大豆胞囊线虫VIGS载体的构建与鉴定

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

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。

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.