Targeted verification of the relationship between Guanling cattle MSTN gene 3'-UTR and bta-miR-27b

【Objective】This paper clarified the relationship between the binding site and targeted regulation between bta-miR-27b and myostatin gene（MSTN）in Guanling cattle， to provide a theoretical basis for improving the meat production of Guanling cattle by regulating miRNA， and a new entry point to molecular genetic improvement of local cattles in Guizhou.【Method】The experiment used TargetScan to predict the possible miRNA binding region and potential interaction miRNA at the 3'-UTR of the bovine MSTN gene， and compared the physiological phenotype， expression differences of MSTN gene and its 3'-UTR end miRNA between Guanling cattle and different Guanling cattle hybrids， and then selected bta-miR-27b to analyze its target site specificity in different Guanling cattle hybrid populations. After that， the MSTN gene 3'-UTR sequence wild type（WT）and the 3'-UTR sequence mutant type（MUT）were cloned into a pMIR-REPORT Luciferase（H306）vector to form a dual-luciferase reporter gene vector，which co-transfected with bta-miR-27b in 293T cells to verify the relationship between bta-miR-27b and MSTN gene expression. And the effects of bta-miR-27b on MSTN gene expression were confirmed by real-time fluorescence quantitative PCR.【Result】Results showed that seven potential miRNA binding sites were found at the 3'-UTR end of MSTN gene. The relative expression levels of miRNAs（bta-miR-27a-3p， bta-miR-27b and bta-miR-128）of Guanling cattle hybrid breeds were extremely significantly higher than that of Guanling cattle（P＜0.01，the same below），and the relative expression level of MSTN gene was extremely significantly lower than that of Guanling cattle. But their body weight，body height，body diagonal length，chest circumference and other growth indicators were greatly better than that of Guanling cattle. There were no mutations or losses in the bta-miR-27b binding region at 3'-UTR end of MSTN gene in Guanling cattle and its hybrid cattle. The fluorescence value of 293T cells co-transfected with MSTN-3'-UTR（WT）and bta-miR-27b mimics decreased by 53.80%，which was extremely significantly lower than that of MSTN-3'-UTR（MUT）+bta-miR-27b mimics co-transfection. After transfection with bta-miR-27b mimics， bta-miR-27b was successfully overexpressed in Guanling cattle primary myoblast cell line（N2），while the relative expression of MSTN gene showed extremely significant downward trend，indicating that bta-miR-27b could inhibit MSTN gene expression.【Conclusion】The binding region of bta-miR-27b and MSTN gene is located at the 62-69 base of 3'-UTR， and the target site is highly conserved. Bta-miR-27b has a strong target regulation effect on MSTN gene，indicating by bta-miR-27b can inhibit MSTN gene expression.