30309.杜氏鹽藻(dunaliellasalina)cdna文庫構建及表達序列標簽分析

1、華中科技大學碩士學位論文杜氏鹽藻（Dunaliellasalina）cDNA文庫構建及表達序列標簽分析姓名：張翅申請學位級別：碩士專業(yè)：微生物學指導教師：紅凌20090520II華中科技大學碩士學位論文AbstractDunaliellasalina，belongingtoVolvocalesakindofgreenunicellularalgaewithnocellwall.Asitcangrowinverylargesalinity

2、rangefrom0.05molLto5.5molLDunaliellasalinaisconsideredtobeamiracleofsaltresistantinautotrophiceukaryoticganisms.Withthesimplecellstructureitbecomesamodelextremophilicalgatoresearchsalttolerantmechanismsearchfsaltresistan

3、tgenes.RecentlyDunaliellasalinaasasourceoflipidssubsequentbiodieselproductionwhichcanalsofixatmosphericcarbondioxidereduceglobalwarminghasbecomeresearchfocus.Atpresent，theDunaliellasalinagenomicprojecthasnotfinishedyetth

4、ereareonlyabout3936ESTs213nucleotideintheGenbankofNCBI.Therefethegenemessageisnotenoughfthefurtherresearch.However，cDNAlibraryisanefficientmethodtoscreenanalyzevaluablegenes.BasedonsuccessfulculturingofDunaliellasalinain

5、thelabatythecellgrowthcurveat1.5molLNaClsalinitywasstudied.HighqualitycDNALibraryfromDunaliellasalinaculturedat1.5molLNaClwasconstructedbyusingTakaracDNALibraryConstructionKit.ThecDNAwassynthesizedbyMMLVwithpurifiedmRNAa

6、fterbluntingthecDNAterminiEcIAdapterswereligateddigestedbyNotIremovedtheshtfragmentbypassingspincolumnthenthecDNAwasligatedwithpAP3neovectelectropatedtheEscherichiacoliDH10BStrain.ThetiterofprimarycDNAlibrarywas1.2106cfu

7、mLtherateofrecombinantwasabove95%.ThecolonyPCRresultsshowedthatthelengthsofmostswerelargerthan500bp.BysearchingtheNCBINucleotidecollection(nrnt)Nonhuman、NonmouseESTsdatabases13of66cloningsequenceshighlyhomologoustotheDun

8、aliellasalinaESTinNCBIGenbank.thenovelESTsprobablyencodenovelgenes.ThesenovelESTslaidthefoundationffurthercloningofthesalttolerancegenesaddedmegeicinfmationfDunaliellasalina.Keywds：DunaliellasalinacDNAlibraryExpressedSeq