Mir444f Regulates Rice Root Development Through Gibberellin

Rice is one of the most important food crops in the world.Excellent traits such as semidwarfism of plants are important for rapidly increasing grain yield and meeting human food demand.However,dwarfing of rice is accompanied by reduced utilization of nitrogen fertilizer.How to balance root development for nitrogen utilization and rice plant dwarfing has become one of the hot spots of current researchers’ attention.miR444 is an important class of small molecule non-coding RNAs that play an important role in regulating gene expression in living organisms and can participate in the regulation of various life activities.miR444,as an important member of the miRNA family,also plays an indispensable role in the regulation of root growth and antiviral defense,but there are few studies on miR444.However,there are few studies on miR444,and most of them are focused on miR444a and miR444b.Therefore,in this paper,we used the CRISPR/Cas9 system to edit rice healing tissues and obtained miR444 family knockout mutants(mir444a-3p,mir444a-5p,mir444b,mir444d,mir444e,mir444f)by genetic transformation.We selected the mir444f mutant as genetic material for subsequent study and analysis.Through root observation at 3,7 and 15 days,we found that the mir444f mutant mainly showed shortened lateral roots and reduced number of mid-column sheath cells in terms of root phenotypes,and to explore its molecular regulatory mechanism,we first used raw signal analysis to predict MIR444f’s regulation of downstream target genes We first used bioconductivity analysis to predict the regulation of MIR444f on the expression of downstream target genes MADS27 and MADS57,verified the regulatory relationship of MIR444f on MADS27 and MADS57 by transient transfer of Ben’s tobacco,verified the expression of MADS27 and MADS57 in MIR444f mutant leaves by q-PCR,and found the downstream target gene of MADS27,SLR1,which is the only DELLA protein in rice.Therefore,we determined the gibberellin signaling pathway in the mir444f mutant and quantified the relevant proteins by Wsetern blot,and analyzed the gibberellin content in the mutant leaves by mass spectrometry.This study revealed that MIR444f regulates the expression of MADS27 and MADS57 transcription factors,which in turn regulate the gibberellin signaling process.In this study,we identified a new pathway of gibberellin regulation of root development,which provides a new idea for further understanding of rice root development mechanism.The main innovations of this thesis are:to reveal that miR444f regulates the root development of rice plants through gibberellin from many different perspectives such as molecular,genetic,cellular and metabolic,and to find the downstream target genes of MIR444f,MADS27 and MADS57,to try to explain the signaling pathway of miR444f in rice.The mir444f knockout mutant was also explored for nitrogen utilization and salt-growing resistance.We hope that this topic can provide theoretical support and help for rice breeders in later molecular breeding design.