基于转录组测序对油茶角鲨烯合酶基因的克隆及分析

油茶是中国重要的木本油料树种,而角鲨烯在茶籽油中含量多少是茶油品质的重要指标,为提高油茶角鲨烯含量,以油茶种子转录组测序为基础,根据Unigene设计SQS基因RACE引物,克隆出基因全长共1 490 bp,开放阅读框1 266 bp,编码422个氨基酸,将氨基酸序列与其他植物SQS进行比对,构建进化树,分析物种间进化关系,进行蛋白质的生物信息学分析,包括蛋白质结构特点、理化性质、氨基酸组成及稳定性分析; 跨膜区域分析; 信号肽识别位点; 磷酸化位点; 二级结构及功能; 结构域分析。通过转录组测序和荧光实时定量分析了SQS基因在油茶种子发育各时期表达量变化规律,并提取各时期油脂,测定角鲨烯含量,发现两种基因表达量分析方法的结果有一致的变化趋势,且与各月份间角鲨烯含量有相同的变化规律。证明了转录组测序的有效性,并推测油茶角鲨烯合酶基因的表达量变化与角鲨烯含量的多少有直接关系,为后续从分子手段提高茶籽油中角鲨烯含量提供了理论基础。

Squalene synthase gene cloning and analysis of Camellia oleifera based on RNA-Seq

Camellia oleifera is a very important woody oil tree,the content of squalene is one of most important factor to evaluate quality of in the C. oleifera oil,to increase the content of squalene from molecular biology method. Changes of SQS related Unigene among seeds development were analyzed based on RNA-Seq,changes of the suqalene content in C. oleifera oil among seed development also tested. The result showed the same variation law. So SQS gene RACE primers were designed according to Unigene,and the full length SQS of C. oleifera was cloned,including 1 490 bp nucleotide,and the ORF is 1 266 bp long,encoding 422 amino acids sequence,which was blasted with that of other plants,the phylogenetic tree were mapped and analyzed. Then bio-informatics of SQS were studied,including structure,physicochemical properties,amino acid composition,and trans-membrane domain,signal peptide recognition,phosphorylation sites,function of secondary structure and domain analysis. The gene expression changes from August to October were similar to those of squalene content in seeds. It is concluded that the gene sequence encodes to be SQS of C. oleifera theoretically, which was a basis for the further study of functional verification and improving suqalene content in C. oleifera seed oil.