Study Of Bovine IFNT Gene Function And Transcriptional Regulation By TEAD Family And YAP1Gene

The object if this study was to research the structure and function of bovine IFNT paralogous,further to research the structure and function of bovine IFNT protein. Additional, examined thepotential role of TEAD family and YAP1in controlling IFNT expression.Prosite data base was used to compare the amino acid sequence between IFNT1and IFNTc1protein. A casein kinase2(CK2) domain was found on IFNTc1amino acid sequence, but there is nothe CK2domain on its paralogous, IFNT1protein. In order to study the function of CK2domain,IFNT1and IFNTc1mutation plasmid were constructed, in which the CK2domain of IFNTc1wasdeleted; conversely, IFNT1has the CK2domain. Recombinant IFNT1and IFNTc1protein wereused to treatment the endometrial luminal epithelium, endometrial stromal cells and MDBK cells.Several classical ISGs’ Expression were detected by PCR analysis. The result indicated thatISG12、ISG15、Mx1gene expression in endometrial luminal epithelium, endometrial stromal cellsand MDBK cells can be induced by IFNT1and IFNTc1directly; Mx2gene expression inendometrial luminal epithelium can be induced by IFNTc1; The IFNT1mutation also can inducethe Mx2gene expression in endometrial luminal epithelium; but the IFNTc1mutation also caninduce the Mx2gene expression in endometrial luminal epithelium. These results indicated that theexpression of Mx2gene maybe regulated by CK2domain on IFNTc1protein directly, further studyis needed to clarify the mechanism by which the CK2domain regulate the Mx2gene expression.PCR analysis was performed to detect the expression of TEAD family and YAP1in bovineconcepus and organs. The result indicated that little TEAD2gene expressed in bovine concepusand organs; TEAD1, TEAD3, TEAD4and YAP1gene can be expressed commonly in bovineconcepus, heart, liver, spleen, kidney, muscle, skin, breast and placenta. Real-Time PCR analysiswas performed to detect the expression of TEAD family and YAP1in bovine concepus duringpre-implantation stage. The result indicated that TEAD family and YAP1expression weresignificantly up-regulated (P<0.05) after implantation.5’-deletion constructs representing various lengths of the upstream regions of bIFNT gene hadbeen prepared, and constructs with one point mutation at AP-1or CDX2or potential TEADbinding sites also had been prepared. These constructs were based on the pGL3Basic Vectorconsisting of a firefly luciferase (Luc) gene. Through the IFNT Luciferase reporter gene analysis,effect of TEAD family and YAP1on IFNT gene expression was detected in JEG3cells. The result indicated that TEAD1and TEAD3had no significant effect on IFNT gene expression; TEAD4canup-regulated the IFNT gene expression significantly (P<0.05) on-631bp-IFNTc1-Luciferasereporter; YAP1can up-regulated the IFNT gene expression significantly (P<0.05), and5’-deletioncannot limit this upregulation; there were no significant synergistic effect between TEAD familyand YAP1.This study worked on the the structure and function of bovine IFNT protein. Detecting theexpression of TEAD family and YAP1in bovine concepus and organs and effect of TEAD familyand YAP1on IFNT gene expression for the first time. In order to provide the challenge to increaseour knowledge of endometrial function and conceptus development. Further to improvereproductive efficiency by preventing embryonic losses that occur during the peri-implantationperiod of pregnancy in domestic ruminants.