| Alcohol dehydrogenase（ADH,EC1.1.1.1）is a zinc-containing enzyme which is composed of two subunits,one has a catalytic effect located in the active center of the enzyme,the other subunit functions in stabilizing the quaternary structure.ADH catalyzes the reversible reaction between primary alcohol and aldehyde by nicotinamide adenine dinucleotide as coenzyme,and is one of the members of the Dehydrogenase/reductase（MDR）protein gene superfamily.In plants,ADH is a small gene family.It plays an important role in stressing conditions such as flood,chilling and drought,and in plant indirect defense reaction by catalyzing the synthesis of green leaf volatiles.So far,no ADH gene has been cloned in tea plant and the biological role of CsADH was poorly understood.In this paper,using the transcriptomic data of tea plant fed by Ectropis obliqua in our laboratory and homologous analysis,three homologous sequences of ADH gene were obtained from the transcriptomic data.Then two full length cDNA sequences of homologous ADH genes were isolated successfully in tea plant by RACE（Rapid Amplification of cDNA Ends）technology.Through the prokaryotic expression system,CsADH proteins encoding by the CsADH genes were expressed in E.coli.The CsADH proteins were purified and the enzyme catalyzing function was validated.By northern blot and qRT-PCR analysis,expression models of the CsADH genes in tea plant after Ectropis obliqua feeding and cold treatment were investigated.There are the main results as follows:1.Two full length cDNA sequences of CsADH genes were isolated successfully in tea plant by RACE technology,which was named CsADH1 and CsADH2separately.The full length cDNA sequence of CsADH1 was 1476 bp,the length of 5’UTR and 3’UTR was 94 bp and 194 bp and the ORF（open reading frame）length of CsADH1was 1188 bp,encoding 395 amino acid.The full length cDNA sequence of CsADH1 was 1505 bp,the length of 5’UTR and 3’UTR was 83 bp and 282 bp and the ORF（open reading frame）length of CsADH1was 1140 bp,encoding 379 amino acid.2.Multiple homologous alignment showed that the homology between CsADH1and CsADH2 was 54%.Protein structure prediction analysis showed that both CsADH1 and CsADH2 proteins contained the three conserved domains of ADH protein,which was the zinc binding kinetic site,the ADH＿N domain with the Gro ES structure,and the Rossmann folded NAD（P）（+）binding protein domain,respectively.3.ORF frames of the two CsADH genes were ligated into the prokaryotic expression vector pMAL c2X,and then transferred into prokaryotic expression strain BL21（DE3）.After induction with 0.2 mg/ml IPTG at 28℃for 12 h,a large amount of Mal E-CsADH fusion proteins were obtained,and the molecular weights were about 80 k Da.The enzymatic properties of CsADH were obtained by measuring the absorbance of NADH at 340 nm by using the full-wave length microplate reader.Results showed that the optimum pH is pH 11.0,and the optimum temperature is37℃,when the catalyzing characters of CsADH proteins were tested.The maximum reaction rate Vmax（NAD）of CsADH1 was 2.803 U/mg,Km（NAD）is 0.86 m M。The maximum reaction rate Vmax（NAD）of CsADH2 was 3.625 U/mg,Km（NAD）is 0.87 m M。4.According to the qRT-PCR results,we found that the expression level of CsADH1 gene was up-regulated at 6 h and 9 h after feeding by Ectropis obliqua,and the expression level of CsADH1 gene was up-regulated about 2 times when compared with that of control.After 12 h of the Ectropis obliqua feeding,the expression level of CsADH1 gene was down-regulated and had no difference compared with the control.As to CsADH2,the expression of CsADH2 reached the maximum at 6 h after feeding,and then decreased slightly.which was down-regulated at about 24 h.The results of Northern blot analysis showed that CsADH1 had the highest expression at 6h and CsADH2 at 9h,which was consistent with the results of qRT-PCR. |
