Tobacco PDS gene fragment-containing cucumber mosaic virus RNA2 attenuated mutant type plasmid vector and application thereof

A technology of cucumber mosaic virus and plasmid vector, applied in the fields of plant virology and molecular biology, can solve the problems of easy loss of resistance of disease-resistant varieties and long breeding cycle

Active Publication Date: 2018-09-04
SHANDONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For the prevention and treatment of plant virus diseases, the most effective method is the screening and cultivation of disease-resistant varieties, but it has the disadvantages of long breeding cycle and easy loss of resistance of disease-resistant varieties.

Method used

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  • Tobacco PDS gene fragment-containing cucumber mosaic virus RNA2 attenuated mutant type plasmid vector and application thereof
  • Tobacco PDS gene fragment-containing cucumber mosaic virus RNA2 attenuated mutant type plasmid vector and application thereof
  • Tobacco PDS gene fragment-containing cucumber mosaic virus RNA2 attenuated mutant type plasmid vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Example 1. Intermediate vector pCB301-CMV Fny2 -build of del2b:

[0040] According to CMV Fny Infectious clone RNA2 sequence, design primers Fny2-del2b 3'-BSST-2662-F and Fny2-del2b 3'-B-2661-R, the PCR amplification product of this pair of primers is to construct CMV visible attenuated virus Linearized intermediate vector for mutant plasmid vectors.

[0041] in CMV Fny Infectious cloning plasmid pCB301-Fny2 was used as a template (CMV Fny The construction of the invasive cloning plasmid pCB301-Fny2 refers to "Agrobacterium-mediated CMV invasive cloning and construction of 2b deletion mutants", Yao Min et al., Chinese Agricultural Sciences, 2011, 44(14): 3060-3068) , under the action of PrimeSTAR HS DNA Polymerase (Takara), carry out reverse PCR amplification, the primer pair is Fny2-del2b 3'-BSST-2662-F and Fny2-del2b 3'-B-2661-R; PCR conditions: 98 Denaturation at ℃ for 10s, annealing at 55°C for 5s, extension at 72°C for 8min, 7 cycles; denaturation at 98°C for ...

Embodiment 2

[0045] Cloning of embodiment 2.PDS partial fragments:

[0046] Using TransZol Up (TransGen) to extract the total RNA of Nicotiana benthamiana plant at the 6-leaf stage, under the action of ReverseTranscriptase M-MLV (Takara), carry out reverse transcription reaction, the reverse primer is PDS-BamH1-531-R, the reaction conditions: in Denaturation at 80°C for 3 minutes without adding enzymes; reaction at 42°C for 1.5 hours after adding reverse transcriptase;

[0047] Then use the reverse transcription product cDNA as a template, and carry out PCR reaction under the action of 2×Es Taq MasterMix (Dye) (CWBIO), the primer pair is PDS-Sma1-333-F and PDS-BamH1-531-R; PCR conditions : Pre-denaturation at 94°C for 2min; denaturation at 94°C for 30s, annealing at 53°C for 30s, extension at 72°C for 10s, 30 cycles; extension at 72°C for 2min; storage at 4°C; the PCR product is a 200bp conserved sequence fragment in N. benthamiana PDS. The PCR product was recovered by a DNA recovery kit ...

Embodiment 3

[0049] Example 3. pCB301-CMV Fny2 -PDS-Q build:

[0050] The intermediate vector pCB301-CMV Fny2 -del2b was double digested with BamH I and Sma I, and the digested product was recovered by a DNA recovery kit; the 200bp conserved sequence fragment in Nicotiana benthamiana PDS was double digested with BamH I and Sma I, and the digested product was recovered by a DNA recovery kit . The intermediate vector pCB301-CMV with the same double digestion Fny2 -del2b and PDS fragments at T 4 Under the action of DNA ligase, ligate overnight at 16°C; transform the competent cells of Escherichia coli DH5α with the ligated products, coat LB plates with 100 μg / mL kanamycin, and confirm positive clones by colony PCR screening, enzyme digestion identification and DNA sequencing pCB301-CMV Fny2 -PDS-Q.

[0051] Result analysis: After the ligation product was transformed, the product was first screened by colony PCR, and the plasmid was extracted for enzyme digestion identification. BamH I a...

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Abstract

The invention discloses a tobacco PDS gene fragment-containing cucumber mosaic virus RNA2 attenuated mutant type plasmid vector and application thereof. The plasmid vector contains a CMVFny strain RNA2 complete sequence, and a tobacco PDS gene 199bp fragment is inserted behind a 2a protein termination codon. The plasmid vector and a wild CMVFny RNA1 and wild CMVFny RNA3-containing plasmid are inoculated to nicotiana benthamiana through an agrobacterium infiltration method, so that weakly pathogenic symptoms can be displayed, and the capacity of inducing gene silencing can be judged with nakedeyes without depending on an instrument; great convenience is provided for deep researches on attenuated vaccines.

Description

technical field [0001] The invention relates to the technical fields of plant virology and molecular biology, in particular to a cucumber mosaic virus RNA2 attenuated mutant plasmid vector containing a tobacco PDS gene fragment and an application thereof. Background technique [0002] Cucumber mosaic virus (CMV) is one of the plant viruses with the widest host range known, which can infect more than 100 families and more than 2000 species of plants. Cause a variety of plants with important economic value (crops, garden crops, etc.) to appear symptoms such as yellowing, dwarfing, deformity, etc., seriously affecting their yield and quality. For the prevention and treatment of plant viral diseases, the most effective method is the screening and cultivation of disease-resistant varieties, but it has the disadvantages of long breeding cycle and easy loss of resistance of disease-resistant varieties. Another method is attenuated virus cross protection, which is to use attenuated...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/66A01H5/00A01H6/82
CPCC07K14/005C07K14/415C12N15/8218C12N15/8283C12N2770/14022
Inventor 原雪峰刘珊珊曹欣然于成明
Owner SHANDONG AGRICULTURAL UNIVERSITY
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