从小麦-簇毛麦易位系TAC文库中筛选Hv-S/TPK基因  

Screening Hv-S/TPK from TAC Library of a Triticum aestivum-Haynaldia villosa Translocation Line

作　　者：孙玉磊[1] 曹爱忠[1] 杨学明[1] 王晓云[1] 陈佩度[1] 

机构地区：[1]南京农业大学作物遗传与种质创新国家重点实验室,南京210095

出　　处：《生物工程学报》2008年第8期1327-1332,共6页Chinese Journal of Biotechnology

基　　金：国家高新技术研究发展计划(Nos.2004AA222140 and 2006AA10A104);国家自然科学基金(No.30700503);江苏省自然科学基金(No.BK2007163);长江学者和创新团队发展计划项目(No.10418)~~

摘　　要：本实验室已经通过基因芯片技术筛选到一个白粉菌诱导后上调表达的抗病相关基因Hv-S/TPK,并获得了它的全长cDNA序列。利用Hv-S/TPK的特异引物筛选小麦-簇毛麦6VS/6AL易位系基因组可转化人工染色体(Transformation-competent artificial chromsome,TAC)文库,获得了阳性TAC单克隆,并进一步获得了含有Hv-S/TPKcDNA序列的5160bp(GenBank Accession No.EU153366)的亚克隆。对亚克隆的序列分析结果表明,Hv-S/TPK基因在起始密码子和终止密码子之间有3个内含子和4个外显子,4个外显子序列与簇毛麦上已得到的Hv-S/TPK的cDNA序列100%同源。对起始密码子上游序列分析结果表明,该基因的调控序列中,含有W-Box、OCS-element等与抗病相关的元件。以TAC克隆为探针与小麦-簇毛麦6VS/6AL易位系有丝分裂中期染色体进行荧光原位杂交(Fluorescence in situ hybridization,FISH),结果表明含有Hv-S/TPK基因的TAC克隆来自于簇毛麦。Hv-S/TPK gene, a resistance related gene to powdery mildew, was cloned by using genechip, and its expression was upregulated after the inoculation of Blumeria graminis to Haynaldia villosa. Using the specific primers of Hv-S/TPK to screen a genomic TAC （Transformation-competent artificial chromosome） library of translocation line 6VS/6AL, a positive TAC was screened. A 5-kb fragment containing Hv-S/TPK was subcloned and identified. This 5160-bp fragment （GenBank Accession No. EU153366） was determined by specific primer walking. The analysis of Hv-S/TPK genomic sequence showed three introns and four extrons between start code and stop code. In the promoter region of Hv-S/TPK, there were W-box and OCS-like elements which were the elements related to disease resistance. In this study, the positive TAC clone was used to as probe in situ hybridized to mitotic metaphase chromosomes of translocation line. The result of fluorescence in situ hybridization （FISH） indicated that the TAC clone containing Hv-S/TPK was from Haynaldia villosa chromosome.

关 键 词：小麦-簇毛麦易位系 可转化人工染色体文库 Hv-S/TPK基因 荧光原位杂交 

分 类 号：S512.1[农业科学—作物学]