Location: Sunflower and Plant Biology Research
Project Number: 3060-21220-033-005-T
Project Type: Trust Fund Cooperative Agreement
Start Date: May 1, 2019
End Date: Dec 31, 2020
Objective:
The three objectives of this grant proposal are: 1) Continue identifying and analyzing clusters of co-regulated genes that show differences in accumulation following cold deacclimation; 2) Establish procedures needed to transform and regenerate canola; and 3) Begin to confirm the efficacy of the genes we identify in regulating cold deacclimation processes using CRISPR technology.
Approach:
1. Identify genes differentially regulated during deacclimation processes.
Previously, gene expression studies on two canola lines with differing temporal and intensity in their responses to deacclimation processes were subjected to transcripomic analysis by RNAseq technologies and resulted in identification of about 3000 differentially expressed genes. We will repeat these studies to confirm differential expression following deacclimation at differing times (0, 1 or 2 weeks) and different temperatures (5C, 10C, and 15C). Genes that are differentially expressed consistently under these conditions will be analyzed to identify probable transcription factor binding sites common to clusters of coordinately regulated genes.
2. Develop standard protocols for canola transformation. We will use methods previously described in the literature to transform hypocotyl sections with one of several different Agrobacterium T-DNA-based binary vectors. We will use these to confirm that we can transform, regenerate, and select for kanamycin-, basta-, and/or hygromycin-resistant plants that express genes of interest. We have several such constructs in the lab such as pBI121 and several pCAMBIA-derived binary vectors. We will also design CRISPR constructs to knock out test genes with easily screened phenotypes such as FLOWERING LOCUS C or common screenable markers such as GFP or GUS.
3. Functional analysis of target genes using CRISPR technology and mutant analysis. Based on previous GWAS and RNAseq studies, we have identified seven target genes that likely have a role in deacclimation processes. We will initially screen the list of putative target genes by obtaining lines of arabidopsis that contain knockout mutations, and lines that over express the target genes, to test their impact on deacclimation in this model species. For genes that impact deacclimation in arabidopsis, we will use gene editing CRISPR technology (Bortesi and Fischer 2015). For knockout vectors, we will use the backbone vector (psk-AtU626) provided by Dr. Steven H. Strauss, Oregon State University, for cloning sequences to induce deletions in target genes associated with cold deacclimation in winter canola. Different constructs containing a single guide RNA or two guide RNAs will be generated using standard cloning procedures. The guide RNAs will be purified after restriction digestion and ligated into a binary vector (2×35S-AtU626-Nos, also obtained from Strauss lab) containing the Cas9 nuclease. For over-expression of target genes, target sequences will be linked to CRISPR backbone constructs containing a transcriptional inducer. Genetic transformation with these binary vector constructs will be accomplished using Agrobacterium strain GV3101 for transformation into winter canola varieties and altered phenotypic responses in cold deacclimation and freezing tolerance processes will be accessed. Additionally, we may also choose to use CRISPR technology to simply change the gene sequence in the plants that were susceptible to freezing damage after deacclimation to the version from the plants that had high freezing survival after deacclimation.
