Functional Characterization Of Y₂SK₂ And SK₃ Type Dehydrins From Agapanthus Praecox Under Abiotic Stresses

Dehydrins are involved in plants'adaptation to water-deficit stress.Two dehydrins from Agapanthus praecox?ApY₂SK₂ and ApSK₃?showed important protective effect under complex stresses.The full-length cDNA and coding protein sequence of ApY₂SK₂ and ApSK₃ gene have been obtained in previous research.This research provided evidence on the specific in vivo and in vitro protection function of ApY₂SK₂ and ApSK₃ protein under various stresses,The wild-type,ApY₂SK₂ and ApSK₃ gene overexpressed transgenic Arabidopsis thaliana plants were treated with different stresses.Phenotype analysis showed that transgenic A.thaliana plants had better stress resistance,larger rosette size,fresh weight and root length under stresses.Physiological detection showed ApY₂SK₂ and ApSK₃ overexpressed plants had lower plasma membrane damage and ROS level,higher antioxidant activity,proline accumulation and photosynthesis capability under salt,osmotic,cold and drought stresses compared with the wild-type.ApY₂SK₂ and ApSK₃ can improve the survival of Escherichia coli under various stress.Plants and E.coli overexpressing ApY₂SK₂ showed better improvement on stress tolerance.ApY₂SK₂ and ApSK₃mainly located in the cytoplasm and cell membrane,ApY₂SK₂ even can locate at the nucleus.The optimum induction conditions for prokaryotic expression of recombination ApY₂SK₂-His/ApSK₃-His proteins in E.coli Transetta?DE3?cells were induced by 1.0⁰.5 mM/0.2⁰.5 mM isopropyl b-D-thiogalactopyranoside at 25°C/35°C for 5⁶ h,respectively.Recombination proteins were purified by nickel affinity chromatography and the boiling bacterial pellets method,revealed the boiling stability of the ApY₂SK₂ and ApSK₃ proteins,provided a foundation for simplification of the dehydrin purification and further in vitro protein function test.In vitro tests indicated that ApY₂SK₂ and ApSK₃ can effectively protect the enzyme activity during the freeze-thaw process,and ApY₂SK₂ also has this function during desiccation treatment.Furthermore,ApY₂SK₂ and ApSK₃ can significantly inhibit hydroxyl radical generation.These two dehydrins can bind metal ions with the binding affinity as Co²⁺>Ni²⁺>Cu²⁺>Fe³⁺,and ApSK₃ binding affinity was higher than ApY₂SK₂.Thus,ApY₂SK₂ has better protective effect on enzyme activity,and ApSK₃ has stronger metal ions binding function and effect on ROS metabolism.Moreover,plant cryopreservation evaluation test indicated that overexpressing ApY₂SK₂ and ApSK₃ gene can enhance the seedling survival ratio from 23%to 47%and 55%respectively;adding recombinant ApY₂SK₂ and ApSK₃ protein to plant vitrification solution may increase the survival ratio of wild-type A.thaliana seedlings from 24%to 50%and 46%.This study suggested that ApY₂SK₂ and ApSK₃ can effectively improve the cell stress tolerance,and have great potential for in vivo or in vitro application.