Construction Of Wuzhishan Pig BAC Library And The Study Of Flavor Characteristic Genes Of Beijing Fatty Chicken

High-molecular-weight(HMW)DNA was prepared form blood of a male Wuzhishan pig ,with a concentration of 3.0×108 cells/ml cell suspension mixed with an equal volume of liquefied 1% low-melting-point agarose, partially digested with HindⅢand fractionated using double size selection. Digestion DNA fragments in the range of 100～400kb was recovered by elctro-elution and ligated into pBeoBAC11 vector, and then was used to transform DH10B competent cells.After incubated individual white colonies were picked and stored in -70℃.We constructed a high-redundancy bacterial artificial chromosome(BAC) library of an important chinese pig species, the Wuzhishan pig. A total153,600 clones were generated in this library and constructed in vector pBeloBAC11(ordered in 40superpools of 400×384 well plates).The average insert size of the BAC clones was estimated to be 152.3kb from 370 randomly isolated clones, and the ratio of no insert was 1.3%,indicating that the library to be approximately 7.4-fold genome coverage. 78% colonies were more than 100kb, representing a 99.99% statistical probability of obtaining at least one clone containing a unique DNA sequence in the library. We constructed the Wuzhishan pig BAC library, which will contribute to a high-resolution physical map for this species and will assist in comparative genomics studies.A fibroblast line from Chicken embryo of the 8-day-age was successfully established by direct culturing of explants. Biological analysis showed that the population doubling time (PDT) for reviving cells was approximately 48 h. Measurement of lactic dehydrogenase (LDH) and malic dehydrogenase (MDH) isoenzymes showed no cross-contamination among the cells. Tests for bacteria, fungi, viruses and mycoplasma were negative. Every index of the Luxi cattle cell line meets the quality control standards of the ATCC (American Type Culture Collection). Not only has the germline of this important chicken breed been preserved at the cell level, but also valuable material had been provided for genome, postgenome and somacloning research. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level.This research use the RT-PCR method to amplify the Adenylosuccinate lyase (ADSL)gene from Beijing fatty chicken cDNA PCR library. The results of subclone and sequences analysis shows: the open reading frame of 1455 nucleotides that encode a protein of 485 amino acid residues. The complete open reading frame of ADSL gene was inserted into expression vector pGEX-4T-1 to construct Beijing fatty chicken ADSL gene fusion expression vector pGEX-ADSL, and transformed into E. coli BL21 (DE3), screening positive cloning, expression induced by IPTG. By SDS-PAGE electrophoresis showed that there is a specific bands in the molecular weight of about 83.5kD which have to do with the expected molecular weight of the same size and isoelectric point of 6.79.The expression of the protein was increased as the increase of induced time, the maximum value of 5 h, reached 26.9% of total cells protein, and mainly in the form of insoluble inclusion bodies of existence. Through the optimization of conditions, succeeded in obtaining a soluble fusion protein, ADSL were purified by Glutathione Sepharase 4B gel-purified, and by Western blotting analysis showed that it is ADSL for Beijing chicken protein. The research is the basis for its further research with biological function of the foundation and its application identification.The specific expression of purH gene in 8 different tissues of Beijing fatty chicken was investigated by RT-PCR in this study. The full length of purH cDNA was inserted into fusion expression vector pEGFP-N3,pEYFP-N1 and pDsRed1-N1 multiple cloning sites between EcoR I and BamH I, and construct recombinant eukaryotic expression vector pEGFP-N3- purH,pEYFP-N1- purH and pDsRed1-N1- purH with GFP as reporter gene. We used lipofectin method to transfect the recombinant vectors into Beijing fatty chicken fibroblast cells. After the G418 screening and resistant colonies was picked and subcultured until use. The results showed: 24,48 and 72h after transferring, the expression efficiency of 3 kind of recombinant fusion protein genes were between10.3%～53.2%, and the fluorescence could be observed in cytoplasm and nucleus well-distributed except cryptomere vesicle; Through the G418 drug screening and monoclonal training, three cell lines of stable expression of pEGFP-N3-purH , pEYFP-N1-purH and pDsRed1-N1-purH fusion protein was cloned. RT-PCR and western blot both confirmed the pEGFP-N3-purH,pEYFP-N1-purH and pDsRed1-N1-purH have been integrated into the Beijing chicken fibroblast cell genome, and access to the normal fusion protein expression. The research is important to genetic mark, nuclear transplantation and transgenic animal clone etc.We constructed a high-redundancy bacterial artificial chromosome(BAC) library of an important chinese pig species, conserved the its whole genome resources, and supplied the research material for genome and post-genome research. Not only has the germline of this important chicken breed been preserved at the cell level, but also valuable material had been provided for gene expression. Moreover, the establishment of this technical platform may provide both technical and theoretical support for storing the genetic resources of other animals and poultry at the cell level. These cloning cell lines of 1 ADSL and 3 purH gene would provide important material for the transgene animal cloning in order to cultivate transgene chicken and divided whether ADSL and purH are the main genes controlling the flavor characteristic or not.