木薯淀粉合成关键酶AGPase小亚基基因全长克隆

目的]克隆木薯腺苷二磷酸葡萄糖焦磷酸化酶（AGPase）小亚基基因cDNA全长序列,为木薯高淀粉品种的分子育种提供依据.方法]根据木薯AGPase小亚基基因已知片段设计特异性引物,以木薯根、茎、叶为材料,利用PCR及RACE克隆木薯AGPase小亚基基因cDNA全长序列.运用生物信息学方法对其核苷酸序列和推导氨基酸的理化性质、蛋白质三级结构进行系统分析.结果]克隆获得木薯淀粉合成关键酶AGPase小亚基cDNA全长1566 bp,其中包括1566 bp的完整ORF,编码一个含521个氨基酸的多肽.其理论蛋白质分子量57.01 kDa,等电点6.1,呈酸性.多重序列比较分析结果显示,木薯淀粉合成关键酶AGPase小亚基核苷酸序列与蓖麻、麻风树和杨树相应序列的相似性分别为87％、87％和86％.结合系统进化树分析结果推测,木薯淀粉合成关键酶AGPase小亚基在不同物种及进化过程中具有高度的保守性.蛋白三级结构分析结果表明,木薯AGPase小亚基蛋白具有15个α-螺旋、24个β-折叠和多个转角.结论]木薯AGPase小亚基基因cDNA全长序列与蓖麻、麻风树和杨树等具有较高的同源性.

Cloning and expression analysis of small subunit gene full-length in cassava AGPase

Objective]The small subunit gene cDNA sequence of cassava AGPase was cloned in order to provide references for genetic transformation of cassava starch and molecular breeding of high-starch varieties.Method]According to known small subunit gene segments of cassava AGPase,the specific primers was designed.By using cassava roots and leaves as materials,cDNA full-length sequence of cassava AGPase small subunit gene was cloned using PCR and RACE technologies.The physical and chemical properties,protein tertiary structure of nucleotide sequence and amino acid were analyzed using bioinformatics method.Result]The cloned cDNA full-length in small subunit of cassava AGPase was 1566 bp,which included 1566 bp of complete ORF,and encoded a polypeptide containing 520 amino acids.The theory protein molecular weight was 57.01 kDa,isoelectric point was 6.1,and it showed acidic.Multiple sequence comparison analysis results showed that cassava AGPase small subunit nucleotide sequence shared 87％,87％ and 86％ of similarity with that of castor,jatropha and poplars,respectively.Phylogenetic tree analysis results showed that cassava AGPase small subunit was highly conservative in different species and in the process of evolution.The protein tertiary structure analysis results indicated that cassava AGPase small subunit protein have 15 alpha helix,24 beta folding and multiple corner.Conclusion]The cDNA full-length sequence of cassava AGPase small subunit gene have higher homology with castor,jatropha and poplar.