Response Analysis Of Rice OsMIR1850 Under Salt Stress

Soil salinization is becoming more and more serious all over the world,which threatens the production safety of main crops including rice.It is of great significance for improving rice stress tolerance to excavate the important genes involved in salt stress response and study the internal mechanism of their participation in salt tolerance response,so as to maintain rice food security.However,at present,the knowledge about salt stress signal transduction is mainly obtained from the model species Arabidopsis thaliana,and focuses on the coding genes.There are few studies on the involvement of miRNA in the regulation of salt tolerance in crops.As a key component of the gene regulatory network,miRNA also plays an important role in plant response to stress.OsMIR1850 was identified in developing seeds.Later,a small amount studies found that OsMIR1850 expression levels changed under drought,high temperature and cold conditions,suggesting that OsMIR1850 was responsive to abiotic stress in rice.In this study,we identified different genotypes of OsMIR1850 knockout mutants created by CRISPR-Cas12 a technology,and investigated the important agronomic characters and quality characters;abiotic stress analysis of salt;physiological index of the 22 bp deficient material of OsMIR1850 mutant under salt stress;RNA sequencing and analysis of OsMIR1850-1 mutant;quantitative PCR analysis of related genes under salt stress.In order to clarify the response mode of OsMIR1850 under salt stress.The main results of this paper are as follows:1.Based on the investigation of important agronomic characters and quality of OsMIR1850 knockout mutant materials,it was found that there were no significant differences in grain number per panicle,seed setting rate,1000-grain weight,grain length,grain width,ratio of length to width,chalky grain rate and starch structure between the mutant materials and the wild type of Nipponia.However,the seed amylose content of the mutant was significantly higher than that of the wild type.2.The OsMIR1850 knockout mutant was treated with salt stress at seedling stage.It was found that the second leaf of wild type seedlings curled more than the mutant after three days of 100 m M Na Cl stress.After 15 days of stress,seedlings were carried out normally,and only the mutant could restore growth.OsMIR1850 mutants showed better salt tolerance.3.The physiological indexes of OsMIR1850-1 and wild type of Nipponia were analyzed under salt stress.It was found that under normal conditions,compared with the wild type,OsMIR1850-1 showed higher chlorophyll content and lower MDA content,but there was no difference in proline content between the two materials.After 3 days of salt stress treatment,the chlorophyll content of OsMIR1850-1 was still significantly higher than that of the wild type.The content of MDA increased in both materials,but the content in salt-treated OsMIR1850-1 was lower than that in salt-treated wild type.The content of proline increased in both materials,but there was no difference between mutant and wild type.4.RNA-seq analysis of OsMIR1850-1 mutant showed that there were 723 upregulated and 350 down-regulated genes in the mutant compared with the wild type.GO enrichment analysis of differentially expressed genes showed that 5 positive regulatory genes in salt stress response were up-regulated and 9 genes about abscisic acid response were differentially expressed.Among them,LOC＿Os01g66120 not only as a positively regulator of salt stress response,but also as a predicted target gene up regulates in OsMIR1850-1.KEGG analysis of differential genes showed that there were 35 differential genes in plant hormone signal transduction pathway.Small RNA sequencing showed that the knockout of OsMIR1850 caused the down-regulation of 6 miRNAs and the up-regulation of 5 miRNAs.6.Quantitative PCR detection of some differentially expressed genes in OsMIR1850-1 mutant materials under salt stress showed that salt stress response positive regulatory genes LOC＿Os01g66120(Os NAC6)and LOC＿Os03g60080(Os NAC9),and ABA response genes LOC＿Os01g58420(Os ERF3)and LOC＿Os01g61080(Os WRKY24)were significantly responsive to salt stress in normal 21-day-old seedlings and in the aboveground parts of seedlings treated with salt for 3 days.