OsMIR398 Targeted Mutagenesis And Mutants Identification

Rice,an annual gramineous herbaceous plant,is one of important grain crops and the gramineous pattern plants.In order to meet the global growing demand for food,the researchers are committed to creating new rice varieties with good characters through genetic improvement.Using genome editing technology to create rice mutants is an important methods for rice genome function research and germplasm innovation.MicroRNA(miRNA)is a kind of small non protein encoding RNA with 21-25 nucleotides,which plays the important roles in plant organ differentiation,tissue growth and stress response by post-transcriptional gene silencing.In the study of plant miRNA,miR398 was a member of plant miRNA which was found to have corresponding ability to oxidative stress signal.Based on the CRISPR-Cas9 technology,we constructs the single gene,double gene knockout vectors and over-expression vectors for rice miR398 family members OsMIR398 a and OsMIR398 b,and through Agrobacterium-mediated transformation in rice.Then selected and identified OsMIR398 knockout mutants and OsMIR398 over-expression transgenic materials,analyzed their genetic stability,stress phenotype,etc.,so asto provide reliable mutant materials and experimental data for indepth analysis of OsMIR398 biological function.The main resultsof this article are as follows:1.T0 generation one targeted knockout mutants and two targeted knockout mutants of OsMIR398 a and OsMIR398 b were obtained: test 29 regenerated rice individuals of OsMIR398 a one targeted knokout mutants(pTL03)and 26 of OsMIR398b's(pTL04).The results of PCR-SSCP and sequencing verification showed that all the tested plants had edited in target locations,and the efficiencies ofmutantions both were 100%;As for OsMIR398 a and OsMIR398 b double genes superimposed CRISPR-Cas9 knockout vectors(pZJP030,pZJP031)the mutation efficiency of OsMIR398a-sgRNA1 was 67.7%,and so do OsMIR398b-sgRNA1,and the efficiency of simultaneous mutation of both sites was 51.6%;the mutation efficiencies of OsMIR398a-sgRNA2 and OsMIR398b-sgRNA2 in pZJP031 were 60.0% and 60.0%,the efficiency of simultaneous mutation of both sites was 40.0%.2.Based on the obtained OsMIR398 a and OsMIR398 b double gene superposition knockout rice mutants,genetic screening were carried out on the T1 generation materials,228 T1 individuals were detected from the T0 generation simultaneous mutants,and OsMIR398 a and OsMIR398 b mutant genotypes could be steadily inherited.42 individuals without Cas9 carriers were identified,132 homozygous mutants were obtained at the OsMIR398a-sgRNA1 site and 118 homozygous mutants were obtained at the OsMIR398b-sgRNA1 site.At last,16 individuals with no CRIPR-Cas9 carriers and both homozygous in two sgRNA sites were identified.3.The OsMIR398 a and OsMIR398 b two targetedknockout mutants(without CRISPR-Cas9 carriers)and OsMIR398 a,OsMIR398b over-expressed T1 generation self-seeds,were tested for salt stress response.Compared with control materials,the OsMIR398 a and OsMIR398 b over-expressed transgenic plants showed salt resistance.The stress tolerance of OsMIR398 a and Os MIR398 b single gene and double gene knockout were significantly reduced,and the tolerability of double gene knockout material was more obvious.