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Developing MicroRNA Vectors for Gene Suppression in Agricultural Plants
G. Tang
Department of Plant and Soil Sciences
Project Description
We have developed an optimized miRNA vector using the miR168 backbone. The optimized portion is focused on the "stem" region of the pre-miRNA. Specifically, a G-U wobble was introduced at the position 1 from the 5' end of the mature miRNA on the miRNA duplex. This significantly enhanced an asymmetric assembly of the mature artificial miRNA into RISCs. The production of artificial miRNAs was confirmed by small RNA Northern blot analysis. We have applied this optimized miRNA vector to silencing dozens of endogenous genes in Arabidopsis plants before its application in crops. The transgenic Arabidopsis plants with artificial miRNA directed gene silencing are now under further evaluation. Preliminary results showed that artificial miRNAs that target the Arabidopsis AGO2 were produced in large quantity. Similar work is under way for other target genes. The construction of an artificial miRNA vector is much simplified. One can complete the construction for silencing many genes (~10-96 genes) simultaneously within two weeks in a high throughput manner. A dedicated website of miRNA information with artificial miRNA designer was developed (http://mirnainfo.com/miRNADesignOne.aspx). This artificial miRNA designer will be optimized based on our coming experimental results.
Impact
Our objective is to develop high throughput tools (miRNA vectors in this project) for functional genomics of crop plants. We not only provide the optimized miRNA vectors, but also develop a workable and simplified protocol to help users to fulfill their goal to silence any gene of interest in any crop. Now the framework for this has been established.
Publications
Tang, G., Galili, G., and Zhuang, X. 2007. RNAi and microRNA: Breakthrough technologies for the improvement of plant nutritional value and metabolic engineering. Metabolomics. 3: 357-369
Wang, W., Gaffney, B., Hunt, A.G., and Tang, G. 2007. microRNAs and Plant Development. Encyclopedia of Life Sciences.
Xiang, Y. and Tang, G. (2006) RISC Biology, microRNA: Biology, Function and Expression edited by Clarke, N.J and Sanseau, P, DNA Press. pp 29-69.