A preparation method of citrus CsbHLH92 gene and its application in citrus
By overexpressing the CsbHLH92 gene in citrus, the problem of insufficient resistance to bacterial ulcer disease is solved, significantly reducing the area and incidence of lesions, and improving the resistance of citrus.
Patent Information
- Application Number
- CN202410242732.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2024-03-04
- Publication Date
- 2024-12-10
- Estimated Expiration
- 2044-03-04
AI Technical Summary
Citrus has weak resistance to bacterial ulcer disease, resulting in problems such as falling leaves, dead heads, weak tree potential and falling fruit, which seriously affects yield and quality.
The citrus CsbHLH92 gene was prepared and used, and the citrus plants were introduced through steps such as RNA extraction, cDNA synthesis, PCR amplification and overexpression vector construction to enhance disease resistance.
Significantly reduce the area of lesions of citrus bacterial ulcer disease, reduce the incidence, and improve the disease resistance of citrus.
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Abstract
Description
Technical Field
[0001] The invention relates to the field of agricultural biological genes, and in particular to a preparation method of a citrus CsbHLH92 gene and an application thereof in citrus. Background Art
[0002] Citrus is the largest fruit in southern my country and an important economic crop in the world. However, citrus bacterial canker (CBC) has seriously hindered the healthy development of the citrus industry. Citrus canker is caused by Xanthomonas citrisubsp. citri (Xcc), which originated in India, Java and other places. The main citrus producing areas in my country, such as Fujian, Hunan and Guangdong, are seriously affected by citrus canker. The canker bacteria mainly infect citrus leaves, branches, fruits, etc., among which seedlings and young trees are more seriously affected. Diseased trees will experience leaf fall, dead branches, weak tree vigor and fruit drop, which seriously affects the yield and quality of citrus. There are dozens of Rutaceae plants affected by citrus canker, most of which are economic cultivars. Studies have found that sweet orange is the most susceptible to the disease, followed by sour orange and grapefruit. At present, more than 30 countries and regions in the world have listed citrus bacterial canker as an important plant quarantine object.
[0003] bHLH transcription factors are widely present in plants and are the second largest transcription factor family in plants. The bHLH domain consists of approximately 60 amino acids and has two functionally different regions, namely the basic region and the HLH region. The basic region contains 13-17 amino acids and is located at the N-terminus of the bHLH domain. It has the function of specifically recognizing and binding to the DNA motif of the target gene promoter. In contrast, the HLH region is located at the C-terminus and contains a hydrophilic and lipophilic α-helix. The two α-helices are separated by connecting regions (loops) of different lengths to form an HLH structure. bHLH transcription factors form homologous or heterologous dimers through the interaction between the two α-helices, thereby binding to different parts of the target gene promoter. bHLH transcription factors are located in the cell nucleus, and some amino acids in their basic region can specifically recognize and bind to the G-box in the promoter region of the target gene, thereby regulating the expression of the target gene.
[0004] bHLH transcription factors are not only involved in many growth and development processes of plants, but also in plant biosynthesis processes, such as the biosynthesis of anthocyanins. Arabidopsis bHLH phytochrome interacting factor 1 (PHYTOCHROME-INTERACTINGFACTOR 1, PIF1) is a regulator of chlorophyll biosynthesis that inhibits seed germination in the dark. Light induces PIF1 degradation, thereby promoting photomorphogenesis; anthocyanin biosynthesis in poplar is induced through pathways regulated by PdTT8 (a bHLH transcription factor) and PdMYB118 (a MYB transcription factor). In addition, bHLH transcription factors also play a role in signal transduction and responses to biotic or abiotic stresses (drought, salt, etc.). bHLH122, which is highly expressed in guard cells, can enhance the drought resistance of Arabidopsis by inhibiting the catabolism of ABA, thereby increasing the content of ABA (Liu et al., 2014); overexpression of OsbHLH148 in rice regulates the function of the JA pathway and OsJAZ (jasmonic acid ZIM domain) protein, thereby enhancing the drought resistance of plants; PtrbHLH66, a bHLH family gene cloned from Citrus aurantium, plays a positive regulatory role in Arabidopsis drought resistance by regulating root growth and ROS clearance; transgenic plants obtained by overexpressing AtNIG1 in Arabidopsis have higher salt tolerance than the corresponding knockout mutants.
[0005] In general, the functional research of bHLH transcription factors provides a richer theoretical basis for disease resistance molecular breeding. However, there is little research on the role of bHLH transcription factors in citrus bacterial canker.
[0006] In view of this, this application is filed. Summary of the invention
[0007] The technical problem to be solved by the present invention is that the existing citrus has weak resistance to ulcer disease, and often suffers from leaf drop, dead branches, tree weakness and fruit drop, which seriously affects the yield and quality of citrus.
[0008] The present invention is achieved through the following technical solutions:
[0009] The present application proposes a method for preparing a citrus CsbHLH92 gene, comprising the following steps:
[0010] S1: Total RNA was extracted from citrus leaves using an RNA extraction kit;
[0011] S2: Synthesize cDNA from total RNA of citrus leaves;
[0012] S3: Use primers to amplify the CsbHLH92 gene from cDNA and recover the CsbHLH92 gene.
[0013] Preferably, S2 comprises synthesizing cDNA from total RNA of citrus leaves using the Recombinant DNase I method, and storing the cDNA at a temperature of -25 to -15°C.
[0014] Preferably, the primers in S3 include OE-F and OE-R.
[0015] Preferably, the amplification system in S3 comprises 10X PCR mix, OE-F, OE-R, cDNA and ddH2O.
[0016] In order to solve the above problems, the present invention also provides an application of a citrus CsbHLH92 gene in citrus, wherein the CsbHLH92 gene is prepared by the above preparation method and introduced into a citrus plant, comprising the following steps:
[0017] S01: Construction of overexpression vector of CsbHLH92 gene;
[0018] S02: introducing the overexpression vector into Agrobacterium;
[0019] S03: Transform citrus plants with Agrobacterium carrying the overexpression vector.
[0020] Preferably, the construction process of the overexpression vector in S01 includes double digesting the CsbHLH92 gene and the overexpression vector pLGN with restriction endonucleases BamHI and SalI and then recovering them;
[0021] The cleaved CsbHLH92 gene was ligated to the overexpression vector pLGN using a T4 DNA Ligase kit and the ligation reaction was carried out overnight;
[0022] The ligation product is transformed into Escherichia coli DH5α, and the transformation method is heat shock transformation or electroporation transformation;
[0023] Positive clones were screened and plasmids were extracted to obtain the overexpression vector pLGN-CsbHLH92 of CsbHLH92.
[0024] Preferably, in S02, the Agrobacterium is Agrobacterium tumefaciens, and the introduction process comprises the following steps:
[0025] Prepare Agrobacterium tumefaciens EHA105 competent cells;
[0026] The pLGN-CsbHLH92 was introduced into the competent cells and then introduced into the Agrobacterium tumefaciens EHA105.
[0027] Preferably, the method for introducing the pLGN-CsbHLH92 into the competent cells is electroporation, which includes thawing the frozen Agrobacterium tumefaciens EHA105 competent cells, adding the constructed overexpression vector plasmid, mixing by pipetting on ice, and transferring the mixture to an electroporation cup.
[0028] Preferably, the conversion comprises the following steps:
[0029] Obtaining epicotyls of citrus seedlings, preparing Agrobacterium culture solution and transforming citrus epicotyl stem segments.
[0030] Preferably, in S03, before the transformation, 28-32 wt % sterile glycerol is added to the Agrobacterium carrying the overexpression vector and then stored in an incubator at -80°C.
[0031] Compared with the prior art, the present invention has the following advantages and beneficial effects:
[0032] The present invention uses citrus as material to study the gene function of bHLH transcription factors, so as to explore the influence of CsbHLH92 on citrus bacterial canker and analyze its value in citrus disease-resistant breeding. Through the research of the present invention, it can be known that overexpression of CsbHLH92 can significantly reduce the lesion area of citrus bacterial canker and alleviate the incidence of citrus bacterial canker. BRIEF DESCRIPTION OF THE DRAWINGS
[0033] In order to more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following briefly introduces the drawings required for use in the embodiments. It should be understood that the following drawings only illustrate certain embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other relevant drawings can be obtained based on these drawings without creative work. In the drawings:
[0034] Figure 1 A line diagram of the CsbHLH92 overexpression vector provided in an embodiment of the present invention;
[0035] Figure 2 A transgenic verification diagram of the CsbHLH92 transgenic plant provided in an embodiment of the present invention;
[0036] Figure 3 GUS staining diagram of CsbHLH92 transgenic plants provided in the embodiment of the present invention;
[0037] Figure 4 A histogram of gene expression levels of CsbHLH92 transgenic plants provided in an embodiment of the present invention;
[0038] Figure 5The phenotype diagram of the CsbHLH92 transgenic plant provided in the embodiment of the present invention, wherein A is OE-3, B is OE-9, and C is OE-16;
[0039] Figure 6 A comparison diagram of leaves of CsbHLH92 transgenic plants for xcc resistance evaluation provided in an embodiment of the present invention;
[0040] Figure 7 A comparison diagram of the lesion area of the xcc resistance evaluation of the CsbHLH92 transgenic plant provided in the embodiment of the present invention;
[0041] Figure 8 This is a comparison chart of the disease index for xcc resistance evaluation of CsbHLH92 transgenic plants provided in an embodiment of the present invention. DETAILED DESCRIPTION
[0042] In order to make the purpose, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments.
[0043] Therefore, the following detailed description of the embodiments of the present invention provided in the accompanying drawings is not intended to limit the scope of the invention claimed for protection, but merely represents selected embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.
[0044] It should be noted that similar reference numerals and letters denote similar items in the following drawings, and therefore, once an item is defined in one drawing, further definition and explanation thereof is not required in subsequent drawings.
[0045] It should be noted that: the embodiment of the present invention uses Wanjin orange as the test object. In practical applications, the method can also be used to improve the resistance of other citrus varieties to ulcer disease.
[0046] Example 1
[0047] Cloning of the coding sequence of the CsbHLH92 gene from Citrus
[0048] 1. RNA extraction and cDNA synthesis
[0049] Total RNA from citrus (Wanjincheng) leaves was extracted using an RNA extraction kit (Adlai, CAT: RN09), and the RNA quality was verified by agarose gel electrophoresis, and its concentration was measured by a concentration meter. Recombinant DNase I was used to synthesize cDNA (TAKARA), and the cDNA was stored at -20°C for future use.
[0050] 2. PCR amplification of the coding sequence of the CsbHLH92 gene
[0051] The CsbHLH92 fragment (encoding the amino acid sequence "MDEFLGDQFHGEGDIFFWLDNEALPVNQSAFVAYANRPRTEFVSQNAGNGSNSLSANKRMIQFLRKIQPAKMEHPELDSERGHRHVINERMRRERERQNYLALHSILPPGTKNDKNSIVKTTANMIHELQCCKQEERKNYELESNLGLKNGGTKVRVNVDNPTSGVDSMLEVLKCLKDLGSSTRSIQAEFSDQKLTAVLDIETQMGAGAVEKDVQRALQKVEDKLLFRHYQEG") was amplified from citrus cDNA using primers OE-F (CGGGATCCATGGATGAGTTCTTGGGAGA) and OE-R (GCGTCGACCTAGCCTTCCTGGTAGTGAC). The fragment length was 705 bp and the amplified fragment was confirmed to be the coding sequence of the citrus CsbHLH92 gene by sequencing analysis.
[0052] The amplification system is: 10X PCR mix: 2.5 μL; Primer OE-F (5 μmol / L): 1 μL; Primer OE-R (5 μmol / L): 1 μL; cDNA about 60 ng; add ddH2O to 25 μL.
[0053] The amplification program was as follows: 98°C, 5 min; 98°C, 15 s, 58°C, 30 s, 72°C, 1 min, 35 cycles; and extension at 72°C for 3 min.
[0054] 3. DNA fragment recovery
[0055] Under UV light, use a clean blade to cut out the agarose gel block containing the target fragment, and use a kit (Adlay) to recover the fragment.
[0056] Example 2
[0057] Construction of overexpression vector and transformation of Agrobacterium
[0058] Vector construction flow chart Figure 1 All restriction endonucleases were purchased from THERMO and operated according to the instructions.
[0059] The specific operation is as follows: CsbHLH92 gene fragment and overexpression vector pLGN were double digested with restriction endonucleases BamHI and SalI, recovered and ligated overnight, and the ligation was carried out using T4 DNA Ligase kit (TAKARA). The ligation product was transformed into Escherichia coli DH5α, and the plasmid was extracted from the positive clone to obtain the overexpression vector pLGN-CsbHLH92 of CsbHLH92. The plasmid was extracted using a kit (Adlai).
[0060] The constructed overexpression vector was introduced into Agrobacterium tumefaciens EHA105 by electroporation. Take the frozen EHA105 Agrobacterium competent cells (50 μL) in advance, melt them on ice, add 2 μL of the constructed overexpression vector plasmid to the competent cells, mix them by blowing, and place them on ice for 5 min. Transfer the mixed solution to the bottom of the previously dried electroporation cup, put the electroporation cup into the card slot and adjust it to the correct position. Adjust the electroporation device to the "Agr" gear, press the electroporation button, and check the electroporation data to ensure that the electroporation is successful. Add 1 mL of LB liquid culture medium to the electroporation cup, blow and mix it with a pipette, transfer it to a sterile centrifuge tube, and shake and culture it at 260 r / min and 28℃ for 60 min. Centrifuge the bacterial solution at 10000 r / min for 1 min, discard the supernatant (about 100 μL of resuspended bacteria remains), resuspend and spread, and incubate it in the dark at 28℃ for 2 days. After the plaques grow, pick a single colony for PCR verification.
[0061] Example 3
[0062] Genetic transformation of citrus (Late Jin orange)
[0063] 1. Obtaining epicotyls of citrus seedlings
[0064] Fresh citrus (Wanjin orange) fruits were washed, surface disinfected with 70% alcohol, seeds were taken out under sterile conditions, the seed coats were peeled off, and the seeds were germinated on seed germination medium. They were cultured in the dark at 28°C for 2 weeks, and then cultured under 16 h light / 8 h dark conditions for 1 week. The epicotyls of the germinated seedlings were cut into 1 cm stem segments under sterile conditions for genetic transformation with Agrobacterium tumefaciens.
[0065] 2. Preparation of Agrobacterium tumefaciens culture solution
[0066] The Agrobacterium liquid (containing the CsbHLH92 overexpression vector) used for transfection was added with 30% sterile glycerol and stored in an ultra-low temperature incubator at -80°C. Before transfection, streak culture on LB solid medium containing 50 mg / L kanamycin. Pick a single colony and inoculate it in 25 ml LB liquid medium containing the same antibiotics, and shake and culture it at 28°C overnight. After measuring the concentration, dilute the liquid to OD=0.1 and shake it twice. After about 3 hours, when the liquid OD=0.5, centrifuge it at 5000 r / min for 10 min, discard the supernatant, and resuspend it in MS liquid medium with pH 5.4 for transfection.
[0067] 3. Transformation of citrus epicotyl stem segments
[0068] The citrus epicotyl stem segments were soaked in Agrobacterium solution for 13 min and then wiped dry. The stem segments were transferred to the co-cultivation medium and cultured in the dark at 26°C for 2 days.
[0069] 4. Screening of transformants
[0070] After co-culture, the epicotyls were transferred to the screening medium and cultured in the dark at 28°C for 7 days. The explants were cultured at 28°C with 16 h light / 8 h dark, subcultured every two weeks, and then preliminarily identified by GUS staining.
[0071] 5. Seedling culture of transformants
[0072] When the seedlings grow to more than 1 cm, they are cut and grafted onto late orange seedlings in sterile test tubes and cultured in seedling medium; when the seedlings grow to about 5 cm, they are grafted onto orange seedlings and cultured in a greenhouse.
[0073] Example 4
[0074] Transgenic plant verification
[0075] 1. PCR detection of exogenous gene integration
[0076] 100 mg of plant leaves obtained in the initial screening were used to extract genomic DNA using a DNA extraction kit (Adele, CAT: DN15), and PCR was used to detect the integration of the CsbHLH92 gene in the citrus genome. The detection primers were 35S-bHLH92-F (CCACTGACGTAAGGGATGAC) and 35S-bHLH92-R (GCCTTCCTGGTAGTGACGAA).
[0077] The PCR amplification program was as follows: 94°C, 3 min; 94°C, 30 s, 58°C, 30 s, 72°C, 1 min, 30 cycles; and extension at 72°C for 3 min.
[0078] PCR results are shown in Figure 2, a 785 bp amplified fragment could be obtained from the positive plants, but no amplification was obtained from the WT plants.
[0079] 2. GUS staining identification of transgenic plants
[0080] Cut a small amount of transgenic plant leaves and use GUS dye to perform initial screening and identification of positive seedlings. Figure 3 As shown, the leaves of positive plants turned the dye solution blue, while the leaves of WT plants did not show color; a total of 3 transgenic plants were identified and labeled as OE-3, OE-9 and OE-16.
[0081] 3. Analysis of CsbHLH92 gene expression
[0082] Total RNA was extracted from citrus leaves (Adlai, CAT No: RN09), and cDNA was synthesized using Recombinant DNase I. qRT-PCR was used to detect the expression of the target gene, and the detection primers were RT-F (TTCATGAGCTGCAATGCTGT) and RT-R (TCCCCCATTCTTCAGACCCA); the detection primers of the internal reference gene Actin were Actin-F (CATCCCTCAGCACCTTCC) and Actin-R (CCAACCTTAGCACTTCTCC).
[0083] The reaction volume was 20 μL, and the reaction procedure was: 95°C, 3 min; 94°C, 10 s, 56°C, 10 s, 72°C, 10 s, 40 cycles; 72°C for 10 min, and the experiment was repeated three times.
[0084] Adoption 2 -△△Ct The relative expression of CsbHLH92 gene in transgenic plants was calculated by the following method: the water-treated sample was defined as the reference factor, i.e., its CsbHLH92 expression level was 1, and then the multiple of the gene expression in transgenic citrus relative to the reference factor was calculated. -△△Ct , which is the relative expression level. The test results are shown in Figure 4 The results showed that the CsbHLH92 gene was expressed at a higher level (>40 times) in the transgenic plants compared with the wild-type plants.
[0085] 4. Phenotypic observation of transgenic plants
[0086] The phenotypes of the three transgenic plants were observed and analyzed. Figure 5 As shown, the three overexpressing plants grew shorter than the wild type during the same period, and black corky protrusions appeared on the leaves, petioles and stems.
[0087] Example 5
[0088] Resistance evaluation of transgenic plants
[0089] After cleaning, the mature leaves were disinfected with 75% alcohol and rinsed with sterile water, and placed on a clean bench; the leaf veins were punctured with needles in the center, with six needles in a group and two groups on each side. The ulcer pathogen liquid was spotted with a pipette, and 1 μL (1 X 105 CFU / mL) was spotted on each pinhole. The leaves were cultured in a 28°C constant temperature and light incubator (16 h light / 8 h dark). The leaves were photographed 10 days after the bacteria were spotted, and the lesion area was counted using Image J V1.47 software. The disease severity was calculated according to the disease index formula. The disease was divided into 0-7 levels according to the lesion area, with the letter R representing the lesion area, and 0 (R≤0.25 mm 2 ), level 1 (0.25 mm 2 <R≤0.5mm 2 ), level 2 (0.5 mm 2 <R≤0.75mm 2 ), level 3 (0.75 mm 2 <R≤1mm 2 ), level 4 (1.0 mm 2 <R≤1.25mm 2 ), level 5 (1.25 mm 2 <R≤1.5mm 2 ), level 6 (1.5 mm 2 <R≤ 1.75 mm 2 ), 7th level (R>1.75 mm 2 ); The disease severity was calculated according to the formula: DI = 100 X Σ [number of lesions at each level X corresponding level value] / (total number of lesions X maximum level).
[0090] Overexpressing plants and WT plants grafted at the same time were selected and inoculated with canker bacteria by needle puncture. Ten days after inoculation with canker bacteria in vitro, the plants inoculated with canker bacteria all developed disease to varying degrees, and there were certain differences in the size of the lesions ( Figure 6 The above experiment was repeated three times to ensure the accuracy of the results.
[0091] The size of the lesions on leaves inoculated with citrus canker bacteria was statistically analyzed. The analysis found that the lesion area of the transgenic plants was significantly smaller than that of the WT plants, with OE-3 having the smallest lesion area ( Figure 7 ). The disease severity of transgenic plants was statistically found to be significantly lower than that of WT plants, especially OE-3 ( Figure 8 ).
[0092] It can be seen that overexpression of CsbHLH92 can significantly reduce the lesion area of citrus bacterial canker and alleviate the severity of citrus bacterial canker.
[0093] In the present invention, the culture medium used for Agrobacterium tumefaciens transformation is:
[0094] Seed germination medium:
[0095] MS + 30 g / L sucrose + 2.5 g / L Gelrite, pH 5.8.
[0096] Co-culture medium:
[0097] MS + 2 mg / L BA + 0.5 mg / L IAA + 1 mg / L 2,4 – D + 100 μmol AS + 30 g / L sucrose + 2.5 g / L Gelrite, pH 5.8.
[0098] Screening medium:
[0099] MS + 2 mg / L BA + 0.5 mg / L IAA + 500 mg / L Cef + 50 mg / L Kan + 30 g / L sucrose + 2.5 g / L Gelrite, pH 5.8.
[0100] Seedling medium:
[0101] MS + 30 g / L sucrose, pH 5.8.
[0102] The specific implementation methods described above further illustrate the objectives, technical solutions and beneficial effects of the present invention in detail. It should be understood that the above description is only a specific implementation method of the present invention and is not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.
Claims
1. A use of the citrus CsbHLH92 gene in citrus bacterial canker, characterized in that: The use includes reducing the lesion area of citrus bacterial canker by overexpressing the citrus CsbHLH92 gene, thereby reducing the incidence of citrus bacterial canker; The amino acid sequence encoded by the citrus CsbHLH92 gene is: MDEFLGDQFHGEGDIFFWLDNEALPVNQSAFVAYANRPRTEFVSQNAGNGSNSLSANKRMIQFLRKIQPAKMEHPELDSERGHRHVINERMRRERERQNYLALHSILPPGTKNDKNS IVKTTANMIHELQCCKQEERKNYELESNLGLKNGGTKVRVNVDNPTSGVDSMLEVLKCLKDLGSSTRSIQAEFSDQKLTAVLDIETQMGAGAVEKDVQRALQKVEDKLLFRHYQEG.
2. The use of a citrus CsbHLH92 gene in citrus bacterial canker according to claim 1, characterized in that: The preparation method of the citrus CsbHLH92 gene comprises the following steps: S1: Total RNA was extracted from citrus leaves using an RNA extraction kit; S2: Synthesize cDNA from total RNA of citrus leaves; S3: using primers to amplify the CsbHLH92 gene from cDNA, and recovering the CsbHLH92 gene; The primers described in S3 include OE-F and OE-R; The amplification system described in S3 includes 10X PCR mix, OE-F, OE-R, cDNA and ddH2O.
3. The use of a citrus CsbHLH92 gene in citrus bacterial canker according to claim 2, characterized in that: S2 includes synthesizing cDNA from total RNA of citrus leaves using the Recombinant DNase I method, and storing the cDNA in an environment with a temperature of -25 to -15°C.
4. The use of a citrus CsbHLH92 gene in citrus bacterial canker according to claim 1, characterized in that: The method comprises introducing the citrus CsbHLH92 gene into citrus plants, and comprises the following steps: S01: Construction of overexpression vector of CsbHLH92 gene; S02: introducing the overexpression vector into Agrobacterium; S03: Transform citrus plants with Agrobacterium carrying the overexpression vector.
5. The use of a citrus CsbHLH92 gene in citrus bacterial canker according to claim 4, characterized in that: The construction process of the overexpression vector in S01 includes digesting the CsbHLH92 gene and the overexpression vector pLGN with restriction endonucleases BamHI and SalI to recover the gene; The cleaved CsbHLH92 gene was ligated to the overexpression vector pLGN using a T4 DNALigase kit and the ligation reaction was carried out overnight; The ligation product is transformed into Escherichia coli DH5α, and the transformation method is heat shock transformation or electroporation transformation; Positive clones were screened and plasmids were extracted to obtain the overexpression vector pLGN-CsbHLH92 of CsbHLH92.
6. The use of a citrus CsbHLH92 gene in citrus bacterial canker according to claim 5, characterized in that: In S02, the Agrobacterium is Agrobacterium tumefaciens, and the introduction process comprises the following steps: Prepare Agrobacterium tumefaciens EHA105 competent cells; introduce the pLGN-CsbHLH92 into the competent cells and then into the Agrobacterium tumefaciens EHA105.
7. The use of a citrus CsbHLH92 gene in citrus bacterial canker according to claim 6, characterized in that: The method for introducing the pLGN-CsbHLH92 into the competent cells is an electroporation method, which includes thawing the frozen Agrobacterium tumefaciens EHA105 competent cells, adding the constructed overexpression vector plasmid, mixing by pipetting on ice, and transferring the mixed solution to an electroporation cup.
8. The use of a citrus CsbHLH92 gene in citrus bacterial canker according to claim 4, characterized in that: The transformation comprises the following steps: obtaining epicotyls of citrus seedlings, preparing Agrobacterium bacterial liquid and transforming citrus epicotyl stem segments.
9. The use of the citrus CsbHLH92 gene in citrus bacterial canker according to claim 8, characterized in that: In S03, before the transformation, 28-32% sterile glycerol is added to the Agrobacterium carrying the overexpression vector and then stored in a -80°C incubator.