草石蚕块茎膨大相关SSH文库构建及分析  

Construction and Analysis of SSH Library in Chinese Artichoke (Stachyssieboldii Miq.) During Tuber Enlargement

作　　者：耿广东[1] 杨祎 张素勤[1] Geng Guangdong;Yang Yi;Zhang Suqin(College of Agriculture Guizhou University,Guiyang,550025)

机构地区：[1]贵州大学农学院,贵阳550025

出　　处：《分子植物育种》2020年第17期5650-5657,共8页Molecular Plant Breeding

基　　金：贵州省科技支撑计划项目(黔科合支撑(2018)2330号);贵州省科技厅重大专项计划(黔科合重大专项字(2016)3002号)共同资助。

摘　　要：为挖掘草石蚕块茎膨大的关键基因,研究其分子机理,以快速膨大的草石蚕块茎为测验方,幼嫩块茎为驱动方,构建了草石蚕块茎膨大相关正向SSH文库。从文库中随机挑选阳性单克隆,利用引物Nest-1和Nest-2R对其进行验证,获得400个克隆,插入片段的大小为200~1 500 bp,主要集中在500 bp左右片段。随机挑取65个阳性克隆测序后得到60条ESTs,经过聚类、拼接和冗余去除后获得单拷贝序列33条,其中序列最长的片段为1 037 bp,最短片段为182 bp。通过序列比对,发现33条单拷贝序列中来源于蚕豆萎蔫病毒基因占39.4%,植物基因占36.4%,其它未知基因占24.2%。根据同源基因的来源分析,来源于茄科植物的基因所占比例最大(41.67%)。序列编号20 (632 bp)的EST与clone cSTB39G13 CENP-C mRNA基因有63%同源性;序列编号25 (378 bp)的EST与光敏色素A基因有84%同源性,这两个ESTs可能与草石蚕块茎膨大相关。In order to explore the important genes and molecular mechanism of Chinese artichoke(Stachys sieboldii Miq.)tuberous stem enlargement,a positive suppression subtractive hybridization(SSH)library related to tuber enlargement was constructed by using the rapidly expanding tuber as Tester and the initial tuber as the Driver in the present study.The positive monoclones were randomly selected from the library and validated by primers Nest-1 and Nest-2R,and 400 clones were obtained.The size of insertion fragments ranged from 200 bp to 1500 bp,mainly concentrated about 500 bp.Sixty-five positive clones were sequenced and 60 ESTswere contained.After clustering,splicing and removing redundancy,33 single copy sequenceswere obtained,ofwhich the longest sequencewas 1037 bp,and the shortest one was 182 bp.By Blast comparison,39.4%of the genes originated from broad bean wilt virus,36.4%of the genes originated from plant genes,and 24.2%genes were unknown.According to the source of homologous genes,the largest proportion(41.67%)came from solanaceous plants.One EST(No.20,632 bp)showed 63%homology with clone cSTB39G13 CENP-C mRNA gene.The other EST(No.25,378 bp)had 84%homology with phytochrome A gene.The two ESTs might be closely related to tuberous stem enlargement in Chinese artichoke.

关 键 词：草石蚕(Stachys sieboldii Miq.) 块茎膨大 SSH文库 表达序列标签 

分 类 号：S644.5[农业科学—蔬菜学]