Expression and Activity Analysis of Non-Structural Protein 2 (NS2) of Bombyx mori Densovirus Zhenjiang Strain

The gene of the non-structure protein 2 （NS2） was cloned by PCR from the genome ofBombyx mori densovirus Zhenjiang strain （BmDNV-Z）, inserted into prokaryotic expression vector pET28a to construct recombinant plasmid pET28a-NS2 and then expressed in bacteria Escherichia coli BL21 （DE3）. The expressed recombinant protein was identified by SDS-PAGE and Western blot analysis. Then, the recombinant protein was purified by Ni-NTA column, renatured and tested for enzyme activities. The purified NS2 protein exhibited a helicase activity unwinding double-stranded DNA substrates into single-strand primers, and higher unwinding activity to polarity substrate. Similarly, the purified NS2 protein possessed an ATPase activity and its enzyme activity was 0.276 μmol gg^-1 h^-1 in this study. The results indicated that the non- structure protein which encoded by the gene of BmDNV-Z NS2 possesses the biological activities of helicase and ATPase, and the helicase prefers to polarity substrates. Based on these results, it is speculated that the gene of BmDNV-Z NS2 plays an important role in the viral DNA replication.

SSR based linkage and mapping analysis of C, a yellow cocoon gene in the silkworm, Bombyx moil

The yellow color of the cocoon of the silkworm Bombyx mori is controlled by three genes, Y （Yellow haemolymph）, 1 （Yellow inhibitor） and C （ Outer-layer yellow cocoon）, which are located on linkage groups 2, 9 and 12, respectively. Taking advantage of a lack of crossing over in females, reciprocal backcrossed F1 （BC1） progeny were used for linkage analysis and mapping of the C gene using silkworm strains C 108 and KY, which spin white and yellow cocoons, respectively. DNA was extracted from individual pupae and analyzed for simple sequence repeat （SSR） markers. The C gene was found to be linked to seven SSR markers. All the yellow cocoon individuals from a female heterozygous backcross （BC1F） showed a heterozygous profile for SSR markers on linkage group 12, whereas individuals with light yellow cocoons showed the homozygous profile of the strain C108. Using a reciprocal heterozygous male backcross （BC1M）, we constructed a linkage map of 36.4 cM with the C gene located at the distal end, and the closest SSR marker at a distance of 13.9 cM.

Cloning and characterization of the gene encoding an ubiquitin-activating enzyme E1 domain-containing protein of silkworm, Bombyx moil

Bombyx mori cytoplasmic polyhedrosis virus （BmCPV） is one of the major viral pathogens for the silkworm. To date, the molecular mechanism of BmCPV invasion has been unclear. We cloned the full length complementary （c）DNA which encodes the ubiquitin-activating enzyme El-domain containing proteinl （UbE1DC1） ofBombyx mori by using suppression subtraefive hybridization （SSH） and rapid amplification of com- plementary （c）DNA ends （RACE）. The full-length eDNA of UbE1DClgene is 1 919 bp, consisting of a 100 bp 5＇ untranslated region, a 637 bp 3＇ untranslated region and an 1 182 bp open reading frame （ORF）, encoding a 393 amino acid protein. The protein contained the THiF_MoeB_hesA_family domain, an adenosine triphosphate binding site, which belongs to the family of ubiquitin-activating enzyme El. Reverse transcription - polymerase chain reaction analysis from the silkworm tissues, namely silk gland, hemo- cyte, fat body, gonad and midgut revealed that UbE1DC1 was expressed in all the five tissues. The real-time quantitative polymerase chain reaction analysis indicated that the relative expression of UbE1DC1 in the normal midgut was approximately 9.78-fold of that in the BmCPV-infected midgut. It is implicated that UbEIDCI may play an important role in the interaction between the host and BmCPV invasion.