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【目的】通过对草菇组学数据的分析,获得一个谷胱甘肽S-转移酶(GSTs)编码基因(vv-gto1),对其基因结构、序列特征及表达情况进行分析,探索GSTs在食用菌生长发育过程中的作用机制,并为其他食用菌GSTs编码基因的发现提供依据。【方法】应用ZOOM软件将基因组与转录组的测序读段(reads)与基因组拼接序列进行定位确定基因全长及其序列准确性,并对基因结构进行可视化分析。采用MEGA 5.1进行多序列比对及进化树分析,对草菇GTO1进行功能归类及同其他真菌的亲缘关系分析。结合表达谱、蛋白质组数据分析该基因在草菇不同生长时期的差异表达,并应用荧光定量PCR进行验证。【结果】vv-gto1全长2083bp,含有11个外显子、10个内含子,编码356个氨基酸;5’端非翻译区长度为305bp、包含1个内含子区域,3’端非翻译区为86bp;RNA加工过程中有两个内含子存在保留现象,由内含子保留所形成的转录本均无法翻译形成正确的保守功能域。vv-gto1全长序列都获得超过50个reads的准确定位,证明该区段的基因组测序及组装结果正确可靠。进化树结果显示,草菇GTO1属于Omega类第I小类谷胱甘肽S-转移酶,与黄孢原毛平革菌的GTO1、GTO2亲缘关系最近。数字基因表达谱、荧光定量PCR及蛋白质组的分析结果表明,vv-gto1在异核体菌丝H1521的表达量最高。【结论】本研究首次在草菇中获得一个Omega类谷胱甘肽S-转移酶编码基因,该基因在异核体菌丝生物学功能的行使过程中可能起到重要作用,同时也暗示草菇异核体菌丝具备较强的抗逆性。另外,vv-gto1可以通过形成不同的可变剪接体来调节基因的转录和翻译,最终影响蛋白质的功能行使。
【Objective】 Glutathione S-transferase (GSTs) encoding gene (vv-gto1) was obtained through the analysis of Herbivora japonica. The gene structure, sequence characteristics and expression of GSTs were analyzed. Edible mushroom growth and development of the mechanism of action, and for other edible fungi GSTs gene to provide the basis for the discovery. 【Method】 The ZOOM software was used to locate the genome sequence and transcriptome sequencing reads and genome splicing sequences to determine the full-length of the gene and its sequence accuracy. The gene structure was visualized. MEGA 5.1 was used for multiple sequence alignment and phylogenetic tree analysis to classify the fungi GTO1 and analyze the genetic relationship with other fungi. The expression profiles and proteomic data were used to analyze the differential expression of this gene at different growth stages of Volvariella volvaceae and the results were verified by real-time PCR. 【Result】 The full length of vv-gto1 was 2083bp, containing 11 exons and 10 introns, encoding 356 amino acids. The 5 ’untranslated region was 305 bp in length and contained 1 intron region and 3’ The translation region is 86bp. There are two introns in the RNA processing process. The transcripts retained by intron can not be translated to form the correct conserved domains. The accurate positioning of more than 50 reads of the full-length vv-gto1 sequence proved that the genome sequencing and assembly results of the segment were correct and reliable. Phylogenetic tree analysis showed that GTO1 belongs to glutathione S-transferase class I of Omega class I, and has the closest genetic relationship with GTO1 and GTO2 of Phanerochaete chrysosporium. Digital gene expression profiling, fluorescence quantitative PCR and proteomic analysis showed that vv-gto1 had the highest expression level in heterokaryotic hyphal H1521. 【Conclusion】 This study is the first to obtain a Omega-type glutathione S-transferase gene in grass mushroom. This gene may play an important role in the biological function of heterokaryon mycelium, and also implies that grass Mushroom heterokaryotic mycelium with strong resistance. In addition, vv-gto1 can regulate the transcription and translation of genes through the formation of different alternative splicing and ultimately affect the function of the protein.