StCDPK29和StNOXC在ASM促进马铃薯块茎愈伤中的功能研究

Wound healing plays an important role in reducing postharvest disease of potato tubers; however, the duration is longer for wound healing naturally. Our previous results indicated that acibenzolar-S-methyl (ASM) promoted wound healing of tubers, and StCDPK29 and StNOXC had a significant contribution; however, the detailed mechanism remains unclear. In this study, the expressions of StCDPK29 and StNOXC genes by over expression and interference are used to clarify the function of the two genes and their relationship during wound healing of tubers elicited by ASM. In addition, the interaction of StCDPK29 and StNOXC is assayed by the methods of yeast two-hybrid, bimolecular fluorescence complementation, pull down and Co-IP. The phosphorylation level of StCDPK29 and StNOXC is analyzed by using polyclonal antibody in vivo. The fusion proteins of StCDPK29 and StNOXC are obtained by prokaryotic expression, which are used for analysis the their phosphorylation level in vitro. Moreover, the phosphorylation sites of StCDPK29 and StNOXC are identified by mass spectrometry. Based on the above experiments, the functions of StCDPK29 and StNOXC induced by ASM in wound healing of potato tubers will be clarified at molecular, biochemical and cell levels. The results will provide a theoretical basis for further understanding mechanism of wound healing in potato tubers promoted by ASM.

愈伤在减轻马铃薯块茎采后腐烂中具有重要作用，但自然愈伤所需时间较长。我们研究发现，苯丙噻重氮(ASM)可促进块茎愈伤，StCDPK29和StNOXC发挥了重要作用，但详细机制不清。本研究拟通过过表达和干扰表达StCDPK29和StNOXC基因，明确这两个基因在ASM诱导块茎愈伤中的功能及其相互关系。利用酵母双杂交、双分子荧光互补、pull down及Co-IP等方法，分析StCDPK29和StNOXC的相互作用；通过制备多克隆抗体，分析StCDPK29和StNOXC的体内磷酸化水平；利用原核表达获得StCDPK29和StNOXC融合蛋白，进行体外磷酸化试验，并利用质谱技术鉴定StCDPK29和StNOXC的磷酸化位点。通过以上研究，在分子、生化和细胞水平解析ASM诱导马铃薯块茎愈伤中StCDPK29和StNOXC的功能，为深入揭示ASM促进的马铃薯块茎愈伤机理提供理论依据。

愈伤是马铃薯块茎的重要采后特性，通过在伤口处形成愈伤组织可有效减少水分蒸腾，抑制病原菌通过伤口侵染。但马铃薯块茎自然愈伤速率较慢，需要采取措施加速块茎愈伤。我们前期研究发现，苯并噻重氮(ASM)可通过激活活性氧代谢来促进马铃薯块茎愈伤，但详细分子机理不明。本项目用100mg/L ASM处理‘陇薯3号’马铃薯块茎，在观察愈伤期间伤口处Ca2+分布和胞内Ca2+浓度，分析StCDPKs和StRbohs的表达情况的基础上，选取关键基因StCDPK29和StRbohC构建过表达和干扰表达载体，通过农杆菌侵染转化法获得转基因植株和块茎，在亚细胞、生化和分子水平上研究了不同转基因块茎愈伤过程中软木脂的积累以及ROS的产生，并验证了StCDPK29和StRbohs之间的互作情况，同时以StCDPK29为诱饵蛋白筛选了愈伤组织cDNA文库，为揭示ASM处理促进马铃薯块茎快速愈伤的分子机理提供了相关依据。结果表明：（1）ASM诱导了愈伤组织胞内Ca2+浓度，促进了CDPKs的转录水平，增加了CDPK激酶浓度和H2O2的积累；（2）通过农杆菌侵染转化法成功获得StCDPK29和StRbohC的转基因植株和块茎，发现过表达块茎损伤部位的软木脂和木质素积累明显高于野生型，且StRbohs的表达发生上调变化，同时O2-和H2O2含量也显著高于野生型，而干扰表达后，愈伤组织中的软木脂和木质素沉积受到抑制，StRbohs不同程度地下调表达，O2-和H2O2含量也相对低于野生型；（3）互作分析发现，StCDPK29与StRbohB在细胞膜上互作，而与StRbohC在细胞核内互作。短截StRbohB和StRbohC的EF-手型结构域分别与StCDPK29互作结果表明，StCDPK29可在StRbohB和StRbohC N-端的EF-手型结构域发生互作；（4）以StCDPK29为诱饵蛋白，筛选核（膜）蛋白酵母双杂交cDNA文库，分别获得了15个蛋白，这些蛋白主要涉及生物或非生物胁迫、木质素生物合成、脂肪酸代谢和能量代谢过程以及软木脂单体的转运等。综上所述，ASM处理可通过诱导马铃薯块茎伤口处组织的Ca2+信号来激活下游的ROS产生元件参与马铃薯块茎的快速愈伤。