The Design And Verification Of ShRNA In Sheep BMPR-1B Gene

BMPs(Bone morphogenetic proteins) belongs to the TGF(transforming growth factor-β) superfamily, it plays an important role in both vertebrate and invertebrate skeletal and organ formation development, it participates development of animal embryos, the immune system, follicle and reproduction, and it plays a physiological function and inseparables from the bone morphogenetic protein receptor. Mutations in the coding region of the BMPR-1B gene (A746G)led to the249th amino acids mutated from glutamine to arginine of the receptor intracellular kinase signal region, it caused the receptor partial inactivation, and it influenced steroid production role in the reaction of the recognition of the ligands of GDF-5and BMP-4, which result to the granule cells of ewe carrying the Fec gene in accelerating differentiation, and which make the follicle mature faster, and it increases the number of ovuLation. This also means that the Q249R mutant inhibits the function of BMPR-1B gene, and decreases inhibition of the mutation in the BMPR-1B gene produced on the ovarian granulosa cell, so in the type of mutations sheep, a high number of ovulation will be caused.In order to obtain the effective shRNA molecules of interfering sheep BMPR-1B gene,1515bp was encoded of BMPR-1B gene cDNA sequence, which was implyfied by sheep ovaries and added the HA tag to this cDNA, then the cDNA with HA tag was inserted into the plex-mcs to build Plex-BMPR-1B lentiviral expression vector. The Plex-BMPR-1B lentiviral expression vector transfected HEK293cells, and cotransfected293T cells with two packaging plasmids to packag virus, and infect HEK293cells with recombinated lentivirus. In the same time,7shRNA molecules were recombinated with PLL-LentiLox3.7vector, and cotransfected293T cells with3packaging plasmids to packag virus, then recombinated lentivirus with shRNA molecules were obtained. At the same time, granulosa cells of the sheep and HEK293cells with Plex-BMPR-1B were infected by recombinated lentivirus, and qRT-PCR and Western blot were performed. The results showed:1、BMPR-1B gene was cloned1515bp in length, coding sequence of1509bp, encoding503amino acids, it according with the anticipate.2、The recombinated BMPR-IB protein was expressed in HEK293cells which was recombinated with Plex-BMPR-1B.3、Through two lines of cell mRNA detection, the most effective shRNA molecules are sh-BMPR-1B-1306and sh-BMPR-1B-1475, respectively99.86%and99.78%.4、Through two detected of qRT-PCR and Western blot,7shRNA molecules in this experiment could inhibit the expression level of sheep BMPR-1B gene by68.3%-99.86%. BMPR-1B-shRNA-1306and BMPR-1B-shRNA-1475shRNA molecules were the most effective shRNA molecules.PLL-BMPR-1B-1306and PLL-BMPR-1B-1475interference effect is better than others, interference efficiency were99.86%and99.78%, and the interference position in sheep BMPR-IB protein serine and threonine protein kinase catalytic domain and kinase region, There is very important signigicant of the location of the PLL-BMPR-1B-1306interference is as same region as Q294R amino acid. The research provides theoretical foundation for multiple births breeding and transgenic research, but it needs further study of the interference of BMPR-1B gene on upstream and downstream signaling molecules and the entire BMP/SMAD signaling pathways influence.