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目的采用ISSR标记技术从分子水平探讨皱边石杉内生真菌资源的遗传多样性,建立皱边石杉内生真菌资源遗传分化指纹图谱,为筛选可产石杉碱甲的目标菌株提供快捷的判别依据。方法以从皱边石杉中分离的100株内生真菌为材料,建立其ISSR优化反应体系并分析其遗传多样性;TLC、HPLC检测发酵产物。结果优化筛选的10条ISSR引物对皱边石杉内生真菌进行遗传多样性分析,共扩增出3 975条清晰条带,多态性条带占100%。遗传相似系数为0.59～0.96,在0.64水平,皱边石杉内生真菌可分为11类;在0.67水平,第I类又可分为5个亚类。采用引物UBC868对13号菌株及皱边石杉基因组DNA扩增,在500、200 bp均具有清晰的扩增条带,发酵产物经TLC和HPLC检测,发现13号菌株与宿主植物皱边石杉同样可产生石杉碱甲。结论皱边石杉内生真菌资源遗传多样性高,遗传距离较远,遗传基础较宽,与13号菌株同属一类的内生真菌可作为石杉碱甲生产的潜力菌株进行重点筛选和诱变。
OBJECTIVE: To explore the genetic diversity of endophytic fungi from the genus Ruxin by means of ISSR technique at molecular level and to establish the genetic differentiation fingerprinting of endophytic fungi from the genus Huperzia, which can provide a quick and reliable basis for the screening of the target strains producing Huperzine A. . Methods 100 endophytic fungi isolated from the genus Huperzia were used as materials to establish their ISSR-optimized reaction system and analyze their genetic diversity. The fermentation products were detected by TLC and HPLC. Results Ten ISSR primers were selected to analyze the genetic diversity of endophytic fungi from the genus Rubus. A total of 3 975 clear bands were amplified and the polymorphic bands accounted for 100%. The genetic similarity coefficients ranged from 0.59 to 0.96. At the level of 0.64, the endophytic fungi could be divided into 11 groups. At 0.67 level, class I could be divided into 5 subgroups. The UBC868 primer was used to amplify genomic DNA of strain No.13 and Gentiana scabra, with clear amplification bands at 500 and 200 bp. The fermentation products were detected by TLC and HPLC. Huperzine A can also be produced. Conclusion Endophytic fungi from the genus Rutilus have higher genetic diversity, longer genetic distance and broader genetic basis. Endophytic fungi belonging to the same genus as strain 13 can be selected and mutagenized as potential strains for the production of huperzine A .