[关键词]
[摘要]
目的 克隆膜荚黄芪苯丙氨酸解氨酶(PAL)基因家族成员,分析它们在不同组织中的表达模式与毛蕊异黄酮葡萄糖苷含量变化,为揭示膜荚黄芪毛蕊异黄酮葡萄糖苷积累的分子机制提供理论依据。方法 以膜荚黄芪总RNA为模板,采用同源克隆法和RACE技术克隆PAL基因并进行生物信息学分析;利用实时荧光定量PCR技术测定PAL基因在根、茎、叶中的表达量;采用HPLC法测定了根、茎、叶中的毛蕊异黄酮葡萄糖苷含量。结果 从膜荚黄芪中克隆了3个PAL基因AmPAL1、AmPAL2、AmPAL3,Genbank登陆号分别为KY086279、KY086280、KY086281。AmPAL1、AmPAL2和AmPAL3的全长cDNA长度分别为2 508、2 401、2 498 bp,均含有2 157 bp的完整开放阅读框,编码718个氨基酸。蛋白质序列分析表明,它们均含典型的PAL酶活中心序列,与植物PAL蛋白同源,且与同属于豆科的植物PAL蛋白相似性最高。系统进化树表明,AmPAL1与AmPAL2和AmPAL3聚为不同的亚类。实时荧光定量PCR分析发现,这些基因在根、茎、叶中表达模式不同,在检测的所有组织中AmPAL1的表达量最高、AmPAL2的表达量次之、AmPAL3的表达量最低,只有AmPAL2的表达水平与毛蕊异黄酮葡萄糖苷积累变化一致(即根 > 茎 > 叶)。结论 从膜荚黄芪中克隆的AmPAL1、AmPAL2和AmPAL3是典型的PAL基因家族成员,推测它们在膜荚黄芪各组织发育过程中起着不同的作用,且AmPAL2可能参与了毛蕊异黄酮葡萄糖苷的积累。
[Key word]
[Abstract]
Objective To disclose the molecular mechanism of calycosin-7-O-β-D-glucoside (CG) accumulation in Astragalus membranaceus, we cloned PAL genes and analyzed the expression patterns of them and changes of CG contents in different tissues of A. membranaceus. Methods PAL genes were cloned with the methods of homology cloning and RACE technique using the total RNA as template and the analysis of bioinformatics on the cloned genes was carried out, gene expressions in root, stem, and leaf were determined with real-time PCR method, and CG content in root, stem, and leaf were analyzed by HPLC methods. Results Three PAL genes were cloned from A. membranaceus. The genbank accession number was KY086279 (AmPAL1), KY086280 (AmPAL2), and KY086281 (AmPAL3), respectively; The full-length cDNA of them was 2 508 bp, 2 401 bp, and 2 498 bp, respectively; And they all consisted of 2 157 bp open reading frame encoding 718 amino acids. Deduced AmPAL proteins had typical active sequences of PAL proteins, they were homology with other PAL proteins, and they shared the highest identities with PAL proteins of leguminous plants. Phylogenetic tree analysis showed AmPAL1 belonged to the different sub-class with the sub-class of AmPAL2 and AmPAL3. Real-time PCR analysis indicated that expression levels of AmPALs were different from each other, the expression level of AmPAL1 was the highest, the expression level of AmPAL2 was the next, and that of AmPAL3 was lowest in all detected tissues, and only the expression levels of AmPAL2 was similar to the changes of CG contents in different tissues (root > stem > leaf). Conclusion The cloned AmPAL1, AmPAL2, and AmPAL3 from A. membranaceus were typical genes of PAL, each might have different function in developing of different tissues, and AmPAL2 might involve in CG accumulation in different tissues.
[中图分类号]
R282.12
[基金项目]
国家自然科学基金资助项目"延边地区膜荚黄芪生物合成毛蕊异黄酮葡萄糖苷的分子机制研究"(21462044);国家自然科学基金资助项目"转PAL基因膜荚黄芪种质改良研究(30860036)";吉林省自然科学基金项目"膜荚黄芪cDNA文库构建与次生代谢功能基因挖掘"(201115228)