Exploration And Functional Verification Of Drought-responsive MicroRNA In Agropyron Mongolicum Keng

Agropyron mongolicum Keng is perennial diploid grasses species of Triticeae,which is stronger drought resistance.It plays an important role on reseeding of natural pasture in drought and semi-desert area of north china and seeding of artificial grassland,as well as have valuable genetic resources for the genetic improvement of wheat crops.MicroRNAs are one class of noncoding RNA and ubiquitous in plants,its has been involved in response to a wide variety of stresses including drought,salinity,chilling temperature et al.In response to the blank of drought-responsive miRNA in A.mongolicum,small RNA library of A.mongolicum under drought stress was constructed in this study,the drought-responsive miRNAs were screened with Solexa high throughput sequencing and bioinformatics analysis;The temporal expression of 7 drought related miRNA by stem-loop real-time PCR,and their target genes were predicted by Target Finder software and psRNATarget database;The over expression vectors of miRNAs were constructed,and using the function of miRNA drought resistance were verified via agrobacterium-mediated method transfer into Arabidopsis thaliana,which could be lay the foundation of the molecular mechanism for drought tolerance of miRNA in A.mongolicum and molecular breeding of forage grass.The main research results are as follows:1.A total of 114 new miRNA were screened in A.mongolicum,53 conservative and61 unconservative miRNA,there are 17 differential expression miRNA of them.2.7 differential expression miRNA of A.mongolicum were predicted 17 target genes in A.mongolicum and 60 target genes in A.thaliana;and most of the target genes responses to drought stress.3.amo-miR21,amo-miR82,amo-miR62,amo-miR5 and amo-miR77 were up-regulated,while amo-miR44 and amo-miR17 were down-regulated under drought stress,and the expression of 7 miRNA in A.mongolicum were consistent with sequencing data.4.7 miRNA of A.mongolicum expressing vectors(pBI121-amo-miR21,pBI121-amo-miR5,pBI121-amo-miR62,pBI121-amo-miR44,pBI121-amo-miR82,pBI121-amo-miR17 and pBI121-amo-miR7)were successfully constructed.5.Transgenic Arabidopsis thaliana plants were obtained,3 drought-responsivemiRNA(amo-mi R21,amo-miR5 and amo-mi R62)in A.mongolicum can steadily inherited,and the regulation of expression trend is the same as that in A.mongolicum.