## 2 Results
### TrHSFB2a Negatively Regulates Drought Tolerance in Transgenic Overexpression Arabidopsis  
The detection of _TrHSFB2a_ expression under abiotic stress observing its role in stress responses in detail. Therefore, the overexpression vector of _TrHSFB2a_ was constructed and transferred into _Arabidopsis thaliana_, and thereby, constructed the _TrHSFB2a_ overexpression Arabidopsis. After developing homozygous T3 lines, the relative expression of _TrHSFB2a_ was analyzed in transgenic _Arabidopsis_ (Figure 4g), and two lines highly expressing _TrHSFB2a_ were selected for a comparative analysis along with the wild type and T-DNA lines of _Arabidopsis_ gene _AtHSFB2a_, purchased from _Arabidopsis_ share platform \"[[https://www.arashare.cn/index/](https://www.arashare.cn/index/)]([https://www.arashare.cn/index/](https://www.arashare.cn/index/)) (accessed 18 December 2020)\", in which _TrHSFB2a_ has been silenced. Under normal conditions, there was no significant difference in plant root length among groups after 7 d of transplanting in 1/2 MS medium (Figure 4d,e,h). However, the soil-grown overexpression _Arabidopsis_ were significantly lower in fresh and dry weight compared to wild-type _Arabidopsis_, and T-DNA lines of _Arabidopsis_ were significantly higher in fresh and dry weights compared to wild type (Figure 4i,j), suggesting that _TrHSFB2a_ produced negative effects on early plant growth and development, which was also obvious in phenotypic observations (Figure 4f), suggesting that overexpression or silencing of _TrHSFB2a_ at least do not cause detrimental effects on growth and developments of _Arabidopsis_. Two overexpression lines (OE3 and OE4), two T-DNA lines (denoted as T77 and T95) and the wild type (Wt) were subjected to different drought treatments. Figure 3: Relative gene expression pattern of _TrHSFB2a_ after drought, heat and salt treatments. (**a**–**c**) Display expression level of _TrHSFB2a_ under polyethylene glycol 6000 15% \(w/v\), heat (35 \({}^{\circ}\)C) and salt (NaCl 200 mM) stresses, respectively, with time course 0, 3, 6, 12, and 24 h after treatments. Error bars represent the standard deviation (SD) of three independent biological replicates. Initially, 5-day-old seedlings of _Arabidopsis_ (wild type and transgenic) were transferred to 1/2 MS plates containing 200 or 300 mM mannitol. After two weeks, the overexpression lines (OE-3 and OE-4) under 200 mM mannitol showed a significantly higher leaf-yellowing rate compared to the wild type, while most of the plants of overexpression lines under 300 mM mannitol dried; the wild type although had curled leaves, all were green (Figure 5a,b). Moreover, root length was significantly higher in T-DNA lines and was significantly lower in overexpression lines under 200 mM mannitol stress, indicating that _TrHSFB2a_ possibly negatively regulates drought stress (Figure 5c-e). Three-week-old wild-type and transgenic _Arabidopsis_ plants were also subjected to drought stress in nutrient soil by withholding water. Plants were well watered, and then, irrigation was stopped. After 12 days of water withholding, all the leaves of overexpression lines OE3 and OE4 wilted  
Figure 4: Homologous relationship of _TrHSFB2a_ based on conserved domains and construction of overexpression lines of _TrHSFB2a_. (**a**) Homologous relationship of _TrHSFB2a_ with most related proteins, with most conserved regions shaded by red color (**b**) Phylogenetic relationship of _TrHSFB2a_ based on conserved domains, (**c**) selection of overexpression lines of _TrHSFB2a_ on 1/2 MS medium containing 50 mg/L Kanamycin for developing homozygous lines. Only transformed plant could grow on selection media, while non-transformed turned into yellow and died (**d,e**) Show plant stature and root growth of wild type (Wt), RNA interference T-DNA lines (T-77 and T-95) and overexpression lines (OE3 and OE4) grown on 1/2 MS medium containing 3% sucrose and 0.7% agar.