里氏木霉连续基因打靶系统的设计以及DNA修复相关基因的研究

发布时间：2018-05-12 09:07

本文选题：里氏木霉 + Cre/loxP　；参考：《华东理工大学》2016年博士论文

【摘要】：丝状真菌是一类在工业生产,农业加工,医药开发研究中有着重要价值的真核微生物。而里氏木霉则是其中一种主要用来生产纤维素酶的工业菌株。虽然2008年里氏木霉全基因组测序信息被解析,但是受困于丝状真菌分子生物学和遗传工程方法的落后,里氏木霉的研究在深度和广度以及研究的规模上都跟真菌模式菌株酵母有很大的差距。因此为了能够更透彻研究里氏木霉的重要功能基因,高产机理,蛋白分泌机制,生长代谢特质等,我们在后基因组时代更加迫切的需要高效的基因操作手段来深入挖掘里氏木霉的分子机制。对于工业菌株来说,基因组遗传性状的稳定性十分重要。但是细胞中的DNA时常面临到内源和外源的损伤危险。受到损伤的DNA能否精准而迅速的完成修复,对于基因组的完整性和稳定性都有着重要的意义。然而工业宿主里氏木霉关于DNA修复机制领域还有待深入研究。因此本课题主要由以下两部分研究内容组成：第一部分为高效可控的连续基因打靶新工具的构建。1、碳源诱导型筛选标记自切除系统LML2.0：基于Cre/loxP重组酶系统,将Cre表达盒以及筛选标记表达盒共同置于两个同向野生型loxP位点之间,通过碳源诱Cre酶表达一步法完成筛选标记去除的同时也没有Cre的残留。本研究创新性的使用了改造后的cre基因,将内源的基因片段或内含子序列融合进cre的编码序列中,而这样的结构并没有损失重组酶的切除反应效率,通过木糖碳源的诱导,我们获得了80%以上自切除效率。我们还比较了用来表达Cre重组酶的xynl诱导型启动子在不同碳源诱导下的重组效率。这些结果证明LML2.0系统可以在原核和真核中稳定存在,而且在木糖的诱导下可以产生稳定有效的筛选标记回收效果。2、位点突变型筛选标记自切除系统LML2.1：在LML2.0的基础上,将野生型loxP更换成突变型lox位点。分别选择了左臂突变型lox和右臂突变型lox各三种,两两组合成九种结构,其中自切除效率最高的是IoxJT15, loxJTZ17这对组合,能达到91%,其他的组合Cre识别效果都不理想。这说明突变了反向重复序列的lox位点影响了Cre的识别。我们也利用loxJT15和loxJTZ17组合产生的lox32进行了自切除的实验,发现几乎不能被Cre识别。因此我们认为loxJT15和loxJTZ17构建而成的LML2.1a可以高效回收筛选标记的同时不会造成遗留位点产生的基因组不稳定风险。3、以LML2.1a为筛选标记系统的连续基因打靶应用：我们用LML2.1a构建的打靶载体成功敲除了原始菌QM6a中的tku70基因,获得高效的同源重组效率。以QM6aAtku70缺陷株作为受体菌,使用相同的打靶载体对七个里氏木霉内源基因进行两到三轮的敲除。结果证明我们的打靶载体缺失可以高效的进行连续的基因打靶操作,而且每一轮的同源重组率和筛选标记的自切除效率都很稳定。另外我们还发现由于敲除转录因子Xyrl会导致xynl启动子转录受阻,致使Cre表达受到影响。4、光控诱导型筛选标记自切除系统LML3.0：用光作为诱导剂的打靶载体中筛选标记可以达到40-70%左右的切除效率,这就避免了前几代系统中碳源诱导性启动子对碳源的依赖。而且由于光诱导的机制和宿主代谢无关,因此我们的光控系统也摆脱了对宿主的依赖,可以成功应用到其他宿主中去,这一点在其他丝状真菌Neurospora crassa, Aspergillus niger和Metarhizium anisopliae中均有体现。这种光控的筛选标记自切除系统是丝状真菌可以利用的精确控制基因表达新工具。5、非同源末端连接途径(NHEJ)人工可控开关系统OFN1.0：我们利用不同方向的loxP位点构建了可以人工控制tku70基因翻转的表达盒,通过诱导Cre的表达完成基因的开关切换。根据TATA/ox以及loxN位置的不同构建了四种不同翻转效果的表达载体(OFN1.0A-D),通过对翻转不同的表达状态的转录水平分析,得出OFN1.0D关闭状态的严谨程度最好。而且我们从不同表达状态对紫外的敏感程度分析也看出,OFN1.0D可以有效的恢复tku70的表达。因此我们的开关系统的设计可以实现对基因精确可控的干预,在丝状真菌的表达调控上有着一定的应用潜力。第二部分为DNA修复相关基因功能的鉴定。1、发现蓝光可以诱导tku70基因以及其上下游未知基因tre78582和tre108087的转录水平在1-2个小时内提高2-3倍。并且通过分析这三个基因的启动子序列发现含有多个光控转录因子的结合位点。说明这三个基因可能作为一个功能基因簇共同对蓝光响应,受到蓝光的调控。2、通过序列比对以及敲除和回补实验,首次鉴定了tre78582就是里氏木霉亚铁螯合酶基因,我们将该基因命名为hem8。通过敲除实验发现了该基因为敲除致死型,经过了外加血红素更换培养等方法,完成了从异核体到同核体的筛选。通过改变培养基中的碳源说明外加的糖类碳源如葡萄糖和乳糖都容易使菌株的卟啉积累加重而导致敲除菌死亡。另外还利用敲除菌卟啉物质的积累检测其自发红色荧光的方法筛选hem8缺陷株,而由此也可将hem8基因开发成一种血红素营养缺陷型筛选标记,结合自发荧光的检测,这也提供了筛选出转化子的可视化的新方法。3、通过蛋白序列同源比对和系统进化树分析,我们鉴定tre108087编码的是一类新的Zn(Ⅱ2Cys6家族的蛋白,它的相关功能从未报道过。我们通过比较tre108087敲除菌,tku70敲除菌,双敲除菌以及回补菌对各类DNA损伤剂的敏感度变化,得出结论：tre108087基因编码的蛋白是一种DNA修复相关的蛋白,它可能与DNA合成或者拓扑异构酶Ⅰ发生相互作用,而且参与了DSB缺口的修复过程。这些结果也为日后更深入的研究里氏木霉的DNA修复机制奠定了基础。
[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.

【学位授予单位】：华东理工大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：Q78;Q93

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