大豆二酰甘油酰基转移酶基因（GmDGAT1A）的表达与功能分析

发布时间：2018-02-02 04:05

本文关键词： 大豆油脂合成二酰甘油酰基转移酶(DGAT) 基因表达遗传转化　出处：《山西农业大学》2016年硕士论文　论文类型：学位论文

【摘要】：大豆是我国及世界的重要油料作物,解析种子油脂合成积累分子机制和培育种子油脂及蛋白含量双高的新品种始终是大豆遗传改良的一个活跃领域。三酰甘油(triacyglycerol,TAG)是植物油脂的主要储存形式。二酰甘油酰基转移酶(diacylglycerol acyltransferase,DGAT)是TAG形成的最后一步反应的关键催化酶。有关大豆(Glycine max) DGAT酶基因的发育调控以及在种子油脂合成积累中的功能未见详尽的研究报道。本文应用基因表达分析和遗传转化等技术解析大豆DGAT酶基因(GmDGATIA)的生物学功能,这为全面理解大豆油脂合成积累机制及调控网络提供了新知识。主要研究结果如下：(1)实时定量PCR检测相关基因表达表明,GmDGATIA在大豆根、茎、叶、花和幼嫩种子中均表达,其中在幼嫩种子中表达量最高。在大豆种子发育过程中,GmDGATIA基因持续表达,在种子发育第3阶段达到其表达峰值。(2)种子含油量的动态检测显示,油脂快速合成积累发生在种子发育第3阶段到第4阶段,其后缓慢增长。(3)比较种子发育过程中GmDGATIA表达和种子油脂积累的动态变化发现,GmDGATIA快速上升表达时期与种子油脂(TAG)含量急剧增加时期密切相关,预示着GmDGATIA基因在大豆种子油脂合成积累过程起重要作用。(4)成功构建GmDGATIA种子特异表达载体,为通过超表达途径获得突变体,解析GmDGATIA的生物学功能提供了必备的重组DNA元件。(5)以大豆品种“Jack”为转基因受体,利用优化的基于大豆体细胞胚发生的基因枪法导入目的基因,经筛选和分子鉴定获得了GmDGATIA超表达的大豆体细胞胚及转基因株系。(6)对大豆GmDGATIA转化体的表型检测揭示,种子特异表达GmDGATIA的材料含油量比未转化的对照提高了1.8%,蛋白含量与对照相比未减少,种子萌发特性等表型发育正常。本文数据证明大豆GmDGATIA具有时空表达特征,且在大豆种子油脂合成积累中起重要作用。种子特异超表达GmDGAT1A促进油脂合成积累,对贮藏蛋白积累无负影响,可打破种子油脂与蛋白含量负连锁表型。
[Abstract]:Soybean is an important oil crop in China and the world. The molecular mechanism of seed oil synthesis and accumulation and the cultivation of new varieties with high seed oil and protein content have always been an active field of genetic improvement in soybean, triacyglycerol triacyglycerol. Tag is the main storage form of vegetable oil. Diacylglycerol acyltransferase is a kind of diacylglycerol. Dat is the key catalytic enzyme in the last step of TAG formation. On Glycine max. The regulation of DGAT gene development and its function in seed oil synthesis and accumulation have not been reported in detail. In this paper, gene expression analysis and genetic transformation were used to analyze soybean DGAT gene. The biological function of GmDGATIA. This provides a new knowledge for the comprehensive understanding of the mechanism and regulatory network of soybean oil synthesis and accumulation. The main results are as follows: 1) Real-time quantitative PCR detection of related gene expression shows. GmDGATIA was expressed in soybean roots, stems, leaves, flowers and young seeds, and the highest expression was found in young seeds. GmDGATIA gene was expressed continuously during the development of soybean seeds. Dynamic detection of oil content in seeds at the third stage of seed development showed that rapid oil synthesis and accumulation occurred from the third to the fourth stage of seed development. The dynamic changes of GmDGATIA expression and seed oil accumulation during seed development were compared. The period of rapid increase of GmDGATIA expression was closely related to the rapid increase of seed oil content. It indicates that GmDGATIA gene plays an important role in the synthesis and accumulation of soybean seed oil. The biological function of GmDGATIA provides the necessary recombinant DNA element. 5) Soybean variety "Jack" is used as the transgenic receptor. The target gene was introduced by the optimized gunshot method based on somatic embryogenesis in soybean. GmDGATIA overexpression somatic embryos and transgenic lines. 6) were obtained by screening and molecular identification. The phenotypic detection of soybean GmDGATIA transformants was revealed. The oil content of the seed specific GmDGATIA expression material increased 1.8% compared with the untransformed control, and the protein content was not decreased compared with the control. The characteristics of seed germination and other phenotypic development were normal. The data showed that soybean GmDGATIA had the characteristics of spatiotemporal expression. Seed specific overexpression of GmDGAT1A promoted oil synthesis and accumulation, but had no negative effect on storage protein accumulation. It can break the negative phenotype of seed oil and protein content.
【学位授予单位】：山西农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S565.1

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