Preliminary Study On Molecular Mechanism Of CCP1 Interaction Protein TUBB4B In Mouse Testis Development

Objective:To investigate the expression and localization of TUBB4B protein and identify the molecular mechanism of TUBB4B interacting with CCP1 in the mouse testis development.Method:1.q PCR and Western blot methods were used to detect the expression of Tubb4b in pcd^3J?+/+?mice at m RNA and protein level,respectively.2.The expression of TUBB4B in the sperm of pcd^3J?+/+?mice was detected by immunofluorescence staining.3.Immunohistochemical method was used to detect the expression of TUBB4B in wildtype mouse testis and pcd^3J?-/-?mouse testis.4.The expression of Tubb4b in mouse sertoli cells?15P-1 cells?,leydig cells?TM3 cells?,and type B spermatogonia?GC1 cells?was detected by PCR.5.The expression of TUBB4B in the testes of pcd^3J?-/-?mice and wild-type mice of P15,P18,P30,P35 was detected by Western blot.6.The polyglutamylation?poly E?level of tubulin was detected by CCP1 protein using Western blot.7.Immunohistochemical method was used to detect the difference of?-Tubulin and?-Actin protein in the testis of pcd^3J?+/+?mice and pcd^3J?-/-?mice.8.The q PCR and Western blot methods were used to detect the difference in expression of TUBB4B in the CCP1 knock-down GC1?GR?cells and negative control?GY?cells at m RNA and protein level.9.Differential expressed proteins of GR cells and GY cells was identified and analyzed by i TRAQ LC-MS/MS.Results:1.The q PCR experiment results showed that the Tubb4b gene was widely expressed in most tissues of pcd^3J?+/+?mice,especially highly expressed in the testis.Western blot test results showed that the TUBB4B protein was also highly expressed in the testis.2.Immunofluorescence staining of spermatozoa and epididymis from mature Balb/c mice showed that TUBB4B was expressed in the sperm head and tail,especially in the mitochondrial sheath of the sperm head and midsection.3.Immunohistochemical results showed that TUBB4B was expressed in the sertoli cells and spermatogenic cells of pcd^3J?-/-?mice and wildtype mice.The expression pattern was also confirmed by detection in mouse testis related TM3,15P-1,and GC-1 cells.This result was consistent with the expression results in mouse testis.4.The expression of TUBB4B in the testis of wildtype mice gradually increases with age.The expression of TUBB4B decreased at P18 in pcd^3J?-/-?mice,and increased at P30Compared with pcd^3J?+/+?mice,the expression of TUBB4B in pcd^3J?-/-?mouse testis was down-regulated from P18.5.Western blot experiment results showed that the expression of TUBB4B was increased and poly E level of proteins were decreased in the p EGFP-CCP1 transfected HEK293T cells.6.The abnormal cytoskeletal structure was observed in the testis of pcd^3J?-/-?mouse.7.There was no difference in the expression of TUBB4B in GR cells and GY cells.8.i TRAQ quantitative proteome analysis showed that 59 proteins were differentially expressed,of which PFN1 protein and CORO2Bprotein are related to microtubules and actin.Conclusion1.TUBB4B is expressed in mouse testis and sperm.2.In pcd^3J?-/-?mouse testis,TUBB4B expression is abnormally decreased from P18?late spermatocyte stage?,which is related to the decrease of germ cells in pcd^3J?-/-?mouse testis.3.TUBB4B depolyglutamylation can be performed by CCP1 protein.4.The abnormal cytoskeletal structure of testis of pcd^3J?-/-?mice has been showed.5.The PFN1 and CORO2B protein in GR cells are related to microtubules and actin and may play function in the maintenance of cytoskeleton in mouse testis.The specific molecular function needs to be explored further.