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Cucumber mosaic virus-induced gene silencing system and its application

A technology of cucumber mosaic virus and gene silencing, applied in the field of plant genetic engineering, can solve the problems of unsuccessful application of corn and no CMV, etc., and achieve the effect of simple method and wide range of hosts

Active Publication Date: 2017-09-26
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although CMV has been engineered into a VIGS vector and successfully applied to plants such as soybean and snapdragon (Nagamatsu et al., 2007; Kim et al., 2011), it has not been successfully applied to maize, and no CMV has been used to silence maize gene reporting

Method used

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  • Cucumber mosaic virus-induced gene silencing system and its application
  • Cucumber mosaic virus-induced gene silencing system and its application
  • Cucumber mosaic virus-induced gene silencing system and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Construction of CMV-ZMBJ invasive cDNA clone

[0030] In this example, firstly, the CMV-ZMBJ isolate, which was firstly isolated from natural infecting corn in my country’s corn production fields, was used as the material to construct an invasive cDNA clone of CMV-ZMBJ, and the skeleton vector for constructing the invasive clone was pCass-Rz .

[0031] The specific implementation method is as follows:

[0032] (1) Extract the virus particles of CMV-ZMBJ expanded in Nicotiana occidentalis, all operations are performed at 0-4°C or on ice;

[0033] a. Put the infected tissue, pre-cooled buffer A (5mM EDTA; 0.5M trisodium citrate, citric acid to adjust pH 6.5-7.0; add 0.5% thioglycolic acid before use) and chloroform in a pre-cooled mixer press 1g : 2ml: 2ml ratio mixing;

[0034] b. Centrifuge these mixtures in a large polypropylene tube, 4°C, 10,000 rpm, 15 min;

[0035] c. Remove the water layer and filter through a layer of pre-moistened (water-moistened) filter cloth (...

Embodiment 2

[0101] Example 2pCMV201-2b N81 -Construction of A vector

[0102] F1-I and R1-A amplify the 1844-2658nt upstream fragment of CMV-ZMBJ RNA2. F2-A and CMV23R-BamHI amplify the downstream fragment of CMV-ZMBJ RNA2 (2753-3054nt). Both the 3'end of the upstream fragment and the 5'end of the downstream fragment are introduced by primers with multiple cloning sites (5'-GGTACCTTGAGCTAGCAGGCCTCTAGA-3') so that the upstream and downstream fragments can be fused together by fusion PCR. In this way, the RNA22659-2752nt region was replaced with multiple cloning sites. The fusion PCR product was ligated back to pCMV201 through Nco I at 1847bp and BamH I at the 3'end.

[0103] The modified pCMV201 was named pCMV2-2b N81 -A (plasmid map such as figure 1 (Shown), the multiple cloning site is used to insert foreign gene fragments, together with pCMV101 and pCMV301 to form a CMV-ZMBJ VIGS vector.

[0104] Table 2 pCMV201-2b N81 -Primer used for vector construction

[0105]

[0106] Note: The italics ...

Embodiment 3

[0107] Example 3pCMV201-2b N81 -A: Construction of gene fragment vector to be studied and Agrobacterium infiltration

[0108] (1)pCMV201-2b N81 -A: Construction of NbPDS vector

[0109] Using Benshengyan cDNA as template, the target fragment NbPDS (NbPDS gene ORF467-764bp, 288bp) was obtained by PCR amplification with primer pair NbPDSf and Nb-PDSr, and NbPDS was cloned into pCMV2-2b using Kpn I and Xba I N81 -PCMV2-2b is obtained from the A vector N81 -A: NbPDS, verified by sequencing. PCMV2-2b with inserted 260bp GFP fragment N81 -A(pCMV2-2b N81 -A: GFP 260 ) As a negative control.

[0110] (2)pCMV201-2b N81 -A: Construction of ZmPDS vector

[0111] Using the cDNA of B73 corn as a template, the target fragment ZmPDS was amplified by PCR using primer pairs ZmPDS1191F and ZmPDSR2, ZmPDS1415F and ZmPDS1665R, respectively N (ZmPDS gene ORF1191-1424bp, 234bp) and ZmPDS C (ZmPDS gene ORF1415-1665bp, 250bp), then use Kpn I and Xba I to separate ZmPDS N And ZmPDS C Clone to pCMV2-2b N81 -A ...

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Abstract

The invention provides a cucumber mosaic virus induced gene silencing (CMV-ZMBJ VIGS) system. The RNAs (ribonucleic acids) of an virion of the isolate CMV-ZMBJ which is separated from CMV naturally infecting corn in the corn field are used as raw materials, after the full-length cDNAs of RNA1, RNA2 and RNA3 in a genome are amplified by RT-PCR (reverse transcription-polymerase chain reaction), the full-length cDNAs are linked to binary expression vectors of a plant, such as pCass-Rz, and then is transformed in agrobacterium to achieve infectivity, and thus CMV-ZMBJ infected cDNA cloned pCMV101, pCMV201 and pCMV301 are constructed successfully. The constructed CMV-ZMBJ VIGS system is suitable for researching the functions of a variety of plant genes and can be used for silencing the genes easily, quickly and efficiently at a low cost only by inserting a part of segments of plant genes to be researched into a vector without carrying out genetic transformation. In addition, a CMV-ZMBJ VIGS vector can be used for researching the functions of corn genes, and compared with the existing BMV (brome mosaic virus) VIGS vector which is usually used for researching the functions of corn genes; the CMV-ZMBJ VIGS vector is applicable to more hosts, including corn varieties such as Va35, B73, Zheng 58 and Mo17.

Description

Technical field [0001] The invention belongs to the technical field of plant genetic engineering, and specifically relates to a gene silencing system induced by cucumber mosaic virus and its application. Background technique [0002] Virus induced gene silencing (VIGS), as an effective reverse genetics technique, has been widely used in the study of plant gene function. Its principle of action is that plants will initiate RNA silencing when they resist virus infection ( RNA silencing) mechanism. Dicer-like protein 4 in plants can recognize dsRNA produced during virus replication and cut it into 21-24 nt siRNAs (small interfering RNAs). One strand of siRNAs combines with proteins such as Agronaute (AGO) to form RNA-induced silencing The RNA induced silencing complex (RISC) specifically binds to the homologous target mRNA and finally leads to the degradation of the target mRNA. This process is known as virus-induced gene silencing (VIGS) (Waterhouse and Fusaro, 2006; Ding and Voin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/82C12Q1/68
Inventor 周涛王蓉王廿陈晖范在丰
Owner CHINA AGRI UNIV
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