《植物生理学报》 2017, 53(9): 1619-1628

油茶CoPDAT基因的克隆与表达分析

赵广, 宋志波, 刘美兰, 龙洪旭, 李泽, 张琳^*

中南林业科技大学, 经济林培育与保护教育部重点实验室, 经济林育种与栽培国家林业局重点实验室, 经济林培育与利用湖南省2011协同创新中心, 长沙410004

通信作者：张；E-mail: triwoodtim918@126.com

摘　要：

磷脂:二酰甘油酰基转移酶(PDAT)能够催化磷脂酰胆碱(PC) sn-2位的酰基转移至二酰甘油(DAG)上, 形成三酰甘油(TAG)和溶血磷脂酰胆碱, 这是另外一条不同于Kennedy途径的三酰甘油合成方式, 故明确油茶(Camellia oleifera) PDAT基因的功能对阐明油茶种子油脂合成机制具有重要意义。本研究通过cDNA末端快速扩增(RACE)技术克隆油茶CoPDAT基因的全长cDNA序列, 该序列长2 773 bp, 开放阅读框为2 022 bp, 编码673个氨基酸。亚细胞定位分析确定CoPDAT基因所编码蛋白质定位于内质网上。实时荧光定量PCR分析结果表明, CoPDAT在油茶不同组织/器官中均有表达, 在种子中不同发育时期的相对表达量呈“上升-下降-上升-下降-上升”的表达特征, 在花后42周表达量最高。CoPDAT基因与油茶种子油脂含量的灰色关联度值为0.8356, 表明该基因与油茶种子油脂的合成积累关系密切。本研究为阐明CoPDAT基因在油茶油脂合成过程中的功能以及为油茶的分子遗传改良提供参考。

关键词：基因克隆; 生物信息学分析; 亚细胞定位; 表达模式; 灰色关联度

收稿：2017-04-26   修定：2017-08-10

资助：国家自然科学基金(31470684)。

Cloning and expression analysis of a phospholipid: diacylglycerol acyltransferase (PDAT) gene in Camellia oleifera

ZHAO Guang, SONG Zhi-Bo, LIU Mei-Lan, LONG Hong-Xu, LI Ze, ZHANG Lin^*

Key Laboratory of Cultivation and Protection for Non-wood Forest Trees, Ministry of Education; Key Lab of Non-wood Forest Products of State Forestry Administration; Cooperative Innovation Center of Cultivation and Utilization for Non-wood Forest Trees of Hunan Province; Central South University of Forestry and Technology, Changsha 410004, China

Corresponding author: ZHANG Lin; E-mail: triwoodtim918@126.com

Abstract:

Phospholipid: diacylglycerol acyltransferase (PDAT) can catalyze the sn-2 acyl of phosphatidylcholine (PC) to transfer to the diacylglycerol (DAG), forming triacylgilcerol (TAG) and lysophosphatidyl choline. PDAT-based TAG synthesis pathway is different from the well-known Kennedy pathway, therefore it is of great significance to clarify the function of PDAT gene in Camellia oleifera. In this study, a specific band of about 800 bp was amplified from C. oleifera seeds by reverse transcription PCR. The full-length cDNA sequence of PDAT from C. oleifera was cloned by RACE (rapid amplification of cDNA ends) technology. CoPDAT (C. oleifera PDAT) was 2 773 bp in length and included an open reading frame of 2 022 bp, encoding 672 amino acids. By subcellular localization analysis, CoPDAT was located on the endoplasmic reticulum. Real-time quantitative PCR (qPCR) analysis showed that the CoPDAT was expressed in varied tissues/organs of C. oleifera. Relative expression amount in the developing seeds exhibited a pattern of “increase, decrease, increase, decrease and increase”, and the highest expression was at 42 weeks after flowering (WAF). Gray correlation value between CoPDAT and the seed oil content was 0.8356, indicating that the expression of CoPDAT had a close relation with seed oil content. This study lays an solid foundation for elucidating the function of CoPDAT in the seed oil synthesis and provided scientific guidance for molecular genetic improvement in C. oleifera.

Key words: gene cloning; bioinformatic analysis; subcellular localization; expression pattern; gray correlation analysis