基于CRISPR/Cas9介导抗旱耐盐PagHyPRP1基因编辑杨树获得及抗性评价1

[Objective]Due to the adverse ecological environment in arid,semi-arid and saline-alkali areas,afforestation and vegetation restoration are difficult.It is urgent to create new varieties of stress-resistant trees to realize the cultivation and utilization of difficult sites.[Method]The CRISPR/Cas9 system was used for multilocus genome editing of PagHyPRP1(including alleles PagHyPRP1A and PagHyPRP1B),a negative regulator of salt and drought stress response in the important cultivated varieties Populus alba×P.glandulosa(84K poplar).PCR and single gram sequencing were used to analyze the editing type of editing targets.Oxidative damage level,antioxidant and osmoregulation capacity were measured in leaves to evaluate the tolerance to drought and salt stress of PagHyPRP1mutants.Key ion flux and related genes expression in roots were measured and analysed to investigate the ROS signalling alteration of the mutant in response to drought and salt stress.[Result]9 mutant plants with 5 genotypes were obtained with 60%mutation efficiency,including two homozygous,two allelic,and five chimeric mutant lines.1 bp insertion or 1-4 bp deletions that led to frameshift mutations were detected in mutants.The mutation type of four mutants(allelic mutant:prp-1;homozygous mutant:prp-2,prp-4;chimeric mutant:prp-6)with 100%mutation is stably,without the occurrence new mutations through asexual propagation,which were selected for drought and salt tolerance evaluation.Compared with 84K,the expression level of PagHyPRP1 in mutants was significantly lower under normal growth conditions,salt and drought stress,which was opposite in OEs,suggesting that PagHyPRP1 play the negative regulatory rol in poplar under drought and salt alkali stress.Under salt and drought stresses,the paghyprp1 mutant lines showed significantly increased plant height,stem diameter,stem weight,root dry weight and root to shoot ratio than the wild-type 84K poplar,with significantly enhanced salt and drought tolerance.In addition,the paghyprp1 mutant lines exhibited higher superoxide dismutase and peroxidase activities and proline content;lower accumulation of hydrogen peroxide,superoxide anions,and reactive oxygen species;higher Na+ efflux(under salt stress),Ca2+ influx(under drought stress)and H+ influx;and less K+ efflux,while the overexpression lines showed the opposite results.All above indicate that the paghyprp1 mutant lines had improved drought and salt tolerance by reducing ROS accumulation,promoting root growth and maintaining root ion homeostasis.[Conclusion]PagHyPRP1 was confirmed as a negative regulator of the salt and drought stress response in poplar,and the paghyprp1 mutant lines had improved drought and salt tolerance by reducing ROS accumulation,promoting root growth and maintaining root ion homeostasis.This study provides candidate genes with important breeding value for forest tree stress resistance gene editing and provides empirical evidence and technical reference for the application of CRISPR/Cas9 system to create new germplasm for breakthrough stress resistance in forest trees.