【作者】 胡林；

【导师】 裴炎； 肖月华；

【作者基本信息】 西南大学 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 棉花是世界上最重要的经济作物。棉花的纤维品质和产量与棉花生产的效益和产值直接相关,均是棉花育种的主要目标。由于遗传基础狭窄,产量和品质相互间存在负相关等原因,利用传统育种手段同时改良棉花纤维产量和品质相当困难。在克隆棉花纤维发育关键基因的基础上,利用基因工程技术改良棉花纤维产量和品质是目前研究的新策略和热点。赤霉素(Gibberellins, GA)是一种非常重要的植物激素,参与调控植物生长发育的各个过程。前期研究表明,GA在棉花纤维发育过程中有着重要的作用。植物体内生物活性GA水平是合成代谢和分解代谢平衡调节的结果。GA2-氧化酶催化的C-2羟基化使活性GA失活并进入分解代谢,是调控活性GA关键步骤。为了解GA2-氧化酶对棉花纤维发育的影响,本论文对棉花纤维中的GhGA2ox2基因进行了初步的生物功能研究。首先在烟草中超量表达该基因,验证其生物学功能。其次,构建种皮特异启动子BAN控制下的GhGA2ox2RNAi,并转化棉花,观察GhGA2ox2基因表达下调对棉花纤维发育的影响。主要结果如下：1、构建了35S::GhGA2ox2超量表达载体,转化烟草得到转基因植株。与野生型相比,转基因植株表型出现矮化、节间缩短、开花延迟等GA缺失性状,同时转基因烟草植株的性状变化与GhGA2ox2基因表达水平密切相关。说明该基因编码产物具有GA2-氧化酶活性,并参与了烟草体内的GA代谢过程中。2、构建种皮特异启动子BAN控制下的GhGA2ox2RNAi载体,并转化棉花得到转基因植株。RT-PCR分析表明,对野生型对照相比,在0DPA的转基因植株的胚珠中和15DPA的纤维中,GhGA2ox2基因表达下调。扫描电镜观察发现,转基因植株与野生型相比,在0DPA的胚珠表皮细胞突起无明显差异。成熟纤维检测结果表明转基因植株的纤维性状,与野生型相比无明显差异。但在15和18DPA,转基因植株纤维中次生壁信号基因的表达发生提前升高,推测是内源GA含量增加促进次生壁合成提前。更多还原

【Abstract】 Cotton is one of the most important economic crops in the world. Fiber quality and yield, directly related to the benefit and outputvalue of cotton production, have been the main breeding targets of cotton. Due to the narrow genetic basis and negative relatedness of quality nand yield traits, it is vey difficult to improve these traits simultaneously by traditioanal breeding methods. Recently, many efforts are focused on a new strategy to improve fiber quality and yield by bioengineering techniques on the bassis of cloning genes key to fiber development.GA (Gibberellins, GA) is an essential plant hormone involved in regulation of various processes of plant growth and development. It was previously reported that GA played an important role in the development of cotton fiber. Plants control the endogenous level of bioactive GA by anabolism and catabolism. GA2-oxidase represents a crutial step in GA catabolism, for it catalyzes the hydroxylation on C-2 to deactivate GAs and convert GA pathway to degradation. To understand the functions of GA 20-oxidase in fiber development, biological functions of a GA 2-oxidase gene, GhGA2ox2, which is preferentially expressed in elongating fibers, were studied. Firstly, GhGA2ox2 was overexpressed in tobacco. Secondly, an RNAi construct of GhGA2ox2 was linked downstream to a seed-specific promoter (BAN), and transformed into cotton.The main results were as followings:1. The overexpression vector 35S::GhGA2ox2 was constructed and transformed into tobacco. Compared with wild-type tobacco, transgenic plants showed GA-deficient phenotypes, such as dwarfism, shortened internodes and delayed flowering time. Meanwhile, the GA-deficient phenotypes were consistent with GhGA2ox2 expression levels in the transgenic plant. These results suggested that the GhGA2ox2 gene encoded a functional GA2-oxidase, which participated in GA metabolism in transgenic tobaccos.2. The seed-specific BAN-driven GhGA2ox2RNAi vector was constructed and transformed into cotton. RT-PCR analysis showed that the expression level of GhGA2ox2 genes were down-regulated in 0-DPA ovules and 15-DPA fibers, compared to wild-type cottons. Scanning electron microscopy revealed no significant difference on the surface of 0-DPA ovule between transgenic and wild-type plant. At maturaturation, no ditiguishable difference in fiber traits was found between the transgenic and the wild-type cottons. When comparing the expression of several secondary wall signal genes in fibers of 15 and 18 DPA, the expression level of some genes incrased earlier in the transgenic fibers, implying that increased endogenous GA level promoted secondary wall synthesis.更多还原