Dataset Information

Cloning and Expression Analysis of One Gamma-Glutamylcysteine Synthetase Gene (Hb?-ECS1) in Latex Production in Hevea brasiliensis.

ABSTRACT: Rubber tree is a major commercial source of natural rubber. Latex coagulation is delayed by thiols, which belong to the important type of antioxidants in laticifer submembrane, and is composed of glutathione (GSH), cysteine, and methionine. The rate-limiting enzyme, ?-ECS, plays an important role in regulating the biosynthesis of glutathione under any environment conditions. To understand the relation between ?-ECS and thiols and to correlate latex flow with one-time tapping and continuous tapping, we cloned and derived the full length of one ?-ECS from rubber tree latex (Hb?-ECS1). According to qPCR analysis, the expression levels of Hb?-ECS1 were induced by tapping and Ethrel stimulation, and the expression was related to thiols content in the latex. Continuous tapping induced injury, and the expression of Hb?ECS1 increased with routine tapping and Ethrel-stimulation tapping (more intensive tapping). According to expression in long-term flowing latex, the gene was related to the duration of latex flow. Hb?ECS1 was expressed in E. coli Rosetta using pET-sumo as an expression vector and the recombinant enzyme was purified; then we achieved 0.827?U/mg specific activity and about 66 kDa molecular weight. The present study can help us understand the complex role of Hb?-ECS in thiols biosynthesis, which is influenced by tapping.

SUBMITTER: Fang W

PROVIDER: S-EPMC4935901 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Cloning and Expression Analysis of One Gamma-Glutamylcysteine Synthetase Gene (Hbγ-ECS1) in Latex Production in Hevea brasiliensis.

Fang Wei W Qiao Luo Shi LS Ming Wu W Jian Qiu Q Feng Yang Wen YW Hua Gao Hong GH Zhou Xiao Xian XX

BioMed research international 20160623

Rubber tree is a major commercial source of natural rubber. Latex coagulation is delayed by thiols, which belong to the important type of antioxidants in laticifer submembrane, and is composed of glutathione (GSH), cysteine, and methionine. The rate-limiting enzyme, γ-ECS, plays an important role in regulating the biosynthesis of glutathione under any environment conditions. To understand the relation between γ-ECS and thiols and to correlate latex flow with one-time tapping and continuous tappi ...[more]

PMID: 27419133

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Project description:BackgroundSmall RNAs modulate plant gene expression at both the transcriptional and post-transcriptional level, mostly through the induction of either targeted DNA methylation or transcript cleavage, respectively. Small RNA networks are involved in specific plant developmental processes, in signaling pathways triggered by various abiotic stresses and in interactions between the plant and viral and non-viral pathogens. They are also involved in silencing maintenance of transposable elements and endogenous viral elements. Alteration in small RNA production in response to various environmental stresses can affect all the above-mentioned processes. In rubber trees, changes observed in small RNA populations in response to trees affected by tapping panel dryness, in comparison to healthy ones, suggest a shift from a transcriptional to a post-transcriptional regulatory pathway. This is the first attempt to characterise small RNAs involved in post-transcriptional silencing and their target transcripts in Hevea.MethodsGenes producing microRNAs (MIR genes) and loci producing trans-activated small interfering RNA (ta-siRNA) were identified in the clone PB 260 re-sequenced genome. Degradome libraries were constructed with a pool of total RNA from six different Hevea tissues in stressed and non-stressed plants. The analysis of cleaved RNA data, associated with genomics and transcriptomics data, led to the identification of transcripts that are affected by 20-22 nt small RNA-mediated post-transcriptional regulation. A detailed analysis was carried out on gene families related to latex production and in response to growth regulators.ResultsCompared to other tissues, latex cells had a higher proportion of transcript cleavage activity mediated by miRNAs and ta-siRNAs. Post-transcriptional regulation was also observed at each step of the natural rubber biosynthesis pathway. Among the genes involved in the miRNA biogenesis pathway, our analyses showed that all of them are expressed in latex. Using phylogenetic analyses, we show that both the Argonaute and Dicer-like gene families recently underwent expansion. Overall, our study underlines the fact that important biological pathways, including hormonal signalling and rubber biosynthesis, are subject to post-transcriptional silencing in laticifers.

Project description:BACKGROUND:Expression patterns of many laticifer-specific gens are closely correlative with rubber yield of Hevea brasiliensis (para rubber tree). To unveil the mechanisms underlying the rubber yield, transcript levels of nine major latex metabolism-related genes, i.e., HMG-CoA synthase (HMGS), HMG-CoA reductase (HMGR), diphosphomevalonate decarboxylase (PMD), farnesyl diphosphate synthase (FPS), cis-prenyltransferase (CPT), rubber elongation factor (REF), small rubber particle protein (SRPP), dihydroxyacid dehydratase (DHAD) and actin depolymerizing factor (ADF), were dertermined, and the relationship between rubber yield with their expression levels was analysed. RESULTS:Except HbHMGR1, HbPMD and HbDHAD, most of these genes were predominantly expressed in latex, and bark tapping markedly elevated the transcript abundance of the analyzed genes, with the 7th tapping producing the greatest expression levels. Both ethephon (ETH) and methyl jasmonate (MeJA) stimulation greatly induced the expression levels of the examined genes, at least at one time point, except HbDHAD, which was unresponsive to MeJA. The genes' expression levels, as well as the rubber yields and two yield characteristics differed significantly among the different genotypes examined. Additionally, the latex and dry rubber yields increased gradually but the dry rubber content did not. Rubber yields and/or yield characteristics were significantly positively correlated with HbCPT, HbFPS, HbHMGS, HbHMGR1 and HbDHAD expression levels, negatively correlated with that of HbREF, but not significantly correlated with HbPMD, HbSRPP and HbADF expression levels. In addition, during rubber production, significantly positive correlations existed between the expression level of HbPMD and the levels of HbREF and HbHMGR1, between HbSRPP and the levels of HbHMGS and HbHMGR1, and between HbADF and HbFPS. CONCLUSIONS:The up-regulation of these genes might be related to the latex production of rubber trees under the stress of bark tapping and latex metabolism. The various correlations among the genes implied that there are differences in their synergic interactions. Thus, these nine genes might be related to rubber yield and yield-related traits in H. brasiliensis, and this work increases our understanding of their complex functions and how they are expressed in both high-and medium-yield rubber tree varieties and low-yield wild rubber tree germplasm.

Dataset Information

Cloning and Expression Analysis of One Gamma-Glutamylcysteine Synthetase Gene (Hb?-ECS1) in Latex Production in Hevea brasiliensis.

Publications

Cloning and Expression Analysis of One Gamma-Glutamylcysteine Synthetase Gene (Hbγ-ECS1) in Latex Production in Hevea brasiliensis.

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