【作者】 陈杰；

【导师】 王沫；

【作者基本信息】 华中农业大学 ， 农药学， 2013， 硕士

【摘要】 茯苓(Wofiporia cocos (Schw.))是担子菌门的一种腐生真菌。茯苓的干燥菌核在东亚广泛用于传统药材,在中国,其菌核更是享有万能药的美誉。茯苓有消炎抗肿瘤等多重功效,三萜类化合物是茯苓的主要活性成分之一。但是茯苓中该类化合物的生物合成却罕有研究。本文着力研究茯苓活性成分三萜类化合物茯苓酸的生物合成途径及该途径中关键酶的作用。本试验以第二代高通量测序为平台,对同一株茯苓的菌丝和菌核分别进行转录组测序。主要研究结果如下：1)通过高通量测序,在菌丝样品中得到了38722186个Reads,共3484996740bp的核苷酸序列,其中质量高于20的比例为96.21%,不确定的碱基比例为0.01%,碱基G、C含量比为58.48%。在菌核样品中得到了39710244个Reads,共3573921960bp的核苷酸序列,其中质量高于20的比例为96.52%,不确定的碱基比例为0.01%,碱基G、C含量比为58%。通过有效Reads的组装,在菌丝中得到了60354个Contig,共30359057bp的核苷酸序列,每个contig的平均长度为503bp,N50是第765个congtig。在菌丝中得到42037个Unigene,其中可识别的序列簇为1098个,可识别的单一序列为40939个；共27146481bp的核苷酸序列,平均每个Unigene平均长度为646bp,N50是第814个Unigene o在菌核中得到了56938个Contig,共33305946bp核苷酸序列,每个contig的平均长度为585bp,N50是第991个congtig,在菌核中得到38533个Unigene,其中可识别的序列簇为1313个,可识别的单一序列为37220个；共29546864bp核苷酸序列,平均每个Unigene平均长度为767bp,N50是第1037个Unigene.2)通过Unigene功能注释,27325个Unigene中,11796个Unigene注释到GOC库中,其中有25个功能分组；2574个Unigene注释到GO库中,其中分3大类进行功能注释,30个功能分组；16386个Unigene注释到KO库中,其中有108个功能分组；27143个Unigene注释到NR库中;17469个Unigene注释至IJSWISSPROT库中。3)通过Unigene代谢通路分析,得出茯苓中三萜类化合物茯苓酸的前体物质羊毛甾醇的生物合成途径。4)通过荧光定量PCR验证,得出在菌丝阶段表达量上调的酶的编码基因为Unigene12366、Unigene20430、Unigene14106、Unigene21656。Unigene6395。5)通过对同一株茯苓不同时期(菌丝和菌核)总三萜和茯苓酸含量的测定,得出茯苓菌丝和茯苓菌核中的总三萜和茯苓酸含量存在差异。其中菌丝总三萜含量为5.36%,茯苓酸含量为0.458%；菌核总三萜含量为1.43%,茯苓酸含量为0.174%。6)通过Realtime-PCR技术,检测茯苓酸的生物合成途径中对关键酶起调节作用的基因表达量,与转录组测序分析得到结果相一致。更多还原

【Abstract】 Wolfiporia cocos (Schw.) is a saprophytic fungus in the Basidiomycetes. In the East Asia, its dried sclerotium is widely used as a traditional medicinal material. In China, its dried sclerotium is regarded as a all-purpose crude drug. Wolfiporia cocos has good reputation from all its excellent functions. Wolfiporia cocos’ main functions are to diminish inflammations and to defeat cancers. Triterpenoids is one of the active compounds in W.cocos and it plays a fatal role in its famous functions, but there are rare researches about biosynthetic pathway of triterpenoids in W. cocos. In this study, the author chooses the illumina HiSeq2000to sequence the mycelia and sclerotium which belong to different periods in the same unit of W. Cocos. In this thesis, author takes strength to discover the biosynthetic pathway of pachymic acid which is important active compound of W. cocos. The main results show as follows:1) With the high technology of Illumina HiSeq2000, sequencing is a main technique which is used to get more effective data. And at last,3484996740bp from38722186high quality sequence reads are obtained in the mycelia.96.21%of these sequence reads have a higher quantity than20.58.48%bases are considered G and C. And only0.01%of bases are uncertain. Meanwhile,3573921960bp from39710244high quality sequence reads are obtained in the sclerotium.96.52%of these sequence reads have a higher quantity than20.58%bases are considered G and C. And only0.01%of bases are uncertain. With assembly results of clean reads,60354contig which contained30359057bp and42037unigene which contained27146481bp are obtained in the mycelia. The mean length of contig is503bp. And the mean length of unigene is646bp. There are1089distinct clusters and40939distinct singletons in the unigene. Meanwhile,56938contig which contained33305946bp and38533unigene which contained29546864bp are obtained in the sclerotium. The mean length of contig is585bp. And the mean length of unigene is767bp. There are1313distinct clusters and37220distinct singletons in the unigene.2) With the unigene function annotation,11796in27325of unigene are annotated to GOC, and they are separated to25function groups.2574in27325of unigene are annotated to GO, and they are separated to30function groups which belong to3parts.16386in27325of unigene are annotated to KO, and they are separated to108function groups.27143in27325of unigene are annotated to NR.17469in27325of unigene are annotated to SWISSPROT.3) With the unigene metabolic pathway analysis, the biosynthetic pathway of lanosterol which belongs to precusor substance of pachymic acid of triterpenoids in W. Cocos. shows.4) With detection of realtime PCR, some enzymes and their coding genes’ expression have had up-regulation in the period of mycelia. They are Unigene12366、 Unigene20430、Unigene14106、Unigene21656、Unigene6395.5) With assaying of pachymic acid and triterpenoids in different periods in the same unit of W. Cocos, it shows that the pachymic acid content are divers between mycelia and sclerotium which is0.458%in the mycelia and0.174%in the sclerotium. As to triterpenoids, the results show the same trend. There are5.36%in the mycelia and1.43%in the sclerotium.6) There are several fatal genes in the pachymic acid biosynthetic pathway. They regulate the expressions of enzymes. Some genes are chosen to be verified by realtime-PCR. It shows the same result with the result of the transcriptome sequencing analysis.更多还原