大豆胞囊线虫<strong>VIGS</strong>载体的构建与鉴定

doi:10.3969/j.issn.1000-7091.2012.03.043

华北农学报 ›› 2012, Vol. 27 ›› Issue (3): 223-226. doi: 10.3969/j.issn.1000-7091.2012.03.043

所属专题： 油料作物；

大豆胞囊线虫VIGS载体的构建与鉴定

张怡, 阮九霄, 马晓萌, 傅露露, 乔月娥, 段素娟, 孟娉, 李成伟

周口师范学院生命科学系, 植物遗传与分子育种重点实验室, 河南周口 466001

收稿日期:2012-02-13 出版日期:2012-06-28
通讯作者: 李成伟(1972－), 男, 河南商丘人, 教授, 博士, 主要从事植物与病原体互作、抗性分子育种及生物反应器制药等研究。
作者简介:张怡(1985－), 女, 河南洛阳人, 助教, 硕士, 主要从事植物与微生物互作研究。
基金资助:
河南省教育厅自然科学研究计划项目(2011B180060);周口师范学院青年科研基金项目(zknuqn201046B);周口师范学院实验室开放项目(201115)

Identification and Construction of the Recombinant Vector of Virus Induced Gene Silencing of Soybean Heterodera glycines Ichinohe

ZHANG Yi, RUAN Jiu-xiao, MA Xiao-meng, FU Lu-lu, QIAO Yue-e, DUAN Su-juan, MENG Ping, LI Cheng-wei

Key Lab of Plant Genetics and Molecular Breeding of Department of Life Science in Zhoukou Normal University, Zhoukou 466001, China

Received:2012-02-13 Published:2012-06-28

摘要/Abstract

摘要： 拟克隆大豆胞囊线虫肌钙蛋白(Hg-tnc)和酰胺样多肽(Hg-flp)基因的部分片段,构建胞囊线虫的病毒诱导基因沉默体系(VIGS),以期为大豆抗胞囊线虫抗性品种的培育及胞囊线虫基因功能验证的研究奠定理论基础。利用RT-PCR方法从大豆胞囊线虫中克隆出目的基因,电泳检测表明,克隆的Hg-tnc基因和Hg-flp基因部分片段分别约为1 000 bp和700 bp。将目的基因连接到烟草脆裂病毒载体pYY13上,转化大肠杆菌,利用PCR进行初步鉴定后进行序列测定。结果表明,大豆胞囊线虫Hg-tnc基因和Hg-flp基因已插入到pYY13载体上,VIGS载体构建成功。

关键词: 病毒诱导的基因沉默, 胞囊线虫, 肌钙蛋白, 酰胺样多肽, 烟草脆裂病毒

Abstract: In this research，the partial cDNA of Heterodera glycines Ichinohe troponin C(Hg-tnc)gene and FMRFamide-like peptides(Hg-flp)were cloned and construct virus induced gene silencing(VlGS)vectors. The target?genes Hg-flp and Hg-tnc can be obtained from H. glycines by RT-PCR and then ligated to tobacco rattle virus(TRV)?vector pYY13. Then the constructed vectors were transformed into DH5α and identified by PCR and sequencing. Sequence analysis indicates that the sequences of H. glycines Hg-tnc and Hg-tnc were approximately 1 000 bp and 700?bp in length and we successfully constructed recombinant vectors pYY13-Hg-flp and pYY13-Hg-tnc. This work laid?the foundations for soybean resistance breeding and the function reseach on Hg-flp and Hg-tnc.

Key words: Virus induced gene silencing(VlGS), Heterodera glycines Ichinohe, Troponin C, FMRFamide-like peptides, Tobacco rattle virus(TRV)

中图分类号:

S435.651

张怡, 阮九霄, 马晓萌, 傅露露, 乔月娥, 段素娟, 孟娉, 李成伟. 大豆胞囊线虫VIGS载体的构建与鉴定[J]. 华北农学报, 2012, 27(3): 223-226. doi: 10.3969/j.issn.1000-7091.2012.03.043.

ZHANG Yi, RUAN Jiu-xiao, MA Xiao-meng, FU Lu-lu, QIAO Yue-e, DUAN Su-juan, MENG Ping, LI Cheng-wei. Identification and Construction of the Recombinant Vector of Virus Induced Gene Silencing of Soybean Heterodera glycines Ichinohe[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2012, 27(3): 223-226. doi: 10.3969/j.issn.1000-7091.2012.03.043.

http://www.hbnxb.net/CN/Y2012/V27/I3/223

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