Genetic Transformation And Functional Analysis Of Os-miR172

MicroRNAs (miRNAs) are19-24nt, highly conserved non-coding RNAs in eukaryotic cells that guide target mRNA degradation or inhibiting their translation by complementary pairing with target mRNA so as to regulate gene expression levels. MiRNAs play an important role in plant growth and development and interaction between plant and environment. miR172family, one of highly conserved miRNA families in higher plants, is involved in flowering time and floral organogenesis by regulating the expression of AP2-like transcription factors. In rice, miR172family has four members (Os-miR172a-d), which are located in chromosome Ⅸ, Ⅰ,Ⅶ,Ⅱ, respectively. Our previous studies showed that viral infection (for example, rice stripe virus)can significantly change the expression patterns of rice endogenous miRNA, in which the expression level of Os-miR172family is more significantly affected. In order to further analysis the function of Os-miR172in rice development and the relationship between rice and virus infection, we firstly amplified and cloned miR172family genes from the genome of Nipponbare, a japonica cultivar of rice; sequencing analysis showed that the cloned nucleotide sequences are identical to those in the GenBank database. Based on these Os-miR172clones, we successfully constructed the plant transformation vectors, including over-expression and function inhibition vectors. Then these vectors were introduced into rice plant by agrobacterium-mediated transformation method. Molecular detection indicated that the interested genes had been integrated into the rice genome and efficiently expressed in transgenic rice plants. Phenotype analysis showed that the transgenic plants over-expressing Os-miR172did not significantly differ from non-transgenic plants in the agronomic traits such as height and tiller number. However, the transgenic plants over-expressing Os-miR172displayed abnormal spikelets, missing or distorted paleae and lemmas, and infertility during the reproductive period. It was observed that the pistils and stamens also were abnormal through the stereoscope and that most pollen was deformed and inviable or sterile by Alaxanders staining. Transgenic plants containing the vector of function inhibition developed slowly at or after the stage of tillering. At the heading stage, panicles were incapable of heading from sheath. However, floral organ developed and produced seeds normally. After artificial inoculation of Rice stripe virus(RSV), the transgenic plants over-expressing miR172displayed typical stripe symptoms, the development of some spikelet became normal and produced seed normally. The transgenic plants containing the vector of function inhibition headed normally but produced more shrunken grains. These results laid the foundation for further analyzing the functions of miR172in growth and development and viral pathogenesis in rice plants.