Transcriptome Annotation And Gene Expression Differences Analysis Of The Recombinant Yeast Strain

The recombinant strain were constructed by recursive DNA shuffling transferred the entire genome of Candida intermedia into the S.cerevisiae,which can ferment xylose and glucose to ethanol.At present,the important research direction is to use the lignocellulosic biomass to produce the bioethanol.Variation in gene expression holds a key to uncovering the mechanism of cell regulation and Environmental adaptability.In recent years,transcriptome sequencing,also called RNA-Seq,provides a new technique to analyze the expression levels of the whole transcriptome.It can be used to reveal the expression of heterologous genes,the rearrangement transcription,the xylose metabolic pathways and other biological problems.In this study,total mRNA from each yeast strain sample which were cultured under xylose condition were sequenced on Illumina platform,then 125 bp paired-end reads and a total of ten GigaByte were generated.Clean reads were obtained through the removal of reads containing adapter and low quality reads.From the result of the quality control,the data were further assembled into 8,496 unigenes with a mean length of 2,661 nt,GC contents were 41.48%,the length of N50 is 4,651 nt,the average length of the unigenes is 2,661 nt.The total of all unigenes were annotated to the NR,SwissProt,KEGG,COG databases and so on.There were 6,777(79.77%)unigenes which had appropriate match.But the longer unigenes,which more than 500 bp,were more likely to have BLAST matches in the protein databases.We applied the differential expression analysis between the recombinant strain and the Candida intermedia strain,there were 5,266 genes differentially expressed,4,951 were up-regulated in the recombinant yeast,the up-regulated genes related to high temperature tolerance and alcohol tolerance were all expressed at high levels.Then,we analyzed the gene expression levels in the xylose metabolism pathway,found that XYL1,XYL2 genes were expressed at high levels in recombinant yeast during xylose fermentation,and its expression were induced more than 7.9 and 3.5 fold,respectively.Other genes such as FBP1,RPE1,RKI1,PYK1,PGK1,were also expressed at high levels,and these genes play important roles in the pathway of xylose fermentation.These up-regulated genes can promote the metabolic rate of xylose fermentation and the product yield of alcohol.Through the analysis of the transcript sequence,it was found that there were three kinds of rearrangements pattern in the recombinant yeast.That is the rearrangement from the two parental genes,and the gene rearrangement from the individual parents.But the rate of the recombinant unigenes was 1.4% when compared to all unigenes.According to the different repeat type,we found 3,696 SSR in the 8,496 unigenes.In the sequences with SSRs,the numbers of Mono-nucleotide repeat type(A/T)is 2,724(73.70%),and the number of Di-nucleotide repeat type(AT/AT)is 359(9.71%).The number of other repeats type(Tetra-nucleotide,Penta-nucleotide,Hexa-nucleotide)is a bit.By comparison with the distribution of SSRs in parental transcript sequence,the recombinant yeast found a combination of the two parents in the features of SSRs sequences.The recombinant yeast transcriptome was interrogated by RNA-Seq technology in this research.The results provided much information about transcripts function,differential expression transcripts,xylose metabolic pathways and rearrangements pattern,which were valuable for such studies in the recombinant yeast as fundamental biological research,gene engineering and industrial applications.