It is reported that β-galactosidase is universally expressed in animals, plants, and microorganisms. However, studies on β-galactosidase in insects are limited, and specifically, it has not been reported in the silkworm, Bombyx mori. Through comparison on NCBI, silkworm EST database, silkworm genome database, and transcriptome database, etc. [18, 19], we found that the complete cDNA sequence of BmGal gene was 2154bp in length, comprising 11 exons, containing an ORF of 1821bp, and encoding a protein of 606 amino acid residues. The protein had an N-terminal signal peptide of 24 residues according to the analysis by SignalP 4.0 Server (http://www.cbs.dtu.dk/services/ SignalP/) . SWISS-MODEL prediction showed that the protein can form a basket-shaped structure comprising three conserved domains. The largest domain had a TIM barrel constructed by α helices and β sheets.
Homology analysis of β-galactosidase amino acid sequences from different species revealed that β-galactosidase in various insects shared a high identity at about 40% in conserved regions and identity in other regions was rather low. Phylogenetic tree constructed using β-galactosidase amino acid sequences in 26 species (Neighbor-Joining method) showed that β-galactosidase in vertebrates and invertebrates significantly differed from each other. What’s more, the evolutionary relationship between BmGal and β-galactosidase in Plutella xylostella was the closest. However, there is no clear report on β-galactosidase in invertebrates. Therefore, the report on BmGal in this paper can provide a theoretical basis for the study of β-galactosidase in invertebrates.
The endogenous β-galactosidase activity was found in various organs at larval, prepupal, pupal and adult stages of Drosophila melanogaster reared under axenic condition by applying X-gal staining methods, such as larval intestine, pupal cellular epidermis, adult pericardial cells and so on . In this study, the BmGal gene was widely expressed in all organs no matter at larval stage, pupa stage, or adult stage according to the mRNA distribution in silkworm tissues. Overall, the expression level of BmGal in the pupal stage was higher than that in the moth stage and higher in the larval stage. In the larval stage, the expression level of BmGal in testis was the highest and the lowest was in the terminal silk gland; in the pupal stage, the highest expression level was detected in the head and the lowest was in the midgut; The expression level of BmGal in the moth stage was relatively less, expression in the Malpighian tubule became the highest and the lowest expression level occurred in the antenna. In conclusion, BmGal was a widely distributed gene and its mRNA level in all organs at larval, pupal, and adult stages of silkworm was quite high.
Testis was chosen for BmGal gene Time-course expression analysis because its expression level of the BmGal gene was the highest among the organs in the fifth instar larva. The expression level increased gradually from the newly molted larva to the seventh day at the fifth instar. However, from the seventh day of the fifth instar to the day before the spinning stage, the mRNA level emerged a rapid decrease of 100 fold in 48 hours. And in the 48 hours from the spinning stage to the day before pupation, the mRNA level increased by 120 fold and reached the peak. In recent years, β-galactosidase has been found to play an essential role in various biological processes. For example, Senescence-associated β-galactosidase staining has now been employed to identify the presence of senescent cells . Su et al. found that the level of β-galactosidase in B cells and serum of rheumatoid arthritis (RA) patients is higher and revealed that altered β-galactosidase may be implicated in the progression of inflammation . Moreover, Streptococcus thermophilus can inhibits colorectal tumorigenesis through secreting β-galactosidase . Interestingly, the results showed that the high expression level and dramatic variation in testis may relate to its morphology changes in the metamorphosis period. And we inferred that the BmGal gene may participate in the development of testis and the procedure of spermatogenesis.
The expression experiments showed that BmGal expressed in the prokaryotic system had no activity, either did it in Pichia pastoris. Therefore, we expressed it in the silkworm expression system, and high enzyme activity was detected. The optimal pH and temperature for β-galactosidase of many bacteria and fungi were found in the range of 6.5-4.5 and 40-60 ºC, respectively [3, 7, 28, 29]. However, the optimum pH and temperature of some β-galactosidase are not in this range, for example, β-galactosidase from Aspergillus lacticoffeatus . In this study, we found that the optimum pH and temperature of BmGal were, respectively, 8.4 and 40℃. Effects of metal ions and organic reagents play important roles in the activity of β-galactosidase. Ba2+, Fe2+, Li+, and K+ can inhibit the activity of Aspergillus lacticoffeatus β-galactosidase, while additives (except ascorbic acid) and detergents can promote its activity . Most metal ions promoted the activity of β-Galactosidase from Alteromonas sp. QD01, especially Mn2+ and Mg2+ . According to the analysis of influences caused by metal ions to BmGal, we found that iron ions can stimulate the activity of the enzyme while cobalt, nickel, or lead ions can inhibit its activity significantly. At present, many β-galactosidases are found to function in the form of oligomers. For example, Thermotoga maritima β-galactosidase is arranged in the form of a peculiar octamer . Structural characterization revealed that β-galactosidase from Bacillus subtilis was a homotrimer in solution . β-galactosidase from the Deep-sea bacterium Alteromonas sp. ML52 is active as a homotetrameric protein . The active phospho-β-galactosidase from Bacillus velezensis was identified to be a homodimer . According to non-degeneration gel electrophoresis of BmGal and Escherichia Coli β-galactosidase, we found that the molecular weight of active BmGal was even larger than that of Escherichia Coli β-galactosidase. As Escherichia Coli β-galactosidase functioned in tetramer mode, we inferred that the enzyme showed activity in oligomer mode, even at least in tetramer mode. This well explained why the expression products in Escherichia Coli or Pichia pastoris didn’t show enzyme activity and indicated that baculovirus–silkworm expression system can express a foreign gene that functioned in multimer mode.