利用酵母同源重组系统快速构建柑橘叶斑驳病毒的侵染性克隆  

Construction of Citrus leaf blotch virus Infectious cDNA Clone by Yeast Homologous Recombination System

作　　者：崔甜甜 晏建红 宾羽 李中安[1] 周常勇[1] 宋震 CUI TianTian;YAN JianHong;BIN Yu;LI ZhongAn;ZHOU ChangYong;SONG Zhen(Citrus Research Institute, Southwest University~Chinese Academy of Agricultural Sciences/National Citrus Engineering Research Center, Chongqing 400712)

机构地区：[1]西南大学/中国农业科学院柑桔研究所/国家柑桔工程技术研究中心,重庆400712

出　　处：《中国农业科学》2018年第9期1695-1705,共11页Scientia Agricultura Sinica

基　　金：国家重点研发计划(2017YFD0202002);重庆市两江学者计划项目;国家重点研发计划政府间国际科技创新合作重点专项(2017YFE0110900)

摘　　要：【目的】构建柑橘叶斑驳病毒(Citrus leaf blotch virus,CLBV)中国分离株的侵染性克隆,为从分子水平解析其致病机理打下基础。【方法】以Gene Art?p YES1L Vector为模板,PYES2117F、PYES2117R为引物扩增含有酵母相关复制起始位点的片段p YES1L-2117,利用限制性内切酶Sac II单酶切双元载体DK1317-2并回收其大片段,利用In-Fusion HD Cloning Kit重组连接双元载体DK1317-2骨架和片段p YES1L-2117,得到可以在酵母-农杆菌-大肠杆菌中复制的三元穿梭载体pCY。以马铃薯X病毒(Potato virus X,PVX)全长cDNA侵染性克隆为模板,pCY-PVX-F、pCY-PVX-R为引物扩增PVX全长,采用限制性内切酶Stu I、Sma I酶切质粒pCY,采用醋酸锂转化法将获得的PVX基因组全长cDNA与线性化的pCY载体共转化酵母菌YPH501,通过同源重组获得PVX基因组全长cDNA克隆。通过农杆菌介导接种本生烟验证所构建克隆的侵染性,从而建立基于酵母同源重组的病毒侵染性克隆快速构建体系。在此基础上,以CLBV中国分离物(CLBV-HBYD)全长cDNA为模板,分段扩增其基因组,得到片段CLBV-1、CLBV-2;利用所建体系对CLBV-1、CLBV-2及pCY载体片段进行同源重组。得到重组质粒pCY-CLBV后,经农杆菌介导接种本生烟和锦橙幼苗,并利用RT-PCR和Northern blot检测其侵染性。【结果】建立了酵母-大肠杆菌-农杆菌的三元穿梭载体PCY,该载体全长10 347 bp,含有酵母、农杆菌、大肠杆菌的复制位点,能在酵母-农杆菌-大肠杆菌稳定复制,可用于在酵母中通过同源重组快速构建病毒基因组全长cDNA克隆,也可用于通过农杆菌介导直接接种植物寄主。利用该体系获得了CLBV中国分离株的基因组全长cDNA克隆16个。随机选取1个克隆进行了序列测定(Gen Bank登录号:MG572236),其基因组全长8 747 nt,包含3个开放阅读框(open reading frame,ORF),ORF1为5 889 nt、ORF2为1 089 nt、ORF3为1 092 nt。全基因组序列比对显示,CLBV-HBYD与已登录的【Objective】 The objective of this study is to construct infectious cDNA clone of Citrus leaf blotch virus（CLBV） isolated from China and lay a foundation for studies on the pathogenesis of CLBV at the molecular level.【Method】Using Gene Art？ p YES1 L Vector as a template, a 2.1-kb fragment containing the yeast replication origin and Trp-1 gene were amplified by primer pair PYES2117 F/PYES2117 R. Plasmid of a binary vector DK1317-2 was digested with Sac II. The amplified and digested productions containing expected fragments were then purified with the Gel Extraction Kit and subjected to In-Fusion cloning. In this way a ternary shuttle vector pCY was obtained. Using Potato virus X（PVX） infectious clones as a template, pCY-PVX-F and pCY-PVX-R as primers, the full length cDNA of PVX genome was amplified. Plasmid pCY was digested with restriction endonuclease Stu I and Sma I. The fragment and the linearized vector obtained above were co-transformed into yeast YPH501 by lithium acetate conversion method, and the full-length cDNA clone of PVX genome was obtained by homologous recombination. The infectivity of the resulting constructs was verified by Agrobacterium tumefaciens mediated inoculation on N. benthamiana, and a rapid cloning system based on homologous recombination in yeast cell was established for construction of viral infectious clone. On this basis, two fragments covering the full-length genome of CLBV-HBYD were amplified, and CLBV-1 and CLBV-2 were obtained. The homologous recombination was then performed to assemble CLBV-1, CLBV-2 and pCY vector fragments using the cloning system established. Then, N. benthamiana and Jincheng（C. sinensis） was inoculated with A. tumefaciens carrying the recombinant plasmid pCY-CLBV. RT-PCR and Northern blot were used to detect the inoculated seedlings.【Result】A ternary yeast-E. coli-A. tumefaciens shuttle vector, pCY, was constructed based on DK1317-2 and p YES1 L. The vector is 10 347 bp in length and contains the replication elements of three bact

关 键 词：酵母同源重组 柑橘叶斑驳病毒 三元穿梭载体 侵染性克隆 

分 类 号：S436.66[农业科学—农业昆虫与害虫防治]