3种龙血树PAL基因克隆及分子鉴定  

PAL Gene Cloning and Molecular Identification of Three Dracaena Plants

作　　者：王延谦 李爽 杨春勇[1,2] 王艳芳 里二[1,2] 张丽霞 WANG Yanqian;LI Shuang;YANG Chunyong;WANG Yanfang;LI Er;ZHANG Lixia(Yunnan Branch,Institute of Medicinal Plant Development,Chinese Academy of Medical Sciences&Peking Union Medical College,Jinghong,Yunnan 666100,China;Xishuangbanna Key Laboratory of Dai Traditional Medicine and Southern Chinese Traditional Medicine,Jinghong,Yunnan 666100,China)

机构地区：[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666100 [2]西双版纳州傣药南药重点实验室,云南景洪666100

出　　处：《西北植物学报》2019年第10期1711-1717,共7页Acta Botanica Boreali-Occidentalia Sinica

基　　金：国家自然科学基金(81703638)

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。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 PALgenes 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.

关 键 词：龙血树 龙血竭 苯丙氨酸解氨酶 基因克隆 基源鉴定 

分 类 号：Q785[生物学—分子生物学] Q786