棉花GhGolS1基因在花药和棉纤维次生壁发育中的功能研究

We had cloned GhGolS1 from a cotton gene cluster involved in cotton reproduction development, the gene encodes galactinol synthase and it is the key enzyme in raffinose family oligosaccharides (RFOs) metabolic pathway. Preliminary studying found the function of GhGolS1 is different from other people's research result, it's not involved in resistance physiology, seed physiology or photosynthate translocation, but involved in anther and the secondary cell wall of fiber development, and the result of bioinformatics analysis reveal the gene structure of GhGolS1, GhAPm and GhNeuF is very conserved in different plant. Based on the above reasons, we plan thorough research GhGolS1. first of all, we will detect anther development, fiber quality and other important agronomic traits of GhGolS1 transgenic overexpress cotton and RNAi cotton; secondly, we will study the GhGolS1 transcription in the transgenic lines and the cotton CMS three lines by the real-time PCR and northern blot in situ hybridization; thirdly, we will use stereomicroscope, scanning electron microscope and high performance liquid chromatography to detect the change of structure of relative tissue and cell and RFOs metabolic pathway resulted by GhGolS1 up-regulated and down-regulated; finally, we will identify the interaction relation between GhGolS1,GhAPm and GhNeuF by bimolecular fluorescence complementation , yeast two hybrid and real-time PCR. Based on the above study, we will can reveal the molecular mechanism of the function of GhGolS1 in the anther and the secondary cell wall of fiber development, and we will identify GhGolS1 interaction relation with GhAPm and GhNeuF. These will lay the foundation for research the whole gene cluster.

我们从棉花生殖发育相关基因簇中克隆了GhGolS1，其是调控棉子糖半乳糖苷系列寡糖RFOs代谢的限速酶，初步研究发现该基因并非像前人发现的那样参与抗性生理、种子生理或光合产物运输，而是与花药和纤维次生壁发育相关，且该基因与基因簇中GhAPm和GhNeuF的排列结构在进化中非常保守，因此本项目拟重点研究GhGolS1，检测该基因的过表达和RNAi干扰转基因棉花花药发育、纤维品质和农艺性状；通过荧光实时定量PCR和原位杂交了解该基因在转基因材料和棉花CMS三系材料中的表达情况，用体式显微镜、扫描电镜和高效液相色谱检测因该基因上下调引起的花药、花粉和纤维结构及RFOs代谢途径的变化，通过双分子荧光互补、酵母双杂交和荧光实时定量PCR验证该基因与GhAPm和GhNeuF的关系。阐明GhGolS1在花药和纤维次生壁发育过程中的分子机理，揭示该基因与其它基因的互作关系，为研究整个基因簇功能打下基础。

我们从棉花生殖发育相关基因簇中克隆了GhGolS1，其是调控棉子糖半乳糖苷系列寡糖RFOs代谢的限速酶，初步研究发现该基因并非像前人发现的那样参与抗性生理、种子生理或光合产物运输，而是与花药和纤维次生壁发育相关，且该基因与基因簇中GhAPm和GhNeuF的排列结构在进化中非常保守，因此本项目拟重点研究了GhGolS1，检测构建了该基因的过表达和RNAi干扰载体并转化了棉花，筛选纯化获得了转基因植株，检测了转基因棉花花药发育、纤维品质和农艺性状；通过荧光实时定量PCR发现其与棉花花粉败育无关，通过双分子荧光互补试验验证了该基因与同一基因簇中的GhAPm和GhNeuF的表达无关，和原位杂交了解该基因在转基因材料和棉花CMS三系材料中的表达情况，用体式显微镜、扫描电镜和高效液相色谱检测了因该基因与花药、花粉和纤维发育以及RFOs代谢之间的关系上下调引起的花药、花粉和纤维结构及RFOs代谢途径的变化，通过双分子荧光互补、酵母双杂交和荧光实时定量PCR验证该基因与GhAPm和GhNeuF的关系。为阐明GhGolS1在花药和纤维次生壁发育过程中的分子机理，揭示该基因与其它基因的互作关系，为研究整个基因簇功能打下基础。