Construction Of Plant Expression Vector Carrying The Gene Involved In Ascorbate Peroxidase From Chinese Wild Vitis And Preliminary Studies On Genetic Transformation

Powder Mildew is one of the main fungus diseases affect the production of grape over the world. We study team have done a great deal of researches with Powder Mildew of the Chinese Wild Vitis Species, and made more progress in the field. The gene of APX we used in this experiment was separated from Baihe-35-1, a highly resistant strain of Chinese Wild Vitis growing in east of China, by mRNA differential display and RACE amplification technology. Compared with multiple sequences, the identity of VpAPX is 79% to 88% to other plants APX genes.We joined the APX gene to expression vector pWR306 form a recombined plasmid pWR-APX and then introduced it into Agrobacterium tumefaciens strain GV3101. The regeneration system was studied using the leaves of Autumn Royal, a seedless table grape released in 1996. We ascertained some affecting factors of this regeneration system of Autumn Royal and did some preliminary study on the Agrobacterium-mediated gene transfer. Conclusions can be deduced as follows:1. The APX gene was obtained from cDNA by polymerase chain reaction (PCR) of special primers. The further cloned it into pGEM-T easy vector and DNA sequencing showed that the gene order is 769bp in length and has an complete open reading frame, which was correspond to our obtained sequence.2. Recombinant plant expression vector pWR-APX was constructed by pGEM-T-APX and pWR306, these first separately double-digested with two restriction enzymes, BamHI and SalI, and then ligated again.3. The recombinant plasmid pWR-APX was introduced into Agrobecterium tumefaciens GV3101 by freezing-thawing method. By screening in medium containing Kanamycin and Gentamycin, and clonoy PCR identification, the results confirmed assuredly.4. By separately use plasmid, colony and Agrobecterium liquid as template, PCR have...