Full-length Cdna Cloning Of Protein Phosphatase Gene From Camellia Oleifera

The discovery of reversible protein phosphorylation is the milestone of human research in the signal transduction work. Protein kinases and protein phosphatases catalyze the reversible protein phosphorylation and dephosphorylation as two kinds of key enzymes. By the dephosphorylation process catalyzed by protein phosphatase, physiological and biochemical characteristics of many key proteins in the signal path can change, such as enzymatic activity and protein subcellular localization, etc., which affect cell proliferation, differentiation and apoptosis as well as individual growth and development. The major findings of this paper is as follows:1) Separation and identification of 4 protein phosphatase genes from the cDNA library of Camellia oleifera. In the constructed cDNA library of Camellia oleifera by our laboratory, there are four protein phosphatase genes:one MPP,1436bp in length; one PTP,1067bp in length, and two PP2Cs,823bp and 1501bp in length. Except the latter PP2C, they each has a poly(A) tail. After translating and comparing with other species on line, it is obvious that the coding section from 194bp to 982bp in the MPP cDNA clone corresponding to the first to the last amino acid of MPP in other species, and the homology is very high. So it can be concluded that the cDNA clone was a full-length MPP gene. We named it CoMPP1; The coding section from 9bp to 878bp in the PTP cDNA clone corresponding to No.16 or No.45 to the last amino acid of PTP in other species, and the homology is very high. So it can be concluded that the cDNA clone was a part of PTP gene, and has a cDNA sequence deletion of untranslated region(UTR) and coding about16 to 45 amino acids at 5'terminal. We named it CoPTP1; The coding section from 1bp to 518bp in the shorter PP2C cDNA clone corresponding to No.240 to the last amino acid of PP2C in other species, and the homology is very high. So it can be concluded that the cDNA clone was a part of PP2C gene, and has a cDNA sequence deletion of UTR and coding about 240 amino acids at 5' terminal. We named it CoPP2C1; The coding section from 2bp to 1162bp in the other PP2C cDNA clone corresponding to No.12 to the last amino acid of PP2C in other species, and the homology is also very high. So it can be concluded that the cDNA clone was a part of PP2C gene, and has a cDNA sequence deletion of UTR in both 5'and 3'terminal and coding about 12 amino acids at 5'terminal.We named it CoPP2C2.2) Full-length cDNA cloning of 2 PPase genes from Camellia oleifera. Use the seed of'Xianglin No.1'which is a excellent clones variety in Camellia oleifera to extract total RNA, and transcript it reversed to cDNA as a template. Using two kinds of special primers GSP and NGSP designed by Primer Premier 5.0 according to the EST and the sequencing results of the three PPase cDNA clones, the full-length 5'terminals of CoPTP1 and CoPP2C1 were obtained by 5'RACE strategy. The 5'RACE fragment was cloned into the vector pMD18-T and then transformed into Escherichia. Coli DH5a and its sequence was obtained by sequencing recombinant plasmid DNA extracted from the transformed strain. After analyzing by Vector NTI 9.0,the full-length of a 1170bp CoPTP1 gene and a 1453bp CoPP2Cl gene cDNA cloning from Camellia oleifera were obtained.3) Bioinformatic analysis of PPase from Camellia oleifera. We use the Vecotr NTI 9.0 to analysis the sequences of PPase genes, the 1436bp CoMPP1 cDNA sequence has a 786bp ORF coding 262 amino acids, a 193bp 5'UTR and a 454bp 3'UTR; the1170bp CoPTP1 cDNA has a 927bp ORF coding 309 amino acids, a 51bp 5'UTR and a 189bp 3'UTR; the 1453bp CoPP2C1 cDNA has a 1113bp ORF coding 371 amino acids,a 48bp 5'UTR and a 289bp 3'UTR. The homology percentage of the three PPase from Camellia Oleifera with other species in Genbank reach up to 84%,71% and 79%. The characteristic of physical chemistry and the second structure of the deduced amino acid sequences were predicted and analyzed by Bio-softwares, and the results for CoMPP1,CoPTP1 and CoPP2C1 are:pI value are 5.17,7.56,5.33; molecular weights are 29.7kDa,34.7kDa,40.2kDa; the instability index are32.82,45.99,53.73;the aliphatic index are77.75, 103.73,80.35; two transmembrane domain; None of the three has any transmembrane region or signal peptide, which shows all of the three PPase belong to non-secretory protein.