温州蜜柑CYVCV的CP基因克隆和序列分析

Cloning and Sequence Analysis in CYVCV CP Gene of Satsuma Mandarin(Citrus unshiu)

柑橘黄化脉明病(citrus yellow vein clearing, CYVCD)是一种危害柑橘的病毒病,克隆分析病原物序列信息对研发高灵敏检测方法具有重要意义。本研究从寻乌县温州蜜柑寄主上获得包含CYVCV的提取物,克隆CYVCV外壳蛋白(coat protein, CP)基因进行扩增测序和生物信息学分析。结果表明,温州蜜柑叶片的CYVCV分离物的CP基因全长978 bp,编码325 aa,温州蜜柑CYVCV的CP基因序列与GenBank中收录的CYVCV的CP基因核苷酸和氨基酸序列相似度在97.1%~99.6%和95.5%~99.8%之间,与同为柑橘属的2种环斑病毒(ICRSV-K1和ICRSV-PU) CP基因的核苷酸序列和氨基酸序列相似性在74.0%~71.1%之间。与α线性科的其他病毒相似度均低于40%。在系统进化树中,系统发育树将CYVCV的CP分为3个类群,寻乌县温州蜜柑的CYVCV的CP蛋白与湖南甜橙和重庆尤里卡柠檬分离物CP蛋白聚成一簇,表明它们之间亲缘关系更近。本研究结果表明CYVCV的CP基因具有一定的分子差异,但变异比较保守,各地不同宿主间CYVCV亲缘性较高,这为进一步检测应用打下基础。

Citrus yellow vein clearing(CYVCD) is a viral disease endangering citrus. Cloning and analyzing the sequence information of pathogen is of great significance to develop a highly sensitive detection method. Citrus yellow vein clearing virus(CYVCV) was isolated from the host of Satsuma mandarin(Citrus unshiu) in Xunwu county. The coat protein(CP) gene of CYVCV was cloned and bioinformatics analysis was carried out to provide the basis for the development of highly sensitive field detection methods. The results showed that the CP gene of CYVCV isolate from Satsuma mandarin leaves in Xunwu County was 978 bp in length, encoding 325 aa. The nucleotide and amino acid sequence similarity of the CP gene of CYVCV isolated from Satsuma mandarin leaves in Xunwu County was 97.1% ~99.6% and 95.5~99.8% with those of CYVCV in GenBank. The nucleotide sequence and amino acid sequence of CP gene shared 74.0%~71.1% similarity with those of two Citrus ringspot viruses(ICRSV-K1 and ICRSV-PU). The similarity of other viruses in the CP gene of α linear family with the CP gene of CYVCV isolated from Satsuma mandarin leaves in Xunwu County was less than 40%. In the phylogenetic tree, the CP of CYVCV was divided into three groups. The CP protein of Satsuma mandarin CYVCV clustered with the CP of Hunan sweet orange and Chongqing Eureka Lemon isolates, indicating that they were more closely related. The results showed that the CP gene of CYVCV had some molecular differences, but the variation was relatively conservative. The genetic relationship of CYVCV among different hosts was high, which laid a foundation for further detection and application.