[关键词]
[摘要]
目的 克隆马蓝邻氨基苯甲酸合成酶(anthranilate synthase,AS)BcASB基因(GenBank登录号QCF61930.1),并对其进行生物信息学分析、时空表达分析以及检测马蓝有效成分靛蓝、靛玉红积累量随时间的变化。方法 通过RT-PCR和RACE技术,克隆BcASB基因全长,应用生物信息学的方法对该基因编码的蛋白进行各种理化性质测定,二级结构和三级结构预测分析以及对核苷酸和氨基酸序列进行比对;利用qRT-PCR技术检测BcASB基因在马蓝不同器官(根、茎、叶)中的表达模式以及在外源诱导子茉莉酸甲酯(methyl-jasmonate,MeJA)、脱落酸(abscisic acid,ABA)、水杨酸(salicylic acid,SA)和乙烯利(ethylene,ETH)诱导下的表达情况;同时运用HPLC测定吲哚类生物碱靛蓝、靛玉红含量的变化情况。结果 克隆获得BcASB基因,开放阅读框(open reading frame,ORF)长度为765 bp,编码256个氨基酸,生物信息学表明,该蛋白不含信号肽且无跨膜区,亚细胞结构定位于叶绿体;qRT-PCR检测结果表明,BcASB基因在马蓝叶和茎中的表达量较高,在根中表达较低;BcASB基因可响应不同外源诱导处理,而影响其转录;HPLC检测结果显示靛蓝、靛玉红含量有着明显的变化。结论 成功克隆马蓝邻氨基苯甲酸合成酶BcASB基因,为进一步阐释该基因在马蓝吲哚类生物碱合成途径的作用及表达调控研究奠定基础。
[Key word]
[Abstract]
Objective To clone the anthranilate synthase (AS) BcASB gene (GenBank accession number AYM45644.1) from Baphicacanthus cusia, and to analyze its bioinformatics and spatio-temporal expression, and detect the changes of indigo and indirubin accumulation in different time periods of B.cusia. Methods The full-length sequence of BcASB gene was cloned by quantitative RT-PCR and RACE techniques. The physical and chemical properties, secondary structure, and tertiary structure of BcASB protein were forecasted and analyzed, and the nucleotide and amino acid sequences were compared by bioinformatics technology. Quantitative real-time PCR was used to detect the BcASB gene expression levels in different tissues (roots, stems, leaves) of B. cusia. And its expression level under methyl jasmonate (MeJA), abscisic acid (ABA), salicylic acid (SA), and ethephon (ETH) was detected by quantitative real-time PCR. Meanwhile, changes in the content of indole alkaloids indigo and indirubin were detected by HPLC. Results The BcASB gene, containing a 765 bp open reading frame (ORF) and encoded 256 amino acids, was cloned. No signal peptide and no transmembrane region were present in BcASB protein. And BcASB protein was located in chloroplast. Relative real-time PCR analysis indicated that BcASB gene showed the higher expression level was in the leaves and stems, while was lower in the roots. BcASB gene could respond to multiple treatments of methyl jasmonate (MeJA), abscisic acid (ABA), salicylic acid (SA) and ethephon (ETH), which promoted its transcription. HPLC test results show that the content of indigo and indirubin with a significant change. Conclusion The BcASB gene was cloned from B.cusia. The results provide a foundation for further elucidating the important role of this gene in the synthetic pathway of B.cusia indole alkaloids and expression regulation.
[中图分类号]
R282.12
[基金项目]
国家自然科学基金面上项目(81573517);福建省自然科学基金面上项目资助(2019J01827);福建农林大学科技创新专项基金面上项目(KFA20011A)