油棕果实中果皮二酰甘油酰基转移酶(DGAT1、DGAT2)基因的功能及分子调控

Oil palm (Elaeis guineensis Jacq) originates from intertropical Africa and imported into South Asia where industrial plantations started about 100 years ago. It is now the most productive world oil crop (3.5 tons/ha/y), with 36% of world production. Current knowledge about oil synthesis was derived mainly from research on seeds in which TAGs accumulate during maturation. Particularly, the use of genomics and the model species Arabidopsis has allowed new insights into seed lipid synthesis and its regulation. Although genes and enzymes involved in some steps of fatty acid (FA) and TAG synthesis still have to be identified or functionally validated, the knowledge was advanced enough to enable oil synthesis pathway reconstruction from transcriptome data in nonmodel species. In contrast, far less is known about the molecular basis of lipid metabolism in the fleshy fruits that accumulate high amounts of TAG in mesocarp of oil palm fruit, the most productive vegetative tissues of plant. For example, the results of Arabidopsis seed microarray data revealed that the fold increase of most genes involved in TAG assembly and storage was significantly higher than that of genes of the core FA biosynthetic machinery. Whether these important characteristics of seed oil synthesis are conserved and function in nonseed tissues such as the mesocarp of the oil palm fruit remains unknown. Actually, whether similar regulatory systems function in oil palm lipid-rich mesocarp during fruit ripening compared with that during seed maturation is still unknown. Triacylglycerols (TAG) are the main components of seeds storage lipids in oil seed plants. TAG biosynthesis in seed cells is thought to occur in endoplasmic reticulum (ER), and TAG accumulates in oil bodies generated through budding off from the outer ER membrane. TAG can be synthesised by enzymes of Kennedy pathway via sequential acylation of the glycerol backbone with three sn-specific acyltransferases, transferring acyl chains from acyl-CoA. Recently, new evidence indicates the importance of phosphatidylcholine in generating the diacylglycerol (DAG) pool for TAG biosynthesis. Diacylglycerol acyltransferase (DGAT) functions in the final step of the pathway by transferring an acyl group from acyl-CoA to the sn-3 position of sn-1, 2-diacylglycerol. DGAT has been proposed to be the rate-limiting enzyme in plant storage lipid accumulation. During our previous research, two DGAT genes (DGAT1 and DGAT2) has been isolated from mesocarp of oil palm, and the expression patterns of the two genes were investigated at different stages of mesocarp development. Base on those results, we want to further elucidate the function and molecular regulation mechanism of DGAT1 and DGAT2 via promoter cloning, cis-regulatory elements identification, transcription factors screening and transgenic function analysis. It will be a deep understand on DGAT genes and regulation manchism of TAG metabolism in mesocarp of oil palm fruit. The results will bring a better understanding of oil accumulation in fruits may present strategies for engineering oil accumulation in other vegetative tissues and provide a practical approach for the genetic improvement on fatty acid related traits of oil palm and possibly other palm crops as well.

油棕是最具特色的热带经济作物之一,生产的棕榈油主要积累在新鲜果实的中果皮组织中，含量达干重的90%，为目前所报道的含油量最高的植物组织类型。但目前，有关植物油脂积累和代谢的研究中几乎没有涉及到以种子之外的组织材料类型中油脂代谢的分子生物学基础。三酰甘油(TAG)是大多数油料作物油脂的积累和储存形式，在植物油脂的合成过程中，二酰甘油酰基转移酶(DGAT)负责催化二酰甘油(DAG)与脂肪酸酰基反应形成TAG的步骤，是TAG合成途径中的的最后一步，也是该途径唯一限速酶。本研究拟采用启动子克隆、顺势作用元件的鉴定、转录因子的筛选和功能基因验证相结合的研究策略，针对油棕果实中二酰甘油酰基转移酶( DGAT1、DGAT2)的功能及表达调控机制展开研究，研究结果将为阐明其在油棕中果皮油脂合成和积累中的作用及调控机制提供理论基础和研究平台，同时为今后油棕乃至棕榈科其他植物脂类代谢研究提供重要理论研究基础。

项目研究采用基因及启动子克隆、顺式作用元件鉴定、转录因子的筛选和转基因功能验证相结合的研究策略，针对与油棕果实中二酰甘油酰基转移酶( DGAT1、DGAT2)的功能及表达调控机制展开研究。通过对该基因在植物油脂代谢中所起的调控作用研究，解析了油棕中果皮油脂代谢途径的中DGAT基因相关的调控途径。其中，通过酵母单杂交实验筛选到的EgMADS21与EgDGAT2启动子存在互作关系，通过原生质体转化、荧光定量等实验皆可证明EgMADS21抑制EgDGAT2的表达，在其稳定表达后，抑制效果更加明显。在油棕中解析了EgMADS21通过抑制EgDGAT2的表达进而调控油棕油脂代谢，为之后代谢通路及调控机制的阐明提供一定的研究基础。通过研究内容严格按照申请书的设计进行，研究结果圆满完成了申请书中所包含的研究内容。