Target gene silencing method for wheatears and grains mediated through recombination barley streak mosaic virus

A technology of mosaic mosaic and gene silencing, applied in the fields of botanical equipment and methods, biochemical equipment and methods, genetic engineering, etc., can solve problems that have not yet been developed

Inactive Publication Date: 2012-07-04
NORTHWEST A & F UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

So far, there is no report on the BSMV-VIGS met...

Method used

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  • Target gene silencing method for wheatears and grains mediated through recombination barley streak mosaic virus
  • Target gene silencing method for wheatears and grains mediated through recombination barley streak mosaic virus
  • Target gene silencing method for wheatears and grains mediated through recombination barley streak mosaic virus

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0016] The applicant adopts the recombinant virus BSMV (Scofield et al., 2005) carrying the 185bpPDS fragment to verify the method of the present invention, the specific method is:

[0017] First, transcribe α, β, γ of BSMV using in vitro transcription method known in the art PDS cDNA obtains its transcripts α, β, γ PDS RNA;

[0018] Second, take three transcripts α, β, γ PDS Each 300ng of RNA was mixed with 25μl of FES Buffer (containing 1% glycine, 0.75% K 2 HPO 4 , 1% diatomaceous earth, 1% bentonite, 1% Na 3 PO 4. 10H 2 O) Mix well between thumb and index finger tip;

[0019] Then, rub gently up and down the ears of wheat in the heading to flowering stage for 5 times.

[0020] The phenotype of ear and seed coat bleaching can be observed after 13 days, that is, the phenotype of endogenous PDS gene silencing, and the phenotype of PDS gene silencing can last until 35 days after inoculation.

[0021] It is foreseeable that according to the wheat ear and grain BSMV-...

Embodiment 2

[0024] The applicant constructed a recombinant γ carrying a 176bp fragment of the wheat seed high molecular weight glutenin subunit (HMW-GS) encoding gene 1Bx14 1Bx14 cDNA. The build method is as follows:

[0025] According to the well-known wheat HMW-GS coding gene 1Bx14 sequence (GenBank accession: AY367771.1) and its homology with other HMW-GS coding genes, the applicant designed upstream primers and downstream primers, wherein:

[0026] Upstream primer: 5′-CGAAGGCGTAGTCTCGCTGGGG-3′;

[0027] Downstream primer 5'-GCGAGCTCCGGAAGCGCG-3';

[0028] Using the plasmid vector pHMW1Bx14 (Liu et al.2011) that has been cloned and constructed by the applicant's research team as a template, a 176bp fragment specific to the 1Bx14 gene (located in the coding region of the 1Bx14 gene) was obtained by PCR amplification methods known in the art. 98th to 273bp of ). Its sequence is as follows:

[0029] 5'-GCGAGCTCCGGAAGCGCGAGCTCGAGGCATACCAACAGGTGGTGGACCAGCAACTCCGAGACGTTAGCCCCGGGTACCGCC...

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Abstract

The invention relates to a target gene silencing method for wheatears and grains mediated through recombination barley streak mosaic virus (BSMV), which achieves silencing of target gene expression of the wheatears and the grains by mixing an equal amount of alpha, beta, gamma, c deoxyribonucleic acid (DNA) in vitro transcript of recombination BSMV carrying target gene fragments and then vaccinating the wheatears ranging from a heading stage to a flowering stage. Compared with the existing ribonucleic acid interfere (RNAi) technical method based on transgenic wheatears and seeds, the target gene silencing method has the advantages of being easy, fast, accurate and good in repeatability.

Description

technical field [0001] The invention relates to a method for silencing target genes of wheat spikes and grains mediated by recombinant barley streak mosaic virus (BSMV). The method can be used to study the biological functions of genes expressed in wheat spikes and developing seeds. Background technique [0002] The rapid development of molecular cloning and next-generation DNA sequencing technology has greatly promoted the isolation and molecular manipulation of a large number of genes and transcripts in cereal crops (http: / / www.ncbi.nlm.nih.gov / guide / dna-rna / ). In addition, the genome sequencing of major crops including rice and corn has been completed, and the genome sequencing of wheat is in progress. The rapid development of structural genomics has made effective methods for gene function analysis, especially agronomic trait-related gene function analysis, a bottleneck in gene function research. In the model plants Arabidopsis thaliana and rice, established gene func...

Claims

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Application Information

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IPC IPC(8): C12N15/83A01H5/00
Inventor 赵惠贤马猛
Owner NORTHWEST A & F UNIV
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