楸子叶片酵母文库构建及甘油二酯激酶MpDGK7互作蛋白鉴定

Construction of Yeast Library in Malus prunifolia Leaves and Identification of Diacylglycerol Kinase MpDGK7 Interacting Proteins

为探究MpDGK7参与调控苹果对非生物胁迫响应的机制,本研究通过构建苹果属植物楸子(Malus prunifolia)叶片的酵母双杂交cDNA文库,筛选与甘油二酯激酶MpDGK7互作的蛋白并进行验证。结果显示,本研究构建的cDNA文库库容达到1.5×107 CFU,重组率100%,平均插入片段长度大于1200 bp。利用MpDGK7构建诱饵载体pBT3-N-MpDGK7,质粒无毒性且不存在自激活,通过共转化方法从文库中筛选到13个与MpDGK7互作的蛋白。选取4个代表性互作蛋白(KAN4、MYC2、BTB/POZ和金属硫蛋白)进行点对点验证,发现它们均与MpDGK7发生互作。双分子荧光互补(BiFC)验证结果表明,MpKAN4与MpDGK7互作主要发生在细胞膜和细胞核上,MpBTB与MpDGK7互作主要发生在细胞膜上。因此,推测MpDGK7可能通过参与KAN4、MYC2、BTB/POZ和金属硫蛋白等介导的胞内信号转导过程来影响苹果对逆境胁迫的响应。本研究结果为进一步阐明DGK基因参与苹果抗逆分子调控的机理奠定了基础。

In order to explore the possible mechanism of MpDGK7 involving in the regulation of apple response to abiotic stress,this study constructed a yeast two-hybrid cDNA library of Malus prunifolia leaves,screened and verified the proteins interacting with the glycerol diesterase kinase MpDGK7.The results showed that the cDNA library constructed in this study reached 1.5×107 CFU,the recombination rate was 100%,and the average inserted fragment length was greater than 1200 bp.The bait vector pBT3-N-MpDGK7 was nontoxic and did not have self-activation.Thirteen proteins interacting with MpDGK7 were screened from the library via co-transformation method.Four representative interacting proteins(KAN4,MYC2,BTB/POZ and metallothionein)were selected for point-to-point verification,and all of them were found to interact with MpDGK7.Bimolecular fluorescent complementation(BiFC)showed that the interaction between MpKAN4 and MpDGK7 mainly occurred on the cell membrane and nucleus,and the interaction between MpBTB and MpDGK7 mainly occurred on the cell membrane.Therefore,it is speculated that MpDGK7 may affect the response of apple to stress by participating in intracellular signal transduction mediated by KAN4,MYC2,BTB/POZ and metallothionein.The results of this study laid a foundation for further elucidation of the mechanism of DGK gene involved in molecular regulation of apple stress resistance.