千层塔内生真菌分离鉴定与产石杉碱甲菌株诱变及分子检测
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摘要
本研究从新鲜千层塔茎段分离、纯化内生真菌；根据菌落形态和孢子等形态特征,结合核糖体基因居间序列(ITS序列)进行菌株鉴定。从千层塔的茎中分离和鉴定出4种内生真菌,分别为枝状枝孢霉、产黄青霉、尖孢镰刀菌和盾壳霉。并对4种内生真菌进行产石杉碱甲的筛选。结果表明：产黄青霉内生真菌SHB在适宜的实验条件下可以发酵合成宿主植物相同的活性成分—石杉碱甲。
以内生真菌SHB为出发菌株,采用微波、紫外线、亚硝酸和硫酸二乙酯对千层塔内生真菌孢子进行诱变。诱变孢子悬液通过在PDA平板继代培养2次,把单菌落孢子转接至PD培养液中进行发酵培养,利用高效液相色谱检测发酵液,筛选出一株遗传稳定,产石杉碱甲能力较高的高产菌株wB3。其发酵产物中石杉碱甲为原始菌株的3.04倍。
为提高千层塔产黄青霉属内生真菌液体发酵产石杉碱甲的产量,通过单因子和正交试验优化了真菌SHB液体发酵的条件。结果表明,千层塔内生真菌wB3液体发酵产石杉碱甲的适宜条件为：温度为28℃,起始pH为6.4,接种量为12%,种龄为48h,转速为160r／min和发酵时间为8d。在此条件下,石杉碱甲的产量可达4.761μg／mL。
千层塔内的内生真菌SHB和突变菌株wB3的发酵液中的菌丝体提取真菌基因组DNA,并应用随机引物扩增多态性DNA技术(Random Amplified Polymorphic DNA,RAPD)进行遗传分析和比较。结果表明：使用随机引物PCR扩增后,扩增产物经琼脂糖凝胶电泳检测发现s450、s452等引物的PCR产物中,在突变菌株wB3和原始菌株SHB之间可以找到明显的差异特征带,为产石杉碱甲功能基因的寻找奠定基础。
In present research the endophytic fungi were isolated and purificated from the stem segment of endophytic fungi of Huperzia serrata. The strains were identified by morphology together with similarity of internal transcribed spacer (ITS) sequence. There are 4 endophytic fungi isolated from Huperzia serrata. The further molecular and morphological identification indicated that these fungi were belonge to Cladosporium cladosporioides (Fres.) de Vries、Penicillium chrysogenum Thom、Fusarium oxysporum Schlechte. emend. Snyd.& Hans and Coniothyrium sporulosum (W. Gams & Domsch) van der Aa respectively. The 4 fungi were screened with fermentating Hup A. Results indicate that Huperzia serrata Endophytic fungi Penicillium chrysogenum Thom SHB can fermentate Hup A that is active ingredient existing in the host plant in suitable experimental conditions.
Mutagenesis of spores from endophytic fungi SHB of Hupezia Serrata was performed by using microwave, ultraviolet ray Co60, HNO2 and DES respectively. The mutated spores were cultivated for two generations on PDA medium, then the well-grown mature spores were transfered to the new PD medium for fermentation. To detecte Huperzine A, The fermentation fluid was purified with HPLC. An strain with stability and the highest level of Hup A mutated Endophytic fungi wB3 was selected. The Hup A is 3.04 doubles of the primitive fungi SHB'ferments.
In order to increase the production of Huperzine A in Huperzia serrata Endophytic fungi fermentation solution, the conditions of fermentation were studied through single factor test and orthogonal test. The results suggested some suitable conditionof fermentation of the fungi wB3 producing Huperzine A, such as culture temperature 28℃, initial pH 6.4, inoculation volume 12%, age of inoculation 48h, rotate speed 160r/min and fermentation time 8d. In this condition, the production of Huperzine A can reach 7.210μg/ml.
DNAs of Endophytic fungi SHB and mutatant wB3 of Huperzia Serrata were derived from hypha in ferment solution, and the genetic analyses were studied by using random amplified polymorphic DNA (RAPD) technique. The PCR producs of the primers s450、s452 can detect the character difference bands between the SHB and wB3, and the results established foundation for seeking the genetic mechanism of generation of Hup A.
以内生真菌SHB为出发菌株,采用微波、紫外线、亚硝酸和硫酸二乙酯对千层塔内生真菌孢子进行诱变。诱变孢子悬液通过在PDA平板继代培养2次,把单菌落孢子转接至PD培养液中进行发酵培养,利用高效液相色谱检测发酵液,筛选出一株遗传稳定,产石杉碱甲能力较高的高产菌株wB3。其发酵产物中石杉碱甲为原始菌株的3.04倍。
为提高千层塔产黄青霉属内生真菌液体发酵产石杉碱甲的产量,通过单因子和正交试验优化了真菌SHB液体发酵的条件。结果表明,千层塔内生真菌wB3液体发酵产石杉碱甲的适宜条件为：温度为28℃,起始pH为6.4,接种量为12%,种龄为48h,转速为160r／min和发酵时间为8d。在此条件下,石杉碱甲的产量可达4.761μg／mL。
千层塔内的内生真菌SHB和突变菌株wB3的发酵液中的菌丝体提取真菌基因组DNA,并应用随机引物扩增多态性DNA技术(Random Amplified Polymorphic DNA,RAPD)进行遗传分析和比较。结果表明：使用随机引物PCR扩增后,扩增产物经琼脂糖凝胶电泳检测发现s450、s452等引物的PCR产物中,在突变菌株wB3和原始菌株SHB之间可以找到明显的差异特征带,为产石杉碱甲功能基因的寻找奠定基础。
In present research the endophytic fungi were isolated and purificated from the stem segment of endophytic fungi of Huperzia serrata. The strains were identified by morphology together with similarity of internal transcribed spacer (ITS) sequence. There are 4 endophytic fungi isolated from Huperzia serrata. The further molecular and morphological identification indicated that these fungi were belonge to Cladosporium cladosporioides (Fres.) de Vries、Penicillium chrysogenum Thom、Fusarium oxysporum Schlechte. emend. Snyd.& Hans and Coniothyrium sporulosum (W. Gams & Domsch) van der Aa respectively. The 4 fungi were screened with fermentating Hup A. Results indicate that Huperzia serrata Endophytic fungi Penicillium chrysogenum Thom SHB can fermentate Hup A that is active ingredient existing in the host plant in suitable experimental conditions.
Mutagenesis of spores from endophytic fungi SHB of Hupezia Serrata was performed by using microwave, ultraviolet ray Co60, HNO2 and DES respectively. The mutated spores were cultivated for two generations on PDA medium, then the well-grown mature spores were transfered to the new PD medium for fermentation. To detecte Huperzine A, The fermentation fluid was purified with HPLC. An strain with stability and the highest level of Hup A mutated Endophytic fungi wB3 was selected. The Hup A is 3.04 doubles of the primitive fungi SHB'ferments.
In order to increase the production of Huperzine A in Huperzia serrata Endophytic fungi fermentation solution, the conditions of fermentation were studied through single factor test and orthogonal test. The results suggested some suitable conditionof fermentation of the fungi wB3 producing Huperzine A, such as culture temperature 28℃, initial pH 6.4, inoculation volume 12%, age of inoculation 48h, rotate speed 160r/min and fermentation time 8d. In this condition, the production of Huperzine A can reach 7.210μg/ml.
DNAs of Endophytic fungi SHB and mutatant wB3 of Huperzia Serrata were derived from hypha in ferment solution, and the genetic analyses were studied by using random amplified polymorphic DNA (RAPD) technique. The PCR producs of the primers s450、s452 can detect the character difference bands between the SHB and wB3, and the results established foundation for seeking the genetic mechanism of generation of Hup A.
引文
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