A new Fe-SOD gene from a native Chinese tobacco germplasm namely HZNH has been successfully cloned and expressed. Full-length cDNA sequences of the Fe-SOD gene was obtained by employing the 5′ and 3′end RACE method ...A new Fe-SOD gene from a native Chinese tobacco germplasm namely HZNH has been successfully cloned and expressed. Full-length cDNA sequences of the Fe-SOD gene was obtained by employing the 5′ and 3′end RACE method from the HZNH′s cDNA library. The full sequence was 1145 bp in length, including 170 bp of 5′untranslated region, 288 bp of 3′untranslated region and 687 bp of coding region. The coding region encoded a peptide of 228 amino acid residues, in which there was a signal peptide with 26 amino acids and a mature peptide of 202 amino acids. The full-length cDNA sequence was compared with all other reported plants’ Fe-SOD genes’. The result of Blast analysis showed that they shared high homology(>80%) ,the highest one was to N. plumbaginifolia’s with the homology as high as 97.69%. This cDNA was constructed into the prokaryotic expression vector, pQE30a/FeSOD, and transformed into E.coli M15 which was induced with IPTG. SDS-PAGE showed that 27 kD proteins was expressed. The soluble proteins showed the Fe-SOD enzyme activities on PAGE-based isozyme spectrum indicating that this expressed soluble protein is indeed the Fe-SOD enzyme.展开更多
文摘A new Fe-SOD gene from a native Chinese tobacco germplasm namely HZNH has been successfully cloned and expressed. Full-length cDNA sequences of the Fe-SOD gene was obtained by employing the 5′ and 3′end RACE method from the HZNH′s cDNA library. The full sequence was 1145 bp in length, including 170 bp of 5′untranslated region, 288 bp of 3′untranslated region and 687 bp of coding region. The coding region encoded a peptide of 228 amino acid residues, in which there was a signal peptide with 26 amino acids and a mature peptide of 202 amino acids. The full-length cDNA sequence was compared with all other reported plants’ Fe-SOD genes’. The result of Blast analysis showed that they shared high homology(>80%) ,the highest one was to N. plumbaginifolia’s with the homology as high as 97.69%. This cDNA was constructed into the prokaryotic expression vector, pQE30a/FeSOD, and transformed into E.coli M15 which was induced with IPTG. SDS-PAGE showed that 27 kD proteins was expressed. The soluble proteins showed the Fe-SOD enzyme activities on PAGE-based isozyme spectrum indicating that this expressed soluble protein is indeed the Fe-SOD enzyme.
