FAD2基因内含子选择性剪接在不饱和脂肪酸合成中的作用

Intron alternative splicing, an important mode in regulating gene expression, is a hot research topic in recent years. Our previous study found that the alternative splicing, resulting in two different splice isoforms in the seeds respectively, occurs in the 5'UTR introns of peanut FAD2A and FAD2B gene, implying that the different splice isoforms may have different expression patterns. FAD2 gene plays an important role in the unsaturated fatty acid synthesis of plant membrane lipids and storage lipids. To clarify the molecular regulation mechanisms of FAD2 gene, it is not only of important theoretical significance but also can help design more reasonable strategies in the genetic improvement of crops. In present study, the expression profiles of the different splice isoforms of FAD2A and FAD2B gene will be investigated first using real time PCR, and then, using GUS reporter system, the effects of different introns from gene FAD2A and FAD2B as well as the cooperation between promoters and introns in the mRNA transcriptional level and gene expression patterns will be characterized, and the crucial regulatory regions or elements in promoters or introns will be identified by the deletion analysis of promoters or introns, and the similarity and difference of regulatory functions of the FAD2 intron between peanut and Arabidopsis or sesame will be explored; further more, using Arabidopsis mutant as transformation acceptor, the different splicing isomers will be transformed into the mutant genome, and the functions of foreign splicing isomers will be analyzed through the detection of composition and contents of fatty acids, and the roles of the different isomers in the synthesis of unsaturated fatty acids of membrane lipids and storage lipids will be discussed, and the molecular regulation mechanisms of peanut FAD2 gene expression will be clarified eventually.

内含子选择性剪接是基因表达调控的重要方式，是近年表达调控研究的热点。我们前期研究发现花生FAD2A和FAD2B基因5′UTR区的内含子存在选择性剪接现象，它们在种子中分别产生2个不同的剪接异构体，推测不同异构体可能具有不同的表达模式。FAD2基因在植物不饱和脂肪酸合成中起重要作用，阐明其调控的分子机制不仅具有重要的理论意义，而且有助于在作物遗传改良中设计更为合理的策略。本研究首先明确不同剪接异构体的表达谱，然后通过GUS表达系统研究不同内含子对mRNA表达水平和表达模式的影响，以及相应的启动子与内含子如何协同调控基因的表达；通过缺失表达分析，进一步明确启动子和内含子中关键调控区域或元件，并比较花生与拟南芥、芝麻FAD2基因内含子调控功能的异同；通过拟南芥突变体功能互补研究，明确不同剪接异构体在膜脂和贮藏脂不饱和脂肪酸合成中的分工，最终阐明花生FAD2基因表达调控的分子机制。

内含子选择性剪接是基因表达调控的重要方式，是近年表达调控研究的热点。本研究通过qRT-PCR检测发现，花生FAD2A 和FAD2B 基因5′UTR 区的内含子确实存在选择性剪接现象，分别产生至少2 个不同的剪接异构体。不同异构体具有不同的表达模式，A2和B2主要在授粉后50天的种子中表达；而A4或B4在检测的所有组织中均有较高水平表达，在根和花中表达量最高。按照项目计划，构建完成了16个用于内含子选择性剪接对表达模式影响研究的GUS基因植物表达载体，并转化了拟南芥，目前大部分转化结构已获得转基因T1代种子，正在进行GUS组织化学检测，部分转化结构已获得T2代纯合株系，并检测了转基因拟南芥GUS 基因表达情况。项目还根据今后调控机理研究的需要，克隆了与AhFAD2基因A2异构体转录起始位点上游200bp调控区相互作用的bHLH转录因子基因序列，并证实了其定位于细胞核中且具有转录激活功能。为进一步明确启动子和内含子中关键调控区域或元件，以及不同剪接异构体在膜脂和贮藏脂不饱和脂肪酸合成中的分工，最终阐明花生FAD2 基因表达调控的分子机制奠定基础。