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, 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.