Cloning And Funtional Characterization Of Dehydration-induced Transcription Factor Gene MfbHLH145 In Myrothamnus Flabellifolia

The woody plant Myrothamnus flabellifolia Welw indigenous to South Africa,which is an important genetic resource need to be excavated.Based on the previous transcriptomic study of Myrothamnus flabellifolia Welw,we cloned a bHLH transcription factor gene MfbHLH145 induced by dehydration,then transformed it into the model plant Arabidopsis thaliana Heynh to know its function.We have get these results through our experiments:1.Cloning and sequence analysis of MfbHLH145 gene fromMyrothamnus flabellifoliaIn this experiment,we extracted the total RNA of Myrothamnus flabellifolia,and designed the primers according to the UniGene with the purpose of amplified the MfbHLH145 gene.The full-length nucleotide sequence of MfbHLH145 gene was determined to be 1020 bp.The amino acid sequence of MfbHLH145 gene was analyzed.The nuclear localization signal sequence PSKKRKL and the typical HLH domain were found and identified it as a bHLH family transcription factor.2.Subcellular localization of MfbHLH145Construct the 2*35S:MfbHLH145:p HB-YFP fluorescent fusion protein and infect A.nicotianae with Agrobacterium.Observing the transient expression of the fusion protein in tobacco leaves to determine the location of MfbHLH145 in the nucleus after dark treatment by laser scanning confocal microscopy,the function of the MfbHLH145 gene can be inherited to the next generation through basic biological functions of cells,and the function of MfbHLH145 can be expressed normally.3.Screening and phenotype identification of transgenic lines of Arabidopsis thaliana HeynhThe 35S:MfbHLH145:pGSA-1403 eukaryotic expression vector was constructed and transformed into model Arabidopsis thaliana Heynh with the method of Agrobacterium transfection.After screening,a pure lineE and lineF of MfbHLH145 gene of T₃ generation were obtained.In this experiment,we used mannitol with 200mmol/L,250mmol/L and300mmol/L to simulate drought stress,100mmol/L and 150mmol/L Na Cl to simulated salt stress on the seedlings to observe the phenotypes of the over-expressed MfbHLH145homozygote plants.In the drought resistance experiment,lineE and lineF showed langer roots than WT in seedling.The number of roots in the seedlings was much more.In the adult plant tests,the homozygote plants can stay longer after hydration,and they were easier to recover after rehydration than WT.In salt stress resistance experiment,lineE and lineF showed a great number of roots and longer roots compared with WT in the seedling experiment.The adult plants experiment showed a longer salt tolerance time and stronger green holding ability.It shows that lineE and lineF are more resistant to drought and salt than WT.4.Determination of physiological indexes of transgenic Arabidopsis thaliana plantsThe water loss rate of leaves,prolineaccumulation and chlorophyll content of Arabidopsis thaliana plants over-expressing MfbHLH145 gene under natural drought conditions were measured.The prolineaccumulation under 300 mM Na Cl stress and the chlorophyll content with 200 mM Na Cl treatment was measured.The results showed that the natural water loss rate of lineE and lineF is lower than that of WT.The prolineaccumulation of lineE and lineF is higher than that of WT under drought and 300 mM Na Cl treatment,the chlorophyll content of lineE and lineF was higher than that of WT with200 mM Na Cl treatment.5.Analysis of the expression in Arabidopsis resistance-related gene in transgenic plantsThe genes related to drought and salt resistance pathways were selected as AtNCED3、AtP5CS5、AtRAB18、AtRD29A、AtRD22,and the RNA and c DNA were extracted from the transgenic lines and WT plants.RT-qPCR was used to compare the relative gene expression of the transgenic plants and WT at the same time points under different treatment conditions.Amounts were measured and it was found that under natural drought and sodium chloride stress,there were differences in the expression levels of some genes in WT and transgenic lines.It is inferred that lineE and lineF have higher resistance to drought and salt stress than WT.