马铃薯卷叶病毒(PLRV)内蒙古分离物基因间隔区(IS)的克隆与序列分析

Cloning and Nucleotide Sequence Analysis of IS Gene of Potato Leaf Roll Virus Inner Mongolia Isolate

根据Genbank已报道马铃薯卷叶病毒(PLRV)基因组序列,分析其基因间隔区(IS)两端的保守区,自行设计、合成一对特异性引物,以PLRV内蒙古分离物总RNA为模板,经RT-PCR扩增得到含PLRVIS的一段369 bp的cDNA,克隆于载体pBS-T中。重组质粒经PCR鉴定、酶切分析和核苷酸序列测定,并进一步与PLRV其他分离物的同源序列作比对。结果表明:克隆的PLRV内蒙古分离物的IS序列与其他全部已发表的13个全基因组中的IS核苷酸序列有很高的同源性,最高达到100%,平均为97.90%,高于这13个PLRV全基因组序列96.81%的同源性,说明IS序列不仅在PLRV的不同株系间比较保守,而且在PLRV的全基因组序列中也是相对保守的。研究结果预示,将IS构建成RNA干扰型结构导入马铃薯,将有可能获得抗PLRV多种株系且抗性更高的转基因植株。

With a pair of specific primers based on Potato Leaf Roll Virus （ PLRV ）genomic conservative sequence lying on the both side of Intergenic Sequence （IS）reported in Genbank,one cDNA fragment （369 bp）containing the IS was amplified by reverse transcription polymerase chain reaction （RT-PCR） using the total RNA of PLRV Inner Mongolia isolate as a template. The fragment was cloned into pBS-T vector and identified by PCR, restrictive enzyme analysis and nucleotide sequence analysis. The cloned IS sequence was compared with that of homologous gene of all other 13 available PLRV isolates .The results showed that it had high homology with the other isolates （the highest homology reached 100% of nucleic acid and the average homology was 97.90% ,while the homology of 13 PLRV complete genome was 96.81% ）.The IS was not only conservative among PLRV strains but also conservative in the sequences of PLRV complete genome. The result implied that transgenic plant with high resistance to PLRV could be obtained through transferring RNA interfere structure of IS into potatoes.