椰子胚乳WRI1类转录因子的克隆及其在脂肪酸代谢调控中的功能研究

Coconut (Cocos nucifera L.), one of the key tropical crops, is a member of the monocotyledonous family Aracaceae (Palmaceae). The pulp of coconut fruit (i.e., the solid endosperm obtained from mature coconuts) is the main part used for fresh consumption and/or processing. The health and nutritional benefits derived from consuming coconut oil have been recognised throughout the World for centuries. Although the benefits of consuming coconut oil have been well documented, the genes and metabolic pathways which make coconut oil an important functional food have remained largely unknown, because of limited knowledge concerning their expression and related molecular biology. Endosperm development in coconut is a unique and poorly characterised process. Few genes and related metabolic processes involved in coconut endosperm development have been investigated. Thus, a large number of unidentified coconut genes provide a great resource for exploring new gene and function. WRI1 is a member of a plant-specific family of transcription factors (AP2/EREBP) that share either one or two copies of a DNA-binding domain called the AP2 domain. It has been demonstrated that WRI1 acts as a transcriptional enhancer of genes involved in carbon metabolism in transgenic seeds overexpressing this transcription factor. Further more, WRI1 promotes the flow of carbon to oil during seed maturation by directly activating genes involved in FA synthesis and controlling genes for assembly and storage of TAG. Although WRI1 was a best-understood example of transcription factors which have been identified in the seeds of A. thaliana, only a few were characterized in other plants and organ/tissues. In fact, in endosperm of palm, the role of WRI1 has never been reported. In previous study, a new member of the WRI1 family was isolated from endosperm of coconut (Cocos nucifera L.), designated CoWRI1, and its expression patterns during nut development were analysed. Considering the great advantage of endosperm as the storage organ of starch and proteins in cereal crops, it is highly valuable to identify and characterize new endosperm-specific WRI1-like transcriptional factor with restricted function only in the endosperm in cereal crops. So, in this project, transcriptional activity and transcriptional interaction with the BCCP2 promoter of CoWRI1 will be verfied by yeast two-hybrid and yeast one-hybrid approach respectively. Based on those result, function of this transcriptional factor will be eluciated by conditional expression in seed of transgenic Arabdopsis and Rice (Oryza sativa) will be carried out via genetic transformation, transgenic plant regeneration, expression analysis and lipid detection. It will be a first glance on related gene and manchism of fatty acid metabolism in endosperm of coconut. The results also provide a practical approach for the genetic improvement on fatty acid related traits of rice and possibly other cereal crops as well.

WRI1(WRINKLED1)类转录因子是在种子发育过程中通过直接激活与脂肪酸合成相关基因的表达影响三酰甘油合成和储存过程，实现碳源向油体分流的转录因子。在前期研究中，通过针对不同发育时期的椰肉差异表达基因的筛选获得了一个椰肉特异性表达的WRI1类转录因子。目前，WRI1类转录因子已被认定为模式植物中发现的第一个具有正向调控脂肪酸合成的转录因子。然而，作为当前植物脂肪酸代谢调控研究的热点，尚无有关WRI1-like类转录因子在棕榈科植物胚乳中的克隆、表达及功能研究的相关报道。因此，本研究针对椰肉中特异表达的WRI1类转录因子在椰子胚乳脂肪酸代谢中所起的调控作用展开研究。研究结果将为今后椰肉中脂肪酸代谢途径的分析和调控研究提供研究基础。同时，获得的WRI1类转录因子将为揭示棕榈科植物中脂肪酸代谢的调控机理，以及最终通过基因工程方式对棕榈科油料作物中脂肪酸代谢调控提供重要的研究基础和技术平台。

椰子属棕榈科椰子属，是热带果树种类中分布和栽培面积最广的果树种类之一，也是仅次于橡胶的热带作物资源和旅游资源。然而，当前国内外有关椰肉发育过程中各类物质的代谢和调控的分子生物学研究几乎处于空白状态。在前期针对椰油脂肪酸代谢相关基因的克隆和鉴定过程中，从不同发育时期椰肉组织的抑制消减杂交文库内筛选得到了一个椰肉组织特异性表达的WRI1类转录因子。利用生物信息学的方法分析发现：该基因含有一个1029bp大小的完整开放阅读框架，编码一段由342个氨基酸残基组成的多肽，其中包含了2个保守的植物所特有的AP2/ERF结构域。与拟南芥AtWRI1转录因子和甘蓝型油菜BnWRI1转录因子有很高的同源性，故将其命名为CoWRI1。荧光定量的结果显示CoWRI1在嫩椰肉中的表达水平相对较高。利用酵母单杂和双杂技术对其转录结合活性和激活活性进行分析表明：CoWRI1具有转录激活和转录结合活性，能够与拟南芥BCCP2基因的启动子序列相结合。转基因功能验证结果表明，在转基因拟南芥中，棕榈酸和硬脂酸的含量呈现出显著性增加。油酸和花生酸的含量显著性降低；棕榈油酸C16:1含量没有显著性的改变。而在转基因水稻植株中，胚乳特异性表达导致转基因植株中油脂的含量呈显著性增加(P<0.05)。 总油脂中不同种类的脂肪酸分析表明：棕榈酸和亚麻酸的含量显著性增加，而油酸水平显著性降低；其他种类的脂肪酸未发现显著性的差异。转基因植株种子中淀粉的含量显著性增加(P<0.01)，蛋白质的含量显著性降低(P<0.01)。转基因植株中不中种类物质的变化说明，CoWRI1基因的表达在降低蛋白含量的同时增加了油脂和淀粉的含量。可能的机制在于该基因对于油脂代谢过程中脂肪酸的增强，影响了种子发育过程中炭的分配。