PPO Gene Clone From Lotus Rhizome Bud And Its Expression

Rhizome is one of three types of Nelumbo nucifera Gaertn. ssp. nucifera, which has the largest cultivation area in 12 kinds of aquatic vegetables and its sales is one of the largest of the 26 kinds of vegetables. Lotus is a food with high medicinal which has shown that lotus has the effect of lowering blood pressure, anti-oxidation, anti-aging, sedation, inhibition of fatty liver, antibacterial and so on.However, lotus would dehydrate and brown when processing and preserving that has a serious negative impact on virtue and its commercial value.Polyphenol oxidase (PPO) is one of the key enzymes that cause lotus browning. In this study, we cloned the cDNA of PPO gene from lotus rhizome bud by using RT-PCR, RACE technology (Gene bank accession number HQ380894), and detected its expression in different tissues by semi-quantitative PCR. The aim is to acquire the enzyme gene information and its genetic background, and to inhibit PPO activity to delay browning by using genetic engineering technique. The main results were as follows:1 Method of extracting RNA from LotusExtraction buffer contained 2% (W / V) CTAB, 2% PVP (W / V), 0.5g / L spermidine, pH 8.0 Tris-HCl and EDTA, and added to the concentration of 2% mercaptoethanol,extracted twice with chloroform, the crude extracts of the RNA by adding an 0.25 volume of LiCl, overnight at -20℃, chloroform extraction once, with 2 volumes of ethanol precipitation 2 hours at -80℃skim supernatant after centrifugal, dissolved RNA precipitate with DEPC water. The results show that the method can obtain pure and complete RNA and remove phenols and polysaccharides and other impurities in the lotus samples effectively.2 Cloned PPO and the full coding region of lotus lotus rhizome buds by RT-PCR and RACEPolyphenol oxidase (PPO) gene was cloned from lotus rhizome (Nelumbo nucifera) by using both RT-PCR and RACE techniques. The full length cDNA of PPO was 2074 bp with open reading frame 1503 bp, encoding a protein of 501 amino acids, 5'UTRs of 160 bp and 3'UTRs of 411 bp (Genbank No.HQ380894). Bioinformatics analysis showed that PPO in lotus had molecular weight 56.8 KD and PI value 8.60, exhibiting tryosinase family characteristics with hydrophilic and transmenbrane properties but no signal peptide. Bothα-helix andβ-turn were found spreading in the whole poplypeptide chain.The results of comparison nucleotide sequence showed that sequences of PPO gene from rhizome buds homology of up to 99 % with same species (Genbank accession number FJ999- 635, but no paper reported) and low homology with other species.3 The expression of PPO in different tissues of LotusThe expression of PPO in different plant tissues was also investigated and the relative expression level were 2.659 (rhizome bud), 2.419 (leaf), 2.02 (rhizome), 1.797 (petal), 1.654 (stem), respectively.