紫苏属药用植物的rDNA ITS区SNP分子标记与位点特异性PCR鉴别  

SNP marker and allele-specific diagnostic PCR for authenticating herbs of Perilla

作　　者：罗玉明[1] 张卫明[2] 丁小余[1] 沈洁[1] 保曙琳[1] 褚必海[1] 毛善国[1] 

机构地区：[1]南京师范大学生命科学学院江苏省生物多样性与生物技术重点实验室 [2]南京野生植物综合利用研究院,江苏南京210042

出　　处：《药学学报》2006年第9期840-845,共6页Acta Pharmaceutica Sinica

基　　金：国家"十五"科技攻关资助项目(2001BA502B05)

摘　　要：目的 分析单种属——紫苏属各变种间rDNA ITS区的序列以及存在的单核苷酸多态性（SNP）现象，设计出位点特异性PCR引物，用于紫苏属各变种间的分子标记鉴别。方法 对紫苏属各变种多个体的rDNA ITS区全序列进行了准确测定，运用Clustral X1．8，MEGA3．0进行排序并进行SNP分析，从而设计出鉴别各变种的等位基因位点特异性PCR鉴别引物。结果 紫苏属各变种（紫苏、白苏、鸡冠苏和耳齿紫苏等）的rDNA ITS区全序列共有615～618bp的长度，ITS1为233～235bp，5．8S为179bp，ITS2为203～204bp，GC含量为61．5％～61．9％。从rDNA ITS区碱基变异的整体情况来看，紫苏属各变种间不仅在非编码的转录间隔区ITS1和ITS2内存在非编码区单核苷酸多态性（ncSNP），而且在保守的5．8S编码区内也存在3个位点的单核苷酸多态性，即编码区SNP（cSNP），所有的SNP均只具2等位多态性。5．8S区cSNP的出现与产生该变异的变种出现的显著形态差异关联。本文还利用这些SNP位点设计出了鉴别紫苏属各变种的位点特异性PCR引物，无需测序即可对紫苏属的原植物及“苏子”、“苏叶”等药材进行有效准确的分子鉴别。结论 紫苏属药用植物rDNA ITS区存在的SNP可用作紫苏属各变种鉴别的分子标记。Aim To authenticate all the varieties of Perilla （single-species genus）, to analyze sequences of rDNA ITS regions and single nucleotide polymorphism （SNP） within them and based on these, to design allele-specific diagnostic PCR primers. Methods The rDNA ITS regions of the Perilla varieties were sequenced and analyzed by Clustal X 1.8, MEGA 3.0. Allele-specific diagnostic PCR primers that can authenticate all the Perilla varieties were designed based on SNPs loci. Results The length of rDNA ITS sequences of Perilla varieties ranged from 612 to 615 bp in size, including ITS1 （230 -232 bp）, 5.8S （179 bp） and ITS2 （203 -204 bp）. The GC content is about 61.5% -61.9%. There is not only SNPs in non-coding region ITS1 and ITS2 （ncSNP） , but also three coding SNPs （cSNP） loci in the conservative region of 5.8S. All the SNPs have only two allele loci polymorphism. The cSNP in 5.8S is related to the morphology variation among the varieties. Allele-specific diagnostic PCR primers have been designed according to SNPs loci to authenticate accurately all the seeds and leaves of Perilla varieties. Conclusion SNPs in rDNA ITS region can be used as an effective molecular markers to authenticate all the varieties of Perilla.

关 键 词：紫苏属 rDNA ITS区 单核苷酸多态性 位点特异性PCR 分子鉴别 

分 类 号：R931.5[医药卫生—生药学]