Cloning、Expression And Functional Preliminary Research On Fur Gene Alr0957of Anabaena Sp. PCC7120

Cyanobacteria is a kind of photoautotrophic phytoplankton, it will trigger algalbloom in the eutrophic water which is rich in nutrients under the appropriatetemperature and light intensity；on the other hand, algae can produce energy materialsSo algae has been playing an important role in both environmental protection andexploiting of new energy sources.We chose a dominant algae Anabaena sp. PCC7120in algal bloom toinvestigate the effect of Fe on growth of the algae and regulation of the fur gene（alr0957）.First of all, we designed a pair of specific primers and amplified a segmentabout450bp by Touch-down PCR from the Anabaena sp. PCC7120genome DNA,then cloned the fur C gene by using TA cloning, and then constructed therecombinant plasmid pET-fur for expression, transformed into E.coli strainBL21（DE3） successfully, and a19kD fusion protein pET-Fur with both T7-tag andHis-tag was induced by IPTG. We optimized the induced temperature, IPTGconcentration and induction time. Then we came to a conclusion that the optimuminduction condition is37℃,0.8mmol/L IPTG and for8hours. However, we need toimprove protein expression by changing the primer sequence.On the other hand, we investigated the influence on the growth of PCC7120under a concentration gradient of Fe³⁺, judging by the growth rate and content ofchlororphyll, total proteins and soluble sugars.It showed that a low level of Fe³⁺concentration can pomote while a high levelmanifested inhibitory. The optimum Fe³⁺concentration ranged from0.5mg/L to0.9mg/L.We can conclude that Fe³⁺concentration affected metabolisms in PCC7120,especially on enzymes in charge of photosynthesis,so that these cultivated algea weredifferent in color.Then we chose three of them（L:0mg/L,M:0.5mg/L,H:2.0mg/L）, extracted totalRNA to analysis transcription diffrences. Compared to sample M, total RNA from theH had much more clear stripes:23S rRNA、16S rRNA and5S rRNA, but a littledegrated;on contrary, RNA from the L showed a weak signal and dispersive, that’sbecause iron deficiency disturbed metabolisms, and retarded growth.At present, researches on iron metabolism regulation focus on marine algae,expression of the ferric uptake regulator gene in vitro and mutants construction weremuch less in fresh algae. In this paper, the fusion recombinant protein pET-Fur wasexpressed, and layed a foundation for protein purifying and mutants construction which would lead us to reveal mechanism that how fur gene regulate iron homeostasisin PCC7120.In this study, we analysised growth, metabolisms and different expression thougha new perspective of ferrum, and concluded the optimum Fe³⁺between0.5mg/L and0.9mg/L. Wether lack or excess of frrum will lead to metabolism disturbance,transcr-iption decrease and growth decline.