Cloning Of Full-length CDNA Of SOD Gene Of Cordyceps Militaris And Its Expression In E.coli And Nicotiana

By comparison of varied kinds of fungi's CuZn-SOD and Mn-SOD amino acid sequences and nucleotide sequences in GenBank, a pair of degenerate primers that was used for RT-PCR to amplify the two SOD genes segments in Cordyceps militaris was designed and PCR product was sequenced. The 5' end and 3' end sequence of the two genes was synthesized by RACE-PCR and sequenced. So, the full-length cDNAs of CuZn-SOD and Mn-SOD genes were gained by overlapping sequenses. The deduced CuZn-SOD amino acid sequence has the similarity of 100% with the sequence Park reported blasted in GenBank and 64% for the Mn-SOD with Emericella nidulans.By RT-PCR, the coding-region sequences of the two genes were amplifyied. Two prokaryotic expression vectors named pGEX-sod!9 and pGEX-sod!4 were constructed by double-digesting the PCR products with Bam HI and Sail and ligating them with pGEX-4T3 vector digesting with the same enzymes. Then E.coli BL21(DE3) was transformed. Induction and SDS-PAGE elctrophoresis showed both vectors expressed GST-fusion proteins. Protein solution analysis showed CuZn-SOD fusion protein was soluble while Mn-SOD counterpart existed in inclusion body. CuZn-SOD was purified by affinity-chromatography that could be used to make antiserum. Mn-SOD fusion protein had specific SOD activity but CuZn-SOD fusion protein didn't by Native-PAGE and NBT stain.Two Plant expression vectors named pBIsod!9 and pBIsodH containing CuZn-SOD and Mn-SOD of Cordyceps militaris were constructed and introduced into Agrobacterium tumefaciens LBA4404. Nicotiana tabacum cv. Yunyan%5 was transformed by leaf disk, expecting to gain SOD overexpression plants. After the selection on the MS media with lOOmg/L Kan, PCR analysis of all CuZn-SOD and Mn-SOD transformed T0 regeneration plants showed that all of them are PCR positive lines, and they are confirmed to be transgenic plants by Southern-blotting.