The Study Of PPO Cloning、Construction Of Sense And Antisense Expression Vector And Prokaryotic Expression Of Pyrus Bretschneideri Cv. Dangshan Su

Pyrus bretschneideri cv. Dangshan Su is one of the four famous pear in china, So far, the planting area has reached333,000hectares, the annual output of2638one thousand tons. In recent years, due to natural environmental changes, variety degradation, extensive field management and other reasons, increased content of pear fruit stone cells, resulting in fruit meat thicken-. taste high residue, which has adversely affected the flavor and quality of Pyrus bretschneideri cv. Dangshan. The cell size and density of stone is to determine the main factors of the pear fruit quality. The stone cells were formed by the lignin biosynthesis, transport and deposition. However, Polyphones oxidase (polyphenol oxidase, PPO) can be involved in the oxidation of chlorogenic acid, coumarin and other phenolic compounds and promoting the lignin synthesis. Therefore, the study the fruit of Pyrus bretschneideri cv. Dangshan polyphenol oxidase has an important significance.In this experiment, using the materials of the fruit of Pyrus bretschneideri cv. Dangshan, we cloned PPO gene and analysized its bioinformatics functions. On this basis, the sense and antisense expression vectors were constructed. At the same time, we construct PPO prokaryotic expression vector and express PPO in vitro. The tissue specificity of the PPO gene expression was also analysized. So, we study the biological functions of the PPO from the different levels.The major findings were as follows:1. PPO gene was1782bp, encoding593amino acids and the GenBank accession No. is JF809859. It has a highly conserved copper ion binding site, each Cu binding site contains three His residues which linked by ligand bond. The conserved residues C179, F348, Y415, Y417, D365, R136, six β-sheet was also found in the N-terminal and C-terminal. Comparing different species of conserved region of homology, we found that the highest homology is apple’s PPO3, up to93.33%, and the PtrPPO13is lowest, only47.30%.It is the closest homologous relationship of Pyrus bretschneideri cv. Dangshan and Apple.2. PPO gene was amplif ied by RT-PCR, and then placed under the35S promoter in either sense or antisense orientation randomly. Through asymmetric restriction enzyme and sequencing identification, the sense and antisense expression vectors of PPO gene were successfully constructed. The sense and antisense expression vector were transformed into Agrobacterium EHA105by freeze-thaw method.3. Analysis of the PPO gene codon,we found that a higher proportion of rare codons reached11.3%in the PPO sequence, as well as three rare codons tandem clusters appear in the sequence. By recombinant DNA technology, the total length of the PPO gene was connected to the prokaryotic expression vector of pET-28a, and successfully constructed the PPO prokaryotic expression vector pET-PPO. Then,the prokaryotic expression vector pET-PPO was transformed into E. coli Rosetta strain,we successfully induced the expression of His-PPO fusion protein.To optimize induced IPTG concentration, temperature, time, we conclude that optimal inducing condition is28℃,1.0mM IPTG,5h.4. After pollination, the expression of PPO continuely decreased. From15d to49d, while fruits underdevelopment. Once fruits were mature, the expression tended to increase. And in49days, the PPO expression of flesh adjacent to peelcore is higher than that of flesh adjacent to core.