【作者】 金志雄；

【导师】 万永继；

【作者基本信息】 西南大学 ， 微生物学， 2011， 博士

【摘要】 植物内生菌(endophyte),是指部分或全部生活史中寄居在健康植物组织内部,对宿主植物不造成明显损伤,合成大量活性产物的微生物。1993年Stierle等首次从红豆杉(Taxus brevifolia)植物内分离得到一株能合成抗癌物质紫杉醇的内生真菌(Taxomyces andreanae),这也提示内生菌能够合成宿主植物的次生代谢产物。近年来,研究者先后从数十种药用植物内生菌的培养物中得到了与宿主植物或其它植物相同或相似的活性成分,包括生物碱类(alkaloids)、甾类(steroids)、萜类(terpenoids)、醌类(quinones)、黄酮类(flavonoid)和肽类(peptides)等。这预示着内生菌将成为药用植物的潜在替代资源,为解决某些药用植物过度消耗、制药工业所致环境污染和化学合成药的副作用等问题提供了新途径。盾叶薯蓣(Dioscorea zingiberensis C. H. Wright)为薯蓣科(Dioscoreaceae)薯蓣属(Dioscorea)单子叶植物,多年生缠绕性藤本,是提取激素类药物的原材料薯蓣皂苷元的主要药源植物。前人研究显示,盾叶薯蓣植物内生真菌Fusarium菌株代谢产物具有较强的抗菌和刺激宿主细胞合成薯蓣皂苷元的能力,这预示相关内生菌在盾叶薯蓣植物的种植、加工和薯蓣皂苷元的生物转化等方面有一定的应用价值。本研究选取砂质和粘质土壤栽培盾叶薯蓣植物的地下茎核心区组织,贴片培养,共分离出48株内生菌,进行了形态结构、生理生化及相关分子生物学鉴定；Bacillus为两种土壤栽培植株的内生优势菌群,Bacillus subtilis所占比例最高。研究发现,内生Fusarium sp. SWM3菌株的发酵液蛋白提取物具有广谱的抗G+和G-细菌的活性并对癌细胞具有显著的毒性。同时,本研究首次获得一株产薯蓣皂苷元类似物的内生B. subtilis SWB8菌株,从其代谢产物中分离并鉴定出薯蓣皂苷元类似物和p-1,3-1,4-葡聚糖酶,二者对癌细胞具有显著的选择性细胞毒性,其中,p-1,3-1,4-葡聚糖酶还具有广谱的抗细菌和真菌的活性。1.盾叶薯蓣内生菌的分离和鉴定选取砂质和粘质土壤栽培植株的地下茎核心区组织,用纯培养的方法分离并鉴定出48株菌,包括46株细菌和2株真菌,分别属于Bacillus、Staphylococcus、Mesorihizobium、Tsukamurella、Citrobacter、Fusarium6个属。沙质土壤栽培植株的内生细菌数量明显高于粘质土壤植株(33：13),其中B. subtilis为两种土壤栽培植株的内生优势菌种(18：48)。内生B.subtilis菌株可生长于蛋白胨、高氏(Gause)和查氏(Czapek)等培养基,且对Staphylococcus等非B. subtilis分离株具有强的抑制作用。有趣的是,内生Staphylococcus细菌同样占有很高比例(9：48),并且多与B. subtilis分布于同一个植株组织的水平断面上。上述内生菌株的成功分离,填补了盾叶薯蓣植物内生细菌研究的空白,为研究有益菌株及盾叶薯蓣植物与内生菌之间的关系打下了良好的基础。2. Fusarium sp. SWM3菌株代谢产物及生物活性的研究Fusarium sp. SWM3菌株发酵液蛋白提取物显示了广谱的抗G+和G-细菌的活性,尤其对金黄色葡萄球菌(17.64±0.6mm)和粪肠球菌(19±1.0mm)抑制效果最为明显,但对白色假丝酵母菌和新型隐球菌无抑制作用。甲基噻唑基四唑(MTT)和流式细胞(FCM)分析显示,蛋白提取物具有明显的诱导人肺腺癌细胞(A549)凋亡的能力,其IC5o值为10.9μg/mL。A549细胞的凋亡与蛋白提取物的浓度成正相关,随着提取物诱导浓度(1.2、2.4、3.6、4.8、6.0μg/mL)的增加,A549细胞凋亡率由0.76%增加至4.29%,而晚期凋亡和坏死率由对照的8.42%增加至20.9%,增幅显著。蛋白提取物处理的A549细胞表现出明显的形态变化,如细胞间隙变宽,细胞变圆、皱缩、脱落等,但未见细胞碎裂和胞质起泡现象。Fusarium sp. SWM3菌株有可能成为一种潜在的抗生素或抗肿瘤药物的来源。3. Bacillus subtilis SWB8菌株代谢产物及生物活性的研究Bacillus subtilis SWB8分离株为革兰氏阳性芽孢杆菌,形成充满内生孢子的单股长链状菌丝或孢子囊结构,需氧或兼性需氧生长,能够利用硝酸盐或亚硝酸盐,生长温度10～50℃(适温32℃),pH6.0-9.0,耐受9.0%NaCl,液体发酵时产生丰富的泡沫。1)β-1,3-1,4-葡聚糖酶的鉴定和生物活性基于有机物萃取、硫酸铵盐析、凝胶色谱、SDS-PAGE和质谱分析,B. subtilis SWB8发酵液蛋白提取物被分离出5个保留时间分别为31.46、32.36、41.26、43.48和52.6 min的蛋白条带(70、56、40、30和25 kDa)；3个蛋白组分(40、30和25 kDa)的部分氨基酸序列与B. cereus (GB:AAK16547)β-1,3-1,4-葡聚糖酶的氨基酸序列相同,被鉴定为β-1,3-1,4-葡聚糖酶。3个蛋白组分拥有相同的氨基酸序列(SAQTYGYGLYEVR),其它氨基酸序列位于B. cereus菌株p-1,3-1,4-葡聚糖酶Ala71到Arg118之间,这提示它们来自于相同β-1,3-1,4-葡聚糖酶前体蛋白的裂解碎片,分别拥有41.26、43.48和52.6 min的保留时间。β-1,3-1,4-葡聚糖酶具有广谱的抗细菌和真菌的活性,尤其对粪肠球菌(21.8±0.3mm)、大肠杆菌(19.0±1.0mm)、白色假丝酵母菌(22.5±0.6mm)和新型隐球菌(24±1 mm)等临床致病菌具有较强的抑制作用。MTT和FCM分析显示,p-1,3-1,4-葡聚糖酶有显著的抑制A549细胞增殖的能力,其IC5o值是11.5μg/mL。A549细胞的早期凋亡率与p-1,3-1,4-葡聚糖酶浓度成正相关,细胞的早期凋亡率由正常对照的4.43%增加至19.2μg/mL的43.1%,而晚期凋亡和坏死率由2.57%增加至14.7%,增幅显著。在β-1,3-1,4-葡聚糖酶的诱导下,A549细胞表现出明显的形态变化,如细胞间隙变宽、变圆、起泡、皱缩和核凝结等。相反,β-1,3-1,4-葡聚糖酶对人骨髓间质干细胞(MSCs)并无明显作用。结果显示,B. subtilis SWB8菌株及代谢产物β-1,3-1,4-葡聚糖酶有可能成为一个潜在的抗菌和抗癌药物的来源。2)薯蓣皂苷元类似物的鉴定和生物活性B. subtilis SWB8发酵液有机相萃取物在200-280 nm和304 nm处具有与标准薯蓣皂苷元相同的紫外吸收峰,并显示出相似的波形。薄层层析显示,萃取物与薯蓣皂苷元Rf值相近(Rf=0.49、0.46)。红外光谱数据显示,萃取物和薯蓣皂苷元在4500～500 cm-1的范围内存在大量相同的官能团。质谱数据显示,萃取物和薯蓣皂苷元有相同的先驱离子(m/z437[M+Na]+、453[M+H2O+Na]+、475、495和569)和碎片离子(m/z 158.6和302.6),提示萃取物具有薯蓣皂苷元相同的骨架结构。然而,与薯蓣皂苷元的碎片离子(m/z 182.5、214.4、225.5、252.7、270.8、347.7、396.5和425.2)相比较,萃取物也产生了不同的碎片离子(m/z 305.3、321.2、337.1、358.2、371.9、377.8和379.8),这提示二者结构存在部分差异。氢谱数据显示,萃取物和薯蓣皂苷元在1.5-5.5 ppm范围内有相对应的氢的信号。综合上述数据,B. subtilis SWB8发酵液有机萃取物被鉴定为薯蓣皂苷元类似物(简称类似物),糖苷配基(骨架结构)的分子式为C27H42O3。抗菌检测显示,类似物具有一定的抑制细菌生长的能力。MTT试验显示,薯蓣皂苷元类似物对A549细胞有显著的选择性抑制作用。A549细胞的抑制率与类似物的浓度成正相关。类似物超过一定的浓度(17.78μg/mL)对MSCs细胞具有一定的细胞毒性,它对MSCs细胞的IC50值(28.8μg/mL)远大于对A549细胞的IC50值(13.09μg/mL).FCM分析显示,用不同浓度类似物(0、2.54、7.62和12.7μg/mL)诱导细胞,A549细胞的早期凋亡率由对照组的3.59%增加至24.4%,而晚期凋亡和坏死率仅由5.44%增加至10.2%。相反,MSCs细胞变化不明显。共聚焦显微镜观察,类似物诱导的A549细胞发生细胞凋亡的形态变化,如细胞间隙变宽、变圆、起泡、皱缩和核凝结等。薯蓣皂苷元类似物主要以诱导凋亡的方式抑制A549细胞,也暗示薯蓣皂苷元类似物可能是一种理想的抗癌药物和植物甾体类物质的替代资源。B. subtilis SWB8菌株在发酵盾叶薯蓣植物生产薯蓣皂苷元方面有重要的应用价值,另一方面,对内生细菌(原核生物)合成宿主植物代谢产物及类似物的机制的研究也具有重要的价值。更多还原

【Abstract】 Endophytes, the microorganisms colonized in the internal tissues of living plants without causing apparent symptoms of disease, synthesize numerous bioactive compounds. In 1993, Stierle and colleagues firstly isolated paclitaxel-producing endophytic fungi from Pacific yew lead to a concept that there is possibility endophytes may produce secondary metabolites of plant. Resent years, more and more bioactive natural products with the same or similar construct of host metabolites, including alkaloids, steroids, terpenoids, quinones, flavonoids, peptides, etc. were obtained from endophytes which isolated from dozens of medicinal plants. These researches indicate that endophytes have gradually become potential alternative sources of medicinal plants. It also provides a new way against over-harvesting of medicinal plants, environmental pollution of drug industry and adverse effects of chemosynthesis drugs.Dioscorea zingiberensis C. H. Wright that belongs to the family Dioscoreaceae, is the medicinal twining monocotyledonae served as the main source of diosgenin that is an important precursor of steroidal drugs. Previously studies demonstrated that endophytic Fusarium strains from D zingiberensis produce active metabolites and stimulate the diosgenin synthesis of host cells which shows the potential value that the endophytes applied in planting, pre-processing and biological transformation of diosgenin.Endophytes were isolated from inner tissues of D. zingiberensis tuber which were planted in the sandy and clay soil, respectively. Bacillus subtilis isolates were the predominant strains in the inner tissues of the plants from either types of soil. All of the 48 endophytic strains were identified by using morphological, physiological, biochemical and molecular biological methods. We have isolated a Fusarium sp. strain SWM3, and the protein extracts showed broad spectrum antibacterial activity. More interestingly, we have isolated a B. subtilis strain SWB8 that producing diosgenin analogue which is the host secondary metabolite. Both of the (3-1,3-1,4-glucanase and the diosgenin analogue secreted by strain SWB8 showed selective cytotoxicity to cancer cell lines. Besides, the bacterial P-1,3-1,4-glucanase also showed spectrum antimicrobial activities against both bacteria and fungi.1. Isolation and identification of endophytesThe inner tissues of D. zingiberensis tuber which planted in sandy and clay soil were collected. Based on pure culture method,48 strains were isolated, including 46 bacteria and 2 fungi, belonged to genus Bacillus, Staphylococcus, Mesorihizobium, Tsukamurella, Citrobacter and Fusarium, respectively. The results showed that the number of endophytes isolated in sandy soil plants was much more than that in clay soil plants (33:13). B. subtilis isolates were the predominant strains in the inner tissues of the plant (18:48). These isolates could grow on peptone, Gause and Czapek medium, and significantly inhibited the growth of other isolates, for instance, the Staphylococcus. But interestingly, the number of Staphylococcus isolates was more than prediction (9:48), and these strains usually co-distributed with B. subtilis isolates in the same cross-section of the plant tissues. These findings made a breakthrough of endophytic bacteria in Dioscorea and also laid a foundation for the further studying of the interaction between endophytes and their plant host.2. The biologic activity of protein extracts of endophytic Fusarium sp. SWM3The protein extracts of Fusarium sp. SWM3 strain shows broad spectrum antibacterial activity. Especially, Staphylococcus aureus (17.6±0.6 mm) and Enterococcus faecalis (19±1.0 mm) were very sensitive to the extracts. In contrast, the inhibition effect could not be observed in Candida albicans and Cryptococcus neoformans.Methyl thiazolyl tetrazolium (MTT) and flow cytometry (FCM) assay showed that the protein extracts induced apoptosis of A549 cells with IC50 value of 10.9μg/mL. The percentage of apoptotic A549 cells treated with the extracts was significantly increased from 0.76% of the control to 4.29% in a dose dependent manner. Meanwhile, the late apoptotic or necrotic ratio was also significantly increased from 8.42% to 20.9%. Morphological changes, such as intercellular space widening, cell rounding, cytoplasm condensation, crimple and abscission were observed but without cytoplasmic blebbing. Fusarium sp. SWM3 strain could be a potential source of desirable antibiotics or anticancer compounds.3. Biologic activity of the metabolites of endophytic Bacillus subtilis SWB8B. subtilis SWB8 was a gram positive, endospore-forming aerobe or facultative aerobe with growth temperature range from 10℃to 50℃(optimum temperature,32℃), pH range from 6 to 9, salinity range 0.5-9.0%(NaCl) and formed rich foam in liquid medium.1) Identification and bioactivity of (3-1.3-1,4-glucanaseBased on gel permeation chromatography (GPC) and SDS-PAGE methods, protein extracts of B. subtilis strain SWB8 showing five peaks with the retention time of 31.46, 32.36,41.26,43.48 and 52.6 min in GPC were separated to five different gel bands of approximately 70,56,40,30,25 kDa. By mass spectrometry, the partial amino acid sequences of 40,30 and 25 kDa proteins showed the same composition with P-1,3-1,4-glucanase of B. cereus (GB:AAK16547). All the three separated proteins were identified as P-1,3-1,4-glucanase, suggesting that the (3-1,3-1,4-glucanase precursor would be cleaved into three fragments of 40,30 or 25 kDa with the retention time of 41.26,43.48 or 52.6 min in GPC, respectively.β-1,3-1,4-glucanase showed broad antimicrobial spectrum activity. E. faecalis (21.8±0.3 mm), E. coli (19.0±1.0 mm), C. albicans (22.5±0.6 mm) and C. neoformans (24±1 mm) were more sensitive toβ-1,3-1,4-glucanase.MTT and FCM assay showed significantly anticancer activity against A549 cells ofβ-1,3-1,4-glucanase with IC50 value of 11.5μg/mL. The percentage of apoptotic A549 cells treated with different concentrations ofβ-1,3-1,4-glucanase was significantly increased from 4.43% of the control to 43.1% of 19.2μg/mL in a dose dependent manner. Meanwhile, the late apoptotic or necrotic ratio was also increased from 2.57% to 14.7%. Morphological changes, such as intercellular space widening, shrinking, cytoplasmic blebbing, cytoplasm condensation and abscission were observed. In contrast, these changes could not be observed in the treated human bone marrow mesenchymal stem cells (MSCs).Perhaps, the B. subtilis strain SWB8 or itsβ-1,3-1,4-glucanase product could be a potential source of desirable antimicrobial and anticancer compounds.2) Identification and bioactivity of diosgenin analogueThe organic extract of B. subtilis SWB8 showed the same UV absorption peak in the range of 200-280 nm and 304 nm and similar waveform as the standard diosgenin. Thin-laver chromatography showed the similar Rf values of the extract (0.49) and standard diosgenin (0.46). Infrared (IR) spectra showed that the extract and standard diosgenin exhibited almost the same stretching and bending vibration and thus revealed the same functional groups in the region from 4500 to 500 cm-1. Electrospray ionization mass spectrum (ESI-MS) of the extract produced an major ion peak of m/z 437.1 [M+Na]+, which is consistent with 437.3 [M+Na]+of diosgenin, both of the samples formed precursor ion groups of m/z 453[M+H2O+Na]+,475,495,569 and fragment ions of m/z 158.6 and 302.6, suggesting the same molecular structural moiety. However, extract formed other different fragment ions of m/z 305.3,321.2,337.1, 358.2,371.9,377.8 and 379.8 in comparing with diosgenin fragment ions of m/z 182.5, 214.4,225.5,252.7,270.8,347.7,396.5 and 425.2. These differences are probably due to the same molecular skeleton but different latent chains. Nuclear magnetic resonance (’H-NMR) spectroscopy analysis showed that the extract and standard diosgenin produced the same hydrogen signals in the range of 1-5.5 ppm, suggesting the similar molecular structure between the analogue and the standard diosgenin.These results validated that B. subtilis SWB8 extract possesses the similar molecular structure of diosgenin and could be designated as diosgenin analogue (C27H42O3). Diosgenin analogue showed certainly antibacterial activity.The results of MTT assay showed selective cytotoxicity of diosgenin analogue that significantly acted on A549 cells. The inhibition ratio of A549 cells indicated positive correlation with the increasing concentrations of diosgenin analogue. A549 cells were intensely inhibited by the extract with an IC50 value of 13.09μg/mL, compared with the higher tolerance of MSCs cells that the IC50 value of 28.81μg/mLFCM analysis showed that the percentage of apoptotic A549 cells which treated with different concentrations of the extract (0,2.54,7.62 and 12.7μg/mL) was significantly increased from 3.59% to 24.4% in a dose dependent manner, but the late apoptotic or necrotic ratio was only increased from 5.44% to 10.2%. In contrast, these changes could not be observed in MSCs cells.Laser scanning confocal microscopy (LSCM) analysis indicated that the apoptosis-like morphologic changes, such as cell shrink, cytoplasmic blebbing and condensation were occurred on induced A549 cells. These results reconfirmed that diosgenin analogue inhibited A549 cells mainly by inducing apoptosis. Perhaps, diosgenin analogue from B. subtilis SWB8 could be a potential source of desirable anticancer compounds and alternative plant steroids.B. subtilis SWB8 has an application value in pre-processing of diosgenin production. On the other hand, it also could be a valuable model to study the mechanism that endophytic bacteria synthesizing the host secondary metabolite.更多还原