Screening Of Oocyte-derived SEPT7 Interaction Proteins And Their Gene Regulation In Bovine

Cleavage is an important developmental event in the early embryonic development process,especially the time and manner of the first embryo division after fertilization has a vital significance for the developmental potential of the embryo and is also an important basis for evaluating the quality of the embryo.Our previous research found that spermatious mi RNA-202 can regulate HDAC6 through the target gene SEPT7,increase the acetylated level of embryonic skeleton protein α-tubulin after fertilization,and enhance the stability of the embryonic skeleton,thereby reducing the occurrence of abnormal ovalofisis.However,the mechanism of SEPT7 regulation of HDAC6 is still unclear,in this study,in order to improve the mechanism of SEPT7 regulating the corresponding pathway signal of HDAC6 and the mechanism of regulating the first embryo division,the interaction candidate proteins of SEPT7 were screened by immunoprecipitation technology combined with mass spectrometry,and the functions of SEPT7 and interaction proteins were discussed,and the following results were obtained:(1)The specific protein binding to SEPT7 antibody was screened by immunocoprecipitation technique and Western blot,and sequenced and analyzed by mass spectrometry.RACK1,RAN,HSP90AA1,HSPA1 A and ACTN1 related to cytoskeleton,embryonic development and cleavage in bovine oocytes or early embryos may interact with SEPT7;(2)The target genes of SEPT7,RACK1,RAN,HSP90AA1,HSPA1 A and ACTN1 were obtained by extracting cell RNA for reverse transcription,eukaryotic expression vectors was constructed respectively,such as 3 × FLAG-cmv-10-SEPT7,p-cmv-HA-RACK1,p-cmvHA-RAN,p-cmv-HA-HSP90AA1,p-cmv-HA-HSPA1 A and p-cmv-HA-ACTN.The overexpressed target gene was transfected by cell liposome,and the overexpressed tag HA and FLAG antibody were combined with agarose beads and target protein to determine whether there was an interaction between them.At the same time,it was verified on embryos by PLA.The results showed that SEPT7 had an interaction with RAN and HSP90AA1,but had no interaction with RACK1,HSPA1 A and ACTN1;(3)qRT-PCR was performed by collecting GV and MII oocytes as well as zygotic and 2-cell embryos.The results showed that there was no significant difference between SEPT7 and HSP90AA1 at transcriptional level;The expression level of RAN increased significantly in MII phase compared with GV phase,decreased significantly in zygote embryo and increased to the same level as GV phase oocytes in 2-cell embryo;(4)By injecting RAN siRNA,HSP90AA1 siRNA and their mixture into zygotic embryos,the results showed that the down-regulation of RAN expression level in early bovine embryos would lead to the down-regulation of HSP90AA1 expression level,but did not affect the expression level of SEPT7,while the down-regulation of HSP90AA1 expression level would lead to the significant increase of SEPT7 expression level,but did not affect the expression level of RAN,the down-regulation of HSP90AA1 and RAN expression level would lead to the significant decrease of SEPT7 expression level.In summary,SEPT7 interacts with RAN and HSP90AA1 in the early embryonic development of cattle,while SEPT7 does not interact with ACTN1,HSPA1 A and RACK1,and RAN plays an important role in embryonic cleavage.In the preliminary study of the regulatory relationship among SEPT7,RAN and HSP90AA1 in early embryos,it was found that there was no significant difference in the expression level of SEPT7 when RAN was knocked down in zygotic embryos;Knockdown of HSP90AA1 significantly increased the expression level of SEPT7,while knockdown of RAN and HSP90AA1 significantly decreased the expression level of SEPT7.It indicates that there is no direct interaction between SEPT7,RAN and HSP90AA1,and there may be other proteins involved.This project is of great significance for improving the regulatory network of early bovine embryo development and explaining the mechanism of cytoplasmic skeleton abnormalities(including prokaryotic formation,spindle structure,division contraction ring structure and abnormal cleavage,etc.)in early bovine embryo development after nuclear transfer.