Application of ClBeclin1 gene in improving resistance of plant to citrus yellow vein clearing virus

Abstract

The invention relates to application of a ClBeclin1 gene to improvement of citrus yellow vein clearing virus resistance of a plant and a method for improving citrus yellow vein clearing virus resistance of the plant, and the method comprises a step of overexpressing the ClBeclin1 gene in the plant. According to the method for improving the citrus yellow vein clearing disease resistance based on ClBeclin1 overexpression, the citrus ClBeclin1 coding sequence is cloned, an overexpression vector is constructed, then a citrus branch is converted to obtain a transgenic hairy root, the virus infection time of the obtained transgenic material is delayed, the virus titer is reduced, and the Citrus yellow vein clearing disease resistance is improved. The titer of the yellow vein clearing virus can be reduced to 52.08% of that of an existing citrus material, and the attack degree of the yellow vein clearing disease can be remarkably relieved. The technical scheme provided by the invention is a potential biological engineering technology for improving the citrus yellow vein clearing disease resistance, and has a great value for citrus yellow vein clearing disease resistance molecular breeding.

Description

Technical Field

[0001] The present invention relates to the field of molecular breeding of fruit trees, and more particularly to the application of ClBeclin1 gene in improving the resistance of plants to citrus yellow vein virus. Background Art

[0002] Citrus yellow vein clearing virus (CYVCV) caused citrus yellow vein clearing disease was first discovered in Pakistan in 1988. It has now spread to major lemon producing countries such as Pakistan, India, Turkey, and Iran. Since the first discovery of citrus yellow vein clearing disease in Ruili, Yunnan in 2009, the disease has occurred in all major citrus producing areas in my country. Lemons and broad-skinned citrus are the most susceptible types, with a production reduction of more than 50% in severe cases. In recent years, nearly one million diseased lemon trees have been cut down in Anyue, Sichuan alone due to infection with citrus yellow vein clearing disease. The area of ​​​​yellow vein clearing disease in Tongnan, Wanzhou and other lemon-dominant counties in Chongqing has also exceeded thousands of acres, and the area of ​​​​yellow vein clearing disease is still expanding. At present, there is no drug that can cure citrus yellow vein clearing disease, and there is a lack of lemon varieties that are resistant to yellow vein clearing disease.

[0003] The long hybrid breeding cycle leads to low breeding efficiency, especially for woody plants, where traditional breeding methods cannot keep up with the speed of citrus diseases caused by environmental changes. With the rise of molecular biology, people began to study the pathogens themselves and the plant's disease resistance response from the perspective of disease resistance genetic engineering. Summary of the invention

[0004] In order to solve the above problems, the present invention provides the use of ClBeclin1 gene in improving the resistance of plants to citrus yellow vein virus.

[0005] In a specific embodiment, the amino acid sequence encoded by the ClBeclin1 gene is shown in SEQ ID NO:2.

[0006] In a specific embodiment, the sequence of the ClBeclin1 gene is as shown in SEQ ID NO:1.

[0007] The present invention also provides a method for improving the resistance of plants to citrus yellow vein virus, comprising the step of overexpressing the ClBeclin1 gene in the plants.

[0008] In a specific embodiment, the amino acid sequence encoded by the ClBeclin1 gene is shown in SEQ ID NO:2.

[0009] In a specific embodiment, the sequence of the ClBeclin1 gene is as shown in SEQ ID NO:1.

[0010] In a specific embodiment, the shoots of the plant are infected with Agrobacterium tumefaciens containing a ClBeclin1 gene overexpression vector, so that the plant produces hairy roots overexpressing the ClBeclin1 gene.

[0011] In a specific embodiment, the plant is citrus.

[0012] The present invention proposes a method for improving resistance to citrus yellow vein disease based on ClBeclin1 overexpression. By cloning the citrus ClBeclin1 coding sequence, constructing an overexpression vector, and then transforming citrus branches to obtain transgenic hairy roots, the obtained transgenic material has delayed virus infection time and reduced virus titer. The yellow vein virus titer can be reduced to 52.08% of the existing citrus material, which can significantly reduce the incidence of yellow vein disease. The technical solution of the present invention is a potential bioengineering technology for improving resistance to citrus yellow vein disease, which has great value for molecular breeding of citrus resistance to yellow vein disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 The bioinformatics characteristics of the ClBeclin1 gene of the present invention are as follows: A is a schematic diagram of the structure of the citrus ClBeclin1 gene.

[0014] Figure 2 The electrophoresis diagram of PCR amplification of ClBeclin1 gene clone of the present invention: CDS represents ClBeclin1 coding sequence; M represents DNA molecular weight standard, the same below.

[0015] Figure 3 The diagram is a structure diagram of the ClBeclin1 plant overexpression vector of the present invention: GUS represents the β-glucosidase gene; CaMV 35S represents the plant constitutive promoter derived from cauliflower mosaic virus; NOS represents the opine synthase gene terminator.

[0016] Figure 4 This is a visualization and identification diagram of transgenic hairy roots overexpressing ClBeclin1 in the present invention.

[0017] Figure 5 This is a PCR identification diagram of the transgenic material of the present invention. + is a positive control; - is a negative control; pNmGFPer:00 is a material with an empty vector; pNmGFPer-ClBeclin1 is a material with overexpressed transgenic hairy roots, the same below.

[0018] Figure 6 This is an analysis diagram of the ClBeclin1 expression level in the transgenic material of the present invention: * indicates a significant difference compared with the control (P=0.05); **** indicates an extremely significant difference compared with the control (P<0.0001), the same below.

[0019] Figure 7 The present invention is an analysis of CYVCV mRNA expression in transgenic hairy root materials that overexpress ClBeclin1 after being infected with yellow vein disease for 15 days.

[0020] Figure 8 The present invention is an analysis of the accumulation of CYVCV CP protein in transgenic hairy root materials that overexpress ClBeclin1 after being infected with yellow vein disease for 15 days. DETAILED DESCRIPTION

[0021] The principles and features of the present invention are described below in conjunction with the accompanying drawings. The examples given are only used to explain the present invention and are not used to limit the scope of the present invention.

[0022] 1. Cloning of the Citrus ClBeclin1 gene

[0023] The citrus ClBeclin1 gene was obtained to encode 515 amino acids, with CC and BARA functional domains at the C-terminus ( Figure 1 ). The CDS sequence of ClBeclin1 gene is shown in SEQ ID NO: 1, and the encoded amino acid is shown in SEQ ID NO: 2.

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

[0025] The ClBeclin1 coding sequence was amplified from citrus cDNA, and the fragment length was 1551 bp ( Figure 2 ), the amplified DNA fragment was sequenced and compared to determine the coding sequence of the citrus ClBeclin1 gene. Under ultraviolet light, a clean blade was used to cut the agarose gel block containing the target fragment, and a kit (BioFlux, CAT: BSC02M1) was used to recover the DNA fragment.

[0026] PCR amplification program: 98°C, 3 min; 98°C, 30 s, 50°C, 30 s, 72°C, 2 min, 29 cycles; extension at 72°C for 10 min.

[0027] 2. Construction of ClBeclin1 overexpression vector and transformation of Agrobacterium

[0028] The homology arm DNA fragment containing KpnⅠ and BamHI restriction sites encoding ClBeclin1 and the overexpression vector pNmGFPer double-digested with KpnⅠ and BamHI and containing the same homology arms were connected at 37°C for 30 minutes. The connection was carried out using ClonExpress II One Step Cloning Kit (Vazyme, CAT: C112). The ligation product was transformed into Escherichia coli DH5α, and the plasmid of the positive clone was 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 the root cancer agrobacterium K599 by heat shock method. The method is as follows: take the frozen Agrobacterium competent cells K599 (50μL) in advance and melt on ice; add 2μL of the plasmid of the overexpression vector to the competent cells, blow and mix, and place on ice for 5min, liquid nitrogen for 5min, 37℃ metal bath for 5min, and ice for 5min. Then add 700μL of TY medium without antibiotics, shake and culture at 260r / min, 28℃ shaker for 2h; centrifuge the bacterial solution at 5000r / min for 2min, discard the supernatant (about 100μL of resuspended bacteria), resuspend and spread on the TY plate medium with streptomycin (strep) and kanamycin sulfate (kan), and culture in the dark at 28℃ for 2 days; after the plaque grows, the single colony is verified by PCR to obtain positive transformants.

[0030] 3. Overexpression of ClBeclin1

[0031] Take 500 μL of Agrobacterium containing pNmGFPer-ClBeclin1 plasmid and add it to 200 mL of liquid TY medium (containing kanamycin and streptomycin) and culture at 28°C and 200 r / min until OD 600 =0.6; then centrifuge at 6000r / s and resuspend with inoculum to OD 600 = 0.8, vacuum infiltrated citrus branches at 0.1 MPa for 30 min, then transferred to vermiculite supplemented with Hoagland nutrient solution and water, and cultured at 26°C with a 16-h photoperiod. After 60 dpi, the transgenic materials were first visually identified using ultraviolet light ( Figure 4 ).

[0032] 100 mg of transgenic material leaves were used to extract genomic DNA using a DNA extraction kit (Adlai, CAT: DN15), and PCR amplification was used to identify positive transformed materials. A 524 bp amplified fragment was obtained from positive plants, while no amplification was obtained from pNmGFPer:00 plants; a 508 bp GFP fragment was obtained from all transformed plants ( Figure 5 ).

[0033] Total RNA (Adlai, CAT No: RN09) was extracted from transgenic materials, and cDNA was synthesized using the reverse transcription kit PrimeScriptRT Master Mix (TaKaRa, CAT No: RR036A). The expression level of the target gene was detected by qRT-PCR. The detection primers were RT-ClBeclin1-F and RT-ClBeclin1-. -△△Ct The relative expression of ClBeclin1 gene in transgenic materials was calculated by the following method: the sample transformed with pNmGFPer:00 empty vector plants was defined as the reference factor, that is, its ClBeclin1 expression level was 1, and then the multiple of gene expression in transgenic materials relative to the reference factor was calculated. -△△Ct , which is the relative expression level. The results showed that the ClBeclin1 gene was expressed at a higher level in transgenic materials than in wild-type plants (up to 5 times that of the control) ( Figure 6 ).

[0034] 4. Evaluation of resistance to yellow vein disease in ClBeclin1 overexpressing materials

[0035] The obtained pNmGFPer-ClBeclin1 transgenic hairy roots were inoculated with crude particles of yellow vein virus by slash, and RNA and protein were extracted 15 days after inoculation to detect the accumulation level of CYVCV. The results showed that 15 days after inoculation with yellow vein pathogen, both the overexpression plants and the pNmGFPer:00 empty-loaded plants could be detected in the plants inoculated with yellow vein pathogen. qPCR and Western Blot analysis found that the CYVCV virus titer of the transgenic material was significantly lower than that of the pNmGFPer:00 empty-loaded plant control, which was reduced to 52.08% ( Figure 7 ) and 83.04% ( Figure 8 ). It can be seen that ClBeclin1 overexpression can significantly reduce the lesion titer of citrus yellow vein disease.

[0036] In summary, the present invention can reduce the lesion titer of citrus yellow vein disease by overexpressing the ClBeclin1 gene. The ClBeclin1 gene provided in the embodiment of the present invention can be overexpressed by a variety of techniques and used for molecular breeding of resistance to yellow vein disease, or can be used together with other disease resistance or disease susceptibility genes to coordinate molecular breeding of citrus resistance to yellow vein disease, which has great application value in breeding of citrus resistance to yellow vein disease.

[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 principle of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of ClBeclin1 gene in improving plant resistance to citrus yellow vein virus.

2. The use according to claim 1, characterized in that: The amino acid sequence encoded by the ClBeclin1 gene is shown in SEQ ID NO:

2.

3. The use according to claim 2, characterized in that: The sequence of the ClBeclin1 gene is shown in SEQ ID NO:

1.

4. A method for improving plant resistance to citrus yellow vein virus, characterized in that: The method comprises the step of overexpressing the ClBeclin1 gene in the plant.

5. The method according to claim 4, characterized in that The amino acid sequence encoded by the ClBeclin1 gene is shown in SEQ ID NO:

2.

6. The use according to claim 5, characterized in that: The sequence of the ClBeclin1 gene is shown in SEQ ID NO:

1.

7. The method according to claim 4, characterized in that: The branches of the plant are infected with Agrobacterium tumefaciens containing a ClBeclin1 gene overexpression vector, so that the plant produces hairy roots overexpressing the ClBeclin1 gene.

8. The method according to claim 4, characterized in that The plant is citrus.

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