Mapping The Mutant Genes Of Ectopia Pupil And White Spot Mutation Mice And Analyzing Candidate Gene Sequence Of The White Spot Mouse

In previously research, a kind of ectopia pupil mouse (named B6-24^#ectopia pupil mouse) was obtained by ENU-induced mutagenesis. Interestingly a white spot mouse (named B6-24" white spot mouse) was found among the offspring of B6-24^# ectopia pupil. Our research focused on the two kinds of mice. At last the two mutation genes were mapped on the chromosome and the mutation site of B6-24^# white spot mouse was successfully identified. The work was divided into two parts.1. Chromosome location for mutation genes of two kind of miceTwo kinds of mutation mice,B6-24^# ectopia pupil and B6-24^# white spot , obtained by ENU-induced mutagenesis from C57BL/6J(B6) mice were of the material in this study . Heredity test indicated that the phenotypes of the two kinds of mutation mice were controlled by different single dominant gene. To map the genes of the two kinds of mice on the chromosome, the mutation F₂[ (B6×D2) F1×B6] ectopia pupil mice and mutation F₂ [ ( B6×D2 ) F₁× D2] white spot mice were produced respectively and microsatellite markers were used to scan genome of mice. Linkage analysis showed that the mutation gene of ectopia pupil mouse linked with D2Mitl7, as 3 cases in 30 F2 mice undergone recombination and the LODS was of 3.27 based on actual recombination ratio. And then, D2Mit398 and. D2Mit259 were selected to test the mutation gene. As the recombination ratio between the mutation gene and these two microsatellites were of (5.45±2.16) % and (0.90±0.90) % respectively, the mutation gene was mapped on chromosome 2, 64.5cM from the centromere. No obvious candidate gene was found in this region. The mutation gene of white spot mouse was linked with D10Mit73, as 4 cases in 34 F2 mice undergone recombination and the LODS was of 4.39 based on actual recombinationratio. And then, D10Mi70 and D10Mit68 were selected to test the mutation gene. As the recombination ratio between the mutation gene and these two microsatellites were of (0.01 + 0.01)% and (0.05 + 0.02)% respectively, the mutation gene was mapped on chromosome 10, 56.8cM from the centromere.2.Sequence analysis of the kitl gene of B6-24*white spot mouseAfter searching for the mouse genome database (MGD) and performing a one-by-one study of all genes located on chromosome subregion, it was believed that kitl gene was an excellent candidate for the B6-24* white spot mouse. We sequenced the two kind of kitl gene (S-kitl and M-kitD of B6-24* white spot homozygous and wild type mice from brain and testicle respectively. To validate the mutation of mRNA, the corresponding genome sequence were amplified and sequenced. At last amino acid changes was predicted by DNAstar. Our results were that : ?the wild type mice had no mutation and the homozygous mice lost 68bp which was the exon 8 of kitl and this also caused a frameshift.? the conserved GT-AG sequence was translate into GC-AG by a mutation in the second base of intron 8 from T to C and this was the reason that caused the skipping of exon 8.(3) the encoded sequence of exon 8 comprised the cytoplasmic domain of kitl and the mutation resulted in the substitution of 28 novel aa which altered the function of kitl. Our work supplied a valuable genetic resource for the function study of kitl. In the research we also found normal kitl sequence in the brain of B6-24# white spot homozygous but not in testicle and so our research also supplied a material for the study of mRNA splicing mechanism.