Identification And Functional Analysis Of MicroRNAs In Response To Drought In Common Wild Rice (Oryza Rufipogon Griff.)

Drought is one of the most important factors that limit the rice productivity.Rice plants will growth slowly during the juvenile phase and decrease the fertility at reproductive stage once drought happens.Common wild rice(Oryza rufipogon Griff.)is the ancestor of cultivated rice(Oryza sativa L.)and with well developed root systems.Previous studies confirmed that common wild rice harboring abundant drought-resistant genes,which provide an opportunity to explore these excellent resources as contributors to improve rice resistance,and productivity.MicroRNA(miRNA)is a class of non-coding,endogenous small RNA that has essential roles in plant development,and environment stress.At present,the study of drought stress in common wild rice is focused on physiological index detection and QTL mapping analysis.However,few studies on miRNAs are involved in drought response and tissue specificity.Thus,we performed high-throughput sequencing to identify the miRNAs in response to drought;and analyzed the molecular mechanism of drought stress in common wild rice.Firstly,we constructed four small RNA libraries,CL and CR from PEG6000-free samples and DL and DR from PEG6000-treated samples('L' indicates the shoot tissue and 'R' indicates the root tissue),to screen the drought-responsive miRNA.There were 103 and 128 miRNA to be differentially expressed during drought stress,including 26 and 43 new miRNA in shoot and root,respectively.Stem-loop qRT-PCR was performed to examine the tissue-specificy of 12 miRNAs.Potential targets were predicted.498 candidate genes were predicted to be potential targets of the 213 miRNAs.88 miRNAs,that were drought responsiveness,were predicted with 197 targets.Further analysis revealed the target genes of miRNAs by 5' RACE.In order to research the function of miRNA in response to drought stress,we selected two miRNAs,miR171 and miR3979,which accumulated preferentially in the root.According to the 5' RACE,miR171 targeted LOM1/LOM2/LOM3.After three generations breeding,we obtained homozygous transgenic lines of miR171 overexpression(miR171-ox)and reducing miR171 activity(STTM-171a)lines in Arabidopsis.The result showed that miR171-ox plants not only caused growth slowly,the shorter primary root,the larger lateral root density,but also induced abnormal shoot branching from the root.Whereas the STTM-171a plants exhibited opposite phenotype.Under drought condition,miR171-ox plants increased drought resistance of Arabidopsis.Further analysis revealed that either miR171-ox or STTM-171a plants increased the sensitivity to ABA treatment,which indicated that miR171 responsing to drought stress by AB A-dependent pathway.BHLH157 has been identified as the target of miR3979 in rice.According to the phenotype of miR3979 transgenic lines(miR3979-ox),overexpressing miR3979 decreased the plant height and root.For the drought treatment,the survival rate of miR3979-ox plants was lower than that of control.Further analysis revealed that miR3979-ox plants increased the sensitivity to ABA treatment,indicating that miR3979 negatively responsed to drought stress in common wild rice.As such,we analyzed the dynamic changes of miRNAs after drought stress in common wild rice,and 2 miRNA that with high expression in root were selected to research.The results revealed the molecular mechanism that miR171 and miR3979 response the drought stress and regulate the root development,providing a valuable resource for genetic and stress studies in common wild rice.