The Construction Of Highly Efficient Chloroplast Expression Vector Of Sugar Beet

A highly efficient chloroplast expression vector of sugar beet were constructed in this thesis. The vector can take the ppdk gene into sugar beet chloroplast well and express highly efficient. It will improve the activation of PEPCase in sugar beet. Then, the produces of sugar beet will increase. At the same time , it will offer a efficient vector for investigating the gene engineering and industrialization.Using the technology of DNA homologous, a kind of highly efficient chloroplast expression vector of sugar beet were constructed The thesis used rps7 and part of rps12 and ndhB as homologous segments, Firstly, using the technology of PCR, two adjacent segments which are about 1.7kb and 1.8kb in length from sugar beet chloroplast genome were cloned. The result of the segments sequence analyse show that the length is 1742 and 1740 nucleic acids. The first segment included part of rps12,rps7 genic coding region and its flanking sequences. the second segment included ndhB gene's introns and the first exons. ,pmi gene that is 1365bp in length were cloned from Ecoil. Then, with the restriction enzymes site rps7,part of rps12 and ndhB gene were transferred to pBluescript SK+ vectors. finally, pmi gene and its expression frame were inserted in rps7,part of rps12 and ndhB as seletion markers. Highly efficient chloroplast expression vector of sugar beet were constructed. The especial vector will be recombined to the distant region between rps 7 and part of rps12and ndhB of sugar beet chloroplast genome and highly efficient expressed. At the same time,CDNA of sugar beet's ppdk gene were cloned with technology of PCR.In this experiment, six middle vectors and one chloroplast expression vector were constrcted:1 Using the site of restriction Enzymes HindⅢand EcoRⅠconnected CaMV 35S promoter,NOS termination and GUS gene to vector pUC18(+),vector pH1 were constructed。2 A middle vector pH2 were constructed after connected pmi gene and expression frame to pUC18.