Project description:Several reports have described the involvement of miRNAs in abiotic stresses. However, their role in biotic stress or to beneficial microbes has not been fully explored. In order to understand on the epigenetic regulation in plant in response to nitrogen-fixing bacteria association, we analyzed the sRNA regulation in maize hybrids (Zea mays – UENF 506-8) inoculated with the beneficial diazotrophic bacteria (Herbaspirillum seropedicae). Deep sequencing analysis was carried out to identify the sRNAs regulated in maize during association with diazotrophic bacteria. For this analysis, maize plants were germinated in wet paper and put in hydroponic system with Hoagland’s solution and then inoculated with H. seropedicae for seven days. Mock and inoculated plants were collected and total RNA from a pool of samples was extracted with Trizol reagent. The two sRNA libraries were sequenced by Illumina. The sequences were filtered to remove adaptors and contaminants rRNA and tRNAs, and sequences with 18-28 nt in length were selected. To identify the miRNAs present in these libraries, we used two strategies using the same website (http://srna-tools.cmp.uea.ac.uk): one to identify novel miRNAs using the maize genome (verson 2) and miRCat pipeline; and other to identify conserved miRNAs using the miRBase database (release 13.0, http://microrna.sanger.ac.uk) and miRProf pipeline. We identified 17 novel putative miRNAs candidates and mapped the precursor of these miRNAs in the maize genome. Furthermore, we identified 25 conserved miRNAs families and the differential expressions were analyzed with miRProf pipeline. The bioinformatics analysis of four up-regulated miRNAs (miR397, miR398, miR408 and miR528) in inoculated plant was validated using stem–loop RT-PCR assay. Our findings contribute to increase the knowledge of the molecular relation between plants and endophytic bacteria.
Project description:Several reports have described the involvement of miRNAs in abiotic stresses. However, their role in biotic stress or to beneficial microbes has not been fully explored. In order to understand on the epigenetic regulation in plant in response to nitrogen-fixing bacteria association, we analyzed the sRNA regulation in maize hybrids (Zea mays M-bM-^@M-^S UENF 506-8) inoculated with the beneficial diazotrophic bacteria (Herbaspirillum seropedicae). Deep sequencing analysis was carried out to identify the sRNAs regulated in maize during association with diazotrophic bacteria. For this analysis, maize plants were germinated in wet paper and put in hydroponic system with HoaglandM-bM-^@M-^Ys solution and then inoculated with H. seropedicae for seven days. Mock and inoculated plants were collected and total RNA from a pool of samples was extracted with Trizol reagent. The two sRNA libraries were sequenced by Illumina. The sequences were filtered to remove adaptors and contaminants rRNA and tRNAs, and sequences with 18-28 nt in length were selected. To identify the miRNAs present in these libraries, we used two strategies using the same website (http://srna-tools.cmp.uea.ac.uk): one to identify novel miRNAs using the maize genome (verson 2) and miRCat pipeline; and other to identify conserved miRNAs using the miRBase database (release 13.0, http://microrna.sanger.ac.uk) and miRProf pipeline. We identified 17 novel putative miRNAs candidates and mapped the precursor of these miRNAs in the maize genome. Furthermore, we identified 25 conserved miRNAs families and the differential expressions were analyzed with miRProf pipeline. The bioinformatics analysis of four up-regulated miRNAs (miR397, miR398, miR408 and miR528) in inoculated plant was validated using stemM-bM-^@M-^Sloop RT-PCR assay. Our findings contribute to increase the knowledge of the molecular relation between plants and endophytic bacteria. Screenning of sRNA transcriptome of maize plants inoculated with Herbaspirillum seropedicae after seven days
Project description:Six bacterial genomes, Geobacter metallireducens GS-15, Chromohalobacter salexigens, Vibrio breoganii 1C-10, Bacillus cereus ATCC 10987, Campylobacter jejuni subsp. jejuni 81-176 and Campylobacter jejuni NCTC 11168, all of which had previously been sequenced using other platforms were re-sequenced using single-molecule, real-time (SMRT) sequencing specifically to analyze their methylomes. In every case a number of new N6-methyladenine (m6A) and N4-methylcytosine (m4C) methylation patterns were discovered and the DNA methyltransferases (MTases) responsible for those methylation patterns were assigned. In 15 cases it was possible to match MTase genes with MTase recognition sequences without further sub-cloning. Two Type I restriction systems required sub-cloning to differentiate their recognition sequences, while four MTases genes that were not expressed in the native organism were sub-cloned to test for viability and recognition sequences. No attempt was made to detect 5-methylcytosine (m5C) recognition motifs from the SMRT sequencing data because this modification produces weaker signals using current methods. However, all predicted m6A and m4C MTases were detected unambiguously. This study shows that the addition of SMRT sequencing to traditional sequencing approaches gives a wealth of useful functional information about a genome showing not only which MTase genes are active, but also revealing their recognition sequences. Examination of the methylomes of six different strains of bacteria using kinetic data from single-molecule, real-time (SMRT) sequencing on the PacBio RS.