Effects of Different Stress Treatments on the Molecular Characteristics of Arabidopsis Thaliana Transgenic Plants Overexpressing Heat Shock Fackor HsfA1a
|Keywords||Arabidopsis thaliana Heat shock factor Heat shock protein Semi-quantitative RT-PCR Western analysis|
Arabidopsis thaliana heat shock factor HsfAla is an important regulatory factor in thermotolerance, but also in tolerance to acid, alkali and H2O2. However, the mechanisms of its stress tolerance are largely unknown. In this thesis, HsfAla overexpression transgenic Arabidopsis plants (OE) were used as study material. On one hand, I used Western analysis to assay the expression levels of heat shock protein Hsp70. The aim is to examine whether HsfAla can be activated in plants by various stresses and play role in the regulation of stress-induced Hsp gene expression.The main results in this study are as follows:(1) Total RNA was extracted from Arabidopsis seedlings priorly stressed by heat, acid, alkali and H2O2for long period, respectively. The mRNA expression levels of heat shock factor HsfAla and heat shock proteins, Hsp70and Hsp18.2were detected by semi-quantitative RT-PCR method. It was found that all stress treatments for long period (more than20min) enhanced the expression of Hsp70and Hsp18.2at mRNA level, in comparison with the plants without stress treatment. But there is no significant difference between OE and wild type plants.(2) Arabidopsis seedlings were stress-treated by heat, acid, alkali and H2O2for short times,1min, and5min, respectively. Total RNAs were extracted and HsfAla, Hsp70, Hsp18.2mRNA expression levels were detected. It was found that Hsp70and Hsp18.2mRNA expression levels were increased by stress treatments for1min, and the induced expression levels of Hsp70and Hsp18.2in transgenic plants were significantly higher than wild-type plants. But after longer time of5min stress treatments, the induced expression levels of Hsp70and Hsp18.2reached the similar levels in both OE and wild type plants.(3) Total protein was extracted from Arabidopsis plants priorly stressed by heat, acid, alkali and H2O2. The expression levels of Arabidopsis heat shock protein Hsp70were detected by Western analysis. It was found that the level of Hsp70expression was increased after stress treatments for1min. With increasing time of stress treatments to5and10min, the levels of Hsp70expression also increased, and the induced Hsp70expression levels in transgenic plants were higher than that in wild-type plants. After stress treatments for20min, the induced Hsp70expression levels continued to increase, however, the levels in transgenic and wild-type plants became similar.(4) These results demonstrate that HsfAla can be activated in plants by heat, acid, alkali and H2O2treatments, and play an important role in the regulation of heat shock protein expression in the early stage of stress treatments. However, the difference in the stress induced Hsp expression between OE and wild type plants is not significant in the late stage. The reason might be that other factors participate in the regulation of Hsp expression in the late stage of stress treatments. These findings offer important insight on the mechanism of HsfAla in stress response.