Application of citrus csgrx9 gene in improving broad-spectrum resistance of citrus Huanglongbing and citrus canker

Abstract

The application discloses application of a citrus CsGRX9 gene in improving broad-spectrum resistance of citrus to Huanglongbing and canker, and belongs to the technical field of molecular breeding. The application discloses for the first time that increasing the expression level of the citrus CsGRX9 gene can significantly improve the resistance of citrus to Huanglongbing and canker; increasing or decreasing the transcription level of the citrus CsGRX9 gene does not obviously affect the phenotype and development of the plant; the citrus CsGRX9 gene provided by the application can be used as a candidate gene for studying broad-spectrum resistance of citrus to diseases, and can be used in molecular breeding of resistance to Huanglongbing and canker of citrus alone or simultaneously, and has important application value.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding technology, and in particular to the application of the citrus CsGRX9 gene in improving broad-spectrum resistance to Huanglongbing and Citrus canker in citrus. Background Technology

[0002] Citrus is the world's largest fruit crop, widely cultivated globally due to its significant economic, nutritional, and medicinal value. However, the high-quality development of the citrus industry is severely hampered by diseases such as Huanglongbing (HLB) and citrus canker. Among these, HLB is the most devastating, causing enormous economic losses to the global citrus industry. HLB is primarily caused by *Candidatus Liberibacter asiaticus* (CLas), a phloem-colonizing bacterium that cannot be cultured in vitro, and is spread through the insect vector, the psyllid (Diaphorina citri), which feeds on citrus trees. The main symptoms of HLB include yellowing or mottling of leaves, upright and hardened leaves, weakened tree vigor, and decreased fruit yield. Currently, there is no cure for this disease; control relies mainly on traditional measures, including preventing psyllid infestation, cutting down diseased trees, and planting disease-free seedlings. Citrus canker is also a bacterial disease caused by *Xanthomonas citri subsp. Citri* (Xcc). This disease primarily affects citrus leaves, twigs, and fruit, and is highly contagious and difficult to eradicate. Current control measures for Huanglongbing (HLB) and citrus canker mainly rely on burning diseased trees and spraying copper-based pesticides and antibiotics. However, these current control measures for both diseases pose risks to human health, require significant human and material resources, and cause environmental pollution. Therefore, given that most commercially cultivated varieties are susceptible to HLB and citrus canker, exploring more economical and effective control measures is of profound significance for the healthy development of the citrus industry.

[0003] For major diseases including Huanglongbing (HLB) and citrus canker, the most effective and fundamental control measures still rely on resistance breeding. However, traditional hybridization breeding suffers from problems such as long breeding cycles, polyembryonic interference, high human and financial costs, and low breeding efficiency. In contrast, modern molecular breeding, through genetic engineering, can synergistically integrate single or multiple gene resources with specific functions into specific commercial citrus varieties, thereby precisely, efficiently, and stably improving traits and providing a driving force for the healthy development of the citrus industry. Currently identified HLB resistance gene resources include CsMYC2, CsTCP15, CsPR5, CsWRKY22, the anti-proteolytic peptide gene APP3, and the antimicrobial peptide gene SAMP, while citrus canker resistance genes include the silkworm antimicrobial peptide D gene, CsBZIP40, and CmOGD2. However, the functions and mechanisms of action of most of the aforementioned genes are not fully understood, limiting their widespread applicability. Furthermore, the technical challenges of multi-gene genetic transformation in citrus make it difficult to create broad-spectrum resistant germplasm through multi-gene synergy. Therefore, high-quality resistance genes for citrus Huanglongbing (HLB) and citrus canker remain scarce, hindering the rapid development of molecular breeding in this field. Identifying candidate genes with broad-spectrum resistance to different citrus diseases and clarifying their functions could provide an economical and effective entry point for molecular breeding of broad-spectrum citrus resistance.

[0004] Glutareoxins (GRXs) are a class of small-molecule oxidoreductases that regulate the function and stability of target proteins through reversible reductive modification, playing a role in cellular redox signal transduction. Based on their amino acid sequences and active site motifs, GRXs are mainly classified into three types: CPYC, CGFS, and CC. The CC type contains the typical CXXC active site motif, unique to terrestrial plants. In Arabidopsis, AtGRXs negatively regulate development and reproductive processes, leading to the accumulation of reactive oxygen species and reduced resistance to fungi. In rice, OsGRX6 negatively regulates plant height and tiller number, as well as flowering time and senescence. Similarly, in cassava, MeGRXC3 negatively regulates plant tolerance to drought stress by reducing reactive oxygen species content and increasing stomatal aperture. However, there are no reports on the function and mechanism of action of citrus GRXs in major diseases such as Huanglongbing (HLB) and citrus canker. Summary of the Invention

[0005] The purpose of this invention is to provide the application of the citrus CsGRX9 gene in improving broad-spectrum resistance to citrus Huanglongbing and citrus canker, so as to solve the problems existing in the prior art.

[0006] To achieve the above objectives, the present invention provides the following solution:

[0007] This invention provides the application of the citrus CsGRX9 gene in any of the following:

[0008] (1) Improve the resistance of citrus to Huanglongbing and / or citrus canker;

[0009] (2) Cultivate citrus fruits with high resistance to Huanglongbing and / or citrus canker;

[0010] The nucleotide sequence of the citrus CsGRX9 gene is shown in SEQ ID NO.6.

[0011] Optionally, the application includes the step of overexpressing the citrus CsGRX9 gene in citrus.

[0012] This invention provides the application of citrus CsGRX9 protein in any of the following:

[0013] (1) Improve the resistance of citrus to Huanglongbing and / or citrus canker;

[0014] (2) Cultivate citrus fruits with high resistance to Huanglongbing and / or citrus canker;

[0015] The amino acid sequence of the citrus CsGRX9 protein is shown in SEQ ID NO.7.

[0016] Optionally, the application includes the step of increasing the content of the citrus CsGRX9 protein in the citrus fruit.

[0017] This invention provides the application of biomaterials containing the citrus CsGRX9 gene in any of the following:

[0018] (1) Improve the resistance of citrus to Huanglongbing and / or citrus canker;

[0019] (2) Cultivate citrus fruits with high resistance to Huanglongbing and / or citrus canker;

[0020] The nucleotide sequence of the citrus CsGRX9 gene is shown in SEQ ID NO.6.

[0021] Optionally, the biomaterial includes a recombinant vector and recombinant bacteria.

[0022] This invention provides a method for improving the resistance of citrus to Huanglongbing and / or citrus canker, comprising the step of overexpressing the citrus CsGRX9 gene in citrus; the nucleotide sequence of the citrus CsGRX9 gene is shown in SEQ ID NO.6.

[0023] This invention provides a method for cultivating citrus fruits with high resistance to Huanglongbing and / or citrus canker, comprising the step of overexpressing the citrus CsGRX9 gene in the citrus fruit; the nucleotide sequence of the citrus CsGRX9 gene is shown in SEQ ID NO.6.

[0024] This invention provides the application of the citrus CsGRX9 gene in citrus molecular breeding, and the nucleotide sequence of the citrus CsGRX9 gene is shown in SEQ ID NO.6.

[0025] The present invention discloses the following technical effects:

[0026] (1) This invention discloses for the first time that overexpression of the CsGRX9 gene can significantly improve the resistance of citrus to Huanglongbing and canker: when the gene is overexpressed, the content of Huanglongbing fungus in citrus remains at a very low level, and the symptoms of Huanglongbing are significantly suppressed; at the same time, after the overexpression material of this gene is inoculated with canker fungus, the leaves hardly develop the disease; this dual resistance to Huanglongbing and canker has been further verified genetically through the interference material of this gene.

[0027] (2) Upregulation and downregulation of the expression of the citrus CsGRX9 gene had no significant effect on plant height and growth;

[0028] (3) The citrus CsGRX9 gene provided by the present invention can be applied to plant molecular breeding and used as a candidate gene for breeding highly resistant varieties of citrus Huanglongbing and canker.

[0029] Therefore, this invention has significant application value, and its implementation can contribute to improving the economic benefits of the citrus industry and reducing environmental pollution caused by traditional control methods such as pesticides and burning. Attached Figure Description

[0030] 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.

[0031] Figure 1 To monitor CLas of the Huanglongbing fungus (HLB) of Egyptian sugar orange and identify genes regulating the early disease progression of HLB; (a) Ct values ​​of CLas content in different months after grafting and transmission, * indicates p < 0.05, t test; (b) HLB symptoms on leaves 16 months after infection, scale bar 1.2 cm; (c) Number of CsGRX9 gene transcripts in healthy and uninfected leaves, significance (FDR) < 0.01, fold change |log2 fold change| ≥ 1;

[0032] Figure 2 The expression level of CsGRX9 gene in the early pathogenesis of Huanglongbing in Egyptian sugar orange was detected by t-test. * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001.

[0033] Figure 3The functional domains and tissue expression patterns of the CsGRX9 gene were analyzed. Among them, (a) was the analysis of conserved domains of the CsGRX9 gene with known homologous proteins of cassava, rice, maize and Arabidopsis; (b) was the analysis of the expression patterns of the CsGRX9 gene in different tissues of Egyptian sugar orange. One-way Duncan ANOVA showed that different letters represented significant differences at the p < 0.05 level.

[0034] Figure 4 Images of the CsGRX9 gene overexpression vector and RNAi interference vector, along with electrophoretic identification of related PCR products, are shown below. (a) shows the core elements of the overexpression vector used for transgenic purposes; (b) shows the core elements of the RNAi interference vector used for transgenic purposes; (c) shows the electrophoretic gel image of the PCR amplification products of the CsGRX9 gene overexpression fragment, where "Repeat 1" and "Repeat 2" represent two replicates, and M represents the DNA molecular weight standard; (d) shows the electrophoretic gel image of the PCR amplification products of the CsGRX9 gene RNAi fragment, with "Repeat 1," "Repeat 2," and M having the same meanings as above.

[0035] Figure 5 To construct CsGRX9 gene overexpression and RNAi interference lines in Egyptian sugar orange; (a) shows the detection of GFP fluorescence signal in leaves of CsGRX9 overexpression and RNAi interference lines in Egyptian sugar orange, WT represents non-transgenic seedlings grown during genetic transformation, and the red scale bar is 2cm; (b) shows the detection of CsGRX9 gene expression level in different transgenic lines; (c) shows the plant height statistics of different transgenic scions of CsGRX9 gene using Ziyang sweet orange as rootstock; in the figures, ns indicates no significant difference, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and t detection is used;

[0036] Figure 6 To evaluate the resistance of CsGRX9 gene overexpression and RNAi interference lines to Huanglongbing (HLB); (a) shows the HLB incidence in non-transgenic (WT) and CsGRX9 gene overexpression and RNAi interference lines 11 months after infection with Cs buds, with red ellipses representing new shoots and leaves, and a scale bar of 6 cm; (b) shows the Cs bacterial cell content in lateral roots and leaves of citrus plants at different months after infection with Cs buds; in the figures, * indicates p < 0.05, ** indicates p < 0.01, *** indicates p < 0.001, and t-test is used.

[0037] Figure 7To evaluate the resistance of CsGRX9 gene overexpression and RNAi interference lines to bacterial canker; (a) shows leaf lesion photographs 12 days after needle inoculation with Xcc in non-transgenic (WT) and CsGRX9 gene overexpression and RNAi interference lines; (b) and (c) show leaf lesion area and disease index, respectively; one-way Duncan ANOVA was used in the figures, and different letters represent significant differences at the p < 0.05 level. Detailed Implementation

[0038] 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.

[0039] 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. Any stated value or intermediate value within a stated range, as well as each smaller range between 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.

[0040] 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.

[0041] 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.

[0042] 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.

[0043] The sequences involved in this invention are shown in Table 1.

[0044] Table 1 Sequence Information

[0045] Example 1: Obtaining the regulatory gene CsGRX9, which is closely related to the early disease progression of citrus Huanglongbing (HLB).

[0046] (1) Infection and monitoring of CLAs content of Huanglongbing bacteria

[0047] Using sweet oranges infected with CLAs (Huanglongbing fungus) preserved by the Fruit Tree Research Institute of Guangdong Academy of Agricultural Sciences as the source of infection (disclosed by Zheng Lanyan et al. in the literature "Evaluation of resistance to CLAs and analysis of resistance-related gene expression in 14 citrus resources" (Zheng Lanyan. Evaluation of resistance to CLAs and analysis of resistance-related gene expression in 14 citrus resources [J]. Journal of Jiangxi Agricultural University, 2025, 47(05): 1278-1288)), infected branches with buds (Ct value of fungal content between 20 and 23; the specific meaning and detection method of Ct value are described below) were pruned into wedge-shaped stem segments 2-3 cm long. Then, Egyptian sugar oranges (Citrus sinensis Osbeck cv. Succari) were grafted 5-10 cm below the stem-rootstock interface by lateral grafting (each graft had 2 grafting points to ensure successful transmission). Finally, TaqMan quantitative PCR technology was used to monitor the monthly changes in CLAs fungal content in the leaves.

[0048] The specific detection method of CLAs content of Huanglongbing fungus: DNA was extracted from leaves using a DNA extraction kit (Magen, CAT: D318702) and diluted to a working solution of 50 ng / μL; then, a 20 μL reaction system was prepared according to the instructions of the probe-based fluorescence quantitative PCR kit (BIO-RAD, CAT: 1725130), and primers HLBas F (SEQ ID NO.1) / HLBas R (SEQ ID NO.2) / HLB Probe (SEQ ID NO.3) and DNA template were added, and TaqMan quantitative PCR reaction was performed to obtain the Ct value; finally, the disease infection and disease development were evaluated according to the standard that a Ct value greater than 35 is negative and less than 35 is positive. For detailed process, refer to the literature "Evaluation of resistance to CLAs and analysis of resistance-related gene expression in 14 citrus resources" (Zheng Lanyan. Evaluation of resistance to CLAs and analysis of resistance-related gene expression in 14 citrus resources [J]. Journal of Jiangxi Agricultural University, 2025, 47(05):1278-1288).

[0049] The above monitoring methods were used for testing, and the test results are as follows: Figure 1 As shown. The results showed that after 11 months, the Ct value of the leaves of the infected plants decreased significantly, and after 16 months, the leaves showed typical Huanglongbing mottled yellowing symptoms, indicating that the transmission of the virus to Egyptian sugar orange was successful. Figure 1 (a) and (b) in the text). Then, asymptomatic new leaves (representing the early stage of the disease) were taken, and transcriptome sequencing was performed on them in comparison with healthy plants to identify the regulatory genes in the early stage of Huanglongbing disease. Among them, the gene LOC102606911 showed significant differential expression between the healthy and infected groups. Figure 1 (c)). Subsequently, the sequence and functional annotation information of the gene were further obtained by searching and comparing the NCBI database (https: / / www.ncbi.nlm.nih.gov / ) and the citrus database (http: / / citrus.hzau.edu.cn / ), and it was named CsGRX9.

[0050] Total RNA was extracted from Egyptian sugar orange leaves using a plant RNA extraction kit (TIANGEN, CAT: B0328B). Then, cDNA was synthesized using a HiScript IV 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, CAT: R412-01) according to the system and procedure outlined in the accompanying instructions. Finally, using the cDNA as a template, a reaction system was prepared according to the instructions of the high-fidelity enzyme Phanta Flash Super-Fidelity DNA Polymerase (Vazyme; CAT: P521-d1). Primers CsGRX9-F (SEQ ID NO.4) and CsGRX9-R (SEQ ID NO.5) were added, followed by conventional PCR amplification. The product was purified and recovered by agarose gel electrophoresis and then sent to Shanghai Bioengineering Technology Service Co., Ltd. for sequencing confirmation. The complete CDS coding sequence of the CsGRX9 gene was finally obtained (SEQ ID NO.6), which is 429 bp in length and encodes 142 amino acids (SEQ ID NO.7).

[0051] Example 2: The CsGRX9 gene was strongly induced to express during the early stage of Huanglongbing (HLB) disease.

[0052] Healthy leaves of Egyptian sugar orange and asymptomatic new mature leaves of Egyptian sugar orange infected with CLas (representing the early stage of the disease, the new leaves should change from light green to dark green, which takes about 15 days) were taken. Total RNA was extracted according to the instructions of the kit in Example 1 and further reverse transcribed into cDNA. Then, the reaction system was prepared according to the instructions of the dye-based quantitative PCR kit SupRealQ Ultra Hunter SYBR qPCR Master Mix(U+) (Vazyme, CAT: Q713-02), and a cycling program was set to detect the expression of the CsGRX9 gene. The quantitative primers for the target gene were CsGRX9-qRT-F (SEQ ID NO. 8) and CsGRX9-qRT-R (SEQ ID NO. 9), and the quantitative primers for the internal reference gene actin were Csactin-qRT-F (SEQ ID NO. 10) and Csactin-qRT-R (SEQ ID NO. 11).

[0053] The results showed that the CsGRX9 gene was strongly induced to express after infection. Figure 2 This indicates that the CsGRX9 gene is related to the pathogenesis of citrus Huanglongbing (HLB).

[0054] Example 3: CsGRX9 protein is a CC-type glutarate reductase, and its encoding gene is mainly expressed in mesophyll and phloem tissues.

[0055] Homologous genes of the citrus CsGRX9 gene in cassava, rice, maize, and Arabidopsis were obtained using the BLAST alignment function of the NCBI database. Then, DNAMAN software was used for protein sequence alignment to analyze the conserved amino acid sequences and functional domains of these homologous genes. Simultaneously, following the instructions for using the kit in Example 2, leaf mesophyll, midrib, flowers, and bark rich in phloem tissue from healthy Egyptian sugar oranges in Example 2 were collected for dye-based quantitative real-time PCR detection to analyze the expression pattern of the CsGRX9 gene in different citrus tissues. The CsGRX9 gene quantitative primers were the same as those shown in Example 2.

[0056] The results showed that the CsGRX9 protein contains the conserved active site motif (Cys-XX-Cys) and A(L / I)W(L / V) motif of CC-type glutreonin, and its encoding gene is mainly expressed in mesophyll and phloem tissues. Figure 3 The above results indicate that CsGRX9 protein is a CC-type glutoreductase and is consistent with the phloem colonization site of CLas, thus suggesting that CsGRX9 protein may be related to the pathogenesis of Huanglongbing (HLB).

[0057] Example 4: Overexpression of Citrus CsGRX9 Gene and Construction of RNAi Repression Vector

[0058] (1) Construction of CsGRX9 gene overexpression vector in citrus

[0059] Using the cDNA from Egyptian sugar orange leaves obtained in Example 1 as a template, PCR amplification was performed according to the standard PCR reaction system and procedure of Example 1 to obtain a CsGRX9 coding sequence product containing the recombinant homologous arm. The primers used were CsGRX9-OE-F (SEQ ID NO.12) and CsGRX9-OE-R (SEQ ID NO.13). The electrophoresis diagram of the amplified product is shown below. Figure 4 As shown in (c); then, following the instructions, using the recombinase (Vazyme; CAT: C117), the recovered product fragment was combined with the overexpression vector Kg2E-35SA-FlAG digested with BsaI (NEB; CAT: R3733V). Figure 4 (a); Vector information can be found in patent publication number: CN118166023A) Homologous recombination reaction was carried out (the reaction system was 10 μL, including 50 ng of linearized vector, 100 ng of product fragment, and 5 μL of recombinase premix); after the recombinant product was confirmed by sequencing by Shanghai Bioengineering Technology Service Co., Ltd., the recombinant plasmid Kg2E-OE-CsGRX9 was obtained.

[0060] (2) Construction of CsGRX9 citrus interference vector

[0061] Following the standard PCR reaction system described above, using the constructed Kg2E-OE-CsGRX9 plasmid as a template, PCR amplification was performed using primers CsGRX9-RNAi-F1 (SEQ ID NO.14) and CsGRX9-RNAi-R1 (SEQ ID NO.15) to obtain RNAi interference fragment 1, with a fragment length of 173 bp, as shown in SEQ ID NO.16. Simultaneously, PCR amplification was performed using primers CsGRX9-RNAi-F2 (SEQ ID NO.17) and CsGRX9-RNAi-R2 (SEQ ID NO.18) to obtain the inverse complementary fragment 2 (SEQ ID NO.19) of interference fragment 1. Figure 4 (d)); then, following the instructions of the restriction endonuclease XbaI (NEB; CAT: R0145V) and recombinase (Vazyme; CAT: C117) kits, respectively, the pGKE1 RNAi vector ( Figure 4 (b) was digested with a single enzyme, and the recovered interference fragment 2 was then recombined into the interference vector; wherein the pGKE1 RNAi vector consists of the backbone fragment of the above-mentioned Kg2E-35SA-FlAG (using Kg2E-35SA-FlAG as a template, Kg2E-F (SEQ ID NO.20) and Kg2E-R (SEQ ID NO.21) as PCR amplification primers, and the product fragment sequence is shown in SEQ ID NO.22) and the RNAi core element fragment (the nucleotide sequence of the RNAi core element fragment is shown in SEQ ID NO.22). As shown in NO.23, the interfering fragment 2 was obtained by recombination and ligation using the above-mentioned recombinase (Vazyme; CAT: C117) (the reaction system was 10 μL, including 50 ng of backbone, 100 ng of RNAi core element fragment, and 5 μL of recombinase premix). After successful sequencing and confirmation by the above-mentioned biotechnology company, the vector after recombination of interference fragment 2 was digested again with the restriction enzyme AbsI (ThermoScientific™; CAT: FD1724) in the same manner to complete the recombination reaction of interference fragment 1. Finally, after confirmation by sequencing again, the complete interference vector pGKE1-RNAi-CsGRX9 was obtained.

[0062] Example 5: Creation of Citrus CsGRX9 Overexpression and RNAi Interference Transgenic Materials

[0063] (1) Construction of transgenic materials

[0064] Referring to the established authorized patent for citrus genetic transformation (patent number: ZL201410555974.6) and the method of Zhang et al. (Zhang et al., A simple and efficient in planta transformation method for pommelo (Citrus maxima) using Agrobacterium tumefaciens, Scientia Horticulture, 2017, 214:174-179), transgenic materials were constructed using in situ genetic transformation technology of citrus seedlings. Specifically, the recombinant plasmids Kg2E-OE-CsGRX9 and pGKE1-RNAi-CsGRX9 were transformed into Agrobacterium tumefaciens EHA105 (VEIDI, CAT: AC1010), respectively. The plasmids were cultured in the dark on KanR resistance plates for 24-36 hours. After the growth of bacterial colonies, two plating activations were performed. Then, the bacterial cells were scraped off, diluted and resuspended with sterile deionized water, and the OD... 600 Adjust the pH to 0.6-0.8, add 20 μM acetylsyringone (Sigma-Aldrich, CAT: D134406), and incubate in the dark for 2 hours. Select healthy Egyptian sugar orange seedlings around 26 days old with 2-3 true leaves. Make a transverse cut on the hypocotyl 2 cm below the first true leaf with a blade. Quickly place a 10 μL white pipette tip saturated with bacterial solution onto the cut surface. After standing for 30 minutes, replace the tip with a new one and incubate in the dark with humidity. After 3 days, replace the tip with one containing KanR resistance and perform selection culture. After 5 days, remove the tip and transfer to normal light conditions (16 h light / 8 h dark, temperature 25℃-28℃, relative humidity 65%-75%, light intensity 25 µmol / m²). -2 s -1 Later, the positive buds were observed and identified using a portable handheld fluorescent lamp (LUYOR, CAT: 3415RG) with GFP fluorescence light source. When the positive buds grew to about 5cm, they were grafted onto Ziyang sweet orange (Citrus junos Sieb. ex Tanaka) as rootstock to propagate transgenic and non-transgenic (control) seedlings to obtain the corresponding propagated plants.

[0065] (2) Molecular detection of transgenic materials

[0066] For the CsGRX9 gene overexpression and RNAi interference citrus materials obtained above, appropriate amounts of leaf tissue were taken, and total RNA was extracted and cDNA was synthesized according to the methods in Examples 1 and 2. Then, the expression level of the CsGRX9 gene was detected by dye-based quantitative real-time PCR, using the same primers as in Example 2. Simultaneously, the plant height of different grafted propagation plants was recorded after six months, and their growth phenotypes were photographed and documented.

[0067] Through the creation and testing of the above-mentioned transgenic materials, two CsGRX9 gene overexpression lines (overexpression lines) and two RNAi interference lines were successfully obtained. Figure 5 In (a) of the two RNAi interference lines, two overexpression lines were designated #13 and #16, and two RNAi interference lines were designated #7 and #9. In the overexpression lines, the expression level of the CsGRX9 gene was increased by more than 6-fold, while in the interference lines, the expression level of the CsGRX9 gene was significantly suppressed, downregulated by about 45%-50%. Figure 5 (b)); at the same time, there were no significant differences in plant height and growth phenotype among different transgenic lines ( Figure 5 (c)). In summary, this invention successfully constructed CsGRX9 gene overexpression lines and RNAi interference lines.

[0068] Example 6 Evaluation of Huanglongbing resistance in citrus CsGRX9 gene overexpression lines and RNAi interference lines (collectively referred to as CsGRX9 transgenic plants)

[0069] After the non-transgenic (control) and CsGRX9 transgenic plants from Example 5 had grown for 10 months, following the method described in Example 1, plants with uniform growth were selected for grafting and virus transmission experiments in a greenhouse (Guangzhou's geographical location: 22°26'-23°56' N, 112°57'-114°3' E; marine subtropical monsoon climate, average annual temperature 21.7℃-23.1℃, average annual precipitation 1923mm, average annual relative humidity approximately 78%, annual sunshine hours approximately 1650-1820 hours). The Ct values ​​of Huanglongbing fungus CLas in citrus lateral roots and leaves were then measured monthly to monitor changes in CLas content. For clarity, the Ct values ​​were further converted to a more direct relative bacterial content using the following formula: Number of CLas cells / ug of citrus DNA = 10^y / m, y = -0.3264x + 12.715, R 2 = 0.9995 Calculate the relative content of Clas bacteria (Log) 10For detailed process, please refer to the literature "Molecular Mechanism of CaLasSDE70 Effector Targeting Host Ubiquitin-Related Protein CsRub2 to Regulate Citrus Huanglongbing Resistance" (Wang Shuai. Molecular Mechanism of CaLasSDE70 Effector Targeting Host Ubiquitin-Related Protein CsRub2 to Regulate Citrus Huanglongbing Resistance [D]. Southwest University, 2023. DOI:10.27684 / d.cnki.gxndx.2023.001975).

[0070] The results showed that at 6 months post-infection, CLas pathogens were first detected in the lateral roots of the control and RNAi interference lines, with the pathogen content in the RNAi interference lines significantly higher than that in the control. For the next two months, the CLas pathogen content in the lateral roots of both the control and RNAi interference lines continued to increase. For the CsGRX9 overexpression lines, a small amount of CLas pathogens was also detected in the lateral roots at 7 months, but thereafter remained at a consistently low level. At 9 months post-infection, similar to the situation in the roots, CLas pathogens were preferentially detected in the leaves of the control and RNAi interference lines, with the pathogen content in the RNAi interference lines significantly higher than that in the control. For the CsGRX9 overexpression lines, the detection of the pathogens was also delayed, with a small amount detectable in the leaves at 10 months. Figure 6 (b)). Eleven months after transmission, both the control and RNAi interference lines showed obvious yellowing or mottled yellowing symptoms on their leaves, with the RNAi interference lines exhibiting more severe symptoms. In contrast, the CsGRX9 overexpression lines showed no obvious symptoms and continued to produce new leaves, indicating good growth. Figure 6 (a) in the middle.

[0071] The results above indicate that overexpression of CsGRX9 can significantly inhibit the proliferation of Huanglongbing fungus, greatly slow down the disease progression, and thus improve the resistance of citrus to Huanglongbing.

[0072] Example 7 Evaluation of canker resistance in citrus CsGRX9 gene overexpression lines and RNAi interference lines (collectively referred to as CsGRX9 transgenic plants)

[0073] Following the inoculation and statistical methods of the authorized patent (patent number: ZL201410555974.6), the resistance of CsGRX9 transgenic plants to citrus canker was evaluated. Specifically, mature leaves from control and CsGRX9 transgenic plants were taken and disinfected on sterile filter paper with 75% alcohol. After the alcohol evaporated from the leaf surface, sterile toothpicks were used to prick the leaves, centered on the veins. Then, a pipette was used to extract the bacterial suspension of citrus canker pathogen (Xanthomonas citrus subsp. *Xanthomonas citrus*, a laboratory-frozen strain, published by Ting Wang et al. in the literature "Editing the CsRIN4 Gene Confers Resistance to Citrus Canker in Citrus sinensis" (Plant Biotechnol. J., 2026, 24: 884-886)). After activation, the OD value was adjusted using deionized water. 600 Adjust the concentration to 0.8 (for experiments), apply 1 μL of bacterial solution to each needle-punched hole; after inoculation, place the leaves in a plant culture chamber (temperature 25℃-28℃, 16h light / 8h dark, relative humidity 65%-75%, light intensity 250µmol / m²). -2 s -1 The cells were cultured, with three replicates for each treatment group. Finally, on day 12 after inoculation, photographs were taken, and the lesion area was calculated using ImageJ software. The area of ​​90-100 lesions in each group was recorded.

[0074] The disease is classified into grades 0-7 based on the area of ​​the lesions, with the lesion area represented by the letter LS: Grade 0 (LS ≤ 0.25mm) 2 Level 1 (0.25mm) 2 < LS ≤ 0.5mm 2 Level 2 (0.5mm) 2 < LS ≤ 0.75mm 2 Level 3 (0.75mm) 2 < LS ≤1.0mm 2 Level 4 (1.0mm) 2 < LS ≤ 1.25mm 2 Level 5 (1.25mm) 2 < LS ≤1.5mm 2 ), Level 6 (1.5mm) 2 < LS≤ 1.75mm 2 Level 7 (LS > 1.75mm)2 ).

[0075] The disease index (DI) is calculated using the following formula: DI = 100 × Σ (number of lesions at each level × corresponding level value) / (total number of lesions × maximum level).

[0076] Depend on Figure 7 It can be seen that 12 days after inoculation with the citrus canker pathogen, the leaves of both the control group and the RNAi interference line showed varying degrees of disease, while the CsGRX9 gene overexpression plants showed almost no obvious symptoms except for pinholes. Statistical analysis showed that the lesion area of ​​the RNAi interference line was 1.39-1.94 times that of the control group, and the disease index was also about 50%-70% higher than that of the control group. Therefore, overexpression of the CsGRX9 gene can significantly reduce the lesion area of ​​citrus canker, alleviate the severity of the disease, and greatly improve the resistance of citrus to citrus canker.

[0077] Therefore, this invention discloses for the first time the novel discovery that increasing the expression level of the citrus CsGRX9 gene can significantly improve the resistance of citrus to Huanglongbing and citrus canker; the increase or decrease in the transcription level of the citrus CsGRX9 gene does not have a significant impact on the phenotype and development of the plant; the citrus CsGRX9 gene provided by this invention can serve as a candidate gene for studying broad-spectrum resistance to citrus diseases, and can be used alone or simultaneously for molecular breeding against citrus Huanglongbing and citrus canker, thus having important application value.

[0078] 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. Citrus CsGRX9 The application of genes in any of the following: (1) Improve the resistance of citrus to Huanglongbing and / or citrus canker; (2) Cultivate citrus fruits with high resistance to Huanglongbing and / or citrus canker; The citrus CsGRX9 The nucleotide sequence of the gene is shown in SEQ ID NO. 6; the application includes overexpression of the citrus fruit in the citrus fruit. CsGRX9 The steps of gene generation.

2. Application of citrus CsGRX9 protein in any of the following: (1) Improve the resistance of citrus to Huanglongbing and / or citrus canker; (2) Cultivate citrus fruits with high resistance to Huanglongbing and / or citrus canker; The amino acid sequence of the citrus CsGRX9 protein is shown in SEQ ID NO.7; the application includes the step of increasing the content of the citrus CsGRX9 protein in citrus fruits.

3. Contains citrus fruits CsGRX9 The application of gene-based biomaterials in any of the following: (1) Improve the resistance of citrus to Huanglongbing and / or citrus canker; (2) Cultivate citrus fruits with high resistance to Huanglongbing and / or citrus canker; The citrus CsGRX9 The nucleotide sequence of the gene is shown in SEQ ID NO.6; The application includes overexpression of citrus fruits. CsGRX9 The steps of gene generation.

4. The application according to claim 3, characterized in that, The biomaterials are recombinant vectors and recombinant bacteria.

5. A method for improving the resistance of citrus to Huanglongbing and / or citrus canker, characterized in that, Including overexpression of citrus in citrus CsGRX9 Genetic steps; the citrus CsGRX9 The nucleotide sequence of the gene is shown in SEQ ID NO.

6.

6. A method for cultivating citrus fruits with high resistance to Huanglongbing and / or citrus canker, characterized in that, Including overexpression of citrus in citrus CsGRX9 Genetic steps; the citrus CsGRX9 The nucleotide sequence of the gene is shown in SEQ ID NO.6.

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