Cloning And Expression Of An Oleosin CDNA From Seeds Of Brassica Napus

Oleosin genes are mainly expressed abundant in the oil-plant seeds. Olsosin, as storage proteins attach specifically to phyospholipid monolayer of the oil bodies, and play important roles in the formation and liquefaction of oil bodies. In the study, we try to establish assays toclone and expression of oleosin cDNA from Brassica napus, which was favourable to discuss the expression of oleosin in time and space, relation with TAG accumulation, and especially apply them to plant genetic engineering.1. RNA of rapeseed was extracted by hot CTAB-LiCl method. The results of ultraviolet spectrophotometer and agarose gel electrophoresis show that the obtained RNA had no obvious degradation and a good sufficient purity. This method can efficiently eliminate the interference of polysaccharide and lipids rich in rapeseed.2. Olsosin cDNA and Perilipin, SREBP cDNA were amplified by RT-PCR in rapeseed. The agarose gel electrophoresis analysis indicated that Perilipin cDNA was not amplified in rapeseed, but SREBP's was amplified, there had a same decompensation in synthetic fat between plant and animal. The results of sequences tested show that oleosin cDNA from Brassica Napus and from Brassica campestris displayed 91% sequence identity. A near full-length oleosin cDNA was amplified with 5'RACE, Blast analysis indicate there was a high degree of sequence similarity between 484bp segment and bete-1,3 glucanase homologue DNA of chromosome IV in A6 from Arabidopis thaliana.3. Northern-blotting was done with amplified cDNA as probe in seeds of different phases and different oil quanlity, which discussed little relation between oleosin mRNA expression profile and different oil quanlity in rapeseed.4. Reading frame of oleosin cDNA was amplified with a new primer and the Internet's primer of 5'RACE from seeds of Brassica Napus and Brassica campestris,and recombined vector pMD18-T. Blast analysis indicated that reading frame of oleosin cDNA from the two forms displays 99% similarity; meanwhile a near full-length new cDNA of oleosin-like-protein from Brassica campestris seeds was registered connecting with previously cloned cDNA.5. Expression vector was recombined with oleosin reading frame cDNA and vectorpTYB2, fusion protein was induced and produced in ER2566, the target protein was purified by chitin pre-packed column. The SDS-PAGE analysis shows that about 93KDa fusion protein and about 27 KDa target protein were produced.