南方水稻黑条矮缩病毒SP7-1植物表达载体的构建及遗传转化

摘要

克隆南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)的SP7-1基因,构建植物表达载体并转化拟南芥,为研究SP7-1致病机理提供基础.根据已发表的SRBSDV SP7-1序列,设计特异性引物,通过RT-PCR的方法从感病水稻植株中克隆到完整的SP7-1基因.通过酶切连接,构建了SP7-1植物表达载体pCHF3/SP7-1,通过农杆菌介导的花序浸润法转化野生型拟南芥,获得转化植株T0代种子.对收获的T0代种子进行卡那霉素筛选,获得了4株阳性植株.阳性植株的PCR和RT-PCR分析检测结果表明,构建的SP7-1载体已成功转化拟南芥,病毒SP7-1已经整合至拟南芥基因组中并能正常表达.%The full-length SRBSDV (southern rice black-streaked dwarf virus) SP7-1 gene was cloned from diseased rice by RT-PCR using a primer designed according to the NCBI GenBank ( accession number :EU784841). SP7-1 plant expression vector pCHF3/SP7-1 was constructed, and were transformed into wild type Arabidopsis thaliana( Col-0) by floral dip method. All four positive transgenic seedlings were identified from To transgenic seeds through kanamycin screening. The results of PCR and RT-PCR showed that SRBSDV SP7-1 gene was integrated into Arabidopsis genome and transcription-al level was higher than that of no-transgenic seedlings. The SRBSDV SP7-1 transgenic Arabidopsis seedlings might provide material for further studies on the functions of SP7-I.