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PLANT GENE REGISTER PGR99-184 |
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Dong Ho Shin, Hunseung Kang, and Kyung-Hwan Han (1999) A Hevea brasiliensis Homolog (HbLAR) of an Arabidopsis Hypothetical Protein Gene YUP8H12R.4 is Expressed Abundantly in Latex and is Not Induced Either by Ethylene or Wounding (Accession No. AF098458). (PGR99-184) Plant Physiol. 121: 1384
Dong Ho Shin, Hunseung Kang, and Kyung-Hwan Han*
Kumho Life and Environmental Science Laboratory, 1 Oryong-Dong, Puk-Gu, Kwangju, 500-712, Korea (DHS, HK). Department of Forestry, 126 Natural Resources, Michigan State University, East Lansing, MI 48824-1222, USA (K-HH).
*Corresponding author: Fax: 517-353-4751;
E-mail hanky@msu.edu
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REPORT |
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Natural rubber (cis-1,4-polyisoprene), an isoprenoid polymer with no known physiological function to the plant, is synthesized on the surface of rubber particles suspended in the cytoplasm of laticifers (latex). The latex of Hevea brasiliensis contains 30-50% (wt/wt) of cis-1,4-polyisoprene. As the site of rubber synthesis, gene expression patterns in the latex have been of interest for the study of rubber biosynthesis. Genes expressed in the latex of Hevea can be grouped into three categories based on the proteins they encode: (1) defense (Broekaert et al. 1990, Sivasubramaniam et al. 1995); (2) rubber synthesis-related; and (3) allergenic proteins (Yeang et al. 1996). Kush et al. (1990) have shown differential expression of several rubber-biosynthesis-related genes in the latex. The rubber elongation factor, an enzyme involved in rubber biosynthesis (Dennis and Light 1989), is highly expressed in laticifers (Goyvaerts et al. 1991). Other rubber-synthesis-related genes such as HMGCoA reductase (Chye et al. 1992) and small rubber particle protein (Oh et al. 1999) have been shown to be highly expressed in the latex.
In the course of our study on the biosynthesis of natural rubber, we have generated a number of expressed sequence tags (ESTs) from a cDNA library prepared from the latex of H. brasiliensis (Han et al., 1999). Comparison of the ESTs with public databases of reported genes identified a cDNA clone that has high sequence homology with Arabidopsis genomic sequences encoding a hypothetical protein YUP8H12R.4. This Arabidopsis protein was reported in the database entry to contain similarity to Saccharomyces cerevisiae hypothetical protein YOR197W (accession no. Z75105). Ten ESTs showing homology to the Arabidopsis sequence have been reported to GenBank from Glycine max. However, the functional activity of these proteins has not been studied.
Because of its abundance among latex ESTs, we named the gene as HbLAR (H. brasiliensis latex-abundant RNA) and were interested in knowing whether this HbLAR protein was involved in rubber biosynthesis. As a first step toward such study, we isolated a full-length cDNA clone encoding HbLAR protein and studied its expression pattern by northern blot analysis. The full-length cDNA clone was obtained by screening the same cDNA library for the ESTs using this EST as a probe. The cDNA clone hybridized to the probe was subjected to in vivo excision according to the protocol provided by the cDNA library kit and sequenced. The sequence contained a 1251-bp ORF encoding a peptide of 417 amino acids with a predicted molecular mass of 45.9 kDa and an isoelectric point of 4.84. The deduced amino acid sequence of the putative protein is highly homologous to those of A. thaliana (59% identity, accession no. 3152600), Schizosaccharomyces pombe hypothetical protein (39% identity, accession no. CAA20128), and S. cerevisiae hypothetical protein YOR197W (38% identity, accession no. S67089). This gene is expressed abundantly in latex compared to leaf tissues. Northern blot analyses also indicated that this gene is not induced by either Ethphone (2-chloroethylphosphonic acid, an ethylene-releasing agent) stimulation or wounding. In order to test the role of HbLAR protein in rubber biosynthesis, we expressed the gene in E. coli using an IPTG-inducible expression vector (pGEX, Pharmarcia). The recombinant protein was purified according to the protocol provided with the kit and used in in vitro rubber assay as described in Oh et al. (1999). The addition of the recombinant HbLAR protein to the in vitro reaction mixture did not affect [14C]IPP incorporation into rubber.
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LITERATURE CITED |
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Broekaert, I., H. I., Lee, A., Kush, N. H., Chua, and N., Raikhel. (1990) Wound-induced accumulation of mRNA containing a hevein sequence in laticifers of rubber tree (Hevea brasiliensis). Proc. Natl. Acad. Sci. U.S.A. 87:7633-7637.
Chye, M. L., C. T., Tan, and N. H., Chua. (1992) Three genes encode 3-hydroxy-3-methylglutaryl-coenzyme A reductase in Hevea brasiliensis: hmg1 and hmg3 are differentially expressed. Plant Mol. Biol. 19:473-484.
Dennis, M. S., and D. R., Light. (1989) Rubber elongation factor from Hevea brasiliensis. Identification, characterization, and role in rubber biosynthesis. J. Biol. Chem. 264:18608-18617.
Goyvaerts, E., M., Dennis, D., Light, and N. H., Chua. (1991) Cloning and sequencing of the cDNA encoding the rubber elongation factor of Hevea brasiliensis. Plant Physiol. 97:317-321.
Han, K.-H., Shin, D.H., Yang, J.M., Oh, S.K., Kim, I.J., and Chow, K.-S. (1999) Genes expressed in the latex of Hevea brasiliensis. Tree Physiology (in Press)
Kush, A., E., Goyvaerts, M. L., Chye, and N. H., Chua. (1990) Laticifer-specific gene expression of Hevea brasiliensis (rubber tree). Proc. Natl. Acad. Sci. U.S.A. 87:1787-1790.
Oh, S. K., H. S., Kang, D. H., Shin, J., Yang, K. S., Chow, H. Y., Yeang, B., Wagner, H., Breiteneder, and K.-H., Han. (1999) Isolation, characterization, and functional analysis of a novel cDNA clone encoding a small rubber particle protein (SRPP) from Hevea brasiliensis. J. Biol. Chem. 274:17132-17138.
Sivasubramaniam, S., V. M., Vanniasingham, C. T., Tan, and N. H., Chua. (1995) Characterisation of HEVER, a novel stress-induced gene from Hevea brasiliensis. Plant Mol. Biol. 29:173-178.
Yeang, H. Y., K. F., Cheong, E., Sunderasan, S., Hamzah, N. P., Chew, S., Hamid, R. G., Hamilton, and M. J., Cardosa. (1996) The 14.6 kd rubber elongation factor (Hev b 1) and 24 kd (Hev b 3) rubber particle proteins are recognized by IgE from patients with spina bifida and latex allergy. J. Allerg. Clin. Immunol. 98:628-639.
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