CAJ | 학술논문

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Gen-Bank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。
위료연구십리향항수분자궤리급기조공궤제。이곡자항수십리향접충12,24,48,72,96 h협편위재료,이용억제차감잡교기술,구건료곡수균유도적SSH문고,사선십리향접충여미접충수균차이표체적기인편단,통과Gen-Bank진행동원비대,대차이표체기인진행공능주석,병이용형광정량PCR기술대부분차이표체편단진행표체분석。수궤도취차감문고중양성극륭측서,공획득368개EST서렬,삽입편단대소위200~750 bp,통과망상GenBank비용여수거고비대분석,발현기중32개EST여항병상관。대여항병상관적EST분석,추측WRKY전록인자、MAPK신호도경、개신호도경、곡광감태-S-전이매、세포색소P450、병정상관단백등가능삼여료십리향여곡수균비친화호작。진일보이용형광정량PCR기술대SSH문고중4개기인주료표체분석,결과표명저사기인균수수균유도표체。통과구건십리향수수균유도적SSH문고,초보명학료십리향삼여항수상관적기인,위하보곡자항수분자육충전정기출。
In order to understand key process controling rust resistance and molecular mechamism,a suppres-sion subtractive hybridizaion( SSH) library was constructed from Shilixiang with young leaves collected from seed-ling 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 com-paring 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 Gen-Bank. By analysing the defence related EST,it was conclued that WRKY transcription factor,mitogen-activated pro-tein kinase cascades,calcium signal transduction,glutathione S-transferase,cytochrome P450,pathogen-related pro-tein,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 molec-ular breeding of rust resistant foxtail millet.