Construction And Identification Of Transgenic Rice Plants Expressing MIR394and Its Target Gene F-BOX

MicroRNAs (miRNAs) are a new class of endogenous non-coding small RNAs (-22nt). MiRNAs base-pair their target genes to repress their translation or induce their degradation. They play important roles in development, hormone response, signal transduction, resistance to plant disease and environmental stresses. Rice (Oryza sativa) is an important food crop in China. In this study, we identified rice miR394and its target a F-box(Os01g69940). The F-box gene coding for one a subunit of SCF complex was involved in ubiquitin degradation pathway. It plays important roles in hormone response, lateral root formation, and light signalling. In order to study miR394function, we constructed35S::miR394,35S::F-box and35S::m5F-box vectors. The vector of miR394gene was then introduced into rice via Agrobacterium tumefaciena. Twelve transgenic lines (To) have been obtained after the processes of pre-culture, co-culture, selective-culture, regeneration and rooting culture. Nine out of the12were identified were positive by GUS staining of the roots and leaves of transgenic seedlings. We also had a detection of DNA and RNA level for transgenic seedlings. DNA analysis showed that9transgenic plants were positive. The expression of pre-miR394in the9transgenic lines was obviously higher than that in wild-type, but the expression of F-box was significantly lower than that in wild-type, indicating that the negative regulation of F-box by miR394. The leaves of35S::miR394transgenic plants were observed to be curled more obvious than wild-type plants during the development. For the analysis of expression pattern of miR394and F-box, promoter-GUSfusion vectors of pmiR394::GUS and pF-box::GUS were constructed.