Functional Analysis Of OsMIR1423 In Regulating Rice Drought Stress Response

Rice is an important food crop in the world and one of the most important food crops in China.In terms of production,rice is the most constrained by water and soil resources,and drought has become the main factor restricting the continuous increase of rice yield in China.Therefore,improving drought resistance of rice has become one of the key problems in rice breeding.The study of abiotic stress resistance of crops is one of the most challenging tasks in the field of plant research.It is of great significance to explore the important genes involved in drought stress response of rice,and further clarify the molecular mechanism of drought response of rice,so as to improve drought tolerance of rice and cultivate new varieties of rice for drought resistance.Current studies on rice drought stress response mainly focus on related protein-coding genes,such as transcription factors and their regulatory mechanisms.There are few studies on how miRNA is involved in the regulation of drought stress in rice.A large number of previous studies have shown that miRNA,as a special class of RNA molecules,plays a key role in plant response to biological and abiotic stresses.Existing literatures and databases suggest that OsMIR1423 may respond to abiotic stresses such as drought and high salinity,and may also be related to plant disease resistance.In this study,we constructed a targeted knockout mutant vector of OsMIR1423 gene based on CRISPR-Cas12 A genome editing system,and successfully obtained regenerated mutant plants through agrobacterium-mediated genetic transformation of rice.Then,three negative homozygous mutant lines with different genotypes were identified and screened,and their agronomic traits,stress analysis and transcriptomic sequencing analysis were carried out to preliminarily explore the function of OsMIR1423 in drought stress response,providing a research basis for in-depth analysis of its molecular mechanism.The main results of this study are as follows:1.A targeted knockout vector p LQ01 based on CRISPR-Cas12 A was designed and constructed for OsMIR1423.Seventeen regenerated plants were obtained by agrobacterium-mediated genetic transformation,and 14 osmir1423 mutant plants were further identified and screened.Three different genotypes were selected through genotype identification for homozygous mutant screening of T1 and subsequent generations,and the mutant material with OsMIR1423 function loss was successfully created.2.The important agronomic and quality traits of osmir1423 knockout mutant were investigated.The results showed that the number of grains per panicle,seed-setting rate and 1000-grain weight of osmir1423 mutant were not significantly different from those of Nipponclear wild type.There was no significant difference in grain length.Both grain width and grain thickness were significantly lower than those of wild type.At the same time,the chalky grain rate of osmir1423 mutant was significantly increased,and the starch grain structure was significantly different from that of the wild type.The amylose content of osmir1423 mutant was significantly increased,but the total starch content remained unchanged.3.The seedlings of osmir1423 mutant were treated with drought stress at seedling stage,and the results showed that the seedlings of osmir1423 mutant under drought stress for 8 days had better drought tolerance than those of The wild type.After rewatering,the speed and condition of the mutant’s recovery growth were significantly better than that of the wild type.The ratio of chlorophyll A/chlorophyll B content of osmir1423 mutant and wild type under drought stress was detected.The results showed that the percentage of chlorophyll A and chlorophyll B content decreased significantly under drought stress than that of wild type,and the ratio of chlorophyll A/chlorophyll B content was higher than that of wild type,showing better drought resistance.4.RNA sequencing analysis of osmir1423 mutant showed that 56 genes were up-regulated and 10 genes were down-regulated in osmir1423 mutant compared with Nippon wild type.GO enrichment of different-expressed genes revealed that four genes related to rice drought resistance were up-regulated,namely LOC＿Os10g39170,LOC＿Os02g47810,LOC＿Os02g50040 and LOC＿Os01g23239.In addition,3 genes related to biological or abiotic stress response were up-regulated and 5 genes were down-regulated.This provides a reference for the follow-up study on the stress response mechanism of OsMIR1423.