热带假丝酵母木糖乙醇发酵相关基因的筛选与分析  

Screening and Analysis of Genes Related to Xylose Fermentation to Ethanol in Candida tropiclis

作　　者：徐勇[1,2,3] 沈翀[1,2] 邱兴天[1,2] 蔡鹏[1,3] 黄敏仁[3] 余世袁[1,3] 

机构地区：[1]南京林业大学化工学院,南京210037 [2]江苏省生物质绿色燃料与化学品重点实验室,南京210037 [3]教育部林木遗传与生物技术省部共建重点实验室,南京210037

出　　处：《中国生物工程杂志》2012年第11期61-69,共9页China Biotechnology

基　　金：江苏省科技支撑项目(BE2011838);国家自然科学基金(31070514);教育部林木遗传与生物技术重点实验室开放基金(FGB200903);江苏省高校优秀学科建设工程(PAPD)资助项目

摘　　要：以木糖代谢模式菌株热带假丝酵母为材料,采用抑制性消减杂交技术构建高质量的木糖、葡萄糖发酵产乙醇的正向、反向SSH-cDNA文库。采用ABI3730系统测定2个文库中的1 013和525条EST序列,共拼接注释得到正向、反向文库中525个和288个差异性表达的已知基因,另有67个和12个可能的新基因,并对已知基因进行了GO分类。通过文库间BLAST比对去除相似序列,再结合经典代谢理论的指导筛选出候选基因,以实时定量PCR对它们进行了验证和转录本的定量比较分析,最终筛选出与部分木糖代谢和乙醇发酵相关的主效基因,包括STP、HAGT、XR2、XDH1、XDH2和ADH。实验为热带假丝酵母及其他木糖发酵菌株的糖代谢和乙醇发酵的转录谱、基因重组、代谢工程及发酵调控等相关研究提供理论指导。The high quality forward and reverse SSH-cDNA libriries were constructed by suppressing subtractive hybridization （SSH） for a model xylose-metabolizing yeast strain Candida tropicalis. About 1 013 and 525 ESTs in two SSH-cDNA libriries were sequenced by Sequencer PRISM ABI3730. From the sequences data,525 and 288 unigene were gotten and annotated and analyzed by GO classification, in addition 67 and 12 unigene were found as potential new genes. Some candidate genes were selected based on combination analysis method of the traditional metabolism theory and common genes deletion by unigene BLAST between two SSH-cDNA libriries, these genes were then tested and detected for relative quantitative comparison of gene transcript by RT- PCR technology. Some key genes related to sugar metabolism and ethanol fermentation were put forward, involving gene STP, HAGT, XR2, XDH1, XDH2 and ADH. It will benefit and provide theoretical guidance for related research on transcript profile, gene recombination, metabolic engineering and fermentation controlling of xylose metabolism and ethanol fermentation in Candida tropicalis and other xylose fermenting strains.

关 键 词：热带假丝酵母木糖乙醇发酵相关基因 抑制性消减文库 实时定量PCR 

分 类 号：Q81[生物学—生物工程]