Construction Of Bacterial Artificial Chromosome Library And Study Of Flavor Characteristic Genes In Beijing Fatty Chicken

In order to preserve precious genetic material of Beijing fatty chicken, we have constructed a bacterial artificial chromosome (BAC) library of Beijing fatty chicken and studied these two genes of chicken, aminoimidazole carboxamide ribonucleotide transformylase and IMP cyclohydrolase (ATIC) and liver fatty acid-binding protein (L-FABP), which involved in meat quality and flavor. The genomic structure of ATIC and L-FABP were determined, and their expression in fibroblasts and Sub-cellular location were studied. Three cell lines that stably expressing pDsRed1-N1-TAIC, pEYFP-N1-ATIC and pEGFP-N3-ATIC fusion protein were cloned successfully.High-molecular-weight (HMW) DNA was extracted from blood of female Beijing fatty chicken, with concentration of 3.0×108 cells/ml cell suspension, and then mixed with equal volume of 1% low-melting-point liquefied agarose, partially digested with HindШand separated using double size selection system. Digested DNA fragments within the range of 100～400kb was recovered by elctro-elution and ligated into pBeoBAC11 vector, and then was used to transform into DH10B competent cells. Individual white colone was picked and stored in -70℃refrigerator after incubation.The Beijing fatty chicken BAC library consists of 115,200 BAC clones totally (conserved in 30 superpools, each in 10384-well plate). 200 clones were selected randomly and analyzed, and the results indicated that the average insert size of the library was estimated to be 158 kb, and the large inserts (>100kb) accounted for more than 70% and the non-recombinant clones was only 0.9%. It was calculated that the genome coverage of the library is 13.7 and the possibility to find a single-copy gene in the library is 99.99%. This Beijing fatty chicken BAC library would serve as a valuable resource in comparative and functional genomic research.The present study established the target cell lines of Beijing fatty chicken and Aletai sheep successfully by direct adherent culture method. In order to study the exogenous gene expression, pEGFP-N3 fluorescent gene was transfected into the fibroblast cells. The results showed that the quality of the cell line met the quality requirements of the ATCC (American Type Culture Collection), and indicated that the cell line was outstanding target cells for gene expression.L-FABP gene was amplified and the specific expression of ATIC gene in 8 different tissues (heart, liver, spleen, lung, kidney, brain, leg muscle and chest muscle) of Beijing fatty chicken was investigated by RT-PCR method in present study. The full length of ATIC cDNA was inserted into fusion expression vector pDsRed1-N1, pEYFP-N1 and pEGFP-N3 between multiple cloning sites of EcoR I and BamH I, while the full length of L-FABP cDNA was inserted into fusion expression vector pEGFP-N3 between multiple cloning sites of EcoR I and Xho I, and recombinant eukaryotic expression vector pDsRed1-N1-ATIC, pEYFP-N1-ATIC, pEGFP-N3-ATIC and pEGFP-N3-L-FABP were constructed fused with GFP as reporter gene. Lipofectin method was used to transfect these recombinant vectors of pDsRed1-N1-ATIC, pEYFP-N1-ATIC, pEGFP-N3-ATIC into Beijing fatty chicken and pEGFP-N3-L-FABP into Aletai sheep fibroblast cells. The resistant colonies were picked and sub-cultured until use after G418 screening. The results showed: at 24h, 48h and 72h after transfection, the expression efficiency of these 4 kinds of recombinant fusion protein were between 10.3% and 53.2%, and the fluorescence well distributed in cytoplasm and nucleus except cryptomere vesicle; three cell lines that stably expressing pDsRed1-N1-ATIC, pEYFP-N1-ATIC and pEGFP-N3-ATIC fusion protein were obtained by the G418 drug screening and monoclonal training. RT-PCR confirmed the pDsRed1-N1-ATIC, pEYFP-N1-ATIC and pEGFP-N3-ATIC had been integrated into the Beijing fatty chicken, fibroblast cell genome, and pEGFP-N3-L-FABP had been integrated into the Aletai sheep fibroblast cell genome, the western blotting results showed that pDsRed1-N1-ATIC, pEYFP-N1-ATIC and pEGFP-N3-ATIC has normal fusion protein expression. On the other hand, there was no significant effect(P>0.05)on cell shape, cell growth and reduplication state when compared with control groups.Above all, The present work not only preserved the germplasm resources of the important Beijing fatty chicken at the gene level but also provided valuable material for genome, post-genome and somatic cell cloning research.