CLONING AND EXPRESSION OF CDNA ENCODING 43KD RUBBER PARTICLE MEMBRANE PROTEIN OF Hevea Brasiliensis

Cloning and Expression of cDNA Encoding 43KD Rubber Particle Membrane Protein of Hevea brasiliensisAbstractA 43KD rubber particle protein was purified from latex yielding a single protein with an apparent molecular mass of 43KD as determined by SDS-PAGE. A degenerate oligonucleotide primer based on the N-terminal amino acid sequence of this purified 43KD rubber particle protein was used to amplify a 1385 bp cDNA by 3'apid amplification of cDNA ends (3'ACE) The structure of the cDNA is consistent with structure of other known polyubiquitin genes. It contains of five repeats in a head-to-hail arrangement without intervening sequences, each encoding an ubiqutin unit of 76 amino acids. At the carboxyl-terminal end of the last ubiqutin unit followed an extra phenylanaline residue. Southern blot analyses revealed that are multiple copies of gene encoding 43KD rubber particle protein in H. Brasiliensis. Western blot demonstrated 43KD rubber particle protein maybe a polyubiquitin. Northern blot analysis of RNA isolated from the latex, young leaves and bark revealed that expression was highest in the latex and lowest in young leaves. The polyubiquitin gene was induced by exogenous ethylene and jasmonic acid treatments. The 5?-UTR was obtained using GenomeWalker. It contained an 1175bp intron immediately 5?to the initiation code for the first ubiquitin-coding. Several sequences similar to eukaryotic cis regulatory element were found in the 5?UTR 430bp proximal 5?flanking sequence. A putative TATA box sequences found at -33bp. At position -79bp, a TCAAT sequence can be observed, which is similar to the CAAT?motif found in a multitude of higher eukaryotic promoters. The G-box element sequence, CACGTG, was found at -127bp. Some sequences 5-nGAAn-3 present many positions, which is similar to the consensus heat-shock element. The A+T rich region, containing 900/a A+T in the 230bp sequence, is present in the 5 flanking region.