CN113999863A

CN113999863A - Method for improving water utilization efficiency of tomato crops

Info

Publication number: CN113999863A
Application number: CN202111283433.9A
Authority: CN
Inventors: 师恺; 王萍; 丁淑婷; 王娇; 喻景权
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-11-01
Filing date: 2021-11-01
Publication date: 2022-02-01
Anticipated expiration: 2041-11-01
Also published as: CN113999863B

Abstract

The invention discloses a method for improving the water utilization efficiency of tomato crops, belonging to the technical field of biology. The method specifically comprises knocking out or silencing SlALD1 gene in tomato crops. The invention obtains a tomato Slald1 gene mutant by using a CRISPR/Cas9 gene editing technology, and finds that the mutant can obviously improve the water utilization efficiency of plants, enhances the resistance to drought, and can be used for the seed selection of tomato germplasm with high water utilization efficiency.

Description

Method for improving water utilization efficiency of tomato crops

Technical Field

The invention relates to the technical field of biology, in particular to a method for improving the water utilization efficiency of tomato crops.

Background

Tomato (Solanum lycopersicum L.), the genus Solanum of the family solanaceae, is one of the vegetables and commercial crops with the widest cultivation area in the world. The lycopene rice has delicious taste, is rich in lycopene with antioxidant health care function, and is widely popular with consumers. However, tomato plants are relatively tall and have continuous flowering and fruit setting, require higher water demand than common green leaf vegetables, and are sensitive to soil moisture conditions.

Fresh water resources are more and more scarce along with global warming, regional rainfall difference, poor drainage and the like. Drought is becoming one of the most serious environmental challenges facing agricultural sustainability. Drought stress alters various physiological, biochemical and morphological characteristics of tomato plants. Drought usually causes closed leaf stomata, significant inhibition of photosynthesis, damage to cytoplasmic membranes, oxidative stress caused by excessive active oxygen, leaf wilting, limited stem elongation, flower and fruit dropping, etc., which seriously affect tomato growth and fruit yield and quality. Tomato, once subjected to drought stress, can cause yield reduction and even abstinence, and is extremely harmful. Therefore, it is very important to cultivate drought-resistant tomato plants with higher water utilization efficiency so as to reduce the yield loss caused by drought to the maximum extent. The plant water utilization efficiency refers to the CO fixed by the water consumed by the plant per unit mass₂The amount of (or dry matter produced), i.e. the ratio of the rate of photosynthetic carbon assimilation to the transpiration rate during physiological plant activity, reflects the water consumption and adaptability of the plant to conditions of water deficit. The water utilization efficiency calculation formula of the single plant of the plant is as follows: plant water use efficiency is biological yield/water consumption. The utilization efficiency of plant water has gradually become an important judgment standard for crop drought resistance and water conservation. Therefore, the creation of germplasm resources with high water utilization efficiency plays an important role in the growth and development of tomato plants with large water demand.

Arabidopsis ALD1 is a transaminase AGD 2-like defense response protein 1, which is capable of removing alpha-amino groups from L-lysine and catalytically synthesizing Pipecolic acid (Pip) (Biochemical peptides and functional enzymes of Pipecolic acid biosynthesis in Plant immunity, Plant Physiol, 2017.). The synthetic pathway of pipecolic acid is conserved among different species, and the same Signaling pathway is found in tomato (amino pathway for N-hydroxy-pipecolic acid synthesis in tomato, Science Signaling, 2019.). In recent years, piperidine acid has been widely studied as a newly found substance capable of significantly improving the resistance of plants to pathogenic bacteria, and the biosynthesis and downstream hydroxylation modification pathways thereof have been studied (Flavin monooxygenic-generated n-hydroxypipecolic acid is a critical element of plant system immunity, Cell,2018.), but there are few reports on the resistance thereof to abiotic stress. The tomato SlALD1 gene is homologous to the Arabidopsis ALD1 gene, and the piperidine acid biosynthetic pathway is also conserved in tomato, but has not been studied for drought resistance.

In recent years, a rapid gene editing technology, namely regularly clustered short palindromic repeats (CRISPR)/CRISPR-associated protein 9(Cas9), is highly valued and becomes the hottest gene editing system at present. The CRISPR/Cas9 gene editing technology can specifically recognize target sites, so that target genes are edited at fixed points to obtain a gene knockout material, progeny can be subjected to selfing to screen a plant line which is edited by the target genes and does not contain Cas9, and the use of the prior controversial transgenic technology for introducing exogenous genes is avoided. The CRISPR/Cas9 gene editing technology precisely knocks out genes, thereby precisely changing crop traits, rapidly obtaining ideal germplasm, and greatly improving the problems of long period, uncontrollable result and the like in the traditional crossbreeding.

Disclosure of Invention

The invention provides a method for improving the water utilization efficiency of tomato crops, and provides a basis for cultivating tomato varieties with high water utilization efficiency.

The invention provides an application of SlALD1 gene in improving the water utilization efficiency of tomato crops, wherein the nucleotide sequence of the protein coding region of the SlALD1 gene is shown as SEQ ID No.1, the length of the protein coding region is 1311bp, and the DNA sequence of the whole gene is shown as SEQ ID No. 6.

Furthermore, the invention also provides application of a protein coded by the SlALD1 gene in improving the water utilization efficiency of tomato crops, wherein the amino acid sequence of the protein coded by the SlALD1 gene is shown in SEQ ID No. 2.

The protein coded by the SlALD1 gene is an enzyme for mediating the biosynthesis of pipecolic acid, consists of 436 amino acids and is transaminase.

According to the invention, firstly, the Arabidopsis thaliana ALD1 gene (gene number: AT2G13810, TAIR website 'https:// www.arabidopsis.org/') is subjected to homologous sequence comparison to obtain the SlALD1 gene (gene number: XM _004250704.4, NCBI website 'https:// www.ncbi.nlm.nih.gov/') with the highest homology in tomato. Sequence analysis is carried out on the SlALD1 gene through a CRISPR P2.0 website (https:// CRISPR. hzau. edu. cn/CRISPR2/), a PAM sequence is searched, a sequence 20bp before NGG is defined as sgRNA, and a sgRNA coding sequence which is positioned on a gene protein coding region and has high specificity is selected, wherein the DNA sequence of the sgRNA of the specific targeting SlALD1 gene protein coding region is shown as SEQ ID NO. 3.

The invention also provides a method for improving the water utilization efficiency of tomato crops, and specifically relates to knocking out or silencing SlALD1 genes in tomato crops.

Preferably, the nucleotide sequence of the protein coding region of the SlALD1 gene is shown in SEQ ID No. 1.

Specifically, the method comprises the following steps:

(1) selecting a target fragment for gene knockout from a protein coding region of a tomato SlALD1 gene, designing a primer, and constructing a vector for knocking out the SlALD1 gene;

(2) constructing agrobacterium gene engineering bacteria containing the carrier for knocking out the SlALD1 gene in the step (1);

(3) transforming the agrobacterium gene engineering bacteria in the step (2) into tomato crop cells, and culturing to obtain homozygous mutant strains which do not contain foreign proteins and are stably inherited.

Preferably, the vector for knocking out the SlALD1 gene is a CRISPR/Cas9 vector.

Further, the nucleotide sequence of the upstream primer for constructing the CRISPR/Cas9 vector is shown as SEQ ID No.4, and the nucleotide sequence of the downstream primer is shown as SEQ ID No. 5.

Specifically, the target fragment for gene knockout is a target fragment containing a PAM structure, and the nucleotide sequence of the first 20 bases of the PAM structure of the target fragment is shown as SEQ ID No. 3.

Preferably, the agrobacterium is agrobacterium GV 3101.

According to the invention, the hydrogen peroxide content in the Slald1 gene mutant plant under the drought condition is found to be obviously reduced compared with that of a control plant by measuring the hydrogen peroxide content in the tomato leaves.

Further, the gene can regulate and control the resistance of the tomato to drought by influencing the content of hydrogen peroxide in plants.

The regulation and control means that the SlALD1 gene changes the steady state of active oxygen in a plant body and weakens the resistance of the plant to drought by influencing the hydrogen peroxide content in the plant body.

The invention finds that the Slald1 gene mutant can obviously improve the water utilization efficiency of plants by determining the water utilization efficiency of the plants under the condition of long-term moderate water deficiency.

Compared with the prior art, the invention has the following beneficial effects:

the invention obtains a tomato Slald1 gene mutant by using a CRISPR/Cas9 gene editing technology, and finds that the mutant can obviously improve the water utilization efficiency of plants, enhances the resistance to drought, and can be used for the seed selection of tomato germplasm with high water utilization efficiency.

Drawings

Fig. 1 is a gene editing site map of T1 generation mutant plants obtained in example 2, in which Slald1#1 was missing one base compared to the control and Slald1#2 was missing 91 bases compared to the control.

FIG. 2 is a plant diagram of a control group and a Slald1 gene mutant tomato under drought stress.

Fig. 3 is a graph of the conductivity of control and Slald1 gene mutant tomatoes under drought stress, wherein the lower case letters a, b represent significant differences at the 5% level between different plants.

FIG. 4 is a graph showing the change of hydrogen peroxide content in control group and tomato with mutant Slald1 gene.

Fig. 5 is a graph showing the water use efficiency of the control group and the solanum lycopersicum mutant with the Slald1 gene under the condition of long-term moderate water deficit.

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. Unless otherwise specified, the technical means used in the examples are well known to those skilled in the art, and the raw materials and kits used are commercially available.

The gene editing tomato background used in the examples below was the conventional wild type variety CR (Condine Red) of tomato, with CR tomato without gene editing as a control.

Example 1

And (3) construction of a CRISPR/Cas9 vector containing the specific sgRNA.

(1) The DNA sequence of SlALD1 gene (GenBank number: XM _004250704.4) is found on NCBI website "https:// www.ncbi.nlm.nih.gov/", the sequence is shown in SEQ ID NO.6, the website "https:// CRISPR. hzau. edu. cn/cgi-bin/CRISPR 2/CRISPR" is input, and the 20bp base sequence (SEQ ID NO.3) which has high on score and GC content of > 40% and is positioned in front of a PAM structure of a protein coding region is found.

CRISPR primers were designed as follows:

CRISPR pre-primer (SEQ ID NO. 4): GATTGTGTTCTCCAAACAATCCCAC, respectively;

CRISPR rear primer (SEQ ID NO. 5): AAACGTGGGATTGTTTGGAGAACAC, respectively;

(2) and taking 5 mu L of each CRISPR front primer and rear primer, uniformly mixing, and annealing into double chains by using a PCR (polymerase chain reaction) instrument for coding single guide RNA (sgRNA), namely a sgRNA coding sequence.

(3) The intermediate vector pMD18-T is subjected to single enzyme digestion by BbsI, purified by a common DNA purification kit, annealed double chains are connected with the vector by T4 ligase, and the double chains are connected overnight at 16 ℃. The heat shock was applied at 42 ℃ to the transformation plates and the resistance of the vector pMD18-T was ampicillin.

(4) Monoclonal colonies were picked and verified by PCR using the CRISPR pre-primer and the vector post-primer (SEQ ID NO. 7).

(5) And (3) sending the bacterial liquid with the correct band size to a company for sequencing, wherein the sequencing result shows that the vector contains a sgRNA coding sequence, the quality-improved particles are subjected to double enzyme digestion by Hind III and Kpn I, and then are connected to a binary expression vector pCAMBIA 1301. And sequencing again to show that the final vector contains a sgRNA coding sequence, the obtained final plasmid is transferred into a GV3101 agrobacterium-infected state by electric shock, and spots are picked for PCR verification after two-day culture at 28 ℃ to obtain the agrobacterium strain which can be used for constructing a CRISPR/Cas9 gene editing material.

Example 2

Preparation and identification of Slald1 gene mutant material.

The sterilized tomato seeds were sown in the sowing medium, and the cotyledons were cut 7 days later. The final plasmid prepared in example 1 is transformed into cotyledons by adopting an agrobacterium infection method, and the totipotency of plant cells is utilized to obtain T₀Tomato is edited by generation genes.

T₀And (5) carrying out generation gene editing tomato seedling detection. Extraction of T by CTAB method₀And (3) generating genome DNA of the plant, taking the genome DNA as a template, designing the following primers at about 200bp before and after the DNA sequence containing the sgRNA coding sequence, and performing PCR amplification sequencing verification:

primer before verification (SEQ ID NO. 8): CATTTCCAGTGAGTTACTCT, respectively;

verified primer (SEQ ID NO. 9): CTTTCTAGAACCCGGGATTT, respectively;

the PCR product obtained was sent to the company for sequencing. Comparing the sequencing result with the original sequence of the gene by using Snapgene software, selecting a plant with sgRNA coding sequence subjected to base deletion and sequencing to display a single peak, selfing and breeding to obtain T₀Seeds of generations.

T above₀Planting seeds in growth chamber to obtain T₁Plants were used and T was detected by the same method as above₁Base editing condition of sgRNA coding sequence of generation plant. Meanwhile, the CRISPR pre-primer (SEQ ID NO.4) and the carrier post-primer (SEQ ID NO.7) are used for carrying out T pair₁And carrying out PCR amplification on DNA of the generation plants to detect whether the DNA contains a Cas9 sequence. Selecting T with sgRNA mutated and without Cas9 protein₁Generation plants, two lines determined as gene editing plants, named Slald1#1 and Slald1#2 respectively, and the gene editing plants are obtained by the following stepsThe resulting editing sites are shown in FIG. 1. Compared with a control plant, Slald1#1 lacks one base, and compared with a control plant, Slald1#2 lacks 91 bases. The two strains T₁After the generation seeds are self-bred, stable genetic T which does not contain exogenous gene Cas9 and has sgRNA mutation is obtained₂And (5) plant generation.

The following examples all use the two homozygous lines T described above₂The plants were used as material for the experiments.

Example 3

Study on drought resistance of Slald1 gene mutant.

Selecting tomato plants with 4 leaves and one core in a tray, and pouring enough water at the bottom of the tray to ensure that the plants are full of water. And pouring out unabsorbed water in the plug after 3-5h, and starting water control and drought treatment. The ambient temperature is controlled at about 25 ℃, the relative humidity of air is about 75 percent, and the light intensity is about 400 mu mol/m²And s. And (5) carrying out drought treatment for 7 days, observing the wilting condition of the plants, and photographing and recording. The wilting condition of the plant can be characterized by the relative conductivity of the leaves, and the high relative conductivity can be used for indicating that the cell membrane permeability is large, the cells are seriously damaged, the wilting degree of the leaves is high, and the plant is more sensitive to drought.

As can be seen from fig. 2 and 3, the Slald1 gene mutant can significantly improve the drought resistance of tomato plants.

Example 4

And measuring the hydrogen peroxide content of the solarium lycopersicum L1 gene mutant and a control group.

(1) Extracting hydrogen peroxide:

grinding tomato leaf 0.3g with liquid nitrogen, adding 3mL HClO₄(1M) homogenizing, and fully vortexing and mixing. Centrifuge at 4 deg.C and 6000rpm for 5min, pipette 2.5mL of supernatant into a new centrifuge tube, and add 4M KOH drop by drop while vortexing until pH is 6-7. 0.05g of activated charcoal was added, vortexed thoroughly for 30s, 4 ℃, 12000rpm, and centrifuged for 5 min. And sucking the supernatant into a clean centrifugal tube, and filtering the supernatant through a filter membrane of 0.22 mu m to obtain the extracted hydrogen peroxide.

(2) Preparing a reaction buffer solution:

0.0548g of ABTS was added to 100mL of potassium acetate solution (100mM, pH 4.4) to prepare a reaction buffer, which was dissolved by sonication and stored in the dark.

(3) Measurement of hydrogen peroxide content:

the sample hydrogen peroxide (1 mL), reaction buffer (1 mL) and POD (catalase) (4. mu.L) were aspirated and mixed by vortexing, the mixture was allowed to stand at room temperature for 3min, and the absorbance of hydrogen peroxide was measured at 412 nm. And calculating according to the standard curve of the hydrogen peroxide to obtain the content of the hydrogen peroxide.

As can be seen from fig. 4, under drought conditions, the hydrogen peroxide content in the Slald1 gene mutant was significantly reduced compared to the control plants, the steady state of active oxygen was protected, the degree of oxidative stress was reduced, and the drought resistance was improved.

Example 5

Under the condition of long-term moderate water deficit, the water utilization efficiency of the Slald1 gene mutant and the tomato of a control group is changed.

Tomato plants growing to 4 leaves and one heart are selected to be subjected to long-term moderate water deficit treatment, and a control group CK (75-85% of relative soil water content) and a moderate water deficit treatment group Drought (45-55% of relative soil water content) are subjected to weighing to monitor the soil water and record the irrigation amount of each time. And (3) putting the roots, stems and leaves of the plant treated by the long-term moderate water deficit treatment at the 30 th day into a 65 ℃ oven to be dried to constant weight, measuring the biological yield of the plant, and calculating the water utilization efficiency of the plant according to a formula. Fig. 5 shows the water utilization efficiency changes of tomato plants of the Slald1 gene mutant and the control group under the condition of long-term moderate water deficit. As shown in fig. 5, under the long-term moderate water deficit treatment, the water utilization efficiency of the solarium lycopersicum 1 gene mutant and the tomato plant of the control group is obviously higher than that of the solanum lycopersicum 1 gene mutant plant of the control group. The result shows that under the long-term moderate water deficiency treatment, the Slald1 gene mutant plant can improve the water utilization efficiency, so that the drought resistance of the plant is improved, and the application value is realized in the actual production.

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aaacagttaa tatgaaatta agaaagtata ttttatctaa caaaatacgt gcaaaaattt 3060

caaagtgaag gttgagggag gagccaagtg aatttggaat atcacttgcg cacgtgtttt 3120

ccctgctttt attagagaag ttattttttt ttttttgaga aactttgtat taacttgagg 3180

gaaacagttt gactaaccaa acatagaaag attcaaacta acgtacttcc tttttattgc 3240

tgaccaaaca aaattaaagt aatattattg attgaatttg acaggaacta agaagagcaa 3300

ttgcagaaac aatatataaa gatcttttgg tagaagaaac tgagatcttt gtgtctgatg 3360

gtgcacaatg tgatctctca agagttcagg tatacttgca tattttcatt tttaatcggt 3420

ttaaaaaaga cataatttac aaacatgatt tttaacttga cgtcatccag taattatgac 3480

atttaacttt ggatatgcac aagtagacat ttaaacttgt ataaaattga acaaatagat 3540

acattcgtcc tacatgacat cctatatgat aattttactt cctacgtggc atcctacgtg 3600

tactatgtca tataggacat gtgtgtttac ttattcattt ttatataagt ttaagtgtct 3660

acttgtgcac actcaaaatt ggaggacata attttccgct gaagtcaagt taagaatcac 3720

gtttatgtat tatgccttta aaaaacaatg ttgtctttct atatttgaaa gtaattgaac 3780

tttaaacttt tatttatcct taatcaataa tttatagtcg ctcaaatatt aaagacaaga 3840

tttataccac aaattctaga gatttttctt attgtactta ctaggggtgt taaaaatgag 3900

gccaattata gataactcat ccaatccgtc caaaattttc agggttgaat ttaggcataa 3960

tacataaata tagcctttaa cttggctttg aatcacattt atacctttca actttgggtg 4020

tgcacaagta gacacttaaa cttgtataaa gttgaacaaa tagacacatg tcctacgtat 4080

catcctacat ttcaaatttt gtcctacgtg tattgtgtca tgtagaattt atgtgtttat 4140

ttatttaaaa gttggatagt taaaatgctt gcttgtgcat tatgaaagtt gaaggtcaaa 4200

gttaaaattt taagtcaagt ttagggtcta atatatgtat tatgccttga atttaagata 4260

atttgaattg actataatct caattctcaa ctcattcgaa tcttacccga tttaaaagaa 4320

aatctttaat tgaatccaac ttcatctcta atttcaaccc attttaaaac ttttaattac 4380

tatattatca catactccct agttgtcatt gacctgcctt gtgccacaac aaattctttc 4440

ttgcagctcc tcttaggttc caatgtgtca attgctgtac aggatccatc atttccagtg 4500

agttactctt gctctcattt gtcttaatta aatttgcttt caacttaatt atttatactg 4560

ttataaacag agatataggt ggaggtaggt ggagttcacg ggttcggacg aatccactag 4620

tttttcgtag attctatatt tgtgttagaa aaatttaaat aaatatatat atatatatta 4680

acatgtgaac ctccaaacta ctaactttgg ctccgcctac agggatatat agattcaagt 4740

gtgattatgg ggcagagtgg tgatttgaag aatgattcag ggagatatgg aaatataaaa 4800

tacatgaaat gcaacctaga gaacgatttc tttccagatc tgtccaaaac tgaaagaaca 4860

gatgttatct tcttctgttc tccaaacaat cccactggtc atgcagcatc taggcaacaa 4920

ttgcagcaac ttgtagagtt tgcacaagta aatggttcaa ttattgtgta tgatgcagct 4980

tattctgcat atatttcaga ctcaagccct aaatcaattt atgaaatccc gggttctaga 5040

aaggtaatat tatgaaactt ttttagtgaa aaatgactcc tcccttccgt tttatatgac 5100

tctgcacgaa gtttaaaaaa gtaaaagaaa cttgtggtcc aaaatgaatg atagaaattt 5160

gtgtgattaa attgtaaatc atttcattaa ggttaaaata aatattttat agttaaatag 5220

ttactaatat aaaaacatgt cattcttttt gaaactaatt aaaaaggaaa gtaagtcaaa 5280

taaattgaga cagaggaaat ataacttata gattcatctt taaactgatt attatttctt 5340

actacaggtt gccattgaga tctcatcttt ctcaaagacg gctggattca caggcgttcg 5400

tctaggatgg actgtagtgc ctaaggagct attttatcta aatggatttc ctgttataca 5460

cgatttcaat cgcataatat gtactagctt taacggtgct tccaatatag ctcaggctgg 5520

tggattggct tgcctatccc cggaaggttt caaggaagtt atgtttaaag tagactacta 5580

taaagagaat gcaaggatct tagttgaaac tttcacttca ctagggtttc gagtttatgg 5640

aggtagcaat gcgccttatg tttgggttca ttttccaggt tcgaaatcat ggaatgtgtt 5700

caattggatt cttgataaga ctcacatcat tacagttcct ggaattggat ttggtccagc 5760

tggtgaagga tacataaggg tttctgcttt tggacgcaga gagaacatct tggaagcatc 5820

taaaagactc ataaccttac tttgtcacac aaattaa 5857

<210> 7

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

ctacttatcg tcatcgtctt tg 22

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

catttccagt gagttactct 20

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

ctttctagaa cccgggattt 20

Claims

The application of the SlALD1 gene in improving the water utilization efficiency of tomato crops is characterized in that the nucleotide sequence of a protein coding region of the SlALD1 gene is shown as SEQ ID No. 1.
The application of the protein coded by the SlALD1 gene in improving the water utilization efficiency of tomato crops is characterized in that the amino acid sequence of the protein coded by the SlALD1 gene is shown in SEQ ID No. 2.
3. A method for improving the water utilization efficiency of tomato crops is characterized in that SlALD1 genes in the tomato crops are knocked out or silenced.
4. The method of claim 3, wherein the nucleotide sequence of the protein coding region of the SlALD1 gene is set forth in SEQ ID No. 1.
5. The method of claim 3, comprising the steps of:

(1) selecting a target fragment for gene knockout from a protein coding region of a tomato SlALD1 gene, designing a primer, and constructing a vector for knocking out the SlALD1 gene;

(2) constructing agrobacterium gene engineering bacteria containing the carrier for knocking out the SlALD1 gene in the step (1);

(3) transforming the agrobacterium gene engineering bacteria in the step (2) into tomato cells, and culturing to obtain homozygous mutant strains which do not contain foreign proteins and are stably inherited.
6. The method of claim 5, wherein the vector for knocking out the SlALD1 gene is a CRISPR/Cas9 vector.
7. The method of claim 6, wherein the nucleotide sequence of the upstream primer and the nucleotide sequence of the downstream primer for constructing the CRISPR/Cas9 vector are respectively shown as SEQ ID No.4 and SEQ ID No.5, respectively.
8. The method of claim 5, wherein the target fragment for gene knockout is a target fragment containing a PAM structure, and the nucleotide sequence of the first 20 bases of the PAM structure of the target fragment is shown as SEQ ID No. 3.
9. The method of claim 5, wherein the genetically engineered Agrobacterium is the Agrobacterium GV3101 strain.