Expression And Regulation Mechanisms Of MiRNAs And Functional Genes In Response To Heat And Drought Stresses In Rice

As the staple food,rice(Oryza sativa)is consumed by more than half of the world population.Drought and heat stresses are the most damaging and widespread abiotic stresses that can severely limit rice production.In recent years,with the development of plant molecular biology,researches have found that many rice micro RNAs(miRNAs)played a key role in stress resistance and acted as a promising tool for improving stress tolerance in rice.However,expression and regulation mechanisms of miRNAs and functional genes in response to heat and drought stresses in rice are not fully understood.In this study,through bioinformatics analysis and experiments,the main molecular functions and signal pathways of miRNAs and their target genes under drought stress were discussed;the analysis and mining of high-temperature genome expression data of rice post-meiosis panicle were carried out based on the rice genome microarray technology platform;the sensitive genes which were differentially expressed in anther and pollinated pistil under heat stress and combined heat and drought stress were found;the sensitive genes sequence visualization and functional analysis were carried out,the possible regulation of miRNAs were predicted.The major results are listed as follows:1.A comprehensive analysis of rice miRNAs and genes transcriptome data under drought stress showed that:(1)Most DE-mi RNA families resisted drought stress by down-regulating their target genes,and there were a degree of cooperation among the mi RNA families and the multiple interaction between mi RNA-regulate targets.(2)Micro RNA-regulated target genes involved in a series of basic metabolic pathways which were related to drought stress,such as sugar and secondary metabolite biosynthesis,peroxisome,plant circadian rhythm,etc.(3)Micro RNAs might play critical roles in regulating genes related to lignin degradation,abscisic acid(ABA)and auxin biosynthesis.(4)The regulation of miRNAs might play an important role in drought signal perceiving and transduction.(5)Four hub miRNAs and six hub target proteins were screened from the miRNAs regulatory network under drought stress.They were mi RNA156,mi RNA529,mi RNA818,mi RNA166 and Pita,PIP5 K,PP2Cs,protein kinases,GLT2,AOX respectively;three of them(mi RNA166,AOX and PP2Cs)were associated with ABA synthesis and signal transduction.2.Microarray analysis was performed on RNA isolated from rice post-meiosis panicles which were treated at 40 centigrade degree for 0 min,10 min,20 min,60 min,and 2 h.A total of 124 miRNAs,corresponding to 37 rice mi RNA families,were identified to be differentially expressed in rice post-meiosis panicle under heat stress.By integrating paired miRNAs and m RNAs expression profiles,we found that the expression levels of 29 mi RNA families were negative correlation to their 178 differentially expressed target genes.Further analysis showed that the majority of miRNAs in 29 mi RNA families resisted heat stress by down-regulating their target genes and a time lag existed between expression of miRNAs and their target genes.Then,GO-Slim classification and functional characteristics analysis of these 178 target genes showed that:(1)Micro RNAs mainly involved in a series of basic biological processes even under heat conditions.(2)Some miRNAs might play important roles in heat resistance(such as osa-mi R164,osa-mi R166,osa-mi R169,osa-mi R319,osa-mi R390,osa-mi R395 and osa-mi R399).(3)Osa-mi R172 might play important roles in protecting rice panicle under heat stress,but osa-mi R437,osa-mi R418,osa-mi R164,osa-mi R156 and osa-mi R529 might negatively affect rice fertility,headingand spikelet development.(4)Osa-mi R414 might inhibit flowering genes expression by down-regulation of LOC＿Os05g51830 to delay the heading of rice.Finally,a heat-induced mi RNA-PPI(protein-protein interactions)network was constructed,and three mi RNA co-regulatory modules were discovered.Among them,for osa-mi R529 centered co-regulatory module,osa-mi R529 might co-express with 11 miRNAs through the target genes and co-regulate 102 high confidence downstream targets.3.A total of 47 sensitive genes were screened from the database in anther and pollinated pistil under heat stress and combined heat and drought stresses through data base.Expression analysis showed that heat shock protein,chaperone and folding enzymes were up-regulated,which could beneficial for plants to resist stress-induced intracellular protein damage.The expression of two calcium-binding protein CML36,which played important role in regulating pollen development and pollen tube growth,have tissue specificity but no stress specificity under two stresses.Under combined heat and drought stress,OMTN4,a positive role in regulating heading and senescence during the reproductive stage of rice,might have specific expression pattern which was different from single heat or drought stress.Down-regulation of WAT1-related protein might be harmful to rice flowering and grain-filling.The network visualization and similarity analysis of the 47 sensitive gene protein sequences showed that the similarities of these sequences were not obvious,but the frequencies of Ala,Gly,and Val in them were higher than other acids.The enrichment analysis of INTERPRO protein domains showed that 47 sensitive genes were mainly enriched in HSP20,HSP70,Tetratricopeptide repeat,CLPA/B,Dna J,FKBP-type peptidyl-prolyl cis-trans isomerase domain and EF hand domains.The functional enrichment analysis of 47 sensitive genes showed that 47 sensitive genes were mainly enriched in some stress-related biological processes and metabolic pathways.In addition,the sensitive genes were also enriched in HSP90 chaperone cycle for steroid hormone receptors and protein methylation etc.Based on ps RNATarget prediction,28 possible miRNAs of 13 sensitive genes were obtained and mi R164,mi R156 and mi R5162 might be the key miRNAs in co-regulatory networks under stress at rice reproductive stage.