| Sperm-mediated gene transfer (SMGT) has been long heralded as a faster and cheaper alternative to more commonly used methods of producing transgenic animals. DNA uptake by the mammalian spermatozoa is a very specific and high regulated phenomenon. The clarification of the molecular mechanism on sperm/DNA interaction is very important to improve the protocols for transgenic underlying SMGT. However, very limited related work has been done about this. As one of the transport systems on the sperm plasma membrane, our previous studies showed that CD4molecule played a distinct role in the process of sperm/DNA interaction. This makes it possible to explain the mechanism of SMGT, which at present is still a mystery in this area. A total of8candidates molecules interacting with CD4molecule was screened by using the yeast two-hybrid system. Among them, RanBP9and CD320was choosen and the genes cloning, sequence analysis, tissue expression and gene polymorphisms analysis were performed in this study,.Ran-binding protein9is a protein that in humans is encoded by the RANBP9gene. This gene encodes a protein that binds RAN, a small GTP binding protein belonging to the RAS superfamily. RanBP9plays an important role in the regulation of spermatogenesis and oogenesis process. CD320is involved in the immune response in the form of receptor that could mediate the transfer of materials and signals. Both genes have not been studied in goat yet. So the specific primers were designed for cloning RanBP9and CD320gene in goat by RT-PCR method, and their nuclear amino acid sequences and protein structures were analyzed. Meanwhile, RanBP9and CD320gene expression levels in different tissues of the goat were detected. Finally, using the polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique, the genetic polymorphisms CD320gene in Dazu Black and Nanjiang Yellow goat ’s also were detected. The main results were as follows:1. The cDNA of goat RanBP9and CD320were obtained and deposited under the GenBank accession number:KF425511.1and KF425510.1, respectively. The length of RanBP9and CD320were1008bp and517bp, respectively.2. The length of RanBP9open reading frame (ORF) is936bp, which located in2-937bp and encoded312amino acid residues, and the applied signal located in937bp and2bp respectively. The ORF length of CD320is459bp, which encoded153amino acid protein, and the463bp of the2bp were added signal.It showed that the amino acid sequence of1-54bp and72-196bp from goat RanBP9is an unknown area by using SMART program analysis, excep that the55-71bp is the low coupling region and197-299bp loated at the smart area; CD320amino acid sequence of100-122bp is at low density lipoprotein receptor domain and18-56bp at the transmembrane region. BLAST sequence alignment with the display, there is similarity of99%between RanBP9genes with rhesus monkeys, gorillas, humans, mice and lizards, however the homology with the cattle, toads and fish Homology were86%,83%and75%respectively; the homology of CD320gene with sheep, cattle homology were98%,96%, and the homology were83%,77%,76%,75%,74%and73%. Comparing with dolphins, tigers, whales, giant pandas, horses and domestic cats homology. Expasy predicted protein with a hydrophobic site, the results showed that there was a high negative peak in RanBP9hydrophobic, which was hydrophilic, and there was positive peak high hydrophobicity in the CD320, which is hydrophobic. The ransmembrane protein was predicted by using TMPRED, the results showed RanBP9was at the281-298bp, CD320transmembrane structures was at103-122, both of which are transmembrane proteins. On the NCBI signal peptide prediction analysis the two gene were not detected signal peptide.3. Collecting tissue samples from Dazu Black goat contains heart, liver, spleen, lung, kidney, pancreas, muscle, testis, ovary, thymusand, and extracte tissues total RNA. The expression of RanBP9, CD32O are detected using semi-quantitative PCR method. RanBP9, CD320was expressed in goats, heart, liver, spleen, lung, kidney, pancreas, spleen, muscle, testis, ovary and thymus tissue, however, CD320is at higher expression levels in the spleen and testis and RanBP9obtain the highest expression in the testis.4. The30blood samples were collected from Dazu Black goat and Nanjiang Yellow goat, respectively and the whole blood DNA is extracted.The polymerase of the CD320and RanBP9was detected using polymerase chain reaction-single strand conformation polymorphism (PCR-SSCP) technique. The results showed that there were two genotypes (AA and AB) about CD320gene in Dazu Black goat and Nanjiang Yellow goat, and the genotype frequencies were0.767and0.233 respectively in Dazu Black goat; A and B gene frequencies were0.883and0.117, the values of He, Ne and PIC were0.207,0.667and0.333; the genotype frequencies in Nanjiang Yellow goat were0.833and0.167respectively; A and B gene frequencies were0.833and0.167; and the values of He, Ne and PIC were0.193,1.386and0.204. The population genetic structure in CD320gene was consistent with Hardy-Weinberg genetic balance law. |
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