皱边石杉内生真菌的ISSR分析及高产HupA功能内生菌的筛选鉴定
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摘要
石杉碱甲是一种高效、可逆、特异性强、毒性低的乙酰胆碱酯酶抑制剂,是治疗阿尔茨海默病的首选药物成分,其主要来源为从石杉科植物中直接提取,但在石杉科植物中的含量低,一般为0.2mg/g左右。石杉科植物为小型低等蕨类植物,有2个属,约150种,稀散分布于世界各地,我国有28种,4个变种。一般高10-30cm,主要生长于海拔300-2600m的原始次生林地,伴生于竹子、杉树、苔藓、红槛木、油茶等植物根系周围,生长环境条件阴暗、潮湿,生长缓慢、资源匮乏。寻找新的石杉碱甲来源,缓解当前用药需求就备受人们关注。由于真菌具有易培养、易控制、生产成本低、生长快等优点,容易实现大规模工业化生产,从植物内生菌中寻找和发现新的活性化合物是当前植物内生菌研究的一个热点。本论文在前期研究的基础上,运用微生物学、现代生物技术和生物信息学等相关知识,对皱边石杉内生真菌进行分离纯化、菌丝体DNA的提取,内生真菌ISSR的遗传多样性分析,内生真菌的种属鉴定,利用现代色谱技术对内生真菌发酵产物进行鉴定,进而利用高速逆流色谱制备高纯度石杉碱甲以及目标高产菌株发酵优化工艺研究,主要研究结果如下：
(1)获得了皱边石杉内生真菌DNA快速有效提取方法。采用氯化苄法提取其总DNA,对比研究了石英砂研磨与超声波振荡2种破壁方式提取DNA的效能。结果表明,采用超声波只需10分钟破壁,较石英砂研磨更快捷、更高效。提取的DNA经琼脂糖凝胶电泳、ITS序列扩增,表明超声波破壁法提取的DNA较石英砂研磨法更能获得良好的PCR扩增结果。超声波提取法可作为植物内生真茵DNA大规模快速提取方法,尤其对质地坚硬的菌落较石英砂研磨更有效。
(2)获得了皱边石杉内生真菌ISSR-PCR最优反应体系、内生真菌遗传多样性及与宿主的亲缘关系。皱边石杉内生真菌ISSR-PCR最优反应体系为：25μL反应体系中,10×PCR Buffer(Mg2+浓度为2.0mmol/L)2.5μL, dNTPs(10mmol/L)0.6μL,引物(10pmol/μL)2.0μL, Taq E(1U/μL)1.5μL, DNA模板(10ng/μL)3μL,无菌ddH2O15.4μL。可获得稳定性高,重复性好,背景清晰的ISSR扩增结果。通过优化筛选的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3975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,可分为11类；在0.67水平,第Ⅰ类又可分为5个亚类,说明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。以UBC868对皱边石杉植物样本DNA进行扩增,在500bp、350bp、200bp处均有清晰扩增条带,其中13号菌株在500bp、200bp均有清晰的扩增条带,可获知13号菌株与宿主植物存在某些基因序列相同的片段,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。
(3)对从皱边石杉分离出的内生真菌发酵产物进行薄层色谱和高效液相色谱分析,并对目标产物进行了高速逆流制备。通过薄层色谱分析,初步推断1、4、5等38个菌株代谢产物中可能含有石杉碱甲或与其结构相似的化学成分。高效液相色谱进一步检测,13、87号菌具有良好的生产石杉碱甲或其类似物的功能,其产量分别为199.6μg/L和18.64μg/L。高速逆流色谱对13、87号菌株发酵液进行检测,分离出了高纯度的目标物质——石杉碱甲。高速逆流分离制备方法为：正己烷-正丁醇-水(3：1：2)为溶剂体系,上相为固定相,下相为流动相,流速为2mL/min,转速为800r/min,进样量为5mL。筛选出了NKA-2大孔吸附树脂为静态吸附的优选树脂,为大规模生产石杉碱甲提供了技术参考。
(4)对薄层色谱检测可能生产石杉碱甲或与其结构与之相似的化学成分的菌株,进一步ISSR分析,去除重复样本得到19个菌株,在形态学鉴定的基础上进行核糖体rDNA ITS序列鉴定, www.ncbi.nlm.nih.gov在线BLAST,与GenBank、EMBL、DDBJ、PDB多个核酸数据库进行同源性比对,绘制系统发育树。用p-distance模式计算遗传距离,邻位相连法进行进化距离分析,确定了19个菌株的种属,其中13号菌为胶孢炭疽病菌(Colletotrichum gloeosporioides)的一种,87号菌为草酸青霉菌(Penicillium oxalicum)的一种。将5、11、12、13、15、16、19、28、37、38、40、44、51、57、65、77、87、88、1号菌株的ITS序列提交GeneBank,获得Accession No分别为：JX230994、JX230995、JX230996、JX230997、JX230998、JX230999、JX231000、 JX231001、JX231002、JX231003、JX231004、JX231005、JX231006、JX231007、 JX231008、JX231009、JX231010、JX231011、JX231012。
(5)以13号菌为供试菌株,对其发酵生产石杉碱甲工艺进行了优化筛选,其最佳发酵条件为：1L马铃薯液体培养体系中加20g麦芽糖,pH值为自然值(5.0),摇床转速150r／min,28℃恒温培养84h,在放大培养后用HPLC检测含量达0.1996gg/mL。
Huperzine A is one of the most important medicine on Alzheimer's disease for it's powerful selective and low toxicity inhibitor of acetylcholinesterase. The main source is extracted from Huperziaceae plant and its concentration is very low, the content is about0.2mg/g. Huperziaceae plant have2genera, about150species, scattered distribution in the world, China has28species,4varieties. It general10-30cm high, major growth in the elevation of300-2600m's in original secondary forest, bamboo, pine, was accompanied by moss, rubrum and other plants around the roots, the growth conditions is dark and damp, mostly grow slowly and demand of especial living conditions and environments. The search for new sources of HupA to alleviate the need for medication has attracted widespread attention. It's a research focus to discover the new active compounds from endophytic fungi for its easy culture, easy control, low cost and short cycle, because of the large scale industrial production, the application of endophytic fungi prospect is very broad. Based on earlier researches, the studies applied Microbiology, Biotechnology and Bioinformatics knowledges, focus on five aspects in this papers as follows:rapid and effective method for isolating total DNA from Huperzia crispata's endophyte fungi, genetic diversity analyzed by ISSR markers, endophyte fungi species classification and identification, chromatographic analysis of fermentation liquid, high purity HupA preparation by HSCCC and fermentation process optimization target.
(1) A time-saving, simple and efficient total DNA extraction method for Huperzia crispata's endophyte fungi mycelia has been developed, In this study, used benzyl chloride method to isolate total DNA of endophyte fungi and comparied quartz sand grind method with ultrasonic break method. Results showed that the DNA extraction efficiency and quality obtained by ultrasonic break method was superior to quartz sand grind method, and it only needed10minutes. The genomic DNA extracted by ultrasonic break treatment was digested by RNaseA and amplified with ITS primers, agarose gel electrophoresis checking results showed that this treatment could obtain good quality PCR effects. It can use for large scale samples a specially for firm texture fungi colony.
(2) The suitable optimal reaction system was established for ISSR-PCR of endophytic fungi of Huperzia crispata, namely25μL reation system containing IO×PCR buffer (2.0mmol/L Mg2+)2.5μL, dNTPs(10mmol/L)0.6μL, primer(10pmol/μL)2.0μL, Taq E(1U/μL)1.5μL, templet DNA(10ng/μL)3μL, aseptic ddH2O15.4μL.10reliable ISSR primers of endophytic fungi of Huperzia crispata analyzed by ISSR markers, based on10primers,99endophytic fungi generated3795clear and reproducible polymorphic bands, of which accounting for100%(PPB=100%). The genetic similarity (GS) among the tested genotypes ranged from0.59to0.96, the test strains were classified into11groups in the GS of0.64. At the GS was0.67, No. I group could be classified into5subgroups. Results showed that higher genetic distance and wider genetic base were existed among endophytic fungi of Huperzia crispata. With primer UBC868to amplify DNA of No.13strain, unambiguous bands were appeared at500bp and200bp. The fermentations products of No.13strain were detected and analyzed by TLC/HPLC and found that No.13strain could produce HupA with the same as host plant Huperzia crispata, the same ISSR category fungi as No.13strain were the most important potential screening and induced mutation strains.
(3) TLC and HPLC analyses were developed on endophytic fungi fermentation liquid of Huperzia crispata, the target product (HupA) was preparated by HSCCC After TLC detection, it can be preliminary estimated that38strains (No.1, NO.4and et al.) fermentation liquid may have HupA or its similar structural of chemical composition. HPLC detection results showed that No.13and No.87endophytic fungi strain have fermentation production of HupA and its similar compounds, the output is199.6μg/L and18.64μg/L. The products of No.13and No.87strain liquid fermentation isolated by HSCCC were high purity HupA. HSCCC method:with a two-phase solvent system composed of hexane-butyl alcoho-water(3:1:2, V/V). The upper phase was used as the stationary phase and the lower phase was used as the mobile phase at a flow rate of2mL/min. The experimental conditions were controlled at800r/min and the sample volume was5mL. The NKA-2macroporous adsorption resin was selected as the best resin for static adsorption, it can be used as large scale HupA industrial production.
(4) On the TLC detection to infer the possible production of HupA and its similar structure of the chemical composition, further ISSR analysesd, after removed duplicate samples obtained19strains, the morphological identification based on rDNA ITS DNA sequence identification, www.ncbi.nlm.nih.gov online BLAST, homology compared with GenBank, EMBL DDBJ and PDB multiple nucleic acid database, then analysesd NJ phylogenetic tree. Used p-distance model to calculate genetic distance, used neighbor joining method for evolutionary distance analysis.19strains of the genus were classified and identified, No.13strain is Colletotrichum gloeosporioides, No.87strain is Penicillium oxalicum.No.5,11,12,13,15,16,19,28,37,38,40,44,51,57,65,77,87,88and No.1strains of ITS GeneBank Accession No. sequences are presented, respectively JX230994, JX230995, JX230996, JX230997, JX230998, JX230999, JX231000, JX231001, JX231002, JX231003, JX231004, JX231005, JX231006, JX231007, JX231008, JX231009, JX231010, JX231011and JX231012.
(5) High purity HupA preparation and fermentation process were optimizated by using No.13strain, the best fermentation process condition is follow:1L potato liquid culture system with20g maltose, The experimental conditions were controlled at pH(5.0), shaking speed800r/min and28℃Incubation for84hours, the output by HPLC detection was0.1996μg/mL
(1)获得了皱边石杉内生真菌DNA快速有效提取方法。采用氯化苄法提取其总DNA,对比研究了石英砂研磨与超声波振荡2种破壁方式提取DNA的效能。结果表明,采用超声波只需10分钟破壁,较石英砂研磨更快捷、更高效。提取的DNA经琼脂糖凝胶电泳、ITS序列扩增,表明超声波破壁法提取的DNA较石英砂研磨法更能获得良好的PCR扩增结果。超声波提取法可作为植物内生真茵DNA大规模快速提取方法,尤其对质地坚硬的菌落较石英砂研磨更有效。
(2)获得了皱边石杉内生真菌ISSR-PCR最优反应体系、内生真菌遗传多样性及与宿主的亲缘关系。皱边石杉内生真菌ISSR-PCR最优反应体系为：25μL反应体系中,10×PCR Buffer(Mg2+浓度为2.0mmol/L)2.5μL, dNTPs(10mmol/L)0.6μL,引物(10pmol/μL)2.0μL, Taq E(1U/μL)1.5μL, DNA模板(10ng/μL)3μL,无菌ddH2O15.4μL。可获得稳定性高,重复性好,背景清晰的ISSR扩增结果。通过优化筛选的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3975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,可分为11类；在0.67水平,第Ⅰ类又可分为5个亚类,说明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。以UBC868对皱边石杉植物样本DNA进行扩增,在500bp、350bp、200bp处均有清晰扩增条带,其中13号菌株在500bp、200bp均有清晰的扩增条带,可获知13号菌株与宿主植物存在某些基因序列相同的片段,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。
(3)对从皱边石杉分离出的内生真菌发酵产物进行薄层色谱和高效液相色谱分析,并对目标产物进行了高速逆流制备。通过薄层色谱分析,初步推断1、4、5等38个菌株代谢产物中可能含有石杉碱甲或与其结构相似的化学成分。高效液相色谱进一步检测,13、87号菌具有良好的生产石杉碱甲或其类似物的功能,其产量分别为199.6μg/L和18.64μg/L。高速逆流色谱对13、87号菌株发酵液进行检测,分离出了高纯度的目标物质——石杉碱甲。高速逆流分离制备方法为：正己烷-正丁醇-水(3：1：2)为溶剂体系,上相为固定相,下相为流动相,流速为2mL/min,转速为800r/min,进样量为5mL。筛选出了NKA-2大孔吸附树脂为静态吸附的优选树脂,为大规模生产石杉碱甲提供了技术参考。
(4)对薄层色谱检测可能生产石杉碱甲或与其结构与之相似的化学成分的菌株,进一步ISSR分析,去除重复样本得到19个菌株,在形态学鉴定的基础上进行核糖体rDNA ITS序列鉴定, www.ncbi.nlm.nih.gov在线BLAST,与GenBank、EMBL、DDBJ、PDB多个核酸数据库进行同源性比对,绘制系统发育树。用p-distance模式计算遗传距离,邻位相连法进行进化距离分析,确定了19个菌株的种属,其中13号菌为胶孢炭疽病菌(Colletotrichum gloeosporioides)的一种,87号菌为草酸青霉菌(Penicillium oxalicum)的一种。将5、11、12、13、15、16、19、28、37、38、40、44、51、57、65、77、87、88、1号菌株的ITS序列提交GeneBank,获得Accession No分别为：JX230994、JX230995、JX230996、JX230997、JX230998、JX230999、JX231000、 JX231001、JX231002、JX231003、JX231004、JX231005、JX231006、JX231007、 JX231008、JX231009、JX231010、JX231011、JX231012。
(5)以13号菌为供试菌株,对其发酵生产石杉碱甲工艺进行了优化筛选,其最佳发酵条件为：1L马铃薯液体培养体系中加20g麦芽糖,pH值为自然值(5.0),摇床转速150r／min,28℃恒温培养84h,在放大培养后用HPLC检测含量达0.1996gg/mL。
Huperzine A is one of the most important medicine on Alzheimer's disease for it's powerful selective and low toxicity inhibitor of acetylcholinesterase. The main source is extracted from Huperziaceae plant and its concentration is very low, the content is about0.2mg/g. Huperziaceae plant have2genera, about150species, scattered distribution in the world, China has28species,4varieties. It general10-30cm high, major growth in the elevation of300-2600m's in original secondary forest, bamboo, pine, was accompanied by moss, rubrum and other plants around the roots, the growth conditions is dark and damp, mostly grow slowly and demand of especial living conditions and environments. The search for new sources of HupA to alleviate the need for medication has attracted widespread attention. It's a research focus to discover the new active compounds from endophytic fungi for its easy culture, easy control, low cost and short cycle, because of the large scale industrial production, the application of endophytic fungi prospect is very broad. Based on earlier researches, the studies applied Microbiology, Biotechnology and Bioinformatics knowledges, focus on five aspects in this papers as follows:rapid and effective method for isolating total DNA from Huperzia crispata's endophyte fungi, genetic diversity analyzed by ISSR markers, endophyte fungi species classification and identification, chromatographic analysis of fermentation liquid, high purity HupA preparation by HSCCC and fermentation process optimization target.
(1) A time-saving, simple and efficient total DNA extraction method for Huperzia crispata's endophyte fungi mycelia has been developed, In this study, used benzyl chloride method to isolate total DNA of endophyte fungi and comparied quartz sand grind method with ultrasonic break method. Results showed that the DNA extraction efficiency and quality obtained by ultrasonic break method was superior to quartz sand grind method, and it only needed10minutes. The genomic DNA extracted by ultrasonic break treatment was digested by RNaseA and amplified with ITS primers, agarose gel electrophoresis checking results showed that this treatment could obtain good quality PCR effects. It can use for large scale samples a specially for firm texture fungi colony.
(2) The suitable optimal reaction system was established for ISSR-PCR of endophytic fungi of Huperzia crispata, namely25μL reation system containing IO×PCR buffer (2.0mmol/L Mg2+)2.5μL, dNTPs(10mmol/L)0.6μL, primer(10pmol/μL)2.0μL, Taq E(1U/μL)1.5μL, templet DNA(10ng/μL)3μL, aseptic ddH2O15.4μL.10reliable ISSR primers of endophytic fungi of Huperzia crispata analyzed by ISSR markers, based on10primers,99endophytic fungi generated3795clear and reproducible polymorphic bands, of which accounting for100%(PPB=100%). The genetic similarity (GS) among the tested genotypes ranged from0.59to0.96, the test strains were classified into11groups in the GS of0.64. At the GS was0.67, No. I group could be classified into5subgroups. Results showed that higher genetic distance and wider genetic base were existed among endophytic fungi of Huperzia crispata. With primer UBC868to amplify DNA of No.13strain, unambiguous bands were appeared at500bp and200bp. The fermentations products of No.13strain were detected and analyzed by TLC/HPLC and found that No.13strain could produce HupA with the same as host plant Huperzia crispata, the same ISSR category fungi as No.13strain were the most important potential screening and induced mutation strains.
(3) TLC and HPLC analyses were developed on endophytic fungi fermentation liquid of Huperzia crispata, the target product (HupA) was preparated by HSCCC After TLC detection, it can be preliminary estimated that38strains (No.1, NO.4and et al.) fermentation liquid may have HupA or its similar structural of chemical composition. HPLC detection results showed that No.13and No.87endophytic fungi strain have fermentation production of HupA and its similar compounds, the output is199.6μg/L and18.64μg/L. The products of No.13and No.87strain liquid fermentation isolated by HSCCC were high purity HupA. HSCCC method:with a two-phase solvent system composed of hexane-butyl alcoho-water(3:1:2, V/V). The upper phase was used as the stationary phase and the lower phase was used as the mobile phase at a flow rate of2mL/min. The experimental conditions were controlled at800r/min and the sample volume was5mL. The NKA-2macroporous adsorption resin was selected as the best resin for static adsorption, it can be used as large scale HupA industrial production.
(4) On the TLC detection to infer the possible production of HupA and its similar structure of the chemical composition, further ISSR analysesd, after removed duplicate samples obtained19strains, the morphological identification based on rDNA ITS DNA sequence identification, www.ncbi.nlm.nih.gov online BLAST, homology compared with GenBank, EMBL DDBJ and PDB multiple nucleic acid database, then analysesd NJ phylogenetic tree. Used p-distance model to calculate genetic distance, used neighbor joining method for evolutionary distance analysis.19strains of the genus were classified and identified, No.13strain is Colletotrichum gloeosporioides, No.87strain is Penicillium oxalicum.No.5,11,12,13,15,16,19,28,37,38,40,44,51,57,65,77,87,88and No.1strains of ITS GeneBank Accession No. sequences are presented, respectively JX230994, JX230995, JX230996, JX230997, JX230998, JX230999, JX231000, JX231001, JX231002, JX231003, JX231004, JX231005, JX231006, JX231007, JX231008, JX231009, JX231010, JX231011and JX231012.
(5) High purity HupA preparation and fermentation process were optimizated by using No.13strain, the best fermentation process condition is follow:1L potato liquid culture system with20g maltose, The experimental conditions were controlled at pH(5.0), shaking speed800r/min and28℃Incubation for84hours, the output by HPLC detection was0.1996μg/mL
引文
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