Characterization Of MiRNAs And Their Target Genes In Response To Low Nitrate Availability In Maize Inbred Line B73

Maize is one of the most important cereal crops. The genetic and physiological basis for nitrogenuse efficiency (NUE) and low nitrogen (LN) tolerance are of great significance for maize productionand breeding studies. miRNAs are a class of about22nt length small non-coding RNA that play animportant role of plant development and adaptation to environmental stresses at the post-transcriptionallevel or at the translation level. Thus, characterizing LN responsive miRNAs, their target genes and theirregulatory role in adaptation to LN stress will found important basis for genetic improvement in maize.In this study, B73, one of maize inbred lines, was analyzed the miRNAs and their target genes in leavesand roots in response to LN stress by combining analysis of the deep sequencing small RNA anddegradome sequencing. The expression pattern of some miRNAs and their target genes were furthervalidated. In addition, homozygote transformation Arabidopsis lines of zma-miR398over-expressionand target mimicry were established in the model system, that found the analysis basis for furtheranalysis on role of miR398in plant adaptation to LN stress. The main results are as follows.⑴Compared with the controls at the optimal nitrate condition, B73seedlings showed the obviousphenotypes at LN stress after15days hydroponic culture.⑵Forty-three known miRNAs from25miRNA familes were detected in response to LN stress byusing a strategy of combining analysis between small RNA and degradome deep sequencing on thelibraries from LN leaves, roots and their optimal N controls, Among them,14species from8families were significantly up-regulated expressed and11species from8families were significantlydown-regulated expression in leaves. Twenty-two species from14families were significantlyup-regulated, and11species from7families were significantly down-regulated expressed in roots.Sixteen of43known species from6families were up-regulated in both roots and leaves. And,7species from5families were down-regulated expressed in both roots and leaves. Whereas,2speciesfrom miR393and miR398families were down-regulated in leaves but up-regulated in roots.⑶Thirty-five new miRNAs responsive to LN stess were identified, and designated as miRC mumbers.According to miRNA annotation criteria, miRC10, miRC40, miRC61, miRC68, miRC69, miRC71,miRC73and miRC77belongs to miR167, miR169, miR395, miR398, miR528and miR2275miRNA families, respectively. Among35new miRNAs in response to LN,30of them weresignificantly differentially expressed in leaves. Among them23of them were up-regulated and7ofthem were down-regulated. Fourteen members were significantly differentially expressed in roots.Among them,8members were up-regulated and6members were down-regulated. miRC50,miRC51, miRC77and miRC78were up-regulated expression both in roots and leaves. And,miRC10and miRC68were down-regulated expression both in roots and leaves. miRC11wasdown-regulated in leaves but up-regulated in roots. miRC60was up-regulated in leaves butdown-regulated in roots.⑷Zma-miRC10and zma-miRC68, two new identified miR169family species, were further identified and analyzed. Sequencing results showed that they were down-regulated expression in both leavesand roots. They were the two highest abundant species of miR169family under optimal nitratecondition, which represented21.1%and53.6%of the total, respectively. And, they weredown-regulated6.0and5.5times, that was the highest the down regulated magnitude withinmiR169family under LN. In addition, miRC10, miRC68and miR169c, r were the highest abundantspecies under optimal nitrate condition, representing the18.0%,17.5%and57.1%of the total,respectively. They were the also the most down-regulated species under low nitrate condition in theroots, which were down-regulated16.3,11.9and7.0times, respectively. The above resultsindicated that miRC10and miRC68may play primary role among zma-miR169species in responseand adaptive to LN.⑸Upon bioinformatics analysis, GRMZM2G078124(Rhodopsin-like receptor) andGRMZM2G165488were the targets of zma-miRC10, and GRMZM2G091964was the target ofzma-miRC68. GRMZM2G165488and GRMZM2G091964are belonging to NYFA (Nucleartranscription factor Y subunit A) transcriptional factor family. Degradome sequencing data showedthat the GRMZM2G078124, GRMZM2G165488and GRMZM2G091964had degraded fragmentsat the predicted target sites, which provided experimental evidence for the target gene prediction. Italso showed that the abundance of GRMZM2G165488mRNA degradation products at a predictedmiRC10cleavage site and the abundance of zma-miRC10showed the positive proportionalrelationship in the maize roots. It indicated that the negative regulation mechanism betweenmiRNAs and their target genes may play a role in LN adaptation.⑹The cleavage sites of the miRNAs, zma-miRC10mediating GRMZM2G078124andGRMZM2G165488, zma-miRC68mediating GRMZM2G091964and zma-miR398mediatingGRMZM2G169890, GRMZM2G058522and GRMZM2G103812_T02were further verified byusing5’-RACE. Quantitative PCR results showed that the expression abundance of zma-miRC10,zma-miRC68, and zma-miR398showed negative correlation with their target genes,GRMZM2G078124and GRMZM2G165488, GRMZM2G091964, and GRMZM2G169890,respectively. It suggested that the negative regulation of miRNA on their target genes are probablyone of the physiological basis for maize adaptive regulation to low nitrogen.⑺Over-expression vector of pCPB-35S::zma-MIR398b and pCPB-35S::zma-MIM398weresuccessfully constructed. We obtained3homozygous transgenic T2Arabidopsis thaliana lines. Thiswork established some material basis for study on the the regulatory role of zma-miR398inadaptation to abiotic stress in plant model system.