Application of CsTCP15 gene in regulation and control of citrus liberobacter asiaticum resistance

Abstract

The invention discloses application of a CsTCP15 gene in regulation and control of citrus liberobacter asiaticum resistance, belongs to the technical field of agricultural biological genetic engineering, and particularly relates to application of the CsTCP15 gene in regulation and control of the citrus liberobacter asiaticum resistance, and the nucleotide sequence of the CsTCP15 gene is shown as SEQ ID NO.1. According to the CsTCP15 gene overexpression, the resistance of the citrus to the citrus huanglongbing is improved, the resistance of the citrus to the Candidate Liberibacter asiaticus (CLA) is improved, the citrus CsTCP15 gene is cloned, overexpression vector construction is carried out, then the citrus is transformed, an obtained transgenic plant can obviously inhibit early infection and colonization of the CLas, and the CsTCP15 gene overexpression vector construction is carried out on the CsTCP15 gene, so that the CsTCP15 gene overexpression vector construction is carried out on the CsTCP15 gene overexpression vector construction is carried out on the CsTCP15 gene overexpression vector construction is carried out on the CLas. The method has a great value for citrus yellow shoot resistance molecular breeding.

Description

Technical Field

[0001] The invention belongs to the technical field of agricultural biological gene engineering, and in particular relates to the application of the CsTCP15 gene in regulating citrus Huanglongbing resistance. Background Art

[0002] Citrus Huanglongbing (HLB) is a devastating disease caused by the pathogen Candidatus Liberibacter. Three pathogenic variants are involved: Candidatus Liberibacter africanus (CLaf), Candidatus Liberibacter asiaticus (CLas), and Candidatus Liberibacter americanus (CLam). CLas is the most widespread, primarily transmitted by the Asian citrus psyllid (Diaphorina citri), posing a serious threat to the global citrus industry. Infection with CLas causes characteristic symptoms such as asymmetric leaf yellowing (mottled chlorosis), thickened leaves, dwarfing, and fruit drop, leading to plant death in severe cases. Because the pathogen cannot be cultured in vitro, research is extremely challenging; only its genome has been sequenced. Given the devastating impact of HLB on agricultural production, identifying HLB-resistant germplasm resources is of great economic and social significance.

[0003] Plants employ a two-tiered innate immune system to defend against pathogen invasion, comprising pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). To counteract these defense mechanisms, pathogens secrete effector proteins, which act as virulence factors to suppress or evade host immune responses. Numerous endogenous citrus proteins are targeted by CLas, thereby suppressing citrus immunity. For example, the activity of citrus papain-like cysteine ​​proteases (PLCPs) is inhibited by the effector protein SDE1 (CLIBASIA_05315), thereby promoting CLas proliferation; the CsACD2 protein is targeted by SDE15 (CLIBASIA_04025), suppressing citrus immunity and promoting CLas proliferation; and the citrus glyceraldehyde-3-phosphate dehydrogenase GAPC directly interacts with SDE3 (CLIBASIA_00420), leading to specific inhibition of the ATG8-mediated immune response. Advances in genetic engineering have provided technical tools for citrus disease-resistant breeding, making the search for disease-resistant genes and the development of disease-resistant varieties of great significance.

[0004] The TCP gene family is named after its three founding members: TEOSINTE BRANCHED1 (TB1) in maize, CYCLOIDEA (CYC) in snapdragon, and PROLIFERATING CELLNUCLEARANTIGEN FACTOR (PCF) in rice. TCP transcription factors, as core regulatory elements, participate in plant development and stress responses, regulating processes such as cell proliferation, phytohormone synthesis, immune responses, and circadian rhythms. Phylogenetically, the TCP family is divided into Class I (PCF subfamily) and Class II, which are further divided into CIN and CYC / TB1 subfamilies. Class I members regulate downstream gene expression by binding to the cis-element GGNCCCAC, while Class II members regulate downstream gene expression by binding to the GTGGNCCC cis-element. Notably, TCP plays a key role in plant responses to abiotic and biotic stresses. TCP transcription factors, as important target proteins of pathogen effectors, positively regulate plant immune responses. However, the role of TCP in citrus defense against CLas has not been reported. Summary of the Invention

[0005] To solve the above technical problems, the present invention proposes the application of CsTCP15 gene in regulating citrus Huanglongbing resistance. By overexpressing CsTCP15 gene, the early infection and colonization of citrus Huanglongbing pathogen is inhibited, thereby improving citrus Huanglongbing resistance.

[0006] To achieve the above object, the present invention provides an application of the CsTCP15 gene in regulating citrus Huanglongbing resistance, wherein the nucleotide sequence of the CsTCP15 gene is shown in SEQ ID NO.1.

[0007] Preferably, the regulation of citrus Huanglongbing resistance is achieved by overexpressing the CsTCP15 gene, thereby inhibiting the early infection and colonization of the citrus Huanglongbing pathogen and improving citrus Huanglongbing resistance.

[0008] The present invention also provides a recombinant expression vector, which overexpresses the CsTCP15 gene.

[0009] The present invention also provides an application of the recombinant expression vector in regulating citrus Huanglongbing resistance, wherein the regulation of citrus Huanglongbing resistance is that the recombinant expression vector overexpresses the CsTCP15 gene to inhibit the early infection and colonization of the citrus Huanglongbing pathogen, thereby improving citrus Huanglongbing resistance.

[0010] The present invention also provides a recombinant strain, which includes the recombinant expression vector.

[0011] The present invention also provides an application of the recombinant strain in regulating citrus Huanglongbing resistance, wherein the regulation of citrus Huanglongbing resistance is that the recombinant strain overexpresses the CsTCP15 gene to inhibit the early infection and colonization of the citrus Huanglongbing pathogen, thereby improving citrus Huanglongbing resistance.

[0012] The present invention also provides an application of the protein encoded by the CsTCP15 gene in regulating citrus Huanglongbing resistance. The amino acid sequence of the protein encoded by the CsTCP15 gene is shown in SEQ ID NO.13. The regulation of citrus Huanglongbing resistance is achieved by increasing the expression level of the protein encoded by the CsTCP15 gene, thereby inhibiting the early infection and colonization of the citrus Huanglongbing pathogen and improving citrus Huanglongbing resistance.

[0013] The present invention also provides a method for improving the resistance to citrus Huanglongbing, which improves the resistance to citrus Huanglongbing by overexpressing the CsTCP15 gene.

[0014] The present invention also provides the use of the CsTCP15 gene in preparing a preparation for inhibiting infection by Candidatus Liberibacter asiaticus. The preparation for inhibiting infection by Candidatus Liberibacter asiaticus is applied to citrus to promote overexpression of the CsTCP15 gene in citrus, inhibit early infection and colonization of Candidatus Liberibacter asiaticus in citrus, and improve citrus resistance to Huanglongbing disease.

[0015] The present invention also provides a preparation for inhibiting the infection of Bacillus asiaticus. The preparation for inhibiting the infection of Bacillus asiaticus comprises an effective component that promotes the overexpression of the CsTCP15 gene in citrus.

[0016] Compared with the prior art, the present invention has the following advantages and technical effects:

[0017] The present invention improves the resistance of citrus to citrus Huanglongbing based on overexpression of the CsTCP15 gene, and enhances the resistance of citrus to CLas, the pathogen of citrus Huanglongbing. By cloning the citrus CsTCP15 gene, constructing an overexpression vector, and then transforming citrus, the resulting transgenic plants can significantly inhibit the early infection and colonization of CLas. Specifically, two months after CLas inoculation, CLas was detected in the transgenic negative strains, but CLas was not detected in the transgenic positive OE-1, OE-2, and OE-3 strains. Four months after CLas inoculation, the amount of CLas in OE-1, OE-2, and OE-3 was significantly lower than that in WT plants. The present invention provides a method for improving citrus Huanglongbing resistance, which is of great value to the molecular breeding of citrus resistance to Huanglongbing. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0019] Figure 1 This is the electrophoresis diagram of PCR amplification of CsTCP15 gene clone. In the figure, Maker represents the DNA molecular weight standard.

[0020] Figure 2 The diagram shows the structure of the CsTCP15 gene overexpression vector. In the figure, GFP represents green fluorescent protein, CaMV 35S represents the plant constitutive promoter, and NOS represents the opine synthase gene terminator.

[0021] Figure 3 This is a flow chart of citrus genetic transformation in Example 1;

[0022] Figure 4 These are GFP fluorescence images of transgenic positive and negative plants, where A is the wild-type Wanjincheng plant, B is the OE-1 transgenic plant, C is the OE-2 transgenic plant, and D is the OE-3 transgenic plant;

[0023] Figure 5 Figure 2 is a PCR identification diagram of transgenic positive and negative plants. In the figure, P represents plasmid pNM-GFPer-CsTCP15-3×FLAG, Maker represents DNA molecular weight standard, WT represents wild-type Wanjin Orange plant, OE-1 represents OE-1 transgenic plant, OE-2 represents OE-2 transgenic plant, and OE-3 represents OE-3 transgenic plant.

[0024] Figure 6 The figure shows the analysis of CsTCP15 expression in transgenic positive and negative plants. In the figure, WT represents wild-type Wanjin Orange plants, OE-1 represents OE-1 transgenic plants, OE-2 represents OE-2 transgenic plants, and OE-3 represents OE-3 transgenic plants.

[0025] Figure 7 The WB identification diagram of transgenic positive and negative plants, WT in the figure represents wild-type Wanjin Orange plants, OE-1 represents OE-1 transgenic plants, OE-2 represents OE-2 transgenic plants, OE-3 represents OE-3 transgenic plants, "**" represents p < 0.005, "***" represents p < 0.001, and "****" represents p < 0.0001;

[0026] Figure 8Figure 2 is a phenotype diagram of transgenic positive and negative plants. In the figure, WT represents wild-type Wanjin Orange plants, OE-1 represents OE-1 transgenic plants, OE-2 represents OE-2 transgenic plants, and OE-3 represents OE-3 transgenic plants.

[0027] Figure 9 Figure 2 shows the detection of CLas pathogen count on leaves of transgenic-positive and -negative plants 2 and 4 months after inoculation. WT represents wild-type Wanjin Orange plants, OE-1 represents OE-1 transgenic plants, OE-2 represents OE-2 transgenic plants, and OE-3 represents OE-3 transgenic plants. “*” indicates p<0.05, “**” indicates p<0.005, and “****” indicates p<0.0001. DETAILED DESCRIPTION

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0029] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each smaller range between any intermediate value within a stated value or stated range and any other stated value or intermediate value within the stated range is also encompassed by the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0030] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of any conflict with any incorporated document, the contents of this specification shall prevail.

[0031] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be exemplary only.

[0032] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0033] The Wanjin orange used in the present invention is sourced from the Citrus Research Institute of Southwest University.

[0034] Example 1

[0035] 1. Cloning of the citrus CsTCP15 gene.

[0036] 1. RNA extraction and cDNA synthesis:

[0037] Total RNA was extracted from citrus (Wanjincheng) leaves using a plant total RNA extraction kit (Adlai, CAT: RN09). RNA quality was verified by agarose gel electrophoresis and concentration was determined using a concentration meter. cDNA was synthesized using a reverse transcription kit, PrimeScript RTMaster Mix (TaKaRa, CAT: RR036A).

[0038] 2. PCR amplification of CsTCP15 gene:

[0039] The DNA fragment of CsTCP15 gene was amplified from citrus cDNA using primers CsTCP15-F (SEQ ID NO.2), CsTCP15-R (SEQ ID NO.3) and high-fidelity enzyme PrimeSTAR Max DNA Polymerase (TaKaRa, CAT: R045Q). The length of the fragment was 1185 bp (including the restriction enzyme site) (e.g. Figure 1 The amplified DNA was sequenced and confirmed to be the citrus CsTCP15 gene (SEQ ID NO. 1). Under ultraviolet light, a clean blade was used to cut out the agarose gel containing the target fragment, and the DNA fragment was recovered using a kit (BioFlux, CAT: BSC02M1).

[0040] PCR amplification program: 94°C, 5 min; 94°C, 30 s, 58°C, 30 s, 72°C, 1 min, 35 cycles; extension at 72°C for 10 min.

[0041]

[0042] The nucleotide sequence of primer CsTCP15-F is shown in SEQ ID NO. 2: SEQ ID NO. 2: ATGATCAATATGGATGATAA.

[0043] The nucleotide sequence of primer CsTCP15-R is shown in SEQ ID NO. 3: CTAGGAATGGTGACTGGTTG.

[0044] 2. Construction of CsTCP15 gene overexpression vector.

[0045] The CsTCP15 gene with homology arms of the pNM-GFPer-3×FLAG vector and the overexpression vector pNM-GFPer were double-digested with restriction endonucleases KpnⅠ and BamH1 (ThermoFisher) and then recovered from the gel using the ClonExpress II OneStep Cloning Kit (Novizen, CAT: C112-01). The ligation product was transformed into Escherichia coli DH5α, and the plasmid of the positive clone was extracted using a plasmid extraction kit (Tiangen, CAT: DP103) to obtain the CsTCP15 gene overexpression vector pNM-GFPer-CsTCP15-3×FLAG (as shown in Figure 2). Figure 2 shown).

[0046] The nucleotide sequence of the primer pNM-GFPer-CsTCP15-3×FLAG-F is shown in SEQ ID NO. 4: SEQ ID NO. 4: TTCATTTGGAGAGGACAGGGTACCATGATCAATATGGATGATAA.

[0047] The nucleotide sequence of the primer pNM-GFPer-CsTCP15-3×FLAG-R is shown in SEQ ID NO. 5: GTAATCCGATCCTCCTCCGGATCCCTAGGAATGGTGACTGGTTG.

[0048] 3. Genetic transformation of citrus (e.g. Figure 3 shown).

[0049] Seed germination medium: MS medium + 30 g / L sucrose + 2.5 g / L Gelrite, pH 5.8.

[0050] Co-culture medium: MS medium + 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.

[0051] Screening medium: MS medium + 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.

[0052] Seedling culture medium: MS medium + 30g / L sucrose, pH 5.8.

[0053] 1. Obtaining epicotyls of citrus seedlings:

[0054] Fresh citrus fruits were washed and surface disinfected with 75% ethanol aqueous solution. The seeds were removed under sterile conditions, the seed coats were peeled off, and the seeds were germinated on seed germination medium. The seeds were cultured in the dark at 28°C for 14 days and then cultured under 16 h light / 8 h dark conditions for 7 days. The epicotyls of the germinated seedlings were cut into 1 cm stem segments under sterile conditions for Agrobacterium tumefaciens-mediated genetic transformation.

[0055] 2. Transformation of Agrobacterium with overexpression vector:

[0056] The constructed CsTCP15 gene overexpression vector pNM-GFPer-CsTCP15-3×FLAG was introduced into Agrobacterium tumefaciens EHA105 (Weidi Biotechnology, CAT#: AC1010) by chemical transformation. The method is as follows: frozen Agrobacterium competent cells EHA105 (50 μL) were taken in advance and thawed on ice; 2 μL of the overexpression vector plasmid was added to the competent cells, pipetted to mix, and then stood on ice for 5 min, liquid nitrogen for 5 min, 42°C for 5 min, and stood on ice for 5 min, and then added 700 μL of LB liquid medium without antibiotics. The culture was shaken at 28°C for 3 h, and the bacteria were collected by centrifugation at 6000 rpm for 1 min. 100 μL of supernatant was retained and the resuspended bacterial block was gently pipetted and smeared on an LB plate containing 50 mg / L kanamycin and 20 mg / L rifampicin, and the plate was inverted and cultured in a 28°C incubator for 3 days. After plaques grew, single colonies were verified by PCR using primers pNM-GFPer-CsTCP15-3×FLAG-F (SEQ ID NO.4) and pNM-GFPer-CsTCP15-3×FLAG-R (SEQ ID NO.5).

[0057] PCR reaction conditions: 94°C for 3 min; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 10 min.

[0058] 3. Preparation of Agrobacterium tumefaciens culture solution:

[0059] Before transfection, streak the Agrobacterium (containing the pNM-GFPer-CsTCP15-3×FLAG vector) for transfection on LB solid medium containing 50 mg / L kanamycin and 20 mg / L rifampicin. Pick a single colony and inoculate it into 25 mL of LB liquid medium containing the same antibiotics. Cultivate with shaking at 28°C for 12 h. Dilute the bacterial solution to an OD of 0.1 and continue culturing to an OD of 0.5. Centrifuge at 5000 rpm for 10 min, discard the supernatant, and resuspend the suspension in MS liquid medium with a pH of 5.4 for transfection.

[0060] 4. Transformation of citrus epicotyls:

[0061] Soak the citrus epicotyl stem segments in Agrobacterium solution for 15 minutes and then wipe them dry. Transfer the stem segments to co-cultivation medium and culture them in the dark at 26°C for 2 days. After the co-cultivation is completed, transfer the epicotyls to the screening medium and culture them in the dark at 28°C for 7 days. Culture the epicotyls at 28°C with 16h light / 8h dark conditions, subculture every 14 days, and observe GFP fluorescence with a handheld fluorescent lamp. Positive plants will show green fluorescence, while negative plants will not.

[0062] 5. Seedling culture of transformants:

[0063] When the seedlings grow to 1 cm, they are cut and grafted onto Wanjin orange seedlings in sterile test tubes and cultured in seedling culture medium; when the seedlings grow to 5 cm, they are grafted onto Citrus aurantium seedlings and cultured in a 28°C greenhouse.

[0064] 4. Verify the transgenic plants overexpressing the CsTCP15 gene.

[0065] 1. GFP fluorescence detection of transgenic plants:

[0066] The transgenic stem segments were tested again using a handheld fluorescent lamp; positive plants showed green fluorescence, while negative plants showed none.

[0067] like Figure 4 As shown, Figure 4 The WT plant shown in A is a negative plant. Figure 4 Middle B, Figure 4 Middle C and Figure 4 OE-1, OE-2 and OE-3 shown in D are positive plants.

[0068] 2. PCR identification of transgenic plants:

[0069] Genomic DNA was extracted from 100 mg of transgenic plant leaves using a DNA extraction kit (Adlai, Cat: DN15). PCR was then performed to detect the integration of the CsTCP15 gene into the citrus genome. The primers used for the detection were ID-pNM-F (SEQ ID NO. 6) and pNM-GFPer-CsTCP15-3×FLAG-R (SEQ ID NO. 5).

[0070] The results are as follows Figure 5 As shown, a 1662 bp amplified fragment was obtained in the positive plants OE-1, OE-2 and OE-3, while no amplification was obtained in the WT plant.

[0071] PCR reaction conditions: 94°C for 3 min; 94°C for 30 s, 58°C for 30 s, 72°C for 30 s, 30 cycles; 72°C for 10 min.

[0072] The nucleotide sequence of primer ID-pNM-F is shown in SEQ ID NO. 6: TCTCAGAAGACCAAAGGGCAAT.

[0073] Transgenic plants were identified by PCR using the forward primer ID-pNM-F within 35S.

[0074] 3. WB identification of transgenic plants:

[0075] 100 mg of transgenic plant leaves were used to extract total protein from citrus leaves using a protein extraction kit (Solebo, CAT. BC3720). The expression of the protein encoded by the CsTCP15 gene (SEQ ID NO. 13) in citrus leaves was detected by Western blotting and incubated with a FLAG tag antibody.

[0076] The amino acid sequence of the protein encoded by the CsTCP15 gene is SEQ ID NO.13: MINMDDKNGIRRPNFPLQLL EKSQQQEASCSFSSSGFPSLTTRIAGGIDDDNNVNEQSSKGFSESSAAAAAKKPVPKRTSTKDRHTKVEGRGRRIRMPAACAARVFQLTRELGHKSDGETIEWLLQQAEPAVIAATGTGTIPANFTSLNISLRSSSGSSMSATHLRNTYFNPNFGSQQLMRNMRSEWERTLIDNNCNSLSFPLTGGNV NAMLQSNKQELRDNNTIATTTTSLDVSEADTGMGRKRRPDQELSQNNQMGSYLVQSSTGSIPASHSTIPATFWMMTNPSNNQVMSGAGESMWTFPSVSNSNMYRGSMSSGGVHFVNLPTPMALLPGQQLGSGIGSSGGTDSHLGMLAALNAYRNISGAAVSESPACASHPHHGAGGDDGQDSTSHHS.

[0077] The results are as follows Figure 6 As shown, the positive plant samples OE-1, OE-2 and OE-3 had a WB band at 47 kDa, while the WT plant sample had no band.

[0078] 4. qRT-PCR analysis of transgenic plants:

[0079] Total RNA was extracted from the leaves of transgenic plants (Adlai, CAT No: RN09), and cDNA was synthesized using the reverse transcription kit PrimeScript RT Master Mix (TaKaRa, CAT No: RR036A). The expression level of the target gene was detected by qRT-PCR. The detection primers were RT-CsTCP15-F (SEQ ID NO.7) and RT-CsTCP15-R (SEQ ID NO.8). -△△Ct Method to calculate the relative expression of CsTCP15 gene in transgenic plants: define the wild-type sample as the reference factor, that is, its CsTCP15 gene expression level is 1, and then calculate the multiple of gene expression in transgenic citrus relative to the reference factor 2 -△△Ct , which is its relative expression level.

[0080] The results are as follows Figure 7 As shown, the CsTCP15 gene was expressed at a higher level in the transgenic plants OE-1, OE-2, and OE-3 than in the wild-type plant WT.

[0081] qRT-PCR reaction conditions: 95°C for 3 min, 94°C for 10 s; 56°C for 10 s, 72°C for 10 s, 40 cycles; 72°C for 10 min.

[0082] The nucleotide sequence of primer RT-CsTCP15-F is shown in SEQ ID NO. 7: SEQ ID NO. 7: GTGTCTGAGGCGGATACTGG.

[0083] The nucleotide sequence of primer RT-CsTCP15-R is shown in SEQ ID NO. 8: TCATCCAAAATGTCGCCGGA.

[0084] 5. Phenotypic observation of transgenic plants:

[0085] like Figure 8 As shown in the figure, the phenotypes of the transgenic plants (OE-1, OE-2, and OE-3) were observed and analyzed, and no obvious abnormalities were found in their appearance and growth. This indicates that overexpression of the CsTCP15 gene did not significantly affect the phenotype and development of the plants.

[0086] 5. Resistance evaluation of transgenic plants overexpressing the CsTCP15 gene.

[0087] After propagation of the transgenic plants, 3-cm citrus branch segments carrying the CLas toxin were grafted onto the transgenic plants. DNA was extracted from leaves near the transgenic toxin source two, four, and six months after grafting, and then tested by qPCR using CLas detection primers.

[0088] The nucleotide sequence of primer RT-18S-F is shown in SEQ ID NO. 9: SEQ ID NO. 9: AATTTGTTGGTCTTCAACGAGGAA.

[0089] The nucleotide sequence of primer RT-18S-R is shown in SEQ ID NO. 10: SEQ ID NO. 10: AAAGGGCAGGGACGTAGTCAA.

[0090] The nucleotide sequence of primer RT-16S-F is shown in SEQ ID NO. 11: SEQ ID NO. 11: TGAGTGCTAGCTGTTGGGTG.

[0091] The nucleotide sequence of primer RT-16S-R is shown in SEQ ID NO. 12: SEQ ID NO. 12: CTGCGCGTTGCATCGAATTA.

[0092] Quantitative PCR (qPCR) was used to analyze the resistance of transgenic plants to Huanglongbing. Transgenic leaves of consistent maturity and growth were harvested at 2, 4, and 6 months after the onset of Huanglongbing infection. DNA was extracted and quantified to 100 ng / μL. qPCR was used to detect the levels of the citrus 18S gene and the CLas 16S gene. The following formula was used: Las copies ( / μg citrus DNA) = [10 (-0.2718×CtCLas16S+10.624) / 10 (-0.2749×CtCs18S+4.0531) ]×10 3(12.7 <CtCLas16S<31.3and8.4<CtCs18S<26.5) The relative content of CLas bacteria (Log10) was calculated. The wild type was used as the control, and the logarithm of the content of Huanglongbing pathogens was used to analyze the resistance level of the transgenic plants. Excel software was used for statistical plotting and SPSS software was used for significance analysis.

[0093] The results are as follows Figure 9 As shown, two months after CLas inoculation, CLas was detected in the WT line, but not in the OE-1, OE-2, and OE-3 lines. Four months after CLas inoculation, the amount of CLas in OE-1, OE-2, and OE-3 was significantly lower than that in the WT plants. This indicates that overexpression of the CsTCP15 gene can significantly inhibit the early infection and colonization of CLas, reducing the severity of HLB in citrus. This gene can be used independently as a molecular breeding agent for disease resistance, or in combination with other resistance or susceptibility genes for molecular breeding of citrus HLB resistance.

[0094] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. The application of CsTCP15 gene in regulating citrus Huanglongbing resistance is characterized by: The nucleotide sequence of the CsTCP15 gene is shown in SEQ ID NO.

1.

2. The application according to claim 1, characterized in that The regulation of citrus Huanglongbing resistance is achieved by overexpressing the CsTCP15 gene, thereby inhibiting the early infection and colonization of the citrus Huanglongbing pathogen and improving the citrus Huanglongbing resistance.

3. A recombinant expression vector, characterized in that: The vector overexpresses the CsTCP15 gene according to claim 1.

4. Use of the recombinant expression vector according to claim 3 in regulating resistance to citrus Huanglongbing, characterized in that: The regulation of citrus Huanglongbing resistance is that the recombinant expression vector overexpresses the CsTCP15 gene, thereby inhibiting the early infection and colonization of the citrus Huanglongbing pathogen and improving the citrus Huanglongbing resistance.

5. A recombinant strain, characterized in that The recombinant strain comprises the recombinant expression vector according to claim 3.

6. Use of the recombinant strain according to claim 5 in regulating resistance to citrus Huanglongbing, characterized in that: The regulation of citrus Huanglongbing resistance is that the recombinant strain overexpresses the CsTCP15 gene to inhibit the early infection and colonization of the citrus Huanglongbing pathogen, thereby improving the citrus Huanglongbing resistance.

7. The use of the protein encoded by the CsTCP15 gene in regulating citrus Huanglongbing resistance as claimed in claim 1, characterized in that: The amino acid sequence of the protein encoded by the CsTCP15 gene is shown in SEQ ID NO.

13. The regulation of citrus Huanglongbing resistance is achieved by increasing the expression level of the protein encoded by the CsTCP15 gene, thereby inhibiting the early infection and colonization of the citrus Huanglongbing pathogen and improving citrus Huanglongbing resistance.

8. A method for improving resistance to citrus Huanglongbing disease, characterized in that: The resistance of citrus to Huanglongbing is improved by overexpressing the CsTCP15 gene described in claim 1.

9. The use of the CsTCP15 gene as claimed in claim 1 in preparing a preparation for inhibiting infection by Candidatus Liberibacter asiaticus, characterized in that: The preparation for inhibiting infection of Bacillus asiaticus is applied to citrus to promote overexpression of the citrus CsTCP15 gene, inhibit the early infection and colonization of Bacillus asiaticus in citrus, and improve the resistance of citrus to Huanglongbing disease.

10. A preparation for inhibiting infection by Bacillus asiaticus, characterized in that: The preparation for inhibiting infection by Bacillus asiaticus comprises an effective ingredient that promotes overexpression of the CsTCP15 gene as claimed in claim 1 in citrus.

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