靶向鸡Stra8基因的miRNA预测及鉴定

发布时间：2018-06-14 16:00

  本文选题：鸡 + miRNA　； 参考：《浙江农业学报》2017年05期

【摘要】：为克隆如皋黄鸡Stra8基因的3’UTR,寻找靶向Stra8基因的microRNA(miRNA),以鸡精原干细胞的c DNA为模板,根据NCBI数据库Stra8的CDS序列设计引物,通过3’RACE技术克隆Stra8基因的3’UTR,并构建相应的荧光素酶表达载体和突变载体;利用生物学预测软件对靶向Stra8 3’UTR的miRNA进行预测,选择评分最高的miRNA进行慢病毒载体构建;以pRL-TK为内参,分别将miRNA与Stra8 3’UTR荧光素酶表达载体和突变载体共转染DF-1细胞,利用双荧光素酶基因报告系统对miRNA进行活性检测。结果表明,采用3’RACE技术成功克隆Stra8基因的3’UTR;Targetscan生物在线软件预测到靶向Stra8基因的4个特异性较高的miRNA,并成功构建其相应的慢病毒载体;双荧光素酶活性检测结果显示,gga-miR-1a、gga-miR-31、ggamiR-218均可通过3’UTR序列抑制Stra8基因的表达,其中gga-miR-31抑制效果最佳。该结果可为后续深入探讨gga-miR-31介导调控的Stra8基因在雄性生殖细胞分化中的调控网络提供依据。
[Abstract]:In order to clone 3 UTRs of Stra8 gene from Rugao yellow chicken, the microRNAs and miRNAs targeting Stra8 gene were searched. Using the cDNA of spermatogonial stem cells as template, primers were designed according to the CDS sequence of Stra8 in NCBI database. The 3G UTRs of Stra8 gene were cloned by 3race technique, and the corresponding luciferase expression vector and mutant vector were constructed, and the miRNA targeting Stra83 UTR was predicted by biological prediction software, and the highest scoring miRNA was selected to construct the lentivirus vector. Using pRL-TK as an internal reference, miRNA and Stra83 UTR luciferase expression vector and mutant vector were cotransfected into DF-1 cells, and miRNA activity was detected by double luciferase gene report system. The results showed that the 3UTRT Targetscan Biosoftware of Stra8 gene was successfully used to predict four highly specific miRNAs targeting Stra8 gene and construct the corresponding lentivirus vector successfully. The results of double luciferase activity test showed that the expression of Stra8 gene could be inhibited by the sequence of 3G UTR, among which gga-miR-31 had the best inhibitory effect. These results may provide a basis for further study on the regulatory network of the Stra8 gene mediated by gga-miR-31 in male germ cell differentiation.
【作者单位】： 扬州大学动物科学技术学院江苏省动物遗传繁育与分子设计重点实验室;
【基金】：国家自然科学基金(31301959) 江苏省自然科学基金(BK20161331) 校大学生学术科技创新基金资助项目(x20160665) 江苏高校优势学科建设工程资助项目
【分类号】：S831.2
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本文编号：2018053