摘要： 为了研究十里香抗锈分子机理及其调控机制。以谷子抗锈十里香接种12,24,48,72,96 h叶片为材料,利用抑制差减杂交技术,构建了谷锈菌诱导的SSH文库,筛选十里香接种与未接种锈菌差异表达的基因片段,通过GenBank进行同源比对,对差异表达基因进行功能注释,并利用荧光定量PCR技术对部分差异表达片段进行表达分析。随机挑取差减文库中阳性克隆测序,共获得368个EST序列,插入片段大小为200~750 bp,通过网上GenBank非冗余数据库比对分析,发现其中32个EST与抗病相关。对与抗病相关的EST分析,推测WRKY转录因子、MAPK信号途径、钙信号途径、谷胱甘肽-S-转移酶、细胞色素P450、病程相关蛋白等可能参与了十里香与谷锈菌非亲和互作。进一步利用荧光定量PCR技术对SSH文库中4个基因做了表达分析,结果表明这些基因均受锈菌诱导表达。通过构建十里香受锈菌诱导的SSH文库,初步明确了十里香参与抗锈相关的基因,为下步谷子抗锈分子育种奠定基础。
关键词:
抑制消减杂交,
谷锈,
谷子,
表达序列标签
Abstract: In order to understand key process controling rust resistance and molecular mechamism,a suppression subtractive hybridizaion(SSH) library was constructed from Shilixiang with young leaves collected from seedling inoculated with Uromyces setariae after 12,24,48,72,96 h.To identify differentially express gene in response to Uromyces setariae,differentially express gene were screened and putative functions were assigned by analysing comparing sequence in non-redundance database from GenBank.The exprssion pattern of partially screened gene were checked by real-time-quantitative PCR.Positive clone were randomly picked out and the fragment were 200-750 bp in length.368 ESTs were obtained and only 32 ESTs were related to defence response by blast analysis in GenBank.By analysing the defence related EST,it was conclued that WRKY transcription factor,mitogen-activated protein kinase cascades,calcium signal transduction,glutathione S-transferase,cytochrome P450,pathogen-related protein,were supposed to involved in the process of incompatible interaction between Shilixiang and Uromyces setariae.Four genes from the SSH library were selected to measure their expression levels by real-time-quantitative PCR and the results showed the four genes were up-regulated in foxtail millet leaves inoculated with Uromyces setariae.Through the construction of SSH library,rust resistant genes were identified,which laid the foundation for the molecular breeding of rust resistant foxtail millet.
Key words:
Suppression subtractive hybridization,
Uromyces setariae,
Foxtail millet,
EST
中图分类号:
李志勇, 贾丽霞, 董立, 王楠, 白辉, 全建章, 刘磊, 董志平. 谷子十里香抗锈抑制消减杂交文库构建及初步分析[J]. 华北农学报, 2014, 29(6): 126-130. doi: 10.7668/hbnxb.2014.06.022.
LI Zhi-yong, JIA Li-xia, DONG Li, WANG Nan, BAI Hui, QUAN Jian-zhang, LIU Lei, DONG Zhi-ping. Construction and Preliminary Analysis of the SSH Library from the Foxtail Millet Rust Resistant Variety of Shilixiang[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2014, 29(6): 126-130. doi: 10.7668/hbnxb.2014.06.022.