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Mutant plasmid combination resistant to both cucumber mosaic virus and potato virus X and application of mutant plasmid combination

A cucumber mosaic virus and mutant technology, applied in the fields of plant virology and molecular biology, can solve problems affecting vaccine safety, mutation repair, length limitation of exogenous virus fragments, etc., achieve superior cross-protection effect and improve stability Sexuality, the effect of improving the control effect

Active Publication Date: 2021-08-24
SHANDONG AGRICULTURAL UNIVERSITY
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  • Abstract
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  • Claims
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AI Technical Summary

Problems solved by technology

[0006] A bivalent attenuated vaccine against cucumber mosaic virus and potato X virus has been developed in the previous research, but due to the limitation of the size of the virus on its own packaging, when constructing a multivalent attenuated vaccine, it has the ability to insert foreign virus fragments. length limit
In the bivalent attenuated vaccine against both cucumber mosaic virus and potato virus X constructed earlier, the length of the inserted potato virus X fragment is 200 bp; although it can play the role of cross protection, some 200 bp insertions have mutation repair Risks that affect the safety of vaccines

Method used

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  • Mutant plasmid combination resistant to both cucumber mosaic virus and potato virus X and application of mutant plasmid combination
  • Mutant plasmid combination resistant to both cucumber mosaic virus and potato virus X and application of mutant plasmid combination
  • Mutant plasmid combination resistant to both cucumber mosaic virus and potato virus X and application of mutant plasmid combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1: Construction of Mutant Plasmids

[0037] mutant plasmid pCC F R2-2bPTII-PX 1967-2066 , pCC F R2-2bPTII-PX 2067-2166 , pCC F R2-2bPTII-PX 5890-5989 and pCC F R2-2bPTII-PX 5990-6089 Build process like figure 1 As shown, the details are as follows:

[0038] 1. Cloning of partial fragments of PVX:

[0039] According to the complete sequences of PVX of 5 different strains published in GenBank (Accession No.: EU571480, EF423572, AB056718, KM659859, JF430080), use DNAMAN software for sequence alignment, find the conserved region of the sequence and design primer PVX-BamHI-1967 -F / PVX-SmaI-2066-R, PVX-BamHI-2067-F / PVX-SmaI-2166-R, PVX-BamHI-5890-F / PVX-SmaI-5989-R, and PVX-BamHI-5990-F / PVX-SmaI-6089-R (see Table 1 for details).

[0040] Table 1: Primers used in the present invention

[0041]

[0042] Total RNA was extracted from PVX-infected tobacco plants, and 100 bp fragments corresponding to 1967-2066, 2067-2166, 5890-5989 and 5990-6089 of the PVX g...

Embodiment 2

[0053] Example 2: Biological Effects of Plasmid Vectors

[0054] Agrobacterium infiltration infecting Nicotiana benthamiana: First, wild-type CMV Fny RNA1, pCC F R2-2bPTII-PX 1967-2066 , pCC F R2-2bPTII-PX 2067-2166 , pCC F R2-2bPTII-PX 5890-5989 , pCC F R2-2bPTII-PX 5990-6089, wild-type CMV Fny Transform the competent cells of Agrobacterium GV3101 with the plasmid of RNA3, smear the LB plates with 50 μg / ml kanamycin and 100 μg / ml rifampicin, and culture at 28°C for 48 hours, pick a single spot for colony PCR verification, A single spot was picked and cultured in 2ml LB medium (containing 50μg / ml kanamycin and 100μg / ml rifampicin) at 28°C and 200rpm for 24h.

[0055] Induction: take 100 μL containing CMV Fny RNA1, pCC F R2-2bPTII-PX 1967-2066 , pCC F R2-2bPTII-PX 2067-2166 , pCC F R2-2bPTII-PX 5890-5989 , pCC F R2-2bPTII-PX 5990-6089 、CMV Fny Add RNA3 bacteria solution to 5ml LB culture solution (containing 50μg / ml kanamycin, 100μg / ml rifampicin), and cultu...

Embodiment 3

[0068] Embodiment 3: the cross protection effect test of plasmid vector

[0069] Utilize the method in embodiment 2, inoculate CMV respectively Fny RNA1 / RNA2-100 Type I / RNA3, CMV Fny RNA1 / RNA2-100 type II / RNA3, CMV Fny RNA1 / RNA2-100 type III / RNA3 and CMV Fny RNA1 / RNA2-100 type IV / RNA3; two mixed CMV Fny RNA1 / RNA2-100 type I+100 type III / RNA3, CMV Fny RNA1 / RNA2-100 type I+100 type IV / RNA3, CMV Fny RNA1 / RNA2 - 100 type II + 100 type III / RNA3 and CMV Fny RNA1 / RNA2 - 100 type II + 100 type IV / RNA3; three-mix CMV FnyRNA1 / RNA2-100 type I+100 type II+100 type III / RNA3, CMV Fny RNA1 / RNA2-100 type I+100 type II+100 type IV / RNA3, CMV Fny RNA1 / RNA2-100 type I+100 type III+100 type IV / RNA3, CMV Fny RNA1 / RNA2 - 100 type II + 100 type III + 100 type IV / RNA3 and four mixed CMV Fny RNA1 / 100 type I+RNA2-100 type II+100 type III+100 type IV / RNA3 for cross protection test.

[0070] Seven days after inoculation of the attenuated mutant, the plants pre-inoculated with the attenuated m...

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Abstract

The invention discloses a mutant plasmid combination resistant to both cucumber mosaic virus and potato virus X. The mutant plasmid combination is prepared from plasmids pCCFR2-2bPTII-PX1967-2066, pCCFR2-2bPTII-PX2067-2166, pCCFR2-2bPTII-PX5890-5989 and pCCFR2-2bPTII-PX5990-6089 which contain a CMVFny isolate mutant RNA (Ribonucleic Acid) 2 according to an equal volume ratio. According to the invention, four mutant plasmids capable of resisting the cucumber mosaic virus and the potato virus X are combined for use and are mixed and inoculated with plasmids of RNA (Ribonucleic Acid) 1 and RNA 3 containing CMVFny isolate, so that the control effect on the cucumber mosaic virus and the potato virus X can be obviously improved.

Description

technical field [0001] The invention relates to the technical fields of plant virology and molecular biology, in particular to a mutant plasmid combination resistant to cucumber mosaic virus and potato X virus and its application. Background technique [0002] Viral diseases are the second largest type of plant diseases after fungal diseases, causing varying degrees of damage to most crops. The prevention and control of plant viral diseases is a research hotspot and difficult issue. Cucumber mosaic virus (CMV) is one of the plant viruses with the widest host range known at present, which can infect more than 1200 kinds of plants in more than 100 families, causing a variety of plants with important economic value (crops, horticulture, etc.) Crops, etc.) appear symptoms such as yellowing, dwarfing, and deformity, which seriously affect their yield and quality (Mochizuki et al., 2012). Potato virus X (PVX) has a wide range of hosts and can infect 240 species of plants in 16 fa...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/40C12N1/21A01H5/00A01H6/82
CPCC12N15/8283C07K14/005C12N2770/14022C12N2770/14034C12N2770/00022C12N2770/00034
Inventor 原雪峰姜文筱刘珊珊于成明
Owner SHANDONG AGRICULTURAL UNIVERSITY
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