Cloning and characterization of cDNA encoding farnesyl diphosphate synthase from rubber tree (Hevea brasiliensis)

Commercially used natural rubber (cis-1,4-polyisoprene) is a secondary metabolite of the rubber tree (Hevea brasiliensis). Previous studies have shown the involvement of a prenyl transferase in the final steps of natural rubber biosynthesis which includes polymerization of isopentenyl pyrophosphate into rubber. Using synthetic oligonucleotides corresponding to the partial amino acid sequences of this protein as probes to screen a laticifer-specific cDNA library, we have isolated a full-length cDNA which encodes a 47 kDa protein with strong homology to farnesyl diphosphate synthases from many species. The catalytic activity of this protein was confirmed by complementing the deletion yeast mutant. In Hevea, this gene is expressed in latex producing cells and in the epidermal region of the rubber plant suggesting a dual role for the protein in the biosyntheses of rubber and other isoprenoids. Although the expression level of this gene is not significantly affected by hormone treatment (e.g. ethylene), regeneration of latex due to tapping increases its expression level.     

A novel cDNA from Parthenium argentatum Gray enhances the rubber biosynthetic activity in vitro

Natural rubber (cis-1,4-polyisoprene) is an isoprenoid compound produced exclusively in plants by the action of rubber transferase. Despite a keen interest in revealing the mechanisms of rubber chain elongation and chain length determination, the molecular nature of rubber transferase has not yet been identified. A recent report has revealed that a 24 kDa protein tightly associated with the small rubber particles of Hevea brasiliensis, therefore designated small rubber particle protein (SRPP), plays a positive role in rubber biosynthesis. Since guayule (Parthenium argentatum Gray) produces natural rubber similar in size to H. brasiliensis, it is of critical interest to investigate whether guayule contains a similar protein to the SRPP. A cDNA clone has been isolated in guayule that shares a sequence homology with the SRPP, thus designated guayule homologue of SRPP (GHS), and the catalytic function of the protein was characterized. Sequence analysis revealed that the GHS is highly homologous in several conserved regions to the SRPP (50% identity). In vitro functional analysis of the recombinant protein overexpressed in E. coli revealed that the GHS plays a positive role in isopentenyl diphosphate incorporation into high molecular weight rubbers as SRPP does. These results indicate that guayule and Hevea rubber trees contain a protein that is similar in its amino acid sequence and plays a role in isopentenyl diphosphate incorporation in vitro, implying that it contributes to the enhancement of rubber biosynthetic activity in rubber trees.     

An intron-free methyl jasmonate inducible geranylgeranyl diphosphate synthase gene from Taxus media and its functional identification in yeast

Geranylgeranyl diphosphate synthase (GGPPS, EC: 2.5.1.29) catalyzes the biosynthesis of geranylgeranyl diphosphate (GGPP), which is a key precursor for diterpenes including Taxol, one of the most potent antitumor drugs. In order to investigate the role of GGPP synthase in taxol biosynthesis, we cloned, characterized and functionally expressed the GGPP synthase gene from Taxus media. A 3743-bp genomic sequence of T. media was isolated by genome walking strategy which contained an 1182-bp open reading frame (ORF) encoding a 393-amino acid polypeptide that showed high similarity to other plant GGPPSs. Subsequently the full-length cDNA of the GGPPS gene of T. media (designated TmGGPPS) was amplified by RACE. Bioinformatic analysis showed that TmGGPPS was an intron-free gene and its deduced polypeptide contained all the five conserved domains and functional aspartate-rich motifs of the prenyltransferases. By constructing the phylogenetic tree of plant GGPPSs, it was found that plant-derived GGPPSs could be divided into two classes, angiosperm and gymnosperm classes, which might have evolved in parallel from the same ancestor. To our knowledge this was the first report that the geranylgeranyl diphosphate synthase genes were free of intron and evolved in parallel between angiosperms and gymnosperms. The coding sequence of TmGGPPS was expressed in yeast mutant (SFNY368) lacking of GGPP synthase activity through functional complementation, and the transgenic yeast showed to have activity of GGPP synthase. This was also the first time to use SFNY368 to identify the function of plant-derived GGPPSs. Furthermore, investigation of the impact of methyl jasmonate (MeJA) on the expression of TmGGPPS revealed that MeJA-treated T. media cultured cells had much higher expression of TmGGPPS than untreated cells.     

Identification and comparison of natural rubber from two Lactuca species

Renewed interest in the identification of alternative sources of natural rubber to Hevea brasiliensis has focused on the Compositae family. In our search for Compositae models for rubber synthesis, we extracted latex from stems of two lettuce species: Lactuca serriola, prickly lettuce, and Lactuca sativa cv. Salinas, crisphead lettuce. Both species contained cis-1,4-polyisoprene rubber in the dichloromethane-soluble portions of their latex, and sesquiterpene lactones in their acetone-soluble portions. The rubber from both species and their progeny had molecular weights in excess of 1,000,000g/mol, and polydispersity values of 1.1. Rubber transferase activity was detected across a range of farnesyl diphosphate initiator concentrations, with decreased activity as initiator concentrations exceeded putative saturation. These results add lettuce to the short list of plant species that produce high molecular weight rubber in their latex. Due to the genomic and agronomic resources available in lettuce species, they provide the opportunity for further dissection of natural rubber biosynthesis in plants.     

Human geranylgeranyl diphosphate synthase. cDNA cloning and expression

Geranylgeranyl diphosphate (GGPP) synthase (GGPPSase) catalyzes the synthesis of GGPP, which is an important molecule responsible for the C20-prenylated protein biosynthesis and for the regulation of a nuclear hormone receptor (LXR.RXR). The human GGPPSase cDNA encodes a protein of 300 amino acids which shows 16% sequence identity with the known human farnesyl diphosphate (FPP) synthase (FPPSase). The GGPPSase expressed in Escherichia coli catalyzes the GGPP formation (240 nmol/min/mg) from FPP and isopentenyl diphosphate. The human GGPPSase behaves as an oligomeric molecule with 280 kDa on a gel filtration column and cross-reacts with an antibody directed against bovine brain GGPPSase, which differs immunochemically from bovine brain FPPSase. Northern blot analysis indicates the presence of two forms of the mRNA.     

巴西橡胶树(Hevea brasiliensis)微管相关蛋白全长cDNA克隆及表达分析

从巴西橡胶树Hevea brasiliensis差减cDNA文库中分离到微管相关蛋白（Microtubule-associated protein,MAPs）基因片段,根据该基因片段序列信息,设计特异引物,采用cDNA末端快速扩增技术RACE（Rapid Amplification ofcDNA Ends）进行差异片段的5＇和3＇端的扩增,获得了长度为788bp的全长cDNA,该基因在GenBank中的登录号为AY461412.序列分析表明该基因包含完整的开放阅读框,编码144个氨基酸,与微管相关蛋白基因家族具有很高的同源性,推测该基因是微管相关蛋白基因.半定量RT-PCR检测证实它在胶乳中的表达强于叶中,胁迫处理（伤害及乙烯处理）使其表达上调.     

Role of lutoid membrane transport and protein synthesis in the regeneration mechanism of latex in different <Emphasis Type="Italic">Hevea</Emphasis> clones

Natural rubber (cis-1,4 polyisoprene) is synthesised in the milky cytoplasm, the latex, of specialized cells called laticifers in the bark tissues of the rubber tree (Hevea brasiliensis). Regeneration mechanism of latex after each tapping (controlled wounding of the bark) was studied in relation to lutoid membrane enzymes and protein synthesis in twelve rubber clones with varying yield potentials during the peak rubber yielding season. High activity of membrane enzymes and better availability of biochemical energy [ATP] were observed in clones viz; RRII 105, RRIM 600, PB 260, RRII 422 and RRII 430. The highest protein biosynthetic capacity was noticed in clone PB 260 and RRIM 600. However, high ATP content, increased invertase activity and protein biosynthesis were observed in the medium yielding clone GT1 compared to clones with low rubber yield potential. Very low sugar content and increased invertase activity in the latex of clone PB 260 indicated intense latex metabolism with high protein turnover that implies fast recouping of the cellular metabolites lost during latex harvesting. Clone PB 217 was characterized by very high sucrose and low ATP concentration and ATPase activity in latex indicating slow metabolism and hence be suitable for inducing latex metabolism using ethylene stimulant. Low rubber yielding clones such as RRII 33 and RRII 38 were consistently recorded a high sucrose content but very low activity of membrane enzymes, reduced ATP concentration and low protein biosynthesis in latex. Among the recently released modern clones (RRII 400 series), latex regeneration capacity was higher in RRII 422 and RRII 430. The significance of lutoid membrane transport and protein synthesis is discussed in relation to general latex metabolism of these rubber clones. The outcome of this study would be helpful to design suitable latex harvesting systems and yield stimulation methods for optimizing latex production in each clone based on metabolic profiling.