水稻HL-CMS细胞质雄性不育基因的体外转录及Northern Blot检测  

In vitro Transcription and Northern Blot of Cytoplasmic Male Sterile Genes in HL-CMS Rice

作　　者：华宇峰 刘京[1] 王春台[1] 刘学群[1] 谭艳平[1] 

机构地区：[1]中南民族大学生命科学学院/生物技术国家民委重点实验室,武陵山区特色资源植物种质保护与利用湖北省重点实验室,湖北武汉430074

出　　处：《安徽农业科学》2014年第12期3497-3498,3501,共3页Journal of Anhui Agricultural Sciences

基　　金：国家自然基金项目(31170296)

摘　　要：[目的]通过基因克隆和体外转录技术获得水稻红莲型细胞质雄性不育(HL-CMS)不育系粤泰A(YTA)中的2个细胞质雄性不育基因orf216和orfH79的RNA,为Northern Blot检测提供阳性定量标准品,并可作为凝胶阻滞试验的底物,用于研究与恢复基因编码产物的相互作用,从而为阐释不育基因与恢复基因的相互作用分子机制奠定基础。[方法]以红莲型胞质雄性不育基因orf216和orfH79的特异引物,利用不育系YTA为材料,PCR扩增获得相应片段,分别连接至pGEM-T Easy质粒并筛选阳性重组质粒,测序鉴定后酶切线性化,补平粘性末端,以rN4为底物、用依赖于DNA的RNA聚合酶进行体外转录,转录产物用DNase处理除去DNA模板,纯化并测定RNA浓度,并用Northern Blot验证。[结果]获得了细胞质雄性不育基因orf216和orfH79各自的RNA,经检测orf216浓度为1.286μg/μl,A260/A280为2.01;orfH79浓度为1.006μg/μl,A260/A280为2.10。[结论]运用体外转录技术获得的RNA片段条带单一,纯度较高,可用于体外大量获取目的 RNA。[ Objective] In order to obtain RNA of orf216 and orfH79 respectively, we performed in vitro transcription experiment of HL-CMS genes orf216 and orfH79. The transcripted RNAs are used as quantitative standard in Northern Blot and substrates of EMSA experiments for the relationship between restore genes and sterile genes. It lays the foundation for explaining the molecular mechanism of CMS/Rf system. [ Method] The primers were synthesized according to the specific sequence of orf216/orf79. The fragments obtained by PCR from YTA were cloned into the pGEM-T Easy vector with SP6 cloning protocol and were linearized. Then, we used blund-ended plasmids to transcription RNA in vitro, purified RNA and measured it by Northern Blot. [ Result ] Specific fragments were amplified from Honglian Cytoplasmic Male Sterile Line YTA. The full length RNAs were gained successfully, the concentration of orf216 is 1. 286 μg/μl, A260/A280 is 2.01 ; concentration of orfH79 is 1. 006 μg/μl, A260/A280 is 2.10. [Conclusion] RNAs of transcription in vitro are high purity product and are used to the study for gene function.

关 键 词：水稻 红莲型细胞质雄性不育 体外转录 NORTHERN BLOT 

分 类 号：S511[农业科学—作物学] Q37[生物学—遗传学]