马铃薯块茎RNA提取及RT-PCR法克隆酸性转化酶基因  

Extraction Total RNA from Potato Tuber and Cloning Acid Invertase Gene by RT-PCR

作　　者：白斌[1,2] 张金文[1] 

机构地区：[1]甘肃省作物遗传改良与种质创新重点实验室,甘肃农业大学,兰州730070 [2]甘肃省冬小麦研究所,兰州商学院

出　　处：《分子植物育种》2005年第4期479-484,共6页Molecular Plant Breeding

基　　金：国家自然科学基金(No.30270843)项目;甘肃省自然科学基金(No.ZS991-A21-029-N)项目资助。

摘　　要：马铃薯块茎由于富含多糖和酚类物质造成RNA提取难度大。通过在SDS-phenol提取缓冲液中加入2%PVP及10mmol/L半胱氨酸以除去大量的酚类物质,利用改进后的SDS-酚法提取的马铃薯块茎总RNA电泳结果表明,总RNA质量高于未经改进的方法。并进行RT-PCR克隆与马铃薯块茎低温糖化反应过程密切相关的块茎酸性转化酶(acidinvertase)基因,经核酸序列对比分析,与公布的已知序列具有98.96%的同源性,为利用反义技术抑制酸性转化酶活性进行马铃薯块茎抗低温糖化研究作好了准备工作。Potato tubers' RNA extraction is difficult for their abounding with hydroxybenzene and polysaccharide. We have extracted the total RNA from potato tuber by the method of SDS-phenol, which is improved by 2% PVP and 10mmol/L Cys added in extraction buffer. Electrophoresis of potato tuber's RNA shows that RNA extracted by improved SDS-phenol method is better than the RNA extracted by unimproved method. Acid invertase gene, which products hydrolyzes Suc to Glc and Fru plays an important role in potato tubers' cold-induced sweetening, was cloned from cold-stored potato tubers by RT-PCR. By sequencing analysis, it has 98.96% similarity with the published sequence. Therefore it well prepared for the researching of the next work of inhibiting potato tubers cold-sweetenning by antisense acid invertase gene.

关 键 词：马铃薯块茎 RNA提取 RT-PCR法 酶基因 酚类物质 总RNA 低温糖化 酸性转化酶 PCR克隆 转化酶活性 半胱氨酸 反应过程 对比分析 核酸序列 反义技术 PVP 缓冲液 同源性 

分 类 号：S532[农业科学—作物学]