Full-length CDNA Cloning Of β-ketoacyl-CoA Synthase 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 edible oil woody plants including Elaeis guineensis, Olea europaea and Cocos nucifera. Very-long-chain fatty acids (VLCFAs) have chain lengths from 18 to 30 carbons, or greater. These fatty acids have a wide physiological functions in Organisms, which participate in the biosynthesis of triacylglycerols, biomembrane and sphingolipids, or act as precursors for the biosynthesis of the epicuticular waxes.The epicuticular waxes is the last barrier in Self-protection of plant,which play a very important role in plant growth,development, and adapt to the external environment.β-ketoacyl-CoA synthase is the rate limiting enzyme in the very long chain fatty acid condensation reaction in endoplasmic reticulum. Cloning ofβ-ketoacyl-CoA synthase gene has an Significant impact on directional control the oil composition of oil crops and provide useful fatty acids. The main results in the paper are as follows:1,Identification ofβ-ketoacyl-CoA synthase gene from the cDNA library of Camellia oleifera. In the constructed cDNA library of Camellia oleifera by our laboratory,we find oneβ-ketoacyl-CoA synthase gene.It is 1147bp in length and a polyA tail. After translating and comparing with other species on line, it is obvious that the coding section from 51bp to 779bp. Except for the vector sequence, it can be concluded that the cDNA clone was a part ofβ-ketoacyl-CoA synthase gene, and has a cDNA sequence deletion of untranslated region and codes about 290 amino acids at 5'terminal,3'end is completed.2,Full-length cDNA cloning ofβ-ketoacyl-CoA synthase gene from Camellia oleifera. Using two special primers GSP1, GSP2 and GSP designed by Primer Premier 5.0 according to the EST sequence of P-ketoacyl-CoA synthase gene, a 1300bp fragment was obtained by 5'RACE strategy to amplify the RNA of seed of fine varieties XiangLin No.1. The 5'RACE fragment was cloned into the vector pMD18-T and then transformed into Escherichia. Coli DH5αand its sequence was obtained by sequencing. After analyzing by Vector NTI 9.0, a 1575bp sequence was formed by splicing EST sequence ofβ-ketoacyl-CoA synthase gene and 5'RACE sequence. According to the sequence,we design for a pair of primers F1,R1 which were located in initiation codon and stop codon. Using RT-PCR product as the template,we found that the new fragment is in line with the size of theory.Then we 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 ofβ-ketoacyl-CoA synthase gene, the full-length cDNA cloning ofβ-ketoacyl-CoA synthase gene from Camellia oleifera was obtained. 3,Function prediction ofβ-ketoacyl-CoA synthase gene from Camellia oleifera. The cDNA sequence is 1891bp in length, and has a 1575bp ORF coding 525 amino acids,45bp untranslated region at 5' end and 268bp untranslated region with a polyA tail at 3' end by using Vector NTI 9.0. The comparability percentage of the cDNA sequence from camellia oleifera with other species in Genbank is over 85%, so we can conclude the sequence is full-length cDNA ofβ-ketoacyl-CoA synthase 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 9.475, molecular weight is 58173.716 Da, four transmembrane domain. Two of them, which are 59aa to 73 aa,97aa to 110 aa,Coincide with the structure of transmembrane,and 254aa to 261 aa,387 aa to389 aa are remote possibility. Based on an analysis of Vector NTI 9.0,there are two main branches of Genetic evolution in different species.One of them is Brassica rapa,Sinapis alba,Brassica oleracea,Crambe abyssinica, the other is Camellia oleifera,Hordeum vulgare,Triticum aestivum,Solanum tuberosum,Arabidopsis thaliana.