Full-length Cdna Cloning Of Aact Gene And Fad6 Gene From Camellia Oleifera And Prokaryotic Expression

As the principal species of woody oil plants in the South of China, Camellia oleifera is one of the four most famous edible oil woody plants.Camellia oil contains oleic acid and linoleic acid UFA mostly, and the average content of UFA is above 90%. In the process of the oil combining. AACT is an enzyme that catalyzes the formation of acetoacetyl-CoA from two molecules of acetyl-COA. Fatty acid desaturase catalyze oleic acid into linoleic acid, linolenic acid and by dehydrogenation. On the base of the cDNA and EST library of Camellia oleifera. We have separatied and identified AACT and FAD6 from the cDNA library of Camellia oleifera by the methods of RNA collect,dengeneration RT-PCR,bioinformatic analysis and Prokaryotic expression, So the study of AACT and FAD6 gene from Camellia Oleifera are important for revealing the lipids biosynthesis patterns and molecular breeding in Camellia Oleifera. The main results in the paper are as follows:1. Full-length cDNA cloning of acetyl-CoA C-acetyltransferase gene from Camellia oleifera. In the constructed cDNA library of Camellia oleifera by our laboratory, there is a AACT gene, the clone number is 6155, the total length is 1414bp and 203bp untranslated region at 3'end. and has a cDNA sequence deletion of untranslated region and codes about 14 amino acids at 5'terminal, Base on sequences information of AACT gene, we designed six pair of primer in AACT conserved regions, a 700bp fragment was obtained by 5'RACE strategy to amplify the RNA of seed of fine varieties clone XiangLin No.1. According to the spliced sequence and analyzing of blast, we can conclude the sequence is part-length cDNA of FAD6 gene, we have submit the sequence to the GeneBank, the receipt number is GU594059.2. Bioinformatic analysis of AACT gene from Camellia oleifera. The cDNA sequence is 1495bp in length, and has a 1227bp ORF coding 408 amino acids,203bp untranslated region at 5'end and 311bp untranslated region with a polyA tail at 3'end by using Vector NTI 9.0. Bioinformatic analysis of AACT gene from Camellia oleifera. The cDNA sequence is 1495bp in length, and has a 1227bp ORF coding 408 amino acids,203bp untranslated region at 5'end and 311bp untranslated region with a polyA tail at 3'end by using Vector NTI 10.0. The homology percentage of the cDNA sequence from camellia oleifera with P. kurrooa in GeneBank is over 86%, so we can conclude the sequence is full-length cDNA of AACT gene. The second structure, isoelectric point, molecular weight, transmembrane domain and signal peptide and so on of the deduced amimo acid sequence was predicted and analyzed by Antheprot 5.0, and the results are:pI value is 6.19, molecular weight is 41628.6 Da, unstabitily ratio is 25.73, belong to a stabitily protein. belong to stabitily protein, it also have two step astride structure region, no singal peptide site, is a unsecretory protein, six transmembrane domain, Other results of AACT have two Thiolases active structure region, and also have many cAMP and cGMP-dependent protein kinase phosphorylation site and Protein kinase C phosphorylation site and Thiolases active site.3. Prokaryotic expression of AACT gene from Camellia oleifera. The fragment encoding the AACT gene was excised from the positive clone pMD18-AACT by Ncoâ… and Xhoâ… and purified by agrose gel fraction method, then the fragment was subcloned into the pET-28a expression vector digested by Ncoâ… and Xhoâ… restriction enzymes, the recombinant expression plasmids were identified by PCR and restriction enzymes analysis. The results indicated the fragment was correctly inserted into the pET-28a expression vector and conformed to a reading frame. The fusion protein were expressed in E.coli BL21(DE3) host with a predicted molecular weight of 41 kDa.4. Full-length cDNA cloning of fatty acid desaturase 6 gene from Camellia oleifera. Base on sequences information of FAD6 gene published in GeneBank, we designed a pair of primer in FAD6 conserved regions, a 486bp fragment was obtained by RT-PCR strategy to amplify the RNA of seed of fine varieties clone XiangLin No.1. After analyzing of blast, we can conclude the sequence is part-length cDNA of FAD6 gene, there is only 11 basic groups indifferently, base on sequences information of the FAD6 gene, a 1451bp sequence was formed by RT-PCR, we have submit the sequence to the GeneBank, the receipt number is GU594060.5. Bioinformatic analysis of FAD6 gene from Camellia oleifera.The cDNA sequence has a 1347bp ORF coding 448 amino acids, the homology percentage of the cDNA sequence from camellia oleifera with CsFAD6 of Camellia sinensis in GeneBank is over 86%, and with the E. gentianoides is below 36%. The second structure, isoelectric point, molecular weight, transmembrane domain and signal peptide and so on of the deduced amimo acid sequence was predicted and analyzed by Antheprot 5.0, and the results are:pI value is 8.67, molecular weight is 51382.3 Da, belong to unstabitily protein, it also have two step astride structure region, no singal peptide site, is a unsecretory protein, four transmembrane domain, Other results of FAD6 has a Cyt-b5 structure region, and also have many cAMP-and cGMP-dependent protein kinase phosphorylation site and Protein kinase C phosphorylation site and N-myristoylation site.