Location: Corn Insects and Crop Genetics Research2012 Annual Report
1a. Objectives (from AD-416):
1. Generate, in collaboration with DOE-JGI, gene expression data (RNA-Seq) for approximately 180 tissues/conditions (30 in year 1, increased to 100 in year 2, and to 180 in year 3); 2. Identify and characterize tissue-specific promoters. Using the gene expression data from Activity 17 above, we will identify up to 20 different promoters in year 1 that mediate tissue or condition-specific gene expression and clone at least 10 such promoters into GUS/GFP expression vectors. These will then be tested transiently in soybean. At the end of year 2 we will continue to identify additional promoters from the gene expression atlas, clone these and test them by transient expression. By the end of year 3 we will reach our goal of screening at least 50 promoter constructs transiently.
1b. Approach (from AD-416):
The initial pilot phase of the project will target 30 conditions. These are yet to be fully defined but will include all of the major tissues of soybean (e.g., leaf, root, seed, stem, apical and root meristem, and nodules). In some cases, these tissues will be analyzed over a set time period. Additional conditions may target various biotic and abiotic stress conditions, etc. In order to maximize the uniformity of these initial samples, all plants will be grown in the Stacey laboratory (using growth chambers) and the mRNA will be isolated by a single lab member before shipping to DOE-JGI for sequencing. We plan to analyze soybean under a wide range of conditions that include various stresses (e.g., drought, pathogen infection, cold, heat, etc.), as well as various hormonal treatments (e.g., auxin, ethylene, cytokiniin, etc.). We plan to utilize the gene expression data, derived from the transcriptional gene index described above, to identify and clone promoter elements that will control gene expression in a tissue-specific or condition-specific way. For example, analysis of the currently available soybean transcriptional index (e.g., http://digbio.missouri.edu/soybean_atlas) led to the identification of roughly 9 genes that show an extremely high level (>1000-fold) of tissue specific expression. The promoters of these genes are, therefore, natural targets for cloning and use to drive tissue specific expression. A natural extension of generating the transcriptional atlas is also the ability to identify and isolate promoters that will be useful for both basic and applied studies in soybean. These promoters will provide the ability to specifically target the expression of transgenes, while also providing tools (e.g., promoter-green fluorescent protein constructs) to analyze the cellular basis of the soybean response to a variety of treatments. We will work closely to make sure that the data are directly integrated into the soybase.org site.
3. Progress Report:
In collaboration with the other partners on the Department of Energy-Joint Genome Institute (DOE-JGI) transcriptome atlas project, the list of metadata to be collected for each RNA sample was finalized. The community decided to focus on one collective experiment to compare and contrast across the multiple species (i.e., soybean, poplar, Setaria, Physicometrella, and Chlamydomonas) whose RNA will be sequenced. This experiment will involve a comparison of growth under three nitrogen sources, nitrate, urea and ammonia. In addition, given that soybean develops a nitrogen fixing symbiosis with soil bacteria, we will also isolate plant material from nodulated plants. Plants have been started to support this experiment and the first of three biological replicates have been isolated. We have completed transcriptome sequencing of plants infected with aphids. This work involves a comparison of aphid resistant plants (Rag2) vs sensitive plants (rag2). RNA was isolated from infected and uninfected leaves over a time course of 0-72 hours and these data are now being analyzed. We have also completed a large experiment to examine the effect of heat treatment on soybean roots and root hairs, again over a time course and these data are also being analyzed. We have harvested tissue representing nine different stages of soybean seed development. Plants are growing for the harvesting of tissues to support RNA-seq of leaves, roots, stems and nodules. Other plants have been started to support the collection of RNA under other growth conditions. All of the transcriptome data collected will be provided to the community as part of the soybean transcriptome atlas.