【摘要】：重排酵母菌,是通過基因組重排技術(shù),將釀酒酵母和間型假絲酵母的原生質(zhì)體遞歸融合,獲得的高效發(fā)酵己糖和戊糖的新型菌株。使用木質(zhì)纖維素類的原料轉(zhuǎn)化生產(chǎn)生物乙醇是目前生產(chǎn)生物質(zhì)原料重要研究方向。研究基因表達(dá)的變化是揭示細(xì)胞調(diào)控代謝機制、適應(yīng)生存環(huán)境的重要手段。近年來,轉(zhuǎn)錄組測序(RNASeq)作為測定全轉(zhuǎn)錄組表達(dá)水平的一種新技術(shù),在生物領(lǐng)域的應(yīng)用越來越廣泛,可用來揭示重排酵母中異源基因的表達(dá)、轉(zhuǎn)錄本重排及木糖代謝途徑等生物學(xué)問題。本研究對重排酵母在木糖培養(yǎng)環(huán)境下的RNA樣品進(jìn)行測序,獲得了約10GB長度為125bp的雙末端數(shù)據(jù),通過質(zhì)量控制,過濾掉低質(zhì)量的reads,使用Trinity軟件進(jìn)行組裝,獲得了8,496條unigenes,總長度為22,614,789nt,GC含量為41.48%,N50長度為4,651nt,平均長度為2,661nt。將所有的unigenes序列比對到NR、Swiss-Prot、KEGG、COG等數(shù)據(jù)庫,共有6,777(79.77%)條序列比對到數(shù)據(jù)庫中,被分配到數(shù)據(jù)庫中的unigenes大部分長度大于500bp,而長度在200~500bp之間的unigenes被注釋到的比例較小。基于RNA-seq數(shù)據(jù),對間型假絲酵母和重排酵母進(jìn)行了基因差異表達(dá)分析,獲得了5,266個差異表達(dá)基因,其中有4,951個基因在重排酵母表現(xiàn)上調(diào),發(fā)現(xiàn)這些高表達(dá)的基因與木糖代謝、耐高溫及耐酒精等特性相關(guān)。對木糖代謝相關(guān)途徑基因表達(dá)量進(jìn)行分析,發(fā)現(xiàn)重排酵母中XYL1、XYL2基因的高表達(dá),表達(dá)量分別提升了7.9、3.5倍,說明在重排酵母中,木糖的吸收和轉(zhuǎn)化效率高于親本間型假絲酵母。在重排酵母中,FBP1、RPE1、RKI1、PYK1、PGK1等基因的表達(dá)量都表現(xiàn)為上調(diào),而這些基因在木糖代謝途徑中參與重要的中間產(chǎn)物的轉(zhuǎn)化過程,這些基因的高表達(dá)可以解釋子代重排酵母的酒精產(chǎn)率的提升。通過對轉(zhuǎn)錄本序列的分析,發(fā)現(xiàn)重排酵母的轉(zhuǎn)錄本存在3種重排方式,即來自于兩個親本基因之間的重排,以及來自于單個親本內(nèi)部的基因重排,但重排unigenes所占的比例較少,約占1.4%。對轉(zhuǎn)錄本中簡單重復(fù)序列(SSR)分析,鑒定了3,696個SSR,其中單堿基型(A/T)含量最多(2,742,占73.70%),雙堿基型(AT/AT)含量次之(359,9.71%),其他類型(四堿基型、五堿基型、六堿基型)個數(shù)都很少。通過與親本轉(zhuǎn)錄本序列中SSRs分布的比較,發(fā)現(xiàn)重排酵母獲得了兩親本中的SSRs的序列特征,預(yù)測到的SSR數(shù)目約為兩親本中SSRs的總數(shù)。本研究通過RNA-Seq技術(shù)解析了重排酵母轉(zhuǎn)錄組,對重排酵母的轉(zhuǎn)錄本功能、差異表達(dá)基因、木糖代謝途徑、重排轉(zhuǎn)錄本的模式及SSR特征等方面進(jìn)行了研究,為重排酵母菌生物學(xué)研究、基因工程和工業(yè)應(yīng)用提供了參考依據(jù)。
[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.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：Q78;Q93

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本文編號：2153849