The Functional Research Of MiRNA398 And Its Target Genes Which Regulate The Heat Resistance In Rice

Heat stress will affect rice productive development and leads to lower yield and quality in rice.The plants can response to a variety of stress by miRNA398.The target genes of mi RNA398 are Cu/Zn superoxide dismutase,it can remove reactive oxygen species in plants and enhance plant resistance for stress.This study which is based on miRNA398 regulating heat resistance in Arabidopsis,predicting the target genes of Osmi RNA398 by Targetfinder and PsRNAtarget,to researh the expression related to high temperature stress of OsmiRNA398 and its target genes.The results were as follows:(1)The molecular identification of STTM398 transgenic rice,the phenotypic analysis and the determination of physiological and biochemical indexes were carried out after heat stress.The result showed that the wildtype(Nipponbare)was more resistant than STTM398 transgenic rice after high temperature stress.Compared with the wildtype(Nipponbare),STTM398 transgenic material has the higher Peroxidase(POD)and Superoxide dismutase(SOD)activity,the Malondialdehyde(MDA)and Proline(Pro)contents are higher than control too after heat stress.Therefore,miRNA398 is related to heat tolerance of rice,and with the weakening of miRNA398 on its target gene,the heat tolerance of rice is weakened.(2)By analyzing the upstream promoter elements of the target genes of OsmiRNA398,homologous comparison with their protein sequence,the result indicated that OsCSD1,OsCSD2 and OsCCS have multiple stress-related elements and highly conserved sequence in a variety of species.(3)By using biochip to analyze the expression characteristics of target genes of OsmiRNA398 under high temperature stress,drought stress as well as light and temperature cycles.Then,verification by using real-time quantitative PCR.The result implied that the target genes of miRNA398 was up-regulated after heat stress,which indicated that the taget of miRNA398 was associated with heat tolerance of rice.And it was a potential heat-regulating element in rice.(4)The expression vector(pCambia2300-OsCCS-GFP)of copper / zinc superoxide dismutase copper chaperone was constructed.The transient expression in tobacco transformed by Agrobacterium mediated method showed that OsCCS expressed on the nucleus.(5)In addition,the pYLCRISPR / Cas9-OsCCS knockout vector was constructed to further study biological function of OsCCS.The study plays an important role in understanding the molecular mechanism of mi RNA398 and its target response to high temperature stress.