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作物学报 ›› 2016, Vol. 42 ›› Issue (05): 658-666.doi: 10.3724/SP.J.1006.2016.00658

• 作物遗传育种·种质资源·分子遗传学 • 上一篇    下一篇

甘蓝型油菜磷脂二酰甘油酰基转移酶(BnPDAT1) cDNA的克隆和功能鉴定

谭太龙,冯韬,罗海燕,彭烨,刘睿洋,官春云*   

  1. 湖南农业大学农学院 / 国家油料改良中心湖南分中心, 湖南长沙 410128
  • 收稿日期:2015-11-13 修回日期:2016-03-02 出版日期:2016-05-12 网络出版日期:2016-03-11
  • 通讯作者: 官春云, E-mail: guancy2011@aliyun.com
  • 基金资助:

    本文由国家自然科学基金项目(31401419)，湖南省教育厅优秀青年项目(13B051)，国家高技术研究发展计划(863计划)项目(2011AA10A104， 2012AA101107)和湖南省作物种质资源与创新利用重点实验室科学基金开放项目(11KFXM08)资助。

Cloning and Characterization of Phospholipids:Diacylglycerol Acyltransferase (BnPDAT1) cDNA from Brassica napus L.

TAN Tai-Long,FENG Tao,LUO Hai-Yan,PENG Ye,LIU Rui-Yang,GUAN Chun-Yun   

  1. College of Agronomy, Hunan Agricultural University / National Oilseed Crops Improvement Center in Hunan, Changsha 410128, China
  • Received:2015-11-13 Revised:2016-03-02 Published:2016-05-12 Published online:2016-03-11
  • Contact: Guan Chunyun, E-mail: guancy2011@aliyun.com
  • Supported by:

    This work was supported by the National Nature Science Foundation of China (31401419), Outstanding youth project of Hunan Provincial Education Department (13B051), the National High Technology Research and Development Program of China (863 Program) (2011AA10A104, 2012AA101107), Science Foundation of Hunan Provincial Key Laboratory for Germplasm Innovation and Utilization in Crop (11KFXM08)

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摘要：

磷脂二酰甘油酰基转移酶(phospholipids:diacylglycerol acyltransferase, PDAT1)是植物三酰甘油(triacylglycerol, TAG)合成的关键酶。本文在甘蓝型油菜湘油15号cDNA中克隆到3个PDAT1全长编码序列(coding sequence, CDS), 经比对分别定位于A02、A10、C09染色体, 分别命名为BnPDAT1-A02、BnPDAT1-A10和BnPDAT1-C09, 其序列长分别为1998、2002和2005 bp, 各自编码665、666、667个氨基酸。预测BnPDAT1基因编码蛋白定位于细胞质膜, 具有典型的PDAT1保守结构域。多序列比对和进化分析表明BnPDAT1基因编码蛋白与甘蓝、拟南芥、亚麻芥PDAT1蛋白具有较高的同源性。酵母互补实验证实该基因编码蛋白具有PDAT1酶活性。BnPDAT1基因在湘油15号中的表达现先上升后降低趋势, 在开花后25~30 d达最大值, 但3个拷贝的表达变化规律存在差异。

关键词: 甘蓝型油菜, 磷脂二酰甘油酰基转移酶, 基因克隆, 酵母互补实验

Abstract:

Phospholipids:diacylglycerol acyltransferase (PDAT1) is a key enzyme in triacylglycerol (TAG) biosynthesis of plants. In this study, three novel PDAT1 coding sequences (CDSs) were isolated from cDNA of Brassica napus L. cv. Xiangyou 15 seeds, which were mapped to the chromosomes A02, A10, and C09, and designated as BnPDAT1-A02, BnPDAT1-A10, and BnPDAT1-C09, respectively. Three BnPDAT1 CDSs were 1998, 2002, and 2005 bp in length and encoded predicted proteins with 665, 666, and 667 amino acid residues, respectively. BnPDAT1 proteins were predicted to be located on the cell membrane and have a typical PDAT1 conserved domain. Multiple sequence alignments and phylogenetic analysis showed that the deduced amino acid sequences of BnPDAT1 were highly homologous to previously reported PDAT1 in Brassica oleracea, Arabidopsis thalian, and Eruca sativa. Furthermore, the catalytic enzyme activity of the cloned BnPDAT1 genes was confirmed by the yeast complementary experiment. The expression level of BnPDAT1s increased gradually in seed development and reached the maximum from 25 to 30 days after flowering. However, three BnPDAT1 copies were also found to be different in expression pattern.

Key words: Brassica napus L, PDAT1, Gene clone, Yeast complementary experiment

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