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

doi:10.11924/j.issn.1000-6850.casb2020-0693

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

所属专题：生物技术

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¹^,²(), 张哲¹^,², 钱红洁¹^,², 张利杰¹^,², 杨艳丽¹^,²()

¹云南农业大学植物保护学院,昆明 650201
²云南省植物病理重点实验室,昆明 650201

收稿日期:2020-11-20 修回日期:2021-04-22 出版日期:2021-10-15 发布日期:2021-10-29
通讯作者: 杨艳丽
作者简介:刘霞,1980年出生,山东青岛人,副教授,博士,研究方向为马铃薯抗性及病害机理研究。通信地址：650201 云南昆明盘龙区金黑路95号云南农业大学植物保护学院,Tel：0871-65228404,E-mail： liuxia_0213@163.com。
基金资助:
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)

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

Liu Xia¹^,²(), Zhang Zhe¹^,², Qian Hongjie¹^,², Zhang Lijie¹^,², Yang Yanli¹^,²()

¹College of Plant Protection, Yunnan Agricultural University, Kunming 650201
²Key Laboratory of Plant Pathology of Yunnan, Kunming 650201

Received:2020-11-20 Revised:2021-04-22 Online:2021-10-15 Published:2021-10-29
Contact: Yang Yanli

摘要/Abstract

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。

关键词: 马铃薯, 致病疫霉, 木瓜蛋白酶, C1A-PH-SCH1基因, 克隆

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.

Key words: potato, Phytophthora infestans, papain-like cysteine proteases, C1A-PH-SCH1 gene, clone

中图分类号:

S435.32

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[J]. 中国农学通报, 2021, 37(29): 13-19.

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[J]. Chinese Agricultural Science Bulletin, 2021, 37(29): 13-19.

图/表 8

图1. PCR扩增图谱 M:DNA marker(DL2000);1:目的片段

图2. 半胱氨酸蛋白酶基因最大ORF核苷酸序列及其编码的氨基酸序列绿色：起始密码子和终止密码子;蓝色：活性位点Cys,His,Asn和Gln;红色：N-糖基化位点

图3. 信号肽预测

图4. 亲水性/疏水性预测结构

图5. 跨膜螺旋结构预测

图6. 二级结构

图7. IPTG诱导大肠杆菌表达C1A-PH-SCH1重组蛋白的SDS-PAGE电泳结果

图8. 生物活性测定

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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

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

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