Isolation, Characterization, and Functional Analysis of a Novel cDNA Clone Encoding a Small Rubber Particle Protein from Hevea brasiliensis

Soo Kyung Oh,Hunseung Kang,Dong Ho Shin,Jaemo Yang,Keng-See Chow,Hoong Yeet Yeang,Birgit Wagner,Heimo Breiteneder,Kyung-Hwan Han

doi:10.1074/jbc.274.24.17132

Abstract

Biochemical evidence reported so far suggests that rubber synthesis takes place on the surface of rubber particles suspended in the latex of Hevea brasiliensis. We have isolated and characterized a cDNA clone that encodes a protein tightly bound on a small rubber particle. We named this protein small rubber particle protein (SRPP). Prior to this study, this protein was known as a latex allergen, and only its partial amino acid sequence was reported. Sequence analysis revealed that this protein is highly homologous to the rubber elongation factor and the Phaseolus vulgaris stress-related protein. Southern and Northern analyses indicate that the protein is encoded by a single gene and highly expressed in latex. An allergenicity test using the recombinant protein confirmed that the cloned cDNA encodes the known 24-kDa latex allergen. Neither ethylene stimulation nor wounding changed the transcript level of the SRPP gene in H. brasiliensis. An in vitro rubber assay showed that the protein plays a positive role in rubber biosynthesis. Therefore, it is likely that SRPP is a part of the rubber biosynthesis machinery, if not the rubber polymerase, along with the rubber elongation factor.

Highlights

Biochemical evidence reported so far suggests that rubber synthesis takes place on the surface of rubber particles suspended in the latex of Hevea brasiliensis
In a separate study using a subtracted cDNA library, we identified an EST whose deduced amino acid sequence matched with the known partial sequence of Hev b 3.2 From the sequence information of the EST clone, we designed a PCR primer corresponding to the sequences from ϩ185 to ϩ211 bp in small rubber particle protein (SRPP)
Sequence analysis showed that the cDNA insert was 910 bp long (Fig. 1) and contained a 612-bp open reading frame flanked by a 63-bp 5Ј-untranslated regions (UTRs) and a 181-bp 3Ј-UTR including a poly(A) tail of 18 bp

Summary

Introduction

Biochemical evidence reported so far suggests that rubber synthesis takes place on the surface of rubber particles suspended in the latex of Hevea brasiliensis. The rubber elongation factor (REF), an enzyme involved in rubber biosynthesis [3], is highly expressed in laticifers [4]. Genes expressed in the latex of Hevea can be divided into three groups based on the proteins they encode: 1) defense-related proteins such as hevein [7], chitinase [8], ␤-1,3glucanase [9], and HEVER [10]; 2) rubber biosynthesis-related proteins such as REF [4], hydroxymethylglutaryl-CoA reductase [11], and farnesyl diphosphate synthase [12]; and 3) latex allergens (proteins) such as Hev b 3 [13], Hev b 4, Hev b 5 [14, 15], and Hev b 7 [16].

Methods

Results

Conclusion