甘蓝型油菜BnMAPK1与BnABA1互作调控油菜抗旱性的分子机理研究

MAPK plays an important role in the development and stress response in plants. At present, the research related to C group of MAPK is rare, resulting in the poor understanding to the corresponding function. The deep research on the gene function and the related regulation mechanism may provide basic information for the MAPK1 function analysis, as well as the theoretical proof for B. npaus breeding.The previous in our lab proved that MAPK1 stimulated the growth and improved resistance against various types of stress in B.napus. Additionally, MAPK1 interacted with ABA1, a protein responded to drought stress. On the aforementioned results, we will confirm the interaction through Pulldown and subcellular localization; explore the influence of the interaction of MAPK1 and ABA1 on drought-resistance by complementary verification test; decipher the mechanism underlying the interaction between MAPK1 and ABA1 adopting yeast two-hybrid system, site-directed mutagenesis, reversible phosphorylation as well as conformation analysis; draw up signal pathway involved in the regulation of BnABA1 on the drought resistance mediated by BnMAPK1 in B. napus through analysis of transcriptome, phenotype, physiological and biochemical index and molecule on B. napus induced by drought.

MAPK在植物生长发育和逆境响应中具有重要功能。目前，MAPK1等C族基因研究很少，其功能也不清楚。研究BnMAPK1基因功能及其调控机制，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。项目组前期研究发现BnMAPK1能促进油菜生长和提高多种抗性，并发现BnMAPK1与抗旱响应蛋白ABA1有互作。本项目将研究MAPK1与ABA1互作调控油菜抗旱的分子机理。用Pulldown、亚细胞共定位技术验证它们互作关系；用突变体互补技术、干旱诱导ABA1转基因以及抗旱和不抗旱油菜比较分析，明确MAPK1-ABA1与油菜抗旱的关系；用限制性片段酵母双杂、定点突变技术、可逆磷酸化蛋白活性及构象分析等，研究MAPK1与ABA1互作的分子机理；用干旱诱导转基因油菜的转录组、表形、生理生化、分子生物学分析等明确BnMAPK1介导BnABA1调控油菜抗旱的信号调控网络分子及分子机制

油菜抗逆性研究对油菜生产具有重要意义，MAPKs基因在提高植物多种抗逆性中起作用。我们已发现BnMAPK1可提高油菜抗旱、抗病、耐高温低温、耐弱光等多种抗性。本项目通过Y2H回转及荧光素酶互补试验验证了BnMAPK1与BnABA1等11个干旱相关蛋白存在互作。通过对干旱胁迫下耐旱和不耐旱油菜、BnMAPK1超量和抑制表达转基因油菜、BnABA1超量表达拟南芥的研究，确定了BnMAPK1和BnABA1基因均有显著提高油菜抗旱性的作用；采用干旱胁迫下BnMAPK1转基因油菜的转录组分析、定量蛋白组学和磷酸化蛋白组学分析，解析了BnMAPK1提高油菜抗旱性的分子机理；采用Y2H法筛选到与BnMAPK1互作的BnABA1A07和BnABA1C07的区段，再用PCR定点突变的方法鉴定出BnMAPK1对BnABA1磷酸化的双位点，BnABA1A07的Tyr607和Thr626，BnABA1C07的Tyr645 和Thr664。Y1H与Y2H试验发现BnMAPK1的转录受RHON1及PIG3调控而响应干旱胁迫应答，而BnMAPK1蛋白及同族的BnMAPK2/7可被上游的BnMKK3/9激活；BnMAPK1能磷酸化转录因子BnABF1、BnABF2、BnAREB3和BnABI5，调控ABA介导的BnABA1 的表达，同时调节BnNCED3 BnCYP707A1 和BnCYP707A2基因表达，从而参与内源ABA 信号调控。采用IP-MASS法研究发现干旱胁迫时BnMAPK1可以通过依赖于ABA信号途径与CYP20-3相互作用对干旱做出响应，同时也可以不依赖于ABA信号途径与ADH2相互作用，通过调控干旱引起的氧化还原过程提高油菜耐旱性。另外，明确了BnMAPK1的耐高温低温、耐荫的机理，BnMAPK2及KAT2基因功能。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。