Evaluation Of Reference Genes In Clostridium Ljungdahlii DSM13528and The Application In Relative Expression Levels Of Target Genes By QRT-PCR

Because the traditional fuels such as petroleum, coal, and gas are limited and environmental consequences become more and more severe due to burning of these fuels, it is urgent to develop sustainable and environmentally friendly fuels. Bioenergy is one alternative which has received more and more attention. Biofuels derived from biomass has shown its great potential. With the advantage of utilizing whole biomass, the technology that fermenting syngas into biofuels is considered to be more attractive than others. Syngas-utilizing bacteria C. ljungdahlii attracted wider range of attention due to its high yields of ethanol and high growth rate. Furthermore, the publication of the genome and establishment of genetic transformation system for C. ljungdahlii DSM13528has made it a model organism for the study on syngas fermentation into biofuels.Clostridium ljungdahlii DSM13528is a promising platform organism for biofuel production from syngas. The gene expression analysis allows better understanding of its important biological characteristics at molecular level, such as carbon fixation and adaptation to solvent. Normalization is prerequisite for accurate gene expression analysis, but no valid reference genes have been proposed for quantitative real-time polymerase chain reaction (qRT-PCR) analysis in C. ljungdahlii DSM13528until now.First, the cultural condition was optimized for C. ljungdahlii with fructose and CO/CO2as the carbon source, respectively. When fructose was used as carbon source, the growth curve of C. ljungdahlii was made and the highest ethanol and acetate production was22.38mM,31.02mM, respectively. When CO/CO2was used as carbon source, the highest ethanol and acetate production was22.72mM,74.8mM, respectively.In this study seven candidate reference genes (gyrA, rho,fotl, rpoA, gukl, recA,16S rRNA) were selected and their expression levels were quantified by qRT-PCR in various culture conditions corresponding to different carbon sources and stresses. Two analytical programs geNorm and NormFinder were used to evaluate their stability. The results showed that gyrA, rho and fotl exhibited the most stable expression levels across all the tested samples and can be confidently used as reference genes to normalize the transcriptional data of target genes by qRT-PCR in C. ljungdahlii DSM13528. This study presented the first attempt to explore the validity of candidate reference genes and provide a set of valid reference gene for normalizing the expression of target genes and transcriptome analysis in C. ljungdahlii DSM13528.At last, the relative expression levels of genes involved in carbon fixation, sugar metabolism, solvent synthesis, quorum sensing, two-component signal system and chemotaxis in C. ljungdahlii cultured in the medium with different carbon source were studied. The result showed that three genes related to carbon starvation expressed higher in C. ljungdahlii cultured in the medium with CO/CO2as the carbon source than fructose as the carbon source. Additionally, genes involved in wood-ljungdal pathway expressed higher in C. ljungdahlii with CO/CO2as the carbon source, however, genes involved in fructose metabolism have lower expression levels in C, ljungdahlii with CO/CO2as the carbon source. The results also above confirmed the accuracy of the reference genes in this study.