【作者】 邵彦春；

【导师】 谢笔钧； 陈福生；

【作者基本信息】 华中农业大学 ， 食品科学， 2007， 博士

【摘要】 红曲霉(Monascus spp．)是我国传统的食用和药用微生物，在我国已有上千年的应用历史。它能分泌产生色素、莫呐可啉类(monacolins)物质、γ-氨基丁酸(γ-Aminobutyric Acid，GABA)、麦角固醇和丰富的水解酶类等多种有益代谢产物，是生产食品发酵剂、着色剂、保健食品、药品或其原料的重要微生物资源。目前，国内外对红曲霉菌种的分类、选育、代谢产物分离纯化及发酵条件的研究较多，而有关红曲霉的分子生物学研究才刚刚起步。GenBank检索结果查见，与红曲霉相关的核酸序列大部分是研究分类和亲缘关系的，有关功能基因的研究很少，而关于红曲霉产色素基因的研究尚未见报道。近年来，利用根癌农杆菌(Agrobacterium tumefaciens)介导T-DNA转化真菌获得突变体的方法成为寻找和鉴定新基因的有效手段。本课题采用根癌农杆菌介导的T-DNA转化法建立了包含5100多株转化子的红曲霉转化子库，从转化子库中筛选得到了24株菌落颜色发生了明显变化的色素突变子，并对其菌落形态及显微结构、潮霉素抗性稳定性及主要代谢产物的分泌能力等进行了分析。在此基础上，采用TAIL-PCR分离到8株色素突变子T-DNA插入位点的左侧序列，并应用RACE技术克隆得到了一株色素突变子805~#T-DNA插入位点的cDNA全长序列，采用基因敲除(gene knock-out)技术对其功能进行了验证。具体研究结果如下：1转化子库的构建及色素突变株的筛选通过优化农杆菌(A．tumefaciens)介导的T-DNA转化红曲霉的条件，构建了包含5100多个转化子的转化子库。T-DNA的PCR验证结果表明，随机挑选的60株转化子均含有T-DNA插入片段。24株随机挑选的转化子的Southern杂交结果表明，T-DNA单拷贝插入率为70.8％。通过对转化子菌落形态的观察，筛选得到53株形态发生明显变化的转化子，其中24株为菌落颜色发生显著变化的色素突变子，其编号分别为189~#、192~#、533~#、635~#、733~#、805~#、959~#、1132~#、1164~#、1263~#、1295~#、2066~#、2606~#、2887~#、3108~#、3257~#、3715~#、3742~#、4081~#、4096~#、4591~#、4698~#、4963~#和4968~#。色素突变子连续传代培养5代后，除733~#、1132~#、3715~#和4591~#外，其它20株色素突变子均能在潮霉素抗性培养基上稳定生长，潮霉素抗性稳定性达83.3％。2色素突变子产红曲色素、monacolin K、GABA和桔霉素的分析采用UV-VIS、TLC、HPLC等方法对上述20株潮霉素抗性稳定的色素突变子的固态发酵产物红曲的色素(包括色价、吸收波长、色素组分)、monacolin K、GABA和桔霉素的产生情况进行了分析。结果表明，与出发菌株相比，色素突变子产代谢产物的能力都发生了不同程度的变化。在产色素方面，色素突变子959~#的色价最高，达2 712，是出发菌株M-7色价1228的2.2倍；1263~#的色价最低，仅为20，只有出发菌株M-7色价的0.01倍。另外，色素突变子红曲乙醇萃取液的UV-VIS扫描图谱和色素组分也发生了显著的变化。在产monacolin K方面，突变子4081~#红曲的含量最高，达1601μg／g，而出发菌株M-7红曲的含量低于0.1μg／g。在产GABA方面，突变子1263~#红曲含量最高，达6183.9μg／g，是出发菌株M-7 436.3μg／g的14.2倍；635~#红曲的含量最低，只有156.1μg／g，约为出发菌株M-7的0.33倍。在产桔霉素方面，突变子635~#红曲的含量最高，达154.57μg／g，是出发菌株0.75μg／g的206倍；1295~#含量最低，为0.10gg／g，为出发菌株的0.1倍。比较上述20株色素突变子产色素和桔霉素的情况，可将其分为4类：第一类，色价和桔霉素都升高的，包括635~#、959~#、1164~#、2066~#、2606~#、2887~#、4081~#和4698~#共8株；第二类，色价和桔霉素都降低的，包括805~#、1295~#、3742~#和4968~#共4株；第三类，色价降低而桔霉素含量升高的，包括189~#、192~#、1263~#和4963~#共4株；第四类，色价升高而桔霉素降低的，包括533~#、3108~#、3257~#和4096~#共4株。从上述四类突变子中挑选出635~#、1164~#、2606~#、805~#、1295~#、189~#、1263~#、4963~#、533~#和3257~#共10株代表性菌株，作为研究红曲霉产色素相关基因的材料。3色素突变子T-DNA插入位点侧翼序列TAIL-PCR扩增及扩增片段功能分析采用TAIL-PCR法对上述10株色素突变子T-DNA插入位点的侧翼序列进行了扩增，得到了635~#、1164~#、2606~#、805~#、3257~#、189~#、4963~#和533~#共8株突变子T-DNA插入位点左侧的DNA序列，扩增的DNA片段长度介于500 bp～1 300 bp之间。FASTA比对分析表明，805~#突变子DNA扩增序列与烟曲霉(Aspergillus fumigatus)Af293的G蛋白信号调节子(regulator for G-protein signaling，RGS)功能域的同源性达84％：其它7株突变子的T-DNA插入位点侧翼的DNA序列未发现有功能明确的相似性序列。突变子533~#、1164~#和2606~#的DNA扩增片段已经登陆Genbank，登陆号分别为DQ861336、DQ861338和DQ1337。4红曲霉G蛋白信号调节子的功能验证以突变子805~#扩增序列的Blast比对分析结果为依据，设计特异性引物，采用RACE技术，扩增得到了对应于该DNA序列的2012 bp的cDNA序列。ORFfin工具分析显示，其包含的最长开放读码框架(open reading frame，ORF)为1851bp，推衍得到包括了RGS和2个DEP(disheveled，Eg1+10，pleckstrin)3个结构域在内的氨基酸序列。以RGS结构域两侧的DNA序列为同源臂，采用基因敲除(gene knock-out)技术对其功能进行了初步研究。研究结果表明，805~#突变子T-DNA插入位点的基因，即编码红曲霉FlbA(nuffy for brlA)的序列对红曲霉色素分泌具有正调节作用。更多还原

【Abstract】 Monascus spp., a kind of fungi, used in food production and traditional medicine for a thousand of years in China, has become an important microorganism resources due to its production of many kinds of metabolites, such as monascus pigment, monacolins,γ-aminobutyric acid(GABA), ergosterol and hydrolyase, which are used as food fermentation starter, food colorant, function food and the ingredients or/and raw materials of medicine. At present, the investigation about Monascus spp. was focused on its classification, breeding, metabolites isolation and fermentation conditions to improve useful metablites, but the study on molecular biology of Monascus spp. is at the beginning stage. It was found that most of the sequences related to Monascus spp. in GenBank were used as classification and identification of the homology, few were functional genes. Until now, there was no report about the gene(s) involved in monascus pigment synthesis.In recent years, an efficient method to find and identify new function genes with Agrobacterium tumefaciens-mediated T-DNA transformation has been developed and widely used. In this paper, a transformant library of Monascus ruber by Agrobacterium turnefaciens mediated T-DNA transformation was constructed and more than 5 100 transformants were obtained. From the library, 24 color-producing mutants, which were markedly different from the original strain M-7 in pigment production, were selected, and the colony morphologies, microscopic structures, the stabilities to hygromycin resistance and the abilities to produce different metabolites of the mutants were analyzed. After that, flanked T-DNA sequences of 8 color-producing mutants were isolated by thermal asymmetric interlaced PCR, according to the bioinformation of isolated sequences, a complete cDNA sequence of color-producing mutants named 805~# was coloned with the method of rapid amplification of cDNA end. Finally, the function of the sequence from 805~# was identified by gene knock-out. The main research contents are as follows:1 The construction of transformant library and selection of color-producing mutantsThe transformant library of Monascus spp., containing more than 5 100 transformants was constructed. The results of PCR amplification of transformants by Agrobacterium tumefaciens-mediated T-DNA transformation suggested that all of the genomic DNA of 60 randomly selected transformants were inserted by T-DNA. The results of southern blot revealed that the single-copy rate of T-DNA into the genomic DNA of 24 randomly selected transformants was 70.8％. By morphology observation, 53 phenotype mutants from the library were selected, among which, there were 24 color-producing mutants and their codes were 189~#, 192~#, 533~#, 635~#, 733~#, 805~#, 959~#, 1132~#, 1164~#, 1263~#, 1295~#, 2066~#, 2606~#, 2887~#, 3108~#, 3257~#, 3715~#, 3742~#, 4081~#, 4096~#, 4591~#, 4698~#, 4963~# and 4968~#, respectively. After five rounds of consecutive incubation, except 733~#, 1132~#, 3715~# and 4591~# the rest of color-producing mutants can grow on the PDA containing 20μg/mL of hygromycin and the stable rate to hygromycing resistance amounts to 83.3％.2 Analysis of monascus pigment, monacolin K,γ-amino butyric acid and citrinin of red fermented rice produced by color-producing mutantsThe contents of monascus pigment, monacolin K,γ-amino butyric acid and citrinin of red fermented rice(RFR) produced by 20 stable color-producing mutants, were analyzed by UV-VIS, TLC and HPLC. The results revealed that the mutants rnetabolites described above were different from those produced by the original strain M-7.In the aspect of pigment, RFR of 959~# was of the the highest pigment-value and amounted to 2 712, which was 2.2 times that of M-7, while the pigment-value of 1263~# was the lowest and was 20, which was just 0.01 times that of M-7. In addition, the UV-VIS spectrum and pigment components of 70％of ethanol solution of RFR produced by 20 of color-producing mutants were markedly different from those of M-7. Regarding to monacolin K, the amounts of monacolin K produced by 4081~# was the higest up to 1 601μg/g while that of M-7 was lower than 0.1μg/g. About GABA, the amounts of GABA of RFR produced by 1263~# was up to 6 183.9μg/g and was 14.2 times that of M-7, the amount of 635~# was the lowest to 156.1μg/g and was just 0.33 times that of M-7. With regards to citrinin, the content of citrinin of RFR produced by 635~# was the higest among of all color-producing mutants, which amounts to 154.57μg/g and was 206 times that of M-7, the content of citrinin of RFR produced by 1295~# was the lowest to 0.10μg/g and was 0.1 time that of M-7.According to the pigment-values and yields of citrinin of RFR produced by color-producing mutants, 20 color-produing mutants were classified into four groups: (1)mutants with higher pigment-value and higher-producing citrinin than those of M-7, they were 635~#, 959~#, 1164~#, 2066~#, 2606~#, 2887~#, 4081~#, and 4698~#, respectively; (2)mutants with lower pigment-value and lower-producing citrinin than those of M-7, they were 805~#, 1295~#, 3742~# and 4968~#, respectively; (3)mutants with lower pigment-value while higher-producing citrinin than those of M-7, they were 189~#, 192~#, 1263~# and 4963~#, respectively, (4)mutants with higher pigment-value while lower-producing citrinin than those of M-7, they were 533~#, 3108~#, 3257~# and 4096~#, respectively. 10 mutants, which were 635~#, 1164~#, 2606~#, 805~#, 1295~#, 189~#, 1263~#, 4963~#, 533~# and 3257~#, were selected from above four groups of mutants and used as materials for further investigating the gene(s) related to monascus pigment synthesis.3 Isolation of the DNA sequences flanked T-DNA by TAIL-PCR and analysis of the function of the isolated sequencesThe DNA sequences flanked T-DNA left border of 10 above color-producing mutants were amplified by TAIL-PCR and the DNA sequences of 635~#, 1164~#, 2606~#, 805~#, 3257~#, 189~#, 4963~# and 533~#, were successfully isolated and the length of isolated DNA fragments ranged from 500 bp to 1 300 bp. By FASTA analysis, the amplified DNA from 805~# carries the RGS functional domain of regulator of G-protein signaling, the similarity of which was up to 84 % with that of Aspergillus fumigatus, but the isolated DNA from the rest mutants had no similarity with DNA sequences in GenBank. The isolated DNA sequences from 533~#, 1164~# and 2606~#, which have been published in GenBank, correspondingly, the recorded No. is DQ861336, DQ 861338 and DQ 861337, respectively.4 Identification of the function of DNA sequence coding for regulator of G-protein signaling of Monascus spp.On the basis of bioinformation from isolated DNA sequence of 805~#, the specific primers were designed and the 805~# cDNA sequence with 2 012bp was amplified by the method of rapid amplification of cDNA end. The cDNA sequence carries a long open reading frame with 1 851bp, which includes RGS domain and two DEP(disheveled, Egl-10, pleckstrin) domains. The sequences of both sides of RGS were designed into homologous borders and the mutants with RGS domain deletion was constructed with gene knock-out. The results showed that the mutants with RGS domain deletion produced little hongqu pigment and the isolated flb A positively regulates the hongqu pigment synthesis.更多还原