Identification And Verification Of MicroRNAs And Their Targets On Response To Low Nitrate In Maize

Maize is an important cereal, fodder and industrial crop, which is the first crop and play a key role in national economic development in China. Nitrogen was the most important fertilizer used most on crop production, but nitrogen recycling efficiency is only 25% and nitrogen use efficiency is low, thereby improving nitrogen use efficiency of maize and low nitrogen resistance has important meaning. Research on transcriptional regulation under low nitrogen treatments could reveal physiological mechanisms of low nitrogen resistance and nitrogen avilibility. MicroRNA (miRNA) is one kind of single strand non-coding RNA with 21-24nt in length. MiRNAs play an important part in regulating and controlling plant growth and development, environment factors and stress tolerance. So it has important research meaning that research on low nitrate inducing miRNA transcriptome, identification on miRNAs and their targets in different tissues responsive to low nitrate, and adaptability regulation and physiology mechanism to nitrate limitation in maize. Hypothesis of regulation network of miRNA and its target gene in maize (nitrogen sensitive maize inbreds Ye478) roots and leaves under low nitrate treatment was proposed based on identifying low nitrate responsive miRNAs by plant miRNA microarray and qPCR verification to miRNA and its target gene expression. Main results are as follows:1. Phenotype identification of hydroponical maize at different nitrate concentrations showed that nitrate treatment has significant effect on biomass set and distribution to roots and stalks. At 0.04mM nitrate concentration, maize seedling of inbreds Ye478showed nitrogen deficiency and the lowest biomass. Root/stalk ration was twice as much as that in other three treatments. At 4mM nitrate concentration, maize seedling showed the bigest biomass, which was three times of that at 0.04mM nitrate concentration and twice of those at 8mM or 12 mM nitrate concentration. Based on these results, low and normal nitrogen treatment was determined as 0.04 mM and 4 mM in maize seedling cultivation of chip cross experiment, respectively.2. Chronic and transient miRNA responding to low nitrogen in maize leaves and roots was verified by two chip systems of SmartArray^TM and GeneChip^?. The results presented that miRNAs have tissue specificity and response characteristic under different time treatments, which was verified by real-time RT-PCR.3. Fourteen chronic miRNAs responding to low nitrate were identified in maize leaves: all of them were down-regulated except that four miRNA were up-regulated. Nine transient miRNAs responding to nitrate limitation were detected in maize leaves. MiR827 were down-regulated and miR169p,miR169i-k were up-regulated, which were opposite to their expression mode under chronic low nitrate condition. miR169e,miR172a-d,miR172e-f,miR397a,b,miR398b,c showed both chronic and transient response to low nitrogen.4. Eight chronic miRNAs responding to low nitrate were identified in maize roots and they were all down-regulated. miR169p,miR169i-k,miR169f-h,miR399d,j,miR408,miR528a,b shared the same expression mode with that in leaves. Nine miRNAs responding to transient treatment were detected in maize roots and they were all up-regulated. miR169p and miR169i-k were detected both in roots and leaves.5. Target genes of low nitrate response miRNAs were identified by bioinformatics approach. Some of the miRNA/targets modules were verified by real-time RT-PCR on the tissue specifity and characteristic under different time treatments. It indicated that miRNA can be involved in the regulation in nitrate limitation condition through the crosstalk with the target genes. A potential regulation network involed in the miRNA/targets modules was proposed base on the function of the target genes. Further more, these targets were divided into three categories. The first were the genes which encode transcription factors. The second were the genes involved in scavenging of the reactive oxygen species (ROS) and the third targets were the genes which encode the crutial enzyme involved in the miRNA biosynthesis.