Study On Salt Tolerance Of ThGRAS Transcription Factor From Tamarix Hispida

GRAS is a family of transcription factors only discovered in higher plant.The origin of its name comes from the GAI(gibberellicacid insensitive)?RGA(repressor of GA1-3 mutant)and SCR(scarecrow).The GRAS family was usually 400?770 amino acids.Up to date,it has been identified 33 GRAS family genes in Arabidopsis thaliana,such as d8,Rht,PAT1,SCL13 and SCL14.However,few researches on function of GRAS transcription factors in woody plants were done.20 ThGRAS genes were identified from the Tamarix hispida transcriptome libraries.Some GRAS transcription factors expression patterns were obtained from seven T.hispida libraries.The results showed that one of the GRAS transcription level was obviously upregulated by salt stress.The expression level was 62.41 fold in leaf after NaCl stress during 12 h.So the gene was selected for the further study and named as ThGRAS1.The ThGRAS1 gene was fused with GFP protein.The subcellular localization in onion epidermal cells was study by using the particle gun technology.The result showed that the ThGRAS1 gene was location in the nucleus.Furthermore,overexpression ThGRASl vector 35s::ThGRAS1 was constructed and transfer into Arabidopsis Thaliana by agrobacterium mediated dipping.The results of qRT-PCR of transgnic lines were showed the ThGRASl were obvious expression in these lines.Then T3 transgenic seedling were selected furthern analyzed.The results of germination rate and growth test showed that the germination rate and biomass accumulation in overexpression ThGRAS1 Arabidopsis lines were obvious higher than the WT after salt stress.The results of histochemical staining showed that overexpression ThGRAS1 Arabidopsis accumulated less reactive oxygen species than wild type under salt stress.And the cell damage is lighter too.The results of physiological index also shows that no difference in MDA content between the transgenic lines and WT at the normal growth condition.But after NaCl stress,the transgenic lines showed lower MDA accumulation,relative conductivity and H2O2 content than its in WT.These results suggested that ThGRAS1 obviously enhanced the ability of salt tolerance in Arabidopsis Thaliana.To confirm the result of ThGRAS1 transcription factors expression in Arabidopsis thaliana,35s::ThGRASl,inhibition expression vevtor,empty vector were transient transfer into Tamarix.The related indexes on these lines under NaCl stress were analyzed.The result is similar to of its in the transgenic Arabidopsis.Suggesting that overexpression of ThGRASl can effectively improve the ability of the salt tolerance in transgenic Tamarix.And it verified the results of heterologous overexpression ThGRAS1 in Arabidopsis at some degree.To analyze the upstream mechanism of ThGRAS1 transcription factor gene.The 1180 bp promoter of ThGRAS1 were cloned by using the genomic walking.The expression vector pBI121-ThGRAS1-GUS were constructed and transfer into the Arabidopsis by flower dipping method.The T4 Arabdopsis in different growth period and different tissue parts were used to GUS staining.The results showed that ThGRAS1 promoter were mainly expressed in mature leaves,root and immature pods.The 1180 bp ThGRASl promoter sequences were analyzed by the PLACE and Plantcare sofeware.The result showed that many elemets were found in the ThGRAS1 promoter,such as CACTFTPPCA1,CAATBOX1,ABRELATERD1,ARR1AT,ASF1MOTIFCAMV,DOFCOREZM,GT1CONSENSUS,MYBCOREATCYCB1,ROOTMOTIFTAPOX1.These cis elements were acted in plants stress resistence.Furthermore,the DOFand NOD cis-elemets were selected study.The results of yeast one hybird showed that ThDofl9 and ThNod2 can specific recognition the DOF,NOD elements in ThGRASl gene promoter,suggested that ThDofl9,ThNod2 may participate in regulating or jointly control plant salt tolerance with ThGRASl.