Development And Analysis Of Os-miR827/1850/3982 Gene Mutants Based On CRISPR-Cas12a In Nipponbare

MicroRNA(miRNA)is a kind of non-coding endogenous small molecule RNA with a length of 19-24 nt in eukaryotes.It regulates the expression level of its target gene mRNA mainly by means of shear degradation,inhibition of translation,and chromatin remodeling.Plant miRNAs play a crucial role when plant growth and development,epigenetics,and response to biological and abiotic stresses.Os-miR827,os-miR1850 and os-miR3982 were obtained by high-throughput sequencing in 2008,2008 and 2011 respectively,and their functions have not been clearly studied.The creation of miRNA mutants is an important and effective way to study their biological functions.In this study,CRISPR-Cas12 a system was used to construct three targeted editing vectors,pCHQ21,pCHQ22 and pCHQ23,for os-miR827,os-miR1850 and os-miR3982,respectively.Agrobacterium tumefaciens was used to mediate the transformation of rice varieties,which of T0 generation were tested for miRNA gene mutants,and stable rice mutant materials with homozygous miRNA gene target mutations were further screened in T1 generation.Salt stress experiments were carried out on osmiR827 and os-miR1850 mutants.The main research results are as follows:1.Three targeted editing vectors pCHQ21,pCHQ22 and pCHQ23 were constructed for rice os-miR827,os-miR1850 and os-miR3982 genes.Through agrobacteriummediated genetic transformation,the transgenic positive rice plants of pCHQ21,pCHQ22 and pCHQ23 were obtained as 11,20 and 10 strains,respectively.The positive rates were all 100%,which proved the high efficiency of rice transformation system.2.SSCP and Sanger sequencing were used to detect the genes of 11 pCHQ21 plants,20 pCHQ22 plants and 10 pCHQ23 plants in the T0 generation.The results showed that the mutation rates of pCHQ21 and pCHQ23 were 100% and 95.2%,respectively.The sequencing results showed that pCHQ21-01,02,03 and 07 were all double allele mutations.The pCHQ22 sequencing showed that pCHQ22-01,02,04 and 21 were double allele mutations and pCHQ22-03,08,10 and 16 were single allele mutations.The pCHQ23 sequencing showed that pCHQ23-01,03,05 and 06 were all double allele mutations.3.Genetic transfer analysis were carried out on pCHQ21-01,pCHQ21-07,pCHQ21-08,pCHQ22-03,pCHQ22-08,pCHQ23-01 and pCHQ23-02 strains,and the genotype separation ratio was 1:2:1,which was in line with Mendelian law of genetic separation,indicating that the mutant could inherit stably.4.In the salt stress experiment,the expression of os-miR827 mutant was consistent with that of the wild type.The os-miR1850 mutant showed tolerance to salt stress.During the salt treatment of pCHQ22-3-1 seedlings,it was observed that on the third day of treatment,part of the tip of the second leaf of the wild type was locally curled and turned yellow,and the first leaflet was completely curled into a tube.However,the tip of the second leaf is relatively normal,and the first leaflet does not appear curly.At the sixth day of treatment,the whole wild type plant was completely curled and yellowish.The mutant was more erect and less curly.On the third day and the sixth day,leaf length and root length of wild type and mutant plants were measured,and statistical results showed that there was no significant difference in root length between the two plants,while the difference in leaf length was significant.