綠竹生長、老化與開花機制之探討─綠竹超氧岐化脢 之抗氧化機制研究

The total superoxide dismutase (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 at least 4 CuZnSOD and MnSOD genes. When supplying 0.05 to 0.2 mM copper or
manganese ion into the bamboo leaf crude protein extract resulted in no activities band-shifting of
isoforms. When denaturing and renaturing of the protein extract with 8 M 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?
By alignment of the published MnSOD cDNA sequences in monocots, the degenerate primers
were designed according to the conserved regions. Two full-length cDNA sequences coding for
green bamboo BoMnSOD were cloned by the RACE and RT-PCR. The deduced amino acid of
BoMnSOD consists of 231 amino acids, including the essential domains of MnSOD and a
mitochondrial transit peptide (27 a.a.) in the N-terminus. The BoMnSOD share 75-89% identity with
MnSODs from other plants. The cDNA coding regions were cloned into the pGEX-6P-1 expression
vector, and expressed in the Escherichia coli BL21 (DE3) strain. Four full-length cDNA sequences
coding for green bamboo BoCuZnSOD also cloned. One full-length BoCuZnSOD cDNA clone of
793 bp consists of 152 amino acids, without transit peptide was analyzed. This cytosolic
BoCuZnSOD shares 81-95% identity with other plant’s.
Recombinant GST-BoCuZnSOD and GST-BoMnSOD remained of the SOD activity, both of
which were stable at alkaline pH and declined to 10% after incubation at 60°C for 20 min. The SOD
activity of recombinant BoCuZnSOD and BoMnSOD were stable more than 3 days incubated at
room temperature. BoMnSOD and BoCuZnSOD cDNA sequences from green bamboo not only
overexpressed in prokaryotes but also remained stable under a broad range of pH, higher
temperature, also very stable in the room temperature. These properties are beneficial for
applications in commercial, such as in cosmetics for skin protection or defending un-esthetic effects
caused by oxygen-containing free radicals.
The promoter of BoCuZnSOD and BoMnSOD were also studied, analysis of the cis-element
within the promoter is proceeding to help us understand the physiological regulation of BoCuZnSOD
and BoMnSOD.