棉花FAD2基因调控花药脂肪酸代谢与花粉外壁发育的机制研究

FAD2 gene encodes the enzyme that is essential for polyunsaturated lipid synthesis. In plants, the expression level of FAD2 determines the content and proportion of polyunsaturated fatty acid composition. It has been found that the specific inhibition of FAD2 expression by RNAi in flowers led to abnormal pollen exine and anther abortion in our previous study. To explore the molecular mechanism of FAD2 gene involved in regulating the content and proportion of different fatty acid composition and pollen exine development during anther development; in this study, the transgenic plants that overexpress FAD2 will be further generated by Agrobacterium mediated-transformation based on RNAi transgenic plant, which had been obtained by our previous study. To clarify the effect of the changes in FAD2 expression levels on anther development, the transgenic plants, RNAi and overexpressed plants, were compared to the control plants. The physiological and biochemical characteristics and dynamic changes of lipid metabolites would be detected and cell morphological would be observed, and tissue distribution of the protein would be also detected during the anthers of different developmental stages. Also, the expression levels of FAD2 and the related key genes will be quantitatively analyzed during anthers development. It would clarify the critical roles of the different fatty acids and their derivatives and the interaction effects on gene expression and the fatty acid synthesis in pollen development. Furthermore, it would elucidate the synergistic effects between the FAD2 and the related key genes or signal molecules during anther development, and it would clear FAD2 gene regulatory pathways involved in the formation of the pollen exine through analysis of transcriptome and differential protein expression profiles. The results would help us to better understand the molecular regulation of plant pollen exine development and the physiological significance of the formation of fatty acid composition.

在植物中，FAD2是多不饱和脂肪酸合成的关键基因，直接决定了植物中多不饱和脂肪酸组分的含量与比例。我们在前期研究中发现，利用RNAi特异性抑制棉花FAD2在花中的表达，导致花粉外壁发育异常，进而花药败育。为探索花药发育过程中，FAD2调控花药脂肪酸代谢与花粉外壁发育的分子机制，本项目在已获得RNAi棉株基础上，拟进一步利用过表达技术获得FAD2过表达棉株。通过从生理生化检测、脂类代谢产物含量的动态变化、细胞形态学观察、蛋白的细胞组织分布和时空表达特点、相关关键基因实时表达水平等方面，深入分析FAD2基因表达水平的改变对花药发育的影响。阐明脂肪酸组分及其衍生物在花药发育中的功能，及其与相关关键基因的互作效应。利用转录组及蛋白表达谱等技术，解析FAD2与花药发育相关调控基因及信号分子的协同作用机制。研究结果将有助于对植物花粉外壁发育的分子调控及植物脂肪酸组分形成的生理学意义有进一步的深入认识。

在高等植物中，FAD2基因是控制单不饱和脂肪酸油酸（C18:1）向多不饱和脂肪酸亚油酸(C18:2)转变的关键基因，直接决定了植物中多不饱和脂肪酸组分的含量与比例。本研究首先分析了棉花FAD2家族基因：GhFAD2基因在A基因组和D基因组中均有4个拷贝。转录组分析表明，GhFAD2-1/2/3/4基因在不同组织中都存在差异，GhFAD2-2和GhFAD2-4基因在陆地棉各组织中都有表达，但表达量很低。GhFAD2-1基因在发育中的种子中特异高效表达，是控制种子中油酸向亚油酸转变的关键基因。而花药中GhFAD2-3的高表达则表明，由GhFAD2-3决定了花药发育中的C18:2的生成。特异性改变GhFAD2-3的表达水平，结果导致花药减数分裂到成熟阶段发育受损，而致雄性不育。GhFAD2-3沉默植株（fad2-3）花药减数分裂期细胞表型异常；与对照相比，花粉成熟期fad2-3花药中C18:1含量为41.48%，相应地增加了5倍。此外，fad2-3花药单不饱和脂肪酸与多不饱和脂肪酸的比例达到5.43，远高于对照中的0.39。研究阐明了棉花花药发育过程中脂肪酸组分的动态变化，表明了C18:2和C18:3是花药的重要结构成分，也是花药发育过程中许多其它类脂生物合成的重要底物。缺乏C18:2和C18:3会对棉花花药功能表皮和花粉外壁的形成有不利影响，这可能是由于fad2-3花粉粒在这些保护壁中的生物合成和/或孢粉素沉积不足造成的。通过对花药表皮成分分析和转录组数据的分析，表明通过调控GhFAD2的表达水平来提高油酸含量，不利于花药的发育。我们的结果研究为进一步研究植株组织的脂肪酸组成对其生长发育的生理意义提供了基础，有助于深入认识花药的发育调控机制，对培育棉花新品种等具有重要的意义。