皱边石杉内生真菌资源遗传多样性的ISSR分析

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。

ISSR analysis on genetic diversity of endophytic fungi of Huperzia crispata

Objective To study the genetic diversity of endophytic fungi of Huperzia crispata using ISSR markers at molecular level, to establish the fingerprint for genetic differentiation, and to provide the reference for screening the endophytic fungi strains which could biosynthesize huperzine A (Hup A). Methods Endophytic fungi (100) isolated from H. crispata were selected as materials, their optimized ISSR reaction systems were established, and the genetic diversity was analyzed. TLC and HPLC were used to detect the fermentation products. Results Ten ISSR primers were screened to analyze the genetic diversity of endophytic fungi of H. crispata. A total of 3 975 bands were amplified and all of them were polymorphic bands. The genetic similarity (GS) among the tested genotypes ranged from 0.59 to 0.96, and the tested strains were classified into 11 groups at GS 0.64. At GS 0.67, group I could be classified into five subgroups. Primer UBC868 was used to amplify No.13 strain and DNA of H. crispata, and unambiguous bands were appeared at 500 and 200 bp. The fermentation products were detected and analyzed by TLC and HPLC and No.13 strain was found to produce Hup A same as the host plant H. crispata. Conclusion Higher genetic distance and wider genetic base exist among endophytic fungi of H. crispata. The same ISSR category fungus as No.13 strain is the most important potential screening and inducing mutation strain.