Study On Buffalo SRY Gene And Preimplantation Embryo SEX Identification

This thesis studies issues of buffalo SRY gene and PCR system for preimplantation embryo sex identification. It is arranged in two parts: part1 includes chapter 1 and chapter 2, which is a review of literatures; part 2 includes chapter 3 and chapter 4, which deals with the experimental works.The objectives of these studies in chapter 3 were to investigate the operation mechanism of SRY gene and design specific primers for buffalo embryos sex identification by cloning, sequencing and Southern blot analysis of SRY gene. SRY gene from Buffalo genome was amplified by polymerase chain reaction (PCR) with primers based on the sequence of Hostein bovine SRY gene. Sequencing and nucleotide sequence analysis revealed that the amplified fragment was 2005 bp including 5' promoter of 1 bp ～ 504 bp, 3'UTR of 1196 bp ～ 2005 bp and open read frame (ORF) of 505 bp ～ 1195 bp. This sequence was submitted to GenBank (Acession No.DQ 417872). BLAST indicated the coding region of SRY gene from Buffalo was highly homologous with other mammals relevant gene (95% ～ 96% homology), especially in the HMG-box region (99% homology), which indicated SRY were highly conservative on evolution. Phylogenetic tree was also constructed after multiple sequence alignment with other mammals. Genomic DNA from male or female buffalos were hybridized with the probe synthesized basing on SRY conserved region, there were only signals on male buffalo genome DNA, which indicated that SRY gene was male-specific.The objectives in chapter 4 was to establish and optimize the PCR system for the sex identification of the buffalo implantation embryos so that this technique can be applied in buffalo production. Two pairs of primers SA1/SA2, SB1/SB2 were designed basing on buffalo SRY gene which obtained from chapter 3. In addition, ZA1/ZA2, GA1/GA2 were designed as control basing on mouse ZFX/ZFY gene and buffalo G3PDH gene, respectively. To select the best primers for PCR amplification, specialties and sensitivity of the primers were tested by amplifying genomic DNA from different mammals and different concentration...