Full-length CDNA And Partial Genome Sequences Cloning Of Pyruvate Kinase Gene From Camellia Oleifera

As the principal species of woody oil plants in the South of China, Camellia oleifera is one of the four most famous woody oil plants. In the growth process of Camellia seeds, as a key glycolytic enzyme, pyruvate kinase(PK) gene controls the formation of pyruvate, which is an important carbon source in fatty acid synthesis, and it also play an important role in the process of photosynthetic product transferring into fatty acids. So the study of PK gene of Camellia Oleifera is of great significance in revealing the influence that glycolysis has on the oil synthesis and molecular breeding in Camellia oleifera. The main results in the paper are as follows:1,Full-length cDNA clone of PK gene of Camellia oleifera. In the constructed cDNA library of Camellia oleifera by the non-wood forest breeding and cultivation key lab of state forestry administration, there is a PK gene fragment that is 900bp in length and has a poly-A tail. After comparing this cDNA sequences with other species on line, we find that the homology is very high.and it has a cDNA sequence deletion of untranslated region and coding many amino acids at 5'terminal. Using three special primers GSP1,GSP2 and GSP3 designed by Primer Premier 5.0 according to the EST sequence of PK. A 1500bp fragment was obtained by 5'RACE strategy to amplify the RNA of seed of fine varieties clone XiangLin No.1. The 5'RACE fragment was cloned into the vector pMD18-T and then transformed into Escherichia. Coli DH5α. Obtaining cDNA sequences of the fragments after bacteria PCR and sequencing. After analyzing by Vector NTI 9.0, a 2114bp sequence was formed by splicing EST sequence of PK and 5'RACE sequence. Two pair primers OEF1,OER1 and OEF2,OER2 were designed according to the spliced sequence, and OEF1 is at the 5'end of spliced sequence, OER2 is at the 3'end, OER1 and OEF2 in the overlapped region, and the opposite overlap 13bp. Using OEF1 with OER1 to amplify 5'RACE fragment and OEF2 with OER2 to amplify EST fragment by overlapped extension PCR strategy, T1 and T2 fragments were obtained respectively. T1 and T2 spliced a longer fragment since there was a same region of 13bp in them. According to the principle of base-pair complementary, a new fragment is amplified by the overlapped region, then to amplify spliced fragments by using OEF1 and OER2, the full Camellia oleifera PK gene cDNA is obtained.2,Partial genome sequences cloning of PK gene from Camellia oleifera. Using staggered primer extension OEF2 and OER2, a 2038bp fragment was obtained by amplify the DNA of leaves of fine varieties clone XiangLin No.1. Determining the Camellia PK gene is part of the genome from the genome sequences by NCBI online comparison. By comparing the patial genome sequences of PK gene from Camellia oleifera with the full-length cDNA, we forecast that the sequences contained a 220bp exon, a 476bp exon and a 1578bp intron.3,Bioinformatic analysis of PK gene from Camellia oleifera. The cDNA sequence is 2114bp in length, and has a 1737bp ORF coding 579 amino acids,108bp untranslated region at 5'end and 269bp untranslated region with a 44bp polyA tail at 3'end by using Vector NTI 9.0. The homology percentage of the cDNA sequence from camellia oleifera with other species in Genbank is quite high, so we can conclude the sequence is full-length cDNA of PK gene. According to the protein sequence deduced by PK gene cDNA sequence, using a series of bioinformatics software applications on the physical and chemical properties, advanced structure, three-dimensional models for predictive analysis, the results shows that the protein isoelectric point (pI) is 5.105, molecular weight is 63766.5Da, stable coefficient is 52.95; it is a kind of unstable protein, has two transmembrane domains, no signal peptide splice site, it is a non-secreted protein; has 37 Serine phosphorylation sites,5 thr phosphorylation sites and 2 tyrosine phosphorylation sites; and it contains 32.30% of the a-helix,20.90% of the (3-sheet. Motif search results indicate that there are a variety of N-glycosylation sites, protein kinase C phosphorylation sites, pyruvate kinase active sites and other functional domains on PK. We can find 12 significant a-helix in homology modeling of the model.