云南碧羅雪山溫泉群之中原溫泉的細(xì)菌物種多樣性分析第第頁(yè)共19頁(yè)云南碧羅雪山溫泉群之中原溫泉的細(xì)菌物種多樣性分析摘要目的：探究云南境內(nèi)碧羅雪山溫泉群之中原溫泉中的細(xì)菌物種多樣性，對(duì)其細(xì)菌物種多樣性進(jìn)行分析，以期獲得一些具有重要藥用價(jià)值的微生物資源。方法：采集中原溫泉底泥樣品，以常規(guī)的微生物可培養(yǎng)化技術(shù)分離純化樣品細(xì)菌，結(jié)合形態(tài)學(xué)和分子生物學(xué)手段對(duì)細(xì)菌菌株進(jìn)行物種鑒定。結(jié)果：在該溫泉底泥中共分離得到45個(gè)屬113個(gè)種250株細(xì)菌。其中，優(yōu)勢(shì)菌為假單胞菌屬和芽胞桿菌，兩菌屬分別獲得14個(gè)種44株菌和11個(gè)種19株菌。結(jié)論：中原溫泉生境內(nèi)細(xì)菌物種多樣性豐富，具有一定的藥用微生物資源開(kāi)發(fā)價(jià)值。關(guān)鍵詞：云南溫泉；物種多樣性；細(xì)菌；中原溫泉BacterialspeciesdiversityanalysisoftheCentralPlainshotspringintheBiluosnowmountainhotspringsgroup,YunnanProvinceAbstractObjective:ToexplorethebacterialspeciesdiversityintheCentralPlainshotspringsoftheBiluosnowmountainhotspringsinYunnanProvince,andanalyzethebacterialspeciesdiversity,inordertoobtainsomemicrobialresourceswithimportantmedicinalvalue.Methods:ThesamplesofsedimentfromtheCentralPlainshotspringwerecollected,andthebacteriawereisolatedandpurifiedbyconventionalculturabletechnology.ThespeciesidentificationofbacteriawascarriedoutbymorphologicalandmolecularbiologicalmethodsResults：250strainsofbacteriabelongingto113speciesand45generawereisolatedfromthehotspringsediment.Amongthem,thedominantbacteriaarePseudomonasandBacillus.44strainsof14speciesand19strainsof11specieswereobtainedfromthetwogenera.:Conclusion:ThediversityofbacteriaspeciesinthehotspringhabitatofcentralChinaisrich,whichhasacertainvalueinthedevelopmentofmedicinalmicrobialresources.Keywords:YunnanHotSpring;speciesdiversity;bacteria;theCentralPlainshotspring前言科技水平的提高和生活質(zhì)量的改善來(lái)源于各種化工能源的開(kāi)發(fā)，而這些能源的開(kāi)發(fā)利用，將會(huì)造成環(huán)境污染、資源匱乏等一系列問(wèn)題。為了找到一條可持續(xù)和綠色發(fā)展的道路，人們開(kāi)始著眼于一些新型能源的開(kāi)發(fā)。近年來(lái)，生物技術(shù)的高速發(fā)展使得各種生物資源得到了一定的開(kāi)發(fā)，獲取資源的手段也明顯進(jìn)步。人們開(kāi)始著眼于微生物分離方法并且聚焦極端環(huán)境中微生物的研究，如海洋、溫泉、鹽湖等，期望獲得一些新的微生物資源和生物活性物質(zhì)[1]。溫泉作為一種極端水生環(huán)境，通常是孤立存在的生境，物理隔離可以使其產(chǎn)生“孤島效應(yīng)”，從而使微生物在進(jìn)化適應(yīng)過(guò)程中形成高度發(fā)散的基因結(jié)構(gòu)和功能特征[2]。研究表明，絕大多數(shù)溫泉的溫度、酸堿度及養(yǎng)分等均異于一般生態(tài)環(huán)境，接近寒武紀(jì)早期環(huán)境條件[3]。因此，溫泉生境作為最接近地球早期環(huán)境的特殊生態(tài)環(huán)境之一，保留了最為原始的菌種資源，空間距離上較為接近的溫泉之間也可能保留了各自的特有物種，存在一些新的微生物資源，尤其是嗜熱菌株。同時(shí)，生存在這種熱泉生態(tài)系統(tǒng)中的微生物，經(jīng)過(guò)長(zhǎng)期的進(jìn)化，容易在過(guò)程中形成它們特有的生物活性物質(zhì)合成途徑或代謝途徑，從而使其次級(jí)代謝產(chǎn)物表現(xiàn)出較好的化學(xué)多樣性及其生物活性[4,5]。作為中國(guó)地質(zhì)構(gòu)造最復(fù)雜、新構(gòu)造最活躍的地區(qū)，云南省溫泉資源幾乎遍布全省，據(jù)現(xiàn)有資料統(tǒng)計(jì)，全省溫泉1015處，約占全國(guó)總數(shù)的三分之一，數(shù)量分布居全國(guó)之冠[6]。近年來(lái)部分特殊生境的溫泉隨著旅游地質(zhì)資源的開(kāi)發(fā)遭到了嚴(yán)重的破壞，因此，藥用微生物資源潛力的盡早發(fā)掘,溫泉生境保護(hù)的加強(qiáng)，變得極為重要。相對(duì)于其它地區(qū)的溫泉微生物研究，云南溫泉生境內(nèi)所含微生物物種多樣性豐富，產(chǎn)酶特性顯著，具有一定研究?jī)r(jià)值。諸多的研究結(jié)果也表明了云南熱泉生態(tài)系統(tǒng)中可培養(yǎng)微生物種類較為豐富，產(chǎn)酶特性突出[7-11]。此外，諸多的研究表明云南溫泉生境中所含微生物物種中還含有極高的嗜酸嗜熱特性[12-15]。另外，云南特殊的熱泉生境也孕育出了一些生理機(jī)制較為特殊細(xì)菌類群，如Xiang等[16]采用純培養(yǎng)方法從騰沖地?zé)釁^(qū)溫泉的水、沉積物混合樣品中分離得到了1株硫化葉菌屬細(xì)菌SulfolobustengchongensisRT8-4，并通過(guò)堿性裂解法提取了一種具有典型綴合功能的新型質(zhì)粒（命名為pTC1）DNA并進(jìn)行了鑒定。研究者還對(duì)騰沖地?zé)岬貐^(qū)的pTC1及其質(zhì)粒變異進(jìn)行了調(diào)查，pTC質(zhì)粒廣泛分布在調(diào)查領(lǐng)域，并發(fā)現(xiàn)宿主生物體已經(jīng)進(jìn)化出一種策略，可以在可移動(dòng)遺傳元件的插入和消除之間保持平衡，從而實(shí)現(xiàn)基因組可塑性，同時(shí)抑制其快速擴(kuò)散。本研究對(duì)云南碧羅雪山溫泉群中原溫泉的菌種物種鑒定與多樣性研究是通過(guò)純培養(yǎng)手段和基于16SrDNA基因序列的系統(tǒng)發(fā)育分析進(jìn)行的，并對(duì)所得到的菌株進(jìn)行菌種保藏。目的在于保護(hù)和開(kāi)發(fā)云南碧羅雪山溫泉群中原溫泉特境微生物資源，發(fā)掘溫泉藥源微生物。1材料和方法1.1材料1.1.1樣品采集樣品采集于云南碧羅雪山溫泉群中原溫泉底部淤泥（溫泉經(jīng)緯度北緯25°52′7，東經(jīng)99°4′56）,將樣品采集后帶回實(shí)驗(yàn)室4℃密封保存。1.1.2培養(yǎng)基LB培養(yǎng)基:胰蛋白胨10.0g/L，酵母提取物5.0g/L，氯化鈉10.0g/L，超純水完全溶解后，用氫氧化鈉調(diào)節(jié)pH值至7.5，超純水定容。121℃高壓蒸汽滅菌21min。用于菌種保藏。R2A培養(yǎng)基:酵母浸出粉0.5g/L，蛋白胨0.5g/L，酪蛋白水解物0.5g/L，葡萄糖0.5g/L，可溶性淀粉0.5g/L，磷酸氫二鉀0.3g/L，無(wú)水硫酸鎂0.024g/L，丙酮酸鈉0.3g/L，瓊脂15.0g/L，25℃下調(diào)整pH值為7.2±0.2。121℃高壓蒸汽滅菌15min。用于菌種分離純化。SGTYP培養(yǎng)基：淀粉5.0g/L，葡萄糖5.0g/L，胰蛋白胨1.0g/L，酵母提取物1.0g/L，蛋白胨1.0g/L，pH值調(diào)至7.6±0.2[17]。用于細(xì)菌發(fā)酵。1.1.3試劑及儀器天根生化科技細(xì)菌基因組試劑盒，無(wú)菌甘油，引物1492R和27F，雙人單面凈化工作臺(tái)，體式顯微鏡，恒溫培養(yǎng)箱，PCR儀，電泳儀，旋轉(zhuǎn)蒸發(fā)儀，粉碎均質(zhì)器，艾柯超純水機(jī)，壓力滅菌鍋。1.2方法1.2.1菌株的分離純化稱取10g溫泉底泥樣品，以10%的濃度配置成樣品混懸水溶液，采用倍比稀釋法將混懸液稀釋至濃度為10-3、10-4、10-5g/mL，分別吸取0.1mL稀釋液均勻涂布于R2A固體培養(yǎng)基上，每個(gè)濃度3個(gè)重復(fù)，于28℃恒溫箱中倒置培養(yǎng)3~4d；體式顯微鏡下挑取單菌落進(jìn)行反復(fù)分離純化，直至獲得純培養(yǎng)物[18]。1.2.2菌種保藏將純化好的細(xì)菌刮下菌體置于無(wú)菌甘油保種液中制成菌懸液，每個(gè)菌種保存3~4管，存于-80℃冰箱中備用。1.2.3菌種鑒定細(xì)菌16SrDNA的提取：主要的提取步驟和注意事項(xiàng)按提取試劑盒內(nèi)相關(guān)說(shuō)明完成。PCR反應(yīng)體系及條件：95℃下預(yù)變性5分鐘，變性30秒，59℃下復(fù)性30秒，72℃下延伸2分鐘，循環(huán)36次；完成后，在72℃環(huán)境中溫育10分鐘，于4℃冰箱中保存。表1PCR反應(yīng)體系ddH2O18μL27F（5'-AGAGTTTGATCCTGGCTCAG-3'）2.5μL1492R（5'-ACGGYTACCTTGTTACGACTT-3'）2.5μL2×Taqmixture25μLDNA模板2μL體系終體積50μLPCR產(chǎn)物的電泳檢測(cè)：PCR產(chǎn)物加入1％瓊脂糖凝膠、1xTAEbuffer及染色劑后取2μL點(diǎn)樣，進(jìn)行凝膠電泳檢測(cè)分析（電壓130V，時(shí)間20-30min），凝膠置于凝膠成像儀下觀察。PCR產(chǎn)物委托上海生物工程股份有限公司進(jìn)行測(cè)序。利用BLAST軟件排除雜峰干擾序列，序列長(zhǎng)度不低于300bp，將得到的DNA序列，與NCBI(NationalCenterofBiotechnologyInformation）的GenBank數(shù)據(jù)庫(kù)中核苷酸序列進(jìn)行比對(duì)，鑒定所分離得到的細(xì)菌，得到其種屬信息。對(duì)于親緣關(guān)系小于97%的菌種，進(jìn)行進(jìn)一步的生理生化測(cè)試，鑒定新種。2.結(jié)果2.1溫泉生境中菌種多樣性如表2所示，從云南碧羅雪山溫泉群中原溫泉底泥中分離得到45個(gè)屬113個(gè)種共250株菌。其中最豐富的是假單胞菌屬（Pseudomonas），有14個(gè)種44株菌，初步判斷為中原溫泉優(yōu)勢(shì)菌，其次為芽孢桿菌屬（Bacillus）共計(jì)11個(gè)種，19株菌，為該溫泉第二優(yōu)勢(shì)菌株。而博斯氏菌（Bosea），短桿菌（Brevibacillus），棒桿菌（Caulobacteraceae），叢毛單胞菌屬（Comamonas），黃桿菌（Flavobacteriales），芽生菌（Gemmobacter），詹氏桿菌（Janibacter），黃體桿菌（Luteibacter），分枝桿菌（Mycolocibacterium），泛菌屬（Pantoea），極單胞菌（Polaromonas），類芽孢桿菌（Paenibacillus），假桿菌（Pseudarthrobacter），鞘脂單胞菌屬（Sphingopyxis），藉伏氏菌屬（Vogesella）等15個(gè)屬在該溫泉中均有分布，但含量較少。表2溫泉分離菌株的數(shù)量及多樣性分布菌株編號(hào)最相似種屬親緣關(guān)系最為接近的菌株NCBI編號(hào)最高相似度（No.ofstrain）（theCloseststrain）(Accessionnumber)(theHighestsimilarity)HSBB77AcidovoraxdelafieldiiMN197564.199.92%HSBB108A.delafieldiiMN449446.199.93%HSBB118A.delafieldiiJQ689177.199.93%HSA3-B47A.delafieldiiKR085770.199.86%HSC-B43A.delafieldiiKR085770.199.22%HSC-B81A.radicisKR085770.199.86%HSC-B90A.soliNR_116740.199.35%HSA2-B18AcinetobacteralbensisMH119668.199.79%HSA2-B42A.albensis\o"ShowreportforMG576176.1"MG576176.199.79%HSA1-B77A.baylyiAB859675.199.21%HSA1-B84A.baylyiNR115042.298.86%HSA1-B89A.baylyiAB859675.199.01%HSA1-B4A.berezinaeJF302885.198.65%HSBB110①A.calcoaceticusMN250321.199.93%HSA1-B75A.guillouiaeMK070050.199.79%HSA1-B96A.guillouiaeKJ147068.198.52%HSA3-B32A.guillouiaeKF749283.199.86%HSA3-B55A.guillouiaeMF521555.199.51%HSA3-B39A.guillouiaeKF749283.199.86%HSA5-B46A.johnsoniiKY767496.199.93%HSBB94A.lwoffiiMN704528.199.72%HSA5-B54A.soliMK874914.199.72%HSC-B91AeromonashydrophilaNC_008570.199.64%HSA5-B34A.hydrophilaMG428979.1100.00%HSC-B10A.salmonicidaLC312129.1100.00%HSC-B39A.salmonicidaNC_009348.199.86%HSC-B58A.salmonicidaNC_009348.199.93%HSC-B82A.salmonicidaNC_009348.1100.00%HSC-B131A.salmonicidaLC312129.1100.00%HSC-B77A.veroniiMN581681.199.86%HSC-B103A.veronii\o"ShowreportforNZ_CDBO01000064.1"CP002607.199.93%HSC-B105A.veronii\o"ShowreportforNZ_CDBO01000064.1"MG063196.199.93%HSA1-B108ArthrobactergandavensisKC514116.199.79%HSA1-B31A.luteolusMH475928.199.79%HSA1-B40-1A.luteolusMH475928.199.79%HSA1-B67A.luteolusMH475928.199.93%HSA1-B72A.luteolusMH475928.1100.00%HSA1-B107A.luteolusMH475928.1100.00%HSA1-B110A.luteolusMH475928.1100.00%HSA1-B111A.luteolusMH475928.199.64%HSA4-B40A.niigatensisHQ236028.199.07%HSA1-B104A.oxydansEU086826.199.33%HSBB87BacillusamyloliquefaciensMK130898.1100.00%HSC-B144B.aryabhattaiMN062938.199.79%HSA1-B73B.circulansMK100762.199.93%HSBB24B.idriensisMN235854.1100.00%HSBB51B.idriensisMK240437.199.93%HSBB122B.idriensisMK240437.1100.00%HSA4-B44B.mycoidesKY038735.1100.00%HSC-B70B.simplesHG424431.1100.00%HSA1-B32-1B.simplexKC764992.199.86%HSA1-B46B.simplexAY833099.199.86%HSA1-B55B.simplexKC764992.199.86%HSA1-B62B.simplexAY833099.199.86%HSBB23B.simplexMK302255.1100.00%HSBB27B.simplexKC764992.199.89%HSA4-B2B.thuringiensisKU179330.1100.00%HSA4-B1B.toyonensisMH921634.1100.00%HSBB33B.velezensisKX129851.199.72%HSBB117B.velezensisCP048002.1100.00%HSA1-B51B.wiedmanniiNR_152692.198.38%HSBB44BacteriumJNKLA11KR027003.199.84%HSA2-B1B.ZPMKJ463378.199.93%HSA2-B14B.ZPMKJ463378.199.86%HSA2-B19B.ZPMKJ463378.199.93%HSA2-B34B.ZPMKJ463378.199.93%HSA2-B4B.ZPMKJ463378.199.93%HSA3-B50B.ZPMKJ463378.199.93%HSC-B99BetaproteobacteriaKY053192.199.55%HSC-B100B.proteobacteriumAF336360.199.15%HSBB6BosearobiniaeMK519072.199.69%HSC-B29BrevibacilluspanacihumiNZ_KI629785.198.30%HSA3-B29BrevundimonasbullataNR_025831.199.92%HSA5-B43B.bullataMG576003.199.85%HSA3-B12B.bullataMG576003.199.93%HSA3-B22B.bullataMG576003.199.92%HSBB32B.intermediaKR085793.1100.00%HSBB82B.intermediaKR811205.198.63%HSBB111B.intermediaKR085793.199.85%HSA4-B3B.intermediaKR085793.199.86%HSA5-B15B.mediterraneaEU379251.199.85%HSA5-B51B.mediterraneaEU379251.199.77%HSA5-B57B.mediterraneaKR085851.199.85%HSA3-B13B.nasdaeMG650163.199.93%HSA4-B51B.staleyiKY047398.199.69%HSBB40B.staleyiMN258890.199.32%HSA4-B9B.vesicularisMK414927.1100.00%HSA4-B19B.vesicularisMH542316.1100.00%HSA6-B1B.vesicularisFJ999941.1100.00%HSA6-B3B.vesicularisKF818658.1100.00%HSA6-B43-1B.vesicularisMK382493.1100.00%HSBB62B.vesicularisMN704395.1100.00%HSBB109B.vesicularisKY501221.199.55%HSC-B106B.vesicularis\o"ShowreportforNZ_CP022048.2"KR085853.1100.00%HSA5-B50B.vesicularisMN252083.1100.00%HSA5-B52B.vesicularisKY501221.1100.00%HSA5-B56B.vesicularisMK382493.1

99.92%HSA3-B43B.vesicularisMH283789.1100.00%HSA3-B60B.vesicularisKT751295.199.93%HSA5-B59B.vesicularisKY501221.1100.00%HSA5-B61B.vesicularisKR085853.1100.00%HSBB31CaulobactermirabilisCP024201.198.92%HSA2-B13C.segnisLC500466.197.81%HSBB71CaulobacteraceaebacteriumKM187497.1100.00%HSBB15ChitinimonastaiwanensisAB731563.199.01%HSA2-B7C.taiwanensisNR_029099.2\o"ShowreportforNR_029099.2"99.57%HSA4-B43ChryseobacteriumaahliNR133722.199.23%HSA4-B50C.aahliNR133722.199.76%HSA5-B8C.ginsengiterraeJX141783.199.71%HSBB29C.glacieiCP015199.199.64%HSA1-B11-2C.rhizosphaeraeMK240433.199.78%HSA1-B23C.rhizosphaeraeMK240433.199.86%HSA1-B43C.rhizosphaeraeMK240433.199.86%HSA1-B44C.rhizosphaeraeMK240433.199.96%HSA1-B45-2C.rhizosphaeraeMK240433.1100.00%HSA1-B86C.rhizosphaeraeMK240433.1100.00%HSBB86ComamonasaquaticaMF354014.199.86%HSA4-B21EnterobacterasburiaeAP019533.199.72%HSA4-B27E.asburiaeAP019533.199.65%HSA6-B15E.asburiaeAP019533.199.72%HSA6-B62-2E.asburiaeAP019533.199.72%HSA6-B63-2E.asburiaeAP019533.199.65%HSA6-B62-1E.cancerogenusLC420103.199.58%HSC-B6Escherichiacoli\o"ShowreportforNC_011751.1"NC_011751.199.79%HSC-B14E.coliNC_011751.199.79%HSC-B73EcoliNC_011751.199.79%HSA3-B56FlavobacterialesbacteriumAY145539.198.55%HSBB9FlavobacteriumcauenseMK396582.199.93%HSA1-B10-2F.johnsoniaeKC122700.198.67%HSA1-B64F.johnsoniaeKC122700.198.67%HSA6-B37F.tistrianumNR_149282.198.85%HSA6-B80-2F.tistrianumNR_149282.198.41%HSA6-B85-2F.tistrianumNR_149282.198.35%HSC-B75F.xueshanenseKR085801.198.78%HSC-B74GemmobacteraquatilisNR_114265.199.70%HSBB7HydrogenophagaatypicaKT345668.1100.00%HSBB78H.bisanensisNR_044268.199.93%HSBB85H.bisanensisNR_044268.198.63%HSBB102H.bisanensisNR_044268.199.78%HSBB63H.defluviiAB638427.199.87%HSBB101H.laconesensisNR_149183.199.64%HSC-B16H.palleroniiMH482328.199.86%HSBB54H.pseudoflavaCP037867.1100.00%HSBB92JanibactermelonisEU333885.199.71%HSA4-B28LuteibacterrhizovicinusCP017480.199.86%HSC-B92*MassiliaflavaCP046904.199.64%HSA1-B57M.haematophilaKU305723.199.57%HSA1-B105M.haematophilaKU305723.199.57%HSA1-B106M.haematophilaKU305723.199.57%HSA4-B66M.haematophilaKU305723.198.50%HSBB11M.variansKF924221.199.93%HSBB41M.variansKF924221.199.93%HSBB43M.variansKF924221.199.86%HSBB65M.variansKF924221.199.93%HSA4-B67MicrobacteriumfoliorumEU834263.199.57%HSA5-B60M.foliorumCP041040.199.93%HSA4-B15MoraxellaosloensisCP024185.299.93%HSBB5M.osloensisFJ357611.199.65%HSBB64M.osloensisAP017381.199.37%HSC-B87*MycolocibacteriummoriokaenseJQ039995.199.57%HSBB42NaxibacteralkalitoleransHQ323424.199.57%HSBB46N.indicaFJ812371.199.36%HSBB49①NoviherbaspirillumaurantiacumNR_118040.199.79%HSBB126N.aurantiacumNR_118040.199.51%HSBB12PaenibacillusglucanolyticusKT427632.199.86%HSA6-B51-2PantoeaeucalyptiMK910226.299.86%HSA2-B16PararheinheimerasoliNR_044294.199.86%HSA2-B23P.soliNR_044294.199.78%HSA2-B32P.soliNR_044294.199.78%HSA2-B35P.soli\o"ShowreportforNR_044294.1"NR_044294.199.79%HSC-B54PedobactercompostiNR_041506.198.93%HSA4-B23P.ginsenosidimutansNR_108685.199.93%HSA4-B48P.ginsenosidimutansNR_108685.1100.00%HSA4-B20P.jeongneungensisNR132685.199.86%HSBB37P.jeongneungensisNR_132685.199.57%HSBB66P.jeongneungensisNR_132685.199.13%HSBB95P.jeongneungensisNR_132685.199.13%HSBB55PolaromonaseurypsychrophilaEU130998.199.87%HSA4-B42PseudarthrobacterchlorophenolicusKU647201.199.22%HSA3-B14PseudomonasabietaniphilaNR_041952.199.86%HSBB70P.aeruginosaJF496542.199.64%HSA3-B58P.aeruginosaJF496542.198.57%HSC-B95P.anguillisepticaKP322343.199.86%HSC-B108P.anguillisepticaKP322343.199.79%HSBB18P.fluorescensFJ588703.199.65%HSC-B30P.fluorescensMF522226.199.79%HSC-B40P.fluorescensHQ874650.199.79%HSC-B41P.fluorescensHQ874650.199.79%HSC-B46P.fluorescensHQ874650.199.79%HSC-B62P.fluorescensHQ874650.199.86%HSC-B80P.fluorescensNC_016830.198.56%HSC-B83P.fluorescensHE659365.199.57%HSC-B89P.fluorescensHE659365.199.71%HSC-B94P.fluorescensNC_016830.198.50%HSC-B101P.fluorescensHQ874650.199.65%HSC-B104P.fluorescensNC_016830.198.51%HSA4-B8P.hunanensisMN177225.199.93%HSA6-B49P.luridaCP015639.199.93%HSA6-B83-2P.luridaCP015639.199.93%HSA4-B30P.moraviensisKT443876.198.35%HSA3-B54P.otitidisMG905275.1100.00%HSA3-B38P.otitidisMG905275.1100.00%HSA3-B35P.otitidisMG905270.1100.00%HSA3-B37P.otitidisMG905276.1100.00%HSA3-B53P.otitidisMG905275.199.93%HSC-B3P.peilMN752883.199.79%HSC-B37P.peilMN752883.199.71%HSC-B19P.peliKC920940.199.71%HSC-B31P.peliKC920940.199.64%HSC-B32P.peliKC920940.199.79%HSC-B38P.peliKU921574.199.79%HSA4-B5P.plecoglossicidaMN208210.1100.00%HSBB22P.plecoglossicidaMN208210.1100.00%HSBB121P.putidKX083527.199.86%HSBB8P.putidaKX279648.1100.00%HSBB60P.putidaMH712982.1100.00%HSBB84P.putidaMG722771.1100.00%HSBB113P.reinekeiKC790251.1100.00%HSBB116P.reinekeiKC790314.199.93%HSC-B8P.rhodesiaeLT629801.199.86%HSC-B78P.rhodesiaeJX994152.1100.00%HSC-B85P.rhodesiaeJX994152.1100.00%HSC-B136P.rhodesiaeJX994152.1100.00%HSA4-B61RheinheimerachironomiEU834263.199.64%HSBB97R.chironomiKF999725.199.64%HSA2-B22R.texasensisLC054835.198.93%HSA2-B39R.texasensisLC054835.199.93%HSA2-B15R.texasensisLC054835.199.86%HSA2-B33R.texasensis\o"ShowreportforLC054835.1"LC054835.199.93%HSBB73RhizobiumnaphthalenivoransMF361887.1100.00%HSBB76R.selenitireducensMK425681.199.85%HSA6-B4SphingobacteriumfaeciumMH119741.198.21%HSA6-B5S.faeciumFJ268960.199.92%HSBB61SphingobiumhydrophobicumCP022746.199.78%HSBB30S.paulinellaeNR_159252.199.85%HSBB75S.paulinellaeNR_159252.199.93%HSA2-B3\o"GotoalignmentforSphingobiumpaulinellaestrainPch-B16SribosomalRNA,partialsequence>gb|JN229904.1|Unculturedbacteriumclone1H3C12116SribosomalRNAgene,partialsequence>gb|KY864399.2|SphingobiumpaulinellaestrainPch-B16SribosomalRNA"S.paulinellaeNR_159252.199.85%HSBB38S.xenophagumMF062659.199.56%HSBB69S.xenophagumMG576011.199.85%HSC-B44SphingopyxisalaskensisNC_008048.198.75%HSBB17StaphylococcusaureusCP047851.1100.00%HSBB39S.aureusMN650918.1100.00%HSA6-B34-1StenotrophomonasmaltophiliaKF378761.1100.00%HSA6-B44S.maltophiliaKF378761.1100.00%HSA6-B53-2S.maltophiliaJX393000.199.86%HSA6-B65S.pavaniiMN709324.1100.00%HSA6-B85-1S.pavaniiMN686363.1100.00%HSBB36①VariovoraxginsengisoliNR_112562.199.72%HSBB105V.ginsengisoliMG576021.198.77%HSBB45VogesellaindigoferaKT983996.199.86%下圖為部分菌種PCR跑膠圖片圖1~4部分菌株的PCR產(chǎn)物跑膠圖注：每個(gè)目標(biāo)條帶長(zhǎng)度為1500bp左右,圖中注釋為Marker所在位置。2.2優(yōu)勢(shì)菌物種多樣性分析根據(jù)16SrDNA基因序列構(gòu)建系統(tǒng)發(fā)育進(jìn)化樹,如下圖（圖5、圖6）所示：圖5芽孢桿菌屬（Bacillus）系統(tǒng)發(fā)育樹圖6假單胞菌系統(tǒng)發(fā)育樹由系統(tǒng)發(fā)育樹（圖5、圖6），我們可以清晰的看出所鑒定的19株芽孢桿菌中主要有11個(gè)種分別為B.aryabhattai，B.circulans，B.idriensis，B.mycoides，B.simples，B.simplex，B.thuringiensis，B.toyonensis，B.velezensis，B.wiedmannii而在這11個(gè)種之間又以B.simplex最多共有6株；44株假單胞菌中主要有14個(gè)種分別為P.abietaniphila，P.aeruginosa，P.anguilliseptica，P.fluorescens，P.hunanensis，P.lurida，P.moraviensis，P.otitidis，P.peil，P.plecoglossicida，P.putid，P.putida，P.reinekei，P.rhodesiae,其中P.fluorescens最多有12株，占所得總假單胞菌的27.3%；而P.abietaniphila，P.hunanensis，P.putid，P.moraviensis這四個(gè)種最少均只有1株。討論共分離純化得到250株菌，共計(jì)113個(gè)種45個(gè)屬。所得到的菌株除編號(hào)為HSA2-B13（Caulobactersegnis）的菌株最高相似度為97.81%，其余菌株最高相似度都在98%以上。在物種多樣性方面，文獻(xiàn)調(diào)研發(fā)現(xiàn)滇西地區(qū)溫泉中的優(yōu)勢(shì)菌多為無(wú)氧芽胞桿菌屬（Anoxybacillus）、芽孢桿菌屬（Bacillus）、棲熱菌屬（Thermus）和亞棲熱菌屬（Meiothermus），而本研究中中原溫泉優(yōu)勢(shì)菌株為假單胞菌屬（Pseudomonas），與云南騰沖等地溫泉物種分布具有較大的差異，這可能與碧羅雪山獨(dú)特的低緯度高海拔、較為劇烈的地殼活動(dòng)地貌有關(guān)，也進(jìn)一步表明了云南溫泉生境中的微生物資源的豐富性。所得到的第一優(yōu)勢(shì)菌種假單胞菌屬種類繁多，功能類群多樣。該菌屬有些是動(dòng)植物病原體，有些對(duì)某些植物病毒具有防治作用，有些具有生物防治能力，而且絕大多數(shù)的假單胞菌屬能分泌脂肪酶。假單胞菌屬脂肪酶性能優(yōu)越，應(yīng)用范圍廣泛。大量的文獻(xiàn)資料表明，假單胞菌屬（Pseudomonas）在農(nóng)業(yè)生物防治、植物生物調(diào)節(jié)、環(huán)境保護(hù)，醫(yī)藥開(kāi)發(fā)方面等領(lǐng)域均有應(yīng)用。值得一提的是，假單胞菌用于醫(yī)藥方面的研究已有報(bào)道，國(guó)外較早時(shí)期有從假單胞菌中分離出具有抗腫瘤活性的大環(huán)類酯類化合物OximidinesI和II[19]，并有利用假單胞菌外毒素與單克隆抗體結(jié)合抗腫瘤的報(bào)道[20]劉國(guó)紅[21]等研究發(fā)現(xiàn)，芽孢桿菌屬在工業(yè)、農(nóng)業(yè)、醫(yī)藥等方面都有其應(yīng)用價(jià)值。在工業(yè)方面，芽胞桿菌可以增強(qiáng)洗滌效果，還可降解水中的污染物，達(dá)到凈化環(huán)境的目的。在農(nóng)業(yè)方面，將其用于養(yǎng)殖業(yè)可以保證動(dòng)物鮮美的肉質(zhì)；用做飼用微生物制劑，可降低飼料成本，同時(shí)減少疾?。痪哂休^強(qiáng)的殺蟲活性可用作生物農(nóng)藥。在醫(yī)藥方面，芽孢桿菌活性制劑可通過(guò)生物拮抗作用來(lái)達(dá)到防治疾病，枯草芽孢桿菌還將有可能成為新型藥物或蛋白載體。這些文獻(xiàn)結(jié)果表明了從云南溫泉生境碧羅雪山溫泉群中原溫泉內(nèi)分離得到的優(yōu)勢(shì)菌無(wú)論是在工業(yè)，農(nóng)業(yè)還是醫(yī)學(xué)方面都具有較大的實(shí)際應(yīng)用價(jià)值，值得進(jìn)一步探索。4.結(jié)論本研究初步表明云南中原溫泉內(nèi)可培養(yǎng)細(xì)菌數(shù)量較多，物種多樣性較為豐富，具有一定的物種特異性。同時(shí)，海拔4500米的碧羅雪山，原始生態(tài)系統(tǒng)保存完整，人跡罕至，地理隔離程度極高，密閉性好，是異域分化產(chǎn)生微生物新物種資源的天然實(shí)驗(yàn)室，其物種多樣性和新穎性有待進(jìn)一步探索。溫泉生境中含有豐富的微生物藥用資源，具有很高的科研與應(yīng)用價(jià)值。本研究對(duì)云南碧落雪山溫泉群中原溫泉底泥中的細(xì)菌資源進(jìn)行了物種多樣性分析，而其抗菌活性、酶活性等其他生理生化活性還有待于進(jìn)一步研究。希望本研究能夠?yàn)樵摰貐^(qū)溫泉來(lái)源微生物的開(kāi)發(fā)以及溫泉生態(tài)系統(tǒng)的進(jìn)一步研究提供數(shù)據(jù)參考。參考文獻(xiàn)LewinA,WentzelA,VallaS.Metagenomicsofmicrobiallifeinextremetemperatureenvironments[J].CurrentOpinioninBiotechnology,2013,24(3):516-525.PapkeRT,RamsingNB,BatesonMM,etal.Geographicalisolationinhotspringcyanobacteria[J].EnvironMicrobiol,2003,5:650-659.YunZ.ThermophilicmicroorganismsinthehotspringsofTengchonggeothermalarea,WestYunnan,China[J].Geothermics,1986,15(3):347-358.DongY,SanfordRA,InskeepWP,etal.Physiology,Metabolism,andFossilizationofHot-SpringFilamentousMicrobialMats[J].Astrobiology,2019.黎唯,李一青,李銘剛,等.極端環(huán)境微生物源活性物質(zhì)的研究進(jìn)展[J].國(guó)外醫(yī)藥(抗生素分冊(cè)),2007(01):5-9+22.梁乃英,劉時(shí)彬,丁建博.云南省志·卷二十五·溫泉志[M].云南:云南人民出版社,1999:190.孫劭靖,路福平,姜楠,等.一株產(chǎn)耐熱普魯蘭酶菌株Anoxybacillussp.LM14-2分離鑒定及酶學(xué)性質(zhì)研究[J].生物技術(shù)通報(bào),2011(09):136-141.WuL,LiuB,HongY,etal..ResidueTyr224iscriticalforthethermostabilityofGeobacillussp.RD-2lipase[J].BiotechnologyLetters,2009,32(1):107-112.殷燕,張波.一株產(chǎn)木糖異構(gòu)酶嗜熱菌的分離鑒定及其培養(yǎng)條件優(yōu)化[J].食品與發(fā)酵工業(yè),2010,36(03):112-116.ChenY,HeY,ZhangB,etal.CompleteGenomeSequenceofAlicyclobacillusacidocaldariusStrainTc-4-1[J].JournalofBacteriology,2011,193(19):5602-5603.ZhangF,YangX,GengL,etal.Purificationandcharacterizationofanovelandversatileα-amylasefromthermophilicAnoxybacillussp.YIM342[J].Starch-St?rke,2015,68(5-6):446-453[12]丁建南,于一尊,何環(huán),等.嗜熱硫氧化硫化桿菌一新菌株的分離與鑒定[J].湖南師范大學(xué)自然科學(xué)學(xué)報(bào),2007(04):104-109.[13]HeH,YangY,XiaJ,etal.Growthands