A Preliminary Study Of Anabaena Sp. PCC7120Iron Regulatory Protein Gene All1691

Cyanophyceae or cyanobacteria is widely distributed,variety,and morphologicaldiversity.The photosynthetic pigment from thylakoids can be revealed by electronmicroscopy.Therefore,cyanobacteria is a kind of photoautotrophic prokaryoticmicroorganisms,which has a higher application value in the aspect of food, resourcesdevelopment,and environmental purification.Reseach on the regulation ofdevelopment and nitrogen fixation mechanism of heterocyst in Anabaena sp.PCC7120have been study detail.Thereinto the ferric uptake regulator protein FurA iscontrolled by the developmental regulation factor NtcA of heterocyst.Each partclosely link to fine network to ensure normal activity of algal cells.On the basis of preliminary study,the first part of this paper selected Anabaena sp.PCC7120as experimental materials and ferric uptake regulator gene fur in the algaeas the research object.In order to clone and express Anabaena PCC7120ferric uptakeregulator gene furA (all1691) and its related gene,and to explore the effect of Fe3+concentration in the environment on growth of algae.Firstly we made an improvementto the traditional phenol chloroform method to extract Anabaena PCC7120chromosome DNA.Specific primers was designed according to the sequenceinformation provided by Cynobase.furA,furB and furC this3fur homologous genefragment was amplificated by Touch-down PCR using total DNA astemplate.Conservation of bacteria liquid pMD-fur was obtained by TA clone.Objective fragment connected with the expression vector pET-28a(+) after plasmidextraction and enzyme digestion.This connecting product was transformed into theexpression host E.coli BL21to construct the recombinant expression plasmidpET-1691.About19KD protein band with the label(T7and histidine tag) fromexpression vector system was detected by the SDS-PAGE in the condition of1mmol/L IPTG inducement and37℃shaking culture for15h. In order to obtain theprotein optimal expression under the experimental conditions,previous studies hadexplored factors inducing temperature,inducer concentration effect,and found that thebest expression under the conditions of37℃,0.8mmol/L IPTG induction.Thisexperiment was conducted to explore the induction time.The result showed12h wasthe best induction time of time setting.In order to predict the protein structure andfunctions of FurA,the method of bioinformatics,such as online software,wasused.What we found was that it was a cell wall-bounding protein and located in thecytoplasm.The3D graph constructing display that its edge have approximately5helical domains,which wrapp Beta folding structure zone in the middle region.Theprotein was purified for future research. The second part of this research were to explore the effect on the growth ofAnabaena sp. PCC7120with different Fe3+concentrations. And these growth index ofalgae cell,such as,protein content,chlorophyll content and total sugar content weremeasured with the change of Fe3+.With the help of pectrophotometry,standard curvemethod and molecular biological methods,growth curve shows that low ironconcentration group (0-0.6mg/L) growth rate increases with the concentrationincreasing.When the concentration reached0.6mg/L,the growth rate wasmaximum.So0.6mg/L was the optimum growth concentration.When theconcentration got0.9mg/L,the growth declined in intermediate iron concentrationgroup(0.6-0.9mg/L).Which was manifested by chlorophyll concentration,total protein,carbohydrate content totally decreased.The algae was hard to bear high concentrationsof iron in high iron concentration group(1.4-4.0mg/L),which reflected that algaegrowth went from bad to worse.RNA bands of Iron deficiency group was the mostbrightness.Cell density was the maximum and RNA concentration was the highestwhen the concentration of Fe3+was2mg/L.These changes were consistent with theexpected results.That was low concentration of iron to promote growth,whereasinhibition.In view of very few freshwater algae research,previous experiments hadachieved FurC expression.In this paper,we choose FurA,which play a major role iniron regulation pathways,as the main object of the research.And combining themethod of bioinformatics,we further improved the basically study of iron uptakemechanism in Anabaena sp. PCC7120,which had a certain significance for theapplication.