嘉兰中酪氨酸脱羧酶基因的克隆与表达分析

Cloning and Expression Analysis of Tyrosine Decarboxylase Gene in Gloriosa superba L.

酪氨酸脱羧酶(EC 4.1.1.28)是高等植物体中秋水仙碱合成的关键酶。为了探索嘉兰秋水仙碱生物合成途径的关键酶基因及其相关功能,本研究根据嘉兰转录组数据库和NCBI数据库,克隆得到一个酪氨酸脱羧酶关键酶基因(GsTyDC1)的CDS序列,并使用RT-qPCR检测GsTyDC1在嘉兰植株中不同组织的表达水平。通过烟草悬浮细胞对GsTyDC1进行稳定转化,并分析其亚细胞定位的结果。结果显示,GsTyDC1基因的CDS区全长为1 545 bp,编码514个氨基酸,蛋白分子量为56.3 kD。生物信息学分析表明,GsTyDC1与水仙、石蒜和芭蕉等百合科植物已报道的Ty DC基因聚在一类,具有类似于与其他已报道物种TyDC基因相似的保守结构区域,并且没有跨膜结构和信号肽。此外,GsTyDC1基因在嘉兰根中表达量最高,花和茎次之,在叶中表达量最低。亚细胞定位显示Gs TyDC1蛋白定位在细胞质中。本研究为下一步验证GsTyDC1基因的功能活性提供一定的基础,为阐明GsTyDC1基因在嘉兰秋水仙碱生物合成途径中的作用提供参考依据。

Tyrosine decarboxylase(EC 4.1.1.28)is the key enzyme for colchicine synthesis in higher plants.In order to explore the key enzyme genes and related functions of colchicine biosynthesis pathway in Gloriosa superba L.,CDS sequences of GsTyDC1 was obtained by screening and homologous cloning according to transcriptome database of Gloriosa superba L.and NCBI database in this study.Specificity of GsTyDC1 expression in different tissues detected by theQuantitative real-time PCR(RT-qPCR).TheGsTyDC1 proteinwas stably transformed by tobacco(BY-2)cells,and the subcellular localization results were analyzed.The results showed that the CDS region of GsTy-DC1 was 1545 bp,and the relative molecular mass of the protein was 56.3 kD,encoding 514 amino acids.Bioinformatics analysis showed that GsTyDC1 protein was clustered with lily plants such as narcissus,garlic and banana,and had conserved domain structure similar to TyDC in other reported species,without transmembrane structure and signal peptide.RT-qPCR analysis showed that the expression of GsTyDC1 was highest in roots,followed by flowers and stems,and lowest in leaves.Subcellular localization analysis showed that GsTyDC1 protein was localized in the cytoplasm. This study provides favorable information for further verifying the functional activity of GsTyDC1 gene,and provides a theoretical basis for clarifying the role of GsTyDC1 gene in the biosynthetic pathway of colchicine.