Transcript Analysis Of Heavy Metal-Regulated Micrornas From Rice (Oryza Sativa) And MiR602a Cloning, Vector Construction And Transformation

MicroRNAs (miRNAs) are endogenous,-21 nt single-stranded RNAs and play important roles in gene expression, development, cell cycles and environmental stresses. Plant miRNAs down-regulate the abundance of their target mRNAs by post-transcription cleavage. More and more attentions have been given to the potential relations of plant miRNAs and abiotic stresses. Growing studies have elucidated the regulating mechanism of some miRNAs under abiotic stresses (e.g. drought, cold and mechanic stresses) However, reports on the responses of miRNAs to heavy metal exposure in various species are rare. Using the cDNA library of small RNAs from rice seedlings exposed to excess Cd, we performed a RT-PCR based assay of several expressions of pre-miRNAs from the roots and shoots subjected to Cd and Cu. The rice pre-miRNAs showed differential patterns of expression. Based on the response to Cd, the expressions of miR159a, miR167d, miR169g were up-regulated; the expression of miR398a was down-regulated and some genes which appeared not to be regulated are miR159b, miR160a, miR164a, miR169g, miR171d, miR393b, miR394a in the shoot.The expressions of miR159a were first up- and then down-regulated; the expressions of miR159d, miR167d, miR398a were down-regulated; some genes which appeared not to be regulated included miR159b, miR160a, miR164a, miR169g, miR171d, miR393b and miR394a in the root. Under Cu stress, expression of miR156a was up-regulated; expression of miR393b was first up and then down; expression of miR398a was down-regulated and some genes which appeared not to be regulated are miR156d, miR159b, miR160a, miR171d in the shoot. Expressions of miR160a, miR171d and miR393b were up-regulated; Expression of miR398a was down-regulated; Expression of miR156a was first up- and then down-regulated. Some genes which appeared not to be regulated were miR156d, miR159b in the root.Our recent studies on rice (Oryza sativa) have found 19 novel miRNAs from the small RNA library. Based on the sequence complementarity to miRNAs, we predicted rice target genes for these new miRNAs. Among these miRNAs, there are three loci respectively located on rice chromosome 1,4 and 8. miR602 was predicted to target xyloglucanendotransglucosylase/hydrolases (XET) genes. XET is a new wall-loosening enzyme which plays crucial roles in cell differentiation, cell expansion and cell wall formation. At present, some XET genes of different functions have been isolated from Arabidopsis, rice, soybean, wheat, maize and other plants. A lot of isoforms of XET express in different species and different organs, which is responsive to many hormones and can be regulated by environmental stresses. In this study, the miR602a gene was transferred into the pCAMBIA1304. The over-expression vector of miR602a gene was then introduced into rice via Agrobacterium tumefaciena. Two transgenic lines (T0) have been obtained after the processes of pre-culture, co-culture, selective-culture, regeneration-culture and rooting culture.