烟草疫霉PnRXLR5863的克隆及信号肽鉴定

Cloning of PnRXLR5863 and Identification of Its Signal Peptide from Phytophthora nicotianae

烟草疫霉引起的剑麻斑马纹病是剑麻生产的主要病害之一。RXLR效应蛋白是疫霉菌中数量最多的一类效应蛋白。为了分离鉴定烟草疫霉中对剑麻产生致病作用的RXLR效应蛋白,本研究基于生物信息分析结合PCR技术克隆候选基因,并利用酵母信号肽捕获系统对信号肽进行功能研究。本研究从烟草疫霉中克隆了一个编码RXLR效应蛋白的基因,命名为PnRXLR5863;该基因长度为402个碱基,编码的蛋白含有134个氨基酸;分子量为15.46 kD,等电点为5.27,是一个疏水性蛋白;SignalP 4.0预测该蛋白含有一个长度为23个氨基酸的信号肽;进化分析结果显示,PnRXLR5863与来自烟草疫霉的蛋白亲缘关系最近;实验结果表明,PnRXLR5863的信号肽,能够使YTK12酵母菌株在CMD-W和YPRAA培养基上生长,并且能将TTC溶液转变为砖红色,表明PnRXLR5863的信号肽具有分泌活性。本研究的结果为进一步研究烟草疫霉侵染剑麻过程中Pn RXLR5863的致病功能提供依据。

Zebra disease,caused by the pathogen Phytophthora nicotianae,is one of the most devastating sisal oomycete diseases.RXLR protein is the most abundant effector in Phytophthora.To isolation and identification the candidate genes from Phytophthora nicotianae,the PCR technology was used to clone the candidate genes based on bioinformatics.The signal peptide of candidate protein was detected via yeast signal peptide capture system.In this study,a candidate gene was cloned and named PnRXLR5863.PnRXLR5863 was 402 amino acids in full length,encoding 134 amino acids with molecular mass of 15.46 kD and pI of 5.27,it is a hydrophobic protein.SignalP 4.0predicted that the protein contained a signal peptide with a length of 23 amino acids.Phylogenetic analysis showed that PnRXLR5863 had the closest relationship with the protein from Phytophthora nicotianae.The experimental results showed that the signal peptide of PnRXLR5863 could make YTK12 yeast strain grow on CMD-W and YPRAA medium,and could convert TTC solution into brick red,indicating that the signal peptide of PnRXLR5863 had secretory activity.These results suggest that signal peptide of PnRXLR5863 might have a signal peptide activity,and play an important role in the secretion of PnRXLR5863 out of the cell.