牦牛"海绵吸附"bta-miR-125b的lncRNA筛选验证及慢病毒载体构建OA北大核心CSTPCD

The aim of this study was to screen for lncRNAs that might adsorb bta-miR-125b during the development of yak follicles,and to construct dual luciferase vectors and lentivirus overexpression vectors,which might provide a basis for studying the function and mechanism of regulating lncRNAs related to yak reproduction.miRanda and RNAhybrid databases were first used to predict lncRNAs that might target yak bta miR-125b,and to select lncRNAs that bta miR-125b had sponge adsorption effect.RNAhybrid predicted binding sites and selected the optimal lncRNA.After collecting the isolated follicles from the ovaries of the yak and extracting total RNA,a cDNA pool was constructed to amplify and screen the obtained lncRNA.Based on this,pmir GLO vector construction technology was used to construct wild-type and mutant dual luciferase reporter vectors for lncRNA,and they were co-transfected with bta miR-125b mimics.Then,293T cells were stained and their dual luciferase activity was detected.Finally,using lentiviral vector construction technology,the correctly sequenced lncRNA was homologously recombined with the fragments of the homologous region of the pHBLV-CMV-MCS-EF1-ZsGreen-T2A uro vector.After pack-aging with the lentiviral packaging system(psPAX2:pMD2G),293T cells were infected,and their infection efficiency and titer were calcu-lated.As a result,it was found that lncRNA-ARS-UCD1.2 and bta-miR-125b in both the databases had tight binding sites.The full-length 1 552 bp sequence of lncRNA-ARS-UCD1.2 was amplified,and the detection of the dual luciferase activity showed that there was a targeting relationship between lncRNA-ARS-UCD1.2 and bta-miR-125b mimic in the yak(P＜0.01).After homologous recombination,enzyme digestion identification and sequencing verification showed that the recombinant plasmid was successfully constructed,with an infec-tion efficiency of(61.520±0.875)%.The infection titer measured by live cell titer calculation was 4×108 TU/mL,indicating that the ln-cRNA-ARS-UCD1.2 overexpressing lentiviral vector was successfully constructed.In summary,this experiment screened the lncRNAs of yak sponge adsorbed bta-miR-125b and verified their targeted binding relationship.At the same time,the overexpression lentiviral vector of this lncRNA was successfully constructed,laying a foundation for further in-depth research on the functional mechanism of lncRNA in cells.