A method for improving citrus canker resistance using csrhy1a interference

Abstract

The application discloses a method for improving citrus canker resistance by interfering with CsRHY1A, and relates to the field of agricultural biological genes. The RNAi fragment of the CsRHY1A coding gene of citrus is cloned, the interference vector is integrated into the citrus, the transcription level of CsRHY1A is reduced, the resistance of the citrus to canker is significantly improved, the phenotype of the transgenic plant is not affected, the canker lesion area of the obtained transgenic plant can be reduced to 85% of that of the existing citrus at the most, the disease index can be reduced to 82% of that of the existing citrus at the most, the disease degree of canker can be significantly reduced, and the phenotype of the citrus is not affected, so that it is concluded that the interference of CsRHY1A can greatly improve the resistance of the citrus to canker; compared with the gene editing technology for silencing the citrus gene, the RNAi technology is more stable, the probability of obtaining a gene-silenced plant is greater, and the RNAi technology is suitable for the high-heterozygosity species of the citrus.

Description

Technical Field

[0001] This invention relates to the field of agricultural biogenetics, specifically to a method for improving resistance to citrus canker by using CsRHY1A interference. Background Technology

[0002] In recent years, my country's citrus industry has developed rapidly, gradually becoming a major economic pillar in the mountainous areas of southern China. However, the development of the citrus industry has been severely hampered by diseases such as citrus canker. Citrus canker is caused by Xanthomonas citrus subsp. *citrus*. Xanthomonas citri subsp. Citri , Xcc Citrus canker is a bacterial disease that affects most major citrus varieties and occurs in all major citrus-producing areas of my country, with the affected area continuing to expand. Therefore, strengthening research on the prevention and control of citrus canker is an urgent need for the development of the citrus industry.

[0003] Despite the high level of attention paid to the prevention and control of citrus canker by governments, producers, and researchers in citrus-producing regions worldwide, the disease remains incurable due to its wide and rapid spread and the difficulty of control. Traditional control methods (such as burning diseased trees and using pesticides) require significant human and material resources and cause substantial environmental damage (Ference et al., 2018). Therefore, the control of citrus canker relies more heavily on the development of new disease-resistant germplasm. Generations of citrus researchers have engaged in long-term hybridization breeding, but the long cycle of hybridization has resulted in low breeding efficiency. Molecular breeding, which can directionally and efficiently cultivate new disease-resistant germplasm, has now seen rapid development and widespread application. In recent years, biotechnology has been used to obtain some citrus resources resistant to citrus canker, such as Jincheng, Xinhui, and navel orange lines transfected with the antimicrobial peptide D gene from silkworms (Chen et al., 1996); late-maturing Jincheng orange lines overexpressing CsBZIP40 (Li et al., 2019); and plants with enhanced resistance to citrus canker obtained by site-directed editing of the CsLOB1 promoter, a citrus canker susceptibility gene (Peng et al., 2015). The RHY1A gene is responsible for guiding the synthesis of E3 ubiquitin ligases in plants and participates in regulating plant stress responses (such as salt tolerance) and hormone signaling pathways.

[0004] However, high-quality candidate genes remain scarce, and their functions and mechanisms of action are not well understood, making it difficult to achieve multi-gene synergistic resistance to citrus canker and hindering the development of molecular breeding for citrus canker resistance. Therefore, it is urgent to specifically identify more genes closely related to citrus canker, deeply analyze their functions and mechanisms, and use them for molecular breeding for canker resistance. Summary of the Invention

[0005] To address the aforementioned shortcomings of existing technologies, this invention provides a method for improving resistance to citrus canker by utilizing CsRHY1A interference.

[0006] To achieve the above-mentioned objectives, the technical solution adopted by this invention is as follows:

[0007] A method for improving resistance to citrus canker by utilizing CsRHY1A interference is provided, comprising the following steps:

[0008] S1: Cloning the RNAi fragment of the citrus CsRHY1A encoding gene as shown in SEQ ID NO: 1, the specific steps are as follows: extract total RNA from citrus, reverse transcribe it into cDNA, and perform PCR amplification on the cDNA to obtain the RNAi fragment of the CsRHY1A encoding gene; the primers used for PCR amplification are CsRHY1A-RNAi-F as shown in SEQ ID NO: 2 and CsRHY1A-RNAi-R as shown in SEQ ID NO: 3;

[0009] S2: Construct the CsRHY1A interference vector using cloned RNAi fragments;

[0010] S3: The constructed CsRHY1A interference vector was transformed into citrus to obtain transgenic citrus plants resistant to citrus canker.

[0011] Furthermore, the specific steps for constructing the CsRHY1A interference vector in step S2 are as follows:

[0012] The PCR products obtained from S1 were divided into two groups. One group was double-digested with AscI and SwaI, and the other group was double-digested with BamHI and SalI. The two fragments recovered from the digestion were ligated into the pUC-RNAi vector to obtain the intermediate vector pUC-RNAi-CsRHY1A.

[0013] The intermediate vectors pUC-RNAi-CsRHY1A and pLGNe were double-digested with KpnI and SalI, respectively. The digestion product containing the RNAi fragment on the intermediate vector pUC-RNAi-CsRHY1A was ligated into the pLGNe vector to obtain the CsRHY1A interference vector pLGNe-CsRHY1A-RNAi.

[0014] Furthermore, the specific steps for converting the CsRHY1A interference vector into citrus in step S3 are as follows:

[0015] The CsRHY1A interference vector was transformed into Agrobacterium tumefaciens by electroporation, and then Agrobacterium tumefaciens was used to transform citrus explants. The genetically transformed explant cells were then cultured in vitro, stained for identification, grafted, and verified by PCR and qRT-PCR to obtain transgenic plants.

[0016] Furthermore, during PCR verification, the primers used were ID-CsRHY1A-F as shown in SEQ ID NO:4 and ID-CsRHY1A-R as shown in SEQ ID NO:5.

[0017] Furthermore, the PCR amplification program is as follows: 3 min at 94℃; 30 cycles: 30 s at 94℃, 30 s at 58℃, 30 s at 72℃; 10 min at 72℃.

[0018] Furthermore, during qRT-PCR verification, the primers used were RT-CsRHY1A-F as shown in SEQ ID NO: 6 and RT-CsRHY1A-R as shown in SEQ ID NO: 7.

[0019] Furthermore, the qRT-PCR validation procedure was as follows: 3 min at 95℃, 10 s at 94℃; 40 cycles: 10 s at 56℃, 10 s at 72℃; 10 min at 72℃.

[0020] The present invention also provides a method for breeding late-maturing orange canker resistant plants, wherein the transgenic late-maturing orange plants obtained by the above method are used as parents to cultivate offspring with stable inheritance.

[0021] The beneficial effects of this invention are as follows:

[0022] This invention clones the RNAi fragment of the CsRHY1A encoding gene in citrus, constructs an interference vector, and integrates it into citrus, reducing the transcription level of CsRHY1A and significantly improving the resistance of citrus to citrus canker without affecting the phenotype of the transgenic plants. The resulting transgenic plants show a reduction in canker lesion area to up to 85% of existing citrus varieties and a disease index to up to 82% of existing citrus varieties, significantly alleviating the severity of citrus canker without affecting the citrus phenotype. Therefore, CsRHY1A interference can greatly improve the resistance of citrus to citrus canker. Compared to gene editing technology for silencing citrus genes, RNAi technology is more stable, has a higher probability of obtaining gene-silenced plants, and is suitable for highly heterozygous species like citrus. It has significant application value in citrus canker resistance breeding and can be used as a candidate gene to conduct canker resistance breeding with multiple canker resistance and susceptibility genes. Attached Figure Description

[0023] Figure 1 This is an electrophoresis image of the RNAi fragment of the CsRHY1A encoding gene in Example 1, obtained through PCR amplification.

[0024] Figure 2 Phenotypic diagrams of the transgenic plants and control plants in Example 3;

[0025] Figure 3This is a diagram showing the results of GUS histochemical staining of the leaf in Example 3;

[0026] Figure 4 This is an electrophoresis image of the plant for PCR identification in Example 4;

[0027] Figure 5 This is a comparison of the relative expression levels of the CsRHY1A gene in plants from Example 5.

[0028] Figure 6 This is a characterization diagram of the plant leaves after inoculation with ulcer pathogens in Example 6;

[0029] Figure 7 This is a comparison chart of the statistical results of the lesion area on the leaves of the plants in Example 6;

[0030] Figure 8 This is a comparison chart of the statistical results of the disease index of the plant leaves in Example 6. Detailed Implementation

[0031] The specific embodiments of the present invention are described below to enable those skilled in the art to understand the present invention. However, it should be understood that the present invention is not limited to the scope of the specific embodiments. For those skilled in the art, various changes are obvious as long as they are within the spirit and scope of the present invention as defined and determined by the appended claims. All inventions utilizing the concept of the present invention are protected.

[0032] Example 1: Cloning and recovery of the RNAi fragment of the CsRHY1A encoding gene

[0033] Total RNA was extracted from the leaves of Late Jin Orange (Chongqing Citrus License No. 2011001) using a plant total RNA extraction kit (Adley, CAT: RN09). RNA quality was verified by agarose gel electrophoresis, and its concentration was determined by a concentration meter. cDNA was synthesized using a reverse transcription kit PrimeScript RT Master Mix (TaKaRa, CAT: RR036A) and stored at -20℃ for later use.

[0034] Using primers such as CsRHY1A-RNAi-F (SEQ ID NO: 2) and CsRHY1A-RNAi-R (SEQ ID NO: 3), and employing the high-fidelity enzyme PrimeSTAR Max DNA Polymerase (TaKaRa, CAT: R045Q), the RNAi fragment encoding the CsRHY1A gene was cloned from the cDNA of *Citrus aurantiacus*. Figure 1As shown, the RNAi fragment length is 300 bp. The PCR amplification program was: 98℃ for 5 min; 98℃ for 30 s, 56℃ for 30 s, 72℃ for 1.5 min, 35 cycles; extension at 72℃ for 10 min.

[0035] Under UV light, the agarose gel block containing the target fragment was cut off with a clean blade, and the RNAi fragment as shown in SEQ ID NO: 1 was recovered using a kit (BioFlux, CAT: BSC02M1).

[0036] Example 2 Construction of CsRHY1A Interference Vector

[0037] The RNAi fragments obtained in Example 1 were divided into two groups. One group was digested with AscI and SwaI, and the other group was digested with BamHI and SalI. The two groups of fragments recovered from the digestion were ligated into the pUC-RNAi vector (Newpro Biotechnology, V015195) using the T4 DNA Ligase kit (Promega, CAT: M1801). The ligation product was transformed into E. coli DH5α, and the plasmids of positive clones were extracted using the plasmid extraction kit (Omega, CAT: D6942) to obtain the intermediate vector pUC-RNAi-CsRHY1A of the CsRHY1A interference fragment.

[0038] The intermediate vector pUC-RNAi-CsRHY1A and the pLGNe vector (Baosai Biotechnology, pLGN-35s) were double-digested with KpnI and SalI, respectively. The digestion product containing the RNAi fragment on the intermediate vector pUC-RNAi-CsRHY1A was ligated into the pLGNe vector to construct the interference vector pLGNe-CsRHY1A-RNAi.

[0039] Example 3: Transformation and preparation of transgenic plants

[0040] The interference vector pLGNe-CsRHY1A-RNAi obtained in Example 2 was introduced into Agrobacterium tumefaciens EHA105 cells (Weidi Biotechnology) using electroporation. Specifically, 50 μL of pre-frozen Agrobacterium tumefaciens competent cells (EHA105) were thawed on ice; 2 μL of the plasmid pLGNe-CsRHY1A-RNAi was added to the competent cells, mixed thoroughly by pipetting, and placed on ice for 5 min; the mixture was then transferred to the bottom of a pre-dried electroporation cup, the cup was placed in the slot and adjusted to the correct position, and the electroporation device (MicroPulser) was inserted. Electroporator (1652100, BIO-RAD) set to "Agr" mode, press the electroporation button, and check the electroporation data to ensure successful electroporation. Add 1 mL of LB liquid medium to the electroporation cuvette, mix well with a pipette, transfer to a sterile centrifuge tube, and incubate at 260 rpm and 28°C for 60 min with shaking. After incubation, centrifuge the bacterial culture at 10000 rpm for 1 min, discard the supernatant, resuspend the bacterial cells in 100 μL of LB liquid medium, spread the resuspended cells, and incubate in the dark at 28°C for 2 days. After the colonies have grown, use primers CsRHY1A-RNAi-F and CsRHY1A-RNAi-R to perform PCR verification on single colonies to obtain transgenic Agrobacterium tumefaciens. The PCR verification reaction conditions were: 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.

[0041] Transgenic Agrobacterium tumefaciens containing the CsRHY1A interference vector was streaked onto LB solid medium containing 50 mg / L kanamycin. Single colonies were picked and inoculated into 25 mL of LB liquid medium containing the same antibiotic, and cultured overnight at 28°C with shaking. The bacterial suspension 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 suspension was resuspended in MS liquid medium at pH 5.4 to obtain the transgenic Agrobacterium tumefaciens bacterial suspension.

[0042] Wash fresh Late Orange fruits, surface sterilize with 70% alcohol, and extract seeds under aseptic conditions. Peel off the seed coat and germinate on seed germination medium. Incubate in the dark at 28°C for 2 weeks, then incubate under 16h light / 8h dark conditions for 1 week. Under aseptic conditions, cut the hypocotyl of the germinating seedlings into 1cm stem segments.

[0043] The seed germination medium consisted of MS medium containing 30 g / L sucrose and 2.5 g / L gelrite, with a pH of 5.8.

[0044] After soaking the epicotyl stem segments of Late Jincheng orange in transgenic Agrobacterium tumefaciens solution for 13 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 two weeks. Then, GUS staining was used for identification to confirm the acquisition of transgenic seedlings.

[0045] The co-culture medium consisted of: MS (Murashige and Skoog medium) + 2 mg / L BA (6-Benzylaminopurine) + 0.5 mg / L IAA (Indole-3-Acetic Acid) + 1 mg / L 2,4-D (2,4-Dichlorophenoxyacetic acid) + 100 μmol AS (Acetosyringone) + 30 g / L sucrose + 2.5 g / L agar, with a pH of 5.8.

[0046] The selection medium consisted of: MS (Murashige and Skoog medium) + 2 mg / L BA (6-Benzylaminopurine) + 0.5 mg / L IAA (Indole-3-Acetic Acid) + 500 mg / L Cef (Cefotaxime Sodium) + 50 mg / L Kan (Kanamycin) + 30 g / L sucrose + 2.5 g / L agar, with a pH of 5.8.

[0047] When the transgenic seedlings grow to more than 1cm, they are cut off and grafted onto the Late Jin Orange seedlings in sterile test tubes and cultured in a seedling culture medium; when the seedlings grow to about 5cm, they are grafted onto the Late Jin Orange seedlings and cultured in a greenhouse at 28℃.

[0048] The seedling culture medium consisted of MS (Murashige and Skoog medium) + 30 g / L sucrose, with a pH of 5.8.

[0049] Transgenic plants (RNAi-CSRHY1A-1, RNAi-CSRHY1A-2, RNAi-CSRHY1A-3) obtained through culture were randomly selected and compared with late-maturing orange seedlings (WT). Their phenotypes are as follows: Figure 2 As shown, by Figure 2 It can be seen that the three transgenic plants showed no obvious abnormalities in phenotype, appearance, or growth.

[0050] Healthy leaves from transgenic plants and common late-maturing orange seedlings were cut into 7mm diameter leaf discs and subjected to GUS histochemical staining for 24 hours. The results are as follows: Figure 3 As shown, by Figure 3 It can be seen that the leaf discs of the cultured transgenic plant (RNAi-CSRHY1A-1 / 2 / 3) show blue edges, while the leaf discs of the WT plant do not show color, proving that the transformation was successful.

[0051] Example 4: PCR identification of transgenic plants

[0052] 100 mg of leaves from the three transgenic plants prepared in Example 3 and the Late Jin Orange seedlings were used to extract genomic DNA using a DNA extraction kit (Adley, CAT: DN15). PCR was then performed to detect the integration of RNAi fragments into the Late Jin Orange genome. The detection primers were ID-CsRHY1A-F (SEQ ID NO: 4) and ID-CsRHY1A-R (SEQ ID NO: 5). PCR reaction conditions were: 94℃ for 3 min; 94℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, 30 cycles; 72℃ for 10 min.

[0053] The results are as follows Figure 4 As shown, by Figure 4 It can be seen that the PCR product of the transgenic plant (RNAi-CSRHY1A-1 / 2 / 3) has a clear electrophoretic band at 636bp, while the PCR product of the late-maturing orange seedling has no electrophoretic band at 636bp.

[0054] Example 5: qRT-PCR analysis of transgenic plants

[0055] 100 mg of leaves from the transgenic plant prepared in Example 3 were used to extract total RNA from the leaves using an RNA extraction kit (Adley, CAT No: RN09). cDNA was synthesized using a reverse transcription kit PrimeScript RT Master Mix (TaKaRa, CAT: RR036A). The expression level of the target gene was detected using qRT-PCR. The qRT-PCR validation program was as follows: 95℃ for 3 min, 94℃ for 10 s; 40 cycles: 56℃ for 10 s, 72℃ for 10 s; 72℃ for 10 min. The detection primers were RT-CsRHY1A-F as shown in SEQ ID NO: 6 and RT-CsRHY1A-R as shown in SEQ ID NO: 7.

[0056] Use 2 -△△CtThe relative expression level of the CsRHY1A gene in transgenic plants was calculated as follows: The water-treated sample was defined as the reference factor, with its CsRHY1A expression level set at 1. The fold increase in gene expression relative to the reference factor in the transgenic plants was then calculated as 2. -△△Ct Its relative expression level. The results are as follows: Figure 5 As shown, by Figure 5 It can be seen that the CsRHY1A gene is significantly suppressed in transgenic plants compared with wild-type plants.

[0057] Example 6: Evaluation of resistance in transgenic plants

[0058] Mature leaves from the three transgenic plants prepared in Example 3 and the late-maturing orange seedlings were washed, disinfected with 75% alcohol, and rinsed with sterile water. They were then placed in a clean bench. Needle punctures were performed centered on the leaf veins, and 1 μL (1 × 10⁻⁶) of ulcer bacteria solution (preserved at Southwest University) was pipetted into each puncture site. 5 CFU / mL); cultured in a 28℃ constant temperature and light incubator (16h light / 8h dark); photographs were taken 10 days after leaf inoculation with the fungus, and the symptoms of the leaves 10 days after inoculation with the causal agent of leaf canker are shown in the image. Figure 6 As shown, by Figure 6 It can be seen that 10 days after inoculation with the ulcer pathogen, the transgenic plants (RNAi-CSRHY1A-1 / 2 / 3) and the late-maturing orange seedlings (WT) all developed the disease to varying degrees in the inoculated plants, and the size of the lesions varied.

[0059] ImageJ V1.47 software was used to count the area of ​​lesions, and the condition was divided into grades 0 to 7 according to the lesion area, where R is the lesion area, grade 0 (R≤0.25mm). 2 Level 1 (0.25mm) 2 <R≤0.5mm 2 Level 2 (0.5mm) 2 <R≤0.75mm 2 Level 3 (0.75mm) 2 <R≤1mm 2 Level 4 (1.0mm) 2 <R≤1.25mm 2 Level 5 (1.25mm) 2 <R≤1.5mm 2 Level 6 (1.5mm) 2 <R≤1.75mm 2 Level 7 (R > 1.75mm) 2 The disease index is calculated according to the formula: .

[0060] The leaf lesion area and disease index were statistically analyzed. The results of the leaf lesion area statistics are as follows: Figure 7 As shown, the statistical results of the leaf disease index are as follows: Figure 8 As shown, by Figure 7 and Figure 8 It was found that the lesion area and disease index on the leaves of the transgenic plants (RNAi-CSRHY1A-1 / 2 / 3) were significantly smaller than those of the late-ripening orange seedlings (WT). The lesion area was reduced to 85% of that of the late-ripening orange seedlings, and the disease index was 82% of that of the late-ripening orange seedlings. Therefore, CsRHY1A interference can greatly improve the resistance of citrus to citrus canker.

[0061] In summary, interference with CsRHY1A can significantly reduce the lesion area of ​​citrus canker and alleviate the severity of the disease. This gene can be used independently for molecular breeding of disease resistance, or it can be used in conjunction with other disease resistance or susceptibility genes for molecular breeding of citrus canker resistance.

Claims

1. A method for improving resistance to citrus canker in late-ripening oranges using CsRHY1A interference, characterized in that, Includes the following steps: S1: Cloning the RNAi fragment of the CsRHY1A encoding gene of Late Orange as shown in SEQ ID NO:

1. The specific steps are as follows: extract total RNA from Late Orange, reverse transcribe it into cDNA, and amplify the cDNA by PCR to obtain the RNAi fragment of the CsRHY1A encoding gene. The primers used for PCR amplification were CsRHY1A-RNAi-F as shown in SEQ ID NO: 2 and CsRHY1A-RNAi-R as shown in SEQ ID NO: 3; S2: Construct the CsRHY1A interference vector using cloned RNAi fragments; S3: The constructed CsRHY1A interference vector was transformed into Wanjin orange to obtain transgenic Wanjin orange plants resistant to Wanjin orange canker disease.

2. The method for improving resistance to citrus canker in late-ripening oranges using CsRHY1A interference according to claim 1, characterized in that, The specific steps for constructing the CsRHY1A interference vector in step S2 are as follows: The PCR products obtained from S1 were divided into two groups. One group was double-digested with AscI and SwaI, and the other group was double-digested with BamHI and SalI. The two fragments recovered from the digestion were ligated into the pUC-RNAi vector to obtain the intermediate vector pUC-RNAi-CsRHY1A. The intermediate vectors pUC-RNAi-CsRHY1A and pLGNe were double-digested with KpnI and SalI, respectively. The digestion product containing the RNAi fragment on the intermediate vector pUC-RNAi-CsRHY1A was ligated into the pLGNe vector to obtain the CsRHY1A interference vector pLGNe-CsRHY1A-RNAi.

3. The method for improving resistance to citrus canker in late-ripening oranges using CsRHY1A interference according to claim 1, characterized in that, The specific steps for converting the CsRHY1A interference vector to Late Orange in step S3 are as follows: The CsRHY1A interference vector was transformed into Agrobacterium tumefaciens by electroporation, and then Agrobacterium tumefaciens was used to transform explants of Citrus aurantium. After genetic transformation, the explant cells were cultured in vitro, identified by staining, grafted, verified by PCR and qRT-PCR to obtain transgenic plants.

4. The method for improving resistance to citrus canker in late-ripening oranges using CsRHY1A interference according to claim 3, characterized in that, For PCR verification, the primers used are ID-CsRHY1A-F as shown in SEQ ID NO:4 and ID-CsRHY1A-R as shown in SEQ ID NO:

5.

5. The method for improving resistance to citrus canker in late-ripening oranges using CsRHY1A interference according to claim 4, characterized in that, The PCR amplification program was as follows: 3 min at 94℃; 30 cycles: 30 s at 94℃, 30 s at 58℃, 30 s at 72℃; 10 min at 72℃.

6. The method for improving resistance to citrus canker in late-ripening oranges using CsRHY1A interference according to claim 3, characterized in that, For qRT-PCR validation, the primers used are RT-CsRHY1A-F as shown in SEQ ID NO: 6 and RT-CsRHY1A-R as shown in SEQ ID NO:

7.

7. The method for improving resistance to citrus canker in late-ripening oranges using CsRHY1A interference according to claim 6, characterized in that, The qRT-PCR validation program was as follows: 3 min at 95℃, 10 s at 94℃; 40 cycles: 10 s at 56℃, 10 s at 72℃; 10 min at 72℃.

8. A method for breeding late-ripening orange resistant plants to citrus canker, characterized in that, Transgenic Late Orange plants obtained by the method described in any one of claims 1 to 7 are used as parents to cultivate offspring with stable inheritance.

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