發(fā)布時間：2018-05-08 07:29

  本文選題：增強子捕獲 + 注解　； 參考：《生物技術通報》2017年05期

【摘要】：為了建立斑馬魚增強子捕獲突變體庫及研究功能基因的表達調控模式,制備了SB轉座子介導的增強子捕獲轉基因斑馬魚(F0),通過繁育建立了組織或器官特異表達報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GFP)與野生型TU系斑馬魚交配,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個發(fā)育階段檢測報告基因綠色熒光蛋白的表達模式;然后通過Splinkerette PCR(SP-PCR)方法檢測SB轉座子在斑馬魚基因組中的插入位置,從而確定捕獲的增強子和功能基因;最后通過胚胎原位雜交驗證內源基因表達模式。結果表明,在F2代胚胎不同發(fā)育階段,GFP表達具有明顯的時空特性,前期在前腦,中腦,后腦三個部位均高水平表達,后期表達部位呈后移趨勢,各個發(fā)育期表達強度基本無變化,結果提示該捕獲增強子具有腦部表達特性。sp-PCR結果分析表明增強子位于基因組1號染色體35、914、498-35、914、621位置,在內源性基因ednraa位點附近。原位雜交結果表明該基因在胚胎24 hpf階段具有轉錄活性。本研究結果提示SB轉座子介導的增強子捕獲技術可高效獲得插入突變斑馬魚,對研究基因功能和獲得具有自主知識產權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】： 揚州大學動物科學與技術學院揚州大學農業(yè)與農產品安全國際合作聯合實驗室;
【基金】：國家自然科學基金項目(31671313,31572364) 揚州現代農業(yè)項目(YZ2016040)
【分類號】：Q78

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本文編號：1860505