Functional Characterization Of The Abiotic Stress Response Mechanisms Of ThbHLH1 Transcript Factor From Tamarix Hispida

bHLH(basic/helix-loop-helix)protein family is the largest family of transcription factors in plants.bHLH proteins contain the structural features of highly conserved DNA binding domain consisting of 60 to 100 amino acids,which contain two functional different regions,i.e.,basic region and HLH region.Most plant bHLH transcription factor participate in variety of physiological processes,such growth,nutrient uptake,signal transduction,and other aspects of resistance to environmental stress response.In this study,a bHLH gene(ThbHLHl)that is response to salt and drought stress was cloned.Based on this study,the promoter region of ThbHLH1 with 2061 bp in length was cloned using TAIL-PCR.The promoter region of ThbHLH1 contains the cis-acting elements related with defense response,including DOF,Ibox and Wbox.The ThbHLH1 fused with GFP gene was construct and transformed into Arabidopsis,subcellular localization study showed that ThbHLH1 protein localized into the nucleus.The ThbHLH1 was transformed into Arabidopsis for overexpression.The two lines of the T3 generation of ThbHLHl transformed plants displayed significantly improved tolerance to salt,Mannitol and ABA.DAB,NBT,Evans blue,DCFH-DA and PI biochemical stanining,measurments of H2O2 content,SOD and POD activity,Electrolytic leakage and water loss rate,were performedto study the ThbHLH1 transformed and WT Arabidopsis plants in response to ABA,Manntiol,and salt treatments.A RNAi vector of ThbHLH1,pFGC5941-ThbHL1,was constructed.Three kinds of transgenic T.hispida plants were generated,including plants overexpressing ThbHLH1(transformed with pROK?-ThbHLH1)RNAi-silenced ThbHLH1 plants(transformed with pFGC5941-ThbHLH1),and control plants(transformed with empty pROK ?).DAB,NBT and Evas Blue biochemical staining suggested that among the three kinds of plants,plants overexpressing ThbHLH1 has lowest O2-and H2O2 content,and suffers lowest cell membrane damage;meanwhile,RNAi-silenced ThbHLH1 plants displayed highest level of O2-and H2O2 content and cell membrane damage.These results suggested that overexpression of ThbHLH1 significantly improved abiotic stress tolerance of plants.There are many DOF motifs in the promoter of ThbHLHl,and yeast one hybrid and ChIP analysis showed that a DOF protein from T.hispida,ThDofl6 can bind to the DOF motifs in the promoter of ThbHLH1,and is the up-stream regulator of ThbHLH1.