油茶甘油二酯激酶基因(CoDGK3)的克隆与表达分析

Diacylglycerol Kinase 3(Co DGK 3) Gene Cloning and Expresion Analysis of Camellia Oleifera

以油茶转录组数据为基础,采用RT-PCR和RACE技术,根据Unigene序列设计油茶甘油二酯激酶基因(Co DGK)RACE引物,分离克隆了DGK基因全长c DNA序列,命名为Co DGK3(Gene Bank登录号:KM068056),基因全长1 568 bp,开放阅读框1 455 bp,编码485个氨基酸;氨基酸同源比对及序列分析表明油茶与其它物种的DGK氨基酸序列具有较高的相似性,其中与麻风树和毛果杨相似性最高,达到78%;利用生物学软件对蛋白质进行生物信息学分析,获得其结构特点及理化性质;分析转录组数据中DGK相关的Unigene序列RTKM值和对Co DGK3基因实施实时荧光定量PCR,结果表明Co DGK3在油茶种子发育各时期表达量变化差异不大,并且发现转录组数据分析和实时荧光定量分析的结果变化规律一致,证明了转录组测序的有效性。

Based on transcriptome Unigene sequence the diacylglycerol Kinase gene named CoDGK3（GeneBank accession number：KM068056） was isolated from Camellia oleifera with the method of RT-PCR and RACE. Bioinformatics approaches are used to analyze and predict the structural characters and physicochemical property. The bioinformatics analysis showed that the CDS of CoDGK3 is 1568 bp in length,OFR 1455 bp and it codes predicted protein of 485 amino acids. Homologous comparison and sequence analysis of CoDGK3 amino acids suggested that the CoDGK3 is highly conserved and showed the highest similarity（78%） with the DGK of Jatropha curcas and Populus trichocarpa.The gene expression changes by q PCR were similar to those of RTKM in transcriptome data indicate that the CoDGK3 expresses stably in different developmental stage of Camellia oleifera seeds and the transcriptome data of Camellia oleifera seeds is valid and reliable.