石油污染土壤中產(chǎn)苦參堿真菌的分離與鑒定

毛東霞, 郭丹丹, 吳玲玲, 田曉雪, 張勝翔, 劉陶, 馬小魁*

(1.陜西師范大學生命科學學院/西北瀕危藥材資源開發(fā)國家工程實驗室,西安710100;2.河南省疾病預(yù)防控制中心,鄭州450000)

石油污染土壤中產(chǎn)苦參堿真菌的分離與鑒定

毛東霞1, 郭丹丹1, 吳玲玲2, 田曉雪1, 張勝翔1, 劉陶1, 馬小魁1*

(1.陜西師范大學生命科學學院/西北瀕危藥材資源開發(fā)國家工程實驗室,西安710100;2.河南省疾病預(yù)防控制中心,鄭州450000)

采用微生物發(fā)酵法獲取天然產(chǎn)物,是一項比植物提取法更為經(jīng)濟的環(huán)境友好技術(shù)。為建立一種微生物生產(chǎn)苦參堿的方法,本文從陜北油田石油污染土壤中分離、篩選和鑒定了一種能合成苦參堿的枝頂孢屬真菌。首先采用薄層層析法、氣相色譜-質(zhì)譜聯(lián)用法等分別確定了在所獲真菌菌株發(fā)酵產(chǎn)物中含有苦參堿物質(zhì);使用光學顯微鏡觀察發(fā)現(xiàn),該菌株菌落呈白色絨毛狀,呈圓形,菌絲分散,背面奶油色至赭黃色;菌絲無色,光滑且具隔膜,孢子囊梗直接從菌絲上長出,呈錐形,多單生;分生孢子無色,光滑,形成簇狀或鏈狀,長紡錘形,兩頭尖,未見厚垣孢子。內(nèi)轉(zhuǎn)錄區(qū)間序列(internal transcribed sequence,ITS)擴增、測序后進行同源性比較發(fā)現(xiàn),該菌株ITS序列與枝頂孢屬的同源性高達99%。構(gòu)建系統(tǒng)發(fā)育樹表明,該真菌與枝頂孢屬菌的遺傳距離最近。綜合形態(tài)學和遺傳進化分析結(jié)果對此菌株進行了種屬分類,確認這株能合成苦參堿物質(zhì)的真菌應(yīng)歸屬子囊菌門(Ascomycota)的枝頂孢屬(Acremoniumsp.)。

苦參堿; 石油污染; 枝頂孢屬; 薄層層析; 氣相色譜-質(zhì)譜聯(lián)用; 鄰接法

苦參堿(matrine)是喹諾里西啶類生物堿中的一類化合物[1],也是農(nóng)業(yè)上常用的廣譜殺蟲劑,因其可以殺滅害蟲而在農(nóng)業(yè)上廣受青睞[2]。據(jù)報道,苦參堿廣泛存在于豆科植物中,是苦參、山豆根、白刺花等藥用植物的主要活性成分之一[3]。苦參堿藥理作用廣泛,主要包括鎮(zhèn)痛、抗感染、抗心律失常、抗腫瘤、抗病毒、免疫抑制及生物調(diào)節(jié)等[4],其中有關(guān)苦參堿抗肝損傷、抗肝纖維化、抗腫瘤、改善心血管系統(tǒng)作用等功效的研究備受關(guān)注[5]。

目前,獲得苦參堿的方法主要包括溶劑提取法、離子交換法、大孔樹脂吸附法、超臨界流體萃取法、超聲萃取法和半仿生提取法等[6-7]。其中溶劑提取、離子交換和樹脂吸附法使用較多。但是,從植物組織中獲得苦參堿往往步驟繁瑣,操作溫度高,流程長,生產(chǎn)效率低,雜質(zhì)較多,產(chǎn)物損失大[8],因此其工業(yè)化程度很低,無法滿足日益增長的市場需求。于是,為了高效獲得大量的苦參堿產(chǎn)品,以滿足其在醫(yī)藥、農(nóng)業(yè)等領(lǐng)域不斷增長的市場需求,建立一種以微生物發(fā)酵技術(shù)為基礎(chǔ)的苦參堿生產(chǎn)方法體系迫在眉睫。

通過微生物發(fā)酵獲得目的產(chǎn)物往往具有易控制、周期短、生產(chǎn)成本低、不受原料和季節(jié)限制、發(fā)酵產(chǎn)物較植物提取產(chǎn)物成分單一、有效成分易分離等優(yōu)點,而且還易于通過基因工程等方法篩選改造高產(chǎn)菌株,進一步提高產(chǎn)物得率[9-10]。石油污染土壤中常含有多種環(huán)狀有機化合物,如苯環(huán)類分子等污染,這些物質(zhì)的存在可能對此類土壤中的微生物種類造成選擇壓力,進而形成可以轉(zhuǎn)化或降解含環(huán)狀有機化合物的微生物區(qū)系?？鄥A類生物堿多為環(huán)狀分子結(jié)構(gòu),有專利報道枝頂孢屬微生物可以轉(zhuǎn)化合成苦參堿類物質(zhì)。本文從篩選分離油田污染土壤中的微生物出發(fā),以期從中篩選獲得可以產(chǎn)苦參堿的真菌,為建立一種微生物合成苦參堿的微生物生產(chǎn)方法體系提供理論基礎(chǔ)。

1 材料與方法

1.1 材料

1.1.1 材料來源 實驗用原油來自中國石油長慶油田分公司;石油污染土壤采自陜北地區(qū)油田油井廢棄鉆井液儲蓄池附近,用無菌采集袋收集并置于4 ℃冰箱中保存待用。

1.1.2 培養(yǎng)基 馬鈴薯葡萄糖固體及液體培養(yǎng)基(potato dextrose agar,PDA)配制參照一般培養(yǎng)基手冊;石油富集固體培養(yǎng)基參照文獻[11]配制;無機鹽培養(yǎng)基參照文獻[12]進行配制;甘油無機鹽培養(yǎng)基是在上述無機鹽培養(yǎng)基中按照10 g/L加入甘油組成。

1.1.3 主要試劑和儀器 苦參堿標準品(貨號:MB6730;CAS號:519-02-8)購自上海源葉生物科技有限公司,其他有機試劑如石油醚、乙酸乙酯、氯仿均為市售分析純。德國徠卡DM2500生物顯微鏡;日本島津公司QP 2010氣相色譜-質(zhì)譜聯(lián)用儀;北京六一儀器廠DYCP-31DN電泳儀。

1.2 污染土壤中真菌分離

將從我國陜西省陜北地區(qū)油田附近采得的污染土壤制成土壤懸液,稀釋成不同梯度,在石油富集固體培養(yǎng)基平板上涂布,置于28 ℃培養(yǎng)箱中培養(yǎng),每天觀察。挑取其中長勢較好的真菌菌絲于新的PDA固體培養(yǎng)基中進行劃線分離,反復(fù)分離培養(yǎng)、馴化,直至得到菌落單一的純化菌株。

1.3 產(chǎn)苦參堿真菌篩選

1.3.1 真菌發(fā)酵產(chǎn)物的薄層色譜檢測 取真菌PDA固體斜面菌種,在無菌條件下接種于已滅菌的PDA液體培養(yǎng)基中,置于恒溫振蕩培養(yǎng)箱中,28 ℃、160 r/min培養(yǎng)后,作為種子液。將種子液按10%的接種量接入甘油無機鹽培養(yǎng)基中,置于恒溫振蕩培養(yǎng)箱中,28 ℃、160 r/min發(fā)酵培養(yǎng)。培養(yǎng)完成后,用濾紙濾除真菌菌絲體,將濾液依次利用石油醚(30-60)、乙酸乙酯、氯仿、正丁醇等體積萃取,濃縮氯仿萃取相并風干氯仿,用一定量甲醇溶解待用。

按常規(guī)方法制備薄層層析(thin-layer chromatography,TLC)G硅膠薄層板(G板),活化備用。配置苦參堿標準品的甲醇溶液(質(zhì)量濃度c=1 mg/mL),將真菌發(fā)酵液提取物與苦參堿標準品分別點樣于G板上,放入V(氯仿)∶V(甲醇)∶V(濃氨水)=5∶0.6∶0.2的展開體系中展開,展開完成后,置于碘缸中顯色,觀察斑點位置,并計算各斑點的比移值(radio frequency value,Rf)。

1.3.2 真菌發(fā)酵產(chǎn)物的氣相色譜-質(zhì)譜聯(lián)用分析

取1.3.1節(jié)中制備得到的真菌發(fā)酵液提取物5 mL,在轉(zhuǎn)速為10 000 r/min下離心5 min,用無水硫酸鈉對上清液除水,然后用0.22 μm微孔濾膜過濾去雜質(zhì),濾液用氣相色譜-質(zhì)譜聯(lián)用(gas chromatograph-mass spectrometer,GC-MS)分析。色譜條件為島津RTX-1MS(30 m×0.25 mm×0.25 μm)非極性石英毛細管柱,柱溫130 ℃,進樣口溫度250 ℃,不分流進樣,進樣量為1 μL,載氣為氦氣,流速1 mL/min。柱溫采用程序升溫:130 ℃,不保留;130～220 ℃,50 ℃/min,保留2 min;220～280 ℃,20 ℃/min,保留15 min。質(zhì)譜條件為離子源溫度230 ℃,接觸面溫度280 ℃,掃描范圍30～550 amu,EI源轟擊電壓70 eV[13]。

1.4 產(chǎn)苦參堿真菌鑒定

1.4.1 分離真菌的形態(tài)觀察 用插片法[14]培養(yǎng)真菌,并在不同時間段取樣制片,在顯微鏡下觀察并記錄真菌菌絲及孢子的形態(tài)。

1.4.2 真菌基因組提取及序列擴增 將篩選所得產(chǎn)苦參堿的真菌菌絲1～2 g放入滅菌的研缽中,用液氮研磨成粉末,使用真菌基因組DNA提取試劑盒提取該菌DNA,采用引物ITS1(5′-TCCGTAGGTGAACCTGCGG-3′)和ITS4(5′-TCCTCCGCTTATTGATATGC-3′)擴增內(nèi)轉(zhuǎn)錄區(qū)間序列(internal transcribed sequence,ITS)。聚合酶鏈反應(yīng)(polymerase chain reaction,PCR)體系為50 μL,反應(yīng)體系為真菌DNA 5 μL,上、下游引物各1 μL,PCR混合液 25 μL,ddH2O 18 μL。反應(yīng)條件:94 ℃預(yù)變性3 min,94 ℃變性30 s,56 ℃退火30 s,72 ℃延伸90 s,共35個循環(huán);72 ℃充分延伸10 min后終止反應(yīng)。對PCR產(chǎn)物進行瓊脂糖凝膠電泳分析后,切取目的片段凝膠進行產(chǎn)物純化回收,將回收的PCR產(chǎn)物送生工生物工程(上海)股份有限公司測序。

1.4.3 真菌同源性比較與遺傳進化分析 登錄美國國家生物技術(shù)信息中心(National Center of Biotechnology Information,NCBI),應(yīng)用BLAST程序?qū)y定的ITS序列與GenBank中的序列進行同源性比較。應(yīng)用MEGA 5.0軟件的Clustal W進行序列比對,采用鄰接法(neighbor-joining method,NJ)構(gòu)建系統(tǒng)發(fā)育樹,自展數(shù)據(jù)集為1 000。

1.4.4 產(chǎn)苦參堿真菌的種屬分類 根據(jù)所獲真菌的形態(tài)結(jié)構(gòu),結(jié)合其遺傳進化特征進行分析,對所獲得的真菌進行種屬鑒定。

2 結(jié)果與分析

2.1 微生物的分離純化

從石油污染土壤中,通過富集、涂布及平板劃線分離篩選,并經(jīng)多次純化后得到1株富集石油的固體培養(yǎng)基及PDA固體培養(yǎng)基上長勢較好且菌落單一的真菌。

2.2 產(chǎn)苦參堿真菌的形態(tài)學鑒定

A：真菌的菌落形態(tài)圖；B：真菌菌絲顯微形態(tài)圖.A: Colony morphology of fungus; B: Microscopy of fungal mycelium.圖1 真菌的菌落形態(tài)及菌絲圖Fig.1 Colony morphology and microscopy of mycelium of fungus

在PDA培養(yǎng)基平板中,可以看到該真菌呈現(xiàn)分散的絨毛狀菌落(圖1A),菌落呈圓形,白色,背面奶油色至赭黃色;菌絲無色,光滑且具隔膜;孢子囊梗直接從菌絲上長出,呈錐形多單生,分生孢子無色,光滑,形成簇狀或鏈狀,長紡錘形,兩頭尖,未見厚垣孢子(圖1B)。查閱文獻并比較，發(fā)現(xiàn)該真菌的菌落形態(tài)特征、菌絲和孢子的顯微形態(tài)與枝頂孢屬(Acremonium)、擬青霉屬(Simplicillium)和頭孢霉屬(Cephalosporium)中的部分菌種較為相似[15-17]。

2.3 產(chǎn)苦參堿真菌篩選

2.3.1 薄層色譜檢測 經(jīng)過薄層層析的定性分析確定,所獲真菌的發(fā)酵液提取物(圖2B)出現(xiàn)與苦參堿標準品(圖2A)顏色和比移值相同的斑點,Rf值為0.68。于是，初步確定該真菌可以發(fā)酵產(chǎn)生苦參堿。

A:苦參堿標準品;B:真菌提取物.A: Marine standard; B: Fungal extract.圖2 真菌發(fā)酵液的薄層層析結(jié)果Fig.2 Thin-layer chromatography result of fungal fermented liquid

2.3.2 GC-MS分析結(jié)果 經(jīng)過氣相色譜-質(zhì)譜聯(lián)用儀分析確定,真菌發(fā)酵液提取物的色譜圖(圖3B)在11.929 min時出現(xiàn)了與苦參堿標準品色譜保留時間(retention time,RT)11.932 min相近的色譜峰(圖3A)。

苦參堿標準品在全離子掃描(selected ion monitoring,SIM)模式下可以得到 4個豐度較高的碎片離子,質(zhì)荷比(m/z)分別為248.2、247.2、150.1和205.1[18]。GC-MS分析結(jié)果多次重復(fù)表明,在所獲真菌發(fā)酵液提取物色譜圖(圖3B)中,保留時間為11.929 min的色譜峰所對應(yīng)的離子碎片質(zhì)譜圖(圖3D)和苦參堿標準品的離子碎片質(zhì)譜圖(圖3C)相類似,并且和質(zhì)譜數(shù)據(jù)庫中苦參堿的特征離子碎片質(zhì)譜圖相匹配(圖3E)。這些結(jié)果進一步確定了該真菌的發(fā)酵液提取物中含有苦參堿成分,說明所獲真菌在液體培養(yǎng)中可以合成苦參堿。

2.4 分離真菌的ITS序列分析與鑒定結(jié)果

經(jīng)瓊脂糖凝膠電泳檢測,PCR擴增產(chǎn)物的片段長度約為600 bp(圖4A)。核苷酸序列測定結(jié)果顯示,ITS片段長度為585 bp,GenBank登錄號為KF803999。 用BLAST軟件比對結(jié)果顯示,該菌的ITS序列與Acremoniumsp. SA-NEX5(JX021664.1)、Acremoniumsp. CB90(HM98995.1)的同源性高達99%。

A:苦參堿標準品氣相色譜圖;B:真菌發(fā)酵液提取物氣相色譜圖;C:苦參堿色譜峰的離子碎片質(zhì)譜圖;D:真菌發(fā)酵液提取物的離子碎片質(zhì)譜圖;E:質(zhì)譜數(shù)據(jù)庫中的苦參堿的質(zhì)譜圖. 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.圖3 苦參堿標準品及真菌發(fā)酵液提取物的氣相色譜-質(zhì)譜圖Fig.3 Gas chromatography-mass spectrometry of matrine standard and the fungus extracts

A：PCR擴增ITS rDNA序列[M:5 000 bp的分子標記;(1～3):PCR產(chǎn)物]；B：基于該真菌rDNA ITS序列的系統(tǒng)發(fā)育樹. 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.圖4 真菌PCR擴增ITS rDNA序列及系統(tǒng)發(fā)育樹Fig.4 Phylogenetic tree and amplification of fungal ITS rDNA by PCR of the fungus

基于該真菌ITS序列與GenBank中同源性序列構(gòu)建的NJ系統(tǒng)發(fā)育樹(圖4B)發(fā)現(xiàn),該真菌與枝頂孢屬的Acremoniumsp. SA-NEX5(JX021664.1)處于同一分支上,而且自檢支持率高達99%～100%。系統(tǒng)發(fā)育分析結(jié)果表明,該真菌與Acremoniumsp. SA-NEX5(JX021664.1)的遺傳距離最近。

綜合形態(tài)觀察、分子鑒定結(jié)果及已有文獻報道,將該真菌歸屬于枝頂孢屬真菌,命名為Acremoniumsp. P0997,并保藏于中國典型培養(yǎng)物保藏中心,保藏號為CCTCCM 2013569。

3 討論

經(jīng)TLC檢測及GC-MS分析確定所獲真菌的發(fā)酵液提取物中含有苦參堿,表明所獲真菌可以合成苦參堿。此外,GC-MS同時檢測了所分離真菌的菌絲體提取物,確定該菌菌絲體中也含有苦參堿物質(zhì)成分。結(jié)果顯示,所獲真菌可能在真菌菌絲內(nèi)以及發(fā)酵液中均可以合成苦參堿,但對于枝頂孢屬菌產(chǎn)生苦參堿的代謝途徑及其相關(guān)合成機制還需進一步研究。

枝頂孢屬真菌(Acremoniumsp.)是一種常見真菌,分布廣泛,目前報道的約有 130余種,主要可分為腐生、植物寄生和自生3種。此類真菌的次生代謝產(chǎn)物中包括酚類、萜類、環(huán)肽和蒽醌類等,多具有良好的生物活性[19]。姚磊等[19]從腐殖質(zhì)中分離得到1株枝頂孢屬菌,并在其代謝產(chǎn)物中檢測到了姜糖酯B、姜糖酯C、D-甘露醇、酒渣堿和枝頂孢素F;余永濤等[20]在1株枝頂孢屬菌的發(fā)酵液中檢測到了苦參堿成分。石油污染土壤中常發(fā)現(xiàn)有環(huán)狀有機分子污染,本研究據(jù)此推測此類污染土壤中可能有可以合成苦參堿類生物堿的微生物分布。研究首次從石油污染土壤中分離得到了產(chǎn)苦參堿的真菌,這為建立苦參堿的微生物發(fā)酵生產(chǎn)方式提供了新的依據(jù)。

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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

陜西省科技計劃資助項目(693102)；中央高校科研業(yè)務(wù)費特別支持項目(1301030208)。

聯(lián)系方式:毛東霞(http://orcid.org/0000-0001-9852-298X)，E-mail:ylainexn@163.com

2015-03-03;接受日期(Accepted):2015-07-01;網(wǎng)絡(luò)出版日期(Published online):2015-09-01

X 13; Q 93

A

*通信作者(Corresponding author):馬小魁(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