参与木聚糖合成的GT43/GT47家族基因在棉纤维次生壁加厚期的功能研究

Cotton is one of the most important fiber crops. Xylan, a dominant secondary cell wall (SCW) component, has been detected in the developing fibers. In addition, the relative abundance of xylan varied between G. hirsutum and G. barbadense, suggesting that these non-cellulosic polysaccharides may be involved in determining final fiber quality. However, the precise roles of xylan in cotton fiber SCW thickening remain elusive and how xylan biosynthetic genes affect fiber development is also unknown. Our previous studies identified three xylan biosynthetic genes belonging to glycosyltransferase family 43 and 47(GT43 and GT47) respectively. They were preferentially and strongly expressed in fiber SCW thickening stage. In this study, to clarify the roles of the three genes which play in xylan biosynthesis, genes were transiently expressed in tobacco (N. benthamiana), in vitro enzyme activity assay was set up and enzyme reaction products were biochemically characterized. In addition, gene overexpression and RNAi-silencing constructs were made and introduced into cotton, the phenotypes of transgenic fiber were assayed to elucidate the roles of xylan which play during fiber SCW thickening stage. Furthermore, to deeply decipher the molecular mechanism of gene regulation, promoters of the three genes were isolated and activities were characterized in transgenic Arabidopsis and cotton. Then core cis-elements were determined by truncated promoter experiments and transcription factors which bind to the promoters were screened via yeast one-hybrid technique. Our findings not only help to understand the complex biosynthesis process of fiber xylan, but also provide new tools for genetically improving fiber quality.

棉花是重要的纤维作物。木聚糖作为一种次生壁组分，已经被发现存在于发育的棉纤维中。而且在陆地棉和海岛棉中，木聚糖的丰度是变化的，显示这种非纤维素多糖参与决定最终的纤维品质。然而木聚糖在棉纤维次生壁加厚期的具体作用还不清楚，木聚糖合成基因如何影响棉纤维发育也未知。我们在前期工作中鉴定了三个在棉纤维次生壁加厚期参与木聚糖合成的基因，分属于GT43和GT47糖基转移酶家族。本项目拟通过在烟草系统中表达纯化蛋白，分析酶活性与酶反应产物，阐明基因在参与木聚糖合成中的具体作用；构建过量表达和RNA干扰载体，转化棉花，分析转基因棉纤维表型，揭示木聚糖在棉纤维细胞次生壁加厚期的功能；分析基因启动子的活性，鉴定核心元件及筛查调控启动子的转录因子，进一步阐释木聚糖合成基因的表达调控机制。本研究结果将有助于理解棉纤维木聚糖合成的复杂生物学过程，也将为棉纤维品质分子育种提供新的理论和技术支持。

本研究系统分析了棉花基因组中GT43家族基因和GT47家族基因，选择了在20dpa纤维中特异表达的Gh_A09G1418（GhGT43A1, IRX9 homolog），GhGT47A（Gh_D13G2434, FRA8 homolog），GhGT47B (Gh_D02G1793, IRX10 homolog)基因，构建了过表达载体和RNAi载体，转化棉花。以GhGT47A RNAi转基因株系为例，和野生型相比，15DPA、18DPA和21DPA GhGT47A RNAi的棉铃不饱满，且显著变小。RNAi沉默株系的成熟棉纤维长度略微变短, GhGT47A的沉默还导致棉纤维细胞壁变薄。木聚糖特异单克隆抗体LM11的免疫荧光实验结果显示，15DPA、20DPA沉默株系棉纤维横切片的荧光信号显著变弱，表明木聚糖含量减少;与野生型相比，沉默株系纤维素荧光染料荧光信号较弱，说明纤维素含量变少。蒽酮法检测了15DPA纤维素的含量，结果显示纤维素含量减少。单糖组分分析结果显示：与野生型相比，沉默株系棉纤维AIR中鼠李糖，木糖在15DPA时较低，20DPA时较高；阿拉伯糖、半乳糖在15DPA，20DPA都较高。15DPA GhGT47A RNAi和WT的棉纤维进行了转录组测序结果显示：和木聚糖主链合成相关的IRX9,IRX10,IRX14的表达量发生下调；和木聚糖主链修饰相关的GUX1，GUX2，RWA2,RWA3，TBL33，GXM1，GXM2发生下调。其次，和次生壁纤维素合成相关的CESA4,CESA7,CESA8的表达量发生下调。此外，我们还发现GhMYB46-D13可激活GhGT43A,GhGT47B和GhGT47A启动子。GhFSN1、GhFSN3均可显著激活GhGT47A 启动子，启动子截短实验表明在GhGT47A 启动子0~ -307 bp，-307~ -627 bp区段上存在GhFSN3的结合元件。GhFSN1的结合元件在GhGT47A 启动子-150bp~ -307bp区域。荧光素酶互补实验检测发现GhGT47A既不能与其自身互作，也不能与GhGT43A1、GhGT47B互作，但GhGT43A1，GhGT43C1，GhGT47B它们之间能形成同源二聚体及异源二聚体。这些结果表明GhGT47A会通过影响木聚糖的合成进而影响纤维素的合成，进而影响纤维品质。

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