Cloning, expression and characterization of alcohol dehydrogenases in the silkworm Bombyx mori

Alcohol dehydrogenases (ADH) are a class of enzymes that catalyze the reversible oxidation of alcohols to corresponding aldehydes or ketones, by using either nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine dinucleotide phosphate (NADP), as coenzymes. In this study, a short-chain ADH gene was identified in Bombyx mori by 5′-RACE PCR. This is the first time the coding region of BmADH has been cloned, expressed, purified and then characterized. The cDNA fragment encoding the BmADH protein was amplified from a pool of silkworm cDNAs by PCR, and then cloned into E. coli expression vector pET-30a(+). The recombinant His-tagged BmADH protein was expressed in E. coli BL21 (DE3), and then purified by metal chelating affinity chromatography. The soluble recombinant BmADH, produced at low-growth temperature, was instrumental in catalyzing the ethanol-dependent reduction of NAD+, thereby indicating ethanol as one of the substrates of BmADH

Mutation of a Cuticle Protein Gene, <i>BmCPG10</i>, Is Responsible for Silkworm Non-Moulting in the 2^nd Instar Mutant

<div><p>In the silkworm, metamorphosis and moulting are regulated by ecdysone hormone and juvenile hormone. The subject in the present study is a silkworm mutant that does not moult in the 2^nd instar (<i>nm2</i>). Genetic analysis indicated that the <i>nm2</i> mutation is controlled by a recessive gene and is homozygous lethal. Based on positional cloning, <i>nm2</i> was located in a region approximately 275 kb on the 5^th linkage group by eleven SSR polymorphism markers. In this specific range, according to the transcriptional expression of thirteen genes and cloning, the relative expression level of the <i>BmCPG10</i> gene that encodes a cuticle protein was lower than the expression level of the wild-type gene. Moreover, this gene’s structure differs from that of the wild-type gene: there is a deletion of 217 bp in its open reading frame, which resulted in a change in the protein it encoded. The <i>BmCPG10</i> mRNA was detectable throughout silkworm development from the egg to the moth. This mRNA was low in the pre-moulting and moulting stages of each instar but was high in the gluttonous stage and in newly exuviated larvae. The <i>BmCPG10</i> mRNA showed high expression levels in the epidermis, head and trachea, while the expression levels were low in the midgut, Malpighian tubule, prothoracic gland, haemolymph and ventral nerve cord. The ecdysone titre was determined by ELISA, and the results demonstrated that the ecdysone titre of <i>nm2</i> larvae was lower than that of the wild-type larvae. The <i>nm2</i> mutant could be rescued by feeding 20-hydroxyecdysone, cholesterol and 7—dehydrocholesterol (7dC), but the rescued <i>nm2</i> only developed to the 4^th instar and subsequently died. The moulting time of silkworms could be delayed by <i>BmCPG10</i> RNAi. Thus, we speculated that the mutation of <i>BmCPG10</i> was responsible for the silkworm mutant that did not moult in the 2^nd instar.</p></div

Assay of the ecdysone titre in the wild type and <i>nm2</i>, as determined by ELISA.

<p>ELISA analysis was repeated at least three times for each set of protein samples. Each point represents the mean value ±SD. **indicates significant difference (p < 0.01) compared with the wild type.</p

The expression and amino acid sequence of the <i>nm2</i> candidate gene.

<p>(A) Expression profiles of <i>nm2</i> candidate genes based on semi-quantitative RT-PCR. 1–13 representing 13 genes: <i>BMgn002598</i>, <i>BMgn002690</i>, <i>BMgn015078</i>, <i>BMgn002599</i>, <i>BMgn002689</i>, <i>BMgn002600</i>, <i>BMgn002688</i>, <i>BMgn002601</i>, <i>BmCPG10 (BMgn002602)</i>, <i>BMgn002603</i>, <i>BMgn002687</i>, <i>BMgn015079</i> and <i>BGIBMGA002685</i>. The <i>Bm-actin A3</i> gene was used as an internal control. Left lane of each map is wild-type and right lane of each map is <i>nm2</i> mutant. (B) Amino acid sequence alignments for wild-type and <i>nm2</i>. The boxes indicate the amino acid deletions in <i>nm2</i>. (C) The top gene structure is the wild-type and the bottom structure is the <i>nm2</i> mutant. The boxes indicate exons for the coding region and the line for the noncoding region. Start and stop codons are indicated by ATG and stop. The deletion of 217 bp in <i>nm2</i> is indicate by a red dotted line. (D) The top is the full -length cDNA of wild-type, and the bottom is the full -length cDNA of <i>nm2</i>.</p

The feeding rescue experiments by 20E.

<p>Each feeding was repeated at least three times for each group of samples (N = 50). The left larvae were fed 20E and moulted in the 2^nd instar. The right larvae were the controls and could not moult in the 2^nd instar with a glossy cuticle phenotype.</p

The results of <i>BmCPG10</i> RNAi.

<p>(A) The developmental condition of silkworm 24 h after injection. The left was injected with EGFP and was moulting. The right was injected with dsRNA of <i>BmCPG10</i> and was pre-moulting. (B) Relative expression levels of <i>BmCPG10</i> 24 h after injection, as determined by quantitative real-time PCR. Each real-time PCR analysis was repeated at least three times for each set of RNA samples. (C) Relative expression levels of <i>BMgn002601</i> 24 h after injection of <i>BmCPG10</i> and <i>EGFP</i> dsRNA, as determined by quantitative real-time PCR. Each real-time PCR analysis was repeated at least three times for each set of RNA samples. Each point represents the mean value ±SD. The relative amounts of <i>BmCPG10</i> were determined using the <i>Bm-actin A3</i> as a standard. **indicates significant difference (p < 0.01) compared with the control.</p

Results of feeding rescue experiments with 20E, cholesterol, and 7dC, using water as a control.

<p>Results of feeding rescue experiments with 20E, cholesterol, and 7dC, using water as a control.</p