调控FAD2不同剪接体参与膜脂和储藏脂合成的机制研究

FAD2 plays an important role in the synthesis of membrane lipid and storage lipid of plant cells. The different copy of FAD2 gene in plant genome has different expression pattern and function, and its regulation can occur at the transcriptional, post-transcriptional or post-translational level, but the specific regulatory mechanism of it is not yet clear until now. Our previous study found that the isoform A2 of AhFAD2A and isoform B2 of AhFAD2B, which may be involved in the synthesis of storage lipid, mainly express in the developing seeds, and a bHLH transcription factor with a seed-specific expression profile can interact with the regulatory regions of them, while isoform A4 and B4 express constitutively, suggesting that they may be associated with membrane lipid synthesis. In this study, we intend to deeply investigate the expression profile of different FAD2 isoforms of both peanut variety with general content of oleic acid and its mutant with high content of oleic acid treated by room temperature and low temperature, and, based on analyzing of fatty acid composition of both membrane lipid in leaves and storage lipid in seeds, preliminarily make a clear of functional divergence of the isoforms. Furthermore, we want to clone the genes of regulatory factors interacting with the regulatory regions of different FAD2 spliceosomes, and to analyze their expression patterns and transcriptional activation characteristics, subcellular localization, and the binding sites with regulatory regions, and identify their functions through transgenic research in both peanut and Arabidopsis, and define the key regulatory factors of FAD2 gene. Finally, the regulatory mechanisms of different isoforms in the synthesis of membrane lipid and storage lipid will be preliminarily clarified, and the molecular biology theory of fatty acid metabolism be enriched, and a foundation for the high oleic acid peanut breeding be made.

脂肪酸去饱和酶FAD2在膜脂和储藏脂合成中具有重要作用。植物中不同拷贝的FAD2基因存在表达模式的差异和功能分工，其调控可发生在转录、转录后或翻译后水平，但具体的机制尚不十分清楚。我们前期研究发现，花生AhFAD2A和AhFAD2B的剪接异构体A2和B2主要在种子中表达，可能参与储藏脂合成，并且初步确定1个种子特异性表达的bHLH转录因子与它们的调控区存在相互作用；而A4和B4为组成型表达，推测可能参与膜脂合成。本研究拟深入分析常温和低温处理下普通油酸含量花生和高油酸突变体不同剪接体的表达模式，结合膜脂和储藏脂脂肪酸组成的分析，初步明确它们的功能分工；进一步克隆调控不同剪接体表达的转录因子基因，详细分析它们的表达及转录激活特征、亚细胞定位和结合位点，并通过花生和拟南芥转基因功能鉴定，明确FAD2表达的关键调控因子。初步阐明不同剪接体参与膜脂和储藏脂合成的调控机制，为花生高油酸育种奠定基础。

花生是重要的油料作物，籽粒含油量在50%左右。我国花生油产量约占植物油产量的25%左右，在保障我国食用油脂安全中具有举足轻重的地位。花生不仅含油量高且具有较好的油脂品质，油酸、亚油酸含量占脂肪酸总量的80%。然而，对于调控花生籽粒油酸、亚油酸含量的关键酶脂肪酸去饱和酶FAD2的表达调控研究不够深入。FAD2不仅参与储藏脂合成，也在膜脂合成中发挥重要作用。植物中不同拷贝的FAD2基因存在表达模式的差异和功能分工，但其调控的具体机制尚不十分清楚。本研究拟通过基因表达模式分析、与FAD2相互作用调控因子鉴定及调控基因转基因功能鉴定，明确调控FAD2参与储藏脂和膜脂合成的关键调控因子。本项目进行了以下几方面研究：利用实时定量RT-PCR（TaqMan探针法）、GUS表达系统和mRNA in situ系统分析了AhFAD2A和AhFAD2B基因不同剪接体表达模式，明确了AhFAD2A和AhFAD2B剪接体A2和B2具有种子特异性表达模式，参与储藏脂去饱和反应；而剪接体A4/B4为组成型表达，在花和根的表达远远高于其他器官和组织；克隆的花生AhFAD2-2基因呈组成型表达的模式，其在非种子器官中的表达远远高于种子中表达，是幼叶中表达的最主要拷贝，明显受冷胁迫诱导，可能参与营养器官膜脂去饱和过程。在前期筛选获得一个种子特异性表达bHLH类转录因子基因AhbHLH1的基础上，利用EMSA和ChIP-qPCR验证了该转录因子能够特异性结合AhFAD2A/2B转录起始位点上游-90 ～ -78nt两个连续的E BOX序列，从而调控其表达。GC法测定3个过表达AhbHLH1和1个抑制表达转基因T2代株系种子脂肪酸组成，发现过表达转基因花生种子主要脂肪酸组成和含量变化不明显；而抑制表达转基因株系种子中油酸含量明显升高，而亚油酸含量相应降低，同时软脂酸含量降低、硬脂酸含量提高。构建了冷胁迫下花生幼叶酵母表达文库及包含冷胁迫调控元件的诱饵菌株，通过酵母单杂交筛选获得与诱饵序列相互作用的候选调控基因，进一步验证工作正在进行。该研究可为花生脂肪酸品质遗传改良提供依据。