Application of CsTCP15 gene in regulating resistance to Huanglongbing in citrus

Abstract

This invention discloses the application of the CsTCP15 gene in regulating resistance to citrus Huanglongbing (HLB), belonging to the field of agricultural bioengineering technology, and specifically relating to the application of the CsTCP15 gene in regulating citrus HLB resistance. The nucleotide sequence of the CsTCP15 gene is shown in SEQ ID NO.1. This invention enhances citrus resistance to HLB by overexpressing the CsTCP15 gene, specifically improving resistance to the HLB pathogen *Candidatus Liberibacter asiaticus* (CLas). The invention involves constructing an overexpression vector for the cloned citrus CsTCP15 gene, followed by transformation into citrus. The resulting transgenic plants significantly inhibit early infection and colonization of CLS, demonstrating significant value for molecular breeding of citrus resistant to HLB.

Description

Technical Field

[0001] This invention belongs to the field of agricultural bioengineering technology, and in particular relates to the application of the CsTCP15 gene in regulating resistance to Huanglongbing in citrus. Background Technology

[0002] Citrus Huanglongbing (HLB) is a devastating disease caused by pathogens of the genus *Candidatus Liberibacter*, primarily involving three pathogenic species: *Candidatus Liberibacter africanus* (CLaf), *Candidatus Liberibacter asiaticus* (CLas), and *Candidatus Liberibacter americanus* (CLam). *CLas* is the most widespread, mainly transmitted by the Asian citrus psyllid (*Diaphorina citri*), posing a serious threat to the global citrus industry. *CLas* infection causes characteristic symptoms such as asymmetrical yellowing (mottled chlorosis), thickened leaves, stunted growth, and fruit drop, leading to plant death in severe cases. Because this pathogen cannot be cultured in vitro, research is extremely difficult; currently, only its genome has been sequenced. Given the devastating impact of HLB on agricultural production, the discovery of HLB-resistant germplasm resources has significant economic and social implications.

[0003] Plants employ a two-layered innate immune system to defend against pathogen invasion, including pattern-triggered immunity (PTI) and effector-triggered immunity (ETI). To counteract these defense mechanisms, pathogens secrete effector proteins as virulence factors to suppress or evade the host's immune response. Numerous citrus endogenous 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), thus 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), specifically inhibiting the ATG8-mediated immune response. Advances in genetic engineering have provided technical means for citrus disease-resistant breeding, making the search for disease-resistant genes and the creation of disease-resistant varieties of great significance.

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

[0005] To address the aforementioned technical issues, this invention proposes the application of the CsTCP15 gene in regulating citrus Huanglongbing (HLB) resistance. By overexpressing the CsTCP15 gene, early infection and colonization of the HLB pathogen are inhibited, thereby improving citrus HLB resistance.

[0006] To achieve the above objectives, the present invention provides the application of the CsTCP15 gene in regulating resistance to Huanglongbing in citrus, and 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, which inhibits the early infection and colonization of the citrus Huanglongbing pathogen and improves citrus Huanglongbing resistance.

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

[0009] This invention also provides the application of the recombinant expression vector in regulating citrus Huanglongbing resistance. The regulation of citrus Huanglongbing resistance involves the recombinant expression vector overexpressing 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 comprising the recombinant expression vector.

[0011] This invention also provides the application of the recombinant strain in regulating citrus Huanglongbing resistance. The regulation of citrus Huanglongbing resistance involves the recombinant strain overexpressing 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 the application of the protein encoded by the CsTCP15 gene in regulating resistance to citrus Huanglongbing (HLB). The amino acid sequence of the protein encoded by the CsTCP15 gene is shown in SEQ ID NO.13. The regulation of citrus HLB resistance is achieved by increasing the expression level of the protein encoded by the CsTCP15 gene, inhibiting the early infection and colonization of the HLB pathogen, and thus improving citrus HLB resistance.

[0013] The present invention also provides a method for improving the resistance of citrus Huanglongbing (HLB) by overexpressing the CsTCP15 gene.

[0014] This invention also provides the application of the CsTCP15 gene in the preparation of a formulation that inhibits the infection of Candidatus Liberibacter asiaticus. The formulation that inhibits the infection of Candidatus Liberibacter asiaticus is applied to citrus to promote the overexpression of the CsTCP15 gene in citrus, inhibit the early infection and colonization of Candidatus Liberibacter asiaticus in citrus, and improve the resistance of citrus Huanglongbing (HLB).

[0015] The present invention also provides an agent for inhibiting Asian phloem infection, wherein the agent for inhibiting Asian phloem infection includes 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] This invention enhances the resistance of citrus to Huanglongbing (HLB) by overexpressing the CsTCP15 gene, specifically improving the resistance of citrus to the HLB pathogen *Candidatus Liberibacter asiaticus* (CLas). The invention involves constructing an overexpression vector for the cloned citrus CsTCP15 gene, followed by transformation into citrus plants. The resulting transgenic plants significantly inhibited early infection and colonization of CLS. Specifically, two months after CLS inoculation, CLS were detected in the transgenic negative lines, but not in the transgenic positive lines OE-1, OE-2, and OE-3. Four months after CLS inoculation, the number of CLS in OE-1, OE-2, and OE-3 was significantly lower than in the WT (uncontrolled) plants. This invention provides a method for improving citrus HLB resistance, which has significant value for molecular breeding of citrus resistant to HLB. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

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

[0020] Figure 2 This is a structural diagram of the CsTCP15 gene overexpression vector. In the diagram, GFP represents green fluorescent protein, CaMV 35S represents the plant constitutive promoter, and NOS represents the crown gall synthase gene terminator.

[0021] Figure 3 This is a flowchart of the citrus genetic transformation process in Example 1;

[0022] Figure 4 The images show GFP fluorescence images of transgenic positive and negative plants. In the images, A is a WT wild-type Late Orange plant, B is an OE-1 transgenic plant, C is an OE-2 transgenic plant, and D is an OE-3 transgenic plant.

[0023] Figure 5 The images show PCR identification of transgenic positive and negative plants. In the images, P represents plasmid pNM-GFPer-CsTCP15-3×FLAG, Maker represents DNA molecular weight standard, WT represents wild-type Late 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 graph shows the expression levels of CsTCP15 in transgenic positive and negative plants. In the graph, WT represents wild-type Late Orange, OE-1 represents OE-1 transgenic plant, OE-2 represents OE-2 transgenic plant, and OE-3 represents OE-3 transgenic plant.

[0025] Figure 7 The images show the Western blot (WB) identification of transgenic positive and negative plants. In the images, WT represents wild-type Late Orange, OE-1 represents OE-1 transgenic plant, OE-2 represents OE-2 transgenic plant, and OE-3 represents OE-3 transgenic plant. "**" indicates p<0.005, "***" indicates p<0.001, and "****" indicates p<0.0001.

[0026] Figure 8The figures show the phenotypic diagrams of transgenic positive and negative plants. In the figures, WT represents wild-type Late Orange, OE-1 represents OE-1 transgenic plant, OE-2 represents OE-2 transgenic plant, and OE-3 represents OE-3 transgenic plant.

[0027] Figure 9 The graph shows the bacterial load on leaves of transgenic positive and negative plants 2 and 4 months after inoculation with CLas pathogen. In the graph, WT represents wild-type Late Jin 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 Implementation

[0028] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of 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 terminology used in this invention is merely for describing particular embodiments and is not intended to limit the invention. Furthermore, with respect to numerical ranges in this invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Every smaller range between any stated value or intermediate value within a stated range, and any other stated value or intermediate value within said range, is also included in this invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0030] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0031] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be apparent to those skilled in the art. This specification and embodiments are merely exemplary.

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

[0033] The late-ripening oranges used in this invention were sourced from the Citrus Research Institute of Southwest University.

[0034] Example 1

[0035] I. Cloning of the Citrus CsTCP15 Gene.

[0036] 1. RNA extraction and cDNA synthesis:

[0037] Total RNA was extracted from citrus (Late Orange) leaves using a plant total RNA extraction kit (Adley, CAT: RN09). RNA quality was verified by agarose gel electrophoresis, and its 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 the CsTCP15 gene:

[0039] The CsTCP15 gene DNA fragment was amplified from citrus cDNA using primers CsTCP15-F (SEQ ID NO.2), CsTCP15-R (SEQ ID NO.3), and the high-fidelity enzyme PrimeSTAR Max DNA Polymerase (TaKaRa, CAT: R045Q). The fragment length was 1185 bp (including restriction enzyme sites). Figure 1 As shown in the image, the amplified DNA was sequenced and identified as the citrus CsTCP15 gene (SEQ ID NO.1). Under UV light, an agarose gel block containing the target fragment was cut off with a clean blade, and the DNA fragment was recovered using a kit (BioFlux, CAT: BSC02M1).

[0040] PCR amplification program: 94℃, 5 min; 94℃, 30 s, 58℃, 30 s, 72℃, 1 min, 35 cycles; extension at 72℃ 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, SEQ ID NO.3: CTAGGAATGGTGACTGGTTG.

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

[0045] The CsTCP15 gene containing the homologous arm of the pNM-GFPer-3×FLAG vector and the overexpression vector pNM-GFPer were digested with restriction endonucleases KpnⅠ and BamH1 (Thermo Fisher) and then recovered using a gel using the ClonExpress II OneStep Cloning Kit (Novitamin, CAT: C112-01). The ligation product was transformed into E. coli DH5α, and plasmids of positive clones were extracted using a plasmid extraction kit (Tiangen, CAT: DP103) to obtain the CsTCP15 gene overexpression vector pNM-GFPer-CsTCP15-3×FLAG (e.g., pNM-GFPer-CsTCP15-3×FLAG). Figure 2 (As shown).

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

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

[0048] III. Genetic transformation of citrus fruits using CsTCP15 gene overexpression vectors (e.g.) Figure 3 (As 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 + 30 g / L sucrose, pH 5.8.

[0053] 1. Obtaining the hypocotyl from citrus seedlings:

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

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

[0056] The overexpression vector pNM-GFPer-CsTCP15-3×FLAG of the constructed CsTCP15 gene was introduced into Agrobacterium tumefaciens EHA105 (Weidi Bio, CAT#:AC1010) using chemical transformation. The method is as follows: 50 μL of frozen Agrobacterium competent cells EHA105 were thawed on ice; 2 μL of the plasmid of the overexpression vector was added to the competent cells, and the mixture was mixed by pipetting. The cells were then incubated on ice for 5 min, in liquid nitrogen for 5 min, at 42℃ for 5 min, and incubated on ice for 5 min. 700 μL of antibiotic-free LB liquid medium was added, and the cells were cultured at 28℃ with shaking for 3 h. The cells were then collected by centrifugation at 6000 rpm for 1 min. 100 μL of the supernatant was collected, and the bacterial blocks were resuspended by pipetting and spread on LB plates containing 50 mg / L kanamycin and 20 mg / L rifampin. The plates were inverted and incubated at 28℃ for 3 days. After the colonies 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℃ for 3 min; 94℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, 30 cycles; 72℃ for 10 min.

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

[0059] Before transfection, Agrobacterium tumefaciens (containing pNM-GFPer-CsTCP15-3×FLAG vector) for transfection was streaked on LB solid medium containing 50 mg / L kanamycin and 20 mg / L rifampin. Single colonies were picked and inoculated into 25 mL of LB liquid medium containing the same antibiotics and cultured at 28 °C with shaking for 12 h. The bacterial culture was diluted to OD = 0.1 and cultured until OD = 0.5. After centrifugation at 5000 r / min for 10 min, the supernatant was discarded and the culture was resuspended in MS liquid medium at pH 5.4 for transfection.

[0060] 4. Citrus epicotyl transformation:

[0061] After soaking the citrus epicotyl stem segments in Agrobacterium tumefaciens solution for 15 minutes and drying them, the stem segments were transferred to a co-culture medium and cultured in the dark at 26°C for 2 days. After co-culture, the epicotyls were transferred to a selection medium and cultured in the dark at 28°C for 7 days. The epicotyls were cultured at 28°C under 16h light / 8h dark conditions, and subcultured every 14 days. Then, the GFP fluorescence was observed by hand with a fluorescent lamp. Positive plants showed green fluorescence, while negative plants showed no fluorescence.

[0062] 5. Seedling culture of transformants:

[0063] When the seedlings grow to 1cm, they are cut off and grafted onto Late Orange seedlings in sterile test tubes and cultured in a seedling culture medium; when the seedlings grow to 5cm, they are grafted onto Trifoliate orange seedlings and cultured in a greenhouse at 28℃.

[0064] IV. Verification of transgenic plants overexpressing the CsTCP15 gene.

[0065] 1. Detection of GFP fluorescence in transgenic plants:

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

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

[0068] 2. PCR identification of transgenic plants:

[0069] Genomic DNA was extracted from 100 mg of leaves of transgenic plants using a DNA extraction kit (Adley, CAT: DN15). PCR was used to detect the integration of the CsTCP15 gene into the citrus genome. The detection primers 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, positive plants OE-1, OE-2, and OE-3 can obtain a 1662bp amplified fragment, while WT plants show no amplification.

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

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

[0073] The forward primer ID-pNM-F was used for PCR identification of transgenic plants within 35 seconds.

[0074] 3. Western blot (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 (Solepro, CAT.BC3720). Western blotting was used to detect the expression of protein encoded by the CsTCP15 gene (SEQ ID NO.13) in citrus leaves, and the leaves were 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, positive plant samples OE-1, OE-2, and OE-3 showed a WB band at 47 kDa, while WT plant samples did not show a band.

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

[0079] Total RNA (Adelaide, CAT No: RN09) was extracted from leaves of transgenic plants and cDNA was synthesized using the PrimeScript RTMaster Mix reverse transcription kit (TaKaRa, CAT: 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). Two... -△△Ct The relative expression level of the CsTCP15 gene in transgenic plants was calculated as follows: Wild-type samples were defined as the reference factor, with a CsTCP15 gene expression level of 1. The fold increase in gene expression relative to the reference factor in transgenic citrus was then calculated as 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 high level in transgenic plants OE-1, OE-2, and OE-3 compared to wild-type plant WT.

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

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

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

[0084] 5. Phenotypic observation of transgenic plants:

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

[0086] V. Evaluation of resistance in transgenic plants overexpressing the CsTCP15 gene.

[0087] After propagation of the transgenic plants, 3cm citrus branch segments carrying the CLas virus were grafted onto the transgenic plants. DNA was extracted from leaves near the transgenic virus at 2, 4, and 6 months post-grafting, and qPCR was performed 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 Huanglongbing (HLB) resistance level of transgenic plants. From the start of HLB grafting transmission, transgenic leaves of uniform maturity and growth status were harvested at 2, 4, and 6 months, and DNA was extracted and quantified to 100 ng / μL. The contents of the citrus 18S and CLas 16S genes were detected by qPCR. 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. Using the wild type as a control, the logarithmic value of the Huanglongbing pathogen content was used to analyze the resistance level of transgenic plants. Data were statistically plotted using Excel software, and significance analysis was performed using SPSS software.

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

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

Claims

1. Application of the CsTCP15 gene in regulating resistance to citrus Huanglongbing (HLB), characterized by: The nucleotide sequence of the CsTCP15 gene is shown in SEQ ID NO.

1. The regulation of citrus Huanglongbing resistance is achieved by overexpressing the CsTCP15 gene, which inhibits the early infection and colonization of the citrus Huanglongbing pathogen and improves citrus Huanglongbing resistance.

2. The application of a recombinant bacterial strain in regulating resistance to citrus Huanglongbing (HLB), characterized in that, The recombinant strain includes an expression vector capable of overexpressing the CsTCP15 gene as described in claim 1. The regulation of citrus Huanglongbing resistance is achieved by the recombinant strain overexpressing the CsTCP15 gene to inhibit the early infection and colonization of the citrus Huanglongbing pathogen, thereby improving citrus Huanglongbing resistance.

3. The application of the protein encoded by the CsTCP15 gene as described in claim 1 in regulating resistance to citrus Huanglongbing (HLB), 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, inhibiting the early infection and colonization of the citrus Huanglongbing pathogen, and thus improving citrus Huanglongbing resistance.

4. A method for improving resistance to Huanglongbing (HLB) in citrus, characterized in that, Overexpression of the CsTCP15 gene as described in claim 1 enhances resistance to Huanglongbing in citrus.

Images