Cloning And Sequence Characterization Of Oleosin And Related Genes From Vernicia Fordii

Vernicia fordii is originated from China, and is well known as one of the four major edible oil woody plants in the world. Tung oil is a kind of high quality dry oil, it is composed of unsaturated fatty acids (UFA) mostly, such as elaeostearic acid, and has widely used for industrial applications. Tung oil is also the high-quality raw material for biodiesel. But tung oil have a problem with high level of unsaturated fatty acid.This research based on the'Duiniantong'developing seeds, genes related to fatty acid synthesis and storage, such as oleosin gene, SAD,FAD3 and FAD9 fatty acid desaturase were cloned by the construction of cDNA library, degenerate PCR,RACE and RT-PCR. We can provide scientific foundation for regulating the fatty acid metabolic pathways by molecular biological methods to improve the tung oil fatty acid.1. Separation and identification of oleosin gene from Vernicia fordii. Two clone were choosed after sequence analysis in the cDNA library and EST library of Vernicia fordii, T3 and T7 universal primer were used to amplify the gene, analysis of sequencing shows that the two genes are full length cDNA of oleosin gene, the cDNA length were 738bp and 838bp, they encoded 137 and 154 amino acids,respectively, they were named VF_oleⅠand VF_oleⅡ. After translating and comparing with other species, we know there is a low conservation of N-terminal and C-terminal, a hight conservation in center, and contain Proline section. Phylogenic tree analysis revealed that VF_oleⅡand VF_oleⅠdisplayed the highest similarity to J.curcas oleosin2 and oleosin3, the identity of amino sequence is 82% and 84%. After submitting these sequences to GeneBank, we received GenBank accession numbers as GU245884andGU245885 accordingly.2. Cloning and sequence characterization of SAD. Based on the alignment of the amino acid sequences and cDNA sequences of SAD from plants of Euphorbiaceae, degenerate primers were designed contain the start codon and stop codon. Total RNA isolated from the riping seed from'duiniantong', the material in the process of the construction of cDNA library was used to generate cDNA by reverse transcription.1179bp coding sequences in length of SAD gene was cloned by degenerate PCR. After submitting the sequences to GeneBank, we received GenBank accession numbers as GU363502. We did some bioinformatic analysis of DNA sequences and amino acids.SAD gene of Vernicia fordii displayed the highest similarity(96%,91%) to Vernicia montana and J. curcas SAD. The SAD protein molecular weight is 45217.7Da, isoelectric points is 5.99,belongs to unstable protein, no transmembrane domain, no signal peptide, the second structure are a-helix and random coil most, there are N-glycosylation site and other domains, and have a FA desaturase 2 domain.3.Full-length cDNA cloning of FAD3 and FAD9 fatty acid desaturase from Vernicia fordii.Based on the alignment of the amino acid sequences and cDNA sequences, degenerate primers were designed in the conservative region. Total RNA isolated from the riping seed from duiniantong, we get cDNA by reverse transcription. Two fragments about 486bp (FAD3) and 360bp (FAD9) in length was cloned by degenerate PCR respectively. Based on the sequence information acquired by degenerate PCR, FAD3, FAD9 fatty acid desaturase gene specific primers were designed for 3'RACE and 5'RACE. Total RNA isolated from the riping seed from duiniantong was used to generate RACE ready cDNA by reverse transcription. The 3'RACE and 5'RACE fragment length of FAD3 gene is 1200bp and 550bp,respectively; the 3'RACE and 5'RACE fragment length of FAD9 gene is 470bp and 900bp.Degenerate fragment and RACE fragment were assembled by us, we designed the primers nearby the region of start codon and stop codon after the Prediction of start codon and stop codon. Complete CDS of the two genes were amplified by RT-PCR. The CDS of FAD3 and FAD9 is 1344bp and 939bp respectively. After submitting these sequences to GeneBank, we received GenBank accession numbers as GU564350 and GU564349 accordingly. We did some Bioinformatic analysis of DNA sequences and amimo acids. The CDS of FAD3 gene displayed the highest similarity(83%) to R. communis fatty acid desaturase(putative), FAD9 gene displayed the highest similarity(84%) to R. communis delta9 desaturase.Protein online analysis results showed that molecular weight of FAD3 protein is 51660.6 Da, isoelectric points is 8.25, belong to stable protein, seven transmembrane domains, no signal peptide, the second structure areα-helix and random coil most, there are N-glycosylation site et al.domains, and Cyt-b5 and FA_desaturase domain. FAD9 protein molecular weight is 36213.5Da, isoelectric points is 8.62, belong to stable protein, five transmembrane domains, no signal peptide, the second structure are a-helix and random coil most, there are N-glycosylation site and other domains, and FA_desaturase domain.