岩黄连DELLA基因编码区的克隆及蛋白转录自激活能力检测

Cloning of coding region of the DELLA genes in Corydalis saxicola and their protein transcriptional autoactivation capacity test

关键词:  岩黄连, DELLA蛋白, 基因克隆, 生物信息学, 转录自激活

Key words:  Corydalis saxicola, DELLA protein, gene cloning, bioinformatics, transcriptional autoactivation

摘要:

岩黄连为罂粟科紫堇属多年生草本药用植物,生长于喀斯特地区的岩缝中,也是国家二级保护植物。DELLA蛋白是植物特有的一类转录因子,在植物生长发育中起重要作用。为探究DELLA蛋白在岩黄连生长发育中的功能,该研究基于岩黄连不同组织转录组及不同组织混样全长转录组数据,克隆获得2个DELLA类基因CsDELLA1和 CsDELLA2的编码区序列(CDS),并进一步对其进行了理化性质、结构、转录本相对表达量,以及转录自激活核心区域的鉴定和分析。结果表明:(1)CsDELLA1和 CsDELLA2的CDS 全长分别为1 902 bp和1 614 bp,分别编码633个和537个氨基酸。(2)实时荧光定量PCR结果显示,两个CsDELLAs转录本相对表达量具有显著的叶组织特异性。(3)氨基酸序列同源性比对及进化树分析结果显示,2个CsDELLAs均具有保守的DELLA结构域和GRAS结构域,并且二者均与罂粟科DELLA家族成员具有较高的相似性。(4)蛋白质结构分析结果表明,α-螺旋是CsDELLA1 和CsDELLA2的主要二级结构,并且二者的蛋白三维构象也相似。(5)2个CsDELLAs均具有转录自激活能力,但激活结构域有差异。CsDELLA1和CsDELLA2,以及截断序列CsDELLA1-N_52-119aa、CsDELLA1-N_120-259aa、CsDELLA1-C_260-633aa和CsDELLA2-C_93-537aa均具有自激活能力; 而二者的N端序列CsDELLA1-N_1-51aa和CsDELLA2-N_1-92aa则无自激活能力。该研究为后续进一步探究CsDELLAs与其他转录因子间的相互作用,以及在岩黄连生长发育中的生物学功能奠定了基础。

Abstract:

Corydalis saxicola, a perennial herbaceous plant of the Papaveraceae family in the genus Corydalis, grows exclusively in karst rock crevices. C. saxicola is also a Chinese national second-class protected plant. DELLA proteins, plant-specific transcriptional factors, play crucial regulatory roles in plant growth and development. In order to explore the function of DELLA proteins in the growth and development of C. saxicola, coding sequence(CDS)of two DELLA-like genes, CsDELLA1 and CsDELLA2, were identified and amplified based on full-length transcriptome and transcriptome data of different tissues of C. saxicola. Molecularly, the physical and chemical properties, structures, the relative expression levels of their transcripts, and key domain transcriptional autoactivation of the two DELLAs were analyzed. The results were as follows:(1)The length of CsDELLA1 and CsDELLA2 CDS were 1 902 bp and 1 614 bp, encoding 633 amino acid(aa)and 537aa, respectively.(2)Quantitative real-time PCR results showed that the relative expression levels of the two DELLA transcripts were exclusively high in the leaves of C. saxicola.(3)Amino acid sequence homology alignment and phylogenetic tree analysis revealed that both CsDELLA1 and CsDELLA2 possessed conserved DELLA domain and GRAS domain, and both had high similarities with members of the DELLA family of Papaveraceae.(4)The analysis results of protein structures revealed that α-helices were important components of the protein structures of CsDELLA1 and CsDELLA2, and their three-dimensional spatial arrangements were similar, too.(5)Both CsDELLAs exhibited transcriptional autoactivation capacities, but the activation domain sites differed. Specifically, CsDELLA1, CsDELLA2, as well as truncated proteins CsDELLA1-N_52-119aa, CsDELLA1-N_120-259aa, CsDELLA1-C_260-633aa, and CsDELLA2-C_93-537aa, had transcriptional autoactivation capacities; while N-terminal sequence of the two CsDELLA1-N_1-51aa and CsDELLA2-N_1-92aa showed no autoactivation capacity. This study lays a foundation for further exploration of the interactions between CsDELLAs and other transcription factors, as well as their biological functions in the growth and development of C. saxicola.