發(fā)布時(shí)間：2018-05-08 07:29

  本文選題：增強(qiáng)子捕獲 + 注解�。� 參考：《生物技術(shù)通報(bào)》2017年05期

【摘要】：為了建立斑馬魚(yú)增強(qiáng)子捕獲突變體庫(kù)及研究功能基因的表達(dá)調(diào)控模式,制備了SB轉(zhuǎn)座子介導(dǎo)的增強(qiáng)子捕獲轉(zhuǎn)基因斑馬魚(yú)(F0),通過(guò)繁育建立了組織或器官特異表達(dá)報(bào)告基因綠色熒光蛋白(Green fluorescent protein,GFP)的品系(F1)。選擇F1代的SK-3系(腦部特異表達(dá)GFP)與野生型TU系斑馬魚(yú)交配,收集受精卵(F2),于24 hpf(Hour post fertilization)、2 dpf(Day post fertilization)、3 dpf、4 dpf、5 dpf、7 dpf 6個(gè)發(fā)育階段檢測(cè)報(bào)告基因綠色熒光蛋白的表達(dá)模式;然后通過(guò)Splinkerette PCR(SP-PCR)方法檢測(cè)SB轉(zhuǎn)座子在斑馬魚(yú)基因組中的插入位置,從而確定捕獲的增強(qiáng)子和功能基因;最后通過(guò)胚胎原位雜交驗(yàn)證內(nèi)源基因表達(dá)模式。結(jié)果表明,在F2代胚胎不同發(fā)育階段,GFP表達(dá)具有明顯的時(shí)空特性,前期在前腦,中腦,后腦三個(gè)部位均高水平表達(dá),后期表達(dá)部位呈后移趨勢(shì),各個(gè)發(fā)育期表達(dá)強(qiáng)度基本無(wú)變化,結(jié)果提示該捕獲增強(qiáng)子具有腦部表達(dá)特性。sp-PCR結(jié)果分析表明增強(qiáng)子位于基因組1號(hào)染色體35、914、498-35、914、621位置,在內(nèi)源性基因ednraa位點(diǎn)附近。原位雜交結(jié)果表明該基因在胚胎24 hpf階段具有轉(zhuǎn)錄活性。本研究結(jié)果提示SB轉(zhuǎn)座子介導(dǎo)的增強(qiáng)子捕獲技術(shù)可高效獲得插入突變斑馬魚(yú),對(duì)研究基因功能和獲得具有自主知識(shí)產(chǎn)權(quán)的新基因具有重要作用。
[Abstract]:In order to establish the catch mutants library of zebrafish enhancer and to study the expression and regulation of functional genes, The SB transposon mediated enhancer was prepared to capture transgenic zebrafish F0, and the strain F1 was established by breeding the tissue or organ specific expression reporter gene Green fluorescent protein (GFP). F1 SK-3 lines (brain specific expression GFPs) were selected to mate with wild type TU zebrafish. The F _ 2s of fertilized eggs were collected, and the expression patterns of green fluorescent protein (GFP) in 6 developmental stages of the report gene were detected at 24 hpf(Hour post fertilization-2 dpf(Day post development stage. Then the insertion position of SB transposon in zebrafish genome was detected by Splinkerette PCR- SP-PCR method to determine the captured enhancer and functional gene, and the expression pattern of endogenous gene was verified by in situ hybridization. The results showed that the expression of GFP in F _ 2 embryos at different stages of development had obvious spatio-temporal characteristics. The expression of GFP was highly expressed in the forebrain, midbrain and hindbrain in the Prophase, and the tendency of the expression was backward in the later stage. There was no change in the expression intensity in all developmental stages. The results showed that the enhancer had the characteristics of brain expression. SP-PCR analysis showed that the enhancer was located in the position of chromosome 1, 35914498-35914621, and located near the ednraa site of endogenous gene. The results of in situ hybridization showed that the gene had transcriptional activity at 24 hpf stage of embryo. These results suggest that SB transposon mediated enhancer capture technique can efficiently obtain inserted mutant zebrafish and play an important role in studying gene function and obtaining new genes with independent intellectual property rights.
【作者單位】： 揚(yáng)州大學(xué)動(dòng)物科學(xué)與技術(shù)學(xué)院揚(yáng)州大學(xué)農(nóng)業(yè)與農(nóng)產(chǎn)品安全國(guó)際合作聯(lián)合實(shí)驗(yàn)室;
【基金】：國(guó)家自然科學(xué)基金項(xiàng)目(31671313,31572364) 揚(yáng)州現(xiàn)代農(nóng)業(yè)項(xiàng)目(YZ2016040)
【分類(lèi)號(hào)】：Q78

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本文編號(hào)：1860505