Gene Cloning And Prokaryotic Expression Of Banana Polyphenol Oxidase

Polyphenol oxidase has a widely application prospect in the enzymatic synthesis thea-flavins,protein cross-linking,black tea quality improvement,tea beverage stability impro-vement and other food aeras.But the resources of PPO are shortage which limited its appli-cation in food industries.In this research,we got banana ppo genes by in silico cloning based on Musa genome information,use the Brazilian banana pulp RNA as template, through RT-PCR test the ppo gene, and expressed in E.coli, finally got the recombinant ppo with activity.This research expand the resources of ppo,and provide a basis for banana ppo study.The research results are as follows:（1） Optimize the extraction method for banana pulp total RNATaking green banana pulp as raw material, effect of some factors including buffer type, ionic strength, pH value, the precipitate temperature and time with different CaCl2concent-ration, the precipitate time with LiCl on the extraction of RNA were studied, with the guide of purity,yield and integrity. The optimizated results are that the extraction buffer was100mM Tris-borate buffer（pH9.0）,use100mM CaCl225℃precipitate20min remove polysaccharose,3M LiCl precipitation RNA12h,can get the high yield,good purity and integrity RNA.When take add same isopropano1-20℃precipitation20min replace3M LiCl precipitation RNA12h, also can get good quality RNA,but the yield was low. The good quality RNA was extracted from ripening banana pulp and peel,green banana pulp and peel,which can used for RT-PCR amplification the380bp MaActin cDNA.（2） In silico cloning of banana ppo and its sequencing analysisUseing the Musa acuminata AAA Group Cavendish banana PPO mRNA sequence （EU880277.1）as a query probe,one highly homologous EST sequence （ES436966） was got by blast in the EST database of NCBI.Then contig₁794（CAIC01022631.1） was discovered by blast the EST sequence in Musa WGS database.One1773bp full length PPO cDNA sequence was attained by analysis the contig with FGENESH software. Through RT-PCR got the gene,which sequence length was1767bp and encoded588aa,sequence analysis indicated that it’s a new encoding banana ppo gene,and has been deposited in GeneBank （Accession No. KF900300.1）（3） The bioinformatics analysis of banana ppoThe banana ppo gene sequence and protein sequence analysed by many Bio-softwares. The results show that banana ppo gene has82%-85%homologous with Poaceae ppo （except Oryza alta）,has nearest affnity with Rhodotypos scandens （DQ851219.1）.Banana ppo most likey located in chloroplastid,which was hydrophilic and no transmenbrane and signal peptide,has a chloroplast transfer peptide with47amino acids. Banana ppo has three conserved domains （Tyrosinase superfamily,PP01_DWL and PPO1_KFDV）,which belongs to the tyrosinase gene family. Banana ppo encoded a total588amino acids,and its molecular formula was C2952H4526N8ooO866S19,molecular weight was65688.3Da,the theor-etical point was6.32. Then analysised the banana PPO second structure and got the tertiary structure model.（4）Construct the prokaryotic expression vector of banana ppo and induce expressionSuccessfully Construct the prokaryotic expression vector BXPPO1-pET22b （+）, BXPP02-pET22b （+）,BXPP01-pQE80Land BXPP02-pQE80L.Then transform BXPPO1-pQE80Land BXPP02-pQE80Linto the M15,transform BXPP01-pET22b （+） and BXPP02-pET22b （+） into the BL21（DE3）.They were induced at different IPTG concentration and different time,however,BXPP01-pET22b （+）,BXPP02-pET22b （+） and BXPP01-pQE80Lcann’t express objective protein, BXPP02-pQE80L express a62KDa objective protein.（5） Renaturation,purification and MS identification of banana PPOBXPP02inclusion bodies was renatured after degeneratation with an enzyme activity of23.61U/mg,which was purified through agarose gel affinity medium （Ni） after degene-ratation.The purified recombinant protein was electrophoresis pure. The recombinant protein was banana ppo by MS identification.