1、大連輕工業(yè)學院碩士學位論文萊茵衣藻氫酶基因克隆及其結構分析姓名：屈建航申請學位級別：碩士專業(yè)：發(fā)酵工程指導教師：張苓花20040301AbstractAbstractChlamydomonasreinhardtiiisagenusofunicellulareukaryoticgreenalgaeandawidelyusedorganismforhydrogenproductionofcurrentresearchThesegreenal

2、gaepossessbotboxygenicphotosynthesisandhydrogenmetabolism，growingthroughphotosynthesisundernormalconditionswithoxygenandlightwhileswitchingtohydrogenasemetabolismteleasehydrogenafteraperiodofanaerobicincubationHydrogenph

3、otoproductionofalgaeprovidesapotentialwayforrenewablehydrogenenergy，whichgetsgreatimportanceindevelopedcountries，Duringthismetabolicprocess，hydrogenaseisthekeyanditscapabililycallbeimprovedthroughgenetictechnologyGenetic

4、engineeringofalgaehydrogenasecomestoplayimportantroleinthisfieldandattractsmoreandmoreresearchersnovInthisworkCreinhardtiiSEwasselectedasmaterialandasepticallycultivatedbyapplicableantibioticsTheaxenicculturewasthenincub

5、atedunderanaerobicconditionsforaperiodoftime，andbyusingTRIzolreagent，totalRNAoftheorganismwasisolatedFromthetotalRNA、hydA2cDNAofCreinhardtiiSEencodinghydrogenase(HydA2)wasobtainedthroughRTPCR。Ontheotherhand，benzylchlorid

6、ewasusedtoextractthegenomicDNAofCreinhardtiiSETheoptimizedoperationalfactorsofthismethodweredeterminedtobepH90extractionbuffer300BLbenzylchlorideper0lgdryalgae，andincubationtemperatureof60℃DNAofJyUA2ofCreinhardtiiSEwasam

7、plifiedbyPCRfromthegenomicDNACreinhardtiiSEhycU2cDNAandCreinhardtiiSEhydA2DNAwereligatedtOpMDl8TvectorandtransformedEcoliJMl09competentcellsbyheatshockrespectiveIBothpositivecloneweiescreenedandselectedfinallyResultsofnu

8、cleotidesequenceanalysisshowthathydA2eDNAofCreinhardtiiSEconsistsof1518nucleosideswithanidentityof982％toCreinhardtii21gr@dA2eDNAreportedbyNationalRenewableEnergyLaboratory(NREL)Atthesametime，砂dA2DNAof(7reinhardtiiSEconta