Differential Expression Analysis Of Pqq And MoxF Genes From Hyphomicrobium Denitrificans

Pyrroloquinoline quinone(PQQ)is a kind of oxidoreductase coenzyme which have important applications in medical treatment,health care and food industry.Pqq and moxF genes play an important role in PQQ biosynthesis and secretion.The objective of this study was to elucidate the synthesis pathway and regulatory mechanism of PQQ,for this purpose,differential expression analysis of pqq and moxF genes from Hyphomicrobium denitrificans was performed by real-time quantitative PCR(qRT-PCR).The selection of candidate reference genes for normalization of qRT-PCR in Hyphomicrobium denitrificans under various growth phases:We used three statistical methods(Bestkeeper,GeNorm,NormFinder)to evaluate the gene expression stabilities of six genes(16S,gapdh,recA,ldh,rpoB,S5).Moreover,the stability of reference genes was verified in two Hyphomicrobium denitrificans strains,these results demonstrated that gapdh and recA genes were the most steadily expressed genes in Hyphomicrobium denitrificans.The analysis of transcript levels of pqq and moxF genes in the batch culture of Hyphomicrobium denitrificans:In the pqqABCDE gene cluster,pqqA gene was determined to be dominantly transcribed over the others,and reached maximum expression at 48 h;meanwhile,pqqE gene expression level is second highest after pqqA,and at the peak at 80 h,it indicated that the formation and modification of PQQ precursor mediated by pqq A and pqqE genes is the rate-limiting step in the synthesis pathway of PQQ.There were different expressive degree of multiple pqqA genes during different Hyphomicrobium denitrificans growth phases,meanwhile,the relative expression level of pqqA3 is the highest.In the moxF gene cluster,the relative expression level of moxF??was the highest,showed that moxF?? genes may play a critical role in methanol oxidation.With the increasing of certain methanol concentration,the expression level of pqqABCDE and moxF?? genes is improving,these results indicated that the transcription of pqqABCDE and moxF?? genes has been demonstrated to be inducible by methanol.The analysis of transcript levels of pqq and moxF genes in different pH culture of Hyphomicrobium denitrificans:Under the condition of pH 7.0,PQQ productivity is up to 742.47 mg/L,which is highest above all pH conditions,Under these conditions,pqqA3 and pqqE was up-regulated,ensured the supply of the precursor PqqA and its modification;whereas moxF?? was down regulation,increased PQQ excretion differentiated from MDH.Notably,pqqABCD genes was up-regulated in low pH,showed that low pH promote the conversion of PQQ from intermediate compounds.In addition,multiple pqqA genes respond differently to pH,moxF? was found to be less correlation with pH conversely,these results indicated multiple pqqA genes were evolved for the adaptation of different survival environment.