Discovery and functional analysis of a new gene (Bm123) in silkworm (Bombyx mori)

Abstract

Previously our research group used the microarray analysis and suppression subtractive hybridization technologies to find a Bombyx mori resistance related gene (NCBI ID: NP_001153678.1) to B. mori nucleopolyhedrovirus (BmNPV) and the gene was named Bm123. But there are no more confirmatory studies about Bm123. In this study, BmNPV resistant strain NB, susceptible strain 306, hybrid group 306♀×NB♂ (resistant strain) and NB♀×306♂ (resistant strain) were analyzed by transcriptomic sequencing and Weighted Gene Co-expression Network Work Analysis (WGCNA) to verify the new gene Bm123 function. Correlation analysis between the WGCNA data and phenotype showed that Bm123 is a gene in ME Turquoise module. This module has a strong correlation with disease resistance phenotype (correlation coefficient is 0.753, P value is 0.0047), indicating that Bm123 is a correlated gene with anti-BmNPV. The full length of Bm123 gene was 691 bp, which is not similar with any sequences of other species in NCBI database. But the Bm123 protein contained the transcriptional activator (multiprotein bridge factor 2, MBF2) domain in the 34 to 122 amino acid sequence, closely to Tribolium castaneum by the evolutionary relationship analysis. The BmNPV resistance function, developmental expression pattern and tissue expression pattern of Bm123 were analyzed by using silkworm resistant strain BC₁₀ (screened by eight backcross and two generation of NB and 306 through hybridization and selfing method, each generation is constructed from the feed by adding BmNPV), NB and sensitive strain 306. It was found that after infection with orally BmNPV, the mRNA and protein levels of Bm123 were up-regulated in the midgut of BC₁₀ and NB, and almost not expressed in 306, indicating that Bm123 was a gene associated with resistance to BmNPV. Bm123 protein expression in various tissues of silkworm (fat body, hemolymph, midgut, epidermis, testis, ovary, malpighian tubule and silk gland) was analyzed. It was found that Bm123 was highly expressed in the midgut and malpighian tubule, while the expression in other tissues was lower. Analysis of Bm123 expression in different development stages of silkworm (eggs, 1^st to 5^th instar larvae, pupae and moth) found that the expression level of Bm123 increased in the 3^rd, 4^th and 5^th instar. The expression level of Bm123 decreased during the pupae and moth stages. It was speculated that the expression of Bm123 was related to the evolution of resistance genes in silkworm. In situ hybridization showed that the Bm123 gene of BC₁₀ was localized in the nucleus of columnar epithelial cells of the midgut, suggesting that Bm123 protein interacts with BmNPV in the silkworm cell nucleus.