小桐子种子油含量关键酶DGAT基因的功能分析

Jatropha (Jatropha curcas L.), a drought-resistant shrub, can fast grow in the poor soil such as terraces of the hills and besides of roads with high oil content in its seeds and has created tremendous interest all over the world for the use of its seed oil as a commercial soucrce of biodiesel. However, due to the unreliability of oil content in its seeds and low yield,planting of jatropha was restricted in agriculture. Investigating the molecular basis of storage lipid accumulation during seed development is an immediate need to understand genetic factors regulating storage lipid biosynthesis in jatropha seeds.Based on our previous studies, we proposed this study focusing on the functional analysis of two key genes JcDGAT1 and JcDGAT2 encoding DGAT enzymes using gene seed-specific over-expression and gene silencing techniques,respectively. With this study, the genetic mechanism of stroage lipid accumulation regulated by JcDGAT1 or JcDGAT2 in developing seeds would be addressed. This study will provide not only molecular evidence to discover the genetic cause of the unreliability of oil content in jatroph seeds,but also critical clues to create improved varieties by genetic engineering.

小桐子因为适应性强、种子油脂经过转换可得到优于0号柴油的生物柴油，可以利用边际土地发展其种植，不与粮食争地，在生物柴油的产业化发展中受到国内外广泛的关注。然而小桐子开发利用历史短，缺乏优良品种，严重限制了小桐子的发展。发展高油含量的小桐子是小桐子育种的一个重要目标。调查小桐子油脂合成的分子机理是实现小桐子基因工程育种的前提。本研究拟通过分子操作技术（基因过表达和基因沉默），集中调查小桐子种子油脂累积过程中关键功能酶基因DGAT对种子储存油脂累积的调控机理，深入研究小桐子JcDGAT1和JcDGAT2基因对种子含油量和脂肪酸成份形成的分子机制，着重阐明JcDGAT1对小桐子种子油含量的调控作用，为理解小桐子种子油脂生物合成的分子机理提供理论依据。

调查小桐子油脂合成的分子机理，是培育优良小桐子品种的基础。本研究原计划通过转基因在获得JcDGAT过表达和反义抑制的转基因阳性苗，进而收获种子分析油脂含量和成分的变化和进行转录组测序，最终解释JcDGAT1和JcDGAT2在小桐子种子油脂合成中的作用。然而因为进展不顺，我们及时的调整了研究方向，增加一些研究内容。目前本研究已经获得JcDGAT1和JcDGAT2转化小桐子的阳性苗，通过异源表达手段考察了JcDGAT1和JcDGAT2过表达对烟草种子油含量和脂肪酸组成的影响，发现二者都能够显著提高烟草种子的油含量，而JcDGAT2对于18C2脂肪酸存在一定的选择性。酵母表达实验证实JcDGAT1更适宜用于油脂生产。斯达油脂酵母(lipomyces strakeyi)能够在胞内积累细胞干重60%的油脂，是一种有重要化工应用前景的微生物。为了探究L.starkeyi油脂积累的分子机制，我们克隆编码乙酰辅酶A羧化酶的基因LsACC1，调查了其在酵母生长周期中的表达规律，在酿酒酵母YA103中过表达LsACC1能够显著提高油含量。我们同时构建了L.starkeyi的转化体系，报告基因LacZ和GUS染色证实我们的体系可以成功地用于L.starkeyi中基因的表达。同时，我们还创造性开发了一种名为IRDL cloning（improved restrction）快速构建表达载体的克隆方法。这些结果为理解储存油脂TAG合成、累积和调控提供了重要的分子证据。