Study On Purification Of Mutant Black-feather Quails And The Relationships Between Genetic Polymorphism Of MHC Class Ⅰ Gene And Immunologic Function

As one kind of extremely complex cell membrane protein with high polymorphism in coded immune system, MHC (major histocompatibolity complex) plays a very important role to pass antigen in the body's immune system. The research of MHC, from which some major issues in immunology are desired to be solved, creates new research fields in immunogenetics. Nowadays, measures such as improvement of raise management, immunifaction, quarrantine of sick birds are used to prevent and cure poultry diseases, but sometimes the effect is not desirable. Numerous researches showed that MHC is closely related to different disease resistance, susceptibility, and production traits, which affects specific and nonspecific immunity of diseases. MHC is important genetic markers of poultry breeding because of its MHC to pass on to offspring stably. And consequently, it becomes a research focus in the genetic field in terms of disease control and prevention. Most of research works aim to improving economic benefit by chioce to strengthen production performance, which results in the side effect decrease of disease resistance. Subsequently, one important purpose of breeding work is to improve the disease resistance and to produce breeds of high resistance immunity. At present, wide and numerous study of MHC in mammalian has been done, which mainly takes chicken as the research object. The quail is an important productive and experimental animal, and quail breeding is known as the future of poultry raising in the 21^th century, but there is no relevant research reports in China and relevant research abroad just focuses on Japanese quail. Based on above reviews, it's significant to study the polymorphism of MHC genes and disease resistances of the quail. The main results of this research are shown as follow:1. With the mutant black-feather quails discovered from hybrizing matching between maroon-feather, white-feather and yellow-feather quails as the experimental material, the research shows that, by hybridization and backcross experiments, the newly-discovered black-feather gene h is located in euchromosome. There is a cross-talk between black-feather gene h and gene locus of B/b and Y/y in sex chromosomes Z. The genotype of male and female of black-feather quails are identified as hhZ^YBZ^YB and hhZ^YBW respectively. The black-feather gene h is different from the Genetic mechanisms of the mutant Japanese black-feather quails, which is the new progress in the study on genetic development of quail's feather. Black-feather quails are purified by three kinds of hybridization and self-bred line experiments, and genealogy of black-feather quails is constructed, which provides an important theoretical basis for cultivating new quail strains for eggs and commercial strains of autosexing breed.2. Twenty-six quails were tested to get blood physiological indexes, and the results can be summarized as below: hemoglobin (HGB) and mean corpuscular hemoglobin concentration (MCHC) are very significant different between male and female quails; packed-cell volume (PCV), total white blood cells (TWBC), and mean corpuscular hemoglobin (MCH) are significant different between male and female quails; biochemical indexes, including total protein (TP), albumin (ALB), globulin (GLO), and triglycerides (TG) are very significant different between male and female quails; sodium (Na)and chloride (Cl) are also significant different between male and female quails; but other indexes are not significant different. Compared with other poultry, there are limited changes in physiological and biochemical characteristics for the mutant black-feather quails.3. The study aimed to research the hatching performance,growth during 0-10 weeks and feed utilization of mutant black-feather quails. The results show that better hatching effect are distributed at the egg weight between 10.4 and 11.3 gram, egg shape index between 0.77 and 0.81;There are not the siginificant difference btween male and female in early ages,they exit significant difference in the 4th week,and extremely significant difference from the 5th to 10th weeks(P<0.01). Female quails reach sexual maturity at about 7-8th weeks, and male quails delay one week. The weekly weight during former seven weeks is extremely significant correlation with cumulative feed intake.4. The five models Logistic, Gompertz, Von Bertalanffy, Brody, and Richards were applied to analyze the growth rule of mutant black-feather quails. The growth curve fitting and analysis were completed by using the body weight data of black-feather quails from 0 to 10 weeks, which included 100 single-feed male and female quails as well as 280 group-feed quails. The results show that there are preferable fitting degrees (R²) for all of the five models. However, Gompertz indicates the highest fitting degree (0.999) and smaller residual sum of square (E). So Gompertz should be the best option to analyze the growth curve of black-feather quails. The turning-point week age are 2.255 weeks, 2.809 weeks, and 2.731 weeks, and turning-point body weight are 45.041 gram, 58.016 gram , and 53.851 gram for single-feed male, female black-feather quails and group-feed black-feather quails respectively.5. Forty-eight mutant black-feather quails were raised at a single cage for each one to investigate the production parameter including age at first egg, primary egg weight, the laying rate, average egg weight, and feed conversion. The results are shown as below: the age at first egg of mutant black-feather quails is 53 days, which is later than other strains; primary egg weight is 8.9 gram;the average laying rate from the age at the first egg to 15^th week is 81%;the average egg weight is 10.9 gram, which is suited for the machining demands;the average feed-gain ratio is 3.01±0.31 from the age at the first egg to 15^th weeks.6. According to GenBank MHC class I mRNA gene sequences of the Japanese quails, the genomic DNA of mutant black-feather quails was refined, from which the PCR was used to clone 1367 base pair fragment of MHC classâ… gene. The results of the gene structure analysis are shown as below: the gene fragments located in 1-241 bp, 391-658bp, 732-873 bp, 954-983 bp, 1143-1175 bp, and 1318-1359 bp are 3^rd, 4^th, 5^th, 6^th, 7^th, and 8^th exons, whose sizes are 241 bp, 268 bp, 106 bp, 30 bp, 33 bp, and 42 bp respectively; the fragments located in other areas are 3^rd, 4^th, 5^th, 6^th, and 7^th introns, whose sizes are 149 bp,73 bp, 116 bp, 159 bp, and 143 bp respectively; there are many mutations in 4^th exon. This experiment also shows that the polymorphism of the gene exon 4 is higher than exon 5, 6, 7, and 8. These results would be helpful to strengthen the molecular biological basis for disease resistance in quail breeding.7. Eighty healthy quails of 40 days were injected with Newcastle Disease Virus (F48E9) for three times, and significant different immunity was shown for this virus among all quails. Subsequently, F48E9 Virus was isolated from sick quails. Genomic DNA was extracted from blood of sick quails. The DNA sequences of 64 quails were detected from exon 4 by PCR products with direct sequenced method. Polymorphism analysis indicated that there were 18 mutational sites in this fragment, 8 A/G mutant sites, 5 T/C mutant sites, 1 G/C mutant site, 1 A/G/C mutant site, 2 G/C/T mutant sites, and 1 A/G/T mutant site. The results show that all mutant sites are the bases alternative sites, and all these sites contain heterozygous sites; all of 18 mutant sites are polymorphic loci by calculating its heterozygosity, polymorphism information content. Corelative analysis between the antibody level in sick quails and the polymorphism in exon 4 shows that there are significant or very significant different in antibody level within genotypes of 2,4,6,13,14,15 mutant sites, so these genes can be used as main candidate genes to improve disease resistance of quails.