Application of ClPsbO gene in improving plant resistance to citrus yellow leaf virus

Abstract

The present application relates to the field of plant antiviral molecular breeding, and particularly relates to application of ClPsbO gene in improving resistance of plants to citrus yellowing vein phasiavirus (CYVCV), and a method for improving resistance of plants to CYVCV, comprising the step of overexpressing ClPsbO gene in the plants.The present application proposes a method for improving resistance of plants to CYVCV based on overexpression of ClPsbO, which comprises the following steps: cloning a coding sequence of ClPsbO of citrus, constructing an overexpression vector, and then transforming a stem section of the citrus to obtain stable transgenic citrus, so that the virus titer of the obtained transgenic material is reduced, the CYVCV titer can be reduced to 37.27% of the existing citrus material, and the disease degree of the CYVCV can be significantly reduced.The technical scheme of the present application is a potential bioengineering technology for improving resistance of citrus to CYVCV, and has great value for molecular breeding of citrus against CYVCV.

Description

Technical Field

[0001] This invention relates to the field of molecular breeding of fruit trees, and more particularly to the application of the ClPsbO gene in improving the resistance of plants to Citrus Yellowing Veining Virus. Background Technology

[0002] Citrus yellow vein clearing virus (CYVCV) is the pathogen causing citrus yellow vein clearing disease. This disease can lead to symptoms such as yellowing of leaf veins, leaf curling, damage to chloroplast structure, and stunted growth in sensitive citrus varieties like lemons, affecting both yield and quality. It is currently prevalent in major lemon-producing countries such as Pakistan, India, Turkey, and Iran. In my country, since the disease was first discovered in Ruili, Yunnan Province in 2009, it has now occurred in all major citrus-producing areas, with sensitive varieties such as lemons and mandarins being the most severely affected, resulting in yield reductions of over 50% in severe cases. In recent years, nearly one million lemon trees have been cut down in Anyue County, Sichuan Province alone due to CYVCV infection. In Tongnan and Wanzhou, Chongqing, major lemon-producing counties, the affected area has exceeded several thousand acres, and the affected area continues to expand. Currently, there is no cure for citrus yellow vein clearing disease, and there is also a lack of lemon varieties resistant to it.

[0003] The long cycle of hybridization breeding leads to low breeding efficiency, especially for woody plants. Traditional breeding methods cannot keep up with the rate of occurrence of citrus diseases caused by environmental changes. With the rise of molecular biology, researchers have begun to study pathogens themselves and plant disease resistance responses through disease-resistant gene engineering. Summary of the Invention

[0004] To address the above problems, this invention provides the application of the ClPsbO gene in improving plant resistance to Citrus Yellowing Veining Virus.

[0005] In one specific embodiment, the amino acid sequence encoded by the ClPsbO gene is shown in SEQ ID NO:2.

[0006] In one specific implementation, the sequence of the ClPsbO gene is shown in SEQ ID NO:1.

[0007] The present invention also provides a method for improving the resistance of plants to Citrus Yellowing Vesmin Virus, comprising the step of overexpressing the ClPsbO gene in the plant.

[0008] In one specific embodiment, the amino acid sequence encoded by the ClPsbO gene is shown in SEQ ID NO:2.

[0009] In one specific implementation, the sequence of the ClPsbO gene is shown in SEQ ID NO:1.

[0010] In one specific implementation, Agrobacterium tumefaciens containing a ClPsbO gene overexpression vector is used to infect the stem segments of the plant, causing the plant to produce positive buds that overexpress the ClPsbO gene.

[0011] In one specific implementation, the plant is a citrus fruit.

[0012] This invention proposes a method for enhancing resistance to Citrus yellow vein disease (CYVD) based on ClPsbO overexpression. The method involves cloning the ClPsbO coding sequence of citrus, constructing an overexpression vector, and then transforming stem segments of citrus yellow flower seedlings to obtain transgenic buds. The resulting overexpressed transgenic material shows a reduction in CYVD virus titer to 37.27% of existing citrus materials, significantly alleviating the severity of CYVD. Furthermore, the function of ClPsbO against CYVD was confirmed through reverse silencing experiments. This invention represents a promising bioengineering technique for enhancing citrus yellow vein disease resistance and has significant value for molecular breeding of citrus CYVD-resistant varieties. Attached Figure Description

[0013] Figure 1 Bioinformatics characteristics of the ClPsbO gene in this invention: A is a schematic diagram of the citrus ClPsbO gene structure.

[0014] Figure 2 The image shows the PCR amplification electrophoresis diagram of the ClPsbO gene clone of this invention: ClPsbO represents the ClPsbO coding sequence; M represents the DNA molecular weight standard, the same below.

[0015] Figure 3 The structural diagram of the ClPsbO plant overexpression and RNAi silencing vector of this invention is as follows: GUS represents the β-glucosidase gene; CaMV 35S represents the plant constitutive promoter derived from cauliflower mosaic virus; NOS represents the crown gall synthase gene terminator; Intron: intron.

[0016] Figure 4 This is a visualization of the transgenic ClPsbO of the present invention.

[0017] Figure 5 The following is a graph showing the transcriptional level analysis of ClPsbO in the transgenic material of this invention: This indicates a significant difference compared to the control (P < 0.01). This indicates a highly significant difference compared to the control (P < 0.001), and the same applies below.

[0018] Figure 6This is a graph showing the abundance of ClPsbO protein in the transgenic material of this invention.

[0019] Figure 7 This study analyzed the CYVCV content in transgenic plants overexpressing ClPsbO 30 days after infection with yellow vein disease.

[0020] Figure 8 This study analyzed the CYVCV content in transgenic plants with silenced ClPsbO 30 days after infection with yellow vein disease, as per the present invention. Detailed Implementation

[0021] The principles and features of the present invention are described below with reference to the accompanying drawings. The examples given are only for explaining the present invention and are not intended to limit the scope of the present invention.

[0022] 1. Cloning of the citrus ClPsbO gene

[0023] The citrus ClPsbO gene, encoding 335 amino acids, contains the photosystem II oxygen-evolving enhancer protein 1 domain. Figure 1 The CDS sequence of the ClPsbO gene is shown in SEQ ID NO: 1, and the encoded amino acids are shown in SEQ ID NO: 2.

[0024] Total RNA was collected from citrus (Eureka lemon) leaves. RNA quality was verified by agarose gel electrophoresis, and its concentration was determined using a concentration meter. cDNA was synthesized using the PrimeScript RT Master Mix (TaKaRa, CAT: RR036A) reverse transcription kit.

[0025] A DNA fragment encoding the ClPsbO sequence was amplified from citrus cDNA, with a fragment length of 1005 bp. Figure 2 The amplified DNA fragment was sequenced and identified as the coding sequence of the citrus ClPsbO gene. Under UV light, an agarose gel block containing the target fragment was cut off with a clean blade, and the DNA fragment was recovered using a kit (BioFlux, CAT: BSC02M1).

[0026] PCR amplification program: 95℃, 5 min; 95℃, 15 s, 54℃, 5 s, 72℃, 40 s, 32 cycles; extension at 72℃ for 10 min.

[0027] 2. Construction and transformation of ClPsbO overexpression and silencing vectors into Agrobacterium.

[0028] The DNA fragment encoded by ClPsbO containing homologous arms of KpnI and BamHI restriction sites was ligated with the overexpression vector pNmGFPer, which was double-digested with KpnI and BamHI and contained the same homologous arms, at 37°C for 30 min. The forward DNA fragment encoded by ClPsbO containing homologous arms of SwaI and AscI restriction sites was ligated with the RNAi vector pNmGFPer-RNAi, which was double-digested with SwaI and AscI and contained the same homologous arms, at 37°C for 30 min. Ligation was performed using the ClonExpress II One Step Cloning Kit (Vazyme, CAT: C112). The ligation product was transformed into *E. coli* DH5α. Plasmids from positive clones were extracted using a plasmid extraction kit (Omega, CAT: D6942) to obtain the ClPsbO overexpression vector pNmGFPer-ClPsbO and an RNAi vector with the inserted forward fragment. Subsequently, the inverted DNA fragment encoding the ClPsbO containing homologous arms of BamHI and SalI restriction sites, and the RNAi vector containing the inserted forward fragment digested with BamHI and SalI and containing the same homologous arms, were ligated at 37°C for 30 min. Transformation and plasmid extraction were then performed according to the above steps. Figure 3 Finally, the overexpression vector pNmGFPer-ClPsbO and the silencing vector pNmGFPer-RNAi-ClPsbO were obtained.

[0029] The constructed vector was introduced into Agrobacterium tumefaciens EHA105 cells using a heat shock method. The method was as follows: 50 μL of frozen Agrobacterium tumefaciens competent cells (EHA105) were thawed on ice; 2 μL of the overexpression vector plasmid was added to the competent cells, and the mixture was thoroughly mixed by pipetting. The cells were then placed on ice for 5 min, in liquid nitrogen for 5 min, in a 37°C metal bath for 5 min, and on ice for 5 min. Then, 700 μL of antibiotic-free LB medium was added, and the cells were cultured at 260 r / min on a shaker at 28°C for 2 h. The bacterial culture was centrifuged at 5000 r / min for 2 min, and the supernatant was discarded (approximately 100 μL of the bacterial cells were resuspended). The resuspended cells were then plated onto LB agar plates containing kanamycin and rifampicin, and incubated in the dark at 28°C for 2 days. After colony growth, single colonies were verified by PCR to obtain positive transformants.

[0030] 3. Overexpression and silencing of ClPsbO

[0031] Preparation of test tubes or culture flasks: Take 41.4 g of MS (Coolaber, PM10121-307-1kg) culture medium powder. Bring the volume to 1 L, melt the explants in a microwave or induction cooker, dispense into test tubes, and autoclave (121℃, 15 min) for later use. Explant culture: Remove two seeds from each culture tube using a scalpel, removing the inner and outer seed coats. Incubate in the dark at 28℃ until approximately 7 cm thick. Then, transfer to a 28℃ environment with 10000 X light for 16 h followed by 8 h in the dark for 5-7 days, until greening occurs after 7 days. Add 500 μL of Agrobacterium tumefaciens culture containing pNmGFPer-ClPsbO and pNmGFPer-RNAi-ClPsbO plasmids to 200 mL of liquid LB medium (containing kanamycin and rifampin), and incubate at 28℃ and 200 r / min until... Then centrifuge at 6000 r / s and resuspend in MS liquid medium (pH=5.4) until... During the inoculum formation process, remove the roots and cotyledons in a clean bench, cut the explant stem segments (1-2 cm), and place them in the pretreatment solution (MS liquid medium, pH 5.8, containing 1 mg / L IAA and 2 mg / L 6-BA), and shake at 28°C / 100 rpm for at least 2 hours. Discard the pretreatment solution in the conical flask containing the stem segments, add the inoculum, and shake by hand for 10-15 minutes. Discard the inoculum, pour the stem segments into a 15 cm glass dish lined with 5 layers each of sterile filter paper and tissue paper, and dry the surface of the stem segments with sterile forceps. Transfer to co-medium (containing pH 5.8, 1 mg / L IAA, 1 mg / L IP, 1 mg / L 2,4-D, and 0.1 M / L AS): Transfer the above stem segments to the co-medium, arrange them neatly, and incubate in the dark at 26°C for 48 hours. Selection medium (containing pH 5.8, 500 mg / L Cef, 0.5 mg / L IAA, 2 mg / L 6-BA, 50 mg / L Kana): Transfer the above stem segments to the selection medium, arrange them neatly, with appropriate spacing (20-30 segments / plate, 12 cm plate), and incubate in the dark for one week. Then transfer to 28℃, light / 16 h, dark / 8 h for further incubation. Second selection medium: After 30-45 days, transfer the above stem segments to a new selection medium, separating sprouted and non-sprouted segments into separate plates, and incubate at 28℃, light / 16 h, dark / 8 h. After 30 dpi, visually identify the transgenic material using UV light. Subsequently, perform shoot tip micrografting (using filter paper bridges) to graft larger seedlings.

[0032] Total RNA was extracted from the transgenic material (Adelaide, CAT No: RN09), and cDNA was synthesized using the PrimeScript RT Master Mix reverse transcription kit (TaKaRa, CAT: RR036A). The expression level of the target gene was detected by qRT-PCR. The detection primers were RT-ClPsbO-F (SEQ ID No: 3) and RT-ClPsbO-R (SEQ ID No: 4). The relative expression level of the ClPsbO gene in transgenic materials was calculated as follows: A sample transgenic with the pNmGFPer:00 empty vector was defined as the reference factor, i.e., its ClPsbO expression level was 1. Then, the fold increase in gene expression relative to the reference factor in the transgenic material was calculated. The relative expression levels were calculated. Results showed that the ClPsbO gene was highly expressed in overexpressing transgenic materials compared to wild-type plants (more than twice that of the control), and lowly expressed in RNAi-silenced transgenic materials compared to wild-type plants (as low as 9% of the control). Figure 5 ).

[0033] Total protein was extracted from transgenic material using RIPA strong lysis buffer (Beyotime, CAT No: P0013B), and the ClPsbO protein content was detected using Abmart's PsbO antibody (Abmart, CAT No: PZ0024) at a dilution ratio of 1:2000.

[0034] 4. Evaluation of resistance to yellow vein disease in ClPsbO transgenic plants

[0035] The obtained transgenic lemons were slash-inoculated with crude CYVCV particles. RNA and protein were extracted 30 days after inoculation to detect CYVCV accumulation levels. Results showed that CYVCV was detectable in both transgenic and wild-type plants 30 days after inoculation. qPCR and Western blot analyses revealed that the CYVCV virus titer in the ClPsbO-overexpressing transgenic material was significantly lower than that in the wild type, decreasing to 37.27% and 53.50% of the wild type, respectively. Figure 7 Conversely, the CYVCV virus titer in the RNAi-silenced ClPsbO transgenic material was significantly higher than that in the wild type, increasing to 199.67% and 143.50% of the wild type, respectively. Figure 8 This demonstrates that ClPsbO overexpression can significantly reduce the viral titer of citrus yellowing vein disease.

[0036] In summary, this invention reduces the lesion titer of citrus yellow vein disease by overexpressing the ClPsbO gene. The ClPsbO gene provided in this invention can be overexpressed using various techniques for molecular breeding of citrus yellow vein disease resistance. It can also be used synergistically with other disease-resistant or susceptible genes for molecular breeding of citrus yellow vein disease resistance, demonstrating significant application value in citrus yellow vein disease resistance breeding.

[0037] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Application of the ClPsbO gene in improving plant resistance to Citrus Yellowing Veining Virus.

2. The application according to claim 1, characterized in that, The amino acid sequence encoded by the ClPsbO gene is shown in SEQ ID NO:

2.

3. The application according to claim 2, characterized in that, The sequence of the ClPsbO gene is shown in SEQ ID NO:

1.

4. A method for improving plant resistance to Citrus Yellowing Veining Virus, characterized in that, The step includes overexpressing the ClPsbO gene in the plant.

5. The method according to claim 4, characterized in that, The amino acid sequence encoded by the ClPsbO gene is shown in SEQ ID NO:

2.

6. The application according to claim 5, characterized in that, The sequence of the ClPsbO gene is shown in SEQ ID NO:

1.

7. The method according to claim 4, characterized in that, The stem segments of the plant were infected with Agrobacterium tumefaciens containing a ClPsbO gene overexpression vector, causing the plant to produce positive buds that overexpress the ClPsbO gene.

8. The method according to claim 4, characterized in that, The plant in question is a citrus (Citrus reticulata).

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