Method for improving citrus liberobacter asiaticum resistance by using CsSARD4 protein and disease-resistant citrus

Abstract

The invention discloses a method for improving citrus liberobacter asiaticum resistance by using CsSARD4 protein and a disease-resistant citrus, and the citrus liberobacter asiaticum resistance of citrus plants is improved by adjusting the expression level of CsSARD4 gene in the citrus plants; the method specifically comprises the following steps: (1) cloning a citrus CsSARD4 gene sequence; (2) constructing an overexpression vector; and (3) transforming the overexpression vector into citrus to obtain a transgenic plant of which the expression level of the CsSARD4 gene is up-regulated. The CsSARD4 gene coding sequence is cloned, the overexpression vector of the citrus CsSARD4 gene is constructed, and then citrus is transformed, so that the content of CLas in the obtained CsSARD4 overexpression transgenic plant is obviously lower than that of a wild type, the attack degree of the citrus huanglongbing can be effectively and obviously reduced, the resistance of the citrus to the citrus huanglongbing is obviously improved, the phenotype of the citrus plant is not influenced, and the application prospect is wide. The gene has a great application value for citrus liberobacter asiaticum-resistant breeding, and can be used as a candidate gene for citrus liberobacter asiaticum-resistant breeding with a plurality of citrus liberobacter asiaticum-resistant and susceptible genes.

Description

Technical Field

[0001] The present invention relates to the technical field of molecular biology, and in particular, to a method for improving the resistance of citrus to Huanglongbing by using the CsSARD4 protein and disease-resistant citrus. Background Art

[0002] Citrus, including mandarins, oranges, grapefruits, pomelos and lemons, etc., belongs to the Rutaceae family and is one of the important fruits with the widest cultivation area and highest yield globally. Rich in beneficial components such as vitamins, flavonoids, coumarins and carotenoids, it is highly favored and widely used in the fields of diet and medicine.

[0003] China is one of the important birthplaces of citrus and also the largest citrus producer in the world. However, in recent years, the citrus industry has faced severe challenges, among which the most prominent is the spread of citrus Huanglongbing (HLB). Citrus Huanglongbing is divided into Asian species (Candidatus Liberibacter asiaticus), African species (Ca.L.africanus) and American species (Ca.L.americanus). The currently known transmission methods of Huanglongbing include grafting transmission and insect vector transmission, etc. This disease is transmitted short distances by the Asian citrus psyllid and long distances by the transportation of diseased seedlings and infected scions. Citrus Huanglongbing has a huge impact on the global citrus industry, causing serious economic losses, and currently no effective pesticides and disease-resistant varieties have been developed.

[0004] Plants have evolved multiple immune systems to cope with pathogen invasion, including pattern-triggered immunity (PTI), effector-triggered immunity (ETI), and induced resistance (IR). More than 100 years ago, the phenomenon of induced resistance was recognized, that is, after plants are locally stimulated by pathogens or chemicals, their basal resistance is enhanced, and they can trigger a defense response to pathogen invasion earlier and faster, which was named systemic acquired resistance (SAR). SAR is induced by the interaction between pathogens and leaves and is related to the accumulation of endogenous SA and the transcriptional activation of pathogenesis-related proteins (PR proteins). SAR has multiple advantages such as broad-spectrum and persistence. Plants can generate broad-spectrum resistance to pathogens by activating SAR, and its occurrence process can be divided into three steps: pathogen invasion activates the host's local acquired resistance (LAR) to limit the infection and spread of the pathogen; at the same time, SAR signal transduction molecules are produced and transported long distances through the phloem tissue to the systemic part to activate SAR, thereby triggering the plant's systemic immune response and inhibiting the pathogen from invading again.

[0005] Multiple studies have demonstrated that SAR is an important immune mechanism for citrus to respond to Huanglongbing infection, and the resistance mediated by it is closely related to the tolerance of citrus to Huanglongbing. Overexpression of CsSAMT1 in sweet orange can promote the generation of the "group immune signal" MeSA and activate the related immunity of adjacent plants to achieve early defense against HLB. Overexpression of CsSABP2 in citrus can increase the accumulation levels of SA, MeSA, and H2O2 after pathogen infection and enhance the dual resistance of late Jincheng orange to Huanglongbing and citrus canker. Using important regulatory genes of SAR such as NPR1 can promote the expression of defense-related genes and enhance the tolerance to Huanglongbing. Therefore, using transgenic technology to enhance the systemic acquired resistance of citrus has great application value in improving the resistance of citrus to HLB.

[0006] Similar to SA / MeSA, Pip (Pipecolic Acid) / NHP (N-Pipecolic Acid) has been a newly discovered signaling molecule in the past five years. It belongs to non-protein amino acids, and its role in the SAR signal has been continuously revealed and has become an established mobile signal for SAR. The precursor of Pip, L-lysine, is catalyzed by Agd2-Like Defense Response Protein 1 (ALD1) to become △1-piperidine-2-carboxylic acid (P2C), and P2C is further catalyzed by SAR-Deficient 4 (SARD4) to become L-Pip. Subsequently, through Flavin-Dependent Monooxygenase 1 (FMO1), it is hydroxylated to form NHP. During the activation of SAR, the amino acid derivative N-hydroxypipecolic acid (NHP) derived from L-lysine and its biosynthetic precursor pipecolic acid (Pip) are synthesized in the leaves inoculated with pathogenic bacteria. NHP and SA coordinate the establishment of SAR by accumulating in systemic leaves. Studies have found that mutating the key enzyme SARD4 for Pip biosynthesis will lead to a decrease in the Pip level and an accumulation of the Pip precursor P2C in Arabidopsis thaliana, a decrease in the expression levels of PR1 and PR2, and a decrease in the SA content, directly affecting the establishment of SAR resistance in distal leaves. Thus, it can be seen the important role of SARD4 in plant SAR resistance. However, currently, there is no research or application on regulating the resistance of citrus to Huanglongbing using the NHP biosynthetic protein SARD4.

[0007] In view of this, the present application is specifically proposed. Summary of the Invention

[0008] The present invention provides a method for improving the resistance of citrus to Huanglongbing using the CsSARD4 protein and disease-resistant citrus to improve the resistance of citrus to Huanglongbing. By integrating the overexpression vector of the citrus flavin monooxygenase CsSARD4 gene into the hairy roots of citrus through Agrobacterium rhizogenes, the expression level of the CsSARD4 protein in the hairy roots of citrus is up-regulated, which can significantly improve the resistance of citrus to Huanglongbing, and the typical symptoms appear later, having great application value in citrus breeding and can be used as a candidate gene for Huanglongbing resistance breeding with multiple Huanglongbing-resistant and -susceptible genes in citrus.

[0009] The present invention is achieved through the following technical solutions:

[0010] In a first aspect, the present invention provides a method for improving the resistance of citrus to Huanglongbing by using the NHP biosynthetic protein CsSARD4 protein. The CsSARD4 protein is a key enzymatic reaction in the NHP biosynthetic pathway, that is, the CsSARD4 protein catalyzes the generation of Pip from P2C. By regulating the expression level of the CsSARD4 protein in citrus plants, the present invention improves the resistance of citrus plants to Huanglongbing. The amino acid sequence of the CsSARD4 protein is shown in SEQ ID NO: 1.

[0011] In a specific embodiment, the gene encoding the CsSARD4 protein is the citrus CsSARD4 gene, and its nucleotide sequence is shown in SEQ ID NO: 2.

[0012] In a specific embodiment, the specific method for regulating the expression level of the CsSARD4 protein is: up-regulating the expression level of the CsSARD4 protein in citrus plants to activate the related signaling pathways of systemic acquired resistance.

[0013] In a specific embodiment, the way to up-regulate the expression level of the CsSARD4 protein in citrus plants is to use an overexpression vector to control the expression of the CsSARD4 gene in citrus, up-regulate the accumulation of the citrus CsSARD4 protein, thereby enhancing the content of Pip and NHP in citrus plants, and ultimately enhancing the plant resistance.

[0014] In a specific embodiment, the method for improving the resistance of citrus to Huanglongbing by using the CsSARD4 protein specifically includes the following steps:

[0015] (1) Clone the coding sequence of the citrus CsSARD4 gene;

[0016] (2) Construct an overexpression vector;

[0017] (3) Transform citrus with the overexpression vector to obtain transgenic plants overexpressing the CsSARD4 protein.

[0018] In a specific embodiment, in step (1), the cloning method of the coding sequence of the citrus CsSARD4 gene is: extract the total RNA of citrus, then reverse transcribe it into cDNA, and finally use high-fidelity enzyme PCR to amplify the DNA fragment of the CsSARD4 gene coding sequence.

[0019] In a specific embodiment, in step (1), the PCR primers used for cloning the coding sequence of the citrus CsSARD4 gene are OE-F and OE-R, and their nucleotide sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4 respectively.

[0020] In a specific embodiment, in step (2), the method for constructing the overexpression vector is as follows: using pNmGFPer as the vector, the pNmGFPer vector carries the CaMV 35S promoter, the CaMV 35S promoter is the cauliflower mosaic virus promoter, and has the nucleotide sequence shown in SEQ ID NO: 5. After digesting the target fragment with SalⅠ and BamHⅠ, it is ligated to the vector recovered by digesting with SalⅠ and BamHⅠ to construct the overexpression vector pNmGFPer-CsSARD4.

[0021] In a specific embodiment, in step (3), the method for transforming citrus with the overexpression vector is as follows: the overexpression vector is transformed into Agrobacterium rhizogenes by heat shock method, and then used to mediate the transformation of citrus explants. The genetically transformed explant cells are cultured in vermiculite and identified by GFP fluorescence to obtain transgenic plants.

[0022] In a specific embodiment, it also includes verifying the transgenic plants by PCR. The primers used are ID-F and ID-R. ID-F is a sequence taken from CaMV 35S on the pNmGFPer vector, and ID-R is designed according to the CsSARD4 gene sequence. The primers have the nucleotide sequences shown in SEQ ID NO: 6 and SEQ ID NO: 7;

[0023] Real-time fluorescence quantitative PCR is used to detect the expression level of the CsSARD4 gene. The primers used are RT-F and RT-R, and their nucleotide sequences are shown in SEQ ID NO: 8 and SEQ ID NO: 9 respectively. The internal reference for real-time fluorescence quantitative PCR is the citrus CsGAPDH gene, and the primers used are GA-F and GA-R, and their nucleotide sequences are shown in SEQ ID NO: 10 and SEQ ID NO: 11 respectively.

[0024] In the second aspect, the present invention provides a citrus with huanglongbing resistance, obtained by the above method, and can have good huanglongbing resistance.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] 1. The method for improving the huanglongbing resistance of citrus using the CsSARD4 protein and the disease-resistant citrus provided by the embodiments of the present invention, by cloning the coding sequence of the citrus CsSARD4 gene, constructing an overexpression vector of the citrus CsSARD4 gene, and then transforming citrus, the CLas content in the obtained CsSARD4 overexpression transgenic plants is significantly lower than that of the wild type, and significantly reduces the incidence of citrus huanglongbing;

[0027] 2. A method for improving the resistance of citrus to Huanglongbing using the CsSARD4 protein and disease-resistant citrus provided by the embodiments of the present invention can significantly reduce the incidence of Huanglongbing in citrus by integrating the overexpression vector of the citrus SAR-deficient protein 4 CsSARD4 gene into citrus, improve the resistance of citrus to Huanglongbing, and does not affect the phenotype of citrus plants.

[0028] 3. A method for improving the resistance of citrus to Huanglongbing using the CsSARD4 protein and disease-resistant citrus provided by the embodiments of the present invention can greatly improve the resistance of citrus plants to Huanglongbing by overexpressing the CsSARD4 gene. This has great application value for citrus breeding for resistance to Huanglongbing, and it can be used as a candidate gene for citrus Huanglongbing resistance breeding in combination with multiple citrus Huanglongbing-resistant and -susceptible genes. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] To more clearly illustrate the technical solutions of the exemplary embodiments of the present invention, the following will briefly introduce the drawings required for the embodiments. It should be understood that the following drawings only show some embodiments of the present invention and should not be regarded as limiting the scope. For those of ordinary skill in the art, other related drawings can be obtained based on these drawings without creative efforts.

[0030] Figure 1 It is a domain alignment analysis diagram of citrus CsSARD4 provided by the embodiments of the present invention: the box indicates "NADP binding site";

[0031] Figure 2 It is a differential expression analysis diagram of the citrus CsSARD4 gene in different tolerance varieties in response to Huanglongbing; the asterisks on the data bars indicate significant differences (*, P < 0.05);

[0032] Figure 3 It is a positive plant identification diagram provided by the embodiments of the present invention: A, Flow chart of the construction of the pNmGFPer-CsSARD4 vector: CaMV 35S, a plant constitutive promoter derived from cauliflower mosaic virus; NOS, terminator of the nopaline synthase gene; B, PCR detection diagram of transgenic plants: P, plasmid of the pNmGFPer-CsSARD4 vector; WT, wild-type control; O1-5, O1-7, and O1-8 represent three transgenic plants; C, qRT-PCR detection diagram of transgenic plants: * indicates significant differences compared with the wild type (P < 0.05), the same below; D, Content change of NHP compared with the control;

[0033] Figure 4 It is a diagram for evaluating the resistance of CsSARD4 overexpressing transgenic citrus to Huanglongbing provided by the embodiments of the present invention. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] To make the objectives, technical solutions and advantages of the present invention more clearly understood, the present invention will be further described in detail below with reference to the embodiments and the accompanying drawings. The illustrative embodiments of the present invention and their descriptions are only used to explain the present invention and are not intended to limit the present invention.

[0035] In the following description, numerous specific details are set forth in order to provide a thorough understanding of the present invention. However, it will be apparent to those of ordinary skill in the art that: the present invention does not have to employ these specific details. In other embodiments, well-known structures, circuits, materials, or methods have not been specifically described in order to avoid obscuring the present invention.

[0036] Throughout the specification, the reference to "one embodiment", "embodiment", "one example" or "example" means that the specific features, structures or characteristics described in connection with the embodiment or example are included in at least one embodiment of the present invention. Thus, the phrases "one embodiment", "embodiment", "one example" or "example" appearing throughout the specification do not necessarily all refer to the same embodiment or example. In addition, the specific features, structures or characteristics may be combined in any suitable combination and / or sub-combination in one or more embodiments or examples. In addition, those of ordinary skill in the art should understand that the drawings provided herein are for illustrative purposes only and are not necessarily drawn to scale. The term "and / or" used herein includes any and all combinations of one or more of the related listed items.

[0037] In the description of the present invention, the orientation or positional relationship indicated by the terms "front", "rear", "left", "right", "upper", "lower", "vertical", "horizontal", "high", "low", "inner", "outer", etc. is based on the orientation or positional relationship shown in the drawings, and is only for the convenience of describing the present invention and simplifying the description, rather than indicating or implying that the device or element referred to must have a specific orientation, be constructed and operated in a specific orientation, and therefore should not be construed as limiting the protection scope of the present invention.

[0038] Example 1

[0039] Bioinformatics analysis of citrus CsSARD4

[0040] Bioinformatics analysis shows that the CDS length of CsSARD4 is 1,008 bp and it consists of 336 amino acids. As Figure 1 shown, the protein sequence alignment analysis shows that CsSARD4 contains the "NADP binding site" domain module, and this module is conserved in both trifoliate orange and sweet orange..

[0041] The amino acid sequence of CsSARD4 protein SEQ ID NO: 1:

[0042] MASTSNFTKNQNPNNPKAPIFISADSLHSILTHSSLIQHFHSSLPKLSPTIQSPIRQNYAVSPHSSLLLMPSWCSSPSLPYIGVKLVTSFPQNSSVNLPGIHASYVLFSSTNGQTLASMDGTVLTLYRTACVSGLASKILARNDSKVLVMIGAGNLAPHLIKAHLAARPSLKRVIIWNRTMKKASDLAEHLSKCGDHNGVCFESNENLEEILELGDIVSCATNSEVPLVRGGKLKVGAHLDLVGSYKHTMRECDDEAVKRGRVFVDNEAALVEAGELVGAFERGVIKEEDIAGNLVELIEGKKMGRRDDDEVTVFKSVGSGVVDLLAAQLVYEAM

[0043] Nucleotide sequence of the CsSARD4 gene: SEQ ID NO.2

[0044]

[0045] Example 2

[0046] Expression analysis of CsSARD4 in citrus

[0047] Expression analysis of citrus CsSARD4 gene in response to different resistant varieties to Huanglongbing

[0048] Compared with Jincheng, Mafenggan is more resistant to Huanglongbing. The CLas content in Mafenggan was significantly lower than that in Jincheng after 4 months of infection. Therefore, this experiment further used RT-qPCR to compare and analyze the expression characteristics of CsSARD4 in Jincheng and Mafenggan after 6 months of infection. Figure 2 As shown, the expression of CsSARD4 gene was significantly up-regulated in roots and down-regulated in leaves of both varieties, suggesting that CsSARD4 is closely related to citrus Huanglongbing resistance.

[0049] Example 3

[0050] Genetic transformation of citrus OE-CsSARD4 gene into Wanjin orange to improve citrus Huanglongbing resistance

[0051] 1. Cloning of Citrus CsSARD4 Gene

[0052] RNA was extracted from Late Jin Orange using an RNA extraction kit (Adlai, CAT: RN09). cDNA was synthesized using Recombinant DNaseI (TAKARA). Primers OE-F (SEQ ID NO: 3) and OE-R (SEQ ID NO: 4) were used to amplify the CsSARD4 fragment from citrus cDNA. The length of the fragment was 1,008 bp, and the sequence was shown in SEQ ID NO: 2. The amplified DNA fragment was sequenced and analyzed to be the coding sequence of the citrus CsSARD4 gene.

[0053] Amplification system: 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.

[0054] Amplification program: 94°C, 5 min; 94°C, 30 s, 56°C, 30 s, 72°C, 1.5 min, 35 cycles; extension at 72°C for 10 min.

[0055] DNA fragment recovery: Under ultraviolet light, use a clean blade to cut out the agarose gel block containing the target fragment. Use a kit (Adlai) to recover the fragment.

[0056] The nucleotide sequence of primer OE-F is SEQ ID No: 3:

[0057] GGATCCATGGCTTCAACATCCAACTTCAC

[0058] The nucleotide sequence of primer OE-R, SEQ ID No: 4:

[0059] GTCGACTTACATAGCTTCATACACCAATTGAG

[0060] 2. Construction of the CsSARD4 overexpression vector and transformation of Agrobacterium rhizogenes

[0061] The vector structure is as shown in Figure 3 Figure A. All restriction endonucleases were purchased from (THERMO) Company and operated according to the instructions.

[0062] The specific operation is as follows: The CsSARD4 gene fragment and the overexpression vector pNmGFPer were double-digested with restriction endonucleases SalⅠ and BamHⅠ and then recovered for ligation. The pNmGFPer vector carries the CaMV 35S promoter, which is the cauliflower mosaic virus promoter and has the nucleotide sequence shown in SEQ ID NO: 5. The ligation was carried out using the 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 CsSARD4 overexpression vector pNmGFPer-CsSARD4. The plasmid extraction was carried out using a kit (Aidlab).

[0063] Take the K599 Agrobacterium competent cells stored in the -80°C refrigerator and insert them into ice until completely melted. Take 1 μL of the plasmid and add it to the bottom of 100 μL of K599 competent cells, and gently stir to mix evenly. Then, let it stand on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min. Add 500 μL of LB liquid medium, and shake it at 220 rpm in a 28°C constant temperature shaker for 2 h - 3 h. Centrifuge the resuscitated bacterial solution at 5000 g for 1 min, discard the supernatant, and leave 100 μL of the resuspended bacterial solution. Pipette and mix evenly, and spread it on the LK plate. Incubate it upside down at 28°C for 2 d. Pick the bacterial colonies and culture them in the LK liquid medium with shaking. Screen the positive clones and store them at -80°C for later use.

[0064] The nucleotide sequence of the CaMV 35S promoter, SEQ ID NO: 5:

[0065] TGGAGTCAAAGATTCAAATAGAGGACCTAACAGAACTCGCCGTAAAGACTGGCGAACAGTTCATACAGAGTCTCTTACGACTCAATGACAAGAAGAAAATCTTCGTCAACATGGTGGAGCACGACACGCTTGTCTACTCCAAAAATATCAAAGATACAGTCTCAGAAGACCAAAGGGCAATTGAGACTTTTCAACAAAGGGTAATATCCGGAAACCTCCTCGGATTCCATTGCCCAGCTATCTGTCACTTTATTGTGAAGATAGTGGAAAAGGAAGGTGGCTCCTACAAATGCCATCATTGCGATAAAGGAAAGGCCATCGTTGAAGATGCCTCTGCCGACAGTGGTCCCAAAGATGGACCCCCACCCACGAGGAGCATCGTGGAAAAAGAAGACGTTCCAACCACGTCTTCAAAGCAAGTGGATTGATGTGATATCTCCACTGACGTAAGGGATGACGCACAATCCCACTATCCTTCGCAAGACCCTTCCTCTATATAAGGAAGTTCATTTCATTTGGAG

[0066] 3. Genetic transformation of citrus (Jincheng orange)

[0067] (1) Preparation and resuspension of bacterial solution. After thawing Agrobacterium rhizogenes K599 stored at -80°C, streak it on an LB plate with a spreading rod and incubate it upside down at 28°C for 2 days. Then pick 4 - 6 single colonies with a pipette tip, add them to 50 ml of LB liquid, and incubate them overnight on a shaker at 220 r / min and 28°C. Measure the OD600 value of the bacterial solution with an ultraviolet spectrophotometer. When the OD600 value is 0.8, transfer the bacterial solution to a centrifuge tube, centrifuge it at 5000 r / min for 10 min, discard the supernatant, collect the bacterial cells, and resuspend them with an equal volume of MS solution as the supernatant.

[0068] (2) Collect diseased citrus branches in the greenhouse, wash them with clean water, and cut them into stem segments about 5 cm long with one or more nodes using a small knife. Put a large amount of vermiculite in a culture pot, add an appropriate amount of tap water and mix well to make the humidity moderate.

[0069] (3) Vacuum infiltration transformation. Immerse the cut ends of the branches in the resuspended liquid and perform vacuum infiltration for 30 min.

[0070] (4) Insert the infected branches into moist vermiculite and place them vertically, with the stem segments inserted into the vermiculite and the leaves exposed to the air. Place them in a constant temperature incubator at 26°C, 16h / d to induce rooting. After one month, most plants will grow roots of 5-10cm.

[0071] Example 4

[0072] Identification of OE-CsSARD4 transgenic plants

[0073] 1. Identification of transgenic plants by GFP green fluorescence

[0074] Use LUYOR portable excitation light source, wear LUV-30A yellow glasses for observation, and choose blue light to irradiate the roots of the plants. Positive plants will appear green, and the roots of negative plants will appear yellow.

[0075] 2. PCR detection of exogenous gene integration

[0076] 100 mg of plant leaves obtained from the initial screening were used to extract genomic DNA using a DNA extraction kit (Adlai, CAT: DN15), and PCR was used to detect the integration of the CsSARD4 gene in the citrus genome. 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. The detection primers were ID-F (SEQ ID NO: 6) and ID-R (SEQ ID NO: 7). The PCR results are shown in Figure 2. Figure 3 As shown in B, the positive plants have amplified fragments, while the WT plants have no amplification.

[0077] The nucleotide sequence of primer ID-F is SEQ ID No: 6:

[0078] TTAGTAGTTGGTAACCTGA

[0079] The nucleotide sequence of primer ID-R is SEQ ID No: 7:

[0080] CGGCGTAGTTTTGGCGGATC

[0081] 3. Analysis of CsSARD4 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 (SEQ ID NO: 8) and RT-R (SEQ ID NO: 9); the detection primers for the internal reference gene GAPDH were RT-F (SEQ ID NO: 10) and RT-R (SEQ ID NO: 11).

[0083] The nucleotide sequence of primer RT-F, SEQ ID NO: 8:

[0084] GGGTAGGCGAGATGATGACG

[0085] The nucleotide sequence of primer RT-R, SEQ ID NO: 9:

[0086] ACACCAATTGAGCAGCGAGA

[0087] The nucleotide sequence of primer RT-F, SEQ ID NO: 10:

[0088] GCTTTCCGTGTACCCACTGT

[0089] The nucleotide sequence of primer RT-R, SEQ ID NO: 11:

[0090] CTCTGACTCCGCCTTGATGG

[0091] The reaction volume was 20 μL, and the reaction conditions were: 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. The experiment was repeated three times.

[0092] Using the 2 -△△Ct method to calculate the relative expression level of the CsSARD4 gene in transgenic plants: Define the sample treated with water as the reference factor, that is, the expression level of CsSARD4 in it is 1, and then calculate the multiple of the relative reference factor gene expression in transgenic citrus 2 -△△Ct , which is its relative expression level. The detection results are as Figure 3 shown in C. The results show that the CsSARD4 gene has a high-level expression in transgenic plants compared with wild-type plants.

[0093] 4. Detection of NHP and Pip contents in transgenic plants

[0094] After chopping 100 mg of citrus tissue, it was quickly frozen in liquid nitrogen, and then ground in a tissue grinder at 50 Hz for 2 minutes. The ground tissue was resuspended in 500 mL of 80% MeOH:H2O (v / v), shaken and mixed evenly at 4°C for 12 h, then centrifuged at 13,000 rpm for 10 minutes. After collecting the supernatant with a 1 mL syringe, it was filtered through a 0.22 μm polyvinylidene fluoride filter membrane, and the changes in NHP and Pip contents in transgenic plants were detected by UPLC-MS / MS. The detection results are as Figure 3 shown in D. The results show that in CsSARD4 transgenic plants, the NHP content is significantly increased in both leaves and roots compared with the empty vector control group, and the Pip content is only significantly increased in roots.

[0095] Example 5

[0096] Evaluation of Huanglongbing Resistance of OE-CsSARD4 Transgenic Plants

[0097] Extract DNA from leaf veins, design primers O11 / O12C for conventional PCR (see Tables 1 and 2) to detect the Huanglongbing virus source. Infect the obtained transgenic plants with the determined virus source by leaf disc grafting. The specific grafting method refers to Xie et al. (Xie et al., 2021). The materials after virus inoculation are cultured in a modified central light incubator, watered, and the symptom changes are observed and recorded regularly. Referring to Li et al. (2006), use quantitative PCR (qPCR)-probe method to detect the content of Huanglongbing pathogen in transgenic plants. At 40, 60, and 80 days after CLas inoculation, pick 3 virus-infected leaves (including transgenic plants and wild-type plants), extract DNA, detect the DNA concentration and quality, and quantify to 10 ng / μL. Use qPCR to detect the CLas content.

[0098] Table 1 qPCR reaction system

[0099]

[0100]

[0101] Table 2 qPCR reaction program

[0102]

[0103] Symptom observation showed that there were obvious phenotypic differences between transgenic plants and wild-type plants. To determine the response of transgenic plants to CLas infection, extract the leaf vein DNA of transgenic plants and wild-type plants at 40, 60, and 80 days after grafting virus inoculation, and use qPCR to detect the growth of CLas in leaf veins. Compared with the wild-type control, the growth of CLas in transgenic plants was slow. At 60 days, although the pathogen content increased, it was still significantly lower than that of the wild-type. Symptom observation found that after 80 days of virus inoculation, the wild-type control showed symptoms such as leaf yellowing and vein swelling, while these symptoms did not appear in transgenic plants. Figure 4

[0104] Thus, overexpression of CsSARD4 can enhance the resistance to Huanglongbing in citrus.

[0105] In the present invention, the media used for Agrobacterium rhizogenes transformation are as follows:

[0106] LB medium: 5 g / L yeast extract + 10 g / L peptone + 10 g / L NaCl, pH 5.8.

[0107] ​MS infection solution: 4.43 g / L MS, pH 5.4.

[0108] The specific embodiments described above further elaborate on the purpose, technical solution, and beneficial effects of the present invention. It should be understood that the above are only specific embodiments of the present invention and are not used to limit the protection scope of the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for improving citrus Huanglongbing resistance by using CsSARD4 protein, characterized in that: The resistance of citrus plants to citrus Huanglongbing disease is improved by regulating the expression level of CsSARD4 protein in citrus plants. The amino acid sequence of the CsSARD4 protein is shown in SEQ ID NO:

1.

2. The method for improving citrus Huanglongbing resistance by using CsSARD4 protein according to claim 1, characterized in that: The gene encoding the CsSARD4 protein is the Citrus CsSARD4 gene, and its nucleotide sequence is shown in SEQ ID NO:

2.

3. The method for improving citrus Huanglongbing resistance by using CsSARD4 protein according to claim 1, characterized in that: The specific method for regulating the expression level of CsSARD4 protein is: upregulating the expression level of CsSARD4 protein in citrus plants.

4. The method for improving citrus Huanglongbing resistance by using CsSARD4 protein according to claim 3, characterized in that: The method of up-regulating the expression level of CsSARD4 protein in citrus plants is to use an overexpression vector to control the expression of CsSARD4 gene in citrus, thereby up-regulating the accumulation of citrus CsSARD4 protein.

5. The method for improving citrus Huanglongbing resistance by using CsSARD4 protein according to claim 1, characterized in that: The specific steps include: (1) Cloning the coding sequence of the citrus CsSARD4 gene; (2) constructing an overexpression vector; (3) The overexpression vector was used to transform citrus to obtain transgenic plants overexpressing CsSARD4.

6. The method for improving citrus Huanglongbing resistance by using CsSARD4 protein according to claim 5, characterized in that: In step (1), the cloning method of the coding sequence of the citrus CsSARD4 gene is: extracting citrus total RNA, then reverse transcribing it into cDNA, and finally using high-fidelity enzyme PCR to amplify the CsSARD4 gene coding sequence DNA fragment.

7. The method for improving citrus Huanglongbing resistance by using CsSARD4 protein according to claim 6, characterized in that: In step (1), the PCR primers used to clone the coding sequence of the citrus CsSARD4 gene are OE-F and OE-R, and their nucleotide sequences are shown in SEQ ID NO: 3 and SEQ ID NO: 4, respectively.

8. The method for improving citrus Huanglongbing resistance by using CsSARD4 protein according to claim 5, characterized in that: In step (2), the overexpression vector is constructed by using pNmGFPer as a vector, wherein the pNmGFPer vector carries a CaMV 35S promoter, which is a cauliflower mosaic virus promoter having a nucleotide sequence shown in SEQ ID NO: 5, and the target fragment is digested with SalⅠ and BamHI and then connected to the vector recovered by digestion with SalⅠ and BamHI to construct the overexpression vector pNmGFPer-CsSARD4.

9. The method for improving citrus Huanglongbing resistance by using CsSARD4 protein according to claim 5, characterized in that: In step (3), the method for transforming citrus with the overexpression vector is as follows: the overexpression vector is transformed into Agrobacterium rhizogenes by heat shock method, and then the citrus explants are transformed by Agrobacterium rhizogenes, and the explant cells after genetic transformation are cultured with vermiculite and identified by GFP fluorescence to obtain transgenic plants.

10. A citrus fruit resistant to Huanglongbing, characterized in that: The method is obtained by any one of claims 1 to 9.

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