Expression And Functional Characterization Of Bm3DE-3β-reductase In Silkworm, Bombyx Mori

Insect growth and development are regulated by molting hormones. The physiological balance of molting hormones depends on its synthesis and degradation. Recent years, the synthesis pathways of ecdysteroid has become increasingly clear. However, few researches were taken on the metabolism of the ecdysone. There are two types of metabolism for ecdysone:directly excrete; convert into the non-active form.Koolman (1989) found that in many insects, especially for Lepidoptera, ecdysone mainly was present in the form of 3-dehydroecdysone (3DE).3DE only showed a very weak activity. Hence, it is considered to be one of main ways for the metabolism of ecdysone in insect. Ecdysone is firstly converted into 3DE by the Ecdysone Oxidase, and then the 3DE is further reduced to 3-epiecdysteroid by an NAD(P)H-linked 3DE 3a-reductase[4].On the other hand, the 3DE is reduced to 3-epiecdysteroid by an NAD(P)H-linked 3DE-3β-reductase. Prothoracic gland is the major organ to secrete ecdysone and 3DE. Previous studies showed that the ratio of 3DE and ecdysone varies in different insects. As 3-dehydroecdysone-3β-reductase takes part in the convertion between 3DE and ecdysone, it may also play an important role in the regulation of molting hormone.The 3DE-3p-reductase enzyme has been purified from the hemolymph of Spodoptera littoralis and Trichoplusia ni. As early as 1996, the 3-dehydroecdysteroid 3β-reductase was purified from Bombyx mori larval hemolymph, and the enzyme parameters were analyzed in vitro. However, the gene encoded 3-dehydroecdysteroid 3β-reductase has not been identified in the silkworm yet. Silkworm has become a model organism in the study of Lepidoptera. The silkworm genome data and gene chip data have been published, all of these provide the information platform for studies of silkworm genes. This study exploits the silkworm genome data to characterize the Bm3DE-3β-reductase gene which is the key gene for the regulation of ecdysone in B. mori. The main results are as follows:1. Bioinformatics analysis of Bm3DE-3β-reductaseBm3DE-3β-reductase was identified based on the data from NCBI and silkworm genome database. Bm3DE-3β-reductase is 1032 bp long, containing 8 exons and 7 introns. Predicted weight of the protein is 39 kDa, PI is 5.69. Bm3DE-3β-reductase in the nscaf 2053 is located on the 2th chromosome. The analysis of protein domain indicated that Bm3DE-3β-reductase (B. mori), Tn-3DE-3β-reductase(T. ni) and Sl-3DE-3β-reductase (Spodoptera littoralis) contain hydrogen aldo-keto domains. Phylogenetic analysis showed that, Bm3DE-3β-reductase has a close relationship with homologs of other Lepidoptera insects.2. Expression pattern of Bm3DE-3β-reductaseThe expression pattern of Bm3DE-3β-reductase was determenied by RT-PCR. It was expressed lowly at the 1st day the 5th instar (5L-1d), but highly in the 7th day the 5th instar (5L-7d) and the initial of the wandering stage (W-0h). The results of the expressions of Bm3DE-3β-reductase during the fifth star are consistent with the time of silkworm molt. Bm3DE-3β-reductase was expressed highly in head, epidermis, fat body, ovary, lowly in hemolymph.3. The prokaryotic expression and polyclonal antibody preparation of Bm3DE-3β-reductaseThe Bm3DE-3β-reductase gene fragment, DNA sequence encoding from the 22nd to 343rd amino acid residues for mature peptide, was chosen and inserted to the prokaryotic expression vector pGEX-4t-1. The recombinant vector was transformed into BL21 (DE3) and induced the expression of the inserted protein coding sequence. The target protein was purified by GST affinity chromatograph. Taking this purified target protein as the antigen, we have prepared the polyclonal rabbit anti-Bm3DE-3P-reductase antibody. The Western blotting analysis of prokaryotic expressed target protein, using this prepared antibody, indicated that the fusion protein has the antigenicity of Bm3DE-3β-reductase in B. mori. and this antibody has high immunologic specificity. 4. Tissue localization of Bm3DE-3β-reductaseWestern blot assay was performed to detect the spatial profile of Bm3DE-3β-reductase gene at the protein level. We found that it was expressed highly in fat body and hemolymph. Further, we detected strong signal in both the organs by immunohistochemistry.5. Eukaryotic expression and functional characterization of Bm3DE-3β-reductaseThe Bm3DE-3β-reductase gene fragment, DNA sequence encoding from the 22nd to 343rd amino acid residues for mature peptide, was cloned and inserted into the eukaryotic expression vector pPIC9K, and then the recombinant vector was transformed into Yeasts (X-33). Methanol was used to induce the expression of the inserted protein coding gene. The target protein was detected by the Western blotting assay. High Performance Liquid Chromatography (HPLC) was used to detect the enzyme activity of the expressed protein. Unfortunately, due to the limite of the substrate, the functional characterization needs to be further studied.