Project description:We sequenced sRNA from the ovular secretions of G. biloba to identify the presence of miRNAs, which provide the first evidence of the extracellular miRNAs function in ovular secretions of gymnosperms .
Project description:MicroRNAs (miRNAs) are emerging as essential, albeit poorly characterized, regulators of biological processes. The miRNA in gymnosperms is under-identified, which limits the progress of miRNA in gymnoperms. Using the high-throughput sequencing, a total of 87 conserved miRNAs were identified from Larix leptolepis. Eighteen novel miRNAs were discovered in our library, most of which were Larix-specific miRNAs.
Project description:In the present study, endogenous cysteine S-nitrosation site and S-nitrosated proteins were identified by iodo-TMT labeling during somatic embryogenesis in Brazilian pine, an endangered native conifer of South America.
Project description:Bursaphelenchus xylophilus is known as the causative agent of pine wilt disease with complex life cycles. In this research, newly published Bursaphelenchus xylophilus genome data were employed to annotate its miRNAs based on deep sequencing technologies. Four small RNA libraries derived from different infection stages of pine wilt disease were constructed and sequenced. Consequently, we obtained hundreds of evolutionarily conserved miRNAs as well as novel miRNA candidates. The analysis of miRNA expression patterns showed that most miRNAs were expressed at extraordinarily high levels during the middle stage of pine wilt disease. Subsequent stem-loop RT-PCR experiments were carried out to validate our results. Functional analysis proved that expression levels of miR-73 and miR-239 were mutually exclusive with their target GH45 cellulase genes., genes known to be responsible for the degradation of the pine cell walls. In addition, another set of atypical miRNAs, termed mirtrons, were identified from B. xylophilus introns. This discovery has expanded the current knowledgebase of such splicing-derived miRNAs into B. xylophilus. Thus, our research has provided detailed characterization of B. xylophilus miRNAs expression patterns during the pathological process of pine wilt disease. The findings will contribute to more in-depth understanding of this devastating plant disease.
Project description:Bursaphelenchus xylophilus is known as the causative agent of pine wilt disease with complex life cycles. In this research, newly published Bursaphelenchus xylophilus genome data were employed to annotate its miRNAs based on deep sequencing technologies. Four small RNA libraries derived from different infection stages of pine wilt disease were constructed and sequenced. Consequently, we obtained hundreds of evolutionarily conserved miRNAs as well as novel miRNA candidates. The analysis of miRNA expression patterns showed that most miRNAs were expressed at extraordinarily high levels during the middle stage of pine wilt disease. Subsequent stem-loop RT-PCR experiments were carried out to validate our results. Functional analysis proved that expression levels of miR-73 and miR-239 were mutually exclusive with their target GH45 cellulase genes., genes known to be responsible for the degradation of the pine cell walls. In addition, another set of atypical miRNAs, termed mirtrons, were identified from B. xylophilus introns. This discovery has expanded the current knowledgebase of such splicing-derived miRNAs into B. xylophilus. Thus, our research has provided detailed characterization of B. xylophilus miRNAs expression patterns during the pathological process of pine wilt disease. The findings will contribute to more in-depth understanding of this devastating plant disease. For the purposes of this study, we classified the pathogenic process associated with PWD into three stages in order to best characterize the expression patterns of microRNAs during the development of this devastating disease. The following describes the first stage (F): about seven days after pine trees are infected with PWNs, the tips of the pine needles begin to turn brown. Next, the middle stage (M) ensues approximately seven days later, when half of the needles on pine trees turn brown. The last stage (L) occurs another 10 days later and pine needles are complete browning. PWNs cultured on Botrytis cinerea grown on PDA medium served as the control stage (C).
Project description:MicroRNAs (miRNAs) are emerging as essential, albeit poorly characterized, regulators of biological processes. The miRNA in gymnosperms is under-identified, which limits the progress of miRNA in gymnoperms. Using the high-throughput sequencing, a total of 87 conserved miRNAs were identified from Larix leptolepis. Eighteen novel miRNAs were discovered in our library, most of which were Larix-specific miRNAs. Identification of small RNA in Larix seedling
Project description:MicroRNAs (miRNAs) are emerging as essential regulators of biological processes. Somatic embryogenesis is one of the most important techniques for gymnosperm breeding programs, but there is little understanding of its underlying mechanism. To investigate the roles of miRNAs during somatic embryogenesis in larch, we constructed a small RNA library from somatic embryos. High-throughput sequencing of the library identified 83 conserved miRNAs from 35 families, 16 novel miRNAs, and 14 plausible miRNA candidates, with a high proportion specific to larch or gymnosperms. qRT-PCR analysis demonstrated that both the conserved and novel or candidate miRNAs were expressed in larch. Several miRNA precursor sequences were obtained via RACE. We predicted 110 target genes using bioinformatics, and validated nine of them by 5’ RACE. Eleven conserved miRNA families including 17 miRNAs with critical functions in plant development and six target mRNAs were detected by qRT-PCR in the larch SE. Stage-specific expression of miRNAs and their targets indicate their possible modulation on SE of larch: miR171a/b might exert function on PEMs, while miR171c acts in the induction process of larch SE; miR397 and miR398 mainly involved in modulation of PEM propagation and transition to single embryo; miR162 and miR168 exert their regulatory function during total SE process, especially during stage 5 to stage 8; miR156, miR159, miR160, miR166, miR167, and miR390 might play regulatory roles during cytoledonary embryo development. These findings indicate that larch and possibly other gymnosperms have complex mechanisms of gene regulation involving specific and common miRNAs operating post-transcriptionally during embryogenesis.
Project description:The significant morphological differences observed during embryo development in angiosperms and gymnosperms are expected to be the result of a differential control of gene expression. We used a loblolly pine (Pinus taeda) cDNA microarray to analyze global transcriptional changes along zygotic embryogenesis in maritime pine (Pinus pinaster). A time-course analysis of the data obtained from the five embryo developmental groups used in this study led to the identification of 4,645 genes whose expression varied along P. pinaster embryogenesis. These transcripts were clustered into six distinct expression profiles. The grouping of these profiles in early, mid-embryogenesis and embryo maturation, according to the developmental period where most of the sequences were up-regulated, evidenced that characteristic transcriptional changes are associated to each developmental period. The application of a cut-off value of 1.95-fold change led to the identification of 1,838 differentially expressed transcripts that were categorized by biological process. Metabolism, interaction with the environment, oxidation-reduction and transport were some of the most represented categories. During early embryogenesis genes putatively involved in phytohormone-mediated signaling were identified, whereas in middle stages the overrepresented genes could be associated with cotyledon formation and induction of somatic embryogenesis. Genes associated with the synthesis of storage products were up-regulated in the latest stages of pine embryo development. It was also during this developmental period that the largest number of sequences putatively encoding transcription factors was identified.
Project description:The significant morphological differences observed during embryo development in angiosperms and gymnosperms are expected to be the result of a differential control of gene expression. We used a loblolly pine (Pinus taeda) cDNA microarray to analyze global transcriptional changes along zygotic embryogenesis in maritime pine (Pinus pinaster). A time-course analysis of the data obtained from the five embryo developmental groups used in this study led to the identification of 4,645 genes whose expression varied along P. pinaster embryogenesis. These transcripts were clustered into six distinct expression profiles. The grouping of these profiles in early, mid-embryogenesis and embryo maturation, according to the developmental period where most of the sequences were up-regulated, evidenced that characteristic transcriptional changes are associated to each developmental period. The application of a cut-off value of 1.95-fold change led to the identification of 1,838 differentially expressed transcripts that were categorized by biological process. Metabolism, interaction with the environment, oxidation-reduction and transport were some of the most represented categories. During early embryogenesis genes putatively involved in phytohormone-mediated signaling were identified, whereas in middle stages the overrepresented genes could be associated with cotyledon formation and induction of somatic embryogenesis. Genes associated with the synthesis of storage products were up-regulated in the latest stages of pine embryo development. It was also during this developmental period that the largest number of sequences putatively encoding transcription factors was identified. Pine immature zygotic embryos were pooled into five different developmental groups according to the collection date (Day0; Day5; Day11; Day15 and Day25). A common reference design was used. Total RNA was extracted from each sample. Three extractions were prepared per sample (biological replicates). A defined ammount from each RNA sample was pooled to use as reference. Messenger RNA was amplified for both test samples and reference. These were hybridized together in two separate experiments (technical replicates). In total, 30 slides were analyzed.