秀珍菇转录组测序和初步分析
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摘要
[目的]本文旨在了解秀珍菇子实体形成的分子机制,为培育优质、高产、抗逆的秀珍菇品种提供理论基础。[方法]构建秀珍菇高质量c DNA文库,采用illumina高通量测序技术对秀珍菇菌丝体和子实体进行转录组测序,并利用生物信息学方法开展基因表达谱的研究以及功能基因的预测。[结果]共获得81 693个非重复序列基因(universal gene,Uni Gene),经BLAST比对NR数据库,可比对上的Uni Gene数量为61 306个。NR数据分析显示,秀珍菇已比对的Uni Gene与糙皮侧耳相似度最高,占总数目的 86.1%。同时,对秀珍菇转录组的Uni Gene进行了生物学通路的注释和预测,共注释14 315个Uni Gene,且部分与糖类代谢、氨基酸代谢、信号传导、翻译相关通路有关,分别为1 683、1 241、1 243和1 372个,表明富集在上述通路中的Uni Gene在秀珍菇子实体形成过程中可能起到重要作用。通过分析SNP突变,发现秀珍菇中分别有10 967和10 683个位点发生C/T转换和A/G转换,同时,有2 102个位点发生A/C颠换。通过查找分析SSR位点发现,共获得7 574个SSR位点,占Uni Gene总数的比例为9.27%。其中,单核苷酸重复在所有SSR重复类型中所占比例最高,为45.14%,其次为三核苷酸重复类型,为31.94%。[结论]通过高通量测序技术获得秀珍菇菌丝体和子实体的转录组数据,结合生物信息学分析,为了解秀珍菇群体遗传多样性、构建遗传连锁图谱和研究其不同生长阶段差异表达基因及其功能奠定基础。
[Objectives]This paper aimed to understand the molecular mechanism of fruiting body formation of Pleurotus pulmonarius and provide theoretical basis for breeding high-quality,high-yield and stress-resistant varieties of P. pulmonarius. [Methods]In this study,on the basis of building high quality c DNA library of P. pulmonarius,the transcriptome of mycelium and fruiting body of P. pulmonarius was sequenced by illumina high-throughput sequencing technology. In addition,bioinformatics methods were used to study gene expression profiles and predict functional genes. [Results]It showed that 81 693 Uni Genes were obtained through sequence splicing and 61 306 Uni Genes were annotated through NR database by BLAST comparison. By statistical species distribution,P. pulmonarius transcriptome had the most similar Uni Genes to Pleurotus ostreatus. Meanwhile,a total number of 14 315 Uni Genes were identified by annotating and forecasting the biological pathways for the transcriptome of P. pulmonarius. Among14 315 Uni Genes,parts of their biological functions were related to carbohydrate metabolism( 1 683),amino acid metabolism( 1 241),signal transduction( 1 243) and translation( 1 372). It was noted that these Uni Genes enriched in afore-mentioned pathway might play a very important role in the process of fruiting body formation of P. pulmonarius. 10 967 C/T and 10 683 A/G conversion,2 102 A/C transversion were found by SNP analysis. Meanwhile,7 574 SSR in 81 693 Uni Genes were found which took 9.27% of the total number of Uni Genes. The characteristic of SSR distribution showed that mono-nucleotide repeat was the highest,which was45.14% of the total,followed by tri-nucleotide repeat,which was 31.94%. [Conclusions]The transcription group data were obtained of the mycelium and fruiting body of P. pulmonarius by high-throughput sequencing technology. Combined bioinformatics analysis,this study laid the foundation for analysing the genetic diversity,constructing genetic linkage map,and researching the differentially expressed genes and its functions at different developmental phases of P. pulmonarius.
[Objectives]This paper aimed to understand the molecular mechanism of fruiting body formation of Pleurotus pulmonarius and provide theoretical basis for breeding high-quality,high-yield and stress-resistant varieties of P. pulmonarius. [Methods]In this study,on the basis of building high quality c DNA library of P. pulmonarius,the transcriptome of mycelium and fruiting body of P. pulmonarius was sequenced by illumina high-throughput sequencing technology. In addition,bioinformatics methods were used to study gene expression profiles and predict functional genes. [Results]It showed that 81 693 Uni Genes were obtained through sequence splicing and 61 306 Uni Genes were annotated through NR database by BLAST comparison. By statistical species distribution,P. pulmonarius transcriptome had the most similar Uni Genes to Pleurotus ostreatus. Meanwhile,a total number of 14 315 Uni Genes were identified by annotating and forecasting the biological pathways for the transcriptome of P. pulmonarius. Among14 315 Uni Genes,parts of their biological functions were related to carbohydrate metabolism( 1 683),amino acid metabolism( 1 241),signal transduction( 1 243) and translation( 1 372). It was noted that these Uni Genes enriched in afore-mentioned pathway might play a very important role in the process of fruiting body formation of P. pulmonarius. 10 967 C/T and 10 683 A/G conversion,2 102 A/C transversion were found by SNP analysis. Meanwhile,7 574 SSR in 81 693 Uni Genes were found which took 9.27% of the total number of Uni Genes. The characteristic of SSR distribution showed that mono-nucleotide repeat was the highest,which was45.14% of the total,followed by tri-nucleotide repeat,which was 31.94%. [Conclusions]The transcription group data were obtained of the mycelium and fruiting body of P. pulmonarius by high-throughput sequencing technology. Combined bioinformatics analysis,this study laid the foundation for analysing the genetic diversity,constructing genetic linkage map,and researching the differentially expressed genes and its functions at different developmental phases of P. pulmonarius.
引文
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[17]Fu Y,Dai Y,Yang C,et al.Comparative transcriptome analysis identified candidate genes related to bailinggu mushroom formation and genetic markers for genetic analyses and breeding[J].Scientific Reports,2017,7(1):9266.
[18]周雁,范秀芝,陈连福,等.SSR在黑木耳和毛木耳转录组中的分布和序列特征[J].菌物学报,2014,33(2):280-288.Zhou Y,Fan X Z,Chen L F,et al.Distribution and sequence characteristics of SSR in the transcriptomes of Auricularia auricula-judae and Auricularia polytricha[J].Mycosystema,2014,33(2):280-288(in Chinese with English abstract).
[19]刘伟,蔡英丽,何培新,等.粗柄羊肚菌转录组的SSR分布和序列特征分析[J].轻工学报,2017,32(2):33-39.Liu W,Cai Y L,He P X,et al.Distribution and sequence characteristics of SSR in transcriptome of Morchella crassipes[J].Journal of Light Industry,2017,32(2):33-39(in Chinese with English abstract).
[20]王冲,周惜时,夏妍,等.铜胁迫下黄花月见草根系蛋白质组学分析[J].南京农业大学学报,2016,39(5):754-762.DOI:10.7685/jnau.201603003.Wang C,Zhou X S,Xia Y,et al.Proteomics analysis of Oenothera glazioviana seedling roots under copper stress[J].Journal of Nanjing Agricultural University,2016,39(5):754-762(in Chinese with English abstract).
[2]周烁红,沈颖越,蔡为明,等.肺形侧耳变温结实相关基因Ppcsl-1的克隆及功能预测[J].菌物学报,2016,35(8):946-955.Zhou S H,Shen Y Y,Cai W M,et al.Cloning and functional prediction of the Ppcsl-1 related to change-temperature fruiting of Pleurotus pulmonarius[J].Mycosystema,2016,35(8):946-955(in Chinese with English abstract).
[3]刘靖宇,江玉姬,谢宝贵,等.草菇子实体不同成熟阶段的比较蛋白质组学分析[J].菌物学报,2014,33(1):55-68.Liu J Y,Jiang Y J,Xie B G,et al.A comparative proteome analysis of Volvariella volvacea fruit-bodies developed at different stages[J].Mycosystema,2014,33(1):55-68(in Chinese with English abstract).
[4]Eastwood D C,Herman B,Noble R,et al.Environmental regulation of reproductive phase change in Agaricus bisporus by 1-octen-3-ol,temperature and CO2[J].Fungal Genetics and Biology,2013,55:54-66.
[5]王伟科,袁卫东,方献平,等.秀珍菇不同发育阶段蛋白质组学分析[J].浙江大学学报(农业与生命科学版),2017,43(5):527-535.Wang W K,Yuan W D,Fang X P,et al.A comparative proteomics analysis of Pleurotus pulmonarius at different developmental phases[J].Journal of Zhejiang University(Agriculture and Life Sciences),2017,43(5):527-535(in Chinese with English abstract).
[6]Lockhart D J,Winzeler E A.Genomics,gene expression and DNA arrays[J].Nature,2000,405(6788):827-836.
[7]房学爽,徐刚标.表达序列标签技术及其应用[J].经济林研究,2008,26(2):127-130.Fang X S,Xu G B.Development and application of expression sequence tags technology[J].Nonwood Forest Research,2008,26(2):127-130(in Chinese with English abstract).
[8]祁云霞,刘永斌,荣威恒.转录组研究新技术:RNA-Seq及其应用[J].遗传,2011,33(11):1191-1202.Qi Y X,Liu Y B,Rong W H.RNA-Seq and its applications:a new technology for transcriptomics[J].Hereditas,2011,33(11):1191-1202(in Chinese with English abstract).
[9]李强,陈诚,熊川,等.杏鲍菇转录组数据SSR位点的生物信息学分析[J].应用与环境生物学报,2017,23(3):454-458.Li Q,Chen C,Xiong C,et al.Bioinformatic analysis of simple sequence repeat(SSR)loci in the Pleurotus eryngii transcriptome[J].Chinese Journal of Applied and Environmental Biology,2017,23(3):454-458(in Chinese with English abstract).
[10]Plaze D F,Lin C W,van der Velden N S,et al.Comparative transcriptomics of the model mushroom Coprinopsis cinerea reveals tissue-specific armories and a conserved circuitry for sexual development[J].BMC Genomics,2014,15:492-509.
[11]Muraguchi H,Umezawa K,Niikura M,et al.Strand-specific RNA-Seq analyses of fruiting body development in Coprinopsis cinerea[J].PLoSOne,2015,10(10):e0141586.
[12]王伟科,周祖法,陈青,等.灰树花菌丝体与原基转录组差异表达分析[J].上海交通大学学报(农业科学版),2016,34(1):74-80.Wang W K,Zhou Z F,Chen Q,et al.Analysis of differentially expressed genes between mycelium and primordium of Grifola frondosa[J].Journal of Shanghai Jiaotong University(Agriculture Science),2016,34(1):74-80(in Chinese with English abstract).
[13]Grabherr M G,Haas B J,Yassour M,et al.Full-length transcriptome assembly from RNA-Seq data without a reference genome[J].Nature Biotechnology,2011,29(7):644-652.
[14]Quevillon E,Silventoinen V,Pillai S,et al.Inter ProScan:protein domains identifier[J].Nucleic Acids Res,2005,6(33):116-120.
[15]Conesa A,Gotz S,García-Gómez J M,et al.Blast2GO:a universal tool for annotation,visualization and analysis in functional genomics research[J].Bioinformatics,2005,21(18):3674-3676.
[16]van der Auwera G A,Carneiro M O,Hartl C,et al.From Fast Q data to high confidence variant calls:the genome analysis toolkit best practices pipeline[J].Current Protocols in Bioinformatics,2014,11(1110):1-43.
[17]Fu Y,Dai Y,Yang C,et al.Comparative transcriptome analysis identified candidate genes related to bailinggu mushroom formation and genetic markers for genetic analyses and breeding[J].Scientific Reports,2017,7(1):9266.
[18]周雁,范秀芝,陈连福,等.SSR在黑木耳和毛木耳转录组中的分布和序列特征[J].菌物学报,2014,33(2):280-288.Zhou Y,Fan X Z,Chen L F,et al.Distribution and sequence characteristics of SSR in the transcriptomes of Auricularia auricula-judae and Auricularia polytricha[J].Mycosystema,2014,33(2):280-288(in Chinese with English abstract).
[19]刘伟,蔡英丽,何培新,等.粗柄羊肚菌转录组的SSR分布和序列特征分析[J].轻工学报,2017,32(2):33-39.Liu W,Cai Y L,He P X,et al.Distribution and sequence characteristics of SSR in transcriptome of Morchella crassipes[J].Journal of Light Industry,2017,32(2):33-39(in Chinese with English abstract).
[20]王冲,周惜时,夏妍,等.铜胁迫下黄花月见草根系蛋白质组学分析[J].南京农业大学学报,2016,39(5):754-762.DOI:10.7685/jnau.201603003.Wang C,Zhou X S,Xia Y,et al.Proteomics analysis of Oenothera glazioviana seedling roots under copper stress[J].Journal of Nanjing Agricultural University,2016,39(5):754-762(in Chinese with English abstract).