a promoter of a jasmonic acid synthesis related gene Acx1 to negatively regulate the transcription of a Acx1 gene and reduce the content of jasmonic acid.

The research of the invention shows that protein encoded by the BZR1 gene negatively regulates the transcription of a Jasmonic Acid (JA) synthesis related gene Acx1(Acyl-CoA oxidase 1), acetyl coenzyme A oxidase encoded by the Acx1 gene is a key enzyme for JA synthesis, and jasmonic acid plays an important role in resisting biological stress of plants. By deleting the function of the BZR1 gene, the resistance of the plant to insect pest stress can be obviously improved.

Further, the application includes: the expression of the BZR1 gene is reduced or deleted by using gene mutation, gene knockout, gene interference or gene silencing technology, so that a mutant plant with enhanced insect pest stress resistance is obtained.

Further, the plant is tomato.

Further, the insect pest stress is cotton bollworm.

The invention carries out sequence analysis on tomato SlBZR1 (gene number: Solyc04g079980), searches a PAM sequence, defines a sequence of 20bp before NGG as sgRNA, and selects the sgRNA sequence which is positioned on a gene protein coding region and has high specificity. Through a gene editing technology and a tissue culture technology, a CRISPR/Cas9 vector of the SlBZR1 gene is constructed, and a homozygous mutant strain which is stable in heredity and does not contain exogenous Cas9 protein is obtained through screening. The mutant plants are found to be capable of obviously enhancing the resistance of tomatoes to the cotton bollworms by inoculating the cotton bollworms.

The whole gene DNA sequence of the SlBZR1 gene is shown as SEQ ID NO.4, the nucleotide sequence of a protein coding region is shown as SEQ ID NO.1, and the length of the protein coding region is 987 bp; the protein coded by the SlBZR1 gene is a key transcription factor in a brassinolide signal channel, consists of 328 amino acids, and has a sequence shown in SEQ ID NO. 2.

Under the stress condition of the cotton bollworm, the jasmonic acid content of the bzr1 deletion mutant is obviously increased compared with that of a Wild Tomato (WT). Therefore, deletion of the tomato Slbzr1 gene is beneficial to improving the resistance of tomato to cotton bollworm.

The SlBZR1 gene coding protein negatively regulates the transcription of JA synthesis related gene Acx1 (gene number: Solyc08g 078390). Furthermore, the nucleotide sequence of the Acx1 gene promoter is shown in SEQ ID NO. 3.

The invention also provides a method for cultivating the cotton bollworm-resistant tomatoes, which comprises the following steps:

(1) selecting a target fragment containing a PAM structure in a protein coding region of the SlBZR1 gene, and designing a primer according to the first 20 bases of the PAM structure of the target fragment to construct a CRISPR/Cas9 vector; the nucleotide sequence of the SlBZR1 gene is shown in SEQ ID NO. 4;

(2) constructing agrobacterium gene engineering bacteria containing the CRISPR/Cas9 vector in the step (1);

(3) transforming the gene engineering bacteria in the step (2) into tomato cotyledons to obtain a stably inherited homozygous mutant strain which does not contain exogenous Cas9 protein and has a target sequence with variation.

Furthermore, the nucleotide sequence of the target fragment PAM 20bp ahead of the structure is shown in SEQ ID NO. 5.

The invention has the following beneficial effects:

the invention discloses the application of the BZR1 gene in regulating and controlling insect pest stress, especially cotton bollworm resistance for the first time, the BZR1 gene function is deleted by a gene editing technology, the cotton bollworm resistance of a plant can be obviously enhanced, and the BZR1 gene can be applied to cultivation of insect pest stress resistant plant varieties.

Drawings

FIG. 1 is a CRISPR/Cas 9-mediated gene editing site and encoded amino acid changes of a mutant plant homozygous for SlBZR1 knockout;

wherein WT is a non-transgenic wild tomato, bzr1 is a mutant tomato knocked out by SlBZR1 gene; compared with WT, the mutant has 10bp base deletion at the position of sgRNA, resulting in premature termination of protein translation.

FIG. 2 shows the phenotype (A) and weight (B) of Helicoverpa armigera 3 days after the tomato SlBZR1 gene knockout mutant plants are inoculated with Helicoverpa armigera;

wherein, the bigger the body type and the weight of the cotton bollworm are, the more the cotton bollworm takes food and the more the tomato plant suffers from insect damage; the lower case letters a, b represent significant differences between different plants at a level of P <0.05 in the T-test.

FIG. 3 shows leaf phenotype (A) and weight (B) of tomato SlBZR1 gene knock-out mutant plants 3 days after inoculation with Helicoverpa armigera;

wherein, the more damaged the tomato leaves and the smaller the weight, the more cotton bollworm is eaten, the more serious the tomato plant is attacked by insect pests; the lower case letters a, b represent significant differences between different plants at a level of P <0.05 in the T-test.

FIG. 4 shows JA content of mechanically wounded bollworm oral secretion treated (W + OS) in tomato SlBZR1 gene knock-out mutant plants after eating by using a simulated bollworm;

wherein, the higher the JA content is, the stronger the insect resistance of the plant is; the lower case letters a, b represent significant differences between different plants at a P <0.05 level in the one-way ANOVA test.

FIG. 5 shows the luciferase reporter gene detection ratio of the SlBZR1 protein regulating Acx1 gene transcription;

wherein the lower case letters a, b represent significant differences between different treatments at the level of P <0.05 in the T-test.

Detailed Description

The present invention will be further described with reference to the following specific examples, which are only illustrative of the present invention, but the scope of the present invention is not limited thereto.

The experimental procedures used in the following examples are conventional unless otherwise specified.

The test materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The tomato variety used in the examples below is the conventional tomato variety Condine Red.

Example 1 preparation and characterization of tomato SlBZR1 deletion mutant plants

1. Construction of CRISPR/Cas9 vector containing specific sgRNA

Firstly, finding out the DNA full-length sequence of tomato SlBZR1 gene on SGN website https:// solgenomics. net/as shown in SEQ ID NO.4, inputting the DNA full-length sequence into http:// CRISPR. hzau.edu.cn/CRISPR 2/website, finding out the sgRNA which has high onscore score and GC content of more than 40 percent and is positioned 20bp base sequence in front of a PAM structure of a protein coding region as a specific targeting gene protein coding region as shown in SEQ ID NO. 5.

And synthesizing a single-chain sgRNA sequence, annealing to form a double-chain sgRNA, wherein the two ends of the sgRNA are provided with Bbs I restriction enzyme cutting sites, and the synthesized fragment is inserted into the Bbs I site of the AtU6-sgRNA-AtUBQ-Cas9 vector to obtain an intermediate vector.

Carrying out double enzyme digestion on the intermediate vector and the pCAMBIA1301 vector by using Hind III and KpnI restriction endonucleases, connecting and transforming escherichia coli DH5 alpha competence, selecting single clone for sequencing, and carrying out electric shock transformation on GV3101 agrobacterium competence by using plasmid with correct sequencing to obtain the agrobacterium strain capable of being used for constructing CRISPR/Cas9 gene editing materials.

2. Preparation and identification of SlBZR1 gene mutant material

The tomato seeds are disinfected and then sown in 1/2MS culture medium, and are cultured in the dark until the seeds germinate, and then are transferred to the light for culture. After the cotyledon grows out, cutting the cotyledon, placing the cut cotyledon in a nursing culture medium, and culturing for 12-24h in the dark. Culturing the prepared agrobacterium tumefaciens for constructing the CRISPR/Cas9 gene editing material to logarithmic phase, centrifuging to remove supernatant, adding MS0.2 for resuspension for infecting cotyledon explants, after infecting for 2.5min in the dark, blotting residual bacterial liquid on the surfaces of the cotyledon explants, placing the cotyledon explants on a nursing culture medium, and co-culturing for two days in the dark. After co-culture, transferring the cotyledon explants to a 2Z culture medium to induce callus, transferring to a 0.2Z culture medium to induce germination after 2-3 weeks, transferring to a new 0.2Z culture medium every 2 weeks until plantlets grow, transferring to an R culture medium to induce rooting, transferring to a substrate and moisturizing after about 2-3 weeks until root systems well develop, and performing survival on T₀And (5) verifying the generation gene editing material.

Taking a small amount of T₀Extracting genome DNA from plant leaves, performing PCR amplification on a DNA fragment containing the sgRNA by taking the genome DNA as a template, performing sequencing comparison on a PCR product, selecting a plant with a sgRNA sequence subjected to base mutation, deletion or insertion, performing self-cross breeding, and obtaining T₀And (5) seed generation.

The verification primers were as follows:

primers before verification: 5'-AGTAAGCAAAACCCACCTGC-3' (SEQ ID NO. 6);

and (3) verifying the primer: 5'-TTCATAATCAATTCTAGATC-3' (SEQ ID NO. 7).

Will T₀Sowing seeds of the generations to obtain T₁Plant generation and detection of T by the above method₁Selecting plants which do not contain Cas9 and have sgRNA variation for seed reproduction and selfing to obtain stably inherited T containing no foreign gene Cas9 under the base editing condition of sgRNA sequences of the generation plants and the existence of Cas9₁Generation homozygous mutant plants 1 line, the gene editing sites are shown in figure 1. The bzr1 mutant was 10 bases less than the control.

Further selfing and breeding the strains, and sowing seeds to obtain stably inherited T which does not contain exogenous gene Cas9 and has sgRNA variation₂And (5) plant generation.

The following examples are given in T₂The generation homozygous lines were used as material for the experiments.

Example 2 identification of the anti-Helicoverpa armigera phenotype of the tomato SlBZR1 Gene

The cotton bollworm is 3-year-old cotton bollworm purchased by Jiyuan Baiyun industry Co., Ltd, Henan, and is subjected to hungry treatment overnight by selecting cotton bollworms with consistent sizes into a 24-hole plate.

When the tomato seedlings grow to have six leaves and one heart, the 2 nd to 4 th leaf blades are selected from the top, the leaf blades (1 +/-0.05 g) with the same mass are weighed and placed in a culture dish filled with 1% agar to prevent the water loss of the leaves in vitro, and the tomato leaves before the butworms are photographed and recorded. Then inoculating the two ends of the starvation-treated cotton bollworms to tomato leaves of a culture dish, covering the culture dish cover and fixing the cotton bollworms by rubber bands to prevent the cotton bollworms from leaving the culture dish. After 3 days of exposure to the light, the weight of the cotton bollworms is detected, and then the cotton bollworms are soaked in 70 percent methanol until the cotton bollworms do not move any more, and are photographed and recorded after being arranged according to the sizes of body types. And correspondingly photographing, recording and weighing the tomato leaves after the pests are killed.

The results are shown in fig. 2 and fig. 3, the bollworm fed with the bzr1 mutant leaf blade is smaller in size and lighter in weight than the bollworm fed with the wild-type leaf blade, the loss of the bzr1 mutant leaf blade after inoculation treatment is lighter than that of the wild-type leaf blade, and the weight of the damaged leaf blade is heavier, which indicates that the bollworm resistance of tomato can be improved by knocking out the SlBZR1 gene.

Example 3 Effect of the tomato SlBZR1 Gene on the resistance hormone JA

When the tomato seedlings grow to have six leaves and one heart, selecting the 3-4 th leaf from top to bottom to carry out W + OS simulated cotton bollworm feeding treatment, collecting leaf samples at 0 and 1h to analyze JA hormone level, and setting 4 times of repetition at each time point.

The processing mode of simulating the feeding of the cotton bollworms by the W + OS is as follows: mechanical wounds are made on the two side faces of the main lobular vein by clamping the main lobular vein with hemostatic forceps once, and then five times of diluted secretion of the mouth organ of the cotton bollworm is smeared on the wounds.

The method for obtaining the secretion of the cotton bollworm mouthparts comprises the following steps: feeding tomato leaves after the cotton bollworm is hungry for 12h, and sucking secretion of mouth organs of the cotton bollworm as stock solution after 12 h.

The JA hormone assay format was: weighing 0.1g of tomato leaves, grinding by liquid nitrogen, adding 1ml of chromatographic grade ethyl acetate, adding 25 μ l of hormone internal standard in a dark place, uniformly mixing by vortex, coating a layer of tinfoil paper to prevent light, and placing in a shaking table at 4 ℃ for overnight with the frequency of 150 rpm. After centrifugation at 12000rpm for 10min at 4 ℃, 850. mu.l of the supernatant was aspirated into a new 10ml tube and stored at 4 ℃ in the dark. Adding 1ml chromatographic grade ethyl acetate into the rest precipitate, placing in a shaking table at 4 ℃ after heavy suspension, shaking for 1h, centrifuging at 12000rpm at 4 ℃ for 10min again, and sucking 900 μ l supernatant. The two supernatants are combined and dried by nitrogen. Add 500. mu.l 70% chromatographic grade methanol, vortex for 30s, transfer the liquid to a 1.5ml tube, aspirate 200. mu.l of supernatant after flash-off into the liner of the flask and measure by high performance liquid chromatography tandem mass spectrometry (Varian320-MSLC/MS, Agilent Technologies, Amstelveen, Netherlands).

The result is shown in fig. 4, 1h after the treatment of W + OS, the JA content of bzr1 mutant plants is obviously higher than that of wild plants, which indicates that the deletion of SlBZR1 gene can improve the JA content of tomato plants after cotton bollworm feeding, thereby improving the cotton bollworm resistance of tomatoes.

Example 4 transcriptional validation of tomato SlBZR1 protein negative control Acx1 Gene

1. Constructing pGreenII 002962-SK vector containing SlBZR1 gene: and carrying out PCR amplification by using tomato cDNA as a template to obtain a SlBZR1 CDS sequence. The primers are as follows:

SlBZR1-SK-F：

5’-cgctctagaactagtggatccATGTGGGAAGGTGGAGGGTT-3’(SEQ ID NO.8)；SlBZR1-SK-R：

5’-gataagcttgatatcgaattcTCACATCCGAGCAGTCCCAC-3’(SEQ ID NO.9)；

the vector and the PCR product are subjected to double enzyme digestion by using BamHI and EcoRI restriction enzymes, and transformed Escherichia coli DH5 alpha is connected.

2. Constructing pGreen II 0800-LUC vector containing Acx1 gene promoter sequence: the method is the same as above, tomato genome DNA is taken as a template, enzyme cutting sites are Not I and Nco I, and the primers are as follows:

pAcx1-LUC-F：

5’-tccactagttctagagcggccgcTTTTTTTCATGTTGGACGGTCTG-3’(SEQ ID NO.10)；

pAcx1-LUC-R：

5’-tgtttttggcgtcttccatggAATAAAATGTTCGTCCACTTTAATATATTC-3’(SEQ ID NO.11)。

3. and (3) transforming the plasmid with correct sequencing into agrobacterium GV3101 competence by electric shock to obtain agrobacterium strain for double-luciferase reporter gene detection experiment.

4. Resuspending the two Agrobacterium strains in an infection solution containing 150. mu. mol/L acetosyringone, and adjusting to optimal concentration (OD)₆₀₀0.75), activating for 3h in an incubator at 28 ℃, and performing activation according to the weight ratio of SlBZR 1-SK: the leaves of Nicotiana benthamiana (Nicotiana benthamiana) are co-infected after pAcx1-LUC is mixed in a ratio of 10:1, the activities of firefly Luciferase and renilla Luciferase are detected after 3 days by using a Dual-Luciferase Reporter Assay System (Dual-Luciferase Reporter Assay System) of Promega corporation in America, and the relation between the fluorescence ratio of the firefly Luciferase and the renilla Luciferase is calculated and the value of pGreenII 002962-SK Empty Vector (Empty Vector) and Acx1 promoter (set as 1).

The results are shown in fig. 5, and compared with the empty vector, the expression of BZR1 enables Acx1 promoter to be inhibited, and the relative LUC/REN value is lower, which indicates that BZR1 inhibits the transcription of Acx 1.

Sequence listing

<110> Zhejiang university

Shandong (Linyi) Institute of Modern Agriculture, Zhejiang University

Application of <120> BZR1 gene in regulation and control of insect pest stress resistance of plants

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actgttttat ggttttctga tctagaattg attatgaaca gcttctgagt ttgtgttttc 1440

tttagttcaa ttgttctttt atgtacttat aatctgttca tgttctcatt tctggataat 1500

tggtatgaat gtatgtagtt taatttgtct gagaatggga gttgtttctg gaaaaaacaa 1560

gaaggaagaa ttgacttgtt cttaatatag aaatggataa gcagggtgtt ttggtgtcta 1620

aggtggggtt tgatgcattt ggttgtggtg tttttagttt atcaactact tttcgagatt 1680

caaagttgca atctgttgaa gttgttatct cagattacag agccttttga tcatttatgt 1740

tcactagttt gatttgaaaa tgtcctcttt tactatgttt tcagggatgc aggccaactc 1800

caatggagat tggaggcact tcagccaaca ttacgccaag ttcttcacga aatccaagtc 1860

ctccctcttc atactttgct agcccgattc catcttacca agttagtcca acatcctcgt 1920

ctttcccaag tccatctcgt ggtgatgcta acatgtcgtc acatccattt gcatttctcc 1980

atagttccat tcccttgtcg ctaccaccat tacgaatatc aaacagtgcc cctgtaacac 2040

cacctctttc atcaccaact agagtcccta agcagatatt taatcttgag actttggcta 2100

gagagtctat gtctgctcta aatatccctt tctttgctgc ttcagcccca actagcccaa 2160

ctcgaggtca gcgattcact cctgctacaa taccagagtg tgacgaatct gattcatcca 2220

ccattgattc tggccagtgg atgagctttc aaaagtacgc agccaatggg atccctactt 2280

ctccgacttt taatcttatt aagcctgtag ctcagagaat tccttctaat gatatgatca 2340

tcgacaaggg taagagcatt gaatttgact ttgagaatgt atcagttaag gcagcatggg 2400

aaggtgaaaa gattcatgag gttggtttag atgatctgga gctcactctc ggaagtggga 2460

ctgctcggat gtgattttca tgattaaaag aactgctcag tgtggattta cagattgaga 2520

ttatccgagc ttgctagctg atgattcact caagcagtcc agtggatgtt cgttacagag 2580

atagatcttg catgttgctg tttaccttat atacagacat tttttttttc cttttcgcct 2640

tatctgatga ggcctttaag tttagtttgg gtcttgtcat aagcatttag gtatagatag 2700

ttgatggttt ctatctcctt gtggttgaga attgtttcaa gtcattgtag actagcagga 2760

gttctcctta aacctgaact atccacagtt atattttcat gattatagta gatccctttg 2820

ttgttatgca actggatttc ttgttttatg atctagcgtc aattcaaaat caatggatgt 2880

gctattcttt aagtctcttt gatgctttat gtccagatca aaacattagt actagttctc 2940

tttgtttaca cacactttca ctataccatt ttaactcata tgtccttaaa ggacaagcaa 3000

tggtgggcca gaaaagactt tgacgactca agggttcata agtgagtgaa agtaagatag 3060

ccctctaaag gaagatctat tcagctcgat catctaattc actccaacag acatgcatga 3120

ttccgtaatc gaaataactt ccatagggct ggccaatggc aagatggatg tcaagaagtt 3180

ggaatttttt gacaatcaaa tcaaagttgt tgaattttct ttgtcactca tttggctcca 3240

ttagtaaact accagtagtc atattaataa taatagcatg ttcatgcaca cgtttgttat 3300

caacacaagc acagacagag aagaagaaag ataaaacaaa tggtgtttaa ctgatttaat 3360

gtacaagacc acggcctaat gtctggatgt ggggttctgt actactgtat tacacatgtt 3420

cctctacagt tactttttca ttattttttt ctcataacgc ggttattgtc acatgtcttg 3480

cttgctgcta tat 3493

<210> 5

<211> 20

<212> DNA

<213> tomato (Solanum lycopersicum L.)

<400> 5

ggaagccatc atggagggaa 20

<210> 6

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

agtaagcaaa acccacctgc 20

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ttcataatca attctagatc 20

<210> 8

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cgctctagaa ctagtggatc catgtgggaa ggtggagggt t 41

<210> 9

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

gataagcttg atatcgaatt ctcacatccg agcagtccca c 41

<210> 10

<211> 46

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

tccactagtt ctagagcggc cgcttttttt catgttggac ggtctg 46

<210> 11

<211> 51

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

tgtttttggc gtcttccatg gaataaaatg ttcgtccact ttaatatatt c 51

Claims (10)

1. The application of BZR1 gene in regulation and control of insect pest stress resistance of plants is characterized in that: the BZR1 gene function is deleted by using a biotechnology means, so that the resistance of the plant to insect pest stress is improved; the coding sequence of the BZR1 gene is shown as SEQ ID NO.1 or has at least 70% homology with the sequence shown as SEQ ID NO.1, and the coded proteins are functionally equivalent.
2. 2. The use of claim 1, wherein the plant is tomato.
3. 3. The use of claim 2, wherein the amino acid sequence of the protein encoded by the BZR1 gene is as shown in SEQ ID No. 2.
4. 4. The use of claim 1, wherein the insect pest stress is cotton bollworm.
5. 5. The use of claim 1, wherein the protein encoded by the BZR1 gene negatively regulates jasmonic acid synthesis.
6. 6. The use of claim 5, wherein the protein encoded by the BZR1 gene is bound to the promoter of jasmonic acid synthesis-related gene Acx1, and the transcription of Acx1 gene is negatively regulated to reduce the content of jasmonic acid.
7. 7. The use of claim 6, wherein the nucleotide sequence of the promoter of Acx1 gene is shown in SEQ ID No. 3.
8. 8. The application of claim 1, wherein the application comprises: the expression of the BZR1 gene is reduced or deleted by using gene mutation, gene knockout, gene interference or gene silencing technology, so that a mutant plant with enhanced insect pest stress resistance is obtained.
9. 9. The method for cultivating the cotton bollworm-resistant tomatoes is characterized by comprising the following steps of:

   (1) selecting a target fragment containing a PAM structure in a protein coding region of the SlBZR1 gene, and designing a primer according to the first 20 bases of the PAM structure of the target fragment to construct a CRISPR/Cas9 vector; the nucleotide sequence of the SlBZR1 gene is shown in SEQ ID NO. 4;

   (2) constructing agrobacterium gene engineering bacteria containing the CRISPR/Cas9 vector in the step (1);

   (3) transforming the gene engineering bacteria in the step (2) into tomato cotyledons to obtain a stably inherited homozygous mutant strain which does not contain exogenous Cas9 protein and has a target sequence with variation.
10. 10. The method for cultivating tomato with resistance to cotton bollworm as claimed in claim 1, wherein the nucleotide sequence of the first 20bp of the target fragment PAM structure is shown as SEQ ID No. 5.

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