苹果锈果类病毒(ASSVd)保定分离物草本寄主鉴定及侵染性cDNA克隆构建  

Identification of Herbaceous Hosts of Apple Scar Skin Viroid(ASSVd)BD-1 and Construction of an Infectious cDNA Clone

作　　者：郗娜娜 李紫腾 杨毅清[1] 江彦军[2] 蒋东帅 孟祥龙 胡同乐[1] 王树桐[1] 王亚南[1] 曹克强[1] XI Nana;LI Ziteng;YANG Yiqing;JIANG Yanjun;JIANG Dongshuai;MENG Xianglong;HU Tongle;WANG Shutong;WANG Yanan;CAO Keqiang(College of Plant Protection,Hebei Agricultural University,Baoding,071001,China;Agricultural Technology Extension Center of Shijiazhuang City,Shijiazhuang,050051,China)

机构地区：[1]河北农业大学植物保护学院,保定071001 [2]石家庄市农业技术推广中心,石家庄050051

出　　处：《病毒学报》2021年第6期1466-1475,共10页Chinese Journal of Virology

基　　金：河北省重点研发计划(项目号:21326506D),题目:河北省苹果病毒病绿色安全高效防控关键技术研究;河北省自然科学基金(项目号:C2019204327),题目:苹果斑点落叶病菌真菌病毒的克隆及特性研究;河北省人力资源和社会保障厅引进留学人员资助项目(项目号:C201839),题目:基于高通量及Sanger测序技术分析五种染病苹果品种ASSVd的vsiRNA及分子变异;河北省高等学校优秀青年基金(项目号:YQ2014023),题目:苹果褪绿叶斑病毒运动相关寄主因子研究。

摘　　要：由苹果锈果类病毒(Apple scar skin viroid,ASSVd)引起的苹果花脸病在我国苹果生产上造成了严重的危害,构建侵染性cDNA克隆是研究病毒基因功能、病毒侵染机制的重要手段。我们首先在温室进行了ASSVd保定分离物(BD-1)草本寄主的鉴定,证明了ASSVd BD-1可侵染油菜(Brassica napus)、黄瓜(Cucumis sativus)、扁豆角(Lablab purpureus)、蚕豆(Vicia faba)、长豆角(Vigna unguiculata)、昆诺藜(Chenopodium quinoa)、苋色藜(C.amaranticolar)共7种草本植物,在油菜上病毒接种成功率最高,达80%,新生叶片表面凹凸不平;其次是黄瓜,达到了77.78%,新生叶片皱缩畸形。通过RT-PCR扩增到ASSVd BD-1全基因组,利用引物引入的方法在基因组中引入T7启动子,构建到pMD18-T simple载体上,通过体外转录获得病毒基因组RNA转录本,在油菜上验证了RNA转录本的侵染性。研究结果为利用草本寄主探究ASSVd生物学特性提供了基础材料,为利用反向遗传学方法分析ASSVd基因功能和侵染机理奠定了基础。Apple scar skin disease,caused by apple scar skin viroid(ASSVd),has caused serious economic losses to apple production in China. Construction of an infectious cDNA clone could help to study the pathogenic mechanism. Here in, we screened herbaceous hosts of ASSVd Baoding isolate(ASSVd BD - 1) in a greenhouse. The results showed that ASSVd BD - 1 could infect Brassica napus,Cucumis sativus,Lablab purpureus,Viciafaba,Vigna unguiculata,Chenopodium quinoa,and Chenopodium amaranticolar. The virus inoculation success rate was highest for B. napus(80%). New leaves of B. napus infected with ASSVd were uneven and wrinkled. C. sativus showed the second highest virus inoculation success rate(77.78%),and malformation and wrinkled new leaves were observed. The ASSVd BD-1 genomic sequence was amplified by reverse transcription PCR(RT-PCR),the T7 promoter was introduced using primers,and genes were cloned into the pMD18-T simple vector. Viral genomic RNA transcripts were obtained by in vitro transcription,and the infectivity of RNA transcripts was verified in B. napus. An infectious cDNA clone of ASSVd BD - 1 was obtained. The results provide a basis for exploring the biological properties of ASSVd in herbaceous hosts,and lay a foundation for analyzing the molecular mechanisms of viral infection and gene function using reverse genetic methods.

关 键 词：苹果锈果类病毒(ASSVd) 草本寄主 侵染性cDNA克隆 

分 类 号：S436.611.1[农业科学—农业昆虫与害虫防治]