| As a specie of woody fuel plants of great importance in the Southern part of China, Camellia oleifera is well known as one of the four major edible oil woody plants in the world. Camellia oil is composed of unsaturated fatty acids (UFA) mostly, such as oleic acid and linoleic acid, and the average content of UFA is above 90%.These kinds of UFA are mainly formed by the way of desaturation and elongation of saturated fatty acids. Plant acyl-acyl carrier protein thioesterases type B (FatB) hydrolyze saturated acyl-ACP thioester bonds, releasing free saturated fatty acids and ACP. FatBs have been shown to play a key role in determining the composition of storage lipids.So the study of FatB gene in Camellia Oleifera is important for revealing the lipids molecular biosynthesis patterns. The main results in the paper are as follows:1.Cloning of partial cDNA sequence of FatB gene from Camellia oleifera. Three FatB genes have been identified from the cDNA library in the constructed cDNA library of Camellia oleifera by our laboratory. According to the results of Blast online, it is obvious that the three FatB genes are all false clones. So "FatB" was used as the keyword in the search in GenBank, and six amino acid sequences and full-length cDNAs of FatB gene in other plants were found.Based on the alignment of the six amino acid sequences and cDNA sequences, a pair of degenerate primers was designed in the conservative region. Total RNA isolated from the riping seed from fine varieties clone'XiangLin No.1'which was also the raw material in the process of the construction of the cDNA library was used to generate cDNA by reverse transcription. A sequence about 700bp in length was cloned by degenerate PCR, after deleting the pair of degenerate primers'sequences we got partial cDNA of 637bp which coded 212 amino acids.2.Cloning of complete cDNA ends of FatB gene from Camellia oleifera.Based on the sequence information acquired by degenerate PCR, Gene specific primers---5-GSP1, 5-GSP2 and 5-NGSP were designed for 5'RACE, and 3-GSP for 3'RACE.Total RNA isolated from the riping seed of from'XiangLin No.1'was used to generate RACE ready cDNA by reverse transcription. Finally, we got six similar but different 5'RACE sequences (each about 900bp in length) and three similar but different 3'RACE sequences (each about 1200bp in length) by RACE amplification.It suggested that CoFatB genes should be assigned to a Gene Family.3.Isolation and Cloning of complete CDS (coding sequence) of FatB Genes from Camellia oleifera.With the result of the alignment of 5'RACE sequences with 3'RACE sequences, we predicted the positions of initiation codon and stop codon. Based on these information, sense primers were designed to land on the upstream of initiation codon and antisense primer was designed to land on the downstream of stop codon. Total RNA isolated from the riping seed of from'XiangLin No.1' was used to produce cDNA by reverse transcription. We got lots of clones by RT-PCR amplification. And then by random sequencing several clones together with bioinformatics analysis, five members were isolated. They were named by co-fatb1,co-fatb2,co-fatb3,co-fatb4, co-fatb5 and their ORF are 1272bp,1311bp,1305bp,1305bp,1263bp in length.After submitting these sequences to GenBank, we received GenBank accession numbers as FJ899670, FJ899671, FJ899672,FJ899673 and FJ899674 accordingly. We discovered an interesting phenomenon that co-fatb1,co-fatb1,co-fatb4 all have complicated initiation codon sequence. Its sequence is'ATGTG',if translation uses ATG as initiation codon, it will stop in advance and there will be no ORF (open reading frame); but if GTG serves as initiation codon, a functional protein will be generate by translation; partial 5'UTR sequence about 150bp of co-fatb5 has three'ATGTG'repeats but none is used for initiation codon. In addition, the amino acids sequence were deeply analysized and some amino acid residues which had something with enzyme activity, substrat specificity and the formation of hot-dog domains were marked. |
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