Project description:MicroRNAs (miRNAs) are non-coding, short, single-stranded RNAs with essential roles in gene regulation in various organisms including higher plants. In contrast to the vast information on miRNAs from many economically important plants, almost nothing has been reported on the identification or analysis of miRNAs from rubber tree (Hevea brasiliensis L.), the most important natural rubber-producing crop. To identify miRNAs and their target genes in rubber tree, high throughput sequencing combined with a computational approach was performed. Four small RNA libraries were constructed for deep sequencing from mature and young leaves of two rubber tree clones, PB 260 and PB 217, which provide high and low latex yield, respectively. 237 miRNAs belonging to 37 known miRNA families were identified, and northern hybridization validated miRNA expression and revealed developmental stage-dependent and clone-specific expression for some miRNAs. We took advantage of the newly released rubber tree genome assembly as well as the genomic databases from leafy spurge and cassava, two species related to rubber tree, and predicted 15 novel miRNAs.
Project description:Tapping panel dryness (TPD) seriously affects the natural rubber (NR) production of Hevea brasiliensis (rubber tree). Several studies have speculated that TPD influences NR biosynthesis in the latex of rubber trees based on the expression changes of NR biosynthesis-related genes. In this study, iTRAQ analysis of latex were carried out to reveal the molecular mechanism of TPD affecting rubber trees NR biosynthesis activity and molecular weight.
Project description:MicroRNAs (miRNAs) are non-coding, short, single-stranded RNAs with essential roles in gene regulation in various organisms including higher plants. In contrast to the vast information on miRNAs from many economically important plants, almost nothing has been reported on the identification or analysis of miRNAs from rubber tree (Hevea brasiliensis L.), the most important natural rubber-producing crop. To identify miRNAs and their target genes in rubber tree, high throughput sequencing combined with a computational approach was performed. Four small RNA libraries were constructed for deep sequencing from mature and young leaves of two rubber tree clones, PB 260 and PB 217, which provide high and low latex yield, respectively. 237 miRNAs belonging to 37 known miRNA families were identified, and northern hybridization validated miRNA expression and revealed developmental stage-dependent and clone-specific expression for some miRNAs. We took advantage of the newly released rubber tree genome assembly as well as the genomic databases from leafy spurge and cassava, two species related to rubber tree, and predicted 15 novel miRNAs. 4 samples examined: PB260 mature leaves, PB260 young leaves, PB217 mature leaves, and PB217 young leaves.
Project description:Nutural rubber (NR) production, latex is harvested by periodical tapping of the trunk bark. Ethylene enhances and prolongs latex flow and latex regeneration. Ethephon, which is an ethylene-releasing compound, applied to the trunk before tapping usually results in a 1.5- to 2-fold increase in latex yield. We investigated gene expression in response to ethephon treatment using Pará rubber tree seedlings as a model system. After ethephon treatment, 3,270 genes showed significant differences in expression compared with the mock treatment. Genes associated with carotenoids, flavonoids, and abscisic acid biosynthesis were significantly upregulated by ethephon treatment, which might contribute to an increase in latex flow. Genes associated with secondary cell wall formation were downregulated, which might be because of the reduced sugar supply. Given that sucrose is an important molecule for NR production, a trade-off may arise between NR production and cell wall formation for plant growth and for wound healing at the tapping panel.
Project description:Ethylene is commonly used as a latex stimulant of Hevea brasiliensis by application of ethephon (chloro-2-ethylphosphonic acid); however, the molecular mechanism by which ethylene increases latex production is not clear. To better understand the effects of ethylene stimulation on the laticiferous cells of rubber trees, a latex expressed sequence tag (EST)-based complementary DNA microarray containing 2973 unique genes (probes) was first developed and used to analyze the latex gene expression changes at three different time-points after ethephon treatment: 8, 24 and 48 h. Transcript levels of 163 genes were significantly altered with fold-change values ≥ 2 or ≤ –2 (q-value < 0.05) in ethephon-treated compared with control rubber trees. Of the 163 genes, 92 were up-regulated and 71 down-regulated. The microarray results were further confirmed using real-time quantitative reverse transcript-PCR for 20 selected genes.
Project description:The extraction of high-purity proteins from the washing solution (WS) of rubber tree latex-producing organelles (also termed rubber particles) in laticifers for proteomic analysis is challenging due to the low concentration of proteins in the WS. Recent studies have revealed that proteins in the WS might play crucial roles in natural rubber biosynthesis. To further examine the involvement of these proteins in natural rubber biosynthesis, we designed an efficiency method to extract high-purity WS proteins. We improved our current borax and phenol-based (BPP) method by adding re-extraction steps with phenol (REP) to improve the yield from low-protein concentration samples. With this new method, we extracted WS proteins that were suitable for proteomics. Indeed, compared to the original BPP method, the REP method improved both the quality and quantity of isolated proteins. By repeatedly extracting from low-protein concentration solutions using the same small amount of phenol, the REP method yielded enough protein of sufficiently high-quality from starting samples containing less than 0.02 mg of proteins per mL. This method was successfully applied to extract the rubber particle proteins from the WS of natural rubber latex samples. The REP-extracted WS proteins were resolved by two-dimensional gel electrophoresis (2-DE), and 28 proteins were positively identified by mass spectrometry. This method has the potential to become widely used for the extraction of proteins from low-protein-concentration solutions for proteomic analysis.
Project description:Ethylene is commonly used as a latex stimulant of Hevea brasiliensis by application of ethephon (chloro-2-ethylphosphonic acid); however, the molecular mechanism by which ethylene increases latex production is not clear. To better understand the effects of ethylene stimulation on the laticiferous cells of rubber trees, a latex expressed sequence tag (EST)-based complementary DNA microarray containing 2973 unique genes (probes) was first developed and used to analyze the latex gene expression changes at three different time-points after ethephon treatment: 8, 24 and 48 h. Transcript levels of 163 genes were significantly altered with fold-change values ⥠2 or ⤠â2 (q-value < 0.05) in ethephon-treated compared with control rubber trees. Of the 163 genes, 92 were up-regulated and 71 down-regulated. The microarray results were further confirmed using real-time quantitative reverse transcript-PCR for 20 selected genes. Analysis used the 8, 24 or 48 h control latex RNA samples comparison to the ET stimulated 8, 24 or 48 h latex RNA samples. Each sample included three independent biological replicates, and each replicate comprised the latex collected from six trees.
Project description:We first report the use of next-generation massively parallel sequencing technologies and de novo transcriptome assembly to gain insight into the wide range of transcriptome of Hevea brasiliensis. The output of sequenced data showed that more than 12 million sequence reads with average length of 90nt were generated. Totally 48,768 unigenes (mean size = 488 bp) were assembled through transcriptome de novo assembly, which represent more than 3-fold of all the sequences of Hevea brasiliensis deposited in the GenBank. Assembled sequences were annotated with gene descriptions, gene ontology and clusters of orthologous group terms. Total 37,373 unigenes were successfully annotated and more than 10% of unigenes were aligned to known proteins of Euphorbiaceae. The unigenes contain nearly complete collection of known rubber-synthesis-related genes. Our data provides the most comprehensive sequence resource available for study rubber tree and demonstrates the availability of Illumina sequencing and de novo transcriptome assembly in a species lacking genome information. The transcriptome of latex and leaf in Hevea brasiliensis
Project description:Purpose: de novo sequencing and comparative analysis of the bark transciptomes of Hevea brasiliensis induced without ethephon (C), with ethephon for 8 hours (E8) and 24 hours (E24) to identify the genes and pathways related to the stimulation of rubber production by ethylene. The goals of this study are to reveal the molecular mechanism behind the stimulation of rubber production by ethylene. Methods: Bark RNA was extracted using the TRIzol® Reagent (Invitrogen) and two cDNA libraries, H (healthy rubber trees) and T (TPD-affected trees), were prepared using the mRNA-Seq 8 sample prep Kit (Illumina). The libraries were deep sequenced using Illumina HiSeqTM 2000 (Illumina Inc., San Diego, CA, USA). Raw reads produced from sequencing machines were resorted to de novo assembly and gene annotation. Results: De novo sequencing and assembly of the bark transciptomes of Hevea brasiliensis induced with ethephon for 8 hours (E8) and 24 hours (E24) were performed. 51,965,770, 52,303,714 and 53,177,976 high-quality clean reads from E8, E24 and C (control) samples were assembled into 81,335, 80,048 and 80,800 unigenes respectively, with a total of 84,425 unigenes and an average length of 1,101 bp generated. 10,216 and 9,374 differentially expressed genes (DEGs) in E8 and E24 compared with C were respectively detected. The expression of several enzymes in crucial points of regulation in glycolysis were up-regulated and DEGs were not significantly enriched in isopentenyl diphosphate (IPP) biosynthesis pathway. In addition, up-regulated genes of great regulatory importance in carbon fixation (Calvin cycle) were identified. Conclusions: The rapid acceleration of glycolytic pathway supplying precursors for the biosynthesis of IPP and natural rubber, instead of rubber biosynthesis per se, may be responsible for ethylene stimulation of latex yield in rubber tree. The elevated rate of flux throughout the Calvin cycle may account for some durability of ethylene-induced stimulation. Our finding lays the foundations for molecular diagnostic and genetic engineering for high-yielding improvement of rubber tree.
Project description:Purpose: de novo sequencing and comparative analysis of the bark transciptomes of Hevea brasiliensis induced without ethephon (C), with ethephon for 8 hours (E8) and 24 hours (E24) to identify the genes and pathways related to the stimulation of rubber production by ethylene. The goals of this study are to reveal the molecular mechanism behind the stimulation of rubber production by ethylene. Methods: Bark RNA was extracted using the TRIzol® Reagent (Invitrogen) and two cDNA libraries, H (healthy rubber trees) and T (TPD-affected trees), were prepared using the mRNA-Seq 8 sample prep Kit (Illumina). The libraries were deep sequenced using Illumina HiSeqTM 2000 (Illumina Inc., San Diego, CA, USA). Raw reads produced from sequencing machines were resorted to de novo assembly and gene annotation. Results: De novo sequencing and assembly of the bark transciptomes of Hevea brasiliensis induced with ethephon for 8 hours (E8) and 24 hours (E24) were performed. 51,965,770, 52,303,714 and 53,177,976 high-quality clean reads from E8, E24 and C (control) samples were assembled into 81,335, 80,048 and 80,800 unigenes respectively, with a total of 84,425 unigenes and an average length of 1,101 bp generated. 10,216 and 9,374 differentially expressed genes (DEGs) in E8 and E24 compared with C were respectively detected. The expression of several enzymes in crucial points of regulation in glycolysis were up-regulated and DEGs were not significantly enriched in isopentenyl diphosphate (IPP) biosynthesis pathway. In addition, up-regulated genes of great regulatory importance in carbon fixation (Calvin cycle) were identified. Conclusions: The rapid acceleration of glycolytic pathway supplying precursors for the biosynthesis of IPP and natural rubber, instead of rubber biosynthesis per se, may be responsible for ethylene stimulation of latex yield in rubber tree. The elevated rate of flux throughout the Calvin cycle may account for some durability of ethylene-induced stimulation. Our finding lays the foundations for molecular diagnostic and genetic engineering for high-yielding improvement of rubber tree. De novo sequencing of the transcriptomes of C (bark without ethephon application), E8 (bark with 1.5%-ethephon treatment for 8 hours) and E24 (bark with 1.5%-ethephon treatment for 24 hours) rubber trees was conducted using Illumina HiSeq 2000.