Transformation And Screening Of T7 RNA Polymerase Gene In Anabaena 7120

As one of the most powerfulgene expression system,T7RNA polymerase/promoter expression system is based on thespecific recognition between the T7RNA polymerase and the corresponding strong promoter.Recently,cyanobacteria has gradually become the new expression hosts for the seasons that its microenvironment,genetic structure and cellular structure are simple.However,expression level of exogenousgenes in the cyanobacteria is low,which limit the development and application of gene engineering.Purpose:To screening and identify the anabaena including T7RNA polymerase which was built by transferringsite integration expression vector of T7RNA polymerase to the Anabaena sp.PCC7120 chromosome.Method:Anabaena sp.PCC7120was carried out the sensitivity test to compare itssensitivity to five antibiotics,to screen resistance gene in the transgenic cyanobacterium.The PCR was performed to obtain the T7RNA polymerase gene with the histidinelabeled,resistance genes Cm and homologous gene fragment F2.The three fragments above together were piecedby overlap extension PCR and linked to carrier of pEASY-BluntZero and sequenced.Then the sequence confiremed were linked up with the pEASY-T1-F1-Tac-NptII-F2 by enzyme digestion and connection to get the site integration expression vector of T7RNA polymerase whichwas linearized andtransferred into the wild type Anabaena,followed the resistance screening and sequence.Finally,the western-blotwas used to detect T7RNA polymerase gene expression.Result:1 Comparison of the Sensitivity of Anabaenasp.PCC7120 to Five Kinds of AntibioticsWild anabaena was coated to theBG11 solid medium with the different concentrationsof antibiotics,cultured in light at the appropriatetemperature.Concentration gradients of Amp were0,5,7,10,12,15?g/mL,Kan were 0,10,15,20,25,30?g/mL,Str were 0,0.002,0.005,0.008,0.01,0.02?g/mL,Nm were 0,0.1,0.5,0.7,1,3?g/mL,Cm were 0,0.01,0.05,0.1,0.5,1 ?g/mL.The results show that all antibiotics can reduce the number of anabaenain a dose-dependent manner.At the concentrationof 5 ?g/mL,Amp can restrain the growth of the anabaena PCC7120,while the high concentrationAmp can lead theanabaena PCC7120 to almost no growth.Similary,20 ?g/mLKan,0.08 ?g/mLStr,0.5 ?g/mLNm,0.01?g/mL Cm can suppress the growth of the anabaena PCC7120.Lethal concentration for Kan,Str,Nm,Cm were 30 ?g/mL,0.02 ?g/mL,3 ?g/mL,1 ?g/mL,respectively.2 Comparison of the Sensitivity of Anabaenasp.PCC7120 to Cm and Nm at the Condition of Liquid-cultureAnabaenawas cultured in the liquid mediums containing the different concentrations of Cm and Nm,respectively.After one week,Nm,at the concentration of 2 ?g/mL,can regress the growth of the anabaena PCC7120 with the yellow color.3?g/mL,Nm can result in the death of cells.4 ?g/mL,Nm can lead to death.While 5 ?g/mL,Cm can regress the growth of the anabaenawith the yellow color and 9 ?g/mL to die.3 Construction of Site Integration Expression VectorT7RNA polymerase gene with His tag,resistance gene Cm and homologous fragment F2 gene were obtained by PCR technique and were sequenced and analyzed.Then we spliced the three genes with correct sequence by overlap-extension PCR to obtain the T7RNAP-Cm-F2 genetic element.Furthermore,the element were cloned to carrier of pEASY-BluntZero followed which the recombinant plasmid was confirmed by digestion and sequence,recyclledg by gel extraction kit.Similarly,the process discussed above were performed again the obtained the pEASY-T1-Fl-Tac.The T7RNAP-Cm-F2 and pEASY-T1-Fl-Tac were linked by T4 ligase,plasmids extracted,enzyme digestion,sequencing to obtain the site integration expression vector.4 Transforming and screening of T7RNA polymerase gene in wild anabaena sp.PCC7120Site-specific integration vector was digested with HindIIIto recycleLinearized Vector.After which,Linearized Vectors were transferred to the wild anabaenasp.PCC7120 in the logarithmic early growthbyelec-transformation.The anabaena containing the transformation vectorwas cultured in the BG-11+Nmedium without the antibiotic in dark condition.After 24h cultured,anabaena was transferred to the BG-11+N solid medium containing different concentrations of Cm in the light.Anabaena was transferred to the BG-11+N solid medium containing higher concentrations of Cm every 3 days.Two weeks later,the anabaena was selected and re-cultured in the 15 ?g/mLCm BG-11+N solid medium for expanding culture.After reaching logarithmic growth phase,the genome DNA was extracted and used as genetictemplate to carry out PCR to identify transgenic anabaena.Also,the preferably voltage for electro-transfer was 1600v.The best methods for extraction of Cyanobacteria genomic DNA was the CTAB methodcompared to the ultrasonic extraction method.CTABcan extracthigh-purity DNA.The results of PCR amplification product sequencing showed that anabaena contain the target gene,indicatingwe successfully obtainedtransgenic cyanobacteria.Conclusion:We developed thesite-specific integration vector which was linearized and transferred into the anabaenasp.PCC7120 and identified by Sequencing,but the further study was conducted to confirm whether the T7RNA polymerasewastransferred into the anabaenasp.PCC7120.