發(fā)布時(shí)間：2018-07-31 08:33

【摘要】：轉(zhuǎn)座子(Transposon, Tn),是在生物界廣泛分布的,能夠在基因組上自主復(fù)制和轉(zhuǎn)移的一段DNA序列。轉(zhuǎn)座子能夠有效介導(dǎo)基因轉(zhuǎn)移,在轉(zhuǎn)基因和基因捕獲等研究中具有很高的應(yīng)用價(jià)值。目前已經(jīng)分離到很多高活性DNA轉(zhuǎn)座子,如PiggyBac(PB)轉(zhuǎn)座子Sleeping beauty (SB)轉(zhuǎn)座子和Tol2轉(zhuǎn)座子等,并已經(jīng)在斑馬魚、小鼠、線蟲等模式生物的轉(zhuǎn)基因和基因捕獲等研究中得到廣泛應(yīng)用,但目前對(duì)PB、SB和Tol2轉(zhuǎn)座子在斑馬魚中的轉(zhuǎn)座特性仍然缺乏深入了解。本研究系統(tǒng)比較了目前研究應(yīng)用最為廣泛的PB、SB和Tol2三種DNA轉(zhuǎn)座子在斑馬魚中的基因轉(zhuǎn)移和基因捕獲效率差異,為轉(zhuǎn)座子在相關(guān)領(lǐng)域的研究和應(yīng)用提供參考。本實(shí)驗(yàn)主要包括：1、為比較PB、SB、Tol2轉(zhuǎn)座子的基因轉(zhuǎn)移效率差異,將含有鯉魚的beta-actin啟動(dòng)子(命名為FAG)和綠色熒光蛋白報(bào)告基因(Green Fluorecent Protein, GFP)表達(dá)盒(FAG-GFP)分別克隆至PB、SB、Tol2轉(zhuǎn)座子框架內(nèi),構(gòu)建成pPB-FAG-GFP、pSB-FAG-GFP、 pTo12-FAG-GFP轉(zhuǎn)基因載體。然后將3個(gè)載體分別與SB,PB及Tol2轉(zhuǎn)座酶mRNA按照5個(gè)質(zhì)量比20ng：10ng,20ng：30ng,20ng：50ng,20ng：80ng,20ng：100ng注射斑馬魚胚胎,獲得3個(gè)轉(zhuǎn)座子和轉(zhuǎn)座酶最佳注射質(zhì)量比例。其中SB和PB為20ng：50ng,Tol2為20ng：30ng。再以最佳注射比例注射斑馬魚胚胎,分別在48hpf和120hpf檢測的斑馬魚FO代胚胎的GFP陽性率,結(jié)果表明：48hpf和120hpf檢測的斑馬魚胚胎中,Tol2的GFP陽性分別為68%和68%(N=872),高于PB(66%和68%,N=885)和SB(64%和65%,N=897),但三者差異不顯著(P0.05)；FO代GFP陽性個(gè)體飼養(yǎng)至成年后與野生型個(gè)體交配獲得F1代,對(duì)F1代的GFP檢測表明：Tol2組的生殖系傳遞效率最高為46.07%(NF0=231),高于PB組的17.86%(NF0=212)和SB組的13.41%(NF0=199)。2、為比較PB、SB、Tol2轉(zhuǎn)座子的增強(qiáng)子捕獲效率差異,將含有斑馬魚krt4基因的基礎(chǔ)啟動(dòng)子和綠色熒光蛋白報(bào)告基因(GFP)的表達(dá)盒(Krt4-GFP)分別克隆至3個(gè)轉(zhuǎn)座子框架中,構(gòu)建成：pPB-Krt4-GFP、 pSB-Krt4-GFP、 pTo12-Krt4-GFP增強(qiáng)子捕獲載體。將3個(gè)載體分別與SB,PB及Tol2轉(zhuǎn)座酶mRNA,按照上文優(yōu)化的最佳比例注射斑馬魚胚胎,分別在48hpf和120hpf檢測斑馬魚F0代胚胎的GFP陽性率,結(jié)果表明：Tol2組增強(qiáng)子捕獲效率最高,48hpf和120hpf時(shí)分別為90.45%和92.71%(N=1524),顯著高于PB組(83.18%和83.32%,N=1276),Tol2組在48hpf高于SB組(88.64%,N=1378),但差異不顯著(P0.05),在120hpf時(shí)顯著高于SB組(88.87%,N=1378)(P0.05)。FO代GFP陽性個(gè)體飼養(yǎng)至成年后與野生型個(gè)體交配獲得F1代,對(duì)F1代的GFP檢測表明：Tol2組生殖系傳遞效率最高達(dá)到55.56%(NF0=165),高于PB組的32.56%(NF0=149)和SB組38.36%(NF0=151)；F1代斑馬魚表達(dá)模式分布分析表明：To12能產(chǎn)生最多的單個(gè)表達(dá)模式的后代,PB則能產(chǎn)生更多表達(dá)模式的后代,SB趨于兩者之間。3、為比較內(nèi)部核糖體插入位點(diǎn)(Internal Ribosome Entry Site, IRES)元件對(duì)轉(zhuǎn)座子介導(dǎo)的基因捕獲效率的影響,將IRES元件插入pTo12-RG-Trap基因捕獲載體的PolyA捕獲構(gòu)件的下游,構(gòu)建成pTo12-GTrap。以不含IRES元件的pTo12-RG-Trap基因捕獲載體為對(duì)照,分別將pTo12-GTrap和pTo12-RG-Trap兩個(gè)載體與Tol2轉(zhuǎn)座酶mRNA,注射斑馬魚胚胎,分別在48hpf和120hpf檢測斑馬魚FO代胚胎的GFP陽性率,結(jié)果表明：pTo12-GTrap組在48hpf和120hpf的胚胎GFP陽性率分別為74.93%和73.91%(N=986),顯著高于pTo12-RG-Trap組(41.86%和41.04%,N=853)(P0.05)表明IRES元件能夠提高基因捕獲效率。4、為比較PB、SB、Tol2轉(zhuǎn)座子的基因捕獲效率差異,以構(gòu)建成pTo12-GTrap為基礎(chǔ),分別用PB和SB轉(zhuǎn)座子框架替換Tol2框架,構(gòu)建成pPB-GTrap和pSB-GTrap.將3個(gè)載體分別與SB,PB及Tol2轉(zhuǎn)座酶mRNA,按照最優(yōu)比例注射斑馬魚胚胎,分別在48hpf和120hpf檢測斑馬魚F0代胚胎的GFP陽性率,結(jié)果表明：Tol2組基因捕獲效率最高(74.93%和73.91%,N=986),顯著高于SB組(66.07%和64.02%,N=1025)和PB組(54.23%和53.26%,N=976),且SB和PB之間差異也達(dá)到顯著水平(P0.05)。綜上所述,本研究表明：在斑馬魚中,TYol2轉(zhuǎn)座子的基因轉(zhuǎn)移效率、增強(qiáng)子捕獲效率、基因捕獲效率和生殖系傳遞效率最高,IRES元件可以增強(qiáng)轉(zhuǎn)座子介導(dǎo)的基因捕獲效率,但在增強(qiáng)子捕獲研究中,雖然Tol2轉(zhuǎn)座子能夠獲得更多的增強(qiáng)子捕獲個(gè)體,而PB能夠獲得較多的表達(dá)模式,在增強(qiáng)子捕獲研究中可能具有更高的應(yīng)用價(jià)值。
[Abstract]:Transposon (Tn), which is widely distributed in the biological community, is a DNA sequence that can be replicated and transferred autonomously in the genome. Transposon can effectively mediate gene transfer and have high application value in the research of transgenic and gene capture. Many highly active DNA transposons, such as PiggyBac (PB) transposon S, have been isolated. Leeping beauty (SB) transposons and Tol2 transposons have been widely used in the studies of transgenic and gene capture in zebrafish, mice, and nematodes, but the transposable characteristics of PB, SB and Tol2 transposons in zebrafish are still lacking in depth. The difference in gene transfer and gene capture efficiency of three DNA transposons of PB, SB and Tol2 in zebrafish can provide reference for the study and application of transposon in related fields. This experiment mainly includes: 1, the difference in gene transfer efficiency of PB, SB and Tol2 transposons, which will contain beta-actin promoter (named FAG) and green fluorescence of carp fish (named FAG) The protein reporter gene (Green Fluorecent Protein, GFP) expression box (FAG-GFP) was cloned into the PB, SB, Tol2 transposon framework and constructed into pPB-FAG-GFP, pSB-FAG-GFP, and pTo12-FAG-GFP transgenic carriers. Then, the 3 vectors were compared with SB, respectively, and transposable enzymes, respectively. The optimum injection mass ratio of 3 transposons and transposes was obtained by injection of 100ng in zebrafish embryos. SB and PB were 20ng:50ng, Tol2 was 20ng:30ng. and zebrafish embryos were injected with the optimum injection ratio. The GFP positive rate of the FO generation embryos of zebrafish detected in 48hpf and 120hpf, respectively, showed that 48hpf and 120hpf detected zebrafish embryos were found. In the fetus, the positive GFP of Tol2 were 68% and 68% (N=872), higher than PB (66% and 68%, N=885) and SB (64% and 65%, N=897), but the three were not significantly different (P0.05), and the FO generation GFP positive individuals were fed to the wild type and obtained the F1 generation. In group 17.86% (NF0=212) and 13.41% (NF0=199).2 of group SB, the enhancers' capture efficiency of PB, SB and Tol2 transposons was compared. The expression boxes (Krt4-GFP) containing the basic promoter of the zebrafish krt4 gene and the green fluorescent protein reporter gene (GFP) were cloned into 3 transposon frames, respectively. Krt4-GFP enhancer capture carrier. The 3 vectors were injected with SB, PB and Tol2 transposes mRNA respectively, and the zebrafish embryos were injected in the optimum proportion. The GFP positive rate of zebrafish F0 generation embryos was detected in 48hpf and 120hpf respectively. The results showed that the capture efficiency of the Tol2 group enhancers was the highest, and the 48hpf and 120hpf were 90.45% and 92.71% respectively. Significantly higher than group PB (83.18% and 83.32%, N=1276), group Tol2 was higher than SB group (88.64%, N=1378), but the difference was not significant (P0.05). At 120hpf, it was significantly higher than group SB (88.87%, N=1378) (P0.05). 55.56% (NF0=165) was higher than that of group PB (NF0=149) and group SB 38.36% (NF0=151). The distribution analysis of F1 generation zebrafish expression pattern showed that To12 could produce the most individual expression patterns, and PB could produce more expression patterns, SB tend to be.3, to compare the internal ribosome insertion site (Internal Ribosome). The effect of Site, IRES) on the gene capture efficiency mediated by transposon, the IRES element was inserted into the downstream of the PolyA capture component of the pTo12-RG-Trap gene capture carrier and constructed into a pTo12-GTrap. with the pTo12-RG-Trap gene capture carrier without IRES elements as the control, and the two carriers of pTo12-GTrap and pTo12-RG-Trap and Tol2 transposin mRNA were taken respectively. The positive rates of GFP in zebrafish embryos were detected by injecting zebrafish embryos at 48hpf and 120hpf respectively. The results showed that the positive rates of GFP in pTo12-GTrap group were 74.93% and 73.91% (N=986) in 48hpf and 120hpf, respectively, which were significantly higher than those of the pTo12-RG-Trap group (41.86% and 41.04%, N=853). The differences in gene capture efficiency of PB, SB and Tol2 transposons were compared. Based on the construction of pTo12-GTrap, Tol2 frames were replaced with PB and SB transposon frames respectively, and pPB-GTrap and pSB-GTrap. were constructed into 3 carriers with SB, PB and Tol2 transposes respectively. The GFP positive rate of the fetus showed that the gene capture efficiency of the Tol2 group was the highest (74.93% and 73.91%, N=986), which was significantly higher than the SB group (66.07% and 64.02%, N=1025) and PB group (54.23% and 53.26%, N=976), and the difference between SB and PB also reached a significant level (P0.05). Hadron capture efficiency, gene capture efficiency and reproductive system transfer efficiency are the highest. IRES components can enhance transposon mediated gene capture efficiency, but in the enhancers capture study, although Tol2 transposons can obtain more enhancers capture individuals, and PB can obtain more expression patterns, may be in the enhancer capture study. Higher application value.
【學(xué)位授予單位】：揚(yáng)州大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78

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