Identification And Expression Analysis Of Mirnas In Immature Maize Root Under The Stress Of Heavy Metal Pb

Development of mining industry, as well as the use of chemical fertilizer and agricultural chemicals, results in the increase of heavy metal in soil tillage layer, which can not only limit the development of crops but also do harm to human and animals'health by food chain. Maize is an important crop grown worldwide, what's more, the stems and leaves are used to be the silage of livestock besides its seeds as food in some areas. At the same time, with high biological yeild, maize is protential to be the new crop restoring soil. miRNA is a newly identified class of 20-24 nt non-protein-coding and endogenous small RNAs, which plays an important role in plant growth, development and response to environmental stresses. Therefore, it is particularly urgent to search for the important miRNAs correlating with heavy metal stress response and preliminarily reveal the molecular mechanism of response to heavy metal stress in maize.In this study, the maize inbred lines 178 and 9782, which were screened and identified high and low accumulation of Pb in previous research, were choosed to be the materials. And through Solexa high throughput sequencing, we obtained a lot of miRNAs expressed in maize roots stressed by Pb, and the target genes were predicted by bioinformatics methods. The important candidate miRNAs were analyzed with Real-time PCR, and semi-quantitative RT-PCR was carried out for further validation of target genes. The main results were as follows:1. Nighty-two known miRNAs and 378 novel miRNAs were detected in maize roots by deeping sequencing. The 92 known miRNAs belonged to 18 miRNA families, and were conserved among maize, rice, Arabidopsis and other species. The 378 novel miRNAs belonged to 185 miRNA families and 20 miRNA families were conserved in gramineous species at different degrees.2. Six miRNAs (zma-root-miR5, zma-root-miR37, zma-root-miR68, zma-root-miR155, zma-root-miR164 and zma-root-miR184) were homo logical to the members of known miRNA169, miRNA164 and miRNA171 family in maize, and there were less than 4 base deviations, which demonstrated that the 6 miRNAs could be the new members of thses miRNA families.3. The results of target genes prediction revealed 54 putative target genes for these known miRNAs. Some of them were involved in the following processes, such as transcription regulation, substance and energy metabolism, electron transport, stress response, and signal transduction through further function prediction.4. For the newly identified miRNAs,489 potential targets were predicted. Of which,453 targets were functionally annotated and the other 36 targets were unknown proteins. The function of these targets could be divided into the following three groups. The first group included some metabolism related genes, the second group was transcription factor and the third important group was transport protein.5. Such cation transporters, such as ABC transporter, low-affinity cation transporter and heavy metal translocating P-type ATPase, were also found to be targeted by some newly identified miRNAs. These targets were previously reported to play important roles in detoxifcation of plant cell under heavy metal stress and these transport proteins seemed to be directly responsed to Pb stress.6. Several stress response proteins, including Manganese superoxide dismutase, peroxidase precursor and phospholipid-hydroperoxide glutathione peroxidase, were predicted in our research. These proteins could scavenge the excrescent ROS caused by Pb stress.7. Combined with target prediction results,6 candidate miRNAs involved in heavy metal stress were analyzed by Real-time PCR. It turned out that the expression of the 6 miRNAs was not obviously different in 178 among different concentrations of Pb stress at 48h stage, but it was not the case at 72h stage when high concentration of Pb induced the expression of the miRNAs. However, it was greater impact in miRNA expression through different concentrations at 48h stage under Pb stress in 9782, and it was much more obvious under the low concentration of Pb.8. The target genes of 6 candidate miRNAs were analyzed by semi-quantitative RT-PCR, including phytochelatins, heavy metal P-type transfer of ATPase, ABC transporter-like protein and NAC1 transcription factor. It was showed that the expression of 6 target genes at 48h stage was significantly higher than that at 72h stage, and less affected by the Pb concentration. However, with the increase of stress concentration, the expression was significantly inhibited in 72h. Combined with Real-time PCR analysis of miRNAs, it was found that target genes of 6 candidate miRNAs were negatively regulated. Consequently it was suggested that the suppressed expression of these target genes mediated by the miRNAs contributed to the high accumulation of Pb in 178.