海岛棉GbHCT基因在棉花纤维发育中的功能及分子机制研究

Poor quality of cotton fiber has become a major obstacle to the sustainable development of the cotton industry. Island cotton contains high-quality fiber genes, which provide gene resources for the improvement of upland cotton fiber. The transcriptome of Gossypium barbadense transcriptome revealed that many genes including HCT in lignin metabolism were expressed in different stages of fiber development. The expression of GbHCT gene was higher in cotton fiber at 5 d and 25 d suggests that GbHCT may be involved in the development of cotton fiber elongation and secondary wall thickening. In this study, the effect of HCT catalyzed product caffeoyl-CoA on the development of cotton fiber was analyzed. The upstream transcription factors of GbHCT gene were identified. The function of GbHCT gene in the formation of cotton fiber quality was clarified by analyzing the expression of GbHCT-overexpressing and GbHCT-suppressing Gossypium barbadense fiber and the expression of related genes of lignin metabolism pathway; Metabolomics was used to analyze the content and composition of lignin H-monomer and G / S-monomer in GbHCT transgenized Gossypium barbadense, and to reveal the changes of GbHCT expression and the composition and content of lignin monomer influences on the fiber quality. Through the above studies, the function and mechanism of GbHCT in cotton fiber development were clarified to provide new candidate functional genes for cotton fiber quality improvement.

棉花纤维品质偏差成为制约棉花产业可持续发展的主要障碍。海岛棉中蕴含着优质纤维基因，为陆地棉纤维改良提供基因资源。前期通过对海岛棉转录组测序发现木质素代谢中包括HCT基因在内的多个基因在纤维发育的不同时期表达，GbHCT基因在开花后5d和25d的棉纤维中表达量较高，暗示其可能参与棉纤维伸长期和次生壁加厚期的发育。本研究分析HCT催化产物咖啡酰辅酶A对棉纤维发育的影响；鉴定GbHCT基因的上游转录因子；通过分析过表达和抑制GbHCT的海岛棉纤维品质及木质素代谢途径相关基因的表达，明确GbHCT基因在棉纤维品质形成中的功能；利用代谢组学方法分析转GbHCT的海岛棉中木质素H-单体与G/S-单体的含量和组成的变化，揭示GbHCT表达量的变化与木质素单体组成和含量对纤维品质的影响。通过以上研究明确GbHCT在棉花纤维发育中的功能和作用机制，为改良棉纤维品质提供新的候选功能基因。

棉纤维发育过程中存在苯丙烷代谢途径，研究HCT基因引起木质素变化从而探究对棉纤维发育的影响。本研究利用棉花基因组数据库，从全基因组范围筛选鉴定HCT基因并对海岛棉GbHCT基因进行功能验证，以及对上游启动子区域进行分析筛选互作转录因子，主要研究结果如下：.鉴定发现海岛棉中共有15个HCT基因，先后从海岛棉中克隆到GbHCT12、GbHCT13、GbHCT14和GbHCT15基因，转化拟南芥发现GbHCT13、GbHCT15基因使植株生长旺盛，抽薹数和茎秆表皮毛数量显著增加，茎秆初生木质部生长加快，HCT基因代谢通路中其他相关基因表达量发生不同程度波动变化。在棉花中沉默GbHCT13、GbHCT15基因，使得植株中木质素含量总体降低，茎秆切片显示木质部导管细胞数量显著减少，细胞壁木质素积累量减少，茎秆表皮毛数量减少。GbHCT13沉默使4CL基因表达量极显著增加，CAD、CCoAOMT、CCR、PAL表达量降低，GbHCT15基因沉默使通路基因在ND203中呈现出极显著波动变化。在棉花中过表达GbHCT13基因可使的棉纤维明增长，而转GbHCT15基因使得棉纤维长度显著增加。.通过棉花胚珠离体实验发现HCT基因代谢产物咖啡酰辅酶A能够在1μmol/L浓度下达到促进胚珠生长及纤维伸长的最佳效果，咖啡酰辅酶A的表达与GbHCT13基因正相关，而GbHCT15从纤维发育起始至伸长期表达量逐渐降低，在纤维成熟阶段表达量增加，初步推测其主要参与促进纤维发育起始和纤维细胞壁的成熟。.对GbHCT12和GbHCT14基因启动子进行克隆，通过启动子预测分析发现ABA响应元件、MYBHv1结合位点等，通过GUS染色技术确定GbHCT12和GbHCT14启动子具有启动活性。从6份纤维长度极端的材料克隆GbHCT14基因启动子克隆，经序列比对发现相似度为96.54%，在同源区域内有13处差异位点，并且在转录起始位点上游70bp内为没有存在差异碱基。.同时构建了海岛棉cDNA文库，通过酵母单杂交技术，筛选到与GbHCT14基因启动子互作的16个候选转录因子。利用生物信息学软件预测到候选基因参与细胞壁的伸长、泛素化反应、细胞的分裂、花发育以及果实成熟的过程，其中GbM_A09G1247与WAK2为同源基因，WAK2与果胶结合后促进细胞壁的伸长，而HCT基因则是影响果胶合成的因素之一。