辣椒疫霉中一个具有转录因子序列特征的外泌蛋白的分析鉴定

Identification and characterization of a secreted protein with sequence characters of transcriptional factors from Phytophthora capsici

植物病原细菌分泌核调控蛋白进入寄主细胞核调控其基因表达以利于自身的侵染,但目前对卵菌中该类蛋白知之甚少。前期生物信息学分析从辣椒疫霉中获得一个具有DNA结合域的外泌蛋白PcSEP9,利用qRT-PCR检测其在辣椒疫霉侵染前期(菌丝、游动孢子囊、游动孢子、休止孢萌发)和侵染阶段[灌根接种本氏烟(Nicotiana benthamiana)后1.5、3、6、12、24、36、72 h]的基因转录水平,发现该基因在辣椒疫霉的生长发育和侵染阶段均表达上调;对该基因进行TA克隆测序,对其编码的蛋白质进行结构域预测,发现其含有信号肽、核定位信号、DNA结合域和锌指RBZ结构域;将其信号肽的核苷酸编码序列克隆至酵母信号肽诱捕载体pSUC2T7M13ORI上,并转化到酵母蔗糖酶缺陷菌株YTK12中,发现重组菌株能成功分泌蔗糖酶,促使棉籽糖分解成单糖,且在YPRAA培养基上可正常生长,同时生成的单糖能与2,3,5-triphenyltetrazolium chloride反应产生红色不溶于水的氯化三苯基四氮唑,明确了该蛋白质的信号肽具有分泌活性;在本氏烟中进行瞬时表达,发现该蛋白质全长和成熟肽均能稳定表达,但对辣椒疫霉的毒力无明显影响。这一研究拓宽了对卵菌外泌蛋白质的认识,为进一步探索该类蛋白质的生物学功能及其作用机制提供了重要的试验数据。

Plant pathogenic bacteria secrete nucleomodulins into the plant nucleus to manipulate host gene expression for facilitating their infection and colonization.At present,little is known about the oomycetic nucleomodulins.Our previous bioinformatic work detected one secreted Phytophthora capsici protein PcSEP9 containing DNA-binding domains.In this study,transcriptional profile of the gene Pcsep9 was analyzed using qRT-PCR technique during the pathogen development(mycelia,sporangia,zoospores and germinating cysts)and host plant infection(1.5,3,6,12,24,36 and 72 h post-inoculation of Nicotiana benthamiana roots)stages.The results showed that the gene was up-regulated during the pre-infection and infection stages.After full-length cloning,analysis of the derived amino acid sequence demonstrated that the protein contained the signal peptide(SP),one nuclear localization signal,two DNA-binding domains and one zinc finger RBZ domain.The predicted SP sequence together with the subsequent two amino acids was fused in-frame to the mature sequence of yeast invertase(SUC2)in the vector pSUC2 T7 M13 ORI,which was then transformed into yeast invertase deficient strain YTK12.The result showed that the fusion of SP to SUC2 enabled the successful secretion of invertase,thus enabling the yeast cells to hydrolyze raffinose into monosaccharides and grow on YPRAA medium.The monosaccharides can be detected by 2,3,5-triphenyltetrazolium chloride,forming red precipitation.The data confirmed that the SP was functional.The protein with or without its SP was transiently expressed in N.benthamianaleaves using agroinfiltration and detected by Western blot.However,its ectopic expression didn’t affect the pathogen’s virulence on N.benthamiana.This study expands our knowledge about oomycete secreted proteins,and provides essential experimental data for further understanding the roles of these proteins involved in the pathogenicity of the pathogens.