大豆疫霉细胞凋亡相关基因的鉴定与表达分析

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

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。

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, p FAM 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 q RT-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.