| Carotenoids Oxygenase is one kind of oxidases, which help to generate the Vitamin A( VA) from carotenoids. In animals, products of carotenoids play an important role in the process of visual formation, cell differentiation, immunity, gene expression and regulation, embryonic and reproductive development. The relevant research in Drosophila melanogaster shows that in the absence of the Carotenoids Oxygenase visual mutant, ninab, vitamin A and retinal pigment content reduce significantly than the wild type, causing the absence of visual. The research of carotenoids oxygenase has not been reported in silkworm. In this study, we identified the silkworm Bmnina B gene and analysised the expression pattern by RT-PCR, Real time PCR, and Western blot. Also, we studied the gene function by RNAi experiment.The main results are as follows:1. Cloning and identification of Bmnina B gene in the Silkworm, Bombyx mori.Through the homologous comparison with reported Nina B gene in the Drosophila melanogaster and Galleria mellonella, we located the homologous gene in the silkworm genome database named BGIBMGA005193 on chromosome 12 nscaf2825. But the result from EST evidence analysis and sequencing result shows that the Gene starting position annotated in the silkworm genome is wrong. There are two extron lost. Finally,we found two kinds of shear in the silkworm m RNA of Bmnina B gene: the first kind of shear Bmnina B-1 contains 14 exons and 13 introns, full-length m RNA for 2153 bp,coding regions for 1536 bp, coding 511 amino acids. And the second one codes 1422 bp,causing 111 bp lost from the position of 496 to 606. For this reason, The 34 th amino acid mutate from T to N, and lacking 37 amino acid residues from 35 th to 71 th.2. The expression pattern analysis of Bmnina B geneAccording to the full length of the BmninaB gene, we designed the primer. Using RT-PCR and Real-time fluorescent quantitative PCR, we have analyzed the expression pattern of Bmnina B genes in embryonic, larva, pupal, and moth stage. We found that Bmnina B gene express highly in the unfertilized eggs and the first day of embryonic period, after the first day, the expression level decreasing to a low level. In the larval stage, the expressing is low. Additionally, Male and female are different during pupal and moth stage, the amount of expression in the female is significantly higher than male.The study in day 3 of 5th instar stage showed that the m RNA content is higher in gonad and head than other 8 tissues.3. The prokaryotic expression, purification and polyclonal antibody preparation of Bmnina B gene.The full length of Bmnina B gene was subcloned into expression plasmid p ET28 a for prokaryotic expression. There are only a small amount of soluble protein found. We immune rabbit with purified Bm Nina B to obtain polyclonal antibodies. The titer of serum antibody is over 1:12800, and the result of Western blot shows a good spacificity of it. Further research of Bm Nina B express pattern in different tissues showed that the cotent of Bm Nina B is high in head and ovary, also detected in nerve, fat body and midgut.4. Silencing the BmninaB gene of silkworm by RNAi to research the function of the geneIn vitro, we synthesized ds RNA for interference Bmnina B gene. After injecting ds RNA in the day 4 and day 6 of the wandering stage, we observed and counted the number of eggs of the female pupa, founding that the number of eggs has no different between the experiment groups and the control group in the day 6(group 80μg, 150μg,220μg, respectively). And remarkably, the number of eggs reduced in the groups of day4 injected(group150μg,250μg, respectively). Besides that, we also found that the ratio of unfertilized and dead eggs increased. Further study shows that the content of Bmnina B m RNA and protein is reduced significantly compared with control group. |
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