| Investigation of mutations in the silkworm has been one of the most important parts in silkworm genetics researches in the last 100 years. From analyzing the mutants heredity comes the laws of genetics, and from linkage analysis comes the linkage map, both of these laid the foundation for silkworm genetics research and breeding. They are also the cornerstones of the molecular linkage map construction, and play an important role in cloning the silkworm mutant gene.Translucent silkworm is a peculiar mutant. Relatively low level of uric acid is accumulated in the larval epidermis, which makes its skin translucent like a piece of oilpaper. So far,40 translucent silkworm mutations have been found, the genes responsible are distributed on 15 different chromosomes. Sex linked translucent silkworm(os) is the first translucent silkworm reported (Tanaka, 1917), which has low level of translucent epidermis, and is sex linked. The gene responsible for this mutation is located on 1-0.0 of the silkworm linkage map. The silkworm strains used in this study-the mutant strain Dazao-os (Near-isogenic line of os) and wild-type(WT) strain C108 were obtained from the silkworm gene bank in Southwest University. By positional cloning, we tried to identify the candidate gene and make a contribution for elucidating the molecular basis of the os mutant. The following are what we have achieved:(1) Selecting silkworm strains:Two kinds of silkworm strain selection were practiced in this research. Firstly, the linkage analysis strain:female "Dazao-os" silkworms(ZosW) were crossed with C108 males(Z+Z+), F1(Z+W, Z+Zos) were obtained; female F1(Z+W) were backcrossed with male "Dazao-os" silkworms(ZosZos), Backcrossl(BC1F) were obtained. Secondly, recombination analysis strain:male "Dazao-os" silkworms(ZosZos) were crossed with C108 females(Z+W), F1(ZosW,Z+Zos) were obtained; male F1(Z+Zos) were backcrossed with female "Dazao-os" silkworms (ZosW), Backcross1(BC1M) were obtained.(2)Mapping of the os locus6 SSR markers on the 1st linkage in the silkworm SSR molecular linkage map were used in this study. Using 20 BC1F individuals and 156 BC1M individuals, SSR-PCR was performed. By using mapping software(Joinmap 4.0, LOD=5.0), we constructed the linkage map of the os locus and SSR markers. The map we constructed is 55.0cM in length, the nearest SSR marker S0105 is 38.1cM from the os locus.(3)Use of new marker and further mapping of the os locusBased on the result of the previous mapping, we found the os locus is located in nscaf2734 of the fine linkage map of silkworm or the gap zone. By using Go classification and cluster analysis, we singled out the genes that are involved in nitrogen metabolism, GTP synthesize and transportation as the candidate gene for os mutation. Some of these genes were used as new markers for further study. Using SSR-PCR and mapping software (Joinmap 4.0, LOD=5.0),20 BC1F individuals and 156 BC1M individuals were analyzed. We found the os locus is located between SSR marker S0105 and new marker Vp. The nearest marker Vp is 1.6cM from the os locus.(4) Metabolism analysis of the os mutantUsing HPLC, we found 114.92μg uric acid was contained in 0.5g silkworm feces from the WT Dazao, whereas no uric acid was detected in the mutant (os) silkworm feces. Based on this finding, we predict that the os mutation is owing to uric acid synthesis deficiency.(5) Initial screening of candidate genes of Sex linked translucent silkworm(os)By integrating the first molecular marker linkage group map and genome physical map,we initial screening the candidate gene scope of:nscaf3040:3800k-end-nscaf2734:1400k-end. On account of Molecular markers mapping and results of physiological analysis, we screened three candidate genes:BGIBMGA012225, BGIBMGA012226 and BGIBMGA012356 for the initial candidate gene identified genes within the Go classification, molecular and functional analysis of the way and biosynthetic analysis of biological information.
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