一种特殊虫草与冬虫夏草的表型和DNA条形码特征比较分析及鉴别方法研究

Comparative Study on Phenotype and DNA Barcode Characteristics Between The Special Cordyceps and Ophionocordyceps sinensis(BerK.)Sacc.

目的:通过对子座和虫体部分进行表型与DNA条形码序列分析,探讨一种发现于四川的特殊冬虫夏草(以下简称特殊虫草)与常见冬虫夏草的区别。方法:体视显微镜观察虫草样品的外部特征;聚合酶链式反应(PCR)扩增得到真菌部位内转录间隔区(ITS)序列和宿主蝙蝠蛾昆虫的细胞色素氧化酶Ⅰ(COⅠ)序列;利用CodonCode Aligner和MEGA软件进行序列分析,建立邻接(NJ)系统进化树;对ITS序列进行限制性内切酶图谱分析,寻找具有鉴别意义的酶切位点,并通过PCR-限制性片段长度多态性(RFLP)鉴别进行验证。结果:特殊虫草与常见冬虫夏草相比,虫体头部的颜色明显不同;子座真菌的ITS序列存在7个差异位点,虫体COⅠ序列存在56个差异位点,基于ITS序列及COⅠ序列的NJ系统进化树分析均显示,两者不能聚为一支;在两者差异区域,冬虫夏草存在1个XhoⅠ的酶切位点,因此其ITS序列的PCR产物可被限制性内切酶XhoⅠ切割为2个片段,特殊虫草不能被XhoⅠ切割,所以酶切前后的PCR产物在琼脂糖凝胶图上没有变化,通过该方法可对两者进行鉴别。结论:发现了一种特殊虫草,其与常见冬虫夏草的真菌子座和宿主蝙蝠蛾昆虫部分均有明显不同,并为鉴别两者提供了有效方法。

Objective:To analyze the differences between the special cordyceps found in Sichuan Province and the typical O.sinensis based on the phenotype and DNA barcode sequence analysis of the stroma and host insect.Methods:The morphological characteristics were observed by the stereomicroscope.The internal transcribed spacer(ITS)sequence and the cytochrome oxidaseⅠ(COⅠ)sequence of the host insect were amplified by polymerase chain reaction(PCR).CodonCode Aligner and MEGA software were used for sequence analysis to establish neighbor-joining (NJ) phylogenetic tree. The restriction enzyme map of the ITS sequence was analyzed to find out the restriction sites of ITS sequences, and the identification was verified by PCR-restriction fragment length polymorphism (PCR-RFLP). Results: The color of the head of the host insects was apparently different between O. sinensis and the special cordyceps. There are 7 different sites within the ITS sequence of the fungus and 56 within the COⅠ sequence of the insects. The NJ tree based on ITS and COⅠ showed that the O. sinensis and the special cordyceps cannot be clustered as an individual branch separately. In the different regions of O. sinensis, there was one XhoⅠ site, so the PCR products of the ITS sequence could be cut into two fragments by the restrictive enzyme Xho Ⅰ. There was only one Xho Ⅰ site within ITS region of O. sinensis but not the special cordyceps, so the PCR products could be cut into two fragments by XhoⅠ while that of the special cordyceps could not be. Conclusion: A special cordyceps was found, which is dramatically different from O. sinensis both in the fungus and in the insect body. The method for identifying the special cordyceps from O. sinensis was also established.