Heat Tolerance Comparison Between Cowpea Varieties And Transformation Of Tobacco And Cucumber With AtHSF1a Gene

High temperature is one of the most common environmental stresses,which affect the growth of plants and especially the productivity of crops.To study on the effect of high temper on the electrolytic leakage of membrane,the content of malondialdehyde(MDA)and the activities of antioxidant enzymes,we used 8 cowpea(Vigna unguiculata W.)wild type seedlings as material. Seedlings of heat-tolerant cowpea variety,zhijiang28-2,was used to study the inducible proteins among the leaf-cell proteins.We also constructed a eukaryotic expression vectors including overexpression ofAtHSF1a,and carried out tobacco and cucumber transformation.The main results were as follows:1.As time of heat-stress went by,heat index,relative conductivity and MDA content of cowpea increased.But the heat index,relative conductivity and MDA content of zhijiang28-2 were lower apparently than others.It showed that under high temperature stress,the damages to plasma membrane of heat-tolerant cowpea variety were not more serious than to heat-sensitive variety.2.Under high temperature stress,SOD,POD and CAT activities of the 8 varieties increased at first,then decreased.But the SOD,POD and CAT activities of zhijiang28-2 were higher than others.The result showed that under high temperature stress,the protective enzymes activities of heat-tolerant cowpea variety were higher.3.A high quality 2-DE profile was obtained.The proteins were separated very well in both dimensions.Employing PDQuest 8.0 software to analyze 2-DE gels,up to 500 protein spots were detected on the gel of zhijiang28-2 leaf total protein.Compared with the control gel,25 novel protein spots appeared,23 spots disappeared,34 spots were up-regulated and 24 spots were downregulated on the sample gel of 45℃5 h treatment.4.According to the sequence of the promoter fragment of heat shock factor AtHSF1a,two specific primers were designed.Target fragment AtHSF1a from Arabidopsis thaliana Columbia DNA was amplified by PCR with PrimStar HS DNA polymerase.The PCR products were purified and cloned into pMD18-T vector then pMD-AtHSF1a was constructed.By white-blue screening, colony PCR and enzyme digest analysis,the result showed the plasmid containing AtHSF1a fragment was transferred into E.coli XL-Blue.The sequencing of the single recombinant containing the inserted AtHSF1a fragment was carried out by Sangon Bio-engineering CO.Ltd. The result showed that the fragment was 1676bp,the same as the sequence of AtHSF1a from Arabidopsis thaliana.5.Plasmid pMD-AtHSF1a and Plasmid pV-HSP70bGUS were double-digested by Xba I+Sac I.The target fragment were collected,purified and ligated,then plant expression vectors pV-70bAtHSF1a was constructed.6.The leaves of tobacco and Agrobacterium of EHA105/pV-70bAtHSF1a were cultured for 2 days in dark.Through culture induction and resistance selection,callus and adventitious bud induction grew.The adventitious bud induction cultured on rooting culture(MS+2.5 mg/L 6-BA +0.2 mg/L NAA+150 mg/L Kan.+300 mg/L Cb+3%sucrose+0.7%agar pH5.8).Then we got the Kan.-resistant plants of transgenic tobacco.Assayed by PCR amplification,the targeted bands with 1676bp was observed respectively form DNA of regenerate plants.Through analyze the relative conductivity,MDA content and SOD activities of transgenic tobacco,we found the No 5 has better heat tolerance.7.The leaves of cucumber were inoculated with Agrobacterium of EHA105/pV-70bAtHSF1a for 8～10 minutes.Through culture induction for 2 days in dark,postculture for one week on culture medium,and transforming to screening-culture medium with 80mol/L Kan.,we get resistant callus and bud.With fresh medium changed every two weeks,resistant bud was transformed into rooting culture medium.Assayed by PCR amplification,the targeted bands with 1676bp was inspected respectively form DNA of regenerate plants.