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Virus-induced gene silencing vector, application thereof and citrus disease prevention and control method

A gene silencing and vector technology, applied in the field of agricultural biology, achieves the effects of environmental friendliness, low cost, and good canker disease prevention and control effects

Pending Publication Date: 2022-04-12
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Citrus yellow vein bright virus can be efficiently transmitted among citrus through citrus whitefly (Dialeurodes citri), spiraea aphid (Aphis spiraecola) and contaminated knives, which poses a major threat to the citrus industry. Virus

Method used

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  • Virus-induced gene silencing vector, application thereof and citrus disease prevention and control method
  • Virus-induced gene silencing vector, application thereof and citrus disease prevention and control method
  • Virus-induced gene silencing vector, application thereof and citrus disease prevention and control method

Examples

Experimental program
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Effect test

Embodiment 1

[0028] Embodiment 1: Construction of recombinant plasmid vector

[0029] The recombinant plasmid vectors pCLBV201-lob376, pCLBV201-CY297, pCLBV201-CY311, pCLBV201-CY310 and pCLBV201-CY313 were respectively constructed based on the plasmid vector pCLBV201 with citrus infectivity.

[0030] The plasmid vector pCLBV201 used in this example is a citrus infective clone constructed by the applicant of the present invention based on citrus leaf mottle virus, which has the DNA sequence shown in SEQ ID No.1.

[0031] Using LOB376F / LOB376R (see Table 1 for the sequence) as amplification primers and Eureka lemon cDNA as a template, the target gene fragment was obtained by PCR amplification using the high-fidelity kit PrimeStar. The target gene fragment is a partial fragment of the citrus lob gene, which has the DNA sequence shown in SEQ ID No.2. For the convenience of description, it will be referred to as the lob367 gene fragment hereinafter. The lob367 gene fragment was subjected to ...

Embodiment 2

[0048] Embodiment 2: Recombinant plasmid vector transforms Agrobacterium and inoculates citrus

[0049] The recombinant plasmid vectors constructed in Example 1 were respectively transformed into Agrobacterium, and then the transformed Agrobacterium was used to inoculate Eureka lemon.

[0050] 1. The recombinant plasmid is transformed into Agrobacterium, and the specific operation process is as follows:

[0051] 1) In the ultra-clean workbench, pre-cool the electroporation cup on ice for 5 minutes, and place Agrobacterium competent cells GV3101 on ice for 5 minutes;

[0052] 2) After the competent cells are fused, add 3uL of the recombinant plasmid vector, gently pipette and mix, then transfer to the electric shock cup, be careful not to generate air bubbles, and place on ice for 30-60min;

[0053] 3) Put the electroporation cup into the electroporation instrument, and adjust it to Agr mode electroshock transformation;

[0054] 4) After the electric shock is completed, imm...

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Abstract

The invention provides a virus-induced gene silencing vector, application thereof and a method for preventing and controlling citrus diseases. The gene silencing vector is constructed on the basis of infectious clone plasmids of the citrus leaf mottle virus. The gene silencing vector is a recombinant plasmid vector constructed by integrating a target gene segment into a CLBV infectious cloning plasmid pCLBV201, and the target gene segment is preferably and reversely connected between a CLBV genome cp gene terminator and 3 'UTR in the plasmid vector pCLBV201. The gene silencing vector of the targeted citrus lob gene provided by the invention blocks the combination of citrus canker pathogen and the target gene, can achieve a good canker prevention and control effect, and is low in cost and environment-friendly. The constructed gene silencing vector targeting different ORFs of the CYVCV genome can effectively inhibit transcription expression of CYVCV in plants, and then prevention and control of the citrus yellow vein disease are achieved.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a virus-induced gene silencing carrier and its application and a method for preventing and controlling citrus diseases. Background technique [0002] Citrus is one of the most widely grown fruits in the world. Citrus canker and citrus yellow vein disease are common citrus diseases. [0003] Citrus canker is a highly destructive bacterial disease caused by Xanthomonas citri ssp.citri (Xcc). Citrus leaves, branches, and fruits infected with the fungus have symptoms of canker lesions, resulting in tree vigor decline, leaf drop and fruit drop, which seriously affect the development of the citrus industry. At present, citrus canker mainly relies on a large number of pesticides such as copper preparations for prevention and control, resulting in increased planting costs for farmers and environmental pollution, which seriously restricts the healthy development of ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/29C12N15/40A01H5/00A01H6/78
Inventor 宋震马志敏宾羽许建建周常勇
Owner SOUTHWEST UNIV
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