| This study was designed to investigate the relationship between variability of ZP gene family(ZP2, ZP3, ZP4) and litter size of hu sheep. In this study, Mixed sample sequencing technologies were used to detect SNP locus with Hu sheep, Han sheep, Mongolian sheep and Tan sheep as experimental animals. The data of number of 100 Hu lambs was collected, and we selected preliminarily some locus which have great influence on the litter size by analyzing the relationship between the SNP locus and litter sizes. Bioinformatics approach was used to analyze the primary, secondary and tertiary structure of ZP. At the same time, the relationship among ZP gene mutation sites, ZP protein structure and natural selection were also analyzed, for explaining the link between structure and function of ZP gene. In addition, we used ZP gene sequences collected from NCBI public database to explore the driving forces underlying evolutionary mechanisms to make this rapid evolution of ZP by analyzing the differences of natural selection between mammals, avian and fish. The results as follows:1. 28 SNP locus were detected by mixed sample sequencing technologies. However, ZP2, ZP3, and ZP4 contained 4, 7, 17 locus, respectively. Linkage analysis showed that the 4 SNPs of ZP2 formed a chain block, ZP3 contained 2 chain blocks(T678C and A859C、T7781G and G7804A) and 3 independent SNP locus. There were 3 chain blocks and 4 independent SNPs on ZP4. Association analysis of chain blocks and independent SNPs with the firstborn litter size and total litter size, repectively, found that the loci of T8176 C of ZP3 and 3 SNPs of ZP4(A7499C, T10223 C and G10383A) have significant impact on the total number of Hu lambs. Evolution tests among 4 populations indicated that ZP3 and ZP4 were subjected by positive selection.2. Bioinformatics analysis of sheep ZP gene showed that ZP2 gene coding product was a hydrophilic trans-membrane protein containing 713 amino acid residues which has a signal peptide of containing 36 amino acid residues. Signal peptide located on 1-36 area and transmembrane domain located on 608-704 area while 1-608 area located on external of membrane. Secondary structure and prediction and conservative domain analysis showed that N-terminal and C-terminal were variation region and the middle part was conservative. ZP3 is an unstable hydrophilic transmembrane protein containing 381 amino acid residues which do not has obvious signal peptide. The secondary structure was primarily composed of loop, β-fold and random coil, ZP-domain located on 3-261 area and transmembrane domain located on 341-363 area which structure is α-helix. Meanwhile, we also found a non-ordinary secondary structure and most of the amino acid residues were exposed. The test result of positive selection indicated that ZP3 was subject to nature selection and most of the positively selected sites were distributed on the non-ordinary secondary structure. Phosphorylation tests showed that a serine residue of 371 loci was modified by phosphonation. Results of sequence alignment indicated that there had high similarity between human and sheep, which suggested they may have similar functional domain. ZP4 gene coding product was unstable hydrophilic transmembrane protein containing 534 amino acid residues which had a signal peptide located on 1-36 area. The secondary structure: α- helix accounts 4.68%, β-sheet accounts 32.02%, random coil accounts for 63.30%, the transmembrane domain zone locate on 508-530. Conserved domain analysis showed that P- domain zone located 141-183, ZP domain zone located on 188-460.3. ZP of monospermy(mammals and fish) has a faster mutation rate and subjected greater selection pressure compared with bird. Monospermy have experienced rapid evolution, while five ZP genes of avian have no significant positive selection signature. Otherwise, we found that most of the positive selection sites located onto the three ZP-N domains of the ZP2(NTR domain) and sperm-egg recognition and binding area(SE domain) of ZP3. |
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