Project Number: 1931-21000-017-10
Project Type: Reimbursable

Start Date: Sep 01, 2007
End Date: Aug 31, 2011

Objective:
Current approaches for transgene containment require the tissue-specific expression of a bacterial-derived cytotoxic gene (either DT-A or Barnase) to kill the plant reproductive gamete or organ, but these gene products are reported to be harmful to animal and human cells, which raises concerns about the potential risk of the transgenic plants to animals, insects, and other organisms. Hence, developing a safe alternative is imperative. Gene silencing technologies [e.g. RNA interference (RNAi) and artificial microRNA (amiRNA)] provide new tools for engineering reproductive sterility, as well as, agronomic traits. RNAi is robust but ineffective at lower temperatures, whereas, amiRNA is effective at low temperatures but less robust overall. RNAi and amiRNA appear mechanistically complementary to each other, thus, integrating both mechanisms would create an ideal technology for engineering environment-friendly and -stable sterile trait. The goal of this research is to develop an integrated RNAi-amiRNA (inRAM) silencing technology and use it to engineer, via silencing of a meiosis-critical gene, stable male, and female sterility for remedying the spread of transgene and invasive species. Specific objectives for the research are: 1. Evaluate the efficiency of RNAi- and amiRNA-mediated engineering of the sterility and integrate (when working) RNAi and amiRNA (inRAM) together to create a robust silencing technology and 2. Evaluate genetic and environmental stability of the sterility engineered by inRAM to ensure the sterile trait is effective for transgene containment under field conditions.

Approach:
The engineering of sterility through silencing of the meiosis-critical genes (MCG) by artificial miRNA or RNAi will be first tested and characterized. Two Arabidopsis genes (270 -300 bp) coding for the 21-nt microRNAs miR159a and miR172a will be isolated, and their native 21-nt miRNA sequences will be replaced by the same length of artificial sequences complementary to the 5¿ coding regions of the chosen MCG genes, respectively, creating several amiRNA gene constructs. In parallel, RNAi constructs will be made by inserting a 300 - 400-bp fragment from the 3¿ MCG coding region as an inverted repeat into the pHellsgate vector, which expresses double-stranded RNA species, a potent inducer of silencing. The inverted repeat is separated by a silencing enhancer intron (pdk) that will serve as a host site for the future insertion of the amiRNA gene. Since amiRNA and RNAi constructs target the silencing of the same gene at different regions of sequence, they are expected to generate a similar sterile phenotype in plants. This will be confirmed by analyses of the gene silencing profiles and sterile phenotypes of amiRNA and RNAi transgenic plants. Once the amiRNA and RNAi are confirmed as competent silencing inducers, the amiRNA genes will be physically inserted into the pdk intron in the RNAi constructs to create inRAM constructs. Each inRAM construct in plants is expected to be transcribed as a single RNA species that will subsequently be spliced by distinct mechanisms into 21-nt amiRNAs and siRNAs, both of which target the same MCG transcript. These small RNAs are anticipated to reinforce each other¿s silencing strength and render the resulting sterile phenotype more stable and effective. The analysis of stability of the sterile trait inRAM transgenic plants under different temperature regimes or in different somatic generations derived from tissue culture will address whether expressed amiRNAs and siRNAs are able to make the engineered sterile trait more effective and stable under physiological and genetic stresses.