Cloning Mstn And Igf-ir Genes And Association Analysis Between Snps And Growth Traits In Yak

In order to improve the traits of growth and development and apply molecular breeding technology in Yak, the MSTN and IGF-IR genes that were the major candidate genes for genetic markers in livestock are researched in four native yak breeds and one improved breed, which are respectively Xinjiang Bazhou yak, Tianzhu White yak, Gannan yak, Qinghai Plateau yak as well as Datong yak. Important contents are that MSTN and IGF-IR genes of yaks were cloned and sequenced with molecular biology technology, the sequence of MSTN and IGF-IR genes of yaks and other species are compared in bioinformatics software, the primary, secondary and tertiary structure and characteristics of coded protein are presumed, the polymorphisms of MSTN and IGF-IR genes are examined with PCR-SSCP, in addition, the correlation of polymorphisms of MSTN, IGF-IR genes and the traits of growth and development in yak is analyzed. The main results are as follows:1. MSTN gene of yak is amplified by fractional PCR on the base of specific primer, then, the object segments are cloned and sequenced. The sequence of MSTN gene whose length is 6613 bp is obtained through the molecular biological software splicing, logging in number is EU926670 in Genbank. Comparing sequence of MSTN gene of yak with other species, the sequence of MSTN gene consists of 3 exons and 2 introns, the length CDS sequence is 1128 bp, logging in number is EU926669 in Genbank, coding 375 amino acid. The size of 3 exons is respectively 373 bp, 374 bp and 381 bp. The size of 2 introns is respectively 1843 bp and 2028 bp.2. A single base conversion (C→T) located at the site of 417 was found in Yak Myostatin gene coding region compared to cattle, which did not cause changes in amino acids. The nucleotide sequence of the coding region and deduced amino acid sequence among different species were quietly conservative. The deduced amino acid sequence of mature protein is closely related to bovine and sheep, homology rates of nucleotide sequences of the coding region of Myostatin gene between the yak and cattle, sheep, pig, mouse, human, dog, house, rabbit, chicken and chimpanzee were 99.9 %,96.5 %,94.3 %, 89.1 %,91.9 %,91.3 %,93.6 %,91.7 %,82.0 %,92.0 %, respectively, while the homology rates of amino acid sequences were 100 %, 93.3 %, 95.5 %, 92.5 %, 94.1 %, 93.3 %, 94.9 %, 94.4 %, 88.0 % and 94.4 % when compared with cattle, sheep, pig, mouse, human, house, rabbit, chicken and chimpanzee, respectively.3. Bioinformatics software analysis showed that the theoretical molecular weight of Yak Myostatin gene was 42.6 kD and PI was 6.14. The secondary structure of coded protein was mainlyα-helix,β-sheet and randomly twist. There is fewα-helix in protein coded by the third extron. Protein coded by MSTN gene in yak is transmembrane protein. The transmembrane domain is from AA6 to AA23. Moreover, the protein had a secretion signal peptide structure, MQKLQICVYIYLFMLIVA, which probably related with TGF-βprotein family. The tertiary structure has been gained by mock construction of molecular confirmation which showed the 98.17% homology with FS 288. It showed that the close relationship between the myostatin of yak and FS 288 gene of other mammals is belong to the familyTGF-β.4. IGF-IR gene was cloned and sequenced with RT-PCR approach to obtain the complete nucleotide sequences of mRNA. This cDNA sequence received from these PCR products contained the entire coding region, covered 4350 bp with 3'and 5'-untranslated regions and an open reading frame of 1367 amino acids.5. 20 base changes is discovered in the nucleoside arrays comparison between yak and cattle, in which 9 transitions for C-T, 2 transition for G-A, 5 transitions for T-C, 3 transitions for A-G and 2 transitions for G-T substitution, respectively. Homology rate of nucleoside acid arrays of IGF-IR between yak and cattle is the highest, reaching 99.8%. However, only in site6, 29 and 30 replacement were responsible for a Gly–to- Arg, Thr -to- Ile and Ser -to- Ile substitution at amino acid, respectively. Comparing with pig, human, dog, mouse, chimpamzee, chicken, the comparability between amino acid sequence coded by IGF-IR is 98.3%, 97.6%, 92.5%, 95.0%, 97.0% and 82.3% respectively.6. Bioinformatics software analysis showed that the theoretical molecular weight of Yak IGF-IR protein coding gene was 154949.3 and PI was 5.71. The secondary structure of IGF-IR protein coding gene were mainly composed byα-helix,β-sheet,β-turn and randomly twist. The IGF-IR protein was predicted to have 3 potential transmembrane domains and located at 6-26,938-958 and 1188-1209. The splice site of signal peptide sequence appear in the adjacent sites around 30-31 amino acids. The nucleotide sequences of IGF-IR gene were compared with human IGF-IR; as a result, the homology was 98.68%.7. The polymorphism of MSTN gene in yak is examined by PCR-SSCP, at the same time, the correlation polymorphism of MSTN gene with growth traits is analyzed. The results indicated, 2 polymorphic locus are discovered, which located at extron 1 and intron 2 respectively. There is a single base mutation at 176 bp site of amplified segment of extron 1, namely G to A. There is a single base mutation at 192 bp site of amplified segment of intron 2, namely A to G. Two different alleles (allele A and B) and three genotypes were found in the locus intron 2 of MSTN gene. The X2-test showed that genotype distribution of intron 2 was in the equilibrium of Hardy-Weinberg within breeds of Gannan yak,Tianzhu White yak and Qinghai Plateau yak, while Datong yak and Xinjiang Bazhou yak obviously was unequilibrium. The least square analysis showed that the SNP of MSTN intron 2 had a significant effect on partial traits of growth including body slanting length and body weight(P<0.05), and the genotype did not affected on body height, heart girth and cannon circumference significantly(P>0.05) for yearly yak. In addition, the SNP of MSTN intron 2 had a significant effect on partial traits of growth including heart girth and body weight(P<0.05), and the genotype did not affected on body height, cannon circumference and body slanting length significantly(P>0.05) for adults yaks.8. The polymorphism of IGF-IR gene in yak is examined by PCR-SSCP, at the same time, the correlation polymorphism of myostation (MSTN) gene with growth traits is analyzed. Results showed, A novel nucleotide polymorphism (SNP)-C-A (242bp) was identified located in Exon 1 in the coding region of the IGF-IR gene. Two alleles (A and B) and three genotypes designed as AA, AB and BB were detected in the PCR products. The SNP loci were in Hardy-Weinberg equilibrium in Datong yak, Gannan yak, Tianzhu white yak and Qinghai Plateau yak populations (P<0.05) except for Xinjiang yak (P>0.05). Least squares analysis revealed that there was a significant effect on Height, body weight and body sleeting length (P<0.05). Individuals of yearly animals with various genotypes had no significant difference (P<0.05) on heart girth and cannon circumference. In addition, no significant was found on height, body weight, heart girth, body sleeting length and cannon circumference for adults yaks (P>0.05).