Seven synthesized shRNA (short hairpin ribonucleic acid) molecules for inhibiting expression of BMPR-1B (bone morphogenetic protein receptor-1B) genes

Abstract

The invention provides a synthesized shRNA molecule for inhibiting expression of BMPR-1B (bone morphogenetic protein receptor-1B) genes. According to sheep BMPR-1B genes (a NM_001009431.1 sequence) in a Gene Bank, a shRNA (short hairpin ribonucleic acid) molecule group interfering BMPR-1B gene transcription is designed by shRNA design software, 8 shRNA sequences aiming at different loca are screened from 34 designed shRNA molecules according to structures and positions and are synthesized; two synthesized complementary single-stranded shRNA molecules form double strands after being annealed and are connected to pLL3.7 slow virus interference vectors respectively to construct 8 slow virus interference RNA plasmids (pLL-BMPR-1B-shRNA), the interference RNA plasmids and BMPR-1B expression vectors produce toxin by a liposome transfection 293 T method, and 7 shRNA molecules which obviously inhibit the BMPR-1B genes are obtained after being detected and expressed by a fluorescence PCR (polymerase chain reaction) and a Western blot. The inhibition efficiency reaches more than 90 percent in an mRNA (messenger RNA) level, and the inhibition rate of BMPR-1B proteins reaches more than 80 percent.

Description

technical field [0001] The invention relates to the design, synthesis and evaluation method of the small interference RNA molecule capable of significantly inhibiting the bone morphogenic protein receptor-1B (BMPR-1B) gene and the inhibitory effect on oocytes. Background technique [0002] The BMPR-1B gene is a regulatory gene that increases ovulation rate and belongs to the TGF-? superfamily member, also known as ALK6 gene; A conserved intracellular kinase domain, nucleotide 830 (A830G), causes arginine to replace glutamine (Q249R); its dominant phenotype is early follicular development and multiple births. Literature search disclosure: (1) Biol.Reprod.64, 1225-1235 Author: Wilson, T. et al published the content of the article "A mutation of ALK6 gene expressed in oocytes and granulosa cells in Booroola sheep showing multiple fecundity traits" is to carry Fec B Homozygous ewes had an increased ovulation rate, accompanied by a large number of cystic follicles than Fec B+...

AI Technical Summary

Problems solved by technology

[0003] Regarding the study of BMPR-1B gene shRNA molecules, the mice with ALK6 gene interference did not show multiple fecundity traits like Booroola ewes, and the follicle development and ovulation rate were normal, but their cartilage formation was affected and skeletal muscle development was defective , indicating that there are species differences in ALK6 mutations, and no reports have been reported for other livestock species, such as sheep, goats, and pigs