水稻寡分蘖短根矮秆基因msd1的克隆与功能分析

Rice architecture is a key trait affecting rice yield. Cloning and functional identification of the genes controlling rice architecture are of biological significance in improving rice yield. A mutant with monoculm, no tiller, short roots, and dwarf was characterized and thus designated as msd1 (monoculm, short-root, dwarf 1). Genetic analysis indicates that msd1 mutant is genetically controlled by a recessive single gene. Combination the map-based cloning strategy and phenotyping both the T-DNA insertion lines that requested from Korea (http://signal.salk.edu/cgi-bin/RiceGE) and silenced plants by RNA interference of the candidate genes in the target interval, a sucrose synthesis-related gene was taken as the putative candidate gene for the msd1. Based on these data, the full-length WT MSD1 and mutant msd1 will be isolated and the sequence difference between the WT MSD1 and mutant msd1 will be obtained by BLAST; then the WT MSD1 complementation construct will be generate to carry out the complementation test in the mutant.Following the target gene conferred, the promoter region of the WT MSD1 will be cloned and inserted into the expressional vectors with GFP or GUS reporter gene, respectively and transgenic plants are generated to carry out the analysis of the expression patterns and the biological function for the WT MSD1. Meanwhile, the differential content of the main intermediates of the sucrose biosynthesis and the starch as well as the differential form of the starch granule between the WT and mutant are analyzed to confirm that the phenotype of the mutant msd1 is caused by deficiency in synthesizing sucrose. These studies will better understand the formation of mutant msd1 phenotype and meanwhile improve genetic breeding of yield trait in rice.

水稻株型是构成水稻产量的重要性状。克隆和鉴定控制水稻株型的重要功能基因对提高水稻产量具有重要的理论和现实意义。本申请项目在获得一份由隐性单基因控制的水稻寡分蘖短根矮秆突变体msd1的基础上，综合运用图位克隆技术以及T-DNA插入突变分析和RNA干涉分析，确定了一个蔗糖合成相关的基因是msd1的目标基因。在此基础上，本申请项目拟克隆野生型MSD1和突变体msd1基因全长并获得野生型和突变体基因间的序列差异信息；构建MSD1的互补载体转化突变体的转基因遗传互补确认得到msd1基因；克隆野生型MSD1启动子，构建GUS和GFP报道基因载体进行遗传转化分析MSD1基因表达模式及生物学功能；通过比较野生型和突变体间蔗糖合成及转化途径的主要中间产物含量差异和淀粉粒的大小及形态变化，确认突变体的蔗糖合成功能缺陷。通过本项目的研究期望解析突变体msd1株型的成因，为水稻产量性状遗传育种提供理论指导。

水稻株型是构成产量的重要因素，是育种家广为关注的性状。水稻的分蘖、根长和株高是水稻株型的重要组成部分。本研究建立在通过田间观察所获得的一份根极短、只有主茎而无任何分蘖、植株矮小等自发突变体基础上，通过基因定位、T-DNA插入突变、RNA干涉分析、MSD1的功能互补、MSD1基因表达，确定控制突变体性状的基因是一个蔗糖合成相关基因，根据该基因的编码产物预测，添加外源的蔗糖及其分解产物葡萄糖和果糖回复了突变体的表型，这些结果暗示msd1植株的蔗糖合成可能受阻。但野生型和突变体之间的蔗糖含量以及蔗糖合成前体物淀粉和蔗糖分解产物葡萄糖和果糖的含量没有差异。这些与预期相反的结果促使我们通过构建淀粉粒会被灰飞虱取食而减少这样的平台以及应用基因芯片分析水稻应答灰飞虱取食间接验证msd1突变体中MSD1的编码产物不是作为碳源为细胞提供合成新化合物的碳骨架以及为细胞的呼吸代谢提供底物与能源，MSD1编码的产物可能是糖信号传递途径中的重要一员，而后的野生型和突变体水稻基因表达芯片结果也佐证了这一推测。本项目的实施，为理解水稻植株形态的建成提供了新的认识，也将扩展我们可溶性糖调控水稻株型的认识。