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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。
A high-quality xylose-glucose and ethanol fermentation forward and reverse SSH-cDNA library was constructed by using the inhibition subtractive hybridization technique using the xylose metabolism model strain Candida tropicalis as material. A total of 1 013 and 525 ESTs from two libraries were determined by ABI3730 system. A total of 525 and 288 differentially expressed known genes in forward and reverse libraries were obtained by splicing, and 67 and 12 possible New genes, and the known genes were GO classification. Candidate genes were screened out by BLAST alignment among the libraries and then selected with the guidance of classical metabolic theory. The real-time quantitative PCR was used to validate and quantify the transcripts of these genes. Finally, the results were compared with those of partial xylose metabolism and ethanol fermentation Related major genes, including STP, HAGT, XR2, XDH1, XDH2 and ADH. The experiment provides the theoretical guidance for the related research on the sugar metabolism of Candida tropicalis and other xylose fermentation strains, the transcriptional regulation of ethanol fermentation, gene recombination, metabolic engineering and fermentation regulation.