蛇足石杉HsAP2-3基因表达分析及蛋白相互作用检测

Expression analysis of HsAP2-3 gene and interaction detection of HsAP2-3 with other proteins in Huperzia serrata

关键词:  蛇足石杉, APETALA2 基因, 生长发育, 表达分析, 酵母双杂交

Key words:  Huperzia serrata, APETALA2 gene, growth and development, expression analysis, yeast two-hybrid

摘要:

蛇足石杉为石松科石杉属多年生药用植物,其生长速率十分缓慢、野生资源濒临灭绝。为探讨APETALA2(AP2)在蛇足石杉生长发育中的生物学功能,该研究基于转录组和全长转录组数据,筛选且克隆到1个AP2-like基因HsAP2-3(GenBank登录号:OR103132),并进一步对其开展了生物信息学分析、转录本表达分析和蛋白相互作用检测。结果表明:(1)HsAP2-3 CDS全长1 734 bp,编码577个氨基酸; HsAP2-3蛋白相对分子质量和等电点分别为64.21 kDa和8.85,含2个典型的AP2结构域和1个保守的核定位信号位点。(2)进化树分析结果显示,HsAP2-3与参与调控植株生长发育的欧洲云杉AP2-like 蛋白亲缘关系最近。(3)荧光定量PCR结果显示,HsAP2-3 在不同株龄蛇足石杉的根、茎、新叶和成熟叶中均有表达。(4)酵母双杂交结果显示,HsAP2-3可以与其自身发生蛋白-蛋白相互作用。(5)蛋白互作网络分析结果显示,HsAP2-3与调控植物生长发育的转录因子存在广泛的相互作用。综上认为,HsAP2-3很可能通过以同源二聚体或与其他蛋白形成复合体的形式参与蛇足石杉生长发育的调控。该研究结果为进一步深入阐释其生物学功能奠定了基础。

Abstract:

Huperzia serrata, which belongs to the genus Huperzia involved in the family Lycopodiaceae, is a perennial, rare and endangered medicinal plant spread in northwest and southwest of China and also other countries. H. serrata is used extensively for treatment of contusions, strains, swellings, schizophrenia, myasthenia gravis, and organophosphate poisoning. However, it grows extremely slowly, which hampered its clinical investigation and application. It is well known that APETALA2(AP2)transcription factor plays crucial roles in the regulation of plant growth and development. In order to explore the biological function of AP2 in H. serrata, this study screened and cloned one AP2-like gene, HsAP2-3(GenBank accession number: OR103132), based on the transcriptome and full-length transcriptome data. Bioinformatics analysis, transcript expression analysis, and protein interaction assays of HsAP2-3 were further carried out. The results were as follows:(1)The HsAP2-3 CDS was 1 734 bp in length and encoded 577 amino acids, and the relative molecular weight and isoelectric point of the HsAP2-3 protein were 64.21 kDa and 8.85, respectively. HsAP2-3 protein contained two typical AP2 domains and a conserved nuclear localization signal site.(2)Phylogenetic tree analysis showed that HsAP2-3 were clustered within the euAP2 clade of the AP2 subfamily, and had the closest relationship with the Picea abies' AP2-like protein, the latter of which were demonstrated to be involved in the regulation of plant growth and development.(3)Quantitative real-time PCR results showed that HsAP2-3 was expressed in roots, stems, young leaves, and mature leaves of Huperzia serrata at different developmental stages, indicating ubiquitous regulation patterns.(4)Yeast two-hybrid assays demonstrated that HsAP2-3 had not transactivation activity, and could form homodimers in Y2HGold yeast cells.(5)The results of protein-interaction network analysis showed that HsAP2-3 has extensive interactions with transcription factors that regulate plant growth and development, including auxin response factors, abscisic acid-insensitive proteins, late elongated hypocotyl proteins, circadian clock associated proteins and B3 domain-containing transcription factors. Overall, these results suggest that HsAP2-3 may be involved in the growth and development of H. serrata in the form of homodimers or protein complexes with other factors, laying the foundation for further illustrating the biological function of HsAP2-3.