Cloning Of Bos Grunniens HSPA12B Gene And Detection Of Its Expression In Yak IVF Embryos

HSPA12B is an important member in the HSP70 family, and the molecular chaperonea which mainly exists in the mitochondria. HSPA12 B can inhibit the internal ROS of the embryo to play a cytoprotective effect, but also can inhibit the apoptosis of cells with tumor suppressor P53 interact. The biological function of HSPA12 B is not only confined to the mitochondria and immunofluorescence detection of subcellular fragments found that the HSPA12 B is located in other parts of the cell. HSPA12 B resides in multiple subcellular sites including mitochondria, endoplasmic reticulum, cell membrane, cytoplasm and cytoplasmic vesicles. Due to the current understanding of the function of HSPA12 B protein,it is limited to its function to promote the growth of vascular endothelial cells. In this study, The expression of HSPA12 B in early IVF embryos were detected by the methods of Real-time PCR and immunofluorescence at m RNA and protein levels and the HSPA12 B gene were detected by the methods of Real-time PCR.The results show that: 1.The gene sequence can be obtained by cloning(Gene Bank accession number:KR258751) 2.The HSPA12 B sequence was analyzed by bioinformatics.The HSPA12 B in early IVF embryos were detected by the methods of Real-time PCR. The results of bioinformatics showed the coding region of the yak HSPA12 B gene was 705 bp in length,encoding 234 amino acids,and the content of Alanine resdues was the highest(about 10.3%)and the content of Asparagine resduces was the lowest(about 0.9%). According to homology analysis,the similarity of HSPA12 B gene in Bos taurus and cattle was up to 99.5%,showing the HSPA12 B gene was highly conserved.The protein was hydrophilic and had no signal peptide,which was not a transmembrane and secreted protein. 3.Real-time PCR results showed that the HSPA12 B in yak early embryos fertilized in vitro expressed in each period,and the relative expression of HSPA12 B in 2~4 cell stage embryos, respectively is 14.8148、4.2699 and 40.4858 fold compared to the 5~8 cell stage embryos,morulae and blastocysts. The difference of the relative expression of HSPA12 B between 2~4 cell stage embryos and 5~8 cell stage embryos,morula and blastocysts was very significant(P < 0.01). 4.The expression of nucleus was lower than the expression of cytoplasm in 2~4 cell stage embryos, 5~8 cell stage embryos and morula,and the result is opposite in blastocysts,whichsuggests that faced in the environmental stress conditions in vitro,distribution of HSPA12 B protein is shifted between the nucleus and the cytoplasm. 5.HSPA12 B played an important role in the development of embryos in vitro fertilization. HSPA12 B can inhibit cell apoptosis, cell senescence, and regulate embryonic development. The experiments analysis the basic structure and genetic characteristics of HSPA12 B protein, and detected the expression of HSPA12 B in vitro fertilization embryo of in different periodsto discuss the fuction of inhibiting apoptosis,delaying cell senescence, regulating cell developmentin the embryonic development perspective of the heat shock protein. This paper provides a further study on HSPA12 B to cell protection during in vitro fertilization embryo development.