琉璃苣种子γ-亚麻酸高效合成的分子与调控机制研究

γ- linolenic acid (GLA) as an essential fatty acid for human has a variety of healthy effects. Some borage (Borago officinalis L.) varieties have the highest GLA content in their seeds compared to all the other natural vegetable oil species, however it remains to be elucidated that the molecular mechanism of GLA synthesis with a high efficiency in borage seeds and the gene regulation of key enzymes involved in GLA biosynthetic pathway. During the past few years our research group world-widely collected 87 accessions of borage germplasm with quite different GLA content in their seeds. GC-FAMEs results showed that the highest GLA content in some borage varieties was 28.1% in an average of three-year results, while the lowest GLA content in some borage varieties is only 12.9% in average. In our future study for this program, we will employ a high-throughput sequencing platform of Roche 454 (GS FLX+) to deeply sequence the transcriptomes of developing seeds from two borage varieties with obviously and significantly different content of GLA in their seeds. By using the method of RT-PCR we will clone genes of Δ6-desaturase, diacylglycerol acyltransferase family. Δ6- desaturase, DGAT are the key enzymes of metabolic pathway of GLA synthesis and triacylglycerol (TAG) accumulation in plants, respectively. We will also clone the upstream regulatory element sequences and transcription factor genes of these key enzyme genes by TAIL-PCR and RT-PCR, respectively. Yeast one-hybrid system will be employed to identify the transcription factors of key enzyme genes. The function of key enzymes, transcription factors, upstream regulatory element sequences will be identified and analyzed biochemically, molecularly, cellularly and individually to seek a breakthrough to elucidate the molecular mechanism of GLA synthesis in borage seeds with a high efficiency. Our study will provide strong evidence to reveal the complex metabolic regulatory mechanism for the efficient synthesis of GLA and accumulation of TAG in borage seeds. Meanwhile many valuable genes will also be cloned in our future study to be used for genetic modification of other organisms.

γ-亚麻酸(γ- linolenic acid , GLA)作为人体的必须脂肪酸拥有多种医疗功效，而琉璃苣是天然植物油中GLA含量最高的，但目前对其GLA高效合成的分子与调控机制仍不清楚。本研究将应用高通量测序仪对GLA含量差异显著的2个极端类型琉璃苣品种（品均最高为28.1%，平均最低为13.4%）的发育种子转录本文库进行深度测序，并利用RT-PCR、TAIL-PCR方法克隆GLA合成与积累代谢途径的关键酶Δ6-脂肪酸去饱和酶和DGAT家族基因，以及克隆它们的上游调控元件序列和转录因子基因，通过酵母单杂交系统对关键酶基因的转录因子进行鉴定，并从生化、分子、细胞、个体水平上对关键酶、转录因子、基因上游调控元件序列的功能进行鉴定，从而为阐明琉璃苣GLA高效合成的分子机制寻求突破口，为揭示琉璃苣GLA高效合成与积累的代谢调控机制提供依据，同时为异源生物更加高效的合成GLA提供必要的基因资源。

γ-亚麻酸(GLA)作为一种人体必需脂肪酸，具有多种药理作用和保健功能，琉璃苣(Borago officinalis L.)籽油是天然植物油中GLA含量最高的，它是获取天然GLA的极佳来源。本项目阐明了琉璃苣种子发育过程中脂肪酸成分的动态变化规律，比较了GLA含量差异显著的3个琉璃苣品种发育种子的转录组数据，挖掘了大量与GLA生物合成相关的酶基因，解析了关键酶基因的功能，明确了关键酶基因的上游启动序列，系统分析了WRI1转录因子和bZIP转录因子家族，获得了琉璃苣叶绿体基因组全序列信息。本项目的完成为提高我国现有油料作物的GLA含量提供了理论依据及基因资源。取得的主要研究进展：1、琉璃苣籽油脂肪酸的组成成分主要为棕榈酸、硬脂酸、油酸、亚油酸、GLA、α-亚麻酸、花生一烯酸和芥子酸，成熟种子中不饱和脂肪酸含量高(84.05%)，其中亚油酸含量最高(38.20%)、GLA次之(24.59%)，棕榈酸、硬脂酸和α-亚麻酸的相对含量随着种子的发育均呈现不断下降的趋势，其他脂肪酸成分不断增加，相关性分析表明棕榈酸与α-亚麻酸呈显著正相关，二者与其他脂肪酸成分呈负相关；2、对琉璃苣籽油中GLA含量不同的3个品种的开花后15天时的发育种子进行转录组测序，共得到59,970条unigene，其中78.1%的unigene在公共数据库中得到注释，共发掘到545条可能参与脂肪酸和三酰甘油生物合成的unigene；3、获得了Δ6-D和KasΙI二个关键酶基因的启动子序列，BoLPAT1基因受非生物逆境调控且NaCl对其表达的影响最大，BoFatB1的原核表达能够显著提高大肠杆菌脂肪酸含量；4、共发掘到1084个转录因子，这些转录因子分别属于51个转录因子家族，克隆到两条WRI1转录因子，对98条bZIP转录因子(其中27条来自琉璃苣)进行聚类分析分成13小类，其中11条与确定参与油脂合成调控的AtbZIP67和GmbZIP123转录因子聚类在同一分支；5、琉璃苣叶绿体全基因组全长为149,835bp，IRs大小为27,014bp，被IRs区分割的大、小单拷贝区长度分别为78,840bp和16,967bp，成功注释110个基因，20个基因含有内含子，GC含量为37.82%。