The Gene Fuction Research Of PoptrHSFA4a

In plants,heat shock transcription factors(HSFs)play key roles in regulating the heat shock(HS)response.Therefore,the genes encoding HSFs are important for adaptation to high temperatures and for tolerance to other abiotic stresses,for instance,Arabidopsis HSFA2 as a key regulator in response to several types of environmental stress,orthologs of the Class A4 Heat Shock Transcription Factor HSFA4a Confer Cadmium Tolerance in Wheat and Rice,and so on.There have been many studies on these genes in herbaceous plants,but few on the HSFs family in woody plants.In this study,we did some researeh about PoptrHSFs and the result are as follows:We identified 31 HSF genes in Populus trichocarpa and investigated their phylogenetic relationships with HSF genes in Arabidopsis and rice.We analysed chromosomal duplications and gene structure.Changes in gene expression were investigated using exPlot and digital northern analyses and nine of the HSF genes showed significant variations in expression patterns.We evaluated changes in the transcript levels of the HSFs in response to abiotic stresses(heat,cadmium,salt,abscisic acid,and drought stresses)by analyzing log2 fold change data.The transcript levels of the PoptrHSFA4a,PoptrHSFA4b and PoptrHSFA5a genes markedly increased in response to a wide range of stresses.This study served to isolate a PoptrHSFA4a gene from Populus trichocarpa cDNA library.The length of the HSFA4a open reading frame(ORF)was 1221 bp.Bioinformatics analysis of PoptrHSFA4a showed the basic structure that contains the structure of Class A.PlantCARE database prediction analysis suggested that there are many cis-elements which associated with stress resistance of plants in the promoter of PoptrHSFA4a,such as MBS,GARE-motif,GA-element,ABRE,CGTCA-motif and TGACG-motif.The recombinant PoptrHSFA4a-GFP fused gene was transformed into onion epidermal cells by particle gun for subcellular location analysis.The results showed that PoptrHSFA4a is located in nucleus and cytoplasm of cell.We successfully constructed two kinds of expression vectors,that is overexpressing vector which were drived by 35S promoter and RNA interferred PoptrHSFA4a vector,the plant expression vector of pBI121-PoptrHSFA4a was transformed into Arabidopsis thaliana via floral dip protocols.The Transformed lines were examined by PCR confirming that PoptrHSFA4a gene have been transformed into Arabidopsis thaliana and expressed on the level of transcription.In the heavy metal and heat stress test,we did the stress resistance analysis,histochemical stain analysis and physiological index analysis between the transgenic Arabidopsis and control.The T3 transgenic Arabidopsis overexpressing PoptrHSFA4a showed considerable stress tolerance under abiotic stress of heat and Zinc,by comparing with wild type.We speculate that this gene has a very important function to the stress,may enhance the resistant to high temperature and Zn stress.After Zn stress,the control and the transgenic were stained with Zinpyr for quantifying zinc.The result exhibited that the content of zinc in the protoplasts of transgenic was significant less than the control and mainly accumulated on the outside of the membrane.The zinc accumulation in the control and the transgenic indicated that the zinc in the transgenic rarely enter the cell and primarily concentrated in the cell gap.It was demonstrating that PoptrHSFA4a probably response to zinc stress in Populus trichocarpa by regulating downstream zinc transporter that localized on membrane.At last,To prepare for the future research,the transgenic Populus ussuriensis overexpressing PoptrHSFA4a and the transgenic Populus ussuriensis of PoptrHSFA4a suppress expression had been abtained.