本文選題：里氏木霉 + Cre/loxP�。� 參考：《華東理工大學(xué)》2016年博士論文

【摘要】：絲狀真菌是一類在工業(yè)生產(chǎn),農(nóng)業(yè)加工,醫(yī)藥開(kāi)發(fā)研究中有著重要價(jià)值的真核微生物。而里氏木霉則是其中一種主要用來(lái)生產(chǎn)纖維素酶的工業(yè)菌株。雖然2008年里氏木霉全基因組測(cè)序信息被解析,但是受困于絲狀真菌分子生物學(xué)和遺傳工程方法的落后,里氏木霉的研究在深度和廣度以及研究的規(guī)模上都跟真菌模式菌株酵母有很大的差距。因此為了能夠更透徹研究里氏木霉的重要功能基因,高產(chǎn)機(jī)理,蛋白分泌機(jī)制,生長(zhǎng)代謝特質(zhì)等,我們?cè)诤蠡蚪M時(shí)代更加迫切的需要高效的基因操作手段來(lái)深入挖掘里氏木霉的分子機(jī)制。對(duì)于工業(yè)菌株來(lái)說(shuō),基因組遺傳性狀的穩(wěn)定性十分重要。但是細(xì)胞中的DNA時(shí)常面臨到內(nèi)源和外源的損傷危險(xiǎn)。受到損傷的DNA能否精準(zhǔn)而迅速的完成修復(fù),對(duì)于基因組的完整性和穩(wěn)定性都有著重要的意義。然而工業(yè)宿主里氏木霉關(guān)于DNA修復(fù)機(jī)制領(lǐng)域還有待深入研究。因此本課題主要由以下兩部分研究?jī)?nèi)容組成：第一部分為高效可控的連續(xù)基因打靶新工具的構(gòu)建。1、碳源誘導(dǎo)型篩選標(biāo)記自切除系統(tǒng)LML2.0：基于Cre/loxP重組酶系統(tǒng),將Cre表達(dá)盒以及篩選標(biāo)記表達(dá)盒共同置于兩個(gè)同向野生型loxP位點(diǎn)之間,通過(guò)碳源誘Cre酶表達(dá)一步法完成篩選標(biāo)記去除的同時(shí)也沒(méi)有Cre的殘留。本研究創(chuàng)新性的使用了改造后的cre基因,將內(nèi)源的基因片段或內(nèi)含子序列融合進(jìn)cre的編碼序列中,而這樣的結(jié)構(gòu)并沒(méi)有損失重組酶的切除反應(yīng)效率,通過(guò)木糖碳源的誘導(dǎo),我們獲得了80%以上自切除效率。我們還比較了用來(lái)表達(dá)Cre重組酶的xynl誘導(dǎo)型啟動(dòng)子在不同碳源誘導(dǎo)下的重組效率。這些結(jié)果證明LML2.0系統(tǒng)可以在原核和真核中穩(wěn)定存在,而且在木糖的誘導(dǎo)下可以產(chǎn)生穩(wěn)定有效的篩選標(biāo)記回收效果。2、位點(diǎn)突變型篩選標(biāo)記自切除系統(tǒng)LML2.1：在LML2.0的基礎(chǔ)上,將野生型loxP更換成突變型lox位點(diǎn)。分別選擇了左臂突變型lox和右臂突變型lox各三種,兩兩組合成九種結(jié)構(gòu),其中自切除效率最高的是IoxJT15, loxJTZ17這對(duì)組合,能達(dá)到91%,其他的組合Cre識(shí)別效果都不理想。這說(shuō)明突變了反向重復(fù)序列的lox位點(diǎn)影響了Cre的識(shí)別。我們也利用loxJT15和loxJTZ17組合產(chǎn)生的lox32進(jìn)行了自切除的實(shí)驗(yàn),發(fā)現(xiàn)幾乎不能被Cre識(shí)別。因此我們認(rèn)為loxJT15和loxJTZ17構(gòu)建而成的LML2.1a可以高效回收篩選標(biāo)記的同時(shí)不會(huì)造成遺留位點(diǎn)產(chǎn)生的基因組不穩(wěn)定風(fēng)險(xiǎn)。3、以LML2.1a為篩選標(biāo)記系統(tǒng)的連續(xù)基因打靶應(yīng)用：我們用LML2.1a構(gòu)建的打靶載體成功敲除了原始菌QM6a中的tku70基因,獲得高效的同源重組效率。以QM6aAtku70缺陷株作為受體菌,使用相同的打靶載體對(duì)七個(gè)里氏木霉內(nèi)源基因進(jìn)行兩到三輪的敲除。結(jié)果證明我們的打靶載體缺失可以高效的進(jìn)行連續(xù)的基因打靶操作,而且每一輪的同源重組率和篩選標(biāo)記的自切除效率都很穩(wěn)定。另外我們還發(fā)現(xiàn)由于敲除轉(zhuǎn)錄因子X(jué)yrl會(huì)導(dǎo)致xynl啟動(dòng)子轉(zhuǎn)錄受阻,致使Cre表達(dá)受到影響。4、光控誘導(dǎo)型篩選標(biāo)記自切除系統(tǒng)LML3.0：用光作為誘導(dǎo)劑的打靶載體中篩選標(biāo)記可以達(dá)到40-70%左右的切除效率,這就避免了前幾代系統(tǒng)中碳源誘導(dǎo)性啟動(dòng)子對(duì)碳源的依賴。而且由于光誘導(dǎo)的機(jī)制和宿主代謝無(wú)關(guān),因此我們的光控系統(tǒng)也擺脫了對(duì)宿主的依賴,可以成功應(yīng)用到其他宿主中去,這一點(diǎn)在其他絲狀真菌Neurospora crassa, Aspergillus niger和Metarhizium anisopliae中均有體現(xiàn)。這種光控的篩選標(biāo)記自切除系統(tǒng)是絲狀真菌可以利用的精確控制基因表達(dá)新工具。5、非同源末端連接途徑(NHEJ)人工可控開(kāi)關(guān)系統(tǒng)OFN1.0：我們利用不同方向的loxP位點(diǎn)構(gòu)建了可以人工控制tku70基因翻轉(zhuǎn)的表達(dá)盒,通過(guò)誘導(dǎo)Cre的表達(dá)完成基因的開(kāi)關(guān)切換。根據(jù)TATA/ox以及l(fā)oxN位置的不同構(gòu)建了四種不同翻轉(zhuǎn)效果的表達(dá)載體(OFN1.0A-D),通過(guò)對(duì)翻轉(zhuǎn)不同的表達(dá)狀態(tài)的轉(zhuǎn)錄水平分析,得出OFN1.0D關(guān)閉狀態(tài)的嚴(yán)謹(jǐn)程度最好。而且我們從不同表達(dá)狀態(tài)對(duì)紫外的敏感程度分析也看出,OFN1.0D可以有效的恢復(fù)tku70的表達(dá)。因此我們的開(kāi)關(guān)系統(tǒng)的設(shè)計(jì)可以實(shí)現(xiàn)對(duì)基因精確可控的干預(yù),在絲狀真菌的表達(dá)調(diào)控上有著一定的應(yīng)用潛力。第二部分為DNA修復(fù)相關(guān)基因功能的鑒定。1、發(fā)現(xiàn)藍(lán)光可以誘導(dǎo)tku70基因以及其上下游未知基因tre78582和tre108087的轉(zhuǎn)錄水平在1-2個(gè)小時(shí)內(nèi)提高2-3倍。并且通過(guò)分析這三個(gè)基因的啟動(dòng)子序列發(fā)現(xiàn)含有多個(gè)光控轉(zhuǎn)錄因子的結(jié)合位點(diǎn)。說(shuō)明這三個(gè)基因可能作為一個(gè)功能基因簇共同對(duì)藍(lán)光響應(yīng),受到藍(lán)光的調(diào)控。2、通過(guò)序列比對(duì)以及敲除和回補(bǔ)實(shí)驗(yàn),首次鑒定了tre78582就是里氏木霉亞鐵螯合酶基因,我們將該基因命名為hem8。通過(guò)敲除實(shí)驗(yàn)發(fā)現(xiàn)了該基因?yàn)榍贸滤佬?經(jīng)過(guò)了外加血紅素更換培養(yǎng)等方法,完成了從異核體到同核體的篩選。通過(guò)改變培養(yǎng)基中的碳源說(shuō)明外加的糖類碳源如葡萄糖和乳糖都容易使菌株的卟啉積累加重而導(dǎo)致敲除菌死亡。另外還利用敲除菌卟啉物質(zhì)的積累檢測(cè)其自發(fā)紅色熒光的方法篩選hem8缺陷株,而由此也可將hem8基因開(kāi)發(fā)成一種血紅素營(yíng)養(yǎng)缺陷型篩選標(biāo)記,結(jié)合自發(fā)熒光的檢測(cè),這也提供了篩選出轉(zhuǎn)化子的可視化的新方法。3、通過(guò)蛋白序列同源比對(duì)和系統(tǒng)進(jìn)化樹(shù)分析,我們鑒定tre108087編碼的是一類新的Zn(Ⅱ2Cys6家族的蛋白,它的相關(guān)功能從未報(bào)道過(guò)。我們通過(guò)比較tre108087敲除菌,tku70敲除菌,雙敲除菌以及回補(bǔ)菌對(duì)各類DNA損傷劑的敏感度變化,得出結(jié)論：tre108087基因編碼的蛋白是一種DNA修復(fù)相關(guān)的蛋白,它可能與DNA合成或者拓?fù)洚悩?gòu)酶Ⅰ發(fā)生相互作用,而且參與了DSB缺口的修復(fù)過(guò)程。這些結(jié)果也為日后更深入的研究里氏木霉的DNA修復(fù)機(jī)制奠定了基礎(chǔ)。
[Abstract]:Filamentous fungi are an important class of eukaryotic microbes which have important value in industrial production, agricultural processing, and pharmaceutical development. And Trichoderma Richter is one of the main industrial strains used to produce cellulase. Although the whole genome sequencing information of Trichoderma Richter was analyzed in 2008, it was trapped in the molecular biology and heredity of filamentous fungi. The lag of engineering methods, the study of Trichoderma Richter has a great gap with fungal yeast strain yeast in depth and scope and the scale of research. Therefore, in order to be able to study the important functional genes of Trichoderma Richter, the mechanism of high yield, the mechanism of protein secretion, and the characteristics of growth and metabolism, we are more urgent in the post genome era. Efficient genetic manipulation is needed to dig into the molecular mechanism of Trichoderma Richter. For industrial strains, the stability of genetic traits is very important. But the DNA in the cells often faces the risk of endogenous and exogenous damage. The damaged DNA can accurately and quickly complete the repair, the integrity of the genome and the integrity of the genome. Stability has important significance. However, the field of DNA repair mechanism of Trichoderma Richter in industrial host remains to be studied. Therefore, this topic is mainly composed of the following two parts: the first part is the construction of a highly efficient and controllable new tool for continuous gene targeting,.1, carbon source induced screening marker self excision system LML2.0: The Cre/loxP recombinant enzyme system combines the Cre expression box and the screening marker box to two identical wild type loxP loci, which can be removed by the carbon source lure Cre enzyme expression one step method and no Cre residues. This study innovatively used the modified CRE gene to put the endogenous gene fragment or intron sequence into the intron sequence. The columns were fused into the coding sequence of CRE, and this structure did not lose the efficiency of the resected reaction of the recombinant enzyme. We obtained more than 80% excision efficiency through the induction of xylose carbon source. We also compared the recombination efficiency of the xynl inducible promoter used to express the Cre recombinant enzyme in different carbon sources. These results prove that LML2.0 The system can be stable in the prokaryotic and eukaryotes, and can produce a stable and effective screening marker recovery effect.2 under the induction of xylose. The loci mutation screening marker self excision system LML2.1: on the basis of LML2.0, the wild type loxP is replaced by the mutant lox site. The left arm mutant LOX and the right arm mutant LOX are selected. Three and 22 are combined into nine structures, of which the highest self removal efficiency is IoxJT15, and the combination of loxJTZ17 can reach 91%, and the other combination Cre recognition results are not ideal. This shows that the mutation of the LOX loci of the reverse repeat sequence affects the identification of Cre. We also use the lox32 produced by the combination of loxJT15 and loxJTZ17 to self excision. The experiment found that it was almost impossible to be identified by Cre. Therefore, we think that the loxJT15 and loxJTZ17 constructed LML2.1a can efficiently recover the screening markers and do not cause the genomic instability risk.3 produced by the remnants, and the LML2.1a is the continuous gene targeting of the screening marker system: we use the targeting vector constructed by LML2.1a to form the target vector. In addition to the tku70 gene in the primordial strain QM6a, the effective homologous recombination efficiency was obtained. The QM6aAtku70 deficient strain was used as the receptor bacteria and the same target carrier was used to knock out the two to three rounds of the seven Trichoderma rickeus endogenous genes. The results showed that the missing target carrier was able to perform a continuous gene targeting operation with high efficiency. The homologous recombination rate of each round and the self removal efficiency of the screening markers are stable. In addition, we have found that the Cre expression is affected by the hindered transcription factor Xyrl that causes the transcription of xynl promoter, and the optically induced screening marker self excision system LML3.0: the screening marker in the target carrier with light as an inducer can be reached. The removal efficiency around 40-70%, which avoids the dependence of carbon source inducible promoters on the carbon source in the previous generations, and because the light induced mechanism is independent of the host metabolism, so our optical control system is also free from dependence on the host and can be successfully applied to other hosts, this point in the other filamentous fungi Neurospora C Rassa, Aspergillus Niger and Metarhizium anisopliae are all embodied. This optically controlled screening marker self excision system is a new tool for the precise control of gene expression,.5, the non homologous terminal connection pathway (NHEJ) artificial controlled switch system OFN1.0, which we can use in different directions. The expression box that controls the tku70 gene reversal is switched by inducing the expression of Cre. Four expression vectors (OFN1.0A-D) are constructed according to the different positions of TATA/ox and loxN. By analyzing the transcriptional level of the overturned state of expression, it is concluded that the severity of OFN1.0D closure is the best. From the analysis of the sensitivity of the different expression states to the UV, we can see that OFN1.0D can effectively restore the expression of tku70. Therefore, the design of our switch system can achieve precise and controllable gene intervention, and has some potential application potential in the regulation of the expression of filamentous fungi. The second part is a reference for the function of DNA repair related genes. It was found that blue light could induce the transcription of the tku70 gene and its upstream and downstream unknown genes, tre78582 and tre108087, to increase by 2-3 times within 1-2 hours. And the binding sites containing multiple light controlled transcription factors were found by analyzing the promoter sequences of the three genes. These three genes may be used as a functional gene cluster. In response to blue light,.2 is regulated by blue light. Through sequence alignment and knockout and supplementing experiments, tre78582 is the first identification of the ferrochelase gene of Trichoderma REI. We named the gene hem8. by knockout experiments to find the gene as a knockout death type and through the replacement of heme. The screening of the isosome to the nucleosome. By changing the carbon source in the medium, the added sugar carbon source, such as glucose and lactose, can easily aggravate the porphyrin accumulation of the strain and lead to the death of the bacteria. In addition, the hem8 defect strain is screened by the accumulation of knockout porphyrin substance to detect its spontaneous red fluorescence, and thus the hem8 can also be used. The gene is developed into a heme nutritional deficiency type screening marker, combined with the detection of spontaneous fluorescence, which provides a new method for screening the visualization of the transformants,.3. Through the homologous sequence of protein sequences and phylogenetic tree analysis, we identify that tre108087 is a new class of Zn (II 2Cys6 family protein, its related functions have never been found. " It was reported that by comparing tre108087 knockout bacteria, tku70 knockout bacteria, double knockout bacteria and remedial bacteria, we concluded that the protein encoded by the tre108087 gene is a DNA repair related protein, which may interact with DNA synthesis or topologic isomeric enzyme I and participate in the DSB gap. These results also lay a foundation for further research on the DNA repair mechanism of Trichoderma rehmyii in the future.

【學(xué)位授予單位】：華東理工大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：Q78;Q93

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相關(guān)期刊論文 前2條

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本文編號(hào)：1878001