Resaearch On Genes, Proteins And Mechanisms Of Iron Characterization Of JSC-1

In this paper, the ferritic related mechanism, genes and protein structures of the newly discovered Leptolyngbya sp. JSC-1 were studied. The results are as follows:I obtained the axenic JSC-1 and kept it at -80?. It grew best at 45?,2000 lux light intensity, 40 ?M iron concentration. JSC-1 can tolerate iron concentration of 400 ?M.The result showed that the iron mineralization mechanism of JSC-1 is related to its structure. With the iron concentration going up, JSC-1 external forms iron particles at first. Because the JSC-1 external EPS layer contains negatively charged carboxyl groups, they can chelate iron ions and form iron minerals. This is the first line to defense antioxidant of JSC-1. When the iron concentration continues to rise, iron mineralized particles are formed with internal polyphosphates. Polyphosphates have the function of detoxification and can chelate metal ions. This is the second line to defense antioxidant of JSC-1.The result showed that the total iron concentration of JSC-1 increased significantly when we cultured JSC-1 with 400 ?M iron concentration, which is related to its internal formation of mineralized particles. At the same time,the Fe2+ in JSC-1 was also increasing significantly, indicating that high iron concentration causes oxidative stress. The activities of SOD and CAT were significantly increased when cultured at 400 ?M iron concentration. The activity of POD was highest at 200 ?M iron concentration, but decreased at 400 ?M iron concentration. POD is less resistant to iron oxidative stress than SOD and CAT.I used RT-PCR to study the expression of antioxidant related genes of JSC-1 when it was cultured at different iron concentration. I optimized the internal reference genes and chose rps1B and livD genes as double internal reference genes. The result showed that bfr, sod-4 and cat genes significantly went up at high iron concentration. This is the third line to defense antioxidant of JSC-1.The bfr, sod-4 and cat genes knockout vectors were successfully constructed with pUC18 and pUC19 vectors. Firstly I tried to genetically transform genes to JSC-1 by natural transformation and electroporation. But it was failed.I blasted the protein sequence of SOD, CAT and BFR and I found that the conservative domain of BFR was different. So I studied the structure of BFR. I constructed three expression vectors pGEX6p-1-BFR, pET28a-BFR,pET22b-BFR and purified BFR and Apo-BFR. BFR and Apo-BFR proteins were crystallized. I obtained 5 sets of Apo-BFR crystal diffraction data and built initial structure. I got 4 sets of diffractive data of Apo-BFR soaked in Fe2+ of 2.5 min, 2 sets of diffraction data of Apo-BFR soaked in Fe2+ of 65 min, 3 sets of Apo-BFR soaked in Zn2+ and Fe2+ of 5 min. The protein structure is being analyzed.