Construction Of Yeast Two-Hybrid CDNA Library Of Rat Testis And Preliminary Screening Of Proteins Potentially Interacting With JUNO

Fertilization, due to haploid sperm and egg combine to form a new genetically distinct diploid organisms, has a great significance to the species continuation and development. Sperm-egg fusion is crucial for fertilization, the process is complicated as a number of molecules are involved, but its molecular mechanism remains unknown. The study of protein interactions in the process of sperm-egg fusion is of great value to solve existing problems of the process. The interaction between IZUMO1-JUNO is one of the most important protein interactions of the sperm-egg fusion process, the lack of anyone of which were infertile. Therefore, we choose JUNO as the research protein for this study, screening proteins which interacted with it except for IZUM01.1、Construction of yeast two-hybrid cDNA library of Rat testisIn this study, we constructed Rat testis yeast two-hybrid library by using SMARTTM cDNA synthesis technology. The storage capacity of the library is 1.5×106 cfu, the titer is 8×107 cfu/mL. The recombination rate is 78%, and the average length of insert fragment is 0.7 kb. This has a great value for the protein interaction screening.2、The construction of pGBKT7-juno bait vectorpMD\9T-juno, used as a template, was cloned and saved in the laboratory and amplified after removing the signal peptide and connecting to pGBKT7 carrier. Identification, self-activation detection and cytotoxicity testing was then conducted. By using a carrier with c-Myc tagged antigen, western blot detected the expression of pGBKT7-juno bait vector in the yeast cells. Bioinformatics analysed several common mammalian’s JUNO amino acid sequences, drew phylogenetic tree, and analysed the kinship of the evolution.3、Screening proteins which have interactions with JUNO by Yeast two-hybrid systermMaking hybrid between rat testis yeast two-hybrid cDNA library and pGBKT7-juno bait vector, we screened out five specific genes(mkrnl, rps21, irgc, wdr54 and rnaseh2a), and analyzed them later respectively. In this study, we preliminary screened out proteins which have interactions with JUNO in egg, and those proteins provide candidate moleculars for the further research about sperm-egg fusion.