Abstract:

Objective: Heat shock transcription factor (HSF) plays an important regulatory role in plant response to abiotic stress. The aim of this study was to identify the stress resistance function of BpHSFA4 in birch(Betula platyphylla) in order to lay a theoretical foundation for using the gene in tree breeding through genetic engineering. Methods: The BpHSFA4 cDNA sequence was obtained by searching with birch transcriptome libraries. Further, the BpHSFA4 gene was cloned by RT-PCR. And the BpHSFA4 cDNA sequence was analyzed using bioinformatics tools. Quantitative real-time RT-PCR was used to monitor the expression patterns of BpHSFA4 genes in the leaves, stems and roots of birch under different stress treatments. The vector of overexpression pROKⅡ-BpHSFA4 and silencing expression vector of pFGC5941-BpHSFA4 were constructed and transient transformed into birch. The empty pROKⅡ was also transient infected into birch as a control. The MDA content, H₂O₂, SOD activity, POD activity, electrolyte leakage and diaminobenzidine(DAB), nitrotetrazolium blue chloride (NBT) and Evans blue staining under NaCl stress were measured and compared between transgenic (OE, SE) and control (CK) plants. Results: The results showed that the cDNA length of BpHSFA4 was 1 170 bp. The protein encoded by this BpHSFA4 gene was 389 amino acids. The relative molecular weight of BpHSFA4 protein was 44.15 kDa, with isoelectric point 5.14. The typical HSF domain was found in the BpHSFA4 sequence.The results of qRT-PCR showed that the expressions of BpHSFA4 gene were different under abiotic stresses and hormone treatments compared to the control. Furthermore, the expressions were significantly different at least at one stress point time for each treatment. In addition, the expression patterns of BpHSFA4 gene were more significantly changed under NaCl stress. To determine whether BpHSFA4 displayed a successful overexpression(OE) or a suppressed expression (SE), the transcript levels of BpHSFA4 in the three types of transformed birch plants were measured by qRT-PCR. The results showed that the expression of BpHSFA4 was significantly higher in OE plants and clearly lower in SE plants, especially 12 h after transformation, indicating that these transient expression plants were suitable for further study. Under NaCl stress, The NBT and DAB staining and H₂O₂ content also showed that overexpression of BpHSFA4 markedly reduced ${\rm{O}}_2^{\bar \cdot }$ and H₂O₂ accumulation, while suppressed expression of BpHSFA4 led to elevated accumulation of ${\rm{O}}_2^{\bar \cdot }$ and H₂O₂. The SOD and POD activities of transgenic birch plants showed that OE had the highest SOD and POD activities, and that SE plants had the lowest SOD and POD activities. The Evans blue staining and MDA content and electrolyte leakage showed that cell death greatly decreased in the OE plants, but greatly increased in the SE plants. Conclusion: BpHSFA4 gene can respond to abiotic stresses and hormone treatments, and stronger under salt stress, it indicates that the gene may be involved in the response to salt stress in birch trees. All results indicated that overexpression of BpHSFA4 improved the salt stress tolerance by increasing the ability to scavenge reactive oxygen species (ROS), the activities of SOD and POD to reduce the accumulation of H₂O₂ and ${\rm{O}}_2^{\bar \cdot }$, thereby reducing cell damage or cell death and enhancing birch resistance. BpHSFA4 was proved to be a candidate gene responding to stresses.

Key words: Betula platyphylla, HSFA4 transcription factor, gene expression, salt tolerance