甘蓝型油菜BnMAPK1基因促进油菜生长发育的分子机理研究

MAPK plays an important role in the development and stress response in plants. MAPK, belonging to group C, is studied sporadically. The BnMAPK1 gene is the very example. The identification of its function and the corresponding regulation mechanism may provide reference for the analysis of its function, even lay foundation for the utilization of the gene in molecule breeding in B. napus. The previous study results in our lab showed that the gene can obviously promote the growth and development of oilrape. We cloned the gene and obtained the genetics-modified plants carrying the over-expression vector or RNAi vector. Based on the exciting phenomenon, the coming research will focus on the uncovering the molecular mechanism underlying the promotion effect. The downstream-regulating gene of BnMAPK1 will be identified based on the analysis of the gene expression profile. The comparison, aiming at the morphological, cellular and physiological difference between the control and the gene-modified plants, will be finished to disclose the molecular regulation mechanism. The proteins interacted with the BnMAPK1 will be screened out through yeast two-hybrid. The interaction will be tested in vitro and in vivo with GST pull-down and co-IP techniques respectively. The subcellular localization of the corresponding interacted proteins will be carried out to verify the substrates of BnMAPK1 protein.

MAPK在植物生长发育和逆境响应中具有重要功能。目前，MAPK的C组基因研究很少，MAPK1的功能也不清楚。研究BnMAPK1基因功能及其表达调控机制，可为植物MAPK1功能解析提供基础资料，为油菜分子育种提供理论依据。项目组前期从油菜中克隆了BnMAPK1基因，获得超量和RNAi载体转化植株，发现BnMAPK1能明显促进油菜的生长发育。在此基础上，本项目将继续深入研究BnMAPK1促进油菜发育的分子机理：通过表达谱分析鉴定BnMAPK1调控表达的基因；比较对照、BnMAPK1超量和RNAi转基因油菜的形态学、细胞学、生理学特性，揭示BnMAPK1的分子调控机制；用酵母单杂交和EMSA分析BnMAPK1的上游调控基因；用酵母双杂交筛选BnMAPK1互作蛋白，以GST pull-down和co-IP进行体外及体内验证；并对互作蛋白进行亚细胞定位，明确BnMAPK1的作用底物。

甘蓝型油菜是我国最重要的油料作物之一，其生长及抗性是科学家关注的重点。MAPK在植物生长发育和逆境响应中具有重要功能。我们已发现BnMAPK1能明显促进油菜生长。本项目通过研究BnMAPK1超量和抑制表达植株和野生型植株生长发育特点，发现BnMAPK1可促进油菜营养器官生长、提前开花，增加生物量，但对籽粒产量无显著影响；BnMAPK1提高油菜叶片叶绿素含量、增加光合速率和根系活力，提高叶片细胞分裂素和赤霉素水平；显微观察发现BnMAPK1超量表达可增加幼叶的细胞数，促进成熟叶细胞扩大；表达谱分析发现BnMAPK1可调控与细胞增殖和分化相关基因（F18O22_180、CPC等）、组织和花发育基因（DI21、AGL71等）的表达，调控内源激素合成及信号转导，从而促进油菜生长。我们还发现BnMAPK1可提高油菜抗旱性、抗病性、耐热性、耐荫和耐强光性，其主要机理是BnMAPK1能快速响应逆境信号，及时诱导抗性相关基因（LEA、Btg-26等）表达，提高其转录表达水平，参与调控逆境信号传导和激素代谢，促进抗逆物质合成及积累，降低逆境伤害，提高油菜抗逆性。采用酵母单杂技术分析BnMAPK1上游调控因子，确定了BnMAPK1核心启动子（160bp），筛选并验证了4个可与其特异结合的转录因子（BnMAPK1-SnRK、BnMAPK1- Rho、BnMAPK1-ZnB和BnMAPK1-FRL-3），它们分别与植物抗逆性、信号转导、蛋白质合成和开花时间相关。用酵母双杂技术筛选BnMAPK1下游互作蛋白，得到42个与其互作的蛋白，其中12个与生长发育相关，15个与非生物胁迫相关，7个与生物胁迫相关，3个与光强度相关，还有与多种信号传导通路相关的基因。另外，明确了BnMAPK1亚细胞定位（细胞核和细胞质），原核表达得到活性较高的蛋白。本研究可为植物MAPK1功能解析提供基础资料，为油菜分子育种提供理论依据。