大豆脂肪酸脱氢酶FAD2家族酶活性差异的进化与功能研究

Soybean (Glycine max) is an important crop, supplying for more than half of vegetable oil production in the world. The quality of edible vegetable oil is determined by the ratio of different unsaturated fatty acids (oleic acid, linoleic acid and linolenic acid) in oil seeds. Fatty acid desaturase FAD2 is the key enzyme to determine the compositions of unsaturated fatty acids in oil seeds, which has different enzyme activities in different organisms. The enzyme activity of FAD2 is crucial to the quality of vegetable oil. However, the molecular mechanisms and functional divergence of different FAD2 enzyme activities in soybeans remain unknown. Therefore, to untangle the molecular mechanism on divergent involvement of the FAD2 genes in determining the compositions of unsaturated fatty acids, we plan to investigate the evolutionary pattern and functional divergence of soybean FAD2 gene family in this proposal. First, tracing the molecular evolution of FAD2 genes, such as phylogenetic relationship, selection pressure and functional divergence analysis, will be conducted to understand the origin and evolutionary pattern of FAD2 gene family, and to find the positive selection sites and functional divergence sites of this gene family. Second, the FAD2 gene sequences in the population of Glycine max will be systematically analyzed to find out the molecular basis of the different FAD2 enzyme activities. Last, site-directed mutagenesis will be performed to verify our predictions from the above analyses. Ultimately, we aim to uncover the mechanisms underlying the functional divergence of the FAD2 family in soybean. Our research will also provide basis for engineering oil plants and designing yeast strains for high-yield linoleic acid and α-linolenic acid.

大豆是重要的油料作物，全球50%以上的植物油由大豆提供。植物油主要组分油酸、亚油酸和亚麻酸之间的比例是影响其品质的关键。FAD2基因编码的脂肪酸脱氢酶是油酸脱氢形成亚油酸和亚麻酸的关键酶，因此该酶活性的变化是影响食用油品质的重要因素之一。但是大豆中FAD2家族酶活性差异的功能分化情况及其分子机制目前都尚不清楚。据此本课题拟通过追踪FAD2基因家族的进化过程，预测其选择压力和功能分化类型，从源头找出影响FAD2基因功能分化的关键位点及组合；接着对栽培大豆群体中的FAD2基因家族进行系统的比较分析，尝试找出大豆群体不饱和脂肪酸含量差异的分子基础；最后，进行转基因功能验证，定点突变预测的关键氨基酸，分析其性质和功能，阐明大豆中FAD2家族酶活性差异的功能分化情况及其分子机制。这些将为基因工程手段理性设计高品质油料作物和高产亚油酸与α-亚麻酸的菌株和植株提供重要的依据。

植物油由多种不饱和脂肪酸组成，主要包括油酸、亚油酸和亚麻酸等。不同植物中油脂含量及各种脂肪酸的组成差异较大。脂肪酸脱氢酶2（FAD2）是催化油酸生成亚油酸和亚麻酸的关键酶，对于植物种子中脂肪酸的组成至关重要。为了揭示FAD2对不同植物中脂肪酸以及油脂含量的影响，我们分别对FAD2基因家族进行了分子进化分析，结果显示，大多数植物中的FAD2基因没有发生分化形成了单系分支。但在部分双子叶植物中，FAD2基因分化形成了种子特异性表达和组成型表达两个分支。生物催化与定向进化的结果显示FAD2酶的催化活性与种子总含油量显著正相关，且位于GmaFAD2-1的143位点在组成型表达和种子特异性表达的FAD2中分化成酪氨酸和异亮氨酸。进一步对该位点的定向进化分析发现，异亮氨酸可以显著提高组成型FAD2的催化活性，酪氨酸可以使种子特异性GmaFAD2的活性降低。该位点是被子植物，尤其大豆中特异进化的控制FAD2活性的关键位点。此外，不同发育时期种子的转录组比较分析结果发现，在油脂合成和代谢途径的所有关键酶中种子特异性FAD2基因的表达量最高，且在高油品系大豆中表达水平显著高于低油品系，该结果显示FAD2s的表达对于植物种子中油脂的含量也具有重要的作用。综上，FAD2基因家族在进化的过程出现了一定的分化，且植物中总油脂含量的变化是FAD2的催化活性和表达分化共同作用的结果。该研究结果对于油料作物的改良提供重要的理论基础，也为高品质油料作物的育种提供了依据。