【作者】 王海英；

【导师】 谢宝贵；

【作者基本信息】 福建农林大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 本研究通过草菇单孢配对获得实验菌株,探讨了草菇总RNA的提取方法,并运用高通量的数字化基因表达谱测序技术对草菇不同发育时期的菌柄进行标签测序,获得了草菇发育过程中纽扣期菌柄(Nb)、蛋形期菌柄(Db)、伸长期菌柄(Sb)、成熟期菌柄(Cb)四个时期的表达谱。运用生物信息学的方法对四个时期表达谱进行分析。通过高通量测序,各个样本库中我们获得了5.7M-6.2M个标签,各库的标签种类数分布在0.1-0.3M之间。去除低质量的标签,我们在Nb、Db、Sb、Cb四个时期中获得的Clean tag总数分别是5612361、5978522、5972497、5967968;获得的标签种类数分别为90437,97022,87911,83702。将测序标签与参考序列进行比对,在Nb、Db、Sb、Cb四个库中比对到参考基因的转录本数目分别为10610,15329,13815,14335。四个样本的标签拷贝数变化呈现趋势一致。测序标签与反义链也有部分匹配,在Nb、Db、Sb、Cb四个库中,分别约有34.40%,35.83%,31.83%,31.85%的参考基因被特异性标签反向匹配,这些基因有可能是双向调控的基因。在Nb、Db、Sb、Cb四个时期中检测到与生长发育相关的蛋白,主要是几丁质合成酶、丝裂原蛋白激酶、内切葡聚糖酶、肌动蛋白、微管蛋白、驱动蛋白等,在草菇不同的发育时期表达量不同,但变化趋势相似,这些蛋白在Db时期对应的转录本含量都比较高,推测Db时期成长旺盛,需要的能源物质较多。细胞壁合成相关的转录本含量也处于很高的水平,微管蛋白在Db时期和Sb时期转录本含量都比较高,推测该转录本与Sb时期菌柄伸长相关。对差异基因进行GO功能分类,对显著性富集的GO条目进行分析,涉及到的转录本主要是与DNA结合、染色体组成、转移酶活性、核糖体蛋白相关。通过分析推测Db时期与Nb时期相比,DNA复制能力较强,细胞迅速分裂分化,存在较多的信号分子调控作用;Db与Sb时期相比, Db时期核糖体数量较多,推测Db时期蛋白质的合成种类和数量较多,表明在草菇菌柄进入伸长期已经完成分化,蛋白质合成能力与Sb时期相比减弱;Sb时期与Cb时期相比,Cb时期组蛋白合成受阻,推测Cb时期草菇染色体活动不频繁,复制能力减弱,存在着衰老的现象,不需要更多的能量供应。对显著性富集的代谢通路进行分析,主要有DNA复制、Ribosome、Cell Cycle等代谢途径,通过分析发现DNA复制途径中Nb、Db、Sb、Cb四个时期DNA复制能力Db时期强,随着发育逐渐减弱;在Risome途径中,Nb、Db、Sb、Cb四个时期,Nb和Db时期核糖体与Sb、Cb时期相比数量较多;在Cell Cycle途径中,Db时期细胞周期活动较多。综合分析推测在草菇的发育过程中Nb至Db时期生长旺盛,生活能力强,细胞增殖和分化活动较多,存在较多的信号调控活动,在该阶段菌柄的伸长主要依赖细胞分裂增殖,Db至Cb时期菌柄的伸长更多依赖细胞的伸长。Db时期DNA复制能力强,蛋白质合成处于高峰期,该时期代谢旺盛,次生代谢物质含量较高,是草菇营养价值较高的时期。通过对四个库的比较,我们发现Nb、Db、Sb、Cb四个时期中特有的转录本数目分别为22、264、154、129,推测这些特有转录本与草菇维持特定时期的生物学特性有关。更多还原

【Abstract】 The article discusses the methods of extracting total RNA from the stipe tissue of Volvariella volvacea.And we report high-throughput tag-sequencing(tag-sqe) analysis using Solexa Genome Analyzer platform on transcription of Nb stage(the stipe tissue of button stage of Volvariella volvacea ), Db stage(the stipe tissue of the egg stage of Volvariella volvacea),Sb stage(the stipe tissue of the elongation stage of Volvariella volvacea),Cb stsge(the stipe tissue of the mature stage of Volvariella volvacea ). We use bioinformatics to analyse gene expression profiles,in order to understand Gene expression and regulation mechanism in Volvariella volvacea.To this end, we sequenced 5.7–6.2 million tags representing 0.1–0.3 million unique transcripts for each library (Nb, Db, Sb, and Cb). After removal of low quality tags, we obtained a total of 5612361,5978522,5972497 and 5967968 clean sequences that corresponded to 90437,97022,87911 and 83702 distinct tags for Nb, Db, Sb, and Cb, respectively. All clean tags were aligned to the reference genes database, The tag mapping to the database sequences generated 10610,15329,13815 and 14335 annotated genes for Nb, Db, Sb, and Cb libraries, respectively. The distribution of total and distinct tag counts over different tag abundance categories showed very similar tendencies for all libraries. Sequencing tags mapped to the complementary strand of the sense gene suggest that its antisense strand has transcripts and that this gene may use bidirectional regulation. In this study, about34.40%,35.83%,31.83% and 31.85% of the genes were matched by the distinct tags in antisense orientations for Nb, Db, Sb, and Cb libraries.We detect some transcriptions related with growth , For example, chitin synthase, mitogen protein kinase, endoglucanase, actin and microtubules protein and kinesin, the counts of the transcriptions showed very similar tendencies for all libraries.we speculate that it is eugonic in Db stage,a lot of energy were needed. The transcription counts of microtubules protein is high in Db period and Sb period. Maybe the transcription related to elongation of stipe tissue.Analyses of differentially expressed genes revealed the substantial changes in gene type and abundance in the differernt stage of fruiting body of Volvariella volvacea. We analysis the difference expressed genes in expression based on GO and KEGG databases. We analyzed three significant difference pathways,they are DNA replication, ribosome and cell cycle. Through the analysis of the significant difference of GO terms and pathways, we speculate that the abilities of DNA replication, cell differentiation and cell division are stronger from Nb stage to Db stage, there were many signal molecular events at this stage. The elongation of sitpe mainly rely on cell division proliferation at this stage; From Db to Cb period the elongation of stipe depends on elongation of cells. In Db period DNA replication ability is stronger, and the protein synthesis efficiency is at peak, metabolism is exuberant During this period.There are many secondary metabolism substances, nutritional value is higher in this period.Through the comparison of four libraries, we found peculiar transcriptions 22, 264,154 and 129 for Nb, Db, Sb, Cb libraries respectively. we speculate that these peculiar transcriptions have something with maintaining the biological characteristics related to the specific period of Volvariella volvacea.更多还原