毛东霞, 郭丹丹, 吴玲玲, 田晓雪, 张胜翔, 刘陶, 马小魁*

(1.陕西师范大学生命科学学院/西北濒危药材资源开发国家工程实验室,西安710100;2.河南省疾病预防控制中心,郑州450000)

石油污染土壤中产苦参碱真菌的分离与鉴定

毛东霞1, 郭丹丹1, 吴玲玲2, 田晓雪1, 张胜翔1, 刘陶1, 马小魁1*

(1.陕西师范大学生命科学学院/西北濒危药材资源开发国家工程实验室,西安710100;2.河南省疾病预防控制中心,郑州450000)

采用微生物发酵法获取天然产物,是一项比植物提取法更为经济的环境友好技术。为建立一种微生物生产苦参碱的方法,本文从陕北油田石油污染土壤中分离、筛选和鉴定了一种能合成苦参碱的枝顶孢属真菌。首先采用薄层层析法、气相色谱-质谱联用法等分别确定了在所获真菌菌株发酵产物中含有苦参碱物质;使用光学显微镜观察发现,该菌株菌落呈白色绒毛状,呈圆形,菌丝分散,背面奶油色至赭黄色;菌丝无色,光滑且具隔膜,孢子囊梗直接从菌丝上长出,呈锥形,多单生;分生孢子无色,光滑,形成簇状或链状,长纺锤形,两头尖,未见厚垣孢子。内转录区间序列(internal transcribed sequence,ITS)扩增、测序后进行同源性比较发现,该菌株ITS序列与枝顶孢属的同源性高达99%。构建系统发育树表明,该真菌与枝顶孢属菌的遗传距离最近。综合形态学和遗传进化分析结果对此菌株进行了种属分类,确认这株能合成苦参碱物质的真菌应归属子囊菌门(Ascomycota)的枝顶孢属(Acremoniumsp.)。

苦参碱; 石油污染; 枝顶孢属; 薄层层析; 气相色谱-质谱联用; 邻接法

苦参碱(matrine)是喹诺里西啶类生物碱中的一类化合物[1],也是农业上常用的广谱杀虫剂,因其可以杀灭害虫而在农业上广受青睐[2]。据报道,苦参碱广泛存在于豆科植物中,是苦参、山豆根、白刺花等药用植物的主要活性成分之一[3]。苦参碱药理作用广泛,主要包括镇痛、抗感染、抗心律失常、抗肿瘤、抗病毒、免疫抑制及生物调节等[4],其中有关苦参碱抗肝损伤、抗肝纤维化、抗肿瘤、改善心血管系统作用等功效的研究备受关注[5]。

目前,获得苦参碱的方法主要包括溶剂提取法、离子交换法、大孔树脂吸附法、超临界流体萃取法、超声萃取法和半仿生提取法等[6-7]。其中溶剂提取、离子交换和树脂吸附法使用较多。但是,从植物组织中获得苦参碱往往步骤繁琐,操作温度高,流程长,生产效率低,杂质较多,产物损失大[8],因此其工业化程度很低,无法满足日益增长的市场需求。于是,为了高效获得大量的苦参碱产品,以满足其在医药、农业等领域不断增长的市场需求,建立一种以微生物发酵技术为基础的苦参碱生产方法体系迫在眉睫。

通过微生物发酵获得目的产物往往具有易控制、周期短、生产成本低、不受原料和季节限制、发酵产物较植物提取产物成分单一、有效成分易分离等优点,而且还易于通过基因工程等方法筛选改造高产菌株,进一步提高产物得率[9-10]。石油污染土壤中常含有多种环状有机化合物,如苯环类分子等污染,这些物质的存在可能对此类土壤中的微生物种类造成选择压力,进而形成可以转化或降解含环状有机化合物的微生物区系。苦参碱类生物碱多为环状分子结构,有专利报道枝顶孢属微生物可以转化合成苦参碱类物质。本文从筛选分离油田污染土壤中的微生物出发,以期从中筛选获得可以产苦参碱的真菌,为建立一种微生物合成苦参碱的微生物生产方法体系提供理论基础。

1 材料与方法

1.1 材料

1.1.1 材料来源 实验用原油来自中国石油长庆油田分公司;石油污染土壤采自陕北地区油田油井废弃钻井液储蓄池附近,用无菌采集袋收集并置于4 ℃冰箱中保存待用。

1.1.2 培养基 马铃薯葡萄糖固体及液体培养基(potato dextrose agar,PDA)配制参照一般培养基手册;石油富集固体培养基参照文献[11]配制;无机盐培养基参照文献[12]进行配制;甘油无机盐培养基是在上述无机盐培养基中按照10 g/L加入甘油组成。

1.1.3 主要试剂和仪器 苦参碱标准品(货号:MB6730;CAS号:519-02-8)购自上海源叶生物科技有限公司,其他有机试剂如石油醚、乙酸乙酯、氯仿均为市售分析纯。德国徕卡DM2500生物显微镜;日本岛津公司QP 2010气相色谱-质谱联用仪;北京六一仪器厂DYCP-31DN电泳仪。

1.2 污染土壤中真菌分离

将从我国陕西省陕北地区油田附近采得的污染土壤制成土壤悬液,稀释成不同梯度,在石油富集固体培养基平板上涂布,置于28 ℃培养箱中培养,每天观察。挑取其中长势较好的真菌菌丝于新的PDA固体培养基中进行划线分离,反复分离培养、驯化,直至得到菌落单一的纯化菌株。

1.3 产苦参碱真菌筛选

1.3.1 真菌发酵产物的薄层色谱检测 取真菌PDA固体斜面菌种,在无菌条件下接种于已灭菌的PDA液体培养基中,置于恒温振荡培养箱中,28 ℃、160 r/min培养后,作为种子液。将种子液按10%的接种量接入甘油无机盐培养基中,置于恒温振荡培养箱中,28 ℃、160 r/min发酵培养。培养完成后,用滤纸滤除真菌菌丝体,将滤液依次利用石油醚(30-60)、乙酸乙酯、氯仿、正丁醇等体积萃取,浓缩氯仿萃取相并风干氯仿,用一定量甲醇溶解待用。

按常规方法制备薄层层析(thin-layer chromatography,TLC)G硅胶薄层板(G板),活化备用。配置苦参碱标准品的甲醇溶液(质量浓度c=1 mg/mL),将真菌发酵液提取物与苦参碱标准品分别点样于G板上,放入V(氯仿)∶V(甲醇)∶V(浓氨水)=5∶0.6∶0.2的展开体系中展开,展开完成后,置于碘缸中显色,观察斑点位置,并计算各斑点的比移值(radio frequency value,Rf)。

1.3.2 真菌发酵产物的气相色谱-质谱联用分析

取1.3.1节中制备得到的真菌发酵液提取物5 mL,在转速为10 000 r/min下离心5 min,用无水硫酸钠对上清液除水,然后用0.22 μm微孔滤膜过滤去杂质,滤液用气相色谱-质谱联用(gas chromatograph-mass spectrometer,GC-MS)分析。色谱条件为岛津RTX-1MS(30 m×0.25 mm×0.25 μm)非极性石英毛细管柱,柱温130 ℃,进样口温度250 ℃,不分流进样,进样量为1 μL,载气为氦气,流速1 mL/min。柱温采用程序升温:130 ℃,不保留;130～220 ℃,50 ℃/min,保留2 min;220～280 ℃,20 ℃/min,保留15 min。质谱条件为离子源温度230 ℃,接触面温度280 ℃,扫描范围30～550 amu,EI源轰击电压70 eV[13]。

1.4 产苦参碱真菌鉴定

1.4.1 分离真菌的形态观察 用插片法[14]培养真菌,并在不同时间段取样制片,在显微镜下观察并记录真菌菌丝及孢子的形态。

1.4.2 真菌基因组提取及序列扩增 将筛选所得产苦参碱的真菌菌丝1～2 g放入灭菌的研钵中,用液氮研磨成粉末,使用真菌基因组DNA提取试剂盒提取该菌DNA,采用引物ITS1(5′-TCCGTAGGTGAACCTGCGG-3′)和ITS4(5′-TCCTCCGCTTATTGATATGC-3′)扩增内转录区间序列(internal transcribed sequence,ITS)。聚合酶链反应(polymerase chain reaction,PCR)体系为50 μL,反应体系为真菌DNA 5 μL,上、下游引物各1 μL,PCR混合液 25 μL,ddH2O 18 μL。反应条件:94 ℃预变性3 min,94 ℃变性30 s,56 ℃退火30 s,72 ℃延伸90 s,共35个循环;72 ℃充分延伸10 min后终止反应。对PCR产物进行琼脂糖凝胶电泳分析后,切取目的片段凝胶进行产物纯化回收,将回收的PCR产物送生工生物工程(上海)股份有限公司测序。

1.4.3 真菌同源性比较与遗传进化分析 登录美国国家生物技术信息中心(National Center of Biotechnology Information,NCBI),应用BLAST程序将测定的ITS序列与GenBank中的序列进行同源性比较。应用MEGA 5.0软件的Clustal W进行序列比对,采用邻接法(neighbor-joining method,NJ)构建系统发育树,自展数据集为1 000。

1.4.4 产苦参碱真菌的种属分类 根据所获真菌的形态结构,结合其遗传进化特征进行分析,对所获得的真菌进行种属鉴定。

2 结果与分析

2.1 微生物的分离纯化

从石油污染土壤中,通过富集、涂布及平板划线分离筛选,并经多次纯化后得到1株富集石油的固体培养基及PDA固体培养基上长势较好且菌落单一的真菌。

2.2 产苦参碱真菌的形态学鉴定

A：真菌的菌落形态图；B：真菌菌丝显微形态图.A: Colony morphology of fungus; B: Microscopy of fungal mycelium.图1 真菌的菌落形态及菌丝图Fig.1 Colony morphology and microscopy of mycelium of fungus

在PDA培养基平板中,可以看到该真菌呈现分散的绒毛状菌落(图1A),菌落呈圆形,白色,背面奶油色至赭黄色;菌丝无色,光滑且具隔膜;孢子囊梗直接从菌丝上长出,呈锥形多单生,分生孢子无色,光滑,形成簇状或链状,长纺锤形,两头尖,未见厚垣孢子(图1B)。查阅文献并比较，发现该真菌的菌落形态特征、菌丝和孢子的显微形态与枝顶孢属(Acremonium)、拟青霉属(Simplicillium)和头孢霉属(Cephalosporium)中的部分菌种较为相似[15-17]。

2.3 产苦参碱真菌筛选

2.3.1 薄层色谱检测 经过薄层层析的定性分析确定,所获真菌的发酵液提取物(图2B)出现与苦参碱标准品(图2A)颜色和比移值相同的斑点,Rf值为0.68。于是，初步确定该真菌可以发酵产生苦参碱。

A:苦参碱标准品;B:真菌提取物.A: Marine standard; B: Fungal extract.图2 真菌发酵液的薄层层析结果Fig.2 Thin-layer chromatography result of fungal fermented liquid

2.3.2 GC-MS分析结果 经过气相色谱-质谱联用仪分析确定,真菌发酵液提取物的色谱图(图3B)在11.929 min时出现了与苦参碱标准品色谱保留时间(retention time,RT)11.932 min相近的色谱峰(图3A)。

苦参碱标准品在全离子扫描(selected ion monitoring,SIM)模式下可以得到 4个丰度较高的碎片离子,质荷比(m/z)分别为248.2、247.2、150.1和205.1[18]。GC-MS分析结果多次重复表明,在所获真菌发酵液提取物色谱图(图3B)中,保留时间为11.929 min的色谱峰所对应的离子碎片质谱图(图3D)和苦参碱标准品的离子碎片质谱图(图3C)相类似,并且和质谱数据库中苦参碱的特征离子碎片质谱图相匹配(图3E)。这些结果进一步确定了该真菌的发酵液提取物中含有苦参碱成分,说明所获真菌在液体培养中可以合成苦参碱。

2.4 分离真菌的ITS序列分析与鉴定结果

经琼脂糖凝胶电泳检测,PCR扩增产物的片段长度约为600 bp(图4A)。核苷酸序列测定结果显示,ITS片段长度为585 bp,GenBank登录号为KF803999。 用BLAST软件比对结果显示,该菌的ITS序列与Acremoniumsp. SA-NEX5(JX021664.1)、Acremoniumsp. CB90(HM98995.1)的同源性高达99%。

A:苦参碱标准品气相色谱图;B:真菌发酵液提取物气相色谱图;C:苦参碱色谱峰的离子碎片质谱图;D:真菌发酵液提取物的离子碎片质谱图;E:质谱数据库中的苦参碱的质谱图. A: Gas chromatogram of matrine standard; B: Gas chromatogram of the fungus extracts; C: Fragmentation pattern of matrine standard; D: Fragmentation pattern of the fungus extracts; E: Fragmentation pattern in the database of mass spectrum.图3 苦参碱标准品及真菌发酵液提取物的气相色谱-质谱图Fig.3 Gas chromatography-mass spectrometry of matrine standard and the fungus extracts

A：PCR扩增ITS rDNA序列[M:5 000 bp的分子标记;(1～3):PCR产物]；B：基于该真菌rDNA ITS序列的系统发育树. A: Amplification of fungal ITS rDNA by PCR [M: 5 000 bp marker; (1-3): PCR product]; B: Phylogenetic tree based on rDNA ITS region sequences of the fungus.图4 真菌PCR扩增ITS rDNA序列及系统发育树Fig.4 Phylogenetic tree and amplification of fungal ITS rDNA by PCR of the fungus

基于该真菌ITS序列与GenBank中同源性序列构建的NJ系统发育树(图4B)发现,该真菌与枝顶孢属的Acremoniumsp. SA-NEX5(JX021664.1)处于同一分支上,而且自检支持率高达99%～100%。系统发育分析结果表明,该真菌与Acremoniumsp. SA-NEX5(JX021664.1)的遗传距离最近。

综合形态观察、分子鉴定结果及已有文献报道,将该真菌归属于枝顶孢属真菌,命名为Acremoniumsp. P0997,并保藏于中国典型培养物保藏中心,保藏号为CCTCCM 2013569。

3 讨论

经TLC检测及GC-MS分析确定所获真菌的发酵液提取物中含有苦参碱,表明所获真菌可以合成苦参碱。此外,GC-MS同时检测了所分离真菌的菌丝体提取物,确定该菌菌丝体中也含有苦参碱物质成分。结果显示,所获真菌可能在真菌菌丝内以及发酵液中均可以合成苦参碱,但对于枝顶孢属菌产生苦参碱的代谢途径及其相关合成机制还需进一步研究。

枝顶孢属真菌(Acremoniumsp.)是一种常见真菌,分布广泛,目前报道的约有 130余种,主要可分为腐生、植物寄生和自生3种。此类真菌的次生代谢产物中包括酚类、萜类、环肽和蒽醌类等,多具有良好的生物活性[19]。姚磊等[19]从腐殖质中分离得到1株枝顶孢属菌,并在其代谢产物中检测到了姜糖酯B、姜糖酯C、D-甘露醇、酒渣碱和枝顶孢素F;余永涛等[20]在1株枝顶孢属菌的发酵液中检测到了苦参碱成分。石油污染土壤中常发现有环状有机分子污染,本研究据此推测此类污染土壤中可能有可以合成苦参碱类生物碱的微生物分布。研究首次从石油污染土壤中分离得到了产苦参碱的真菌,这为建立苦参碱的微生物发酵生产方式提供了新的依据。

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Isolation and identification of a matrine-producing fungal strain from petroleum-contaminated soil.

Journal of Zhejiang University (Agric. & Life Sci.)， 2015，41(5):586-592

Mao Dongxia1, Guo Dandan1, Wu Lingling2, Tian Xiaoxue1, Zhang Shengxiang1, Liu Tao1, Ma Xiaokui1*

(1.CollegeofLifeSciences,ShaanxiNormalUniversity/NationalEngineeringLaboratoryforResourceDevelopmentofEndangeredCrudeDrugsinNorthwestofChina,Xi’an710100,China; 2.HenanProvincialCenterforDiseasePreventionandControl,Zhengzhou450000,China)

Matrine is one of the quinolizidine alkaloids. It is widely used as broad-spectrum agricultural pesticides, which exists mainly in roots of leguminous plants. Matrine has great prospects of research and development as it has very extensive pharmacological effects, mainly including analgesia, anti-inflammatory, anti-arrhythmic, antitumor, antiviral, immunosuppression and biological control. The raw material of obtaining matrine is mainly the traditional Chinese medicine sophora. But the extraction method of matrine from plants often has its drawbacks, such as the cumbersome extracting steps, the high extraction temperature, the complex operation process, and using plenty of organic solutions. In addition, the production efficiency is not high enough, and the product has many impurities. Also, this production process often results in a large number of loss in resource. As a consequence, the matrine production derived from plants has challenged with the requirements of industrial production, as it cannot meet the increasing market demand. The production of matrine by microbial fermentation, however, would be an alternative for the production obtained from plants, and must have more advantages over the extracting from plants, with less labor and time consumed. However, there are no many reports concerning microbes with capacity to produce matrine. Hence, it is very necessary to obtain matrine-producing microorganisms for establishing the bio-production by microbial fermentation.

The study presented hereby was carried out to isolate and identify the fungus with the ability to synthesize matrine from petroleum-contaminated soil samples. The fungal isolate with the capability to synthesize matrine was classified by morphological and molecular biological methods. The fungal isolates were firstly obtained from samples of oil-contaminated soil in North Shaanxi, China, through spread-plate technique and plate streaking. The chemical compositions in fermentation broth of the obtained fungus were detected by thin-layer chromatography and gas chromatograph-mass spectrometer. Subsequently, the isolate was observed under the light microscope and the classification of the isolate was performed preliminarily according to the morphological characteristics. The internal transcribed sequence (ITS) of the isolated fungus was amplified and sequenced. Homology comparison was then conducted and the phylogenetic tree was established based on the sequences of other strains from previous reports. Finally, the matrine-producing fungus was classified according to the combined results of both morphology observation and phylogenetic analysis.

The experimental results showed that a fungal strain was isolated from the samples of petroleum-contaminated soil. The results of both thin-layer chromatography and gas chromatograph-mass spectrometer indicated that the fungus had the capacity to produce matrine. The morphological observation showed that the colony of this fungus was white villous, circular, abaxially cream to reddish brown, with mycelia scattered. The hyphae was colorless, smooth and septate. The sporangiophores were conical, mostly solitary, showing to grow directly from the hyphae. The conidia were colorless, smooth, showing clusters or chains, with long spindle shaped, pointed at both ends and there was no chlamydospore observed. These morphological characteristics of this fungal strain were highly similar to that ofAcremoniumsp. andSimplicilliumsp. reported previously. The results of the fungal homology comparison showed that the fungus was more closely related toAcremoniumsp. with 99% homology and the phylogenetic analysis indicated that there was no significant difference in genetic distance between this strain andAcremonium. Integrating these above results, the fungus should belong toAcremonium, with the name ofAcremoniumsp. P0997 in this study.

As there are not so many reports aboutAcremoniumsp. to produce matrine, our study has provided much information about this species with the capacity to produce matrine. These results would contribute to establishing matrine production by microbes. Therefore, this study concerning forAcremoniumsp. with the capacity to synthesize matrine has a promising application prospect for matrine production by microbes. Further researches are ongoing about the metabolites of the genus in our laboratory. The study would provide an important scientific basis for establishing matrine production by microorganisms.

matrine; petroleum-contaminated;Acremoniumsp.; thin-layer chromatography; gas chromatograph-mass spectrometer; neighbor-joining method

陕西省科技计划资助项目(693102)；中央高校科研业务费特别支持项目(1301030208)。

联系方式:毛东霞(http://orcid.org/0000-0001-9852-298X)，E-mail:ylainexn@163.com

2015-03-03;接受日期(Accepted):2015-07-01;网络出版日期(Published online):2015-09-01

X 13; Q 93

A

*通信作者(Corresponding author):马小魁(http://orcid.org/0000-0002-7842-7632)，Tel:+86-29-85310266;E-mail:biomarkuis@gmail.com

URL:http://www.cnki.net/kcms/detail/33.1247.s.20150901.0954.002.html