天仙子中参与托品烷生物碱生物合成的ArAT基因的克隆及功能鉴定

发布时间：2018-06-27 11:38

本文选题：天仙子 + 芳香族氨基酸氨基转移酶　；参考：《药学学报》2017年01期

【摘要】：托品烷类生物碱是临床上广泛应用的抗胆碱药物,其生物合成涉及到苯丙氨酸的转氨基反应。根据天仙子(Hyoscyamus niger)侧根和叶的转录组数据,筛选到3条功能注释为芳香族氨基酸氨基转移酶的基因,命名为HnArAT1、HnArAT2和HnArAT3。通过序列同源性分析,发现HnArAT3与颠茄的AbArAT4的氨基酸序列同源性最高,两者为直系同源。组织表达谱显示,HnArAT3基因在根中特异性表达,与其他3个合成途径基因(PMT、TRI和H6H)表达模式相同。用病毒诱导的基因沉默(VIGS)方法在天仙子植株中鉴定HnArAT3的功能,对感染了病毒的植株,用实时荧光定量PCR检测基因的表达量,HPLC法测定托品烷类生物碱的含量。结果表明,在pTRV2-HnArAT3干扰的植株中,HnArAT3基因的表达量有显著下降,3种托品烷类生物碱莨菪碱、山莨菪碱和东莨菪碱含量也有显著的降低。以上结果表明HnArAT3是天仙子中参与托品烷类生物碱生物合成的苯乳酸支路途径基因。HnArAT3基因的克隆为进一步研究托品烷类生物碱的生物合成和代谢调控奠定了基础,也为开展托品烷类生物碱的代谢工程研究提供了新的候选靶基因。
[Abstract]:Tropine alkaloids are widely used as anticholine drugs in clinic. Their biosynthesis involves the transamino reaction of phenylalanine. Based on the transcriptional data of lateral roots and leaves of Hyoscyamus niger, three genes, named HnArAT1, HnArAT2 and HnArAT3, were identified as aromatic amino acid aminotransferases. The amino acid sequence homology between HnArAT3 and AbArAT4 of belladonna was the highest, and the homology between HnArAT3 and AbArAT4 was linear. Tissue expression profiles showed that HnArAT3 gene was specifically expressed in root, which was similar to the other three genes (PMTTRI and H6H). Virus induced gene silencing (VIGS) method was used to identify the function of HnArAT3 in the plant of Tianxianzi. For the infected plants, the expression of genes was detected by real-time fluorescence quantitative PCR and the content of tropine alkaloids was determined by HPLC. The results showed that the expression of HnArAT3 gene in the plants interfered with pTRV2-HnArAT3 decreased significantly, and the contents of scopolamine and anisodamine also decreased significantly. These results suggest that HnArAT3 is a branch pathway gene of phenyllactic acid involved in the biosynthesis of tropine alkaloids. The cloning of HnArAT3 gene lays a foundation for further study on biosynthesis and metabolic regulation of tropine alkaloids. It also provides a new candidate target gene for the metabolic engineering of tropine alkaloids.
【作者单位】：西南大学生命科学学院三峡库区教育部生态环境教育部重点实验室;西南大学-西藏农牧学院药用植物联合研发中心;西南大学药学学院;西藏大学农牧学院;
【基金】：国家863计划资助项目(2011AA100605) 国家自然科学基金资助项目(31370333) 教育部新世纪优秀人才计划资助项目(NCET-12-0930) 中央高校基本科研业务费专项资助项目(XDJK2013A024) 中央高校基本业务费专项资助项目(XDJK2014C064)
【分类号】：R284.3

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