| DDK syndrome is a reproduction phenomenon with early embryonic death in mice.Specific performance: When DDK females are mated with males of other strains,they are mostly infertile,the embryo died during the 3-5 days of gestation due to blastocyst formation;while DDK males exhibit normal fertility in the reciprocal crosses.Ovum mutant(Om)is considered to be the cause of the syndrome mutation.However,the gene sequence and mechanism are still unclear.The aim of this study was to observe the effects of different concentrations of arginine on the DDK syndrome and the relationship between arginine metabolism and DDK syndrome;At the same time,we searched the modified gene of DDK syndrome to reveal the cause of embryonic mortality deviated from the semi-lethal rate after heterozygous female mice with Om gene backcross with C57BL/6(B6)male.In present study,we explored the influence of DDK syndrome by both nutrition and gene,which laid the foundation for the final explanation of the mechanism of DDK syndrome.This study was divided into three parts:In the first part,effects of mice dietary supplement different concentrations of arginine on DDK syndrome.This study used single factor completely randomized design.A total of 120 F1(B6 ? × DDK ?)mice were randomly divided into 3 groups.The diets supplemented with arginine were 1.10%,1.34% and 1.59% respectively.The mice were dissected on the 12 day of pregnancy,observed the number of live fetus and implantation,detected the nitric oxide content in the uterus.The results showed that the number of implantation of 1.34% arginine group was significantly higher than the 1.10% and 1.34% group(P <0.05).At the same time,the number of live fetus in the 1.34% group was the highest,but there was no significant difference compared with the other two groups(P>0.05).The results of nitric oxide in the placenta and uterus showed that the addition of 1.34% and 1.59% arginine in the mouse diet was significantly higher than that in the 1.10% group(P <0.05).The experimental results showed that the addition of 1.34% arginine to the diet of mice could improve the survival rate of DDK syndrome,but the difference was not significant;The detection of nitric oxide in the placenta showed that there was no direct relationship between DDK syndrome embryo death and arginine deficiency and nitric oxide metabolism.The second part is to initial locating the modifier gene of DDK syndrome.We used the microsatellite markers D11Mit36?D11Mit66 and D11Mit247,which are closely linked to the Om locus,selected the heterozygosis(Om/+)N2 female mice to construct QTL mapping population.Selected the microsatellite markers,which covering 19 autosomes of mice.Screened the polymorphism molecular markers between DDK and B6 strains by gel electrophoresis,and used these polymorphism molecular markers to whole-gene scan the locating population.The number of live fetuses and the number of implantation at 12 days of gestation of heterozygosis(Om/+)N2 female mice were collected as phenotypic data.Finally,we used the CIM of Win QTLCart2.5 software to find the modifier gene of DDK syndrome.The results showed that,in this study,we totally screened 289 N2 heterozygotes female for QTL mapping,screening ratio was 23.94%.I totally screened 67 polymorphic microsatellite markers through comparing the DNA gel amplification results of F1(B6 ? x DDK ?),DDK and B6 mice,screened ratio was 25.97%.On average,there were 3.53 markers on each chromosome,the average distance between the markers was 18.56 c M.Collected the phenotypic data of reproduction showed that live fetus averaged 2.57 and implantation averaged 3.59,they are normal distribution.Combined with the results of 19363 whole genome scans and phenotypic data to QTL analysis.We found that implantation had a peak on chromosome 9 with an LOD of 2.3,which was less than the 5% significance threshold level of 2.5,by the 1000 permutation test at ?=0.05.Therefore,there was no locus of implantation on chromosome 9.The significance threshold of the whole-genome scan was found to be 2.9 for live fetus.The LOD scores(3.5)indicated that a modifier gene locus of live fetus was located on chromosome 9.We named this site as aggravate modifier gene of Ovum mutant 1(Amom1).The site was located in the 38.9-51.9c M range of chromosome ninth in mice.These results showed that we found a major modifier gene locus in B6 strain background,this loci could aggravate DDK syndrome,caused the survival rate of embryos were further reduced.The results of this study explain the reasons why embryonic mortality deviated from the semi-lethal rate after Om heterozygous females backcross with the B6 male.The third part is to fine mapping the Amom1 locus.Fine mapping the Amom1 by increasing the locating population on the basis of preliminary localization and adding high-density polymorphic molecular markers on the chromosome 9,especially the confidence interval of Amom1 locus.The candidate genes in the confidence interval of Amom1 locus were identified by reference to the mouse database,and the candidate genes were further screened by bioinformatics methods.The search for candidate genes layed the foundation for the final cloning of DDK syndrome modifier gene.The results showed that 28 polymorphic microsatellite markers were screened by the comparison of the gel electrophoresis.The screening rate was 21.88% and the average distance between markers in Amom1 locus was 0.93 c M.Added 56 effective samples on the preliminary mapping population,which made the fine mapping population reach 345.Fine mapping of Amom1 locus was performed by Win QTLCart2.5 software.The mapping results showed that the maximum LOD value of the Amom1 gene locus was 4.4,which was located at the position of 47.9c M on chromosome 9.Based on the significant confidence interval of ?=0.05,we used the 1.5 LOD-drop method to estimate confidence interval of Amom1 gene locus,which made the confidence level up to 98.3%.The results showed that the interval of Amom1 gene locus was located from 47.7 to 49.1 c M on chromosome 9,about 1.4 c M.The contribution rate of Amom1 locus was 10.56%,the additive effect of Amom1 locus was-1.46,and the dominant effect of Amom1 locus was 12.72.Analysis of Amom1 interval corresponding 90700-94500 Mb by NCBI and MGI database,A total of 19 genes were found in these two databases,12 of them were unknown genes,4 were the Plscr family genes,2 were the Zic family genes and the 1 was the Plod family gene.The next steps need to be further screening the 19 genes,and to be verifying the candidate genes.Based on the above results the addition of 1.34% arginine in the diet of mice slightly alleviated DDK syndrome,fetal death of DDK syndrome has no direct relation with dietary arginine metabolism,but it has a direct relationship with the modified genes Amom1,which in the genetic background of B6 strains.This modifier gene through regulated the express of Om to further increase the embryonic mortality,in other words it aggravated the DDK syndrome.The study revealed a relationship between dietary arginine and the embryonic mortality of the DDK syndrome,and located the modifier gene for the DDK syndrome laid the foundation for a comprehensive explanation the mechanism of DDK syndrome. |
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