The Study Of Genetic And Function Analysis Of CD320 And RanBP9 Molecules In Goat(capra Hircus)

Sperm-mediated gene transfer(SMGT) is simple and efficient way of producing transgenic animals. The spontaneous capability of sperm cells binding exogenous DNA molecules and internalizing them into nuclei can be exploited by using spermatozoa as vectors for delivering foreign genetic information to eggs during fertilization. But there is randomness and uncertainty for SMGT in mammals and its mechanism is unclear yet, which limits the widely application of the technology. The interaction of various types of molecular is necessary in the process of SMGT, in which two candidate moleculars, CD320 and RanBP9 interacted with CD4, that is one of the important molecular invoved in SMGT were identified via yeast two-hybrid system,and verified interactions with CD4 molecules between CD320 and RanBP9 present in the eukaryotic cell by Co-immunoprecipitation. However, their function in sperm transfection exogenous DNA are unclear. Caprae CD320 and RanBP9 gene sequence were cloned and deteceted their expression levels in different tissues. Here, we analysed genetic polymorphism of CD320 and RanBP9 molecules in three goat population(Dazu black goats, Hechuan white goats and Jining grey goats), andanalyzed these expression in testis and epididymis tissues of goat, verified the role in sperm transfection exogenous DNA, based on the existing experiment. These works lay the foundation of clarifying the mechanism of SMGT. The main research methods and results are as follows:1. We studied the CD320 gene exon 3 and 3’ untranlated region polymorphisms in three goat breeds(Dazu black goats, n = 40, Hechuan white goats, n = 44, Jining grey goats, n = 33) by PCR-SSCP. CD320 gene exon 3 was notdetected SNPs sites, although there are SNPs sites of 3’ untranlated region.The polymorphic sites are located in noncoding section, then did not cause the change of amino acid. The order of PIC, He, Ne of two types(AA, AB genotypes) were detectedand are Hechuan white goat, Dazu black goat and Jining grey goat in this three goat breeds.2. We studied the relative expression level of CD320 gene in goat testis, caput epididymis, corpus epididymis, caudal epididymis by Q-PCR. The results showed that CD320 gene relative expressionis significantly higher in caudal epididymisthan than other tissues(P < 0.01). CD320 protein was mainly expressed in epithelial columnar cells of corpus epididymis and caudal epididymis by immunofluorescence. And CD320 protein was located in the spermatozoa acrosome cap, and other parts of the spermatoma not expression.3. In the experiment of sperm transfection exogenous DNA, spermatozoa with CD320 antibody were incubated inexperimental group, the transfection rate of positive spermatozoa was 9.79%, the transfection rate of positive spermatozoa was 16.08% in control group, This indicated that the efficiency of sperm transfection exogenous DNA is significantly reduced after CD320 was blocked by antibody.4. We studied the RanBP9 gene exon 11 and 3’ untranlated region polymorphisms in three goat breeds(Dazu black goats, n = 40, Hechuan white goats, n = 44, Jining grey goats, n = 33) by PCR-SSCP and RanBP9 gene did not detected SNPs sites.5. We studied the relative expression level of RanBP9 gene in goat testis, caput epididymis, corpus epididymis, caudal epididymis by Q-PCR. The results showed that RanBP9 gene relative expression is significantly higher in corpus epididymis than other tissues(P < 0.01). And RanBP9 protein was mainly expressed in epithelial columnar cells of corpus epididymis and caudal epididymis by immunofluorescence. RanBP9 protein was located in spermatozoa head section in addition to the equator area, and in the main part of spermatozoa tail by spermatozoa immunofluorescent.6. In the experiment of sperm transfection exogenous DNA, spermatozoa with RanBP9 antibody were incubated in experimental group, the transfection rate of positive spermatozoa was 9.40%, the transfection rate of positive spermatozoa was 16.08% in control group, This indicated that the efficiency of sperm transfection exogenous DNA is significantly reduced after RanBP9 was blocked by antibody.