本文選題：豬　切入點：CLTC　出處：《浙江農(nóng)林大學(xué)學(xué)報》2017年03期

【摘要】：網(wǎng)格蛋白介導(dǎo)的胞吞是病毒侵入細(xì)胞的重要途徑,網(wǎng)格蛋白重鏈(clathrin heavy chain,CLTC)是形成網(wǎng)格蛋白小窩結(jié)構(gòu)的重要組成部分。針對CLTC基因的轉(zhuǎn)錄后調(diào)控特別是調(diào)控豬Sus scrofa CLTC的miRNA目前還不太清楚。本研究旨在篩選出調(diào)控豬CLTC基因的miRNA。利用生物信息學(xué)方法預(yù)測出6個靶向豬CLTC基因的miRNA,將豬CLTC基因3′UTR克隆至雙熒光素酶報告基因載體psi CHECK2中獲得雙熒光素酶報告基因重組載體psi CHECK2-CLTC-3′UTR。將預(yù)測得到的miRNA分別和重組載體psi CHECK2-CLTC-3′UTR共轉(zhuǎn)染到細(xì)胞中,以亂序序列作為陰性對照(NC),檢測miRNA對重組質(zhì)粒熒光素酶活性的影響。結(jié)果發(fā)現(xiàn)miR-205,miR-1,miR-129-5p和miR-206均能夠顯著抑制熒光素酶活性(P0.05)。在豬腎上皮細(xì)胞系PK15細(xì)胞中超表達miR-1和miR-129-5p后,定量PCR(q-PCR)結(jié)果顯示:豬CLTC基因的表達量顯著下調(diào)。突變了psi CHECK2-CLTC-3′UTR載體中這4個miRNA的種子序列的結(jié)合位點發(fā)現(xiàn):miR-1對突變質(zhì)粒中的熒光素酶無顯著抑制作用。表明miR-1與豬CLTC基因有直接的靶向關(guān)系,并通過其種子序列抑制CLTC基因的表達。
[Abstract]:Grid protein mediated endocytosis is an important pathway for virus invasion into cells, and grid protein heavy chain clathrin heavy chain1 (CLTCc) is an important component of the formation of grid protein nest structure.The posttranscriptional regulation of CLTC gene, especially the miRNA that regulates porcine Sus scrofa CLTC, is unclear.The aim of this study was to screen out the miRNA that regulates the porcine CLTC gene.Six miRNAs targeting porcine CLTC gene were predicted by bioinformatics. The porcine CLTC gene 3'UTR was cloned into double luciferase reporter gene vector psi CHECK2 to obtain double luciferase reporter gene recombinant vector psi CHECK2-CLTC-3 UTR.The predicted miRNA and the recombinant vector psi CHECK2-CLTC-3'UTR were cotransfected into the cells respectively, and the sequence sequence was used as negative control to detect the effect of miRNA on the luciferase activity of the recombinant plasmid.The results showed that both miR-205 miR-1 miR-129-5p and miR-206 could significantly inhibit luciferase activity (P0.05).After overexpression of miR-1 and miR-129-5p in porcine renal epithelial cell line PK15, the results of quantitative PCRQ-PCRR showed that the expression of porcine CLTC gene was significantly down-regulated.The binding sites of the four miRNA sequences in the psi CHECK2-CLTC-3'UTR vector were mutated. It was found that the luciferase in the mutant plasmid was not significantly inhibited by 1: miR-1.The results showed that miR-1 had direct targeting relationship with porcine CLTC gene, and the expression of CLTC gene was inhibited by its seed sequence.
【作者單位】： 浙江農(nóng)林大學(xué)動物科技學(xué)院;
【基金】：國家自然科學(xué)基金青年基金資助項目(31501921) 浙江省自然科學(xué)基金青年基金資助項目(LQ15C170001) 浙江農(nóng)林大學(xué)科研發(fā)展基金資助項目(2014FR068)
【分類號】：Q78
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本文編號：1694668