采用多基因联合方法鉴定福建长乐和福清产区马铃薯Y病毒株系组成

发布时间：2019-06-02 23:14

【摘要】：【目的】马铃薯Y病毒(Potato virus Y,PVY)是马铃薯生产上危害较为严重的病毒,也是制约马铃薯可持续发展的主要病毒之一。论文旨在开发一套简便、准确、快速的PVY分子鉴定技术,并采用该技术及时查明福建省部分产区PVY病害的发生、分布及PVY株系组成。【方法】采用ELISA方法对采自福建省长乐市、福清市马铃薯种植区疑似受PVY感染的样品进行检测,并根据文献报道的PVY P1、VPg和CP基因保守区设计3对简并引物,对ELISA检测后的阳性样品进行基因扩增、克隆,并将获得的序列进行核苷酸序列一致性、重组位点、基因型分布和系统发育分析。【结果】ELISA检测结果表明,17份样品中有13个样品与PVY抗体呈阳性反应,其他呈阴性反应。13个阳性样品均能成功扩增出3个与P1、VPg和CP基因预期大小一致的特异片段。BLAST比对分析显示P1、VPg和CP基因与文献报道的已知PVY分离物的核苷酸序列一致性分别为72%—99%、85%—99%和88%—99%。P1、VPg和CP 3个基因联合序列分析显示,FQ01分离物与PVY~(N-Wi)株系的核苷酸序列一致性最高,FQ08分离物与PVYE株系的核苷酸序列一致性最高,CL01、CL02、CL05和CL13 4个分离物与PVY~(NTN-NW)株系SYR-I型的核苷酸序列一致性最高,CL03、CL04、FQ02、FQ06、FQ09、FQ11和CL12 7个分离物与PVY~(NTN-NW)株系SYR-II型的核苷酸序列一致性最高。重组分析显示,除CP基因外,长乐市和福清市两个产区的PVY分离物的P1和VPg基因中均检测到显著的重组信号。基因型统计分析结果显示,长乐产区的P1基因为N型(60%)和N×O重组型(40%),VPg基因均为N×O重组型,而CP基因均为O型,而福清产区的P1基因为N型(25%)和N×O重组型(75%),VPg基因为N×O重组型(87.5%)和O型(12.5%),而CP基因除了一个分离物为N型外,其他均为O型。系统发育分析显示分离物FQ01与PVYN-Wi株系聚为一簇,FQ08与PVYE株系聚为一簇,CL01、CL02、CL05和CL13与PVY~(NTN-NW)株系SYR-I型聚为一簇,CL03、CL04、FQ02、FQ06、FQ09、FQ11和CL12与PVY~(NTN-NW)株系SYR-II型聚为一簇,表明在系统发育关系上,分离物FQ01与PVYN-Wi株系最近,FQ08与PVYE株系最近,CL01、CL02、CL05和CL13与PVY~(NTN-NW)株系SYR-I型最近,CL03、CL04、FQ02、FQ06、FQ09、FQ11和CL12与PVY~(NTN-NW)株系SYR-II型最近。【结论】PVY在福建省长乐市、福清市马铃薯种植区普遍存在,重组株系已成为田间的主流株系,且PVY~(NTN-NW)已成为优势重组株系。
[Abstract]:[objective] Potato Y virus (Potato virus Y, PVY, is a serious virus in potato production, and it is also one of the main viruses that restrict the sustainable development of potato. The purpose of this paper is to develop a set of simple, accurate and rapid PVY molecular identification technique, and to find out the occurrence and distribution of PVY diseases and the composition of PVY strains in some producing areas of Fujian Province in time. [methods] ELISA method was used to identify PVY diseases collected from Changle City, Fujian Province. Samples suspected of PVY infection in potato growing areas of Fuqing City were detected. According to PVY P1, VPG and CP gene conserved regions reported in the literature, three pairs of degenerate primers were designed to amplify and clone the positive samples detected by ELISA. The obtained sequences were analyzed by nucleotides sequence consistency, recombination sites, genotypic distribution and phylogeny. [results] the results of ELISA showed that 13 of the 17 samples were positive for PVY antibody. Other negative reactions. Three specific fragments of VPG and CP genes were successfully amplified from 13 positive samples, which were consistent with the expected size of P1 and VPG genes. Blast comparison analysis showed that P1, The nucleotide sequence consistency of VPg and CP genes with known PVY isolates reported in the literature was 72% 99%, 85% 99% and 88% 99%, respectively. P1. The combined sequence analysis of VPG and CP genes showed that The nucleotide sequence consistency between FQ01 isolate and PVY~ (N-Wi) strain was the highest, that between FQ08 isolate and PVYE strain was the highest, and that between FQ08 isolate and PVYE strain was the highest. The nucleotides of CL05 and CL13 isolates were the highest with PVY~ (NTN-NW) strain SYR- I, and CL03,CL04,FQ02,FQ06,FQ09,FQ11 and CL12 isolates were the highest with PVY~ (NTN-NW) strain SYR- II. The recombinant analysis showed that significant recombinant signals were detected in P1 and VPg genes of PVY isolates from Changle City and Fuqing City except CP gene. The results of genotypic statistical analysis showed that the P1 gene in Changle producing area was N type (60%) and N 脳 O recombinant type (40%), VPg gene was N 脳 O recombinant type, while CP gene was O type). The P1 gene in Fuqing area was N type (25%) and N 脳 O recombinant type (75%), VPg gene was N 脳 O recombinant type (87.5%) and O type (12.5%), while CP gene was O type except one isolate was N type. Phylogenetic analysis showed that the isolates FQ01 and PVYN-Wi, FQ08 and PVYE, CL01,CL02,CL05 and CL13 and PVY~ (NTN-NW) SYR- I, CL03,CL04,FQ02,FQ06,FQ09, were grouped into one cluster, one cluster with PVY~ (NTN-NW) strain, one cluster with PVY~ (NTN-NW) strain SYR- I, and one cluster with PVY~ (NTN-NW) strain SYR- I, CL03,CL04,FQ02,FQ06,FQ09,. FQ11 and CL12 and PVY~ (NTN-NW) lines SYR- II cluster, indicating that in the phylogenetic relationship, the isolates FQ01 and PVYN-Wi, FQ08 and PVYE, CL01,CL02,CL05 and CL13 and PVY~ (NTN-NW) were the closest, CL01,CL02,CL05 and CL13 and PVY~ (NTN-NW) lines SYR- I, respectively. CL03,CL04,FQ02,FQ06,FQ09,FQ11 and CL12 and PVY~ (NTN-NW) lines SYR- II were recent. [conclusion] PVY is common in potato growing areas of Fuqing City, Changle City, Fujian Province, and the recombinant strain has become the mainstream strain in the field. And PVY~ (NTN-NW) has become the dominant recombinant strain.
【作者单位】：福建农林大学植物病毒研究所/福建省植物病毒学重点实验室;
【基金】：国家现代农业马铃薯产业技术体系(CARS-10)
【分类号】：S435.32

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