Title：Molecular Cloning and Characterization of Copper/Zinc Superoxide
Dismutase and Manganese Superoxide Dismutase from Green
Bamboo
M.S. Student： Tsung-Han Wu(R92B42024)
Advisor：Dr. Tsung-Luo Jinn
Abstract：
By alignment of the published MnSOD (manganese superoxide dismutase)
cDNA sequences in monocots, the degenerate primers were designed according to the
conserved regions. Two complete cDNA sequences coding for green bamboo MnSOD
were cloned by the RACE and RT-PCR. The deduced amino acid of MnSOD cDNA
consist of 231 amino acids, containing the essential domains of MnSOD, including a
mitochondrial transit peptide (27 a.a.) in the N-terminal. Which shared 75-86%
identity with those of the MnSOD from other plant. Both cDNA coding regions were
cloned into the pGEX-6P-1 expression vector, and expressed in the E.coli BL21 (DE3)
strain. Most of the expressed GST-MnSOD fusion protein aggregated in inclusion
body, but without SOD activity after purification.
A full-length CuZnSOD cDNA clone with 793 bp was also cloned. The deduced
amino acid sequence consists of 152 amino acids, without transit peptide was
discovered. This cytosolic CuZnSOD shows 81-90% identity with other plant. We
designed primers to amplify the complete cDNA by RT-PCR, 4 different CuZnSOD
complete cDNA were cloned. Two of them were also expressed in E.coli as shown
above. Both of the expressed GST-CuZnSOD fusion proteins remained SOD activity,
which was stable in pH>5 and declined to 10% after incubation at 60℃ for 20
minutes.
The total SOD isozymes activity in green bamboo were analyzed, at least 7
isoforms of CuZnSOD and 1 MnSOD were identified. Southern blotting analysis
suggested that there are 5 CuZnSOD and 4 to 5 MnSOD genes. When supplying
0.05-0.2mM copper ions into the bamboo leaf crude extract resulted in no
band-shifting of isoforms on the native PAGE after SOD activity stain. When
denaturing and renaturing the crude extract with 8M urea followed by dialysis, shown
the same result as copper ions treatment. The abundance of green bamboo SOD genes
and the complexity of CuZnSOD isoforms suggested that green bamboo has a more
complex regulation in its antioxidant system. Which may tribute to the
physiologically special properties of green bamboo under oxidative stress.