Study Of The Multiprotein Complexes On The Rubber Particle Membrane In Hevea Brasiliensis

In this research, the rubber particle membrane proteins from latex of Hevea brasiliensis cultivar RRIM600have been determined by the Blue native polyacrylamide gel electrophoresis technique (the BN-PAGE).The first, we prepared the antibodys of rubber elongation factor (REF), the small rubber particle protein (SRPP) and the rubber transferase enzyme (HRT2) by the following steps: Amplification the REF, SRPP and HRT2gene from the latex of Hevea brasiliensis through RT-PCR followed by cloning to pEASY-E1expression vector. The positive clones were identified by PCR. At last the positive clones were confirmed by gene sequencing. The proteins obtained from positive clones which induced by1mM IPTG were followed by purification with Ni-chelating column(Ni-NTA). The antisera against REF, SRPP and HRT2were obtained from the New Zealand white rabbits which challenged with purified REF, SRPP and HRT2protein respectively. ELISA and Western blotting showed that we have obtained specific polyclonal antibodies against REF, SRPP and HRT protein respectively with high efficiency.The second, we determined the effect of different centrifugation speed on the extraction of rubber particle membrane proteins. By checking the rubber particle with microscopy showed that the minimum centrifuge speed for isolating the large rubber particles and the small rubber particles is12,000rpm and30,000rpm respectively. The results showed that1%SDS is the best detergent for eluting the large rubber particles have been determined by SDS-PAGE. The best detergent for eluting different components in the latex has been determined by the BN-PAGE. The results showed that1%Digitonin,0.1%Triton X-100and1%Triton X-100is better detergent for eluting the large rubber particles, small rubber particles and the lutoids membrane proteins respectively. The study also showed that REF and SRPP proteins expressed differentially among rubber particle membrane proteins that likely have relationships with the formation and development mechanism of the rubber particles.The third, a membrane protein complex with strong signal on the large rubber particles has been resolved efficiently by BN-PAGE/SDS-PAGE. Western blotting by using antibody against REF showed that the protein complex probably consists of REF and other high molecular weight protein.