Development And Analysis Of OsMIR394 Targeted Mutants In Rice

microRNA(miRNA)is a type of endogenous non-coding small RNA with a length of 19-24 nts that widely exists in eukaryotes and viruses.It is a key component of a complex gene regulatory network in an organism.In plants,miRNAs are approximately21 nt in length,and they can regulate the growth and development of plants,epigenetics,biological and abiotic stress response by directly cutting target gene mRNAs.This study based on the CRISPR-Cas12a genome editing system targeted knockout of rice OsMIR394 gene to create a homozygous and stable inherited OsMIR394 function deletion mutant material at the target site.sRNA-Seq analysis,as well as further agronomic character determination and abiotic stress experiments,initially explored the function of OsMIR394,the main research content and results are as follows:1.We designed and constructed a directed editing vector pYLJ24 based on CRISPR-Cas12a genome editing system for rice OsMIR394.Agrobacterium-mediated rice stable genetic transformation method was used to obtain 16 independent regenerated plants with a PCR positive rate of 100%.Further SSCP rapid detection and Sanger sequencing results of T0 generation plants showed that a total of 11 OsMIR394 mutant plants were obtained,and 7 different genotypes were identified.The gene mutations were mainly indels of 1-22 bp,including 6 single point mutations,double 2strains with site mutation.2.The genetic stability analysis of OsMIR394-1-2,OsMIR394-9-2 and OsMIR394-10-2 mutant lines was carried out.Further isolate and identify three vector-free homozygous mutant lines from the T1 generation,OsMIR394-1-2-5(-10bp/-10bp);OsMIR394-9-2-13(-22bp/-22bp);OsMIR394-9-2-17(+ 1,-9bp/+ 1,-9bp).3.The stable growth of the OsMIR394-1-2-5(-10 bp /-10bp)strain(the strain lacks5 bp at the mature miRNA)T2 generation field growth and T3 generation seed agronomic traits were measured.The results showed that the field growth and development of OsMIR394 knockout mutants were not significantly different from those of wild type,while the seed size,grain length,and grain width of OsMIR394 knockout mutants were significantly higher than those of wild type,and thethousand-grain weight was significantly increased compared with wild type.Scanning electron microscopy results on the surface of the grain showed that the length of the epidermal cells of the OsMIR394 knockout mutant was significantly higher than that of the wild type,which was consistent with the increase in seed length.4.The OsMIR394 knockout mutants were further tested for seed germination,seedling growth,and tolerance to abiotic stresses such as drought,salt,and copper ions.Compared with wild-type controls,the mutants had drought,There was no obvious difference under salt and copper ion stress conditions.5.RNA-Seq analysis of OsMIR394 knockout mutants.Transcriptome analysis showed that a total of 27 differential genes were detected in the OsMIR394 knockout mutants,26 were up-regulated genes,and 1 was down-regulated genes.At the same time,four down-regulated miRNAs were identified in the OsMIR394 knockout mutants.The above results lay the foundation for further understanding of the impact of OsMIR394 on gene expression at the genome-wide level.