Identification And Functional Analysis Of MiRNAs And Their Target Genes In Response To Heat Stress At Booting Stage In Rice (Oryza Stativa)

Rice(Oryza sativa)is the most important food crop in the world.With global warming,heat stress has become one of the main problems to be solved in rice growing areas in China.Recently studies suggested that miRNAs play an greater role in regulating growth,development and stress response in rice.However,the regulation mechanism of miRNAs in rice under heat stress is very complexity,and the related research is not clear.In this study,high throughput sequencing technique was used to sequencing the small RNA using young spikes of heat sensitive variety 9311 and heat resistance variety N22,and miRNAs and their target genes were identified in response to heat stress at booting stage in rice.The regulatory mechanism between miR398 and it’s target gene Os CSD2 in rice was analyzed under heat stress by molecular biological methods.1.Eight s RNA libraries of rice varieties 9311 and N22 under heat stress were successfully constructed.The results of sequence quality and s RNA length distribution show that there were 17,157,478(80.44%)and 17,874,788(83.51%)clear reads in 9311 and N22 spikelets,respectively,which ensures the high quality and data reliability of the sequencing sequence.Through bioinformatics study,a total of 933 miRNAs were obtained,including464 known miRNAs and 469 unknown miRNAs(including 125 new miRNAs),and we predicted 14780 candidate target genes.There were 165 differentially expressed miRNAs were obtained,of which 95 were up-regulated and 70 were down regulated.GO enrichment analysis showed these targets genes regulated by differentially expressed miRNAs participated in ATP binding,cellular components,transcriptional regulation,DNA binding,metabolic processes,metal ion binding,cellular processes,etc.KEGG annotation analysis found that these target genes were assigned to phenylalanine metabolism,and in this pathway,there were 10 target genes were regulated by four miRNAs.We further analysed the expression of 10 miRNAs and their target genes in rice 9311 and N22 under heat stress for 0h,3h,6h,and 12 h using q RT-PCR.The results showed that the expression of miR156f-5p,miR166k-5p were substantially decreased,and the expression of their target genes significantly increased;but the miR397 b,miR398 b,miR399 d,miR408-3p,miR528-5p,miR5496 and novel＿miR＿15 were substantially increased,and their target genes significantly decreased;however,the miR164 c was substantially decreased,its target genes significantly increased.The expression level of miRNAs and their target genes show a dynamic trend under heat stress at different time.This study provided the basis for our further study on the function of miR398 and its target genes in rice.2.We obtained STTM398 mutant lines by short tandem targets mimic(STTM).The over-expressing vector of OE-MIR398 a was constructed,and the transgenic lines of T2 generation were obtained using the genetic transformation system of rice(Oryza sativa),reporter gene examination,GUS staining verification and rice breeding.The analysis of agronomic characters showed that the average plant height,spike length and setting rate of wild type were significantly lower than OE-Mi R398 a transgenic lines,but higher than those of STTM398 mutan lines.The 1000-grain weight,grain length,grain width and grain length/width of OE-Mi R398 a transgenic plants lines were not significantly distinct from wild type,while the 1000-grain weight,grain length and grain width of STTM398 mutant lines were significantly lower than wild type lines.These results indicated that miR398 a may regulated the rice growth and development.3.The results showed that the survival rates of OE-Mi R398 a overexpression and wild type lines were significantly higher than that of STTM398 mutant lines under heat stress at seedling stages,and the heading and flowering time of OE-miR398 a overexpression and wild type lines were significantly earlier than that of STTM398 mutant lines at booting under heat stress stage.The results of DAB staining,SOD,POD,CAT,MDA related physiological indexes,and pollen viability analysis showed that the leaf damage degree of STTM398 mutant lines were significantly greater than that of wild type and OE-Mi R398 a overexpression line,and the iodine staining rates of wild type and OE-miR398 a overexpression lines were significantly higher than that of STTM398 mutant lines.q RT-PCR analysis showed that the relative expression of miR398 a in over expression transgenic lines were significantly higher than that of wild type at seedling and booting stages under normal conditions,while the relative expression of miR398 a in mutant plant lines were significantly lower than that of wild type.These results indicated that the heat tolerance of rice was increased to a certain extent by overexpression of miR398 a,while the heat tolerance of rice was decreased by silencing of miR398.This suggests that miR398 may be involved in regulating rice response to heat stress.4.The cleavage sites between miR398 a and it’s target gene Os CSD2 were validated through RLM-5’RACE.The results showed that osa-miR398 a was complementary with the target Os CSD2 in specific site,and Os CSD2 was cleaved at two different specific site.By CRISPR/Cas9 genome-editing technology,target sequences were designed in the two different cleavage sites,and the CSD2-T1、CSD2-T2 and CSD2-T1T2 knockout vectors were constructed.T-DNA-free mutant lines of T2 generation in rice(Oryza sativa)were obtained by the genetic transformation system and detection.After heat stress treatment and q RT-PCR,the results showed that the survival rate of Os CSD2 mutant lines in rice was significantly lower than that of wild type lines,and the relative expression of Os CSD2 gene in mutant lines were significantly lower than wild type lines under normal conditions.These results suggestion that Os CSD2 gene was induced by heat stress,and participates in the regulation of heat tolerance in rice.