钩藤钩发育相关SEPALLATA-like基因的克隆与表达分析

Cloning and expression analysis of hook development-related SEPALLATA-like genes in Uncaria rhynchophylla

关键词:  钩藤, SEPALLATA-like, 基因克隆, 亚细胞定位, 转录激活活性, 表达分析

Key words:  Uncaria rhynchophylla, SEPALLATA-like, gene cloning, subcellular localization, transcriptional activation activity, expression analysis

摘要:

钩器官是药用植物钩藤的特殊结构,也是主要药用部位,与花序被认为属于同源器官,但对于钩是如何发育而来的尚不清楚。为解析钩器官的来源及其发育机制,该研究通过RT-PCR及RACE技术克隆了3个钩藤MADS-box转录因子家族SEPALLATA(SEP)-like基因的完整编码区序列,并采用生物信息学预测、亚细胞定位、转录激活实验、RT-qPCR方法初步分析其功能。结果表明:(1)UrSEP1、UrSEP3.1、UrSEP3.2的全长cDNA序列长度分别为931、981、1 097 bp,编码区长度分别为738、726、729 bp,分别编码245个、241个、242个氨基酸,蛋白序列中均包含保守的MADS结构域、K-box、SEP Ⅰ 和SEP Ⅱ 基序。(2)系统进化树分析显示,UrSEPs与小粒咖啡CaSEPs的亲缘关系最近,其中UrSEP1属于LOFSEP亚类中的SEP1/2分支,UrSEP3.1和UrSEP3.2同属于SEP3亚类。(3)亚细胞定位发现,UrSEPs均定位于细胞核,说明UrSEPs在核中发挥作用,其中UrSEP1和UrSEP3.2具有转录激活活性,提示UrSEP1和UrSEP3.2能够直接结合DNA序列。(4)UrSEPs基因的RT-qPCR结果表明了钩与花序的同源性和二者发育上的分歧,UrSEP3.1和UrSEP3.2在钩中的转录水平均显著高于根、茎、叶,并且在钩与花序两种发育过程中具有类似的表达模式; UrSEP1在钩芽中的表达量明显上升,高于其他营养组织及花序芽,并在钩与花序发育过程中具有相反的表达模式。综上认为,UrSEP1、UrSEP3.1和UrSEP3.2参与了钩的发育,并可能在钩和花序早期发育命运分歧中发挥作用。该研究从基因层面为揭示钩藤钩器官的来源提供了线索,为解析其形态建成的分子机制奠定基础。

Abstract:

The hook is a distinctive structure of the medicinal plant Uncaria rhynchophylla, and serves as its primary medicinal part. Although the hook and inflorescence are considered to be homologous organs, the developmental mechanism of the hook remains unclear. To investigate the origin and the developmental mechanism of the hook, this study cloned the complete coding sequences of three SEPALLATA(SEP)-like genes, belonging to the MADS-box transcription factor family, by using RT-PCR and RACE techniques. The functions of these SEP-like genes were preliminarily characterized through bioinformatic prediction, subcellular localization, transcriptional activation assays, and RT-qPCR methods. The results were as follows:(1)The full-length cDNA of the three genes, UrSEP1, UrSEP3.1 and UrSEP3.2, were 931, 981, 1 097 bp in length, which contained the coding sequences(CDS)of 738, 726, 729 bp, encoding 245, 241, and 242 amino acid residues, respectively. All proteins contained conserved domains, including MADS domain, K-box, SEP Ⅰ and SEP Ⅱ motifs.(2)Phylogenetic tree analysis revealed that UrSEPs exhibit the closest relationship to CaSEPs from Coffea arabica. UrSEP1 clusters within the SEP1/2 clade of the LOFSEP subgroup, while UrSEP3.1 and UrSEP3.2 belong to the SEP3 subgroup.(3)Subcellular localization found UrSEPs all localized in the nucleus and confirmed the nuclear activity of UrSEPs. Transcriptional activation assays suggested that UrSEP1 and UrSEP3.2 might exhibited transcriptional activation activities, which remindering can bind DNA directly.(4)Results of RT-qPCR revealed potential homology between hook and inflorescence, alongside developmental divergence. UrSEP3.1 and UrSEP3.2 showed higher transcriptional levels in hook than in root, stem or leaf, with similar expression patterns in inflorescence; the expression level of UrSEP1 peaked in hook bud, surpassing levels in other vegetative tissues and inflorescence bud, and displayed opposing expression patterns during hook versus inflorescence development. In summary, the three UrSEPs genes encode nuclear-localized proteins from SEP1/2 and SEP3 clades, implicating their roles in hook development and early-stage fate divergence between hook and inflorescence. These results provide genetic insights into origin of the hook and establish a foundation for elucidating the molecular mechanisms underlying hook morphogenesis in Uncaria rhynchophylla.