乌桕DGAT1和DGAT2调控油脂积累的分子机理解析

Sapium sebiferum(Chinese tallow tree) which originates from China is well known as an important source of oil. While the oil content is up to 65.76%, the Sapium sebiferum is one the most ideal raw material for producing bio-diesel. But the mechanism of high oil-content of Sapium has not been reported so far. DGAT enzyme was the most important factor on oil accumulation in plants. This project intends to clone the key oil synthesis enzyme genes DGAT1 and DGAT2 of Sapium. The relationship between the mRNA level of those two genes and lipid accumulation will be analyzed by quantitative PCR. Moreover, the correlation between the protein abundance of those two genes and lipid accumulation will be determined by western blot. Meanwhile, promoter analysis will be proceeded to clarify how those two genes regulate lipid accumulation in spatial and temporal. Moreover, these genes will be expressed in Brassica napus with reverse genetics method to investigate the main function of them in the process of lipid accumulation. This project aims to explain the molecular mechanism of DGAT regulating lipid accumulation in multiple levels, including gene transcription, regulation and protein expression, all of which will provide theoretical basis of molecular mechanism of lipid accumulation in woody fuel species and technical support for improving Sapium clones by genetic engineering.

乌桕是原产我国的重要木本油料树种，果仁含油率高达65.76％，乌桕梓油是制造生物柴油的理想原料，有关乌桕含油率高的机理，至今未见相关报道。DGAT酶是促进油脂积累的重要因子。本项目拟采用同源克隆的方法从乌桕克隆出油脂合成的关键酶基因DGAT1和DGAT2。通过荧光定量PCR从转录水平明确这两个基因的表达与油脂积累的关联性关系；采用western blot从翻译水平确定这两个蛋白的表达丰度与油脂积累的关联性关系；通过启动子分析阐明这两个基因的时空表达模式与油脂积累的调控关系。采用反向遗传学的方法在甘蓝型油菜中表达这两个基因，明确其在油脂积累过程中的关键作用。最终从基因的转录、调控和蛋白质的表达等多层面解析DGAT调控乌桕油脂积累的分子机制。研究成果为木本油料树种油脂积累的分子机理研究奠定理论基础，并为乌桕品种改良提供技术支持。

乌桕梓油是制造生物柴油的理想原料。有关乌桕含油率高的机理，至今未见相关报道。.DGAT酶是促进油脂积累的重要因子。本项目采用RACE技术从乌桕种子中克隆出了调控油脂合成的两个关键酶基因SsDGAT1和SsDGAT2。SsDGAT1的全长CDS序列为1524bp, 编码507aa。SsDGAT2的全长CDS序列为1101bp, 编码336aa。通过酶切连接的方法构建了pYES2-SsDGAT1和pYES2-SsDGAT2酵母表达载体，将这两个载体分别转进DGAT缺失的酵母突变体中，结果都能弥补TAG合成的缺陷，说明SsDGAT1和SsDGAT2都具有DGAT酶的活性。亚细胞定位结果显示SsDGAT1和SsDGAT2都定位在内质网中。以不同发育时期的乌桕种子为材料，通过荧光定量PCR从转录水平明确了这两个基因的表达量是先随着种子的发育是先增加后降低，SsDGAT1的表达峰值在种子开始形成后100天左右，SsDGAT2的表达峰值在种子开始形成后90天左右。采用Western blot从翻译水平明确了随着这两个蛋白的表达丰度的是随着种子的不断发育先增加后降低，SsDGAT1在10月中旬的乌桕种子表达量最高，SsDGAT2在9月下旬的乌桕种子表达量最高。采用染色体步移的方法克隆了SsDGAT1和SsDGAT2的启动子，通过Gateway克隆的方法分别构建了这两个基因的启动子驱动GUS表达的载体，借助农杆菌介导的方法转化了甘蓝型油菜，通过GUS染色分析发现SsDGAT1和SsDGAT2在种子中的时空表达模式是先增加后降低，而乌桕种子油脂积累随着种子的发育不断增加；采用反向遗传学的方法在甘蓝型油菜中表达这两个基因，明确了SsDGAT1在油菜种子开始形成的20天左右表达量达到峰值，SsDGAT2在油菜种子开始形成的15天左右表达量达到峰值。SsDGAT1转基因油菜种子的含油量增加了6%-7%，SsDGAT2转基因油菜种子的含油量增加了3%-4%。.研究成果为乌桕分子育种奠定理论基础。