关键词: 巴西橡胶树, 胶乳, SNAREs蛋白, Ykt6

Abstract:

The plant endomembrane system or secretory pathway is critical for biosynthetic, response to stress, and other various physiological functions. Although it is clear that some mechanisms of protein trafficking are conserved between all eukaryotes, plants appear to possess a unique and highly complex vacuolar—targeting pathway. The molecular mechanisms responsible for targeting within these complex pathways have only recently been explored. The specificity of vesicle docking and fusion is mediated by proteins called SNAREs (soluble N-ethylmaleimide-sensitive factor (NSF) attachment protein receptor). Characterizing such factor would help to understand the endomembrane system or secretory pathway. Recently, we have obtained several cDNA clone related to secretory pathway by screening of a subtracted cDNA library. The subtracted cDNA library was constructed by suppressive subtractive hybridization (SSH), in which tester was latex poly(A+)RNA while driver was leaf poly(A+)RNA . In order to identify markers responsible for vesicle docking and fusion within the secretory pathway in rubber laticiferous vessels cell, one of these cDNA clone which is highly homologous to the gene SNAREs was isolated. According to its sequences information, we obtained a novel full-length cDNA termed R295 by using rapid amplification of cDNA ends (RACE) strategies. R295 is 1 070 bp long containing a 600 bp ORF, flanked by a 93 bp 5’-UTR and a 377 bp 3’-UTR, which are available from the GenBank databases under the accession numbers AY605930. Full-length gene of the R295 has predicated to contain 199 amino acid residues and to be of molecular masses of 22.47 kD. The deduced protein has a pI of 7.83. Hydropathy and transmembrane motif analysis of deduced amino acid sequences indicated that R295 does not possess a transmembrane spanning domain, but it contains a carboxyl-terminal CAAX motif suggesting that it is post-translationally modified by the addition of a 15- or 20-carbon isoprenoid. The result of the conserved domains analysis indicated that R295 not only has a highly conserved region, SNARE coiled-coil domain, a signature motif for vesicle-associated membrane proteins (VAMP), but also contains a longin domain and a prenyl group binding site (CAAX box), it is implied that R295 would be an YKT-like SNAREs. Phylogenesis analysis revealed that R295 is the most closely related to the SNARE of N. tabacum. The transcripts of R295 were observed by RT-PCR. R295 was high expressed in the latex than in leaves.

Key words: Hevea brasiliensis, SNAREs, latex, Ykt6