Cloning and sequence analysis of CP genes of PRSV from pumpkin

中国人. 利用电镜和酶联免疫法在云南省采集到的5份南瓜病样中检测到番木瓜环斑病毒（Papaya ring spot virus，PRSV）。为了进一步从分子水平确定云南省南瓜病毒病原种类，并为下一步转基因育种提供抗性基因，采用反转录PCR（RT-PCR）方法扩增了5个分离物的外壳蛋白（coat protein, CP）基因片段，并克隆到pGEM-T载体中。核苷酸序列测定表明，番木瓜环斑病毒石屏分离物(PRSV-SP)和番木瓜环斑病毒蒙自分离物(PRSV-MZ)的CP基因长873 nt,编码290个氨基酸，番木瓜环斑病毒峨山分离物(PRSV-ES)、番木瓜环斑病毒版纳分离物(PRSV-BN)和番木瓜环斑病毒宾川分离物(PRSV-BC)，3个分离物CP基因长867nt，编码288个氨基酸。PRSV 5个分离物核苷酸序列的同源性在94%以上，氨基酸序列的同源性在96%以上。与国内外17个分离物相比，核苷酸序列同源性为89.6%～98.7%，氨基酸序列同源性为86.5%～99.6%。其中PRSV-SP和来自于越南分离物PRSV-V47无论是核苷酸序列，还是氨基酸序列同源性都达到了最高，而5个分离物与来自于巴西（PRSV-BR）、美国（PRSV-USA）、墨西哥（PRSV-Y）核苷酸序列同源性均低于90%。

英语. Papaya ring spot virus (PRSV) was tested with electron microscopy and ELISA from 5 diseased pumpkin samples collected in Yunnan Province. To further determine pathogen types of pumkin viral disease in Yunnan Province on a molecular level and provide anti-disease genes for trans-gene breeding, CP gene fragment was amplified by RT-PCR from 5 isolates and cloned to pGEM-T vector. Nucleotide sequence showed that CP gene of PRSV Shiping isolate (PRSV-SP) and PRSV Mengzi isolate (PRSV-MZ) contains 873 nt encoding 290 amino acid, while that of PRSV Eshan isolate (PRSV-ES), PRSV Banna isolate (PRSV-BN) and PRSV Binchuan isolate (PRSV-BC) contains 867 nucleotide encoding 288 amino acid. The nucleotide and amino acid homology of the 5 isolates is higher than 94% and 96% respectively. Compared with sequence of other 17 foreign and domestic isolates, the homology of nucleotide is between 89.6% and 98.7%, the homology of amino acid is between 86.5% and 99.6%. It was also revealed that PRSV-SP was shared highest similarity with sequence of isolate PRSV-V47 from Vietnam, but these five isolates were shared low similarity with sequence of isolate PRSV-BR from Brazil, PRSV-USA from USA and PRSV-Y from Mexico, among which the homology of nucleotide is less than 90%.