鞘脂及GhKDSRs基因在棉花纤维伸长中的功能和调控机制

As a class of essential membrane components and bioactive molecules, sphingolipids are important for fluidity and permeability of membrane, and the function of membrane-binding proteins. Cotton fibers are extremely elongated single cells. Fiber elongation entails membrane expansion and a series of physiological and biochemical activity enhancement. Our previous studies indicated that inhibiting sphingolipid biosynthesis through FB2 treatment, a sphingolipid synthetic inhibitor, severely suppressed fiber elongation on cultured cotton ovule. Furthermore, the GhKDSR1gene was isolated from elongating fibers, which encodes a 3-ketodihydrosphingosine reductase (KDSR) involved in sphingolipid biosynthesis and specifically expressed in fiber cell, particularly in rapid elongating fiber. The ligon lintless-1(li-1) mutant, characterized by abnormal lint fiber development, bears very short lint fibers similar with the fuzz fibers and was employed as an ideal material to study fiber elongation. The expression level of GhKDSR1 gene was remarkably down-regulated in 10 DPA fibers of li-1 mutant compared with its isogenic wild-type line, TM-1. These results revealed that the GhKDSR1 gene and sphingolipids play important roles in fiber elongation. However, the functions and regulatory mechanism of sphingolipids and GhKDSR1 gene in fiber elongation are largely unknown. This project, to address this issue, we plan to determinate the content of major sphingolipids at various developmental stages of fiber cells, up-regulate or down-regulate the expression level of GhKDSR1 in transgenic fibers, characterize the GhKDSR1 promoter by 5'-deletion assay and screen the transcription factors binding to the promoter, and analyze digital gene expression profiling in transgenic fibers and wild type fibers. These studies would reveal the functions and regulatory network of sphingolipids and GhKDSR1 gene in fiber elongation. The results could be beneficial to further clarify the molecular mechanism of fiber development. The gene and the fiber-specific promoter might be useful in gene engineering to improve the fiber quality.

鞘脂是生物膜重要的组成成分，对膜的流动性、通透性和膜结合蛋白功能具有调控作用。纤维细胞是一个极度伸长的单细胞，纤维伸长过程中伴随着细胞膜面积的急剧扩张和一系列代谢活动的增强，鞘脂在纤维伸长中可能具有重要作用。课题组前期研究发现，抑制纤维中鞘脂合成能够显著抑制纤维伸长，进而筛选到纤维中特异表达的鞘脂合成酶基因GhKDSR1，该基因在超短纤维突变体的纤维伸长阶段表达量明显降低，表明该基因和鞘脂与纤维伸长关系密切。本项目拟通过棉花遗传转化和检测纤维发育过程中鞘脂成分及含量的动态变化，分析转基因纤维伸长期基因表达谱变化和GhKDSR1启动子表达特性，明确纤维伸长中基因表达调控-GhKDSR1-鞘脂合成-纤维发育-响应基因及代谢途径的对应关系，阐明鞘脂及GhKDSR1在纤维伸长中的功能和调控机制。为深入解析纤维发育调控机制提供新的数据，为纤维改良基因工程提供纤维特异启动子和目标基因。

棉花是重要的天然纤维作物，在我国国民经济中占有重要地位。我国棉花纤维的品质偏低、类型单一，严重阻碍了我国棉花产业的发展。棉花纤维是胚珠外珠被表皮细胞经极性伸长和次生壁加厚而形成的单细胞纤维，极性伸长和次生壁加厚过程是纤维产量和品质的形成过程，但其发育的调控机理还知之甚少。本研究分析了细胞膜的重要组成成分——鞘脂在纤维发育过程中的动态变化以及抑制纤维中鞘脂合成对纤维发育的影响；通过克隆棉花纤维中特异或优势表达的鞘脂合成酶基因GhKDSR1-1和GhKDSR2-1以及GhGCS1，构建植物表达载体和棉花遗传转化，获得超量表达和抑制目标基因的转基因棉花，分析了转基因纤维的性状变异和纤维细胞中鞘脂组分和含量的变化，以及基因表达谱的变化；进一步检测了转基因纤维中植物固醇合成酶基因的表达变化和调控植物固醇关键合成酶基因的转基因纤维中鞘脂相关基因的表达变化。.通过这些研究证明含有VLCFA的复杂鞘脂分子在纤维伸长期高量积累，在次生壁合成期降低，抑制鞘脂的合成严重抑制纤维细胞伸长。超量表达GhKDSR1-1使纤维长度增加15.12%，相反，抑制该基因表达导致纤维长度减少21.12%；超量表达GhGCS1可是纤维长度增加达26.01%，相反，抑制GhGCS1表达导致纤维长度降低10.47%。超量表达GhGCS1基因促进纤维细胞鞘脂合成，两种鞘脂终产物GIPC和GCS的含量显著增加，上游产物显著减少。在超量表达GhGCS1的纤维细胞中植物固醇和油菜素内酯合成代谢关键基因显著升高，相反，在抑制GhGCS1基因的纤维中植物固醇、油菜素内酯合成代谢及信号传导关键基因的表达显著降低。同样，在植物固醇变化的转基因纤维细胞中鞘脂合成酶基因的表达也发生显著变化。这些研究结果揭示了鞘脂在纤维伸长中具有重要作用，其作用机制是通过影响植物固醇的合成以及油菜素内酯的合成代谢和信号而调控纤维细胞发育。本研究结果不仅揭示了纤维发育过程中鞘脂-植物固醇-细胞膜-植物激素之间的内在关系，而且为改良棉花纤维产量品质提供了优良目标基因和转基因材料。