Application of ClBeclin1 gene in improving resistance of plants to citrus tatter leaf phyllody-associated virus
By cloning the citrus ClBeclin1 gene and constructing an overexpression vector, citrus branches were transformed to produce hairy roots, thereby achieving gene overexpression. This solved the problem of insufficient resistance to Citrus Yellowing Vein Virus, significantly reduced the virus titer, and improved the disease resistance of citrus.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- SOUTHWEST UNIV
- Filing Date
- 2024-10-21
- Publication Date
- 2026-05-29
AI Technical Summary
Existing technologies are insufficient to effectively improve the resistance of citrus to Citrus Yellowing Veining Virus (CVV), and traditional breeding methods are inefficient and cannot meet the needs of disease outbreak speed.
By cloning the ClBeclin1 gene in citrus, an overexpression vector was constructed and transformed into citrus branches to produce hairy roots that overexpress the ClBeclin1 gene, thereby achieving gene overexpression to enhance disease resistance.
This study significantly reduced the titer of Citrus Yellowing Veining Virus, alleviated the severity of the disease, and improved the resistance of citrus to the virus, providing an efficient molecular breeding method.
Smart Images
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Abstract
Description
Technical Field
[0001] This invention relates to the field of molecular breeding of fruit trees, and more particularly to the application of the ClBeclin1 gene in improving plant resistance to Citrus Yellowing Veining Virus. Background Technology
[0002] Citrus yellow vein clearing virus (CYVCV), a disease caused by the citrus yellow vein clearing virus, was first discovered in Pakistan in 1988 and is now rampant 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 losses of over 50% in severe cases. 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 ClBeclin1 gene in improving plant resistance to Citrus Yellowing Veining Virus.
[0005] In one specific embodiment, the amino acid sequence encoded by the ClBeclin1 gene is shown in SEQ ID NO:2.
[0006] In one specific embodiment, the sequence of the ClBeclin1 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 ClBeclin1 gene in the plant.
[0008] In one specific embodiment, the amino acid sequence encoded by the ClBeclin1 gene is shown in SEQ ID NO:2.
[0009] In one specific embodiment, the sequence of the ClBeclin1 gene is shown in SEQ ID NO:1.
[0010] In one specific implementation, Agrobacterium tumefaciens containing a ClBeclin1 gene overexpression vector is used to infect the branches of the plant, causing the plant to produce hairy roots overexpressing the ClBeclin1 gene.
[0011] In one specific implementation, the plant is a citrus fruit.
[0012] This invention proposes a method to enhance resistance to Citrus yellow vein disease (Cyclocarya citrus) based on ClBeclin1 overexpression. The method involves cloning the ClBeclin1 coding sequence of citrus, constructing an overexpression vector, and then transforming citrus branches to obtain transgenic hairy roots. The resulting transgenic materials exhibit delayed virus infection time and reduced virus titer; the Cyclocarya citrus virus titer can be reduced to 52.08% of that in existing citrus materials, significantly alleviating the severity of Cyclocarya citrus disease. This invention represents a promising bioengineering technology for enhancing resistance to Cyclocarya citrus and has significant value for molecular breeding of citrus resistant to the disease. Attached Figure Description
[0013] Figure 1 Bioinformatics characteristics of the ClBeclin1 gene in this invention: A is a schematic diagram of the ClBeclin1 gene structure in citrus.
[0014] Figure 2 The image shows the PCR amplification electrophoresis diagram of the ClBeclin1 gene clone of this invention: CDS represents the ClBeclin1 coding sequence; M represents the DNA molecular weight standard, the same below.
[0015] Figure 3 The structural diagram of the ClBeclin1 plant overexpression 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.
[0016] Figure 4 This is a visual identification diagram of the hairy roots of the transgenic plants overexpressing ClBeclin1 in this invention.
[0017] Figure 5 This is a PCR identification diagram of the transgenic materials of this invention. + indicates a positive control; - indicates a negative control; pNmGFPer:00 indicates materials transfected with empty vector; pNmGFPer-ClBeclin1 indicates materials overexpressing transgenic hairy roots, and so on.
[0018] Figure 6 The following is a graph showing the expression level analysis of ClBeclin1 in the transgenic material of this invention: This indicates a significant difference compared to the control group (P=0.05). This indicates a highly significant difference compared to the control (P < 0.0001), and the same applies below.
[0019] Figure 7 This study analyzed the expression level of CYVCV mRNA in the transgenic hairy root material overexpressing ClBeclin1 15 days after infection with yellow vein disease, as per the present invention.
[0020] Figure 8 Analysis of CYVCV CP protein accumulation in transgenic hairy root material overexpressing ClBeclin1 15 days after infection with yellow vein disease. 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 ClBeclin1 gene
[0023] The citrus ClBeclin1 gene encodes 515 amino acids and contains CC and BARA functional domains at its C-terminus. Figure 1 The CDS sequence of the ClBeclin1 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 ClBeclin1 sequence was amplified from citrus cDNA, with a fragment length of 1551 bp. Figure 2 The amplified DNA fragment was sequenced and identified as the coding sequence of the citrus ClBeclin1 gene. Under UV light, an agarose gel block containing the target fragment was cut with a clean blade, and the DNA fragment was recovered using a kit (BioFlux, CAT: BSC02M1).
[0026] PCR amplification program: 98℃, 3 min; 98℃, 30 s, 50℃, 30 s, 72℃, 2 min, 29 cycles; extension at 72℃ for 10 min.
[0027] 2. Construction of ClBeclin1 overexpression vector and transformation of Agrobacterium tumefaciens
[0028] The ClBeclin1-encoding DNA fragment 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. Ligation was performed using the ClonExpress II One Step Cloning Kit (Vazyme, CAT: C112). The ligation product was transformed into E. coli DH5α, and plasmids from positive clones were extracted using a plasmid extraction kit (Omega, CAT: D6942) to obtain the ClBeclin1 overexpression vector pNmGFPer-ClBeclin1. Figure 3 ).
[0029] The constructed overexpression vector was introduced into Agrobacterium tumefaciens K599 using a heat shock method. The method was as follows: 50 μL of frozen Agrobacterium tumefaciens competent cells (K599) 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 TY 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 plated onto TY plates containing streptomycin and kanamycin sulfate, 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 of ClBeclin1
[0031] Add 500 μL of Agrobacterium tumefaciens culture containing the pNmGFPer-ClBeclin1 plasmid to 200 mL of liquid TY medium (containing kanamycin and streptomycin), and incubate at 28℃ and 200 r / min until OD. 600 = 0.6; then centrifuge at 6000 r / s and resuspend in the inoculum to OD. 600 = 0.8, citrus branches were vacuum-soaked at 0.1 MPa for 30 min, then transferred to vermiculite supplemented with Hoagland's nutrient solution and water, and cultured at 26℃ for 16 h photoperiod. After 60 dpi, the transgenic materials were first visually identified using ultraviolet light. Figure 4 ).
[0032] Genomic DNA was extracted from 100 mg of leaves of transgenic material using a DNA extraction kit (Adley, CAT: DN15). Positive transformation materials were identified by PCR amplification. Positive plants yielded a 524 bp amplified fragment, while pNmGFPer:00 plants showed no amplification; all transformed plants yielded a 508 bp GFP fragment (…). Figure 5 ).
[0033] 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-ClBeclin1-F and RT-ClBeclin1-. 2 -△△Ct The relative expression level of the ClBeclin1 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 ClBeclin1 expression level was 1. Then, the fold increase in gene expression relative to the reference factor in the transgenic material was calculated as 2. -△△Ct The relative expression level was calculated. Results showed that the ClBeclin1 gene was expressed at a high level in transgenic materials compared to wild-type plants (up to more than 5 times that of the control). Figure 6 ).
[0034] 4. Evaluation of resistance to yellow vein disease in ClBeclin1 overexpression materials
[0035] The obtained pNmGFPer-ClBeclin1 transgenic hairy roots were slash-inoculated with crude CYVCV particles. RNA and protein were extracted 15 days after inoculation to detect CYVCV accumulation levels. Results showed that CYVCV was detectable in both overexpressing plants and pNmGFPer:00 empty vector plants 15 days after inoculation. qPCR and Western blotting analyses revealed that the CYVCV virus titer in the transgenic material was significantly lower than that in the pNmGFPer:00 empty vector control, decreasing to 52.08% of the control. Figure 7 ) and 83.04% Figure 8 Therefore, it can be seen that ClBeclin1 overexpression can significantly reduce the lesion titer of citrus yellowing vein disease.
[0036] In summary, this invention reduces the lesion titer of citrus yellow vein disease by overexpressing the ClBeclin1 gene. The ClBeclin1 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.
Claims
1. Application of the ClBeclin1 gene in improving plant resistance to Citrus yellowing virus, wherein the amino acid sequence encoded by the ClBeclin1 gene is shown in SEQ ID NO:2, and the plant is citrus.
2. The application according to claim 1, characterized in that, The sequence of the ClBeclin1 gene is shown in SEQ ID NO:
1.
3. A method for improving plant resistance to Citrus Yellowing Veining Virus, characterized in that, The method includes the step of overexpressing the ClBeclin1 gene in the plant, the amino acid sequence of which is shown in SEQ ID NO:2, and the plant is a citrus.
4. The method according to claim 3, characterized in that, The sequence of the ClBeclin1 gene is shown in SEQ ID NO:
1.
5. The method according to claim 3, characterized in that, The plant branches were infected with Agrobacterium tumefaciens containing a ClBeclin1 gene overexpression vector, causing the plant to produce hairy roots overexpressing the ClBeclin1 gene.