油葵DGAT1和DGAT2基因调控含油量和脂肪酸组分的分子机制

The obtained HaDGAT1 and HaDGAT2 gene from sunflower can significantly increase oil content and change fatty acid composition. In order to study the molecular mechanism and the function differences of the two genes, HaDGAT1 and HaDGAT2 were overexpressed in Columbia wild type of Arabidopsis thaliana and DGAT gene mutant under CaMV35S and seed-specific overexpression promoter. And the Saccharomyces cerevisiae mutant disrupted in endogenous DGAT and PDAT enzyme activities was used to express HaDGAT1 and HaDGAT2. the function and the regulation of HaDGAT1 and HaDGAT2 in oil accumunation were studied by measuring seed oil content and fatty acids composition in transgenetic A. thaliana and yeast. DGAT enzymes assay in vitro were used to evaluate the activity of each DGAT enzyme and their substrate binding selection were studied.The activity of DGAT enzyme and other related physiological and biochemical metabolism in transgenetic A. thaliana were measured during seed oil synthesis. Oil content and fatty acid with different seed development stages between low and high oil of sunflower were studied, along with the expression characteristics of HaDGAT1 and HaDGAT2, which revealing the mechanism of HaDGAT1 and HaDGAT2 on regulating seed oil content and fatty acids composition.

已克隆的油葵HaDGAT1和HaDGAT2基因，能显著提高油脂含量及改变脂肪酸组分。为了研究其分子机制与 功能差异，项目分别用组成型、种子特异型启动子调控HaDGAT1和HaDGAT2在拟南芥Col0和DGAT基因突变体中表达。利用半乳糖诱导型启动子在酵母突变株中表达。通过测定转基因拟南芥和酵母TAG含量和脂肪酸组分，分析HaDGAT1和HaDGAT2基因在调控油脂积累过程中的作用。通过转基因酵母的体外DGAT酶活性测定，分析2个基因对不同脂肪酸底物的偏好。通过测定转基因拟南芥DGAT酶活性等相关生化指标，研究HaDGAT1和HaDGAT2调控油脂合成代谢的生理生化基础。同时，结合高、低含油量油葵种子不同发育时期的脂肪酸和总油脂含量的动态变化，以及HaDGAT1和HaGAT2的表达特征分析，揭示HaDGAT1和HaDGAT2调控油脂和脂肪酸组分的可能分子机制与功能差异。

二酰甘油酰基转移酶（DGAT）是生物体内TAG合成的关键酶，其作用是催化二酰甘油加上脂肪酸酰基形成三酰甘油。本项目结合分子生物学、生物信息学等方法与技术，对油葵DGAT基因家族（HaDGAT1和HaDGAT2）参与种子油脂与脂肪酸合成调控的分子机理进行了初步研究。首先从油葵种子中克隆了1个HaDGAT1和2个HaDGAT2基因（HaDGAT2-1和HaDGAT2-2），采用qRT-PCR分析了各个基因在不同组织、种子发育不同时期的表达模式，结果显示3个基因在油葵不同组织中均有表达，HaDGAT1可能在根和种子油脂积累初期发挥重要作用，HaDGAT2-1在子叶和种子油脂积累过程中均具有重要作用，HaDGAT2-2对于油葵种子发育后期油脂积累发挥关键作用；分别将HaDGAT1和HaDGAT2基因转化酿酒酵母、拟南芥或烟草中异源表达研究基因对油脂和脂肪酸合成的影响，结果表明，HaDGAT1和HaDGAT2基因超表达能够提高转基因酵母和烟草TAG含量，改变脂肪酸组分；采用GC-MS方法分析了油葵种子发育过程中油脂和脂肪酸动态变化规律，结果表明，开花后17天为油葵种子油酸、亚油酸、硬脂酸和软脂酸积累高峰期，随后4种脂肪酸含量急剧下降；克隆了HaFAD2基因并研究了其功能，以期进一步研究HaDGAT和HaFAD2基因在脂肪酸合成中的协同作用；建立了油葵的组织培养再生体系；通过基因工程方法将油葵HaDGAT2-1基因转化油葵自交系，进行种子特异性表达，筛选获得阳性转基因油葵植株。研究结果为油葵油脂合成，以及亚油酸合成机理的解析提供理论参考，并为油葵的遗传改良提供潜在的基因资源，从而提高油葵的单位面积产量和调控脂肪酸组分的含量。