Cloning Of Polyphenol Oxidase Gene And Its Regulatory Sequence Of 5' Terminal In Nai(Prunus Salicina Lindl.var.cordata J.Y.Zhang Et Al.)

There exists commonly browning of pulps in Nai (Primus salicina Lindl.var. cordata J.Y.Zhang et al.\ which decreases the edible quality and storage ability of fruit and even loses fruit commercial value. Browning of pulps results from enzymatic reaction by integration of polyphenolics, polyphenol oxidase (PPO) and oxygen. To resolve the problem of browning of pulps by anti-sense RNA technology, the PPO gene and its regulatory sequence of 5' terminal were isolated from buds and pulps of Nai by RT-PCR, RACE and chromosome walking etc in the study for genetic transformation, which would create a new way of genetic modification of browning of pulps in Nai. The main results were as follows.1. The method for extraction and purification of genomic DNA from buds of Nai was developed.To remove secondary substances such as polyphenolics, polysaccharides and pigments etc in the tissues of Nai, the young buds were grinded with 10% (WAV) Polyvinylpolypyrrilodone (PVPP) together, and the concentration of β-mercaptoethanol in extraction buffer was 1% (V/V); the lysis solution was extracted twice with 24:1 chloroform : isoamyl alcohol; the aqueous phase obtained was mixed even with 1/10 volume NaCl/CTAB solution preheated at 65 ℃, then extracted twice again with 24:1 chloroform : isoamyl alcohol; in roughsolution of DNA, 2/3 volume of 5M sodium chloride and three volume of absolute ethanol were added to precipitate DNA. The results showed that the impurities such as polyphenolics and polysaccharides etc were effectively removed from the samples by the method, and the pure and intact DNA sample obtained was directly used in successive PCR amplification, digestion of endonucleases and chromosome walking etc.2. The method for extraction and purification of total RNA from buds of Nai was developed.The buds were grinded with 10% (WAV) PVPP together, and 1% (V/V) p-mercaptoethanol was added in Trizol extracting buffer; absolute ethanol was added to 20% (V/V) final concentration in homogenization solution to precipitate polysaccharides; Lithium Chloride was added to final concentration (3M) to precipitate RNA in the aqueous phase obtained after extracting with chloroform; in the rough solution of RNA, 1/30 volume of 3M sodium acetate (pH5.2) and 1/10 volume of absolute ethanol were added to precipitate polysaccharides. The results showed that the pure and intact RNA sample obtained was directly used in the successive RT-PCR and RACE reactions.3. The method for extraction and purification of total RNA from pulps of Nai was developed.To neutralize organic acid in pulps of Nai, in the extraction buffer, pH value of Tis-HCl and EDTA was 8.8, the concentration of CTAB was 2.5% (W/V), 0.5% SDS (W/V) was added; absolute ethanol was added to 20% (V/V) final concentration in the pulps lysis solution, and then extracted twice with 24:1 chloroform risoamyl alcohol; 1/30 volume of 3M sodium acetate (pH5.2) and 1/10 volume of absolute ethanol in rough solution of RNA to precipitate polysaccharides twice. The resultsshowed that the impurities such as polyphenolics, polysaccharides, and organic acids etc were effectively removed from pulps of Nai by the method, that the pure and intact RNA sample obtained was directly used in the successive RT-PCR and RACE reactions.4. The coding regions of PPO genes were cloned from buds and pulps of Nai by the techniques of RT-PCR, RACE and chromosome walking etcThe coding region of PPO gene cloned from Nai's buds contained 1770 nucleotides coding for 589 amino acid residues; the coding region of PPO gene cloned from pulps of Nai contained 1794 nucleotides coding for 597 amino acid residues; each sequence of the amino acids contained one thylakoid-transit region and two copper-binding sites, i.e., CuA and CuB. Alignments for nucleotide sequences and deduced amino acid sequences of PPO from buds and pulps of Nai to those of other plants with DNAMAN software showed that the two PPO gene were higher homologous to some Rosaceae fruit trees, the phylogenetic tree was drawn according to the results of alignments for the sequences of the amino acids of PPOs in plants, the result showed that the PPOs of pulps from Nai, apricot and apple were clustered into one group, and that the PPOs from buds of Nai, peels and leaves of apple, and pulps of pear were clustered into another group, therefore, the PPO genes from buds and pulps of Nai were two members among the gene family, the sequences of nucleotides and amino acids of PPO from buds of Nai were repectively 62.7% and 50.4% homologous compared with those from the pulps. The intron was found in PPO gene of pulps, but PPO gene of buds had no intron.5.The regulatory sequence of 57 terminal in the PPO gene from buds of Nai was cloned by chromosome walking.