利用酵母双杂交筛选木薯果胶甲酯酶抑制因子MePMEI1的互作蛋白

Screening of Pectin Methylesterase Inhibitor Me PMEI1-interactive Proteins in Cassava(Manihot esculenta) Using Yeast Two-hybrid System

果胶甲酯酶抑制因子(PMEI)在植物抗逆过程中具有重要作用。为了探究木薯MePMEI1的生物学功能及其在蛋白互作网络中的作用,本研究通过构建诱饵载体pGBKT7-MePMEI1,通过酵母双杂交方法从木薯cDNA文库中筛选Me PMEI1的互作蛋白。结果表明,酵母双杂交共筛选出57个阳性克隆,阳性率约为32.6%,测序鉴定出37种含完整开放阅读框(ORF)的候选互作蛋白,包括非特异性丝氨酸/苏氨酸蛋白激酶/苏氨酸特异性蛋白激酶、叶绿素a/b结合蛋白、电压依赖性阴离子通道蛋白(VDAC2)、乙二醛酶Glyoxalase-2、氮素调节蛋白、木糖葡萄糖转移酶(TCH4)等,并通过回转验证检验其蛋白互作的真实性。验证结果表明Me PMEI1与VDAC2、Glyoxalase-2蛋白具有相互作用。这进一步补充了其蛋白互作网络,为探究MePMEI1蛋白的互作网络提供新的依据。

Pectin methylesterase inhibitor(PMEI) plays an important role in the process of plant stress resistance.In order to explore the biological function of MePMEI1 in cassava and its role in protein interaction network, in this paper, the bait vector pGBKT7-MePMEI1 was constructed in cassava and the interaction proteins of MePMEI1 were screened in cassava c DNA library by yeast two-hybrid. The results showed that a total of 57 positive clones were screened, and the positive rate was about 32.6%. About 37 kinds candidate interaction proteins with complete open reading frame(ORF) were identified by sequencing, including non-specific serine/threonine protein kinase, threonine specific protein kinase, chlorophyll a/b binding protein, voltage dependent anion channel protein2(VDAC2), nitrogen regulatory protein, xyloseglucose transferase(TCH4), Glyoxalase-2 protein, etc. The authenticity of protein interaction was verified by rotary verification. The results showed that MePMEI1 interacted with VDAC2, glyoxalase-2 protein, which further supplemented its protein interaction network, and provided new evidence for exploring the interaction network of MePMEI1 protein.