深海热液区非培养微生物基因资源的功能选与的研究

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1、——————————————————————羔堕L——————一一克隆子，这些混合克隆子具有一定的细胞毒性。研究发现，滤纸片琼脂扩散法和双层琼脂平板法相结合，对文库进行高通量筛选，可以有效避免出现活性克隆假阳性和漏筛；文库构建前，对样品进行放线菌和真菌等的富集，有助于提高筛选效率。关键词：深海热液区；宏基因组文库；转座突变；功能筛选ⅡAbstractDeep．seahydrothermalvents，amongthemostextremeenvironmentsone酬【n，arcch跚．acteriz

2、edbythehightemperature，highpressure，sharpchemicalgradients，pHgradientsandSOon，containinglotsofspecialmicroorganisms·Inordertoadapttosurviveintheextremeenvironments，thesemiroorganismsformthemselfuniquemech锄ism柚dproducesomespecialsecondarymetabolites，which

3、contributetoexploremarinenaturaldrugsandnewbioactivemolecule·Molecularecologicalstudiesrevealedthatonlytinyfractionoftotalmicroorganismsinmarinehavebeenidentifiedandcharacterized,becausethemajorityofthemhavenotbeencultivated．Aconcept，metagenome，represent

4、sthetotalmicrobialgenomeinnaturalecosystemconsistingofgenomesfrombothcuIturablemicroarg觚ismsandviablebutnon-culturablebacteria·ItcanovercometheIimitationofpureculnJmlmethod，andconducestostudingonmicrobialgeneticresourcesfromdeep··seahydrothermalvents·Int

5、hisreSearch，sevenmetagenomiclibrariescontaining26600membersintotaI，havebeenscreenedtoselectantibacterialactiveclonesorantifungalactiveclonesorantumouractiveclonesbyfunction-basedscreening·UsingBnciH潞s¨bliliSandStaphylococcus删Fell$astheindicatorbacteria,f

6、iveactivecloneswercscrcenedfromFosmidlibrary4143-1bydouble—agarplatemethod，tenclonesfromFosmidlibrary5-35．17andthreeactiveclonesfromFosmidlibrary3-11．8．withtheactivepercentagetoclonesinitselflibraryWas0．0635％，0·15％andO．153％，respectively．Using溉，妇m砚妒份cerev

7、isiaeandCandidaalbicansastheindicatorfungi，nine锄ti亿ngalcloneswereselectedoutofabovefifteenactiveclonesbyfilterpaperagardiffusionmethod，andthefiveantifungalclonescanproducepigment-UsingHTl080andBEL7402astumourcellslinemodel，threeclonesoutofnineprodueingpi

8、gmentactivecloneshavestrongactivityantitumourbyCCK-8method·Illthepaper,someactivecloneswrefoundoutthattheyhadtheabilityofproducepigment,unexpectedly．fiveactivecloneswhichCanproducepigmientmAbstractsteadilywereselectedfortr