Profiling ethylene-responsive genes expressed in the latex of Hevea brasiliensis using cDNA microarray
ABSTRACT: 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. 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: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.
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:ngs2014_07_hevea-hevea_tpd-seq-RNAseq analysis of latex samples from healthy and Tapping Panel Dryness-affected trees.-Identification of Tapping Panel Dryness (TPD)-affected trees in a polyclonal trials grown under standard condition. Trees were tapped since November 2010 every 2 days. Latex yield and TPD occurrence were monitored as well as latex RNA samples were collected twice a year for further analysis. At the end of the experiment, gene expression in latex of healthy and TPD trees were compared. Overall design: 21 samples with 3 biological replicates. genotype comparison, normal vs disease comparison
Project description:Murine bone marrow derived macrophages were infected with Leishmania major or Leishmania donovania promastigotes, allowed to phagocytose latex beads or not treated. Gene expression profiles were compared to identify i) the effect of Leishmania infection; ii) the differences in effects between L. major and L. donovani; and iii) the effect of pahgocytosis of latex beads.
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:Wood stiffness is the most important wood quality trait of forest trees for structural timber production. We investigated genes differentially transcribed in radiate pine trees with distinct wood stiffness using bulked segregant analysis (BSA) and cDNA microarrays. Transcript accumulation in earlywood (EW) and latewood (LW) of high (HS) and low stiffness (LS) trees in two progeny trials was compared. Radiata pine trees used for microarray experiment were selected from two progeny trials planted at Flynn and Kromelite, Australia. Based on the IML-based MOE measurement, five families with highest and lowest MOE each were selected from each trial, which represented two segregant populations with contrasting wood stiffness. Two individuals from each selected family were further sampled. Developing xylem tissues of selected trees in Flynn trial were sampled in spring (October) and autumn (April), representing earlywood (EW) and latewood (LW) of juvenile aged trees, respectively. Collection of xylem tissues from Kromelite trial was arranged in summer (late November) when latewood (LW) was formed. The xylem tissues were scraped at breast height with a sharp chisel after the bark was removed. In Flynn trial EW and LW tissues were collected from the same sampled trees on opposite sides of the trunk. Transcript accumulation was compared in trees with highest (HS) and lowest stiffness (LS) using xylem samples from Flynn collected in spring (EW) and autumn (LW), as well as Kromelite in summer (LW), respectively. Bulked segregant analysis (BSA) was used for the experiment design. Total RNA samples extracted from the five trees with HS were pooled at equal amount, and compared to the bulked five individuals with LS. This pooling strategy can partly minimize the genetic variation among different genotypes. Dye swaps were applied in each biological replicate.
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:Wood density is a foundamental quality trait for structural timber, bioenergy and pulp industries. We investigated genes differentially transcribed in radiate pine juvneile trees with distinct wood density using cDNA microarrays. Radiata pine trees were selected from a progeny trial planted at Flynn, Australia. Based on the gravitical measurement of wood cores, 12 families with highest and lowest density each were selected, representing two groups of trees with contrasting wood density. One individual with higher or lower density were further sampled in each selected family. Developing xylem tissues of selected trees were sampled in autumn (April) when latewood (LW) was formed. The xylem tissues were scraped at breast height with a sharp chisel after the bark was removed. Wood cores of the sampled trees were further measured using SilviScan 2. Total RNA extracted from ten developing xylem tissues with confirmed distinct density in each tree group were pooled into two bulks (five trees each), and the two bulks of HD were compared with two LD bulks in the microarray experiment (named the bulk experiment). Six developing xylem tissues with the most distinct density from each tree group were further chosen. Six xylem tissues with HD were individually compared with bulked six xylem tissues with LD in the second microarray experiment (named individual experiment). These two different pooling strategies can partly minimize the genetic variation among different genotypes. Dye swaps were applied in each biological replicate.
Project description:Background: Witches’ broom disease of Mexican lime (Citrus aurantifolia L.), which is caused by the phytoplasma “Candidatus Phytoplasma aurantifolia”, is a devastating disease that results in significant economic losses. Plants adapt to abiotic stresses by regulating gene expression at the transcriptional and post-transcriptional levels. MicroRNAs (miRNAs) are a recently identified family of molecules that regulate plant responses to environmental stresses through post-transcriptional gene silencing. Methods: Using a high-throughput approach to sequence small RNAs, we compared the expression profiles of miRNAs in healthy Mexican lime trees and in plants infected with “Ca. Phytoplasma aurantifolia”. Results: Our results demonstrated the involvement of different miRNAs in the response of Mexican lime trees to infection by “Ca. Phytoplasma aurantifolia”. We identified miRNA families that are expressed differentially upon infection with phytoplasmas. Most of the miRNAs had variants with small sequence variations (isomiRs), which are expressed differentially in response to pathogen infection. Conclusions: It is likely that the miRNAs that are expressed differentially in healthy and phytoplasma-infected Mexican lime trees are involved in coordinating the regulation of hormonal, nutritional, and stress signalling pathways, and the complex interactions between them. Future research to elucidate the roles of these miRNAs should improve our understanding of the level of diversity of specific plant responses to phytoplasmas. Small mRNA profiles of healthy (H) and Phytoplasma-infected Mexican lime trees were generated by deep sequencing, six replicate, using Illumina Hiseq2000
Project description:Effect of the presence of fruits on the expression of genes possibly involved in floral induction in the terminal meristem of spur bourse shoot. Investigation on mecanisms involved in Biennial Bearing in mature apple trees cultivar Royal Gala. Two-condition experiment : 'ON' trees (unthinned control) & 'OFF' trees (deflowered) for comparison. Three comparisons at sampling dates : 28, 48 and 119 days after full bloom (DAFB). Two dye switch biological replicates for each treatment and sampling date.