Studies On Cloning Of Chicken Fatty Acid Binding Protein Genes And Their Relationships With Intramuscular Fat Content

In order to screen chicken heart type fatty acid-binding protein (H-FABP) gene genomic DNA library was constructed in A phage. Four positive clones containing chicken H-FABP gene were obtained. Six Chinese local chicken breeds, including Beijing Oil chicken, Dwarf chicken, Taihe silky fowl, Chongren Ma chicken, Luyuan chicken, Xiayan chicken, and a Arbor Acre broil were used as experimental populations. Chicken H-FABP gene and adipocyte fatty acid-binding protein (A-FABP) gene were studied as two candidate genes which having effects on intramuscular fat (IMF%) content. PCR-RFLP and PCR-SSCP analysis were used to study genetic variations in candidate genes. The associations between genetic markers and performance traits were analyzed by using general linear model (GLM). The results were as follows:1. Genomic DNA library from Beijing Oil chicken was constructed in EMBL3 phage with a titer of 3.84+105, which could secure the representation and integrality of the library.2. P1 was designed to amplify the 2kb fragment of the second intron of chicken H-FABP gene after alignment of conservative sequences in H-FABP genes from different species. P2 was designed to amplify a 221bp fragment in chicken H-FABP gene intron 2 based on the result of sequence analysis. It was used to screen the genomic DNA library by PCR to determinate the location of sub-library where positive recombinants containing chicken H-FABP gene located. Random primers were used to obtain the probe using the second intron of chicken H-FABP gene as template. DIG-labeled probe was obtained after the labeling reaction. Four positive reconstructed phages were selected after Southern hybridization.3. Two polymorphic sites, Hha I and Msp I -RFLPs, were detected in the second intron of chicken H-FABP gene using PCR-RFLP method. The Hha I site had three alleles, designated as A,B and C. Two T-C transitions were revealed at 386 and 723 position respectively by sequencing analysis. The Msp I site had two alleles, designated as M and N. A T-C transition was revealed at 246 position. Pa was designed to amplify a fragment containing the third intron of chicken A-FABP gene according to the sequence of chicken A-FABP gene exon4 and intron2. A Hinf I -RFLP was discovered in chicken intron3 with two alleles designated A and B. A G-alleles were analyzed in a total of 505 samples from Beijing Oil chicken and Dwarf chicken. In the two populations Hinf I -RFLP site was in Hardy-Weinberg equilibrium, while Msp I site was in disequilibrium. In Beijing Oil chicken population Hha I -RFLP site was in equilibrium contrary to that of the Dwarf chicken population. A chi-square analysis suggested allele frequencies and genotype frequencies differed significantly at these loci in Beijing Oil chicken and Dwarf chicken populations (P< 0.01) .4. Specific primers P4, P5 and P6 were designed to amplify fragments from chicken A-FABP gene. Three PCR-SSCP loci, designated as P4, P5 and P6-SSCP, were discovered, each had two alleles, designated as A and B. Sequence analysis revealed p4 and P6-SSCP loci were generated because of C-T transition, while P5-SSCP was caused by a G-A transition. The distribution of corresponding genotypes and alleles at three PCR-SSCP loci was analyzed in a total of 728 samples in seven chicken populations tested. A chi-square analysis suggested allele frequencies and genotype frequencies differed significantly at these loci in the seven chicken populations tested(P<0. 01) . Except the Pe-SSCP locus in Xiayan chicken, all the three loci reached Hardy-Weinberg equilibrium in the populations tested.5. The relationships between three PCR-RFLPs loci, three PCR-SSCP loci and performance traits (intrmuscular fat%, IMF% esp.) were analyzed in a total of 505 samples from Beijing Oil chicken and Dwarf chicken by GLM. Results showed that different locus differed significantly in performance. We suggest that chicken H-FABP gene and A-FABP gene are worthy of being further studied as candidate genes having effects on IMF%.