A cDNA that encodes a sigma class glutathione S-transferase (GST; bmGSTS2) from the silkworm (Bombyx mori) was cloned by reverse transcriptase polymerase chain reaction and sequenced. The deduced amino acid sequence revealed 68%-63% identity with the sigma class GSTs from other insects. bmGSTS2 mRNA was widely distributed in various tissues. The recombinant enzyme was functionally overexpressed as a soluble form in Escherichia coli, purified to homogeneity, and characterized. The optimum pH of bmGSTS2 was approximately pH 7.0, and bmGSTS2 retained >75% of its original activity after incubation for 12 h at pH 6.0-8.0. Incubation for 30 min at temperatures below 40°C did not affect the enzymatic activity. bmGSTS2 was able to catalyze the reaction of glutathione with hydrogen peroxide, and 4-hydroxynonenal. These results indicate that bmGSTS2 may play a role in antioxidant defense in the silkworm.
Genome editing is a powerful tool for the functional analysis of targeted genes. We have previously found that a novel knock-in system, precise integration into target chromosome (PITCh), allows the integration of a donor vector harboring the hsp90 promoter and GFP into the silkworm biogenesis of lysosome-related organelles complex 1, subunit 2 gene in a precise and efficient manner. Here, we examined whether this technique can be used for the knock-in of other silkworm genes. The ku80 gene was selected as a target and was efficiently mutagenized using a pair of transcription activator-like effector nucleases (TALENs) that were constructed for its specific cleavage. Knock-in was then carried out using these TALENs. Microinjection of TALEN mRNAs mixed with the donor vector resulted in significant expression of the GFP marker in G0 larvae. Importantly, GFP expression was also detected in G1 individuals, suggesting that the integrated donor vector can be inherited in the next generation. Genotyping analysis showed that the donor vector was inserted into the targeted ku80 locus in four individuals. It was further found to be inherited in the G2 generation in a Mendelian manner. We argue that the PITCh system offers a versatile tool for the knock-in of various genes, and should contribute to the further promotion of sericultural studies.
The behavior and horizontal dispersal of larvae of the wild mulberry silkworm Bombyx mandarina, the domesticated silkworm B. mori, and their F1 hybrid were compared in the laboratory. B. mandarina larvae moved out of an experimental arena faster than B. mori and the hybrid. They tended to remain motionless for a period, and then quickly move out of the arena, whereas F1 hybrids spent a longer time moving around before leaving the arena, and B. mori larvae showed frequent and short movements without changes in position. These results suggest that domestication has led to behavioral changes that affect horizontal dispersal.
Bacillus thuringiensis (Ishiwata, 1901; Berliner, 1915) has been extensively utilized as a microbial insecticide because it has insecticidal activity. The search for useful strains of this bacterium has been conducted using Cry gene sequences of each strain as an index. Therefore, we could more efficiently search for useful strains by obtaining the sequence of Cry genes for use as a search index. In this study, for the purpose of establishing an efficient classification method, we developed a genetic marker that detects useful strains of B. thuringiensis. We performed the phylogenetic analysis of B. thuringiensis using the genome profiling (GP) method. As a result, correlations between phylogenetic relationships and H serotypes were different, and these results were the same as those reported previously (Enomoto et al., 2015). In addition, by comparing the many profiles obtained with the GP method, the specific sequence of the H1 serotype was provided. We designed new primers that could amplify the specific sequence of H1 serotype strains, and on performing the polymerase chain reaction, a common band was obtained only from H1 and H24ab serotype strains. Consequently, we designed a genetic marker capable of searching H1 and H24ab serotype strains more efficiently.
We modified hydroxyapatite (HA)-coated nonwoven polyethylene/polypropylene fabrics by coating with silk fibroin (SF) to improve them as a three-dimensional substrate for culturing human hepatocellular carcinoma-derived FLC-5 cells. After 25 days of culture, FLC-5 cells cultured on nonwoven fabrics coated with HA and HA plus SF (HA-SF) partially formed multicellular aggregates, whereas the cells cultured on tissue culture plates formed monolayers. FLC-5 cells cultured on nonwoven fabrics and tissue culture plates for 25 days were subjected to quantitative assay for cell number and albumin secretion. The lowest cell number and the largest amount of albumin were found for nonwoven fabrics coated with HA-SF. Normalizing albumin values to cell number data demonstrated that albumin secretory function per cell on 2 kinds of nonwoven fabrics was remarkably higher than that on tissue culture plates. Moreover, albumin secretory function per cell on nonwoven fabrics coated with HA-SF was twice as high as that on HA-coated nonwoven fabrics. These preliminary results suggest that modification of HA-coated nonwoven fabrics by SF coating induced functional improvement of FLC-5 cells.
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