侵染番茄的番茄褪绿病毒山东泰安分离物的分子鉴定和序列分析

摘要

2012年秋季，在山东泰安番茄主要种植区中采集到叶片褪绿，叶脉颜色变深的疑似番茄褪绿病毒病和番茄侵染性褪绿病毒病的番茄样品。利用番茄褪绿病毒（Tomato chlorosis virus ，ToCV）的特异引物 ToCV1/ToCV2和番茄侵染性褪绿病毒（Tomato infectious chlorosis virus ，TICV）的特异引物 TICV1/TICV2分别对样品进行扩增，最后仅得到利用引物 ToCV1/ToCV2扩增的101 bp 的核苷酸序列，对该核苷酸序列克隆并测序。序列比对表明，山东泰安地区分离物与已登录的番茄褪绿病毒（ToCV）分离物相似性都在99％以上。随后，对山东泰安种植区ToCV 番茄分离物进行外壳蛋白（CP）及热激蛋白（HSP70）序列的扩增、克隆和测序（GenBank 登录号 KC812620/KC812625），经 NCBI BLAST 比对发现，目的序列与番茄褪绿病毒日本番茄分离物 ToCV-Japan/Tochigi（GenBank登录号 AB513442/AB513443）相似性最高为99％，同属于毛型病毒属的番茄褪绿病毒，这是首次明确山东地区番茄受到番茄褪绿病毒的侵染。%In the autumn of 2012,tomato plants showing symptoms of interveinal chlorosis,leaf curling and nec-rotic fleck on lower leaves,similar to symptoms induced by Tomato chlorosis virus (ToCV)and Tomato infectious chlorosis virus (TICV)(two members of the genus Crinivirus in the family Closteroviridae)were collected in Tai’an, Shandong Province.Reverse transcription polymerase chain reaction (RT-PCR)was performed to test the presence of ToCV and TICVwith the specific primers ToCV1/ToCV2 and TICV1/TICV2 for ToCV and TICV,respectively.Only with ToCV primers,a 101-bp specific fragment was amplified from the symptomatic samples,and no amplification with TICV primers from any samples.Sequence analysis of the amplified fragment shared 99% nucleotide sequence identity with that of ToCV isolates registered in the NCBI.The presence of ToCV was confirmed by using the specific primer targeting the heat shock protein 70 (HSP70)gene and coat protein (CP)gene.The BLAST results showed that the target sequence from the isolates had 99% sequence identity with those from ToCV-Japan/Tochigi isolates (GenBank accession no.AB513442/AB513443),belonging to the genus Crinivirus .It is the first report that ToCV infected tomato in Shandong.