Prokaryotic Expression And Polyclonal Antibodies Preparation Of CrtB And CrtI From Erwinia Uredovora

Carotenoid is an important indicator for nutritional quality of wheat. In order to increase the carotenoid content and explore the metabolic control mechanism of carotenoids in wheat, a carotenoid desaturase gene(CrtI) and a phytoene synthase gene(CrtB) from Erwinia uredovora were successfully transformed into mode wheat variety Bobwhite previously in this laboratory. Transgenic wheat lines containing a single or both genes were obtained, which had different carotenoid content for different expression of CrtB and CrtI at transcriptional level in wheat endosperm of transgenic offsprings plants.In order to further exploring the relationships between the carotenoid content and the expression of the two genes at the translation level, prokaryotic expression vectors of pET32-CrtI and pET32-CrtB were constructed, respectively. These two expression vectors were then transformed into E. coli BL21(DE3) competent cells. Two recombinant fusion protein His-Trx-s-CrtI and His-Trx-s-CrtB were sucessfully expressed after being induced by IPTG. Then the two recombinant proteins were purified and used as antigen to immune rabbits, and the polyclonal antibodies for CrtB or CrtI protein were obtained. By using western-blot, the expressions of CrtB and CrtI in different transgenic offsprings were examined. The results showed that different transgenic offspring plants expressed the exogenous proteins of CrtB and CrtI at different levels. The polyclonal antibodies prepared in this study can be used for "Golden Wheat" study.