含DUF1645保守结构域的水稻粒型、粒重调控基因OsXCL分子作用机理研究

Grain size and shape are important determinants of grain yield and quality in cereal crops, which are usually controlled by non-allelic genes and quantitative trait loci (QTLs). OsXCL, encoding a nucleus-localized DUF1645 protein of unknown function. Overexpression of OsXCL greatly and positively affected an array of traits, including increases in grain length, grain weight and grain number per panicle, resulting in a significant increase in grain yield. In our previous study, the function and molecular analyses of OsXCL were investigated mainly at the transcriptional leve. The obtained data showed that the enhanced OsXCL expression in rice promoted cell division and grain filling. A large number of genes involved in plant cell cycle and cytokinin signaling processes were induced and suppressed in the OsXCL-overexpressing plants, suggesting that OsXCL may regulate cell growth by acting via a cytokinin signaling pathway. OsXCL encode a DUF1645 domain-containing protein.Three putative member of the DUF1645 protein familys are cloned from maize, sorghum and wheat. Similar to OsXCL, Overexpressions of the three OsXCL-like genes are significantly enhanced gain length and grain weight in transgenic rice plants. Further analysis showed that the intactness of DUF1645 domain seems critical for these genes to function in grain length and weight increases. The following experimental studies will be performed in this work: functional analysis of OsXCL by overexpressing and CRISPR/Cas9 gene knockout in transgenic rice lines, yeast two-hybrid, biomolecular fluorescence complementation analysis and EMSA analysis, spatial and temporal gene expression pattern assessment of expression of genes regulated by OsXCL. The main research objectives are to analyze the functions of the OsXCL related to grain size development, explore its mechanism of action, andrevealing the possible function and molecular mechanism of plant conserved DUF1645, and provide a theoretical reference and candidate gene to develop new high quality and yield rice varieties.

粒形是重要的产量性状和品质性状，阐明粒形遗传基础和分子机理有利于同时改良水稻的产量和品质。在已完成的基金委项目中，主要从基因表达调控水平解析水稻OsXCL基因功能，OsXCL正向调控水稻粒长、粒重，改良穗形、株形，改善米质。该基因可能通过CK信号传导途径正调控下游响应基因，促进细胞有丝分裂和轴向生长，调控水稻生长发育。进一步研究发现，该基因编码区包含一个未知功能结构域DUF1645，不同作物编码DUF1645的基因也能正调控水稻粒长、粒重，其完整性对粒长、粒重调控似乎是必须的。因数据库中无合适突变体，本项目拟在现有研究基础上，完成CRISPR/Cas9无义突变株表型及功能分析、酵母双杂交、BiFC、EMSA、OsXCL蛋白质特性分析，进一步阐明OsXCL可能的分子作用机制、DUF1645及其不同模块对粒形、粒重的调控功能，为高产、优质、“理想株型”超级稻分子选育提供新的候选基因和理论参考。

粒形是千粒重最主要的决定因素，千粒重是遗传力最高的水稻产量构成因子；干旱是对我国农业生产影响最大的气象灾害，而水稻生产又是农业生产中受干旱危害最大的农作物。本研究项目主要结果包括：（1）水稻OsSGL基因是一个极为关键的水稻生长发育调控基因，特别是在生殖生长时期，对水稻剑叶形态构成、花粉育性、颖花发育等均起调控作用，抑制该基因的表达，或者降低其表达量并不能影响基因功能，但基因的缺失，蛋白质构象的改变则对水稻生长发育有较大影响。（2）DUF1645结构域中存在粒长、粒重的核心调控区，该结构域不同程度的片段缺失会影响水稻粒长、粒重调控这一生物学功能。其功能域C端结构的完整性对粒长调控这一生物学功能是必须的。（3）结合基因芯片数据和qReal- time PCR两者数据结果，对OsSGL基因过量表达引起的水稻基因组表达水平差异进行分析，初步筛选受OsSGL调控的下游基因；（4）OsSGL基因是水稻中特有且功能极其保守的一个基因，在禾本科植物中含有DUF1645功能域的OsSGL同源基因氨基酸序列相似、表达模式相似，其生物学功能也极其相似。（5）结合转录组和qReal- time PCR两者数据结果，在禾本科植物中含有DUF1645功能域的OsSGL同源基因功能极其保守，并且有相同或极其相近的功能调控分子作用途径。我们首次阐明禾本科植物DUF1645未知功能域有正向控制粒长、粒重、耐旱性及叶片形态构成的保守功能，且有相近的分子作用途径。挖掘新的水稻粒形、耐旱性调控相关基因，并深入相关基因功能及机制的研究将为水稻高产、耐逆分子育种提供候选基因资源和良好的理论基础。