CsKTNp60基因调控黄瓜微株型形成的分子机理研究

Plant type refers to the morphological characteristics and spatial arrangement of the plant and organs, which determines the photosynthetic posture of the plant and directly affects the photosynthetic efficiency of the crop. Researches about the genes related to plant type and their function play important roles in the high-efficient production of crops. Researches about molecular mechanism of cucumber plant type are very few. By chemical mutation method in our previous researches, we obtained a micro-plant type cucumber mutant ‘mp’, which has significantly shortened internode and miniaturized organs. We used Mutmap method to map and identify a candidate gene, CsKTNp60, which encodes the microtubule cleavage protein Katanin p60. Only one nucleotide base mutation was found between wild type and mp. However, the molecular mechanism of this gene regulating the cucumber plant type is still unknown. In this project, we plan to carry out the analysis of gene expression, genetic transformation and TILLING technology to verify the candidate gene CsKTNp60, clarifying its regulation mechanism for micro-plant type of cucumber. Further, through in vivo / in vitro microtubule binding experiments and RNA-seq analysis, we will investigate deeply the biological function of microtubule cleavage protein in cucumber cell and plant architecture formation. The expected results from this project will not only enrich the theoretical knowledge of cucumber plant architecture formation and provide reliable theoretical basis for the ideal plant type breeding of cucumber in future, but also provides an important reference for the study of the metabolism and regulation mechanism of plant microtubule cleavage protein.

株型是指植物形态特征及各器官的空间排列方式，决定着植株受光姿态，直接影响作物的光合效率。株型相关基因及调控机制的研究对作物高效生产具有重要的理论和实践意义。黄瓜株型相关分子机制研究较少，申请者前期通过诱变得到黄瓜微株型突变体‘mp’，植株节间缩短且各器官表现微型化，利用Mutmap方法定位并克隆获得微株型关键候选基因CsKTNp60，该基因可编码微管剪切蛋白Katanin p60，突变体仅一个碱基变异，但该基因调控黄瓜株型的分子机制仍未知。本项目拟通过表达分析、遗传转化及TILLING等技术验证候选基因CsKTNp60，解析该基因的株型调控功能；同时通过体内/体外微管结合实验、转录组分析等方法明确微管剪切蛋白在黄瓜细胞形态建成及株型调控中的生物学功能。研究结果不仅可以丰富黄瓜株型形成的理论知识，为培育黄瓜理想株型新品种提供理论依据，同时也为研究植物微管剪切蛋白的代谢及调控机制提供重要参考。

株型是影响作物光合效率及栽培方式的关键因素。本项目以诱变获得的黄瓜微株型突变体mp为材料，在前期图位克隆的基础上围绕微管剪切蛋白CsKTNp60调控黄瓜微株型形成的分子机理开展了相关研究。利用连锁图谱开发了有利于微株型黄瓜分子辅助育种所能使用的分子标记MSNP-7，可作为微株型黄瓜表型连锁的分子标记应用于实际生产中；荧光定量分析显示CsKTNp60基因在突变体各组织中的表达量显著低于野生型；亚细胞定位发现野生型及突变型CsKTNp60蛋白均表达于烟草叶片细胞的细胞膜、细胞质及细胞核中；CsKTNp60基因的RNAi沉默表达阳性的黄瓜植株表现出矮化等生长发育缺陷表型，表明该基因可调控黄瓜株型的发育；微管体外共沉淀及剪切试验表明CsKTNp60蛋白可在体外结合和剪切微管，且该蛋白氨基酸的突变使得蛋白结构域构象发生改变，致使其结合和切割微管的能力下降；微管相关性试剂体外喷施试验证实微管状态可以影响野生型及mp突变体的株型变化；通过对黄瓜酵母cDNA文库进行筛选获得了5个可能与CsKTNp60互作的蛋白，涉及到萜类物质合成、MAPK信号级联、RNA转运以及甲基化依赖的染色质沉默等多个调控通路；转录组学及代谢组学联合分析表明CsKTNp60调控黄瓜株型发育涉及多基因多代谢物的联合调控，富集分析表明CsKTNp60的突变影响多个与植物生长发育相关的代谢通路，包括萜类物质合成、植物激素积累、细胞生长发育、光合作用等调控通路。本项目研究成果阐释了黄瓜微株型性状产生的遗传机制，为黄瓜微株型的分子设计育种奠定了理论基础，并提供了可直接利用的分子标记，具有重要的育种实践价值；另外提出了CsKTNp60参与黄瓜株型建成的调控模型，并解析了微管剪切蛋白在黄瓜株型调控中的生物学功能，为黄瓜株型改良提供理论依据。