Full-length Cdna Cloning Of Bc Gene And β-ct Gene From Camellia Oleifera

Camellia oleifera is one of the principal species of woody oil plants in the South of China, the seed of Camellia oleifera is used to extract oil which rich in oleic acid and linoleic acid UFA mostly, and the average content of UFA is above 90%, take tea-oil is be beneficial to people's health. Acetyl Co-A carboxylase is a key and speed-control enzyme in Fatty acid synthesis. So the study of ACCase gene from Camellia Oleifera is 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 BC gene 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. According the conserved nucleotide fragment of the BC gene in other species, We use the Primer Premier 5.0 to design the degenerate primer that amplify partial sequence of BC gene, The fragment were cloned into a vector pMD18-T and then transformed into Escherichia. Coli DH5a, BC's sequence was obtained by sequencing. Then we based on the partial sequence, we design the 5'GSP,5'nested GSP,3'GSP to amplify the completed 5'end and 3'end of BC gene. After analysis by Vector NTI 9.0, a 1901bp sequence was formed by splicing BC 5'RACE sequence, degenerate sequence and BC3'RACE sequence. Then three pairs primers were be design to the Overlap Extension PCR of BC, and amplify three fragments:A1,A2 and A3.there are a overlapping area of 19bp between Al and A2,16bp between A2 and A3.Use the primer BC JCF1 and BC JCR3,we get the completed CDS fragment of BC. Collected the product, cloned it into vector, transformed it into E.coli and sequenced it. For the sequencing result was same to the cDNA sequence of BC gene, the full-length cDNA cloning of BC gene from Camellia oleifera was obtained.2. Full-length cDNA cloning of BC gene from Camellia oleifera. The ways to cloneβ-CT gene is similar to these of clone BC gene, the length of BC 5'RACE and BC 3'RACE that had been producted in degenerate PCR are 725bp,691bp,1375bp, and the full-length of.β-CT gene is 1987bp. There is a overlapping region between two RACE fragments, We design two pairs primer to the Overlap Extension PCR ofβ-CT. B1, B2 were obtained respectively, because B1 and B2 was lap over about 23bp, so the completed CDS was be amplify by Overlap Extension PCR. After clone it to vector, transformed to E.coli and sequenced it. We make sure it is the completed CDS ofβ-CT gene.3. Bioinformatic analysis of BC gene from Camellia oleifera. We use the Vecotr NTI 9.0 to analysis the sequence of BC gene, the cDNA sequence is 1901bp in length, and has a 1599bp ORF coding 533 amino acids,81bp untranslated region at 5'end and 218bp untranslated region with a polyA tail at 3'end. The homology percentage of the cDNA sequence from Camellia Oleifera with other species in Genbank is over 87%, so we can conclude the sequence is full-length cDNA of BC gene. The characteristic of physical chemistry, the second structure, and so on of the deduced amino acid sequence was predicted and analyzed by Bio-software, and the results are:pI value is 6.88; molecular weight is 58509.3Da; two transmembrane domain; no signal peptide, it is not a exudation; the instability index is 40.81, the protein as unstable; The second structure might be 31.33% helix, 18% sheet. There are many different prosite in BC as N-glycosylation site, Protein kinase C phosphorylation site, ATP-grasp fold profile and so on; In the three-dimensional model of BC, we find 12 helixes and a cupped area in the central of this protein, maybe it is associated with it's function of combine to the Biotin.4. Bioinformatic analysis ofβ-CT gene from Camellia oleifera. The cDNA sequence is 1987bp in length, and has a 1530bp ORF coding 510 amino acids,454bp untranslated region with a polyA tail at 3'end. The homology percentage of the cDNA sequence from Camellia Oleifera with other species in Genbank is over 87%, so we can conclude the sequence is full-length cDNA ofβ-CT gene. The result of analyzed by Bioinformatic:pI value is 5.17; molecular weight is 58077.9Da; two transmembrane domain; no signal peptide; the instability index is 43.28, the protein as unstable; The second structure might has 27.99% helix,17.85% sheet. There are many different prosite inβ-CT as N-glycosylation site, Protein kinase C phosphorylation site, N-myristoylation site and so on; In the three- dimensional model ofβ-CT, we find the 277 amino acid on the N-end ofβ-CT is unconservative for no tamplate, there are 7 heliex and 5 sheet in it.