Cloning And Expression Study Of COMT Gene In Pummelo

Pummelo[Citrus maxima(Burm.)Merr.]is famous for home and abroad because of thin skin and juicy.But juice sac granulation phenomenon occurred during growth and postharvest storage has become the most prominent problem that influences the quality,and the abnormal accumulation of lignin in the cell walls is one of the main reasons for granulation.In our study,cafffeic acid-O-methyltransferase(cmCOMT)genes which participated in lignin synthysis were cloned,and one of the 5’ regulatory sequence of genes was also obtained.The cDNA,gene sequences equencesand revelant promotor sequence were analysised by bioinformatical methods.Then prokaryotic expression analysis and quantitative expression analysis were conducted.The object of the study is to verify the regulation of caffeic acid-O-transferase gene during juice sacs granulation of pomelo.1 Cloning and quantitative expression analysis of cmCOMTs geneOn the basis of former research results,full-length cDNA sequences of cmCOMT1/2/3,which related to lignin metabolism in ’Guanximiyou’ pummelo were cloned.ORF length of COMT1/2/3 was 1101bp,1092bp,1092bp respectively,and each of their amino acids were 366,363 and 363;’Guanximiyou’ juice DNA was used as template to clone COMT1/2/3 DNA sequences,Each of the full length of cmCOMTl/2/3 was 4030bp,2204bp,3221bp,which were composed by 3 introns and 4 exons,and ecah intron splice sites are in line GT-AG rule eukaryotes.Bioinformatics analysis results showed that three proteins,which were encoded by cmCOMTs genes,belonge to hydrophilic and stable proteins.All proteins had AdoMet_Mtases superfamily and dimerization domain conserved superfamily.Both of them were compose by a-Helix,extension chain and irregular curls,cmCOMT 1/2 which cling to N-terminus outside inclined to the outside by transmembrane,then cmCOMT3 inclined inwardly within the N-terminal outside,and phosphorylation of all proteins occurred maily on serine,which twelve serine of cmCOMT1 was the most,having followed by thyrosine and threonine residues.In this study,The quantitative analysis of cmCOMT1/2/3 gene expressions were dectected by Fluorescence quota PCR during fruit development period.Several periods before 157d almost had no significant difference,the relative expression levels of cmCOMT1 gene almost tend to zero.The cmCOMT1 gene has the highest relative expression levels in 211d(the worst period of granulation during sampling period),However,the relative expression levels of cmCOMT2/3 also had no significant difference during granulation,the expression of cmCOMT2/3 of recent column cell in 117d was higher than other periods and parts of the granulation process,the lowest relative expression levels of cmCOMT2 in 107d juice sac away from the column,also the lowest of cmCOMT3 in 95 juice sac away from the column.By comparing cmCOMTl/2/3 gene expression changes during fruit development of pummelo,the relative expression of cmCOMT1 is significant higher than the expression of two other genes,eapecially the relative expression level reached a peak in 211d,namely the worst period of granulation during sampling period.the relative expression analysis of cmCOMTl/2/3 showed that cmCOMTl was significantly correlated with granulation,cmCOMT2/3 were no significantly correlated with granulation.2 Prokaryotic expression analysis of cmCOMT1 and Cloning,cis-elements analysis of cmCOMT1 promoter in ’Guanximiyou’Had cloned three cmCOMTs genes and conducted the relative quantitative analysis,based on the aboved results,then the cmCOMT1 gene was inserted into pEASY-E1 vector to construct recombinant vector pEAY-E1-cmCOMT1,which was then transformed into Transetta(DE3)strain.The soluble fusion protein was expressed under the induction conditions with 37℃ 5h 0.5mM IPTG.The fusion protein was purified by affinity chromatography and was detected by SDS-PAGE.The results showed that pure fusion protein with 3 9KD molecular weight was obtained.Mass spectrum identification results of COMT1 sequence further proved that was cafffeic acid-O-methyltransferase gene,the coding product of COMT1 was caffeic acid-O-methyltransferase gene.To explore the regulatory function of cmCOMT1 gene promoter in ’Guanximiyou’,the 5’-flanking sequence of cmCOMT1 was isolated by TaKaRa-Genome Walking Kit,The results showed that the length of cmCOMT1 was 949bp.Using online prediction software BDGP,5’-flanking sequence of cmCOMT1 gene may be present three core promoter regions,which scores were 0.85,0.81 and 0.98 respectively.Three core promoter regions located on 244-294bp,508-558bp and 822-872bp.Sequence analysis results indicated that the sequence located between 822-872bp is likely the real core promoter region of cmCOMT1 gene.Bioinformatical softwares PlantCare and PLACE were employed to analysis the 5’-flanking region sequence of cmCOMTl gene.The results suggest that the sequence contained numbers of normal core elements,such as TATA-box and CAAT-box,morever,there were many predicted cis-elements that respond to ethylene,gibberellin,salicylic acid and MYB transcription factors,endosperm(GCN4-motif,Skn-i)etc,especially,a number of unknown or specify elements were also found.