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大豆疫霉细胞凋亡相关基因的鉴定与表达分析

引用本文：陈琳琳,耿雪晶,王雯,张雄,窦道龙,李洪连.大豆疫霉细胞凋亡相关基因的鉴定与表达分析.植物保护学报,2018,45(4):812-818

DOI：10.13802/j.cnki.zwbhxb.2018.2017017

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作者	单位	E-mail
陈琳琳	河南农业大学植物保护学院, 小麦玉米作物学国家重点实验室, 河南省粮食作物协同创新中心, 郑州 450002
耿雪晶	河南农业大学植物保护学院, 小麦玉米作物学国家重点实验室, 河南省粮食作物协同创新中心, 郑州 450002
王雯	南京农业大学植物保护学院, 南京 210095
张雄	南京农业大学植物保护学院, 南京 210095
窦道龙	南京农业大学植物保护学院, 南京 210095
李洪连	河南农业大学植物保护学院, 小麦玉米作物学国家重点实验室, 河南省粮食作物协同创新中心, 郑州 450002	honglianli@sina.com

中文摘要:为探讨大豆疫霉Phytophthora sojae细胞凋亡潜在的调控机制，根据已知的细胞凋亡蛋白，利用在线工具BLASTP、pFAM和SMART在大豆疫霉蛋白组数据库中鉴定细胞凋亡同源蛋白并构建其进化树，通过转录组数据和实时荧光定量PCR技术分析细胞凋亡相关基因在大豆疫霉生长、发育及侵染不同时期的表达情况。结果显示：在大豆疫霉中共鉴定到13个细胞凋亡同源蛋白，包括核酸内切酶G （PsNUC1）、细胞色素c （PsCYCS）、凋亡诱导因子（PsAIF）、丝氨酸蛋白酶（PsHtrA-1、PsHtrA-2和PsHtrA-3）、多聚ADP核糖聚合酶（PsPARP-1、PsPARP-2和PsPARP-3）和TatD核酸酶（PsTatD1、PsTatD2、PsTatD3和PsTatD4）。在进化上，PsNUC1、PsCYCS、PsAIF、PsHtrA-1、PsPARP-1、PsPARP-2、PsPARP-3、PsTatD1和PsTatD2与人及秀丽隐杆线虫Caenorhabditis elegans的同源蛋白亲缘关系较近，而与真菌相关蛋白亲缘关系较远，PsHtrA-2、PsHtrA-3、PsTatD3和PsTatD4与酿酒酵母Saccharomyces cerevisiae相关蛋白相似度更高，说明大豆疫霉细胞凋亡蛋白在进化中发生了较大变异。大豆疫霉细胞凋亡相关基因PsHtrA-1和PsRARP-1在孢子囊阶段诱导表达，PsHtrA-2和PsRARP-2在游动孢子阶段上调表达，PsAIF、PsHtrA-3、PsRARP-1和PsRARP-2在侵染阶段明显诱导表达，PsCYCS在侵染阶段下调表达。细胞凋亡相关基因在大豆疫霉不同阶段的表达模式有较大差异，说明细胞凋亡在大豆疫霉生长、发育及致病过程中具有重要作用。

中文关键词:大豆疫霉细胞凋亡基因鉴定表达分析

Identification and expression analysis of apoptosis-related genes in soybean stem and root rot pathogen Phytophthora sojae

Author Name	Affiliation	E-mail
Chen Linlin	Collaborative Innovation Center of Henan Grain Crops, National Key Laboratory of Wheat and Maize Crop Sciences, College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan Province, China
Geng Xuejing	Collaborative Innovation Center of Henan Grain Crops, National Key Laboratory of Wheat and Maize Crop Sciences, College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan Province, China
Wang Wen	College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, China
Zhang Xiong	College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, China
Dou Daolong	College of Plant Protection, Nanjing Agricultural University, Nanjing 210095, Jiangsu Province, China
Li Honglian	Collaborative Innovation Center of Henan Grain Crops, National Key Laboratory of Wheat and Maize Crop Sciences, College of Plant Protection, Henan Agricultural University, Zhengzhou 450002, Henan Province, China	honglianli@sina.com

Abstract:To investigate the possible mechanisms of apoptosis in soybean stem and root rot pathogen Phytophthora sojae, the apoptosis-related proteins in P. sojae were identified based on known apoptosisrelated protein sequences by using BLASTP, pFAM and SMART. Apoptosis-related homologous neighbor-joining phylogenetic trees were constructed using Mega 4.0, and apoptosis-related genes transcription levels in P. sojae growth, development and infection were examined by qRT-PCR and transcriptome data. The results showed that endonuclease G (PsNUC1), cytochrome c (PsCYCS), apoptosis inducing factor (PsAIF), serine protease (PsHtrA-1, PsHtrA-2 and PsHtrA-3), poly (ADP-ribose) polymerase (PsPARP-1, PsPARP-2 and PsPARP-3) and TatD nuclease (PsTatD1, PsTatD2, PsTatD3 and PsTatD4) were identified by bioinformatics methods in P. sojae. In the phylogenetic trees, PsNUC1, PsCYCS, PsAIF, PsHtrA-1, PsPARP-1, PsPARP-2, PsPARP-3, PsTatD1 and PsTatD2 in P. sojae were close to homolog proteins from Homo sapiens and Caenorhabditis elegans, rather than fungi, while PsHtrA-2, PsHtrA-3, PsTatD3 and PsTatD4 were closer to related proteins from Saccharomyces cerevisiae. These results suggested that apoptosis genes had undergone great changes in P. sojae evolution. Transcriptome data and real-time PCR analysis revealed that PsHtrA-1 and PsRARP-1 were highly expressed in P. sojae sporangia, and both PsHtrA-2 and PsRARP-2 were highly upregulated in zoospores. Four apoptosis-related genes, including PsAIF, PsHtrA-3, PsRARP-1 and PsRARP-2, showed a relatively higher expression level during infection stages, while PsCYCS was downregulated during infection stages. The apoptosis might function in P. sojae growth and pathogenicity.

keywords:Phytophthora sojae apoptosis gene identification expression analysis

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