粗糙脉孢菌组成性毒素合成基因簇的初步研究

发布时间：2018-05-28 00:31

本文选题：粗糙脉孢菌 + 组成性毒素合成酶　；参考：《江苏师范大学》2017年硕士论文

【摘要】：粗糙脉孢菌(Neurospora crassa)是脉孢菌属的一种多细胞丝状真菌,其菌丝透明,并有分隔和分枝,菌丝疏松,呈网状,能产生桔色分生孢子。粗糙脉孢菌生长快,易培养,遗传背景清晰,所以是实验室普遍使用的模式真菌。前期研究发现,粗糙脉孢菌通过合成组成性毒素(constitutive toxins,CT)来抵御昆虫,并形成特定的亚细胞结构储存组成性毒素,避免对自身细胞造成伤害。利用antiSMASH预测,粗糙脉孢菌中合成组成性毒素的基因PKS5(polyketide synthase gene,PKS5)和编码短链脱氢酶还原酶(DH)、转运蛋白超家族(MFS-1,MFS-2)、细胞色素450(CYP-1,CYP450-2)、转录因子(TF)、硫酸根转运蛋白(SULT)等9个基因共同构成了组成性毒素合成基因簇(CTNC)。CYP450是真菌体内含量最多、功能最多的活性蛋白酶,能够降解外源毒物以及自身有毒次级代谢产物。MFS参与调控,能够将菌体产生的毒素排到细胞外发生作用。SULT将硫酸根转运到胞内发生同化反应,合成半胱氨酸,参与电位的调节。转录因子在细胞基因表达过程中起着双重调控作用:1.对丝状真菌次级代谢产物进行调节;2.对不利于自身的外来活性氧自由基信号传导作出反应。在本论文中,开展了以下研究:1.以粗糙脉孢菌Ku70突变株为出发菌株,将CTNC基因簇中的10个基因分别敲除,获得10个突变菌株,通过薄层层析(TLC)检测代谢产物变化情况。2.克隆基因PKS5并将其与pYH-WA-pyrG-KI载体连接,整合到模式真菌构巢曲霉的WA位点,通过PCR技术筛选突变菌株,并对其次级代谢产物进行检测。3.以cDNA为模板克隆TF基因,并将其与pETMALc-H载体连接,转化大肠杆菌蛋白表达菌株BL21,诱导蛋白表达,获得相应蛋白。用获得的蛋白作为抗原注射家兔,从家兔的血清中获得抗体。4.以粗糙脉孢菌Ku70突变株为出发菌株,对其TF基因进行原位超强表达,提取蛋白,通过Western Blot检测TF基因的蛋白表达水平,TLC检测次级代谢产物合成水平。结果显示,粗糙脉孢菌的10株突变菌株中有4株颜色变为白色且不产生孢子,说明被敲除基因与孢子的形成有密切联系。通过TLC检测得出,突变菌株次级代谢产物数量减少。粗糙脉孢菌的PKS5基因整合到构巢曲霉基因组WA位点后成功表达,TLC和HPLC检测结果显示,突变型构巢曲霉的次级代谢产物中有3种新物质产生。大肠杆菌BL21成功表达出转录因子,并通过注射家兔获得相应抗体。提取TF超强表达菌株蛋白,Western Blot结果显示TF超强表达菌株中TF基因的表达水平显著上调,TLC检测显示次级代谢产物的生成量显著增加。
[Abstract]:Neurospora crassault, a multicellular filamentous fungus of the genus Cephalosporium, has transparent hyphae, separated and branched, loose hyphae, reticulate, and can produce orange color conidium. C. crassifolia is a model fungus widely used in laboratory because of its rapid growth, easy culture and clear genetic background. Previous studies have shown that C. crassifera protects against insects by synthesizing a constitutive toxin, and forms specific subcellular structures to store constitutive toxins, thus avoiding damage to its own cells. Using antiSMASH prediction, Nine genes, including PKS5(polyketide synthase gene PKS5), encoding short chain dehydrogenase reductase (DHH), transporter superfamily MFS-1 (MFS-2), cytochrome 450 (CYP-1) CYP450-2, transcriptional factor (TFN), sulfate radical transporter (SULT), etc. The toxin synthase gene cluster CTNCU. CYP450 was the most abundant in fungi. The most active protease can degrade exogenous poison and its own toxic secondary metabolites. MFS is involved in the regulation, and the toxin produced by bacteria can be discharged into the cell to produce extracellular effects. SULT can transport sulfate to the cells to produce assimilation reaction. Synthesis of cysteine, involved in the regulation of potential. Transcription factors play a dual role in regulating cell gene expression: 1. The secondary metabolites of filamentous fungi were regulated. The signal transduction of extraneous active oxygen species is not favorable to itself. In this paper, the following research is carried out: 1. The 10 genes in the CTNC gene cluster were knocked out and 10 mutant strains were obtained. The metabolites were detected by TLC. The gene PKS5 was cloned and ligated with pYH-WA-pyrG-KI vector and integrated into WA site of Aspergillus nestis. The mutant strain was screened by PCR and its secondary metabolites were detected. TF gene was cloned by using cDNA as template and ligated with pETMALc-H vector. The protein expression strain BL21 was transformed into E. coli protein expression strain BL21. The protein expression was induced and the corresponding protein was obtained. The antibody. 4. 4 was obtained from rabbit serum by injecting rabbit with the obtained protein as antigen. The TF gene of Ku70 was expressed in situ and the protein was extracted. The protein expression level of TF gene was detected by Western Blot and the secondary metabolite synthesis was detected by Western Blot. The results showed that 4 of the 10 mutant strains were white and did not produce spores, indicating that knockout genes were closely related to spores formation. TLC analysis showed that the number of secondary metabolites of mutant strains decreased. After the PKS5 gene was integrated into the WA site of Aspergillus nestis genome, the results of TLC and HPLC analysis showed that three new metabolites were produced in the secondary metabolites of Aspergillus mutans. E. coli BL21 was successfully expressed as transcription factor and the corresponding antibody was obtained by injection of rabbit. The results of Western Blot showed that the expression level of TF gene in TF super expression strain was significantly up-regulated. TLC analysis showed that the production of secondary metabolites was significantly increased.
【学位授予单位】：江苏师范大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q936

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