Computational And Cloning Indentification Of Novel MicroRNAs And Analysis Of Their Differential Expression Under Drought Stress In Maize

As a class of negative regulators in eukaryote organism,miRNA(abbreviated to "miRNA") regulates almost reach to all cellular levels of organism.More and more researches about relationships between miRNA and plant stress tolerance,especially in Arabidopsis thaliana and Oryza sativa,have been reported.So miRNA is on of key factors in plant stress tolerance regulations.However,the identified plant miRNAs and their diversity functions in plant stress tolerance are still far from saturation.Zea mays,being a non-model plant,certainly far behind Arabidopsis thaliana and Oryza sativa on miRNA research.There are only 98 identified miRNAs from Zea mays were submitted to miRNA database.For exploring relationship between microRNA and drought stress response mechenism in maize,with polyethylene glycol 6000(18%) simulated drought stress disposing,we used computaional and cloned method to find new miRNAs from maize self lines 200B and 87-1,and target genes of these new miRNAs were subsequently predicted. As a result,some conclusions had been obtained concerning relationship between miRNA regulation and drought stress in maize by the express of miRNA and their functions.The finding of the research are as follows:1 Eighteen new miRNAs had been found.Among them,eleven obtained from coumputational method and the others from cloning method.The lengths of their sequences range from 19 to 21 nt,and 7 of the sequences were began with "U" from 5' termination. This character is similar to what have been reported in other plant miRNAs.2 Most miRNA we found had high conservation in Gramineae.By " blast " genome database of other plants incluing rice,wheat conservation of miRNA was identifed among them.As a result,besides five of the eleven miRNAs which obtained from computational method had conservation in Gramineae,four sequences of cloned miRNAs exhibit conservation in it.3 By RNA-tailing and primer-extension RT-PCR,all of the seven cloning miRNAs and four computaional prediced miRNAs had been detected,which indeicateds their reality in maize.On the other hand,it also proved that the identified miRNAs,under the drought stress condition,expressed in 87-1 are higher than that expressed in 200B.4 The eleven identified miRNA,according to the drought stress material and non-drought stress material,had a key connection with drought in maize,miRNA1,miRAN3,miRNA6,miRNA479,miRNA782,miRNA815a,miRNA 820 only expressed in drought stress maize material of 87-1,which proved that these seven miRNAs had relationship with drought response mechenism.5 Twelve target genes were predicted for 8 of 11 new identified miRNAs by using plant target predict software miRU.Most of these target genes are involve in signal transduction,non-biological stress,biological stress,transmembrane transport,transcription factor,and metabolism.Especially,we discovered that maize drought tolerance had a complicated relationship with CDC2/CDC28 protein kinase,receptor-like protein kinase, extension protein,dolichol,CER1 and CIPKs by analysing target gene functions of the identified miRNAs from drought stress maize material of 87-1.