The Study on Arabidopsis Thaliana Heat Shock Factor HSFA 1d Response to Formaldehyde Stress
|School||Kunming University of Science and Technology|
|Course||Biochemistry and Molecular Biology|
|Keywords||Formaldehyde contamination Heat shock factors HSFAld Excessive expression Formaldehyde resistance|
At present, people had paid close attention to formaldehyde pollution which was harm to environment and human health. The research showed plant can repair formaldehyde pollution, but few researches focused on the genes responsible to formaldehyde tolerance in plant. Based on the results of our laboratory, AtHsfA1d was found to be a transcription factor responsible to formaldehyde stress in Arabidopsis thaliana. The expression analysis of Arabidopsis AtHsfA1d under formaldehyde stress indicates that the expression of AtHsfA1d increased by extending time. We thought AtHsfAld may participate in formaldehyde stress. Therefore, the experiment chose AtHsfAld and investigated formaldehyde stress response of AtHsfA1d. The main results were as follows.First, mRNA expression analysis of AtHsfA1d under 4mM Formaldehyde, formic acid and methanol respectively showed AtHsfA1d was up-regulated by formaldehyde stress.Second, prepare for specific antibody of AtHsfA1d for subsequent experiment. RNA was extracted from Arabidopsis, then amplified it to obtainΔC-AtHsfA1d fragment (specific fragment of AtHsfA1d) by RT-PCR.ΔC-AtHsfA1d gene was subcloned into the prokaryotic expression vector pET32a(+) to construct pET32a(+)-ΔC-AtHsfA1d recombinant plasmid by TA-clone, restriction enzyme digestion and sequence, then recombinant plasmid was introduced into E.coli BL21 to induce the target protein expression. The optimal conditions for expression of target protein were 0.5 mM IPTG, 28℃,8 h which were determined by adjusting the IPTG concentration, induction temperature and induction time, and SDS-PAGE showed the expressed recombinant proteins formed solubility.ΔC-AtHsfA1d recombinant protein was obtained by affinity purification and gel purification method and concentration was 1mg/mL and total content was 2mg, the pure proteins were used as antigens to produce antibody. Immune results showed that protein serum titer achieve 10-5.Third, in order to identify expression and function analysis of AtHsfA1d in saccharomyces cerivisiae under formaldehyde stress. We constructed yeast expression vector pYES3-AtHsfA1d, which was introduced into saccharomyces cerivisiae by electrotransformation and identification of pYES3-AtHsfA1d by colony PCR. Comparison growth condition of transgenic saccharomyces cerivisiae with that of wild type under formaldehyde stress(0 mM, 1mM,1.5 mM,2 mM,4 mM,6 mM), the result showed transgenic tobacco had higher survival rate than Original yeast strains. Original yeast strains grew in the initial concentration (OD6oo=2.0) and the rest had no growth especially in 4mM formaldehyde stress, but transgenic strains growth is good. This showed AtHsfA1d can increase tolerance of saccharomyces cerivisiae to formaldehyde stress.Fourth, to investigate the influence of the formaldehyde tolerance of AtHsfAld in tobacco.We used gateway technology to get pK2GW7-AtHsfA1d recombinant vector that introduced into Agrobacterium tumefaciens pPMP90 by electrotransformation. At last pK2GW7-AtHsfA1d was introduced into tobacco leaves, then transferred them to MS4 nutrient medium after co-culture, to get calli and buds with resistance of tobacco. Buds were tranfered to MS nutrient medium to get transgenic tobaccoes. Transgenic tobaccoes were identified on genome, transcriptional level and protein expression level. PCR analysis showed genome and cDNA of transgenic tobaccoes with AtHsfA1d had a size of 1458bp amplified band, which was consistent with the positive control plasmid. Protein expression showed transgenic tobaccoes were used as antigen, and recognized antibody each other. These showed AtHsfA1d gene had been integrated into the tobacco genome.Then we investigated formaldehyde absorption rate of transgenic tobacco and wild type under formaldehyde treatment (4 mM), and fresh weight and total protein of transgenic tobacco and wild type under formaldehyde treatment (6 mM). The result showed transgenic tobacco had higher absorption rate than wild type under formaldehyde treatment (4 mM), and the fresh weight increased from transgenic tobacco was about twice than the wild type, physiological analysis under 6 mM formaldehyde also showed total protein content of transgenic tobacco increased. These results showed arabidopsis heat shock factor AtHsfAld improve tobacco formaldehyde tolerance.