Studies On Cloning And Activity Of SOD In The Three Species Of Nostoc

As a very important and high economic value Cyanobacteria, Nostoc.sp was used as a good material to research life event under extreme environments (such as drought, cold, hot and UV). It has long been proposed to research its stress response and mechanism of tolerance, to find its mechanism of growth and development, and to accomplish the artifical culture. Our work was performed under these backgrounds. We cloned the SOD gene from different Nostoc and transformed into bacteria. The enzyme activity was measured with fusion proteins. Moreover, we measured and compared the time course pattern of enzyme activity under different stress treatments. Most of the results are as follows:1) We designed the primers based on the Nostoc SOD gene in GenBank and cloned 5 putative gene fragments from genomic DNA of 5 different Nostoc. The PCR products were inserted into vector pUC19 and sequenced. Sequence analysis showed that they exhibit high homology (98¹00% similaritial nuclear acids and 96 ¹00% identical amino acids) with Nostoc SOD gene.2) The PCR products were cloned into vector pET, and the resulting plasmid (pET-SOD) was introduced into bacterial (BL21). A putative Fe-SOD protein was obtained by SDS-PAGE analysis. We purified this protein with Ni exchange column and analyzed its specific activity of enzyme is 2550U/mg.The active pattern of enzyme activity under different stress was analyzed among different transgenic bacterias. The putative expression protein shows a high SOD activity after different treatment. These results suggest that these different Nostoc SOD genes show high commonness.3)We also analyzed enzyme activity under salt stress. The result shows that the Nostoc flagelliforme SOD enzyme activity did not show significant change during 36h after 0.8mol.L^-1 NaCl treatment. After 36h its activity decreased only 15% compared to oh, and this change has no relation with salt content.However, the Nostoc commune and Nostoc sphaeriodes SOD enzymes display different pattern compared with Nostoc flagelliforme SOD enzyme. Both of them show enhanced SOD activity during 12h after lower salt treatment (<0.4mol.L^-1 NaCl). This result suggests that its activity responds to stress treatment. Under high salt stress (>0.4mol.L^-1 NaCl), the SOD activity is obviously decreased. Compared with the lower decreased SOD activity (50% for Nostoc commune and 54% for Nostoc sphaeriodes) under lower salt stress, both SOD show higher decreased activity under higher salt stress (75% and 77% respectively)4)The Nostoc flagelliforme SOD activity shows a slightly enhanced under different pH stress. After treatment at pH5.4 for 36 h, the Nostoc flagelliforme SOD also maintains a high activity level.The Nostoc commune and Nostoc sphaeriodes SOD activity display a higher decreased under higher pH treatment (pH9.4 and pH10.4), and lost the SOD activity after 48h. All these data indicated that the SOD shows a higher activity under acid environment compared with alkalescence environment.5) The Nostoc flagelliforme SOD activity shows a slightly enhanced under different temperature stress. But the Nostoc flagelliforme began to disorganization after treatment at 35℃for 15h so that we could not continue testing the SOD activity. The Nostoc commune and the Nostoc sphaeriodes SOD activity display a different change under different temperature treatment. The Nostoc commune SOD activity displays clearly decreased while Nostoc sphaeriodes SOD activity gone up. We still can find some common character among the Nostoc. SOD activity is the highest when them were treated at 35℃.From the sequence analysis, we found that the SOD gene from different Nostoc.sp display high homology. They encode a same protein and exhibit a similar enzyme activity. However, they also showed the different enzyme activity under difference stress treatments. These results indicated that Nostoc flagelliforme exhibited a higher stress tolerance than other Nostoc, implying that there exists a steady protect system in Nostoc flagelliforme which plays an import role in stress tolerance together with SOD.There has a relationship between Nostoc and human business. To research its gene expression and to analyze the mechanism of activity of life will help us to detect the life event under extreme environments and the corresponding survival mechanism. It also provide theoretical foundation and technical parameters for resources conservation and development and utilization of Nostoc.