| Members of the genus Potyvirus, the largest group of plant viruses, are transmitted by aphids in a non-persistent manner. The HC-Pro protein of plant potyviruses is an important viral product that is involved in many functions during the virus life cycle. Among other well-eatablished functions of the HC-Pro, the aphid transmission process seems to be highly influenced by the biologically active HC-Pro protein. We using the Pichia pastoris express system to obtain this protein.Recombinant DNA technology was used to construct recombinant plasmid, which was transformed into the Pichia pastoris to express the interesting peptide. The biological function of the interesting peptide has been identified. According to the on press PVY-N HC-Pro sequence, one pairs of primers were designed. The total RNAs, purified from tobacco leaves infected by PVY-N was used as templet in RT- PCR, then the interesting gene was obtained. After digested with SnaBI and EcoRI , the obtained DNA fragment was linked with pPIC9K by T4DNA ligase and then the recombinant plasmid transformed into competent DH5a. After positive transformants were sieved out by PCR, digesting analysis and sequencing were also used to confirm the positive result more. After linearization, the recombinant plasmid was transformed into Pichia pastoris by electroporation, which then were cultured in MD plate free of histidine, from which the positive colones were propagated. Multicopy clones were screened from vary levels of G418 -YPD plate and cultured and induced with methanol to secrete interesting peptide, which was identified by SDS-PAGE and Western blotting.The interesting gene fragment with SnaBI and EcoRI were amplified by RT-PCR, which inserted into vector plasmid pPIC9K after digested by SnaBI and EcoRI, and the recombinant plasmid was transformed into competent JM109. Positive clones were screened by PCR from the LB plate with AMP and Kan. Digesting analysis result shows that the interesting gene were inserted into the vector pPIC9K with correct direction. Sequencing map also illuminates the interesting DNA fragment of PVY-N HC-Pro was cloned into the expression vector correctly. Multicopy integrants were screened with G418 from Pichia... |
