Regulation Of Target Gene Expression By AtMIR396Family And Its Possible Role In Root Development

microRNAs (miRNAs) are19-24nt sequence-specific non-coding RNAs which mediate gene silencing at the post-transcriptional levels. siRNAs which are more abundant than miRNA in eukaryotic genomes can mediate gene silencing at both the transcriptional and post-transcriptional levels.In Arobidopsis, the family of miR396contains two members:miR396a and miR396b. They were encoded by two genes:AtMIR396a and AtMIR396b. GRF (growth-regulating factors) gene family comprises nine members and seven of which (GRF1, GRF2, GRF3, GRF4, GRF7, GRF8, GRF9) have been predicted and validated as the targets genes of miR396. GRFs are involved in leaf development through regulation of cell proliferation. miR396, which targets GRF and restricts the expression regions of GRF, also plays a role in leaf development.In this study, transgenic35S:MIR396a and35S:MIR396b lines had been obtained in order to investigate the role of miR396in Arabidopsis. Northern bolt showed that miR396a or miR396b were overexpressed in these lines. Both of them presented smaller leaves than WT. Besides the smaller leaves,35S:MIR396a but not35S:MIR396b showed shorter root phenotype. GUS staining of promoter:GUS transgenic lines revealed that GRFs and MIR396were expressed obviously in root area. Furthermore, different GRFs exhibited different spatial transcriptional patterns in root, which indicated there were different types of expression relationships between miR396and its targets. qRT-PCR showed that the mRNA level of GRFs were reduced in transgenic lines. Since all the grf mutants we detected did not present any morphological changes in root, we proposed that the reduced level of GRF was not the reason for the short root phenotype presented in35S:MIR396a lines.The sequence of miR396a and miR396b are almost identical. The only difference between them is the last nucleotide in the3’terminus, it is a Guanine in miR396a but a Uridine in miR396b. It is generally recognized that this difference is not enough to bring bias in target gene selection and down-regulation. In order to determine whether the stem-loop sequence of MIR396a is a reason for the short root phenotype, we constructed35S:preMIR396a-b (miR396b and miR*396b in MIR396a stem loop) and35S:preMIR396b-a (miR396a and miR*396a in MIR396b stem loop) transgenic plants. We found that almost all the transgenic lines of35S:preMIR396a-b presented short root phenotype as35S:MIR396a, while35S:preMIR396b-a have normal root phenotype as WT. Therefore, we proposed that over-expression of the stem-loop sequence of MIR396a is a reason for the short root phenotype.Using the deep sequencing method, recent researches have showed that some canconical MIR genes could also generate siRNAs at the same sites. Considering MIR396might also generate other siRNAs, we searched miR396related miRNA and siRNA in the public small RNA database. The putative targets of these small RNA were also predicted using the psRNATarget software. We found that AT1G1020can be well matched to sRNA14117which generated from MIR396a. The results of5’RACE experiment showed that the most common5’end of AT1G10120cleavage products mapped to the tenth nucleotide of the miR396and the thirteenth of the sRNA14117. Thus, AT1G10120had been confirmed as a new target gene of MIR396.