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Our recent paper (Joshi et al., 2010) describes the identification of sRNAs from root, seed, flower, and nodules. We are now analyzing data to add sRNAs from B. japonicum inoculated (0-48 HAI) soybean root hair samples and the corresponding stripped root tissues. This includes 30,250, 33,034 and 219,131 sRNA sequences that had unique genome hits in the mock inoculated root hair, inoculated root hair, and stripped root tissues, respectively. A total of 129 miRNAs were identified from root, seed, flower and nodules, including 42 miRNAs that matched previously identified soybean miRNAs or were conserved in other species. However, 87 novel miRNAs were identified. We also predicted the putative target genes of all identified miRNAs with computational methods and verified the predicted cleavage sites in vivo for a subset of these targets using the 5’ RACE method. Finally, we also studied the relationship between the miRNA and expression of the respective target genes by comparison to Solexa cDNA sequencing data. A genome browser was developed (http://digbio.missouri.edu/soybean_mirna/) that allows direct comparison of miRNA and mRNA (from our transcriptome analysis) expression. |
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A manuscript, currently under review (Brechenmacher et al., 2010) describes the polar and non-polar root hair and stripped root metabolites. Metabolites were analyzed after water and methanol/ chloroform extraction by GC-MS and after extraction with 80% methanol by UPLC-MS. A total of 1691 metabolites were identified by combining GC-MS and LC-MS approaches, with 134 responding significantly to inoculation (0-48 HAI). Principal component analysis clearly segregated root hair from stripped root metabolites. |
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We conducted Illumina Solexa cDNA sequencing on 14 different soybean tissues/conditions. This information is contained in a searchable soybean gene expression atlas [(Libault et al., 2010); http://digbio.missouri.edu/~soybean_atlas/]. |
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We mined the soybean genome and identified over 5500 putative transcription factors (Libault et al., 2009). A searchable database focused on the soybean TF genes was developed (http://casp.rnet.missouri.edu/soydb/) (Wang et al., 2009). We utilized both qRT-PCR and Illumina Solexa cDNA sequencing to identify 204 TF genes that responded directly to B. japonicum inoculation. We also showed that RNAi silencing of a specific Myb TF gene significantly reduced soybean nodulation (Libault et al., 2009). |
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We utilized root hair infection by the symbiotic bacterium, Bradyrhizobium japonicum as a tool to perturb cellular function. For example, Libault et al. (2010) utilized Affymetrix DNA microarray hybridization, high-throughput Illumina Solexa cDNA sequencing, and quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) to show that over 48,000 of the 69,000 predicted soybean ORFs were expressed in soybean root hairs. These expression data were used to improve the current soybean genome annotation; identifying new ORFs, defining splice variants, extending genes both 5’ and 3’, and providing transcriptional support for the annotated genes. |
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The success of our project rests on our development of a highly reproducible method for root hair isolation in quantities sufficient for a variety of functional genomic studies (Wan et al., 2005 (see protocol) This method generates highly pure soybean root hair preparations, as well as the comparative stripped root tissue (i.e., roots after the root hairs have been removed). |
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Proteomics is the study of the structure, function and interaction of proteins. Proteins play a critical role in the life of any organism by being involved in many processes within the cell. The proteome, which is the complete collection of proteins in an organism, is much more complicated to study than the genome or transcriptome. An organism harboring 20 000 genes can have more than 1 million different proteins due to alternative splicing and post-translational modifications. Furthermore, the level of transcription of a gene often does not reflect the level of the encoded protein. An mRNA can be present in numerous copies in a cell but the mRNA stability (half life) and efficiency of translation will affect the level of the corresponding protein. A protein can also be present in a cell but inactive until subjected to post-translational modifications like phosphorylation. Finally, many proteins can interact with other proteins and only the resulting complex may have biological function. The measurement of mRNA levels via transcriptomics (e.g., via the use DNA microarrays, see discussion of this subject on our website) is complimentary to proteomics.
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