3种龙血树PAL基因克隆及分子鉴定
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引用本文:王延谦,李 爽,杨春勇,王艳芳,里 二,张丽霞.3种龙血树PAL基因克隆及分子鉴定[J].西北植物学报,2019,39(10):1711~1717
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作者单位
王延谦1,2,李 爽1,2,杨春勇1,2,王艳芳1,2,里 二1,2,张丽霞1,2 (1 中国医学科学院 北京协和医学院药用植物研究所云南分所云南景洪 6661002 西双版纳州傣药南药重点实验室云南景洪 666100) 
基金项目:国家自然科学基金(81703638)
中文摘要:该研究以3种龙血树(剑叶龙血树、海南龙血树和岩棕)的DNA和总RNA为模板,采用同源克隆法克隆龙血竭3种基源植物的苯丙氨酸解氨酶(phenylalanine ammonia lyase)基因PAL,利用DNAMAN、MEGA7等软件进行生物信息学分析,以探讨龙血竭3种基源植物的分子鉴定方法,为生产实际中对不同来源的龙血竭原料的鉴定提供理论依据。结果表明:(1)在龙血竭3种基源植物中分别获得长度为718 bp的 PAL片段,序列分析发现,3种龙血树PAL片段高度一致(99.69%),仅存在5处碱基突变;系统进化分析表明,龙血树PAL片段与百合科植物PAL基因聚为一类。(2)RT PCR分析发现,PAL基因在3种龙血树的根、茎、叶中均有表达,同种龙血树不同组织的表达量差异较小,且PAL基因在岩棕中的表达量较高,在剑叶龙血树和海南龙血树中表达量较低。(3)利用保守引物进行分子鉴定发现,同种龙血树PAL序列一致,3种龙血树PAL片段存在5处碱基突变,且这些碱基突变为稳定遗传,表明3种龙血树PAL基因高度保守。(4)根据突变位点建立了龙血竭3种基源植物的分子鉴定方法,进一步分子鉴定结果表明,剑叶龙血树的碱基位置为GCGGG,海南龙血树的碱基位置为CTGGC,岩棕的碱基位置为GTTTG。该研究结果为龙血竭原材料溯源和质量控制奠定了理论与技术基础。
中文关键词:龙血树  龙血竭  苯丙氨酸解氨酶  基因克隆  基源鉴定
 
PAL Gene Cloning and Molecular Identification of Three Dracaena Plants
Abstract:In this study, the PAL genes of three Dracaena (D.cochinchinensis, D. cambodiana and ‘Aizong’) plants were cloned by homologous cloning using the DNA and total RNA. Bioinformatics of PAL gene were analyzed with DNAMAN and MEGA7 software. The purpose of this study is to explore the molecular identification methods of three Dracaena plants, and provide a theoretical basis for the identification of different Dragons blood sources. The result show that: (1) three 718 bp PAL gene segments were obtained from D.cochinchinensis, D. cambodiana and ‘Aizong’, respectively. The homology of this 3 PAL genes was 99.69%, and just only 5 base mutations in base sequence. Phylogenetic analysis showed that the PAL gene of three Dracaena plants clustered with Liliaceae plants. (2) PAL gene was expressed in roots, stems and leaves of three Dracaena plants by RT PCR analysis. The difference of PAL gene expression in different tissues of Dracaena plants is small, and higher in ‘Aizong’ and lower in D.cochinchinensis and D. cambodiana. (3) Gene sequencing showed that PAL gene sequences were completely consistent in the same species, and just only 5 base mutations among three Dracaena plants. The PAL genes of three Dracaena plants are highly conserved. (4) Molecular identification method for three Dracaena plants was established based on the mutation site. Further molecular identification results indicate that GCGGG base site for D.cochinchinensis, CTGGC for D. cambodiana and GTTTG for ‘Aizong’. The research of molecular identification method is beneficial to the traceability and quality control of dragon’s blood.
keywords:Dracaena  dragons blood  phenylalanine ammonia lyase  gene cloning  base source identification
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