A Screening Research Of Genes On The Differential Expression Of X And Y Sperms Of Dairy Cow

This study pertains to the differential expression of specific genes located at X and Y chromosome-carrying sperms of some Holstein cows. In the experiments, we used a self-optimized methodology of solid-phase differential hybridization to construct the cDNA plasmid library for Y-to-X sperm differential hybridization, analyzed those screened differential genes by means of bioinformatics, and then employed Fluorescence in situ Hybridization (FISH) to test the gene specificity thereof. We concluded that:(â…°) Differential fragments were obtainable from using magnetic beads to extract mRNAs from Holstein X and Y sperms, to synthesize X sperm cDNAs and then to conduct Y-to-X sperm differential hybridization. The fragments obtained were SMART amplified so that a cDNA plasmid library for Y-to-X sperm differential hybridization was constructed, followed by cloning, identification and then gene sequencing. An online homology analysis was done to the sequences of those screened genes. Sequence comparisons revealed that the sequences of screened genes were highly homologous to that of cow genes available at EST database. Among them, one type of fragment was identified as the Sry gene, and others unknown.(â…±) Digoxin-marked probes were prepared by PCR amplification using the primers designed based on the identified Sry gene, with the cDNAs from conventional Straw frozen semen being the template. X sperms, Y sperms and routine sperms were subject to FISH tests at RNA level, respectively. X and Y sperms could be distinctively identified by their fluorescence signals. And the Sry mRNA was positioned at the middle part of the tail of Y sperms. By Fluorescence in situ hybridization, the mean hybridization efficiency was93%.And positive hybridization signals were94.6%,93.3%and50.3%for X-, Y-sort and normal sperm, separately. There was no significant difference (P>0.05) by Chi-square test. The Sry probe FISH-based determination of the purity of Holstein sperms was correct and reliable, providing good technical reference to other methods of sperm purity evaluation.