A Research On Screening Marek's Disease Resistance-Related Molecular Genetic Markers Of Three-Yellow Chicken

Marek's disease (MD) is a malignant T lymphoproliferative disease of chicken and has been causing big losses in the poultry industry. Research on the screening of MD resistance-related molecular genetic markers, with the purpose of establishment some corresponding molecular genetic markers, will enhance the resistance breeding of Three-Yellow chicken, and provide a complementary measure to the vaccination and biosecurity strategies for the effective control of MD.A PCR procedure was developed to amplify exon 2 of major histocompatibility complex B-L Bâ…¡gene (B-L Bâ…¡) from the PBL samples of 120 challenged birds, and the PCR products were digested with restriction endonucleidases Aluâ… , Caiâ… , Cfrâ… , Hinlâ… , Hinfâ… and Rsaâ… respectively. The observed homozygous genotypes at the six loci were utilized to tentatively identify the homozygous birds of B-L Bâ…¡allele genes, and seven homozygous genotypes birds were found by cloning and sequencing (including 5 resistant individuals: A₁₁, C₂₃, D₈, D₁₂, K₈ and 2 susceptive individuals :A₅, B₂₁).And then the B-L Bâ…¡gene sequences of the seven homozygous genotypes birds were compared with the published sequences of haplotypes for B²,B⁶,B¹²,B¹⁹and B²¹. The nucleotide substitutions with high variability were observed in the sequences. But the sequences of resistant individuals A₁₁, C₂₃, D₁₂ and K₈, which showed the high resistance to MD in the challenge, were identical, and have the high homologies of nucleotide and amino acids (98.9% and 97.8%, respectively) with the MD-resistant haplotype B⁶. The nucleotide homology between susceptible individuals A₅ and B₂₁, both showed the high susceptibility to MD in the challenge, is up to 95.5%, and have the high nucleotide homology (96.3% and 94.8%, respectively) and high amino acids homology (91.0% and 89.9%, respectively) with the MD-susceptible haplotype B¹⁹.The nucleotide and amino acids homologies between resistant individuals A₁₁, C₂₃, D₁₂, K₈ and susceptible individuals A₅ is low (91.4% and 83.1%, respectively), and susceptible individuals B₂₁ is low too(93.3% and 85.4 %, respectively), existing significant difference. Therefore, we presume that the B-L Bâ…¡gene sequences of resistant individuals A₁₁, C₂₃, D₁₂ and K₈ is one characteristic sequence for MD resistance; and the B-L Bâ…¡gene sequence of resistant individual D₈ which showed the high resistance to MD is another characteristic sequence for MD resistance. Nevertheless, the B-L Bâ…¡gene sequences of susceptible individuals A₅ and B₂₁ were believed to belong two characteristic sequences respectively for MD susceptibility.A reverse-transcriptase polymerase chain reaction (RT-PCR) procedure was developed to amplify the transcripton of growth hormone gene (GH) from Xiayan chicken, and the purified GH gene product was cloned into pMD-18T vector. The recombinant pMD-18T-GH was digested with restriction endonucleidases EcoRâ… and Hindâ…¢and the digested segment was directionally cloned into EcoRâ… and Hindâ…¢sites of the plasmid pSP72 following the T7 promoter. The linearized plasmid was used as template to synthesize an anti-sense RNA probe by the T7 RNA polymerase. The extracted total RNA samples from PBLs were used to analyze the dynamic expression pattems of the GH gene in the birds of different resistances against MD by rabionuclease protection assay (RPA). The results showed that the relative amount of GH gene transcript in the resistant birds was lower than that of the susceptible birds, and existing significant difference. Therefore, GH gene can be primarily selected as one of the molecular genetic markers in the MD resistance breeding of Three-Yellow chicken.A reverse-transcriptase polymerase chain reaction (RT-PCR) procedure was developed to amplify the cDNA of growth hormone gene (GH), B-F locus of major histocompatibility complex (MHC) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene from Xiayan chicken, and the purified products were cloned into pGM-T vector. That cDNA of GH, B-F and GAPDH was recombined with pGM-T vector was proved by digestion with restriction endonucleidases EcoRâ… and Hindâ…¢and PCR amplification and sequences analysis. GH, B-F and GAPDH was amplified by real-time fluorescence quantitative PCR from the plasmid DNA which was diluted to series standard concentrations, and the standard curves were established which indicate that there is a good linear function in statistics between the Ct value and the concentration gradient of standard plasmid DNA specimen. Analysis of the dynamic curve, under this condition of the reaction, the sensitive degree is 10 copies/uL. It is obvious that the recombinant plasmid and standard curve for GH, B-F and GAPDH real-time quantitative PCR were constructed successfully.The results of the study demonstrated that both the sequences of B-L Bâ…¡gene and the expression of GH gene existed significant difference in the MD-resistant line and MD-susceptible line. Therefore, B-L Bâ…¡gene and GH gene can be primarily selected as the molecular genetic markers in the MD resistance breeding of Three-Yellow chicken. In addition, the constructions of recombinant plasmid and standard curve for real-time quantitative PCR provide the basis for detection of peripheral blood lymphocytes (PBLs) GH,B-F and GAPDH gene expression between the MD-resistant line and MD-susceptible line.