甘蔗赤霉素生物合成途径和信号转导关键组分GA20ox1、DELLA和GID1基因克隆及功能鉴定

Sugarcane is an major sugar crop harvesting stalk, and proper regulation of its stalk elongation is important in commercial production and Sugers . One of the most important functions of gibberellin is to promote stem/stalk elongation and increase plant height. GA20, DELLA and GID1 are the important components in gibberellin biosynthesis, signal recognition and conduction, have been the hot research points at home and abroad. Based on the gene cloning of GA20ox1 and GID1 fragments from sugarcane, this research proposal would like to clone the full-length sequences of GA20ox1, DELLA and GID1 genes from sugarcane (Saccharum officinarum) by homology cloning strategy and rapid-amplification of cDNA ends, and to analyze them with relative bioinformatics methods. We would use real-time fluorescence quota PCR to analyze the gene expression of GA20ox1, DELLA and GID1 in different sugarcane varieties, different tissues and abiotic stress, and to analyze their interactions and relationships between different genes. We would construct sense expression vectors and RNAi expression vector, introduce the sense expression vectors into Arabidopsis and sugarcane, and introduce the RNAi expression Vector into sugarcane. We would analyze the morphologic, agronomic, physiological characteristics and the gene expression of the transgenic plants. The proposed research will provides the important foundation for using genetic engineering means to control stalk elongation in sugarcane.

甘蔗是以收获地上茎为主的糖料作物，适当调控节间伸长的比例是决定产量和糖分的关键。赤霉素最突出的效应是促进茎的伸长。GA20、DELLA和GID1作为赤霉素合成、信号识别和传导的一个重要组成部分，对调节植物节间伸长起着重要作用，一直是国内外研究的热点。为了明确GA20ox1、DELLA和GID1基因功能及其相互作用的网络调控，本申请项目在分离到甘蔗GA20ox1、DELLA和GID1基因片段的基础上，利用同源克隆结合RACE技术扩增GA20ox1、DELLA和GID1基因全长，并对其进行生物信息学分析；定量PCR分析基因在不同甘蔗品种、不同部位和非生物胁迫下的基因表达，并分析不同基因表达调控网络；构建GA20、DELLA和GID1基因正义和hpRNA表达载体，把正义载体导入拟南芥和甘蔗，hpRNA载体导回甘蔗，并对转基因植株进行综合性状分析。为利用基因工程手段调控甘蔗节间伸长提供全新途径。

赤霉素最突出的效应是促进茎的伸长。GA3ox1、GA20ox1、DELLA 和GID1 作为赤霉素合成、信号识别和传导的一个重要组成部分，对调节植物节间伸长起着重要作用，一直是国内外研究的热点。项目通过同源克隆和RACE 技术克隆了甘蔗GA20ox1、DELLA、GID1和GA3ox1基因，它们的基因片段长度分别为1125 bp、1876 bp、1407 bp和1530 bp；对它们进行了生物信息学分析，同源性分析和聚类分析结果表明，甘蔗GA20ox1、DELLA、GID1和GA3ox1因与禾本科单子叶C4植物归为一个进化分支；利用实时荧光定量PCR 分析了它们的表达特性，组织特异性表达分析表明：GA20ox1、DELLA、GID1和GA3ox1基因在甘蔗根、茎、+1叶和幼叶中均有表达，GA3ox1和DELLA基因在+1叶中表达量最高，GID1基因在茎中表达量最高，GA20ox1基因在幼叶中表达量最高 在低温（0℃）、干旱（PEG6000）、多效唑和赤霉素4种非生物胁迫下，甘蔗GA20ox1、DELLA、GID1和GA3ox1基因均被诱导表达，其表达水平也因不同的调控机制而有所差别；成功构建GA20ox1、DELLA、GID1基因过量表达载体，并导入拟南芥，对转基因植株进行综合性状分析。为利用基因工程手段调控甘蔗节间伸长提供全新途径。