Differential Expression Of MicroRNA159 And MicroRNA168 Under Drought Stress In Maize

With the deterioration of ecological environment, various biotic and abiotic stresses have become the obstacles of restricting corn production; The drought stresse is gradual one of the important factors which restrict yield of com. Though it is the most effective mean that reduces loss by improving drought resistance of corn, we still know little about the genetic mechanisms of drought resistance. At the same time, drought resistance is a complex quantitative trait which is controlled by many genes, so some of related genes cloned by using cloning, mutagenesis and mutants techniques and their analysis of express identity in drought stresse just tell me, in part, why. A new class of regulatory factors—microRNAs (miRNAs) which were found in the beginning of this century. miRNAs are small approximately 21-nucleotide RNAs that function posttranscriptionally to regulate gene activity. miRNAs function by binding to complementary sites in target genes causing mRNA degradation and/or translational repression of the target. miRNAs function broadly to control many aspects of plant development and stress response. So the analysis of differential expression of miRNA under drought stress is expected to alternatively reveal the molecular mechanism of plant drought resistance.This study used drought-sensitive inbred lines of maize, Dan340 and 21ES, and drought-tolerant inbred lines,87-1 and 81565, as the materials to detect the differential expressions of miR159 and miR168 under drought stress by miRNA microarray assay. After verifying the reliability of miRNA microarray's results by Locked-nucleic acid (LNA) Norhtern blotting, a web-based computing system, WMD3, was used for prediction of miR159 and miR168 target mRNAs, and DGE experiments were use to analyze the differential express of these predicted target genes between different inbred lines under the same stress conditions, then we explored the potential regulation pattern of miR159 and miR168 under drought stress condition. Our results are mainly as follows:1. Most of the miR159 family members were up-regulated under drought stress. However, the miR159 family members showed different expression patterns either among the three time points, or between the four maize lines. Based on these results, we guessed that the response to the drought stress and regulation of the target genes may have different effect, though the family member of miR159 have mostly similar base sequence.2. The expression levels of two memeber of the miR168 family were inhibited in Dan340,21ES and 87-1 inbred lines. but only the expression levels of miR168b was exchanged in strong drought-tolerant inbred line 81565. It implyed that miR168a and miR168b played different roles on drought stress.3. The results of LNA-Northern blotting that the expression level of miR168 under the same stress condition were completely coincident with the results of miRNA microarray assay. It confirmed that the results of miRNA microarray assay was reliable.4. A total of 32 miR159 potential target genes and 4 miR168 potential target genes were predicted by WMD3, some of which were regulated together by multiple miRNA family memebers, and some of which were regulatied by specific miRNA family members. All of these target genes involved in a variety of upstream of basic regulatory pathway.5. Analysis of the differential expressions of target genes by DGE showed that: The expression levels of miR159 target genes MYB55-like protein mRNA were down-regulated specifically in strong drought-tolerant inbred line 81565. The expression levels of Rac GTPase activating protein 1 mRNA were up-regulated specifically in drought-sensitive inbred lines Dan340 and 21ES. The expression levels of RNA binding S1 mRNA were changed in each of inbred lines, but the expression trend in 81565 was very reverse of other three inbred lines; In four inbred lines the expression levels of miR168 target genes AGO1 mRNA were up-regulated except inbred line Dan340. After 24 h of drought stress, the expression levels of AtIDD2-like protein gene mRNA were extremely up-regulated by reduction of miR168, the degree of up-regulated reached 1379.57 times compared with the untreated control.6. We speculated that the conservative family miR159 and miR168 may be the regulator in upstream of drought response pathway, by using the differential expression of miR159, miR168 and their target genes to analysis the potential regulation pattern of miR159 and miR168.