基于ITS序列差异建立G-四链体DNA传感器鉴别太子参品种的方法研究

ITS sequence with high evolution rate provides plentiful variation sites and information sites, and possesses intraspecific polymorphism.As research, there are different variations in ITS sequence among the different varieties of Pseudotstellaria heterophylla. G-quadruplex, a particular four chain spiral structure formed of G-rich nucleic sequences, has received more and more attentions in the field of sensor design in recent years. In this project, the ITS sequence of the authentic Jiangsu heterophylla is taken as standard, and the "Zhenshen No.1" from Fujian Zherong is taken as an example. Its ITS sequence is compared with the standard, and the sequence containing specific variation sites is considered as target-gene.According to the catalytic activity of the G-quadruplex DNAzyme,a label-free G-quadruplex DNAzyme sensor is designed through combining the hairpin structure of molecular beacon with the novel split mode of G-quadruplex. And the sensor is supposed to recognize the target-gene specifically. In addition, oxided graphene nanoparticle has different binding force with the single-stranded, double-stranded DNA and G-quadruplex, and meanwhile some organic fluorescent molecules can specifically recognize the G-quadruplex structure. The properties are utilized in the design of the label-free G-quadruplex fluorescent sensor for the purpose of low background and high specificity. After that, the molecular biology techniques, such as plant genomic DNA extration, PCR amplification and so on, are utilized in the establishment of G-quadruplex DNA sensor for identifying the varieties of heterophylla. And the research may lay a foundation for the development of a new analytical method for Traditional Chinese Medicine.

ITS序列进化速率较快, 提供丰富的变异位点和信息位点，存在种内多态性。研究发现，太子参不同品种在ITS序列存在着特异的变异位点。G-四链体是由富 G核酸序列形成的独特四链螺旋结构，近年来在传感器设计领域受到了越来越多的关注。本项目以道地江苏太子参的ITS序列为标准，以福建柘荣"柘参1号"为例，分析其ITS序列存在的变异位点，确定靶基因。根据 G-四链体DNA酶的催化活性，结合分子灯塔发夹结构的特异性与G-四链体新型裂分模式，设计特异识别靶基因的非标记型G-四链体DNA酶传感器。另结合氧化石墨烯纳米粒子对单链、双链、G-四链体不同结构的DNA结合力不同，利用特异识别G-四链体结构的有机荧光分子，设计背景信号低的、特异的、非标记型 G-四链体荧光传感器。结合植物基因组DNA提取、PCR 扩增等分子生物技术，建立鉴定太子参混杂品种的G-四链体DNA传感器，为中药鉴定提供新的分析方法奠定基础。

ITS序列提供丰富的变异位点和信息位点，存在种内多态性，太子参不同品种在ITS序列存在着特异的变异位点。本研究根据目标太子参特异的变异位点，结合G-四链体的独特四链结构及其结合hemin形成的类过氧化酶催化活性，构建四个应用于识别目标太子参品种的DNA传感器。（1）按照1：1：1：1 的新型G-四链体裂分模式，结合G-四链体类过氧化物酶的催化活性，构建了一个识别目标太子参的猝灭型可视化传感器，实验发现该传感器耗时短、费用低、具有较好的选择性。（2）实验发现喹哪啶红能够特异识别平行构象的G-四链体结构，结合氧化石墨烯纳米粒子对单链、双链、G-四链体不同结构DNA的结合力不同，构建了一个识别目标太子参的G-四链体DNA荧光传感器。结果发现氧化石墨烯的引入可以降低体系的背景信号，同时该传感器表现出很好的选择性，但灵敏度不够。（3）据报道，Fe3O4磁性氧化石墨烯纳米粒子对不同DNA结构如单链、双链、G-四链体构象的结合力不同，同时应用其磁性分离能力，构建了一个识别目标太子参的磁性氧化石墨烯-G-四链体DNA酶荧光传感器。结果发现，该DNA酶传感器具有很好的灵敏度，对鉴别与目标太子参有三个及以上碱基差异的其他地区太子参有很好的选择性；另外，但在实际样品的检测方面仍需进一步的研究工作。（4）滚环扩增技术是新近发展起来的一种恒温核酸扩增方法，可通过环状DNA模板的体外核酸恒温扩增实现对靶核酸的信号放大。本项目基于超支滚环扩增技术构建了一个识别目标太子参的荧光传感器。结果发现，该传感器具有很好的灵敏度，同时对鉴别目标太子参和其他产地的太子参具有很好的选择性。.总之，基于太子参品种ITS序列的差异，结合G-四链体、荧光分子、石墨烯纳米粒子和超支滚环扩增技术构建的这四个核酸传感器具有较好的选择性和灵敏度，可进一步开发为中药品种鉴定的核酸试剂盒。已发表论文2篇，申请专利1个。