Effects Of NYD-SP27 Interference Gene On Fertilization Efficiency Of Sheep In Vitro

Testis development specific protein gene 27?NYD-sp27?is a subtype of Phospholipase C?Phospholipase C,PLC?,which plays an important role in spermatogenesis,capacity,acrosome reaction,and fertilization.Currently,NYD-sp27 has been reported in the study of humans and mice,but the role of in vitro fertilization of Sheep has not been reported.[Objective]:This study is to clone NYD-SP27 gene and construct interference vector of NYD-SP27 in sheep.This study explored influence of NYD-SP27 gene silencing on in vitro fertilization?ivf?of sheep by using testicular injection and RNA interference of NYD-SP27,providing scientific basis for revealing the function mechanism of nyd-sp27 gene.[Method]:?1?RNA was extracted from Sheep testis and NYD-SP27 gene were transcribed from cDNA by kit.?2?The NYD-SP27 gene was cloned and connected to p LL3.7 and then sent to the company for sequencing.The successfully sequenced NYD-SP27 was cloned into eukaryotic expression vector pLL3.7 for constructing interference vectors.The NYD-SP27interference vectors were extract using endotoxin free kit and were subjected to intratestis injection of Sheep three times.Sheep semen were collected through the electronic semen collector.The semens were fixed with formaldehyde for checking green fluorescence using microscope.?3?NYD-SP27 genes were amplified by PCR and protein electrophoresis were observed for confirming NYD-SP27 gene interference.The disruption of the NYD-SP27 gene in ram semen can be used to study the in vitro fertilization of Sheep.?4?NYD-SP27 gene interference may have effect of in vitro fertilization and early embryo development and can be used for verifying the role of NYD-SP27 gene in regulating endogenous PLC gene.Results and conclusions:?1?The total length sequence of NYD-SP27 gene was successfully cloned and obtained.The sequence analysis of Sheep NYD-SP27 gene showed that the NYD-SP27 gene contained 1 901bp length and only 1617 bp of open reading frame?ORF?,encoding 538 amino acids.Molecular weight was 61995.66.The theoretical value of PI was 6.16,the positive charge residues?Arg+Lys?was 61.The negatively charged residues?Asp+Glu?was 67.The molecular formula was C₂₇₉₈H₄₃₁₉N₇₃₇O₈₁₉S₁₉.The in vitro half-life was 30 h.The instability coefficient was 46.96?unstable?.The total average absorbency was-0.403.?2?The green fluorescence can be observed in formalin fixed semen,suggesting that the NYD-SP27 gene can be effectively expressed in sperm cells.The weak protein bands can be observed in protein electrophoresis.The results showed that NYD-SP27 gene interference effect is obvious,and ram semen by disruption of the NYD-SP27 gene can be used for Sheep IVF.?3?NYD-SP27 interference group and the control group of Sheep semen in vitro fertilization results showed that the fertilization the rate was 20%,which was 56%.The morula rate of the former was 31%.The latter was 55%.Our result showed that inhibition of NYD-SP27 gene promoted in vitro fertilization and early embryo development.