重排酵母菌转录组注释和基因差异表达分析
发布时间：2018-07-29 20:05
【摘要】：重排酵母菌,是通过基因组重排技术,将酿酒酵母和间型假丝酵母的原生质体递归融合,获得的高效发酵己糖和戊糖的新型菌株。使用木质纤维素类的原料转化生产生物乙醇是目前生产生物质原料重要研究方向。研究基因表达的变化是揭示细胞调控代谢机制、适应生存环境的重要手段。近年来,转录组测序(RNASeq)作为测定全转录组表达水平的一种新技术,在生物领域的应用越来越广泛,可用来揭示重排酵母中异源基因的表达、转录本重排及木糖代谢途径等生物学问题。本研究对重排酵母在木糖培养环境下的RNA样品进行测序,获得了约10GB长度为125bp的双末端数据,通过质量控制,过滤掉低质量的reads,使用Trinity软件进行组装,获得了8,496条unigenes,总长度为22,614,789nt,GC含量为41.48%,N50长度为4,651nt,平均长度为2,661nt。将所有的unigenes序列比对到NR、Swiss-Prot、KEGG、COG等数据库,共有6,777(79.77%)条序列比对到数据库中,被分配到数据库中的unigenes大部分长度大于500bp,而长度在200~500bp之间的unigenes被注释到的比例较小。基于RNA-seq数据,对间型假丝酵母和重排酵母进行了基因差异表达分析,获得了5,266个差异表达基因,其中有4,951个基因在重排酵母表现上调,发现这些高表达的基因与木糖代谢、耐高温及耐酒精等特性相关。对木糖代谢相关途径基因表达量进行分析,发现重排酵母中XYL1、XYL2基因的高表达,表达量分别提升了7.9、3.5倍,说明在重排酵母中,木糖的吸收和转化效率高于亲本间型假丝酵母。在重排酵母中,FBP1、RPE1、RKI1、PYK1、PGK1等基因的表达量都表现为上调,而这些基因在木糖代谢途径中参与重要的中间产物的转化过程,这些基因的高表达可以解释子代重排酵母的酒精产率的提升。通过对转录本序列的分析,发现重排酵母的转录本存在3种重排方式,即来自于两个亲本基因之间的重排,以及来自于单个亲本内部的基因重排,但重排unigenes所占的比例较少,约占1.4%。对转录本中简单重复序列(SSR)分析,鉴定了3,696个SSR,其中单碱基型(A/T)含量最多(2,742,占73.70%),双碱基型(AT/AT)含量次之(359,9.71%),其他类型(四碱基型、五碱基型、六碱基型)个数都很少。通过与亲本转录本序列中SSRs分布的比较,发现重排酵母获得了两亲本中的SSRs的序列特征,预测到的SSR数目约为两亲本中SSRs的总数。本研究通过RNA-Seq技术解析了重排酵母转录组,对重排酵母的转录本功能、差异表达基因、木糖代谢途径、重排转录本的模式及SSR特征等方面进行了研究,为重排酵母菌生物学研究、基因工程和工业应用提供了参考依据。
[Abstract]:Saccharomyces cerevisiae is a new strain of high efficient fermentation of hexose and pentose by genomic rearrangement technology, which combines the protoplasts of Saccharomyces cerevisiae and Candida mycelia by recursive fusion. Conversion of lignocellulosic materials to bioethanol is an important research direction in biomass production. Studying the changes of gene expression is an important means to reveal the mechanism of cell metabolism and adapt to the living environment. In recent years, transcriptome sequencing (RNASeq), as a new technique for the determination of total transcriptional expression, has been widely used in the biological field, which can be used to reveal the expression of heterologous genes in rearrangement yeast. Transcription rearrangement and xylose metabolism pathway and other biological problems. In this study, the RNA samples of rearrangement yeast in xylose culture environment were sequenced, and the two-terminal data about 10GB length of 125bp were obtained. Through quality control, the low quality readswere filtered out, and the Trinity software was used to assemble the samples. 8496 unigenes were obtained, the total length was 22614789ntGC, the length of N50 was 4651nt, the average length was 2661nt. 6777 (79.77%) sequences of unigenes sequences were aligned to databases such as NRN Swiss-Protn KEGGG. Most of the unigenes sequences assigned to the database were larger than 500bp, while unigenes with length between 200~500bp were annotated in a small proportion. Based on RNA-seq data, 5266 differentially expressed genes were obtained from Candida and rearrangement yeast. 4951 genes were up-regulated in rearrangement yeast, and these overexpressed genes were found to be involved in xylose metabolism. High temperature resistance and alcohol resistance and other characteristics related. The high expression of XYL1 + XYL2 gene was found in rearrangement yeast, which increased by 7.9 ~ 3.5 times respectively, which indicated that the efficiency of xylose absorption and transformation in rearrangement yeast was higher than that in parent Candida cerevisiae. In rearrangement yeast, the expression of genes such as FBP1RPE1, RPE1RKI1, PYK1PYK1 and PGK1 were up-regulated, and these genes were involved in the transformation of important intermediate products in xylose metabolism pathway. The high expression of these genes could explain the increase of alcohol yield in progeny rearrangement yeast. By analyzing the sequence of transcripts, we found that there are three rearrangements in rearrangement yeast transcripts, that is, gene rearrangement from two parents and gene rearrangement from within a single parent, but the proportion of rearrangement of unigenes is relatively small. About 1.4. The simple repeat sequence (SSR) analysis of transcripts showed that 3696 SSRs were identified. The single base type (A / T) was the most abundant (2 / 742, 73.70%), the double base type (AT/AT) was the second (359 / 9.71%), and the other types (four, five and six bases) were few. By comparing with the SSRs distribution in the parent transcripts, it was found that the SSRs sequence characteristics of the two parents were obtained by yeast rearrangement, and the predicted number of SSR was about the total number of SSRs in the two parents. In this study, rearrangement yeast transcriptome was analyzed by RNA-Seq technique. The function of rearrangement yeast transcripts, differentially expressed genes, xylose metabolism pathway, pattern of rearrangement transcripts and SSR characteristics were studied. It provides a reference for the biological research, genetic engineering and industrial application of yeast rearrangement.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q93
本文编号：2153849
[Abstract]:Saccharomyces cerevisiae is a new strain of high efficient fermentation of hexose and pentose by genomic rearrangement technology, which combines the protoplasts of Saccharomyces cerevisiae and Candida mycelia by recursive fusion. Conversion of lignocellulosic materials to bioethanol is an important research direction in biomass production. Studying the changes of gene expression is an important means to reveal the mechanism of cell metabolism and adapt to the living environment. In recent years, transcriptome sequencing (RNASeq), as a new technique for the determination of total transcriptional expression, has been widely used in the biological field, which can be used to reveal the expression of heterologous genes in rearrangement yeast. Transcription rearrangement and xylose metabolism pathway and other biological problems. In this study, the RNA samples of rearrangement yeast in xylose culture environment were sequenced, and the two-terminal data about 10GB length of 125bp were obtained. Through quality control, the low quality readswere filtered out, and the Trinity software was used to assemble the samples. 8496 unigenes were obtained, the total length was 22614789ntGC, the length of N50 was 4651nt, the average length was 2661nt. 6777 (79.77%) sequences of unigenes sequences were aligned to databases such as NRN Swiss-Protn KEGGG. Most of the unigenes sequences assigned to the database were larger than 500bp, while unigenes with length between 200~500bp were annotated in a small proportion. Based on RNA-seq data, 5266 differentially expressed genes were obtained from Candida and rearrangement yeast. 4951 genes were up-regulated in rearrangement yeast, and these overexpressed genes were found to be involved in xylose metabolism. High temperature resistance and alcohol resistance and other characteristics related. The high expression of XYL1 + XYL2 gene was found in rearrangement yeast, which increased by 7.9 ~ 3.5 times respectively, which indicated that the efficiency of xylose absorption and transformation in rearrangement yeast was higher than that in parent Candida cerevisiae. In rearrangement yeast, the expression of genes such as FBP1RPE1, RPE1RKI1, PYK1PYK1 and PGK1 were up-regulated, and these genes were involved in the transformation of important intermediate products in xylose metabolism pathway. The high expression of these genes could explain the increase of alcohol yield in progeny rearrangement yeast. By analyzing the sequence of transcripts, we found that there are three rearrangements in rearrangement yeast transcripts, that is, gene rearrangement from two parents and gene rearrangement from within a single parent, but the proportion of rearrangement of unigenes is relatively small. About 1.4. The simple repeat sequence (SSR) analysis of transcripts showed that 3696 SSRs were identified. The single base type (A / T) was the most abundant (2 / 742, 73.70%), the double base type (AT/AT) was the second (359 / 9.71%), and the other types (four, five and six bases) were few. By comparing with the SSRs distribution in the parent transcripts, it was found that the SSRs sequence characteristics of the two parents were obtained by yeast rearrangement, and the predicted number of SSR was about the total number of SSRs in the two parents. In this study, rearrangement yeast transcriptome was analyzed by RNA-Seq technique. The function of rearrangement yeast transcripts, differentially expressed genes, xylose metabolism pathway, pattern of rearrangement transcripts and SSR characteristics were studied. It provides a reference for the biological research, genetic engineering and industrial application of yeast rearrangement.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q78;Q93
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