向日葵柄锈菌金属蛋白酶PhMEP1信号肽的结构分析及其功能验证  

Identification of signal peptide secretory function of metalloprotease PhMEP1 from Puccinia helianthi

作　　者：黄家英 景岚[1] 路妍 HUANG Jia-ying;JING Lan;LU Yan(College of Horticulture and Plant Protection,Inner Mongolia Agricultural University,Hohhot 010011,China)

机构地区：[1]内蒙古农业大学园艺与植物保护学院,内蒙古呼和浩特010011

出　　处：《中国油料作物学报》2024年第6期1285-1295,共11页Chinese Journal of Oil Crop Sciences

基　　金：国家自然科学基金(32060598,32160642);旱区作物逆境生物学国家重点实验室2022年开放课题(CSBAA202213);内蒙古自治区高等学校创新团队发展计划(NMGIRT2320)。

摘　　要：向日葵锈病是向日葵生产中的重要病害之一,严重影响向日葵的产量和品质。然而,向日葵锈菌的作用机理与发病机制不清晰,尚没有明确的致病关键因子,难以获得准确的防治靶标。因此,鉴定锈菌致病因子,明确其作用机理,对病害的精准控制具有重要意义。经课题组前期研究发现,向日葵锈菌胞外金属蛋白酶PhMEP1在锈菌致病过程中发挥重要作用,为揭示其致病机理,研究PhMEP1与寄主互作的位点,本文首先利用生物信息学软件对PhMEP1的信号肽序列进行分析,预测信息显示基因ORF序列区域前22个氨基酸为信号肽;然后借助酵母分泌系统信号肽筛选法通过不同的选择性介质和颜色反应来评估信号肽是否具有分泌功能。结果显示,信号肽融合蛋白的酵母转化子可以在YPRAA培养基(YPR agar medium,YPRAA)上正常生长,说明重组菌株能成功分泌蔗糖酶促使棉籽糖分解成单糖;同时将生成的单糖进行2, 3, 5-三苯基氯化四氮唑(triphenyltetrazolium chloride, TTC)显色试验,结果显示,TTC被还原为红色的三苯甲臢(triphenylformazan, TPF)。为了验证该信号肽能否在病菌与植物互作中发挥分泌功能,实验进一步利用蛋白质印迹法,检测信号肽融合蛋白在在烟草细胞中的表达情况。结果显示,在烟草质外体液(apoplastic fluid, AF)中检测到信号肽融合蛋白,说明融合蛋白在具有PhMEP1的信号肽后可向细胞间转移。以上结果均证明,金属蛋白酶PhMEP1的信号肽具有分泌活性,推测PhMEP1是由信号肽引导从胞内分泌到胞外发挥作用。Sunflower rust is one of the most important diseases of sunflower,leads to a serious decline in sun⁃flower output and quality.However,the key disease-associated genes of sunflower rust are still not clear,so,there is no specific targets for prevention.Identifying the pathogenic factors and mechanism of rust is essential for disease control strategies.Our team found that the extracellular metalloprotease PhMEP1 plays an important role in the pathogenesis of disease.In order to further clarify its pathogenic mechanism,in this study,we first identified the sites where the protein interacts with host.The transport of proteins depends on a signal sequence with complex physiological functions.The signal peptide(SP)of PhMEP1 is a signal sequence that can be cleaved,but the protein transport depends on it.The signal peptide sequence of PhMEP1 was analyzed for structure and function using bio⁃informatics software,and the results suggested that the signal peptide was the front 22 amino acids of the ORF re⁃gion.Then,the secretory function of the signal peptide was verified by using the yeast secretion system is to adopt a different media and color reactions whether the signal peptide has secretory function or not.The results showed that the yeast transformants of the signal peptide fusion protein could grow normally on YPRAA medium,the secretion of invertase and the hydrolysis of raffinose into monosaccharides were also confirmed with 2,3,5-triphenyltetrazoli⁃um chloride(TTC),which reacts with monosaccharides to form the insoluble triphenylformazan(TPF).To determine whether the signal peptide has a secretory function in the pathogen-plant interaction,it was used western blot to ex⁃pression of the signal peptide fusion protein in tobacco cells.The results showed that the signal peptide fusion pro⁃tein was detected in tobacco apoplastic fluid(AF),indicating that the fusion protein could be transferred to the inter⁃cellular after having the signal peptide of PhMEP1.These results suggest that the signal peptide of

关 键 词：向日葵柄锈菌 金属蛋白酶 信号肽 分泌活性 

分 类 号：S432.2[农业科学—植物病理学] S435.655[农业科学—农业昆虫与害虫防治]