Separation And Gene Cloning Of Oleosin From Camellia Oleifera Seeds And Its Prokaryotic Expression

Oil tea tree (Camellia oleifera) is the most important ligneous edible oil trees in South China, and its oleosin from seeds plays a key role in the storage of oils in the seeds. With the development of the oil body-based expression system, the cloning and expression of oleosin is getting more and more focused. This impels us to separate and clone the oleosin gene of Camellia oleifera and develop it into a novel, economical, and simple expression system. The main results are as follows:1) Separation and identification of oleosin proteins in Camellia oleifera seedsOil body was extracted from Camellia oleifera mature seeds after floating centrifugation and repetitive washing. The stabilization of oil body in the medium of pH 7.2, 6.8, and 6.2 was preformed by turbidity test and light microscope. The result showed that the oil body has a natural surface and aggregated notability when the pH was lowered to 6.8 and 6.2 respectively, suggesting that oil body of Camellia oleifera has an isoelectric point of about 6.5. The result of SDS-PAGE analysis on oleosin precipitated with cold acetone revealed that Camellia oleifera oleosin displays two molecular mass of about 14.4kD and 18.4kD, according with the ORF forecast described in the text.2) Separation and bioinformatic analysis of oleosin in Camellia oleifera seedsThe bioinformatics methods such as EST technologh, multiple alignment and sequence clustering were used to separate and screen genes of oleosin family from cDNA library and EST library of Camellia oleifera seed. The obtained oleosin genes were clustered into 5 classes, and named as co_olel (including two subclasses A and B), co_olell, co_olelll, co_oleIV, and co_oleV respectively. The result of comparison with oleosin proteins of other species revealed that Co_oleI, Co_oleIV and Co_oleV are high molecular weight (H) isoforms, and Co_oleII and Co_oleIII are low molecular weight (L) isoforms. The genomic DNA sequences of these oleosin genes were obtained by PCR using serial specific primers (containing the start codon and the stop codon) designed according to their corresponding cDNA sequence. The sequence comparison of cDNA and genomic DNA showed that they display difference to a certain extent, and none of...