Cloning And Sequence Analysis Of Polyphenol Oxidase Of Amorphophallus

Amorphophallus are perennial herbaceous plants, and has a long history of cultivation in the southwest mountainous area of our country. Its underground bulb sweet are riched in KGM, Because of its special physical and chemical properties, for high blood pressure, diabetes, obesity, constipation have prevention and auxiliary treatment effect. It also have broad application prospects in the chemical industry, environmental protection, construction and other fields.A. konjac bulb have high water content, the kind of taro is easily damaged and resulting in browning during the process of transportation. It affects the quality of taro and production. In the process of peeling and slicing bulb into powder, the cell material are easily browning under the action of enzymes. It affect the quality of konjac powder. Therefore, controlling the konjac enzymatic browning is a problem urgently to be solved in the production and processing.Polyphenol oxidase (PPO) is one of the key enzymes that cause konjac browning. In this study, we cloned the conservative coding regions sequence of PPO gene from the konjac blade by using the method of homologous cloning. Using the technology of RT-PCR and RACE, we achevied the full-length of polyphenol oxidase gene. The aim is to acquire the sequence information of A. konjac PPO gene and its genetic background, and provide theoretical basis to regulating the expression of PPO and get new varieties that gain resistance to enzymatic browning by using genetic engineering method. The main results were as follows:1. Methods of extraction and purification the A. konjac genomic DNA and RNA in different tissueExtraction of konjac genomic DNA, Extraction buffer contained2%(W/V) CTAB、100mmol/LTris-HCl(pH8.0、1.4mol/LNaCl、20mmol/LEDTA、1%PVP(W/V) and added to the concentration of0.2%mercaptoethanol, extracted twice with chloroform, isopropanol and NaAC(3mol/L, pH5.2)to precipitate DNA,75%ethanol washing impurity2-3times, anhydrous ethanol to dehydration, with ddH2O dissolving precipitation to get DNA samples, saved at-20℃for use. Improved CTAB method can effectively remove impurities such as polysaccharides, polyphenols of konjac pollution, obtaining complete and pure DNA samples and can be used in the subsequent molecular markers, such as resource identification, genetic map construction research.Extraction of konjac RNA of different dissue, Extraction buffer contained2%(W/V)CTAB、100mmol/L Tris-HCl(pH8.0),2mol/L NaCl、25mmol/LEDTA2%PVP(W/V)、0.5g/L spermidine and added to the concentration of0.5%mercaptoethanol. extracted twice with chloroform, add cold anhydrous ethanol in RNA crude extract, blowing blending added to adsorption column of the kit, add Buffer RW1and centrifugation, Buffer RW2and centrifugation, dissolving RNA by Rnase-free water,-80℃saved for use. CTAB reagent kit in combination with phase, which can effectively remove impurities such as polysaccharide, polyphenol of konjac, obtain complete and pure samples of RNA, can be directly used in the subsequent molecular biology operation (such as semi-quantitative analysis, RACE, etc.).2. Cloned PPO gene of the full coding region of konjac blade by homology cloning and RACE techniquesWith A. konjac blade gDNA and cDNA first chain as template, using degenerate primers for PCR amplification, we obtained the DNA and cDNA fragment were763bp and translated a protein of254amino acid. And then, we cloned the PPO gene of the full coding region of konjac blade by RACE techniques. The full length cDNA of PPO was2026bp with open reading frame1452bp, encoding a protein of484amino acids,5’UTRs of384bp and3’UTRs of190bp.Nucleotide sequence alignment results showed that the konjac PPO gene encoding region sequence and triticum aestivum(GenBank accession number:HQ228149.1) had the highest similarity of84%, and the similarity of oryza alta PPO (GenBank accession number: DQ532414.1) was77%, and the similarity with ipomoea batatas(GenBank accession number: AY822711.1)、solanum tuberosum(GenBank accession number:U22922.1) PPO were76%,79%, and rosaceae plants of similarity were between74%and76%. Coding region of amino acid sequence alignment showed that konjac PPO and pineapple ananas comosus(GenBank accession number:AAO16865.1), nelumbo nucifera(GenBank accession number: ADP89908.1), triticum aestivum(GenBank accession number:HQ228149.1), Malus domestica(GenBank accession number:BAA21676.1) plants such as similarity were68%,63%,61%and63%, respectively.3. The analysis of protein structure of A. konjac PPO Bioinformatics analysis showed that PPO in konjac blade had molecular weight55KD and pI value9.32. The PPO gene of konjac belongs to the characteristic of tyrosinase family. The91～108,250～261amino acid residues of konjac PPO were CuA and CuB area respectively. The highly conservative CuA and CuB area were riched in his residues. A. konjac PPO protein was hydrophilic protein and no signal peptide. The homology modeling results of amino acid showed that the protein contains15alpha helix structure,5beta turn structure. Phylogenetic tree showed that PPO protein of A. konjac had the closest phylogenetic relationship to Gramineae(barley, wheat, maize), and they were clustered into one group.