Studies On Tansferring AiiA, TuMV-CP Genes Into Chinese Cabbage By Pollen-tube Pathway

Chinese cabbage (Brassica campestris L. ssp. pekinensis) originated in China. It is one of the most widely distributed and the largest acreage of vegetable crops in China,which plays a decisive role in the "Vegetable Basket Project " in northern China especially. However, in recent years,with the continuous expanding of Chinese cabbage cultivation, soft rot, virus diseases, and many other diseases have increasing trend,which affected the production of Chinese cabbage Seriously.This paper reports the studise on tansferring aiiA, TuMV-CP genes into Chinese cabbage by pollen-tube pathway. The results of the studies are as follows:1. This study cloned aiiA gene according to the known sequence of aiiA gene, and constructed the plant expression vector pBBBast-aiiA on the base of plant expression vector pBBBast+ by restriction, ligation, screening, identification and other operations.2. Prokaryotic expression vector, pET29a-aiiA, was constructed and transformed into bacteria strain of E.coli BL21(DE3) to express AiiA protein. Through optimize the expression conditions such as temperature, IPTG concentration and induction time and detected by SDS-PAGE,the soluble AiiA protein(51.1μg/mL)was the highest content at 20℃for 25h with 0.8 mmol/L IPTG induction.3. Biological activity analysis showed that the liquid supernatant of BL21(DE3)-pET29a-aiiA and AiiA protein have good activity and strong antimicrobial activity for Eriwinia carotovora.4. This study transferred aiiA, TuMV-CP genes into Chinese cabbage'erniuxin'by pollen-tube pathway. PCR and PCR-Southern indicated: acquire 5 aiiA transgenic plants, the transformation activity was 0.0242% and 29 TuMV-CP transgenic plants, the transformation activity was 0.1531%.