| Black-bone chicken is a unique domestic chicken breed in China, and the Silkie has been admitted as an International Standard Breeds because of its unique appearance. Chinese regard Black-bone chicken as an officinal chicken breed for long term. As for the root of difference, it is well known that differential expression of genes leads to various traits of organism. Out of question, the difference between black-bone chicken and other chicken breeds derives from the differential expression of genes. In recent times, DDRT-PCR (differential display reverse transcription polymerase chain reaction) is one of the most widely employed techniques to identify differential expressed genes. And its application is becoming wider and wider in biological area. Through this experiment we evaluated the availability about DDRT-PCR in screening differential expressed genes or expressed sequence tags (ESTs) between black-bone chicken breeds' and general layer and meat breeds' liver, and has obtained genes or ESTs related to unique traits of black-bone chicken through bioinformatic analysis to compare nucleic acid sequences derived from the experiment with Genbank database.High quality total RNA were isolated from adult chicken's liver organ of four chicken breeds: silk fowl (SK), normal plumed black-bone chicken (NP), SuQin 96 layer chicken (SQ) and Abar acer meat chicken (AA). The differential expressed genes in liver organ of above chicken breeds were studied via DDRT-PCR technique for the first time. We analyzed the unitarity of each re-amplified differential displayed cDNA bands cut from the silver stained non-denatured polyacrylamide gels with SSCP. The authenticity of differential expression was identified via reverse Northern dot blotting and Northern dot blotting jointly. Thirteen true differential cDNA fragments of black-bone chicken were obtained from the experiment. All of them were cloned to pBluescrit T vector and converted into TOP10 Ecoli bacteria. Finally, ten differential expressed cDNA fragments of black-bone chickens had obtained positive clone and been sequenced. Ten black-bone chickens differential expressed ESTs were submitted to dbEST of Genbank (Accession number: CN606328-CN606329 and CN606331-CN606338).When compared all ten black-bone differential expressed ESTs with the chicken(Gallus gallus domestics) nucleotide sequences deposited in the nr database and the dbEST database of Genbank via BLASTn tool, three (CN606332, CN606333 and CN606336) of them were found highly similar to two known genes and they were looked as homologous sequences of the two genes. Three (CN606328, CN606331 and CN606338) of other ESTs had their highly similar nucleotide sequences in chicken nucleotide databases but with unknown functions. The other four ESTs had no significant similarity with existing chicken genes or ESTs and they were regarded as new ESTs of chicken.The two genes homologous with black-bone chicken differential expressed ESTs were chicken 18S rRNA gene and chicken heat shock 70 kDa protein 5 (HSPAS) gene. The former was homologous with differential expressed ESTs CN606332 and CN606336, which were identified by reverse Northern dot blotting and Northern dot blotting as only expressed in SK liver or expressed in SK and NP liver. BLASTn was employed to compare the two genes with chicken genomic DNA database (chicken wgs_contig). The results showed that chicken 18S rRNA gene was located on chromosome 4,6,14 and 26. And the HSPAS gene was located on chromosome 17.Three ESTs (CN606328, CN606331 and CN606338) with significant similar nucleotide sequences but unknown function were analyzed via sequence assembling with their homologous gene clusters or related genomic DNA sequences. Two contigs, whose length equaled to 556 bp and 1029 bp, were obtained from the assembling. And one 1374bp ORF (open reading frame) containing CN606338 was obtained from related genomic DNA sequences. Result of functional analysis suggested that the protein sequences translated from the 556bp contig and 1029bp contig were highly similar... |
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