Isolation And Identification Of The Aldehyde Dehydrogenase Gene Poaldh1 Of Pleurotus Ostreatus

Aldehyde dehydrogenase plays a key role in reducing the aldehyde toxicity to cell by taking part in the metabolic pathway of aldehyde.However,there has been no report about aldehyde dehydrogenase gene of Pleurotus ostreatus so far.This study cloned and characterized the aldehyde dehydrogenase gene poaldh1 of P.ostreatus for the first time,which is aimed to reveal the mechanism of the primordial formation of P.ostreatus from the perspective of anti-stress.A gene possibly coding for aldehyde dehydrogenase was obtained by comparative analysis of differentially expressed genes between the dicaryotic mycelium stage and the primordial stage of P.ostreatus,then the obtained gene was named poaldh1 in this study.Several researches on the poaldh1 gene were conducted as follows.Firstly,the complete coding sequence(CDS)of poaldh1 gene was obtained by alignment of the expressed sequence tag(EST)of poaldh1 gene with the transcript of P.ostreatus PC15.Secondly,the CDS region of polaldh1 from P.ostreatus New 831 was cloned by reverse transcription-PCR(RT-PCR).The obtained fragment was then sequenced and analyzed by bioinformatics.The results showed that the length of the complete genomic DNA of poaldh1 gene was 2,016 bp,the CDS was 1,437 bp encoding a putative protein of 478 amino acids.The POALDH1 protein had the highly conserved 10-peptide sequence,the catalytic domain and the active-site glutamate of aldehyde dehydrogenase.The results of the protein function prediction and the GO analysis showed that POALDH1 is a NAD(P)+-dependent aldehyde dehydrogenase.Thirdly,the differential expression of poaldh1 gene between dicaryotic mycelium stage and primordial stage,under light,temperature differences condition related to primordia formation or under different abiotic stress was analyzed by semi-quantitative PCR(sq RT-PCR).The results showed that the expression of poaldh1 in primordia stage was significantly different from that in dicaryotic mycelium stage,and the former was about three times higher than the latter.The condition stress related to primordia formation were simulated,and the results showed that the expression of poaldh1 was significantly upregulated under the stress of light or temperature differences.When exposed to different concentrations of sodium chloride and mannitol,the mycelial growth was inhibited and the expression level of poaldh1 was higher than the control ones.In summary,we speculated that the poaldh1 gene might take part in primordia formation by responding to the external environmental stimulus.The poaldh1 gene was heterologously expressed in E.coli by using the expression vector p ET22b(+)-Ptac,and the POALDH1 protein,about 52 k Da,was successfully induced.The ALDH specific activity of the obtained recombinant strain was 0.58 U/mg.While the acetaldehyde tolerance of recombinant strain carrying p ET22b(+)-Ptac-poaldh1 was significantly high than control strain.Finally,the binary expression vector p Po-GPD containing gpd promoter was successfully constructed in this study.The overexpression vector and antisense silencing vector of poaldh1 gene were constructed by using the binary expression vector p Po-GPD,which will be transformed into P.ostreatus by Agrobacterium tumefaciens-mediated transformation method to construct the poaldh1 gene over-expressing and silencing mutant.