Cloning of DREB1D gene from pitaya and its functional analysis in transgenic Arabidopsis thaliana

【Objective】DREB (dehydration responsive element binding) proteins are widely present in plants and are primarily involved in the abiotic stress response of plants. The two primary DREB transcription factors are DREB1 and DREB2, with DREB1 being mostly associated to low temperature and drought stress and DREB2 being primarily related to drought, salt, and high temperature stress. Pitaya (Hylocereus monacanthus) belongs to the cactus plants, because of its high nutritional value and strong resistance stress, it is popular with customers in karst regions like Guizhou and Guangxi. DREBs were found responsive to drought stress in pitaya, leaving the underlying mechanism unrevealed. This study intends to clone HmDREB1D (HU02G01866.1) gene and verify its biological function.【Methods】The pCambia35s-HmDREB1D-GFP plant overexpression vector was constructed by seamless cloning technology and transformed into Tobacco. The fluorescence signal of HmDREB1D was observed under a laser confocal microscope to determine the subcellular location. The expression vector of pCambia35sHmDREB1D was constructed. The HmDREB1D gene was transformed into A. thaliana, a total of 6 transgenic A. thaliana plants were obtained, and 3 overexpressed transgenic Arabidopsis (OE3, OE4 and OE5) plants were chosen for further biological verification. After surface sterilization, Hm-DREB1D transgenic Arabidopsis (OE3, OE4 and OE5) and the wild type Arabidopsis seeds were sown in 250 mmol · L- 1 mannitol 1/2 MS medium for drought stress treatment (16 h/8 h day/night cycle, 24 ℃ ). The germination rate was counted after 7 days. The seedlings grew in 1/2 MS medium for 7 days, and then were transplanted into 1/2 MS medium containing 250 mmol · L- 1 mannitol for drought stress treatment (16 h/8 h day/night cycle, 24 ℃ ). The root length and fresh weight were measured 7 days after the treatment. Transgenic Arabidopsis (OE3, OE4 and OE5) and the wild type Arabidopsis seeds were surface sterilized, sown in 1/2 MS medium and cultured for 7 days, and then transplanted in pots filled with nutrient soil vermiculite (3∶1) and placed in an artificial climate growth chamber for 4 weeks (16 h/8 h day/night light cycle, 24 ℃ ). Mock drought 20% PEG6000 (3 d), high temperature 42 ℃ (1 d) and low temperature -20 ℃ (1 h) were subsequently applied. Transgenic Arabidopsis and the wild type Arabidopsis leaves were collected before and after stress treatment for determination of physiological and stress gene expression levels. The relative conductivity was measured by Jenco3020. The activities of peroxidase (POD), superoxide dismutase (SOD) and catalase (CAT) were determined by spectrophotometer. The qRT-PCR technique was used to detect the response gene expression level of transgenic Arabidopsis (OE3, OE4 and OE5) and the wild type Arabidopsis under drought (RD20 and RD22), high temperature (HSP70 and HSFA1D) and low temperature (COR47 and COR15A) stress. 【Results】A 723 bp open reading frame codes the 243 aa HmDREB1D, which is categorized in DREB1s subfamily because of the AP2 domain. The results of GFP fluorescence signal showed that the pCambia35s-HmDREB1D-GFP was transformed into Tobacco and distributed in the nucleus, while the pCambia35s-GFP protein was distributed on the cell surface, indicating that HmDREB1D protein was located in the nucleus. The HmDREB1D gene was transformed into Arabidopsis. The HmDREB1D gene responded greatly to the drought stress with 250 mmol·L-1 mannitol treatments. The results showed that seed germination rate and seedling growth status of transgenic Arabidopsis were better than the wild type Arabidopsis under drought stress. The germination rate of transgenic plant remained above 87.8%, while the germination rate of wild type Arabidopsis was only 37.3% . The results showed that HmDREB1D gene enhanced the tolerance of transgenic Arabidopsis to drought stress by increasing the germination rate of transgenic Arabidopsis under drought stress, indicating that the germination rate was positively correlated with stress resistance. After drought, high temperature and low temperature stress, transgenic Arabidopsis had not only a better phenotype than the wild type Arabidopsis, but also a significantly higher survival rate than the wild type Arabidopsis (p＜0.05). The results showed that transgenic Arabidopsis had strong water retention capacity, which improved the survival rate of transgenic Arabidopsis. The relative conductivity of transgenic Arabidopsis was significantly lower than the wild type Arabidopsis (p＜0.05). The damage of transgenic Arabidopsis was less than that of the wild type Arabidopsis under drought, high temperature and low temperature stress, indicating that the stress resistance of transgenic Arabidopsis was higher than that of the wild type Arabidopsis. The antioxidant enzyme activity of the HmDREB1D transgenic Arabidopsis was significantly higher than that of the wild type Arabidopsis (p＜0.05). The results showed that transgenic Arabidopsis could improve the antioxidant capacity of transgenic Arabidopsis through the activity of antioxidant enzymes, and then reduce the harm caused by drought, high temperature and low temperature stress. According to the results of qRT-PCR, the expression of stress response genes in transgenic Arabidopsis showed upward trend under drought, high temperature and low temperature stress, and was significantly higher than that of the wild type Arabidopsis (p＜0.05). The results showed that HmDREB1D gene enhanced the stress ability of transgenic Arabidopsis under drought, high temperature and low temperature stress by inducing the expression ofstress-related genes.【Conclusion】Aforementioned results suggest that HmDREB1D might be positively involved in the stress response of pitaya.