本文選題：雞 + miRNA��； 參考：《浙江農(nóng)業(yè)學(xué)報(bào)》2017年05期

【摘要】：為克隆如皋黃雞Stra8基因的3’UTR,尋找靶向Stra8基因的microRNA(miRNA),以雞精原干細(xì)胞的c DNA為模板,根據(jù)NCBI數(shù)據(jù)庫Stra8的CDS序列設(shè)計(jì)引物,通過3’RACE技術(shù)克隆Stra8基因的3’UTR,并構(gòu)建相應(yīng)的熒光素酶表達(dá)載體和突變載體;利用生物學(xué)預(yù)測軟件對靶向Stra8 3’UTR的miRNA進(jìn)行預(yù)測,選擇評分最高的miRNA進(jìn)行慢病毒載體構(gòu)建;以pRL-TK為內(nèi)參,分別將miRNA與Stra8 3’UTR熒光素酶表達(dá)載體和突變載體共轉(zhuǎn)染DF-1細(xì)胞,利用雙熒光素酶基因報(bào)告系統(tǒng)對miRNA進(jìn)行活性檢測。結(jié)果表明,采用3’RACE技術(shù)成功克隆Stra8基因的3’UTR;Targetscan生物在線軟件預(yù)測到靶向Stra8基因的4個(gè)特異性較高的miRNA,并成功構(gòu)建其相應(yīng)的慢病毒載體;雙熒光素酶活性檢測結(jié)果顯示,gga-miR-1a、gga-miR-31、ggamiR-218均可通過3’UTR序列抑制Stra8基因的表達(dá),其中g(shù)ga-miR-31抑制效果最佳。該結(jié)果可為后續(xù)深入探討gga-miR-31介導(dǎo)調(diào)控的Stra8基因在雄性生殖細(xì)胞分化中的調(diào)控網(wǎng)絡(luò)提供依據(jù)。
[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.
【作者單位】： 揚(yáng)州大學(xué)動物科學(xué)技術(shù)學(xué)院江蘇省動物遺傳繁育與分子設(shè)計(jì)重點(diǎn)實(shí)驗(yàn)室;
【基金】：國家自然科學(xué)基金(31301959) 江蘇省自然科學(xué)基金(BK20161331) 校大學(xué)生學(xué)術(shù)科技創(chuàng)新基金資助項(xiàng)目(x20160665) 江蘇高校優(yōu)勢學(xué)科建設(shè)工程資助項(xiàng)目
【分類號】：S831.2
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本文編號：2018053