紫色马铃薯sAGP基因的克隆及其生物信息学分析

Cloning and Bioinformatics Analysis of sAGP Gene from Purple Potato

腺苷二磷酸葡萄糖焦磷酸化酶(AGPase)是植物内淀粉合成的限速酶。sAGP基因编码AGPase的小亚基。本研究使用同源克隆法,以NCBI中已知马铃薯sAGP基因(GenBank:AY186620.1)序列设计引物,克隆紫色马铃薯‘蓉紫芋5号’的AGPase小亚基基因sAGP的编码区序列。结果显示,克隆的序列与普通马铃薯一致性达99%,成功获得了紫色马铃薯的c DNA;该片段具有一个1 425 bp的开放阅读框,编码475个氨基酸残基;预测蛋白质分子量为52.43 kD,等电点为6.23。sAGP同源性分析表明紫色马铃薯与番茄、辣椒、烟草的同源性均达到90%以上。用氨基酸序列构建三维模型,发现克隆得到的sAGP包含10个α螺旋,25个β折叠,并存在NTP transferase domain与pbH1两个结构域,符合NTP转移酶家族特征,推测其为AGPase小亚基。本研究克隆了紫色马铃薯sAGP基因并对其序列特征进行了初步分析,为今后进一步了解研究紫色马铃薯中的淀粉合成机制提供了科学依据。

AGPase (ADP-glucose pyrophosphorylase) is the rate-limiting enzyme in starch synthesis in plants. sAGP gene encodes the small subunit of AGPase. In this study, homologous cloning method was used to design primers for the known potato sAGP gene sequence (GenBank: AY186620.1) in NCBI to clone the AGPase small subunit encoding gene of purple potato 'Rongziyu 5'. The results showed that the sequence was 99% identical with potato, and the purple potato cDNA was successfully obtained. The fragment had an open reading frame contained 1 425 bp, encoding 475 amino acids. The predicted molecular weight of protein was 52.43 kD, and the isoelectric point was 6.23. Homology analysis showed that the homology of purple potato sAGP with Solanlum tuberosum Linn., Solanum lycopersicum Mill., Capsicum annuum Linn. and Nicotiana attenuate Linn. were higher than 90%. Using the amino acid sequence to construct a three-dimensional model, finding that the sAGP contained 10 alpha-helix, 25 beta-folds, and there were two domains exited: NTP transferase domain and pbH1, which conformed to the characteristics of NTP transferase family, inferring that the cloned sequence is AGPase small subunit. In this study, the sAGP gene of purple potato was cloned and its sequence characteristics were preliminarily analyzed, which would provide a scientific basis for further understanding the starch synthesis mechanism of purple potato in the future.