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Cucumber mosaic virus induced gene silencing system and application thereof

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

Active Publication Date: 2015-03-04
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 application thereof
  • Cucumber mosaic virus induced gene silencing system and application thereof
  • Cucumber mosaic virus induced gene silencing system and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Construction of embodiment 1 CMV-ZMBJ infective cDNA clone

[0030] In this example, firstly, the CMV-ZMBJ isolate that was first isolated from the domestic corn production field and naturally infected corn was used as a material to construct an invasive cDNA clone of CMV-ZMBJ, and the backbone 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 amplified in Nicotiana occidentalis, all operations are carried out at 0-4°C or on ice;

[0033] a. 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 blender by 1g :2ml:2ml ratio mixing;

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

[0035] c. Remove the aqueous layer and filter through a layer of pre-wet (water wet) filter cloth (avoid fil...

Embodiment 2

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

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

[0103] The transformed 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 constitute the CMV-ZMBJ VIGS vector.

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

...

Embodiment 3

[0107] Example 3pCMV201-2b N81 -A: Gene fragment vector construction and Agrobacterium infiltration

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

[0109] Using Nicotiana benthamiana cDNA as a template, the target fragment NbPDS (NbPDS gene ORF467-764bp, 288bp) was amplified by PCR with the primer pair NbPDSf and Nb-PDSr, and NbPDS was cloned into pCMV2-2b using Kpn I and Xba I N81 -A vector to obtain pCMV2-2b 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 maize as a template, the target fragment ZmPDS was amplified by PCR with primer pairs ZmPDS1191F and ZmPDSR2, ZmPDS1415F and ZmPDS1665R, respectively. N (ZmPDS gene ORF1191-1424bp, 234bp) and ZmPDS C (ZmPDS gene ORF1415-1665bp, 250bp), then utilize Kpn I and Xba I respectively to ZmPDS N and ZmPDS C Cloning into pCMV2-...

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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 in particular 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 technology, has been widely used in the study of plant gene functions. Its principle of action is that plants will initiate RNA silencing when they resist virus infection ( RNA silencing) mechanism. Dicer-like protein4 in plants can recognize dsRNA produced during virus replication and cut it into 21-24nt siRNAs (small interfering RNAs). One strand of siRNAs binds to proteins such as Agronaute (AGO) to form RNA-induced silencing Complex (RNA induced silencing complex, RISC), and specifically combined with the homologous target mRNA eventually leading to the degradation of the target mRNA, this process is virus-induced gene silencing (VIGS) (Waterhouse and Fusaro, 2006; Ding ...

Claims

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Application Information

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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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