关键词: 蜡梅, BBX, 逆境胁迫, 转基因

Abstract:

Objective: BBX (B-box) is an important subfamily of zinc finger structural protein transcription factor family, which plays an important regulatory role in plant growth and development, plant signal transduction and stress response. Cloning and functional analysis of the BBX24 gene in Chimonanthus praecox (wintersweet) may help to enrich the understanding of the BBX gene family in plants, and provide theoretical basis for the stress-resistant regulation mechanism of wintersweet. Method: Based on the cDNA sequence of CpBBX24 gene in the wintersweet transcriptome database, we obtained the CpBBX24 gene and analyzed the sequence characteristics and evolutionary tree by DNAStar and MEGA. The expressions of CpBBX24 in different tissues and flowering stages, and the changes of CpBBX24 gene expression under ABA, MeJA, drought, high salt, high temperature and low temperature treatments were analyzed using qRT-PCR. At the same time, the Gateway technology was used to construct plant overexpression vectors, and Arabidopsis was transformed by floral dipping method. Homozygous lines of the T3 generation were used for phenotype observation and abiotic stress tolerance analysis. Result: The cDNA sequence of CpBBX24 is 1374 bp, containing 729 bp open reading frame (ORF), encoding 242 amino acids with molecular weight was 26.54 kD and isoelectric point 4.93. Sequence analysis showed that CpBBX24 protein had two tandem B-box domains at the N-terminus, and its C-terminus did not contain a CCT domain. The results showed that the CpBBX24 gene expressed in the roots, stems, cotyledons, young leaves, mature leaves, and flower tissues such as outer petals, inner petals, pistils and stamens, and the expression level was highest in cotyledons. In different flowering stages, the expression of CpBBX24 fluctuated, and the expression level was the highest in the senescent stage. ABA, MeJA, drought, High salt, high temperature and low temperature could induce the expression of CpBBX24 gene, and the expression trends were different. Under the stress of PEG (30% PEG-6000), high salt (300 mmol·L^?1 NaCl), hot (42 °C) and cold (4 °C) stress, the overall growth state of the transgenic Arabidopsis plants was better than that of the wild type. The values of relative conductivity and malondialdehyde concentration were significantly lower than those of the wild type, indicating that the overexpression of CpBBX24 gene enhanced the drought, high-salt, high-temperature and low-temperature stress tolerance of Arabidopsis. Conclusion: The expression of CpBBX24 was induced by ABA, MeJA, drought, High salt, high temperature and low temperature stress, indicating that it may be involved in the regulation of adversity stress tolerance in wintersweet. The overexpression of CpBBX24 enhanced the stress tolerance of Arabidopsis to drought, high salt, high temperature and low temperature. These results indicated that CpBBX24 gene plays an important role in response to stresses such as drought, high salinity, low temperature and high temperature.

Key words: Chimonanthus praecox, BBX, adversity stress, transgenic