Cloning, Expressing And Characterization Of Fe-SOD From Isoetes Sinensis

According to Ching’s system, Isoetes sinensis belong to Isoephytina. However,some scholar considered that/sinensis should be classified to Lycophytina accordingto molecular data. The palynology and sporophyte on I. sinensis have been wellstudied, though，the plant biology of gametophyte and embryo of that remain lessunderstood. Furthermore, there are few studies on the genetic structure of L sinensisbased on selected molecular marker. So far, the function of genes and the molecularmechanism of I. sinensis were not stuidied. The present investigation successfullycloned Isoetes sinensis Fe-SOD gene, termed IsFeSOD,The total RNA was extracted using I. sinensis containing macrospore andmicrospore as experimental material. Then the cDNA sequence of Fe-superoxidedismutase was cloned by RACE. A pair of degenerate pirmers designed based on thehighly conserved sequences to clone the418bp mid-fragment of SOD gene fromIsoetes sinensis-The full-length cDNA was obtained by the rapid amplification ofcDNA ends (RACE) technique. It consisted of1469nucleotides and an open-readingframe of744bp encoding247amino acids.The bioinformatics analysis was used to compare IsFeSOD with other plants，gene sequences in Gene Bank, which indicating that IsFeSOD also contains TGGGAconservative sequence. We also found that Fe-SOD mRNA sequence are highlyhomologous from100to750with several DNA sequences from Chlamydomonasreinhardtii, Marchantia polymorpha, Barbitla unguiculaia, I. sinensis,Ceraiopteris thalictroides, Pinus pinaster, Oryza saliva, Arahidopsis lhaliana.By the amino acid sequences also found3conserved sequence: FNNA、FGSGW、WEHAYY, and other two: WNHHFF and HWGKH.Furthermore，the recombinant pET32-FeSOD plasmids were transformedinto host E. coli BL21for expression. The recombinant protein about43kDa was induced by IPTG, in line with the expected size. The recombinant proteinwas used for western boltting detection.Ferns as important transition groups in the plant kingdom，is an important andindispensable part to study biological evolution and phylogeny. In addition, there aremany very important plant groups, plenty of valuable medicinal plants, some specialstress-resistant plants. Research of these ferns functional genes and metabolicpathways have great value in medicine, industry, agriculture, horticulture, food and tomaintain ecological balance.DNA molecular markers, gene cloning, microarray and RNAi technology hasjust been applied in studing ferns. But these techniques has become maturity in seedplant molecular operation. The results of this study provide some data and referencefor ferns.