牦牛“海绵吸附”bta-miR-125b的lncRNA筛选验证及慢病毒载体构建

Validation of lncRNA screening and lentiviral vector construction for“sponge adsorption”of bta-miR-125b in yak

旨在筛选在牦牛卵泡发育过程中可能吸附bta-miR-125b的长链非编码RNA(lncRNA),并构建双荧光素酶载体与慢病毒过表达载体,为研究调控牦牛生殖相关lncRNA的功能及机制提供基础。利用miRanda与RNAhybrid数据库预测可能靶向牦牛bta-miR-125b的lncRNAs,筛选出bta-miR-125b具有“海绵吸附”作用的lncRNAs,由RNAhybrid进行结合位点预测,筛选出最优的lncRNA;采集牦牛卵巢分离卵泡并提取总RNA构建cDNA池,以RT-PCR技术扩增筛选所得lncRNA;在此基础上利用pmir-GLO载体构建技术构建该lncRNA野生型及突变型双荧光素酶报告载体,并将其与bta-miR-125b mimics共转染至293T细胞,检测双荧光素酶活性;最后利用慢病毒载体构建技术将测序正确的lncRNA与pHBLV-CMV-MCS-EF1-ZsGreen-T2A-puro载体同源区的片段同源重组,通过慢病毒包装系统(psPAX2:pMD2G)包装后感染293T细胞,计算感染效率及滴度。结果:两个数据库中的lncRNA-ARS-UCD1.2与bta-miR-125b存在紧密结合位点,扩增出lncRNA-ARS-UCD1.2的序列全长为1552 bp;双荧光素酶活性检测表明牦牛lncRNA-ARS-UCD1.2与bta-miR-125b mimic具有靶向关系(P<0.01);同源重组后经酶切鉴定及测序验证表明成功构建了重组质粒,感染效率达(61.520±0.875)%,感染滴度为4×10^(8)TU/mL,表明lncRNA-ARS-UCD1.2过表达慢病毒载体成功构建。综上,本试验筛选出牦牛“海绵吸附”bta-miR-125b的lncRNA,并验证了两者的靶向结合关系,同时成功构建了该lncRNA的过表达慢病毒载体,为下一步深入研究lncRNA在细胞中的功能机制奠定了基础。

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 packaging with the lentiviral packaging system(psPAX2:pMD2G),293T cells were infected,and their infection efficiency and titer were calculated.As a result,it was found that lncRNA-ARS-UCD1.2 and bta-miR-125b in both the databases had tight binding sites.The fulllength 1552 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 infection efficiency of(61.520±0.875)%.The infection titer measured by live cell titer calculation was 4×10^(8) TU/mL,indicating that the lncRNA-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.