Study On Buffalo FSHR Gene And Its Expression

This thesis studies issues related to buffalo FSHR gene and its expression, it is arrangedin two parts: partⅠis a review of literatures; partⅡincludes chapterⅠand chapterⅡ,which deals with the experimental works.The objective of this study in chapterⅠwas to understand the mechanism of function ofFSHR gene and design the specific primers for detecting the buffalo FSHR gene withhalf-quantification methods after the cloning of the buffalo FSHR gene and the sequencesanalyzing. According to the FSHR gene, the buffalo genome DNA and RNA were extractedfrom the buffalo blood and ovaries. After PCR and RT-PCR amplification, these fragmentswere detected, purified and inserted into the vector PMD18-T and sequenced. The results ofsequence showed that the length of FSHR cDNA was 2229 bp with 976 bp in the promoter, 1-64 bp in the 5'-UTR, 2153～2229 bp in the 3'-UTR, 65～2152 bp in the ORF,and 14572152 bp in the exon10 of the gene. Compared with the bovine, the homology of suchsequence was 98%. After the multi-alignment among the exon10 of the gene in buffaloes withother mammals using ClustalX, phylogenetic tree was constructed, which showed thepreferable relationship between bovine and buffalo. 678 amino acids encoded FSHR and thewhole signal peptide sequence of 17 amino acids in N-tail were analyzed using TMHMM, Itwas found that, the length of the transmembrane, intracellular domains and extracellulardomain were consisted of 349, 264 and 65 amino acids, respectively.The study in chapterⅡwas to analyze and compare the expression of bufflogonadotropin receptor mRNA in different tissues and granulas in COCs of different sizes offollicles, different quality grades and different in vitro maturation phases by RT-PCR withspecific primers designed according to the sequence in buffalo FSHR gene, the resultsrevealed that: 1) the FSHR gene was expressed in the ovary and testis with no expression inother tissues; 2) the FSHR mRNA in granulas of COCs from different sizes of follicle haddifferent abilities of expression, the relative abundance of FSHR mRNA from large follicleswas lower than those in medium and small follicles; 3)FSHR mRNA abundance was affectedby grade of COCs, the relative abundance of FSHR mRNA was significantly higher in grade A COCs than in grade C COCs, there were significant differences in three kinds of granulas ofCOCs FSHR mRNA abundance between grade A and B and between grade B and C; 4)gonadotropin receptors mRNA abundance in granulas of COCs was affected by the time ofmaturation, there was a trend towards decreased FSHR mRNA abundance.