我国甘薯E病毒全基因组序列特征及荧光定量检测技术的建立  

Complete Genomic Sequence Characteristics and Establishment of qPCR Detection Technique of Sweet Potato Virus E in China

作　　者：唐伟 张成玲[1] 杨冬静[1] 马居奎 陈晶伟 高方园 谢逸萍[1] 孙厚俊[1] TANG Wei;ZHANG ChengLing;YANG DongJing;MA JuKui;CHEN JingWei;GAO FangYuan;XIE YiPing;SUN HouJun(Xuzhou Institute of Agricultural Sciences in Jiangsu Xuhuai Area/Institute of Sweet Potato,Chinese Academy of Agricultural Sciences/Key Laboratory of Biology and Genetic Improvement of Sweet Potato,Ministry of Agriculture and Rural Affairs,Xuzhou 221131,Jiangsu)

机构地区：[1]江苏徐淮地区徐州农业科学研究所/中国农业科学院甘薯研究所/甘薯育种农业农村部重点实验室,江苏徐州221131

出　　处：《中国农业科学》2023年第20期4010-4020,共11页Scientia Agricultura Sinica

基　　金：国家甘薯产业技术体系(CARS-10);徐州市现代农业面上项目(KC21140)。

摘　　要：【目的】甘薯E病毒(sweet potato virus E,SPVE)是甘薯上新发现的一种病毒。本研究对我国甘薯E病毒江苏徐州分离物(SPVE-XZ)的全基因组序列进行测定,分析该病毒基因组序列特征,并建立针对SPVE的荧光定量(quantitative PCR,qPCR)检测技术,为监测SPVE发生分布、检测种薯种苗中SPVE带毒情况并及时防控该病毒提供理论依据和技术支持。【方法】采用small RNA深度测序,结合RT-PCR和RACE技术,获得SPVE-XZ的全基因组序列,利用MegAlign、MEGA11等软件对获得的全基因组序列进行序列比对、系统发育关系分析。设计SPVE荧光定量检测引物,并通过对退火温度及引物浓度的优化,建立针对SPVE的qPCR检测方法,测定其特异性和灵敏度,应用于江苏省和山东省田间甘薯样品的检测。【结果】除ploy(A)外,所获得的SPVE-XZ全基因组序列全长为10919 nt,包含1个长为10560 nt的开放阅读框(open reading frame,ORF),编码3519 aa的多聚蛋白。5′非翻译区(5′untranslated region,5′UTR)和3′非翻译区(3′UTR)分别为129和230 nt。在P1和P3蛋白内分别包含由移码产生的PISPO和PIPO蛋白。全基因组序列分析表明,SPVE-XZ与韩国SPVE GS分离物(SPVE-GS)全基因组核苷酸一致率最高,为98.6%,多聚蛋白氨基酸一致率为98.5%。利用邻接法基于cp基因核苷酸序列和多聚蛋白氨基酸序列构建的系统进化树发现,SPVE-XZ与SPVE-GS均聚类在一起。建立的SPVE荧光定量检测体系可检测到SPVE,而甘薯病毒2(sweet potato virus 2,SPV2)、甘薯C病毒(sweet potato virus C,SPVC)、甘薯G病毒(sweet potato virus G,SPVG)、甘薯羽状斑驳病毒(sweet potato feathery mottle virus,SPFMV)、甘薯褪绿矮化病毒(sweet potato chlorotic stunt virus,SPCSV)、甘薯潜隐病毒(sweet potato latent virus,SPLV)、甘薯褪绿斑病毒(sweet potato chlorotic fleck virus,SPCFV)、黄瓜花叶病毒(cucumber mosaic virus,CMV)均未能检测到;灵敏度可达3.12×102 copies/μL,是常规PCR 【Objective】Sweet potato virus E(SPVE)is a novel virus infecting Ipomoea batatas.The objectives of this study are to determine the complete genomic sequence of SPVE Xuzhou isolate(SPVE-XZ)in China,analyze the genomic sequence characteristics of SPVE-XZ,and develop the quantitative PCR(qPCR)assay for the specific detection of SPVE.This study will provide theoretical basis and technical support for the detection,monitoring and controlling SPVE in China.【Method】The complete genomic sequence of SPVE-XZ was obtained by using small RNA deep sequencing,combined with RT-PCR and RACE technologies.The sequence alignment and phylogenetic relationship analysis of SPVE-XZ were performed by using software MegAlign and MEGA11.The primers of qPCR for rapid detection of SPVE were designed,and the qPCR detection method for SPVE was established by optimizing the annealing temperature and primer concentration.The specificity and sensitivity of the qPCR were determined.The developed qPCR technology was used to detect sweet potato samples collected from Jiangsu Province and Shandong Province.【Result】The complete genomic sequence of SPVE-XZ was 10919 nt,excluding the 3′-terminal poly(A)tail,containing the typical open reading frame of 10560 nt in length and encoding a putative large polyprotein of 3519 amino acids.The 5′UTR and 3′UTR of SPVE-XZ were 129 and 230 nt,respectively.PISPO and PIPO were produced with the frameshift in P1 and P3,respectively.Genomic sequence analysis showed that the genomic nucleotide sequence identity between SPVE-XZ and Korean SPVE-GS isolate was 98.6%,and the amino acid sequence identity of polyprotein between them was 98.5%.Phylogenetic trees were constructed based on the coat protein gene sequences and polyprotein amino acid sequence using the neighbor-joining method.The result showed that SPVE-XZ was clustered with SPVE-GS in these phylogenetic analyses.The established qPCR method could detect SPVE specifically,however sweet potato virus 2(SPV2),sweet potato virus C(SPVC),sweet potato vi

关 键 词：甘薯 甘薯E病毒 全基因组 系统进化分析 qPCR检测 

分 类 号：S435.31[农业科学—农业昆虫与害虫防治]