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Cucumber mosaic virus mediated gene silencing system and application thereof in plant target gene silencing

A cucumber mosaic virus, gene silencing technology, applied in application, genetic engineering, plant genetic improvement and other directions

Active Publication Date: 2021-11-02
TSINGHUA UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the past 100 years, a large number of CMV strains have been isolated from various host plants, providing a large number of resources for viral pseudo-recombination and / or construction of chimeric viruses. At present, there is no combined chimeric virus based on pseudo-recombination applied in maize. Synthetic CMV Virus Development of VIGS Vectors

Method used

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  • Cucumber mosaic virus mediated gene silencing system and application thereof in plant target gene silencing
  • Cucumber mosaic virus mediated gene silencing system and application thereof in plant target gene silencing
  • Cucumber mosaic virus mediated gene silencing system and application thereof in plant target gene silencing

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1 Construction of Pr CMV-mediated VIGS vector

[0062] 1. Construction of pCMVFZ3 vector

[0063] (1) Using the plasmid pCMVF3 as a template, PF1 and PR1 were used for PCR amplification to obtain a DNA fragment I (1288bp) comprising the Fny-RNA3 sequence 1-1288nt; using the plasmid pCMVZ3 as a template, PF2 and PR2 were used for PCR amplification, Obtain DNA fragment II (957bp) comprising ZMBJ-RNA3 sequence 1271-2218nt;

[0064] Table 1 The primer sequences used to construct the pCMVFZ3 vector

[0065]

[0066] The PCR reaction system (50 μL) is as follows:

[0067]

[0068] The PCR reaction procedure is:

[0069]

[0070] (2) The above-mentioned amplified DNA fragments I and II were mixed at a molar ratio of 1:1, and used as a template, and PF1 and PR2 were used as primers for recombinant PCR amplification to obtain full-length DNA fragment III (2225bp);

[0071] (3) DNA fragment III was digested with BamHI, and inserted into the pCB301 vector doub...

Embodiment 2

[0098] Example 2 Application of Pr CMV VIGS system in corn

[0099] 1. pCMVZ2 2bN81 ::Construction of LUC vector

[0100] (1) Using the plasmid pQLC1454 (preserved in our laboratory) as a template, PF6 and PR6 were used for PCR amplification to obtain a PCR product containing the LUC (firefly luciferase) gene fragment (219bp), and its 5' end was recognized by XbaI digestion site, with a KpnI restriction recognition site at the 3' end;

[0101] Table 3 Construction of pCMVZ2 2bN81 ::Primer sequences used by LUC vectors

[0102]

[0103] (2) Use KpnI and XbaI to double-digest the PCR product, and connect to pCMVZ2 that also uses KpnI and XbaI double-digestion 2bN81 In the vector, transform Escherichia coli DH5α, and the operation method is the same as above;

[0104] (3) Pick positive clones for sequence identification, and the vector with the correct sequence is named pCMVZ2 2bN81 ::LUC, and transformed into Agrobacterium GV3101, the operation method is the same as above...

Embodiment 3

[0149]Example 3 Application of Pr CMV-LIC VIGS system in Nicotiana benthamiana

[0150] 1. pCMVZ2 2bN81 -LIC::NbPDS and pCMVZ2 2bN81 - Construction of LIC::LUC vectors

[0151] (1) Trizol method to extract total RNA from Nicotiana benthamiana leaves, using Oligo(dT) 18 Carry out reverse transcription reaction to obtain the total genome cDNA of Nicotiana benthamiana, and the operation method is the same as above;

[0152] (2) Using the above cDNA as a template, use primers PF13 and PR13 with the LIC adapter sequence to carry out PCR amplification to obtain the Nicotiana benthamiana NbPDS gene fragment (195bp), which contains LIC1 and LIC2 adapter sequences on both sides of the gene fragment respectively; as For the control LUC gene fragment, use pQLC1454 (preserved in our laboratory) as a template, and use PF17 and PR17 for PCR amplification to obtain a LUC gene fragment (202bp) with a LIC sequence linker;

[0153] Table 6 Primer sequences used for target gene amplification...

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Abstract

The invention provides a cucumber mosaic virus mediated gene silencing system and application thereof in plant target gene silencing. The gene silencing system provided by the invention is obtained by modifying a pseudorecombinant chimera cucumber mosaic virus infectious clone, and the infectious clone can efficiently infect plants and does not cause serious diseases. The gene silencing system has various advantages, can efficiently silence target genes, is long in gene silencing duration, is beneficial to large-scale plant gene functional omics research, is simple in operation method, does not depend on special equipment, and has great application prospects.

Description

technical field [0001] The present invention relates to the field of biology. Specifically, the present invention relates to the construction of a pseudorecombinant chimeric cucumber mosaic virus silencing vector and its application in plant target gene silencing. Background technique [0002] Virus-induced gene silencing (VIGS) is a technique developed based on the RNA-mediated antiviral defense mechanism in plants, and has been widely used in the study of plant gene functions and the screening and identification of functional genes . By inserting the plant gene sequence into the virus, with the replication and movement of the virus in the plant, the specific degradation of the plant endogenous gene RNA can be achieved. Compared with other methods of inducing gene deletion, VIGS has the following advantages: simple, rapid and efficient gene silencing; no stable plant transformation process is required; VIGS can be applied to forward and reverse genetics research, thereby ...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/29C12N1/21A01H5/00A01H6/46A01H6/82
CPCC12N15/8205C12N15/8218C07K14/415Y02A50/30
Inventor 刘玉乐李焕改
Owner TSINGHUA UNIV
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