基于CRISPR/Cas9技术的水稻OsASL1.1基因突变体创制及干旱胁迫下的表型分析OA

To investigate the expression characteristics of OsASL1.1 and its role in the drought stress re-sponse,the subcellular localization of OsASL1.1 protein and cis-acting elements in the promoter region of Os-ASL1.1 gene were analyzed.Additionally,Real-time Quantitative PCR(qRT-PCR)was used to examine the ex-pression patterns of OsASL1.1 in response to abscisic acid(ABA),mannitol,and polyethylene glycol 6000(PEG6000).CRISPR/Cas9 gene editing technology was employed to generate Cas9-OsASL1.1 mutants with Kitaake as background,and the mutation types were characterized.The abscisic acid(ABA)sensitivity and drought tolerance of mutants were analyzed.The results indicated that OsASL1.1 protein was primarily localized in plastids.The promoter region of OsASL1.1 contained cis-acting elements responsive to low temperatures and plant hormones(ABA and auxin),including five ABA responsive cis-acting elements(ABREs).OsASL1.1 ex-pression was induced by AB A,mannitol and PEG6000.Two homozygous mutant types,Cas9-6 and Cas9-12,were generated.Cas9-6 harbored a 17 bp insertion that caused premature translation termination,whereas Cas9-12 had a 12 bp deletion that resulted in the loss of four amino acids.Under ABA treatment,root elongation of Cas9-Os-ASL1.1 mutants and wild type were inhibited;however,the inhibition was less severe in the mutants than that in the wild type.Under 150 mmol/L mannitol treatment,the survival rates of both Cas9-6 and Cas9-12 mutants were significantly lower than that of wild type,with Cas9-6 showing the lowest survival rate.In summary,the Cas9-Os-ASL1.1 mutants show reduced sensitivity to ABA and decreased drought tolerance,indicating that the loss of Os-ASL1.1 function diminishes rice sensitivity to ABA and drought tolerance.