Preliminary Study On The Role Of Lilium Pumilum LpbHLH115 In Saline-alkali Response

In the natural environment,plants are constantly threatened by various biotic and abiotic stresses.drought and salt stress conditions hinder plant growth and reduce crop productivity.bHLH gene family is involved in various biological processes in plants,and many of bHLH functions have been revealed,including response to iron ion uptake,salt stress and drought stress,and petal growth.In this study,the open reading frame sequence of the LpbHLH115gene was cloned using bulb cDNA as a template and bioinformatics analysis was performed.The expression of the gene was analysed using real-time fluorescence quantitative PCR.Yeast resistance analysis.The LpbHLH115 gene was successfully infiltrated into tobacco and lilium pumilum by Agrobacterium mediated method and comparative resistance analysis experiments were carried out on transgenic tobacco with LpbHLH115.The LpbHLH115 protein was successfully purified and the LpbHLH115 transcription factor was verified to bind to the LpFRO7 promoter region by yeast single hybridization,bimolecular luciferase,and EMSA experiments.Specific findings were as follows:1.The open reading frame sequence of the LpbHLH115 gene was successfully cloned from the bulb of lilium pumilum and the sequenced,LpbHLH115 gene was 675 bp in length and encoded 225 amino acids.Conservative structural domain analysis showed that the LpbHLH115 gene belongs to the bHLH family.2.Fluorescence quantitative PCR results showed that the LpbHLH115 gene was relatively highly expressed in leaves,LpbHLH115 gene expression was maximized at 12 h after Na₂CO₃stress,and LpbHLH115 gene expression was maximized at 24 h after H₂O₂,NaCl,and NaHCO₃stress.3.The LpbHLH115-pYES2 vector was successfully constructed,transferred into the yeast strain INVSc I,and the yeast broth was diluted and spotted on the medium with added stress,and the results showed that the yeast containing the recombinant gene all grew better than the control on the medium containing H₂O₂,NaCl,Na₂CO₃and NaHCO₃.4.pCAMBIA1300-LpbHLH115 plant expression vector was constructed,and the recombinant plasmids were transferred into tobacco and lilium pumilum by Agrobacterium infestation for resistance screening,and tobacco and lilium pumilum plants overexpressing the LpbHLH115 gene were successfully obtained.The results showed that the LpbHLH115 transgeninc lines were more resistant to stress than the wild type at the germination and seedling stages under saline and oxidative stress.5.The LpbHLH115 transcription factor was verified to bind to the LpFRO7 promoter region by yeast single hybridization,bimolecular luciferin,and EMSA experiments.