| This research contains two parts.Partâ… : The cost of developing replacement nanny goats could be reduced by decreasing age at puberty because nanny goats can be brought into production at earlier age. Jining Grey goat is a sexual precocious breed. The objective of this study was to screen genes related with puberty to look into molecular mechanism of puberty and establish fundamental materials and techniques for further study and genetic selection.Subtracted cDNA libraries of hypothalamus, pituitary, ovary-uterus and adrenal gland of juvenile stage, puberty and the same age control of puberty between Jining Grey goats and Liaoning Cashmere goats were constructed using suppression subtractive hybridization (SSH). And the differential expression genes were analyzed by bioinformatics. The main results obtained were listed as follows:1. The 12 forward subtracted cDNA libraries of hypothalamus, pituitary, ovary-uterus and adrenal gland of juvenile stage, puberty and same age control of puberty between Jining Grey goats and Liaoning Cashmere goats were constructed using suppression subtractive hybridization (SSH). The subtraction efficiency was estimated by GAPDH gene, and the results showed that the GAPDH was subtracted efficiently at 25 folds at least, which demonstrated that differential expression genes were also enriched efficiently. The size of inserted fragments was 250-900 bp identified by PCR and sequencing.2. There were 2046 differentially expressed ESTs in 12 subtracted cDNA libraries, which contained 689 ESTs at juvenile stage, 725 ESTs at puberty and 623 ESTs at the same age control of puberty. The number of differential expressed ESTs from hypothalamus and pituitary was a little more than ovary-uterus and adrenal gland at every stage. The differentially expressed ESTs in each subtracted cDNA libraries were classified to high homology genes, high homology ESTs and novel ESTs according to sequence homology in the GenBank nr and EST database.3. The high homology genes were carried out functional classification at the internet. The high homology genes were related to signal transduction and cell-cell communication, material transport, replication/transcription/translation correlated, cell structures and motion, metabolism, defension of cell and organism according to PANTHER site. And based on COG classification, the high homology genes were related to cellular processes and signaling, information storage and processing and metabolism, three main types, 16 subdivisions.4. Pathway analysis in KEGG pathway database of the high homology genes revealed the most three pathways with most genes involving: metabolic pathways, Parkinson's disease and oxidative phosphorylation.5. Protein interaction analysis of the high homology genes revealed the most dominant network: structure of ribosome/protein translation and oxidative phosphorylation. Partâ…¡:The kisspeptin/GPR54 pathway is crucial in the process of puberty onset. Variations in or near Lin28B reached a genome-wide association with age at menarche. The aim of the current research was to find new genes associated with sexual precocity of goat and understand the mechanism of action, and provide some useful information for the molecular breeding to shorten the timing of puberty of goat.In the first experiment, a DNA fragment with 4118 bp of goat KiSS-1 was obtained, which contains an open reading frame (ORF) of 408 bp and encodes 135 amino acids, having 91.11% and 95.24% identity with the protein sequence of bovine and sheep, respectively, and 60.53%, 58.12%, 59.66%, 72.50% with human, mouse, rat and pig, respectively. The protein was predicted containing a signal peptide of 17 amino acids. There were two mutations (G3433A [A86T] and C3688A) in exon 3, three mutations (G296C, G454T and T505A) in intron 1 and a 18 bp deletion (-)/insertion (+) (1960-1977) in intron 2 and no mutation in exon 2. The genotype distribution of the six mutations didn't show obvious difference between sexual precocious and sexual late-maturing goat breeds and no consistency within the sexual late-maturing breeds. For the 296 locus, the Jining Grey goats with genotype CC had 0.80 (P<0.01) or 0.77 (P<0.01) kids more than those with genotype GG or GC, respectively. No significant difference (P>0.05) was found in litter size between GG and GC. For the 1960-1977 locus, the Jining Grey goat with genotype -/- had 0.77 (P<0.01) or 0.73 (P<0.01) kids more than those with +/+ or +/-, respectively. No significant difference (P>0.05) was found in litter size between +/+ and +/- genotypes. For the other four loci, no significant difference (P>0.05) was found in litter size between different genotypes in Jining Grey goats. The present study preliminarily indicated an association between allele C of the 296 locus and allele - of the 1960-1977 locus in KiSS-1 and high litter size in Jining Grey goats.In the second experiment, a DNA fragment of 4258 bp of goat GPR54 was obtained, which contains an ORF of 1137 bp and encodes 378 amino acids, having 94.57% and 99.58% identity with the protein sequence of bovine and sheep, respectively, and 86.69%, 84.88%, 82.47%, 88.99% with human, mouse, rat and pig, respectively. The protein was predicted to have seven transmembrane regions. There were no base pair variation in exons 1-4 and three base changes (G4014A [G328D], G4136A [G368S] and C4152T [P373L]) in exon 5 by sequencing and the three mutations may have some correlation with sexual precocity in goats. For the 4152 locus, the Jining Grey goat with genotype TT and CT had 1.02 (P<0.01) and 0.84 (P<0.01) kids more than those with genotype CC, respectively. No significant difference (P>0.05) was found in litter size between TT and CT genotypes in Jining Grey goat. For the other two loci, no significant difference (P>0.05) was found in litter size between different genotypes. The present study preliminarily indicated an association between allele T of the 4152 locus in GPR54 and high litter size in Jining Grey goats.In the third experiment, an mRNA sequence with 916 bp of goat Lin28B was obtained, containing a CDS of 744 bp, encoding 247 amino acids, which was predicted having a cold shock domain and a pair of zinc finger domain. The 247-amino acid has 90. 80%, 90.32%, 76.19%, 76.92% and 94.09% identify with the protein sequence of human, macaque, rat, mouse and bovine, respectively. Two alternative transcripts were detected expressing in goat tissues. And the alternative splicing site lies in intron 3, which contain a microsatellite sequence in its 3'- terminal. There was no mutation in exons 1-3 and nine mutations found in exon 4 (G911A, C1026T, A2934T, C3053T, G3248A, C3414G, A3770T, C4478T and G4742A), which were all in 3'-UTR, by sequencing. The genotype distribution of the eight sites detected didn't show obvious difference between sexual precocious and sexual late-maturing goat breeds and no consistency within the sexual late-maturing breeds. For the 2934 locus, Jining Grey goats with genotype TT and AT had 0.83(P<0.01) and 0.48(P<0.05) kids more than the goats with genotype AA, and no significant difference (P>0.05) was found in litter size between TT and AT genotypes in Jining Grey goat. For the other seven loci, no significant difference (P>0.05) was found in litter size between different genotypes in Jining Grey goats. The present study preliminarily indicated an association between allele T of the 2934 locus in Lin28B and high litter size in Jining Grey goats.
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