【作者】 杨莎；

【导师】 陈福生；

【作者基本信息】 华中农业大学 ， 食品科学， 2009， 硕士

【摘要】 红曲菌（Monascus spp.）是用来发酵生产红曲的微生物,它能够产生红曲色素、Monacolin K、γ-氨基丁酸（γ-amino butyric acid,GABA）、麦角固醇等生理活性物质,具有较高的商业价值。目前,国内外关于红曲菌的研究主要集中在菌种分类、选育,发酵条件研究,桔霉素检测等方面,关于其分子生物学的研究刚刚起步,其功能基因研究主要集中于色素、桔霉素和Monacolin K代谢调控途径等方面。双组分系统是细菌、植物与真菌中均存在的一种信号传导途径,其包含组氨酸蛋白激酶与应答调节蛋白两个组分。研究表明,双组分系统在丝状真菌中与氧化应激应答、渗透压应答及孢子形成有关。目前为止,在红曲菌中关于双组分系统的研究尚未见报道。本实验室前期采用根癌农杆菌介导的转化方法建立了包含10000多株T-DNA突变子的红色红曲菌（M.ruber）M-7转化库。本实验从转化库中选出8株T-DNA插入突变子,并对其菌落形态和显微形态进行了观察。在此基础上,采用TAIL-PCR分离得到4株突变子的T-DNA侧翼序列,并利用SON-PCR对其中1株突变子DNA序列进行了延伸,总共获得5个基因,发现其中一个基因mrr的编码蛋白与曲霉属（Aspergillus spp.）压力应答调节蛋白高度同源,利用基因敲除的方法对其功能进行了初步研究,主要研究内容如下:1红色红曲菌突变子T-DNA侧翼序列的克隆通过对143株T-DNA插入突变子与出发菌株M-7菌落形态进行比较,筛选得到8株菌落形态发生变化的突变子并对其显微形态进行了观察。采用TAIL-PCR法对上述8株突变子T-DNA插入位点的侧翼序列进行了扩增,得到了3192^#、3440^#、2341^#、3725^# 4株突变子T-DNA插入位点侧翼序列。其中,突变子3192^#扩增序列726bp,与构巢曲霉（Aspergillus nidulans）压力应答调节蛋白编码基因重叠101bp。利用SON-PCR对此序列进行延伸后获得全长为13649bp的DNA序列。此序列共包含5个基因,其编码氨基酸序列分别与曲霉属Cog6、Nif3蛋白、BolA蛋白、AhpC/TSA家族蛋白及压力应答调节蛋白的同源性达到80%以上。2 mrr基因敲除压力应答调节蛋白编码基因全长2356bp,包含6个外显子,5个内含子,预测蛋白含634个氨基酸,具有一个保守的接受结构域和一个DNA结合区,这两者是应答调节蛋白的基本原件。推测该基因可能是红色红曲菌双组分系统中应答调节蛋白编码基因,将其命名为mrr（Monascus ruber response regulator）。构建mrr基因敲除载体pCMRR,并利用农杆菌介导转化的方法成功获得3株△mrr突变子。3mrr功能初步研究对△mrr突变子的性状进行了初步的研究,发现mrr基因的缺失使得红曲菌的生长速度加快,有性发育滞后。Mrr基因的缺失对于红曲菌耐渗特性影响不显著,但其缺失使得红曲菌对外界氧化压力的敏感性增大,△mrr突变子H₂O₂最大耐受浓度低于0.3mmol/L,而M-7的最大耐受浓度高于0.75mmol/L,推测mrr基因与红色红曲菌氧化应激信号传导和有性繁殖相关。更多还原

【Abstract】 Monascus spp., used for red rice fermentation, is a kind of fungi with high commercial values due to its production of many kinds of physiological active substances, such as monascus pigment, Monacolin K,γ-aminobutyric acid and ergosterol. At present, the investigation about Monascus spp. was focused on its classification, breeding, fermentation conditions and citrinin detection, but the study on molecular biology of Monascus spp. is at the beginning stage. The investigation of functional genes about Monascus spp. was focused on the genes related to the metabolic regulation of pigment, citrinin and Monacolins K.The two component system which exists in bacteria, plants and fungi comprises two protein components, a histidine protein kinase and a response regulator protein. Study showed that the roles of the two component system were involved in oxidative stress response, osmotic response and spore formation in filamentous fungi. Until now, there was no report about the genes involves in the two component system in Monascus spp..The T-DNA inserted transformant library of Monascus ruber M-7 containing more than 10000 transformants was constructed in preliminary studies of our laboratory. In this study, 8 T-DNA inserted mutans were selected, and the colony morphologies, microscopic morphologies were observed. After that, the T-DNA flanking sequences of four mutants were isolated by TAIL-PCR, and one of the four isolated sequence was extended by SON-PCR. 5 genes were cloned and the coding protein of mrr, one of the five, has high homology to the response regulator protein of Aspergillus spp.. Finally, the function of mrr was analyzed by gene knock-out. The main research contents are as follows:1 Isolation of the the T-DNA flanking sequences of Monascus ruber mutantAccording to the comparison between 143 T-DNA inserted mutans and M-7, 8 mutants with different colony morphologies were selected. The microscopic morphologies were observed, and the DNA sequences flanked T-DNA border of 8 mutants were amplified by TAIL-PCR and the T-DNA flanked sequences of 3440^#, 2341^# and 3725^# were successfully isolated. 726bp isolated from 3192^# has a 101bp region of overlap with Aspergillus nidulans stress response regulator. A sequence with length of 13649bp was cloned by extending the flanked sequences of the 726bp region by SON-PCR, and in the sequence, 5 genes were found to have more than 80% homology with Cog6, Nif3, BolA, AhpC/TSA family and response regulator protein, respectively. 2 gene knock-out of mrrThe mrr（Monascus ruber response regulator） encoding the response regulator protein contains 2356bp with 6 exons and 5 introns, and the dedued amino acids sequence of mrr contained 634 amino acids with a receiver domain and a DNA binding domain which both were the basic components of response regulator protein of the two component system. To analyze the funtion of mrr in Monascus spp., the gene knock-out vector pCMRR was constructed and transformated into Monascus ruber M-7 by the method of Agrobacterium tumefaciens mediated transformation. Finally, 3△mrr mutants was obtained.3 Primarily study on the function of mrrThe characters of△mrr mutant were studied and the results show that the growing rate of△mrr mutant was fast than M-7, while the sexual development of△mrr mutant was slower. Mrr was not involved in osmostress resistance but was required for H₂O₂ resistance, the maximum tolerated concentration of△mrr mutant was less than 0.3mmol/L, while the M-7 is higher the 0.75mmol/L, mrr was presumed to be related to oxidative stress signal transduction and sexual reproduction of Monascus spp..更多还原