Cloning Of Small RNAs Related To Nitrogen Nutrition In Maize And Functional Analysis Of MiRNA169

Nitrogen (N) is closely related to crop’s yeild and quality, and it is the most required element of crop, related research has focused on nitrogen uptake and assimilation, while the corresponding regulatory mechanism is still unclear. Lagged study of regulatory mechanism severely limit researchers breed nitrogen efficient use crop by genetic engineering techniques. As an important upstream regulatory factor, the finding of plant miRNAs attracted the attention of molecular plant nutrition biologists. Although recent studies indicated that miRNAs regulate plant adaptive responses to nutrient deprivation, the functional significance of miRNAs in adaptive responses to N-limitation remains to be explored.To elucidate the regulatory mechanism of plant nitrogen uptake and assimilation from the perspective of miRNAs, we use maize and Arabidopsis as model plant, and use the RNA genomics technology, the bioinformatics, and the molecular biology to explore the plant miRNAs that affected by N-limitation. The main results are as follows:1. We discovered a total of99absolutely new loci belonging to47miRNA families by small RNA deep sequencing and degradome sequencing, as well as9new loci were the paralogs of previously reported miR169, miR171, and miR398, significantly expanding the reported150high confidence genes within26miRNA families in maize.2. Through the degradome we detected the potential targets of miR164f*, miR167g*/h*/i*, miR169r*, miR168a*/b*and miR827*, suggesting that miRNA*could play important roles in plant physiological metabolism processes by mediated mRNA cleavage, which are far from former consider.3. Small RNA deep sequencing, Q-PCR, small RNA northern blot and5’-RACE analysis identified eight miRNA families (five conserved and three newly identified) differentially expressed under the N-deficient condition, and we summarized a potential function network of miRNA that responses to N-limitation in maize. The expression of miR169, miR395, miR827and miRC1are down-regulated by N-limitation, they directly involved in maize nitrogen stress adaptation by regulate the expression of their targets; miR.171, miR528, miRC19and miRC37are up-regulated under N-limitation condition, their targets are suggested to involved in the N-limitation singal transduction in maize.4. Further research are carried in Arabidopsis as functional verification, we chose miR169and its target NFYA5, which is a conserved miRNA between plant species. Analysis of the expression of miR169precursors showed that MIR169a was substantially down-regulated in both roots and shoots by N-limitation.35S::MIR169a transgenic plants are hypersensitive to N starvation in hydroponic solution, agar medium and soil, and in all these conditions35S::MIR169a transgenic plants emerge a symptom:the leaves turn yellow. However,35S::NFYA5transgenic plants are more tolerance to the N-limitation, and35S::NFYA5transgenic plants can gain more biomass than wild type, suggesting miR169is involved in the N-limitation responses by regulated the expression of its target in plant. 5. Under N-limiting conditions, the total N contents in roots of35S::MIR169a transgenic plants were c.4.5%, which was lower than that of the wild-type (5%). Furthermore, compare to wild-type, the expression of AtNRT1:1and AtNRT2:1are less in35S::MIR169a transgenic plants. These results provide evidence that miRNAs have functional roles in helping plants to cope with fluctuations in N availability in the soil.