从小麦-簇毛麦易位系TAC文库中筛选Hv-STPK基因(打印版).doc

从小麦-簇毛麦易位系TAC文库中筛选Hv-S/TPK1 南京农业大学作物遗传与种质创新国家重点实验室, 南京 2100952 江苏省农业科学院农业生物技术研究所, 南京 210014: 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单克隆并进步获得了含有Hv-S/TPK cDNA序列的5160 bpGenBank 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(luorescence 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。: 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 Screening Hv-S/TPK from TAC Library of a Triticum aestivum- Haynaldia villosa Translocation Line Yulei Sun1, Aizhong Cao1, Xueming Yang1,2, Xiaoyun Wang1, and Peidu Chen1 1 National Key Laboratory of Crop Genetics and Germplasm Enhancement, Nanjing Agricultural University, Nanjing 210095, China 2 Institute of Agricultural Biotechnology, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China Abstract: 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 tra