蜡梅转录因子CpBBX24基因的克隆及功能分析

Cloning and Functional Analysis of the Transcription Factor CpBBX24 Gene of Chimonanthus praecox

【目的】BBX(B-box)是锌指结构蛋白转录因子家族中一个重要的亚家族,在植物生长发育、植物信号转导及逆境胁迫响应中具有重要的调控作用。对蜡梅BBX24基因的克隆与分析,有利于丰富植物中对BBX基因家族的认识,为蜡梅抗逆调节机制提供新的理论依据。【方法】以蜡梅转录组数据库中获得的蜡梅CpBBX24基因cDNA序列为基础克隆基因全长,使用DNAStar和MEGA进行序列分析和进化树构建。采用qRT-PCR进行蜡梅不同组织及不同花期表达特性分析,以及ABA、MeJA、干旱、高盐、高温和低温等处理后表达分析。同时,使用Gateway技术构建植物表达载体,花絮侵染法转化拟南芥,对T3代纯合系进行表型观察及非生物胁迫耐性分析。【结果】获得CpBBX24的cDNA序列长为1374 bp,包含729 bp的开放阅读框(ORF),编码242个氨基酸,分子量为26.54 kD,等电点为4.93。序列分析表明CpBBX24蛋白在N端有两个串联的B-box结构域,其C端不包含CCT结构域。表达特性分析表明,CpBBX24在蜡梅根、茎、子叶、幼叶、成熟叶各器官,外瓣、内瓣、雌蕊、雄蕊等组织中均有表达,其中在子叶中表达量最高。在不同开花时期,CpBBX24表达呈波动变化,在衰老期表达量最高。ABA、MeJA、干旱、高盐、高温和低温均能诱导CpBBX24基因的表达,表达趋势各有差异。在PEG(30%PEG-6000)、高盐(300 mmol·L^(-1)NaCl)、高温(42℃)、低温(4℃)胁迫下,转基因拟南芥处理后植株总体生长状态优于野生型,相对电导率和丙二醛浓度数值显著低于野生型,说明CpBBX24基因过表达增强了拟南芥的干旱、高盐、高温及低温胁迫耐性。【结论】ABA、MeJA、干旱、高盐、高温和低温等处理诱导了CpBBX24的表达,说明其可能参与蜡梅的逆境胁迫耐性调控。CpBBX24的过表达增强了拟南芥的干旱、高盐、高温及低温等胁迫耐性。这些结果表明CpBBX24基因在干旱、高盐、低温和高温等胁迫响应中发挥重要作用。

【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℃)and cold(4℃)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.