固氮斯氏假單胞菌A1501亞硝酸還原酶編碼基因nirS的研究.pdf

1、中國農業(yè)科學院碩士學位論文固氮斯氏假單胞菌A1501亞硝酸還原酶編碼基因nirS的研究姓名：王麗英申請學位級別：碩士專業(yè)：微生物學指導教師：平淑珍20050601AbstractDenitrificationbyprokaryotesisa11importantpartoftheglobalnitrogencyclePseudomonassm拉eHA1501wasisolatedfromricepaddiesintheSOUth∞are

2、aofChinaBacterialdenitriflcationiSexpressedinresponsetothesingaloflowoxygentensionInthisstody，wepresentphysiologicalandmoleculardataondenitrificatiOninPseudomonasstutzeriAl501ThennirSwasclonedintopSUP202suicideplasmidvec

3、torwhichwascapableofconjugalselftransfertOawidevarietyofGramnegativebacteriaThenkmlacZ丘agmentfrompKOK5wasintroducedintotheencodedregionofnirSandnirSKmLacZfusionplasmid(pSUP202TCKlwasconstructedThentherecombinantplasmidwa

4、s缸ansferedintothewildtypestrainA1501bytriparentalmatingassayandthenitsmutant(M31WasobtainedbyinsertionofthebmcassetteintothegenomebyhomologousrecombinationinthehostgenomeDenitrificationbetweennirSmutantandwildtypeAl501ha

5、ddistinctdifferenceWhennitratewasusedasterminalelectronacceptordenitriflcationabilitydiscreasedWhennitritewasusedasterminalelectronacceptorwhenwildtypeAl501andnirSmutantwereincubated10hours，nitriteintheculturecouldbeabso

6、lutelyconsumedforA1501comparedto20％fornitSmutantItisconcludedthatthereexistsanotherapproachdifferentfromN瞞denilrifieafionprocessandlowmamboHzationactivityinA1501Bydesigningthespecificpnmer$，thenitSpromoterwasclonedbyPCRm

7、ethodThenitWasclonedintopGD926plasmidvectorwhichWasitsextensivehostrangesandwascapableofconjugalselftransfertoawidevarietyofGramnegativebacteriaThefusionvectorWaStransferredintowildtypestrainA1501andrpoNbyconjugation，and

8、thenBgalactosidaseacfivityWasassayedTheresultshowedthattheexpressionofnirSWasrepressedbyoxygen，anddenitrificationacfivityunderanaerobicconditionsWaShigherUnderdifferentnitriteconcentration，theexpressionofnitSwaslowandthe

9、highestactivityarrivedto2600units強eexpressionofnirSWasinducedbynitrateNitrogenaseacridtyWashighestintheprgSellSeofIOmmol／Lnitrateconcentration，about12900UnitsInthepresenceoftoohighnitrateconcentration(10retool／L)ortoolow

10、nitrateconcentration(O1mmol／L)，nitrogenaseactivityisabout3500unitsWhennitratewasusedasterminalelectronaccepterunderanaerobiccondition，theexpressionofnitSinrpoNWaslowerthaninwildtype，wasaboutonefourthinwildtypeItshowedtha

11、tnirspWasainduced(i54dependentpromoterTherewasnocharacteristic054sequenceinnirSpromoteronginSOitWassupposedthatthereexistsother054characteristicsequenceinnirSpromoteroriginA1SOcolOnizationonthesurfaceofriceIX)otofnitSmut