Mapping And Cloning Of The Egg Colour Gene And Mutation Analysis In Sex-Linked Egg Colour Strain Of Silkworm, Bombyx Mori

Sex-linked egg colour silkworm strain(L1) was bred by Strunnikov, a Russian scientist, with irradiation technology. In the females of this strain, there is a translocation fragment in W chromosome, which was from the Chr:10containing the wild-type egg colour gene(+w2), meanwhile there is a homozygous recessive mutation on the Chr.10, which causes white eggs and eyes. In1996, Zhejiang Academy of Agricultural Sciences introduced this strain from Russia and succeed to improve breeding. However, how did the white mutation cause and where the gene located were still unknown. In present research, we performed positional cloning of the w2locus from w2mutant-Ll. Then, bio-informatics was employed as a tool to analyze the regulation mechanisms about egg colour in the sex-linked egg colour strain. The contents and results are as follows:1.Mapping of the mutational gene(w2) in silkworm, Bombyx moriAccording to the genome sequences of two scaffolds Bm_scaf44and Bm_scaf30on the Chr.10in the data banks, we got19polymorphism markers from167specific SSR markers, which were used to examine the DNA divergence between p50and L1. To detect the genotype of these polymorphic markers, we obtained96male moths development by white eggs from a single-pair backcross (BC1) between an L1(w2/w2) female and an F1male(L1×p50).The fine genetic linkage map of w2was constructed after the genotype of the BC1generation male groups. In combination with the position of these markers of the Chr:10’s physical map, we were able to narrow the responsible region within0.45Mb, that was from15.53Mb to15.98Mb on a scaffold Bm_scaf30(Chr.10). 2. Determination of the full-length gene sequence of the normal egg colour gene(+w2)The full-length cDNA sequence was obtained by3’and5’RACE and bio-informatics methods. The cDNA sequence was2053bp long and had one2013bp length ORF which encoded a proten of670amino acids.By determining the genome structure of+w2in the strain p50, it consists of13introns and14exons, with the start codon in the first exon and the stop codon in the last exon. All of the exon-intron boundaries followed the canonical GT/AG rule.Forecast and analysis showed that the+w2protein had a typical domain structure of the ABC transporter superfamily:an ABC_trans domain and an ABC2_membrane domain. Homologous analysis shows that the+w2in silkworm, Bombyx Mori, is an orthologous gene of Drosophila scarlet, and a defect in its conservative regions can causes a’white’ mutation-white eggs and eyes.3.Characterization of the w2gene in the sex-linked egg colour strainWe isolated w2cDNAs from p50and L1separately by RT-PCR, comparison of the nucleotide sequences of the cDNA between them revealed a difference in the3’region. A136nt length fragment was inserted in the last exon of the w2mutant. Then, to determine the origin of abnormal w2cDNA, we examined the genomic sequences containing the14th exon.we found the same insertion fragment for the area, but another fragment (216nt) in3’-non-coding region was lost. That caused16amino acid (FHLLALLALRYRTRRK) of the C-terminal of normal protein were replaced by the new19amino acid (LGRQRLGSAPGIAEVH GRR), which led to a diffreent secondary structure of the new protein.