Screening And Primarily Expression Characterizing Of ESTs Differential Displayed In New-born Four Kinds Of Chicken's Leg Muscle

Silkie is a unique breed in China, it is emphasized by scientists owing to its unique traits, precious officinal and nutritious values. In appearance, Silkie takes on "black" characters in skin, muscle and bone. Furthermore, the meat quality of Silkie is superior to other chickens, yet its growth is slower than others. It is the different kinds of expressing genes or the same gene but in different expressing amount that exhibits diversity in organism' traits. By differential display reverse transcription PCR (DDRT-PCR), we compared the difference of the expressing genes among New-born Silkie (SK), Normal Plume Black-bone(NP) , Nike layer(NK) and Arbor Acres broiler' leg muscle, which will help us to understand the genetic basis of unique character in new-born Silkie's leg muscle.In this experiment, differential expressing genes in leg muscle of chicken mentioned above were studied via DDRT-PCR technique, which firstly adopted 72 primer pairs that piloted by 3 T7 (dT11) N as anchor primers and 24 M13-ARPs as random primers. We obtained 214 differential displayed cDNA bands that were cut from the silver stained non-denatured polyacrylamide gels, 101 cDNA bands were gained in succedent re-amplified PCR and purification. We confirmed the unitarity of 101 cDNA bands by SSCP technique, 87 unitary cDNA bands were obtained. Finally, the authenticity of differential expressing cDNA bands was confirmed via gene chip scanning technique and 6 true differential cDNA fragments were obtained, but only 5 true ESTs were sequenced successfully.As for the 5 ESTs, We gained the following information about D1 and D2 by comparing with nucleic acid sequences which obtained from the experiment and databases derived from Genbank. D1 is homologous with Gallus gallus' COXⅢ gene (cytochrome C oxidase subunit Ⅲ) in mitochondrion genome. D5 is homologous with M-CK gene (muscle creatine kinase) and located at Gallus gallus chromos 32 by e-PCR on NCBI. Using Blastn search, e-PCR, and electronic extension by finding UniGene on NCBI, we gained the following information about unknown functional ESTs about D2, D3 and D4. D2 is similar to hypothetical 18K protein-goldfish gene. D3 have high similarity rate with congtig2 which assembled by Sequence Assembly Program, and the protein encoded by frame=+1 of contig2 is highly similarity rate with predicted HIPK protein in chicken. D4 is highly similar to contig2937 derived from nr database on NCBI using Blastn research, and the protein encode by the frame=+3 of contig2937 is identical to epsilon isoform of 14-3-3 protein. Major fragments of 14-3-3ε gene are located at chromos 19, the other minor are located at chromos 9 by comparison with Chicken Genomic Database on NCBI.These differential expressing genes which having known its function in our experiment are probably related to development of leg muscle and the synthesis and transportation of melanin in chicken. Firstly, COXIII gene is differentially expressed in SK and NK layer, but not in AA and NP, which suggests the differential expression of COXIII gene is probably related to development of leg muscle in new-born chicken breeds. Secondly, M-CK is differentially expressed in AA chicken but not in the other three breeds, which suggests M-CK plays a pivotal role in leg muscle' development and differentiation in new-born chicken. Finally, 14-3-3e gene is differentially expressed in AA, NK and NP but not SK, so we confer that the expression of 14-3-3e gene is related to synthesis and transportation of melanin in new-born chicken' leg muscle.As COXIII and M-CK gene play a concernful role in energy of metabolization and development of muscle, the two differential expressing genes probably are responsible to the difference of development in the four chicken breeds' leg muscle. The synthesis and transportation of melanin in Silkie is a complex process needs multitudinous genes to participate in, so we have many difficulties discovering the "pivotal gene" influencing the reproduction of melanin and comprehend the mechanism of regulation of melanin. The role of 14-3-3￡ gene in melanin may give us a new insight to understand how the melanin is functioned. The relationship between the three differently expressing genes about COXIIL M-CKs 14-3-3e gene and its own unique trait in different chicken breeds is predicted by combining our experiment, bioinformatic analysis and existing research, therefore further study about them is needed. However, going-deep-into validation and investigation toward unknown functional gene is still needed by combining bioinformatic analysis and further experiment.