| Tibetan Chicken (TC) mainly distributed in Tibetan altiplano, is a rare original chicken breed in China. It has many characteristics, such as cold-resistant, disease-resistant, anoxia-resistant, good meat quality, high value for medicine, extensive cμltivation and good fancy. At the same time, it has the disadvantage of slow growth and low-level egg-laying rate. How to utilize Tibetan chicken virtue and get rid of its flaw? And it is so actual merit and significance for us to utilize and improve Tibetan Chicken from its genetic substance.For removing TC demerit, one of our primary tasks is to increase growth rate of TC. First, cloning and identifying some genes potentially related to growth of TC, and then these genes functions are investigated and these genes are applied for production and practice. Combining of bioinformatics and biotechnology, a set of new genes possibly related to TC growth were cloned and discriminated. The main results are as follows:1. Full-length cDNA library of TC leg muscle was constructed by using SMART? (Switching Mechanism At 5' end of RNA Transcript) cDNA Library Construction Kit.The results showed that the titer of the library was 1.1×10~6, the recombinant percentage was 99%, the average inserts length were 1.2-1.5kb and the rate of the integrated gene inserted into this library was 47.36%.2. 200 ESTs were randomly single-pass sequenced from 5' end of full-length cDNA library of TC leg muscle. 80 Contigs and Singletons were assembled by SeqMan program of DNAStar soft. The results of full length cDNA annotation showed that no anyone intact gene existed in EST sequences.3. 22 ESTs corresponding 13 new genes were re-sequenced and assembled. The results of full length cDNA annotation discovered that 2 EST sequences corresponding Serfl and Hypothetical Protein maybe included the whole gene sequences and the length of sequences were 713bp and 2408bp, respectively. 4 ESTs corresponding Unknown gene, Myotilin, Glypican 4 and Cytochrome c oxidase subunit Vic gene maybe included the complete Coding Sequence (CDS) and the length were 462bp, 1268bp, 568bp and 496bp, respectively.4. 40 of 44 EST sequences were effectively electro-extended and assembled into 40 Super Contigs (SC). The results of full length cDNA annotation proved that 10 SC sequences were likely to covering intact genes, and 9 SC sequences were likely to containing complete CDSs.5. The nucleotide sequences of 12 ESTs covering the whole genes were predicted and analyzed on aspects of their basic properties, electronic expressed profiles andelectronic gene mapping. And basic properties, structures and functions of translated protein sequences of CDS of 12 genes were analyzed, too. The above aspects of translated protein sequences of CDS of 9 ESTs were predicted. The results indicated that 21 EST sequences contain some important functional sites and functional structure domains.6. 2 EST sequences corresponding Serfl and eIF3S9 genes were extended and validated by 5' and 3' RACE method and their length are702bp and 884bp, respectively. By comparison between extended Sequences and original EST sequences, the two sequences were extended 53bp and 160bp and homology scores were 99% and 94%, respectively. In addition, "AATAAA" signals and "PolyA" tails were present in 3' end of two sequences.7. A practical method of SNPs detection was constructed, named Modified Allele Specific PCR (M-ASP), and only needs two PCR for distinguishing different genotypes.8. A kit for speedily detecting and diagnosing advantaged genotypes of Ghrelin gene related to TC fast growth was constructed.9. Some SNPs of 3 known genes (GH, IGF-I and Ghrelin genes) and 2 new genes (Serfl and eIF3S9 genes) were detected among TC and recessive white chicken. And association of genes with 17 growth traits of two chicken breeds were analysed. The results showed that: ? SacI-RFLP and M-ASP simultaneously existed in intron 4 of GH gene, and a base pair mutation from C to G took palace in this site. There was a significant association of Genotype CC with body weight at the age of 7 weeks (P<0.05). ? Hinfl-RFL? and Psf I-RFLP existed in 5' UTR of IGF-I gene, and a base pair mutation from A to C took palace in Hinfl identifying site and a base pair mutation from C to T took palace in Pstl identifying site. There were significant associations of Genotype CT with body weight at birth and body length at the age of 7 weeks and of Haplotype genotype A+T/C+T with body weight at birth, body length and shank girth at the age of 2 weeks and shank girth at the age of 16 weeks (P<0.05). (3) M-ASP existed in sites 71, 1215 and 2355 of IGF-I gene. Otherwise, Eco 72I-RFLP existed in Site 1215 of Ghrelin gene. A base pair mutation from T to C took palace in site 71, A base pair mutation from A to G in site 1215 and A base pair mutation from A to G in site 2355. There were significant associations of Genotype CT in Site 71 with body weight and shank length at the age of 2 weeks, body weight and shank girth at the age of 7 weeks and body weight, shank length and shank girth at the age of 16 weeks and of Genotype AG in site 1215 with body weight at the age of 16 weeks (P<0.05). There were significant associations of Haplotype genotype CAA/CAG with body weight and shank girth at the age of 16 weeks (P<0.05). ? MspI-RFLP and Hin61-RFL? existed in eIF3S9 gene, and a base pairmutation from T to C took palace in Mspl identifying site and a base pair mutation from A to G took palace in Hin6\ identifying site. There were significant associations of genotype CT in Mspl identifying site with body weight at birth, body weight and shank length at the age of 7 weeks and body weight, shank length and body length at the age of 16 weeks, of genotype AG in Hin6\ identifying site with body weight at the age of 2 weeks and shank length at the age of 4 weeks and of Haplotype genotype C+A/T+A with body weight and shank length at birth, body length at the age of 7 weeks and shank girth and body length at the age of 16 weeks (P<0.05). (D Tasl-RFLP existed in Serfl gene, and a base pair mutation from C to T took palace in Tasl identifying site. There were significant associations of genotype CT with body weight and shank length at birth and shank length at the age of 4 weeks (P<0.05).
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