产喜树碱喜树内生真菌的筛选及喜树内生真菌的SRAP分析_英文_

产喜树碱喜树内生真菌的筛选及喜树内生真菌的SRAP_英文_ Screening of Camptothecin Production and SRAP Analysis of ophytic Fungi from End * Camptotheca acuminata Decne 1 122 1 1,, LI XiaLIUJ ia-jiaCHENJ ian-huaLUAN inMg-baoYINZ hen-zhenYANGD ong-liang ( 1 School of Chemistry and Chemical Engineering，Central SouthU niversity，Changsha 410083，China) ( 2 Institute of BastF iber Crops，Chinese Academy oAfgr icultural Sciences，Changsha 410083，China) Abstract Endophytic fungi，lived in stems andl eave sof hostp lants，were supposed to beffe cteive or novel sources forthe rapeutic compounds, Camptothecaac uminata Decne is a traditional medical plant in China, About 50e ndophytic fungi were isolated from barks Cof, acuminata, A new fungus pro ducing camptothecin ( CPT) was identified as aP enicillium based oni ts morphological features, Ten endophytic fungi of C, acuminata were chosen to be pli faimed using molecular maker sequence rela ted amplified polymorphism ( SRAP ) , Th e combinations of SRAP primers turned out to pobe lym orphic and a t otal of 1 295 o lpymorphic bands were Key words PenicilliumEndophytic fungusMolecular maker Genetic diversityobtained, These strains were groupedin to 3 main clusters and revealed that SRAP oculd isolate the tsrains of C, medicinal plant，was first found to have 1 Introduction ,6,pharmacological acuminata efficiently, It laso provided evidence for discussing the genetic diversities of psecies in al,agent named camp tothecin by Wall CPT et,7, C, acuminata specifically inhibits DNA topoisomerase I and exhibitsEndophytic fungi，which live within plants' internalwith SRAP molecular method, preferential toxicity to proliferating cells, At tissues or organswi thout causing any apparent symptoroms present，some disease sin the hostpl ants，may produce same iomril sar endophytic fungi with anti-tumor activity werei solated ,8-9,compounds wi th plants， presumably attributed to HCPT,dfirosmcovered to produc e CPT andNew the C, acuminata, Xylaria sp, and F usarium solani werepolyketides with significant inhibition on the ,1,growth ofsymbiotic relationship with their hosts, A number ,10,human-tumor HeLa cells were obtained by Yuan eatl , of Nevertheless， the use of ndop heytic fungi for endophytic fungi with good b ioactivity had been the isolated production of bioactive productsre mains limited, from p lants，such as from Ca ssa spectabs， Artemsaiiliii Camptothec aacuminataDecne， atraditiona Sequence re lated amplified polymorphism，a PCR-l annua，Cynododna ctylon and soo n，since Strobel et al 2,,based marker tesmy，swas a newero lmecular markerf irst applied in genetic diversities of Tricholoma Received April 02, 2011Accepted May , 1 12011 ,11,,12-14,matsutake，introduced by Li et al， and had been obtained a psecies of nedophytic fungi * Supported by N athe tional Natural Science Foundation of Auricularia polytricha and Ganoderm a strains, China successfully ( Taxomyces However， the app lication of SRAP has never ( 3090091) 3 and the Key ResearcIthem s from theMin istry of Education ,3-5,andreanae) produced acplitaxel fromT axus brevifolia, beenof China ( 03126) Corresponding author，E-mail: liujj090@3 163, net,, discussed in endophytic fungi of C, acuminata, Thus，thei solation and CPT opdruction of nedophytic72? , The a mplif ication products were naly zaed by werefungi in C, acuminata as well as SRAP electrophoresis in polyacrylamide gels and tsained by analysis reported,silver ,17, 2 Materials and methods as described by Bassam elt aand photorgaphed, DM 2000 lpus DNA (B ioTeke Corporation，Beijing) wasu sed 2. 1 Source and isolation of endophytic fungi Table 1 Primer sequence usedfo r SRAP analysis as isze markers, Barks oCf , acuminata were gathered Z haatng jiajie me6 TGAGTCCAAACCGGACA em12 GACTGCGTACGAATTCTC-- nature conservation area，HunanPr ovince，China, The me-8 TGAGTCCAAACCGGACT em-19 GACTGCGTACGAATTAGC me-13 TGAGTCCAAACCGGAAG em-23 GACTGC GTACGAATTATT me-16 TGAGT CCTT barks were surface-sterilized according to the TCCGGTAA em-24 GACTGCGTACGAATTACG me-23 CTGGCGAACTCCGGATG em-25 ,15,GACTGCGTACGAATTATG me-24 GGTGAACGCTCCGGAAG em-26 al,dperosccrediubereds by Huang et The barks were icnut to GACTGCGTACGAATTCGG 2 pieces of 0. 5 camnd thepnl aced on potato dextrogasre a ( PDA) supplemented with ampicillin and gentamicin，and purified when incubated on PDA at 28? , 2. 4 PCR-SRAP data analysis 2. 2 Fermentation and analysis of CPT Reproducible DNA bands were score das 1The strains were fermented in liquid potato dextrose mdeium at 25? for 7 days after been prepared ( presence)or 0 ( absence) tfroair nss, Dice' s on PDA slants, The cultures were extractedti m3e s with similarity coefficientchloroform after concentrated under pressure and between trasins were calculated using condensed, The residues were redissolved in methanol, s The ersidues were naalyzed by TLC with developing ,18,the Numerical( NTSYSpc， version 2. 1 ) softwarpe acka ge, The- solvent ( chloroform / methanol，9 ? 1 ) and HPL C using a Taxonom y Multivariate Analysis System reverse-phase Noca pac C18co lumn ( 4 × 150mm，4m) μand SHAN program wapps liaed to thedi stance matrices cluster detection at 254 nm wit h a gradient program as ,19,analysis using UPGMA algorithm，and a dendrogram follows: 30% : 35% acetonitrile for 5 imn，35% :4 0% acetonitrile for 7 inm ( 1ml / min ) , The CPT was produced basedi mpolne smatching matrix, purchased from RonghePha rmaceutical Technology Development Corporation ( Shanghai，China) was used atsa ndsard, 3 Results and discussions 2. 3 DNA extraction and SRAP analysis Total DNA was extracted from 1. 0 g ocefli wa et my3. 1 Isolation and identification of the endophytic using the cetyl trimethylammonium bromide ( CTAB ) method fungus，EFC I-13 ,16,described by Zeng etl a, Six forwardp rimers and About 50 st rains of nedophytic fungi were six reversep rimers ( SangoBni otech，Shanghai) were isolated from barks Cof , acuminata grown in the Zhangjiajie， Hunan porvince，China，non-sporulating fungi( 48.9 % ) ， Alternaria ( 12.6 % ) ，Phomopsis ( 6.9 % ) ，Penicillium ( 6.3 % ) ， Mucor ( 4.6 % ) and F usarium ( 4.6 % ) , Among thefsune gi，the metabolites of one trsain，EFC I- 13，had a spwoitt h the same Rvfa lue of reference CPiTn selected for SRAP analysis ( Table SRAP TLC ( Fig, 1 ) ，and the sameete nrtion time of reference 1 ), amplifications were performeind 25 reaction l μCPT in HPLC ( Fig, 2 ) , This str ain was c onsidered volumes to containing the following reagents: 1. 0 g of μ produce CPT andi dweantsi fied as Penicillium according to genomic its morphological featuresF i(g , 3) , DNA，0. 5l of eachp rimer，1l dNTPs ( 2. 5ol mm/ μμ White mycelium was produced after trthe ain wsas L) ， cultured one day oPDn A at 2 5? ， and green po ress 1. 5 U Taq oplymerase ( BioTeke Corporation，Beijing) ， appearedin the center coof lo nies on the second 2. 5 10 ×PCR buffer，2 of MgCl( 25 mmol / l l μμ2 day, L ) ， Then，apophysis showed upin the center awndhit e aerial and tserile double-distilled water, Amplification Fg. 3 Photos of mcoscopca mophoogca iirilrlil characters of EFC I-13 excision，were rough and appeareliked a hcain, Conidium Fig. 1 Result of TLC was 1. 2 4 :m 1.and shaped aps her iscal or μ ReferenceC PTExtract froEmF C I-13( a)( b) ellipse without transversum, The ersults showed the fun gus， EFC I-13， is a species of Penicillium, This is the f irst time to find a species of Penicillium isolated from C , acuminata that could producec ampothecin, The effect orfi ovuas chemical factors on theCPT biosynthesis could be investigated in futurew ork, To then on-producing CPT fungi，a lot of workc ould be done toiso late some otherc tiave components from thesfeun gi, 3. 2 PCR-SRAP analysis According to d ifferent culture characteristics， 10 endophytic fungi were chosen to be amplified for RSAP analysis ( Fig, 4 ) , Thir ty- six random SRAP i mer prcombinations revealed polymorphisms among tesst trains,The similarity matrix of test trasins used in SRAP analysis showed the Fig. 2 Determination of CPT by HPLC chromatogram similaritCy l ucsoteeffr iAc iceonmtp r ivsaledu e 7 w ages nr oantgyipneg d E fF rC(o m -12，of 0. 52,? ( a) Reference CP T( b) Extract froEmF C I-13 0. 41EFC -13to E70. F7C， in-d14i caEtFedC th-a1t5 ，theEgFeC n eti-c5 1r，elEaFtCi onsh-51ips of ????? 10 ands EtrFaCi ns -12 w )ere different， which was consistent that werdei vided into 4 subclusters -?branche d hypha ewithdiaphragm were observed, at with the morphological method, similarity index value of 0. 65, Within Cluster A，Conidiophores， producedby hyphostroma， weraboe ut strains The pyhlogenetic dendrograums ing cluster analysis 2. 4: 2. 7m with septum，and the topw hoifch were notμ EFC-12 and EFC-13 appeared to be the clmoosselty ?? based on SRAP ( Fig, 4) showed that thetr a10in s scould swelled，but generated u ltmiple branches wh ich shaped related with a similarity index value of 0. was made up of 2 genotEyFpCed -4(0 ，EFC-52)be groupeidn to 3 main clusters at a ismilarity itnhadetx ??like broom, Conidia，generated by conidiophores through 77, Cluster B value Fig. 4 UPGMA dendrogram of 10 strains constructed based on molecular profiles revealed by SRAP markers ,7, Kjeldsen E，SvejstrupJ Q，GromovaI I，et al, Camptothecin could be separate aitmil sarity index value of 0. 75, EFCinhibits both thecl eavag eand erlegation reactions of eukaryotic -30 was theon ly one in cluster C,? DNA topoisomerase I, Journal of Molecular Biology，1992，28 Though there oarnley 10 strains used in the study ( 4) : 102 5-1030, of endophytic fungi of acuminata efficiently ,8, Liu K H，Ding X W，Deng B W，et al, 10-Hydroxycamptothecin C,andSRAP analysis，it identified that the SRAP cou ld manifested that i t's feasible to d iscuss the produced by a nnedowp hyetic Xylaria sp, ，M20，from Camptotheca isolate genetic acuminata, Biotechnology Letter，s2010，32( 5) : 689693,- diversities of psecies using SRAP molecular technology ,9, Soucik K，Sebastian Z，Michael S, An endophytic fungusf rom Camptotheca ac uminata that produces campt othecin by and analogues, Journal of Natural Product，s2009，72( 1) : 2-7, References expanding the number iosfo lates, ,10, Yuan L， Lin X， Zhao P J， et la, New Polyketides from ,1, Petrini O, Fungal endophytes of tree In: AndrewsJ H， Endophytic Diaporthe sp, XZ07, Helvetica Chimica -leave,s Hirano S, Microbial Ecology of leave,s New York : Acta， Spinger- 2009，92( 6) : 184190,- Verlag press，1991, 179197,- ,11, Li G，Qurios C F, Sequencerelated amplified polymorphism- ,2, Strobel G，Stierle A，Stierle ,D Taxol and taxanepro duction ( SRAP) ，a new marker system basedi mopnl e aP CRs reaction: by its application to mppaing and gene itnaggg in Taxomyces andreanae， an nedophytic fungus of Pacific ye w, Brassica, Science，1993，260( 5105) : 21 4-216, Theoretical and AppliedG enetics，2001，103( 2-3) : 455-461, ,3, Silca G H，Teles H L，Trevisan H C，et al, New ,12, Ma D L，Yang G T，Mu L Q，et al, Application of SRAP int he bioactive genetic diversity of Tricholoma matsutakein northeasterCnhi na, metabolites produced byP homopsis cassiae，an endophytic fungus African Journal of Biotechnology，2010，9( 38) : 624 4-6250, in Cassia spectabilis, Journal of theB razilian Chemical ,13, Yu M Y，Ma B，Luo X，et al, Molecular diversity of Society， Auricularia 2005，16( 6B) : 1463-1466, polytricha revealed by inter-simple sequence repeat aqnduen csee- ,4, Lu H，Zou W X，Meng J C, New bioactive metabolites produced related amplified polymorphism markers, Current by Colletotrichum sp, 1，an endophytic fungusin Artemisia annu, Microbiology， Plant Science，2000，151( 1) : 67-73,2008，56( 3) : 240-245, ,14, Sun S ，JGao W， Lin S ,Q Analysis of gneetic diversity ,5, Li Y，Song Y C， Liu J Y， et la, Anti-Helicobacter in pyorli Ganoderma p opulation with a novel molecular marker R ASP,substances fronm dop heytic fungal cultures, World Journal Applied Microbiology and B iotechnology， 2006， 72 ( 3 ) :of variation in terms orfe striction endonuclease,s Proceedings of ,16, ZengF Y，Zhang Y Z, Preparation of deible fungusDN A from the Polysaccharide rich sample, Acta EdulisF ungi，1996，3: 1317,- National Academy of ci enSces ( USA ) ， 1979， 79 ( 10 ) : ,17, BassamB J， Gresshoff P M, Silver staining DNA 52695273,- in ,19, SneathP H A, Numerical taxonomy, San Francisco: Springer polyacrylamide gels, Natural Protocols， 2007， 2 Press，2005, 39-42, ( 11 ) : 26492654, - 产喜树碱喜树内生真菌的筛选及 SRAP 喜树内生真菌的分析 11 2 2 1 1 霞李刘佳佳陈建华栾明宝殷珍珍杨栋梁 ( 1 410083 2 410083)中南大学化学化工学院医药化工所 长沙 中国农业科学院麻类研究所 长沙 。。摘要 生活在宿主植物里的内生真菌是很重要的药用资源喜树是中国的传统药用植物从喜树植 50 ，，物中分离得到了大约 种菌株其中一株产喜树碱的菌株通过形态学鉴定为青霉属这是首次在喜树 。( SRAP) 植物中发现产喜树碱的青霉属菌株为研究简单序列重复相关序列扩增多态性在喜树 ，SRAP SRAP 。内生真菌中应用的可行性选择了十株喜树内生真菌进行 多态性分析引物共扩增出 1 295SRAP ，，。，条带而这些菌株也被分为三大类这些结果明研究喜树内生真菌具有高效性是讨论 。喜树内生真菌的遗传多样性的有效方法 关键词 内生真菌青霉属分子标记遗传多样性 Q75中图分类号 : 2011-04-02: 2011-05-11收稿日期修回日期 * ( 3090091)3 、( 03126 ) 国家自然科学基金国家教育部重点项目资 助项目 ，: liujj090@3 163, net,,通讯作者电子信箱