Identification And Differential Expression Analysis Of Small RNA Related To Drought Tolerance In Rice

MicroRNA plays an important role in plant growth and development and response to environmental stresses.Previous studies have shown that miRNAs are involved in the process of abiotic stress response.Although there are a lot of reported miRNAs in rice,the role and function of miRNAs in response to external environmental stress are still not clear.The specific regulation network and molecular mechanisms need to be further studied.In this thesis,a drought tolereant introgression line（DK151）and its drought-sensitive recurrent parent（IR64）were used to identify the candidate miRNA responsible for drought stress in rice by using microRNA and degradation seqencing,combined with bioinformatics and molecular biology analysis.The main results are as follows:1.DK151 was shown higher yield than IR64 under both drought and irrigated conditions,.The activities of aseorbate peroxidase（APX）,superoxide dismutase（SOD）,and total antioxidant AOC in DK151 were significantly higher than that in IR64 under drought stress,indicating DK151 was more tolerent than IR64 under drought stress.2.In this study,four small RNA libraries were further constructed and analyzed by high-throughput sequencing.There were 254 and 158 differentially expressed known miRNAs in IR64 and DK151 under drought condition,respectively.there were 215 and 265 mi RNAs identified to be differently expressed between DK151 and IR64 under irrigated and drought conditions,respectively.A number 54 miRNAs were detected to be affected by drought stress both in IR64 and DK151,and 104 and 200 miRNAs were found to be specifically differently in DK151 and IR64,respectively.A total of 76 drought induced mi RNAs were identified by differential expression analysis and target genes prediction.Of them,12 mi RNAs were quantitatively confirmed by quantitative RT-PCR.3.Two DK151 degradome libraries were constructed,and 3251 transcripts were detected for 470 mi RNAs（222 known miRNAs and 248 new miRNAs）.A total of 220 target genes were predicated for the 12 candidate miRNAs,but only eight miRNAs including osa-miR1425-5p,osa-miR169i-3p,osa-miR396b-5p,Osa-miR396e-5p,osa-miR398 b,osa-miR5490 osa-mi R5521 and novel_mir₅15 were characterized by detecting its cleavage sites.4.In order to verify whether the target gene of the degradation group was complete,we analyzed one candidate miRNA（osa-mi R398b）and its cleavage sites of two target genes by RLM-RACE technique.We found one target gene LOC_Os03g22810.1 was not detected in the sequencing of the degradome,suggesting that osa-miR398 b may negatively regulates LOC_Os03g22810.1 by reducing its expression.5.In this study,12 knockout vectors and overexpression vectors were constructed for 12 candidate mi RNAs respectively.A total of 24 overexpressed transgenic T0 lines and 16 knockout transgenic lines were obtained,which laid a foundation for further elucidating the molecular mechanism of these mi RNA.