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G蛋白偶联受体(G protein-coupled receptors,GPCRs)广泛存在于动物、植物以及微生物中,是最大的涉及信号转导的膜受体家族。本研究通过基于隐马尔科夫模型的HMMER 3.0软件搜索玉米大斑病菌(Setosphaeria turcica)基因组数据库,鉴定并获得GPCRs超家族基因及其基因组定位;采用MEGA 6.0软件进行系统进化分析;通过通用样品数据系统(generalized sample data system,GSDS)工具进行基因结构分析;利用Inter Pro和SMART工具分析GPCRs的保守结构域。进一步利用q RT-PCR技术,对所确定的GPCR基因在分生孢子发育形成侵染结构的过程中不同阶段的相对表达量进行分析。结果表明,在玉米大斑病菌基因组中至少有9个典型的GPCR,其中3个属氮源感应因子类,信息素受体和碳源感应因子类各2个,类环磷酸腺苷受体和真菌视蛋白类各1个。这些基因散布在玉米大斑病菌基因组中,且结构复杂多样,但所有成员编码产物均由N端、7个跨膜结构域、3个胞内环、3个胞外环及C端组成。每个跨膜结构域包含20~25个氨基酸。以分生孢子为对照,所有基因在附着胞成熟时期接种后12 h(12 hours post inoculation,12 hpi)均显著下调(P<0.05);除基因St Rtc1和Stfdd123以外,全部基因整体变化趋势均呈现上调/持平(3 hpi)-上调(6 hpi)-下调(12 hpi)-上调(24 hpi)的规律,尤其在侵入丝形成时期(24 hpi)显著上调(P<0.05)。在附着胞形成时期(6 hpi),仅St Ste2p和St Rtc2表达显著上调。St Rtc1和Stfdd123在各阶段中的表达量与对照差异不显著或显著降低。本研究为深入解析植物病原真菌GPCR超家族的功能提供了理论依据。
G protein-coupled receptors (GPCRs) are widely found in animals, plants and microorganisms and are the largest family of membrane receptors involved in signal transduction. In this study, HMMER 3.0 software based on Hidden Markov Model was used to search the database of Setosphaeria turcica genome to identify and obtain the GPCRs superfamily genes and their genomes. Phylogenetic analysis was performed using MEGA 6.0 software. (GSDS) tools were used to analyze the genetic structure; the conserved domains of GPCRs were analyzed using Inter Pro and SMART tools. The q RT-PCR technique was further used to analyze the relative expression levels of the identified GPCR genes in different stages of the process of conidial development and infiltration. The results showed that there were at least 9 typical GPCRs in the genome of S. turcica, of which 3 were nitrogen-sensing factors, 2 for pheromone receptors and 2 for carbon-source induction, and 5 for cyclic adenosine monophosphate Fungal opsin each one. These genes are interspersed in the genome of S. turcica and are complex and diverse in structure. However, all the members encode the product consisting of N-terminal, 7 transmembrane domains, 3 intracellular loops, 3 extracellular loops and C-terminal. Each transmembrane domain contains 20-25 amino acids. With conidia as control, all genes were significantly down-regulated at 12 h post-inoculation (12 hpi) at all stages of attachment maturation (P <0.05), except for genes St Rtc1 and Stfdd123 (3 hpi) - up - regulation (6 hpi) - down regulation (12 hpi) - up regulation (24 hpi), especially up - regulation in invasive silk formation (24 hpi) (P <0.05). At the attachment phase (6 hpi), only St Ste2p and St Rtc2 expression were significantly up-regulated. The expression levels of St Rtc1 and Stfdd123 in each stage were not significantly different or significantly decreased compared with the control. This study provides a theoretical basis for further analysis of the function of plant pathogenic fungi GPCR superfamily.