小桐子磷脂二酰甘油酰基转移酶(JcPDAT1) cDNA的克隆与功能鉴定  

Cloning and functioning of phospholipids:diacylglycerol acyltransferase(JcPDAT1) cDNA from Jatropha curcas

作　　者：徐荣华[1] 邱丽俊[1,2] 阳天泉[1,2] 王如玲[1] 田波[1] 刘爱忠[1] 

机构地区：[1]中国科学院西双版纳热带植物园,热带植物资源开放实验室,云南昆明650223 [2]中国科学院研究生院,北京100049

出　　处：《中国油料作物学报》2013年第2期123-130,共8页Chinese Journal of Oil Crop Sciences

基　　金：国家自然科学基金(30871548),国家自然科学基金(青年基金,30900908);中国科学院重点方向性项目(KSCX2-EW-Z-15)

摘　　要：使用简并PCR结合RACE技术从能源植物小桐子种子中克隆了一个PDAT基因的cDNA全长,命名为JcPDAT1(GenBank登陆号为HQ827796)。JcPDAT1全长2 869bp,包含一个长为2 013bp的开放阅读框,编码670个氨基酸。多序列比对和进化分析表明该基因编码蛋白与其他植物PDAT高度同源,具有典型的PDAT结构域。Realtime PCR结果表明JcPDAT1在种子、叶和根里面都有表达,且在种子发育过程中大量表达。酵母互补实验证实该基因编码蛋白具有PDAT酶活性。在与酿酒酵母突变体H1246α的互补实验中发现,TLC层析(薄层层析法)和尼罗红染色结果都显示JcPDAT1的表达使H1246α恢复合成TAG,说明JcPDAT1具有PDAT的功能活性。According to the conserved region of PDAT gene available from GenBank, the cDNA sequence of PDAT gene from Jatropha curcas L. was cloned by degenerative PCR and RACE. The gene was named JcPDAT1 （GenBank accession HQ827796）. The full length cDNA of JcPDAT1 is 2 869bp, encoding 670 amino acids. Sequence alignment and phylogenetic analysis revealed that JcPDAT1 shared high homology with PDAT from other plants, such as RcPDAT1 （Ricinus communis, 75%） and AtPDAT1 （Arabidopsis thaliana, 74%）. The sequence harbored typical functional domains of PDAT. Realtime PCR showed that JcPDAT1 was expressed in different tissues including seeds, leaf, root tip, and was highly expressed in developing seeds, especially at the oil accumulation stage. TLC analysis and Nile red staining showed that JcPDAT1 could restore the TAG biosynthesis and accumulation in Saccharomyces cerevisiae mutant strain H1246？？？which indicated that the JcPDAT1 possessed PDAT enzyme activity.

关 键 词：小桐子 PDAT 简并PCR 基因克隆 荧光定量PCR 酵母互补实验 

分 类 号：Q786[生物学—分子生物学] S565.903[农业科学—作物学]