致病疫霉木瓜蛋白酶亚家族基因C1A-PH-SCH1的克隆与表达分析

刘霞, 张哲, 钱红洁, 张利杰, 杨艳丽

中国农学通报. 2021, 37(29): 13-19

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中国农学通报 ›› 2021, Vol. 37 ›› Issue (29) : 13-19. DOI: 10.11924/j.issn.1000-6850.casb2020-0693
生物科学

致病疫霉木瓜蛋白酶亚家族基因C1A-PH-SCH1的克隆与表达分析

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Cloning and Expression Analysis of the Papain-like Cysteine Proteases Subfamily Gene C1A-PH-SCH1 of Phytophthora Infestans

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摘要

为进一步明确引起马铃薯晚疫病的致病因子,通过克隆马铃薯晚疫病致病疫霉菌分泌蛋白中的半胱氨酸蛋白酶家族基因,构建该基因的原核表达载体,在大肠杆菌BL21中诱导表达。本试验采用TA克隆法,提取来自云南马铃薯产区的致病疫霉菌XH05-5-4的总RNA,反转录cDNA,PCR扩增后将该基因片段连接到载体,转化大肠杆菌BL21并诱导表达。克隆获得半胱氨酸蛋白酶家族基因全长为1176 bp,包含1个最大的开放阅读框1077 bp,编码387个氨基酸。推测蛋白相对分子量41.671 kD,理论等电点4.88;经IPTG诱导和SDS-PAGE检测,所构建的原核表达载体表达的蛋白与预期蛋白相符合。生物活性测定表明:马铃薯叶片出现坏死,类似于晚疫病症状。利用TA克隆技术从致病疫霉菌XH05-5-4中克隆得到了1个半胱氨酸蛋白酶家族基因(登录号:KP938956命名:C1A-PH-SCH1),通过同源性比对发现C1A-PH-SCH1为木瓜蛋白酶亚家族基因,成功构建了原核表达载体,并使其在大肠杆菌中得到了表达,对生物活性测定结果显示出现了类似于晚疫病症状的水渍坏死斑症状,推测C1A-PH-SCH1参与马铃薯晚疫病的发病过程。

Abstract

To further clarify pathogenic factor of potato late blight, we cloned the cysteine protease family gene in the secreted protein of Phytophthora infestans causing potato late blight and express in E. coli BL21. In this study, TA cloning method was used to extract total RNA of P. infestans XH05-5-4 of Yunnan, synthesize cDNA by reverse transcription and connect gene fragment to the vector after PCR amplification. Then we induced the expression of it in E. coli BL21. The full length of cysteine protease family gene is 1176 bp, containing 1077 bp open reading frame (ORF) encoding 387 amino acids. Predicted by bioinformatics analysis, its protein molecular weight is 41.671 KDa, isoelectric point is 4.88. After IPTG induced expression and SDS-PAGE identification, the protein expressed in the constructed prokaryotic expression vector is consistent with the expected protein. The results of biological activity examination show that potato leaves have necrosis and this symptom is similar to potato late blight. A cysteine protease family gene (GenBank Accession: KP938956, name: C1A-PH-SCH1) was cloned from P. infestans XH05-5-4 by TA cloning method. It is a papain enzyme subfamily gene by homology comparison, and the prokaryotic expression vector is successfully constructed and expressed in E. coli BL21. The symptom is similar to watery necrosis of potato late blight, therefore, it is speculated that C1A-PH-SCH1 is involved in the pathogenic process of potato late blight.

关键词

马铃薯 / 致病疫霉 / 木瓜蛋白酶 / C1A-PH-SCH1基因 / 克隆

Key words

potato / Phytophthora infestans / papain-like cysteine proteases / C1A-PH-SCH1 gene / clone

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导出引用
刘霞 , 张哲 , 钱红洁 , 张利杰 , 杨艳丽. 致病疫霉木瓜蛋白酶亚家族基因C1A-PH-SCH1的克隆与表达分析. 中国农学通报. 2021, 37(29): 13-19 https://doi.org/10.11924/j.issn.1000-6850.casb2020-0693
Liu Xia , Zhang Zhe , Qian Hongjie , Zhang Lijie , Yang Yanli. Cloning and Expression Analysis of the Papain-like Cysteine Proteases Subfamily Gene C1A-PH-SCH1 of Phytophthora Infestans. Chinese Agricultural Science Bulletin. 2021, 37(29): 13-19 https://doi.org/10.11924/j.issn.1000-6850.casb2020-0693

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基金

国家重点研发项目“西南区马铃薯化学肥料和化学农药减施技术模式集成与示范”(2018YFD0200808)

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