红肉苹果MdMKK9互作蛋白的筛选与鉴定

Screening and Identification of MdMKK9 Interacting Proteins in Red-Fleshed Apple

MKK9是丝裂原活化蛋白激酶(MAPK)家族重要成员之一,是在植物细胞响应外界胁迫信号转导中发挥重要作用的酶。为深入研究苹果MKK9的相关功能,以红肉苹果‘黛红’为材料,克隆到MdMKK9基因CDS序列,利用酵母双杂交试验筛选苹果cDNA文库,并通过酵母双杂交筛选、体内双分子荧光互补(BiFC)和体外Pull-down试验进行MdMKK9互作蛋白验证。结果表明,通过对苹果cDNA文库的筛选,共获得11个参与调控植物生长发育及应答逆境胁迫的候选互作蛋白;选取花青苷合成相关蛋白MdCHS及氮胁迫调控相关蛋白MdSPX3进行酵母双杂交、体内BiFC及体外Pull-down试验,结果证明MdMKK9可与MdSPX3互作。

MKK9 is an important member of the mitogen-activated protein kinase(MAPK)family,which plays an important role in signal transduction of plant cells in response to external stresses.In order to study the functions of apple MKK9,the MdMKK9 gene was cloned from the red-fleshed apple‘Daihong’,and the apple cDNA library was screened by yeast two-hybrid test.The MdMKK9 interaction protein was verified by yeast two-hybrid verification,in vivo bimolecular fluorescence complementarity(BiFC)test and in vitro pull-down test.The results showed that through screening the apple cDNA library,a total of 11 candidate interaction proteins involved in the regulation of plant growth and development and the response to adversity stresses were obtained.The anthocyanin synthesis-related protein MdCHS and nitrogen stress-related protein MdSPX3 were selected for yeast two-hybrid,in vivo BiFC and in vitro Pull-down assays.The results showed that MdMKK9 could interact with MdSPX3.