Cloning, Expression And Sequence Analysis Of Bmgox, Glucose Oxidase Genes In The Silkworm, Bombyx Mori

Glucose oxidase (GOX) is an aerobic dehydrogenase. It has no side effects and non-toxicity on organisms. GOX is widely distributed in kingdoms of anmimal, plant and microorganism. Compared with the fungal GOX, the insect GOX has not received much attention before. Howeverm, insect GOX has recently been found to play an important role in insect-plant interactions.In this paperâ… investigated the two GOX candidate genes in the silkworm, Bombyx mori.â… cloned and named them as Bmgox63 and Bmgox 72. The main results are as follows:(1) Cloning and sequence analysis of GOX genes.The full length of Bmgox63 gene was 1,710bp, encoding a protein of 566 amino acids, and the predicted molecular weight of this protein was 63kDa, and Isoelectric Point was 5.99. The whole length of the other gene Bmgox72 was 1,830bp, encoding a protein of 606 amino acids, and the predicted molecular weight of this protein was 65kDa, and Isoelectric Point was 5.44. Both of these two genes were located on the 16th chromosome of silkworm, Bombyx mori.(2) Different tissues and stage expression patterns of Bmgox63 and Bmgox72We found that the expression level of Bmgox63 at mRNA level was low. Bmgox72 gene was expressed at all of the development stages and in all tissues more or less. The expression level rose apparently from the fifth-instar 3d, and the expression in both male and female silk glands were higher than other tissues. (3) prokaryotic expression of Bmgox63 and Bmgox72I subcloned coding regions of the two genes into PET-28a (+) expression vector, transformed the recombinant vector into the E. coli (BL21 strain), then induced the expression of protein and purified the product. Subsequently I prepared antibody using the purified protein, and then detected the expression protein through Western blotting, proved that the antibody was correct.(4) The expression of these two genes at protein levels.Consistent with the microarray data, the Bmgox72 protein was highly expressed in silk gland, but the Bmgox63 has no expression signal.In conclusion, the results presented in this study suggested that Bmgox72 may be GOX of silkworm and Bmgox63 may be a psedogene.