Transformation And Identification Of Heavy Metal-Regulated MiR604a And MiR396c From Oryza Sativa

MicroRNAs (miRNAs) are a novel class of short, endogenous non-coding small RNAs that have the ability to base pair with their target mRNAs to repress their translation or induce their degradation in both plants and animals. In plant, most of miRNAs play important roles in development and signal transduction. Recent studies demonstrate that some miRNAs are involved in abiotic stresses (e.g. drought, cold and heavy metal). Many plant miRNAs are evolutionarily conserved crosss the species. At the same time, some non-conserved miRNAs with the similar function may exist in rice (Oryza sativa), suggesting that rice may evolve a special mechanism for regulating the responses to heavy metal toxicity.Osa-miR604a is one of non-conversed miRNAs derived from heavy metal-treated rice sRNA library. In this study, the pre-miR604a gene was linked into the expression vector driven by 35s promotor. The pre-miR604a gene was introduced into rice callus tissue via Agrobacterium tumefaciena. Five transgenic lines (To) were obtained and identified by PCR, GUS and GFP analysis. The seeds of 35s::pre-miR604a seedlings showed some special physiological changes like germination delay. The T1 seedlings show features of anti-toxicity to Cd2+ in term of root-length and fresh weight.We additionally analyzed miR396c by generating transgenic plants which induced expressing miR396c precursor with its promoter. Four lines of To were successfully obtained using the approches indicated above. Analysis with PCR showed that the transformants was positive. Identification of these transgenic seedlings with fluorescence of GFP showed strong up-regulation by Cd-treated. Finally, using computational approche, we predicted MREs (metal-regulatory elements) around the promoter region upstream of miR396c gene. We show that five miRNA promoters contain this element.