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.

Functional analysis of a miRNA‐like small RNA derived from Bombyx mori cytoplasmic polyhedrosis virus

Bombyx mori cytoplasmic polyhedrosis virus(BmCPV)is a major pathogen of the economic insect silkworm,Bombyx mori.Virus‐encoded microRNAs(miRNAs)have been proven to play important roles in host–pathogen interactions.In this study we identified a BmCPV‐derived miRNA‐like 21 nt small RNA,BmCPV‐miR‐1,from the small RNA deep sequencing of BmCPV‐infected silkworm larvae by stem‐loop quantitative real‐time PCR(qPCR)and investigated its functions with qPCR and lentiviral expression systems.Bombyx mori inhibitor of apoptosis protein(BmIAP)gene was predicted by both target prediction software miRanda and Targetscan to be one of its target genes with a binding site for BmCPV‐miR‐1 at the 5′untranslated region.It was found that the expression of BmCPV‐miR‐1 and its target gene BmIAP were both up‐regulated in BmCPV‐infected larvae.At the same time,it was confirmed that BmCPV‐miR‐1 could up‐regulate the expression of BmIAP gene in HEK293T cells with lentiviral expression systems and in BmN cells by transfecting mimics.Furthermore,BmCPV‐miR‐1 mimics could up‐regulate the expression level of BmIAP gene in midgut and fat body in the silkworm.In the midgut of BmCPV‐infected larvae,BmCPV‐miR‐1 mimics could be further up‐regulated and inhibitors could lower the virus‐mediated expression of BmIAP gene.With the viral genomic RNA segments S1 and S10 as indicators,BmCPV‐miR‐1 mimics could up‐regulate and inhibitors down‐regulate their replication in the infected silkworm.These results implied that BmCPV‐miR‐1 could inhibit cell apoptosis in the infected silkworm through up‐regulating BmIAP expression,providing the virus with a better cell circumstance for its replication.

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.