本文選題：黑曲霉　切入點：基因敲除　出處：《合肥工業(yè)大學》2017年碩士論文

【摘要】：黑曲霉(Aspergillus niger),曲霉屬真菌中的一個常見種,既是重要的工業(yè)發(fā)酵菌種,但同時也是具有侵染能力的致病真菌。超氧化物歧化酶(Superoxide dismutase,SOD,EC 1.15.1.1)是生物體內重要的抗氧化損傷的金屬酶類,其主要功能是清除生物體內產生的超氧自由基。在黑曲霉中主要有Cu/Zn型的sodC和Mn型的sodB。為探究超氧化物歧化酶sodC基因對黑曲霉在生理代謝方面所起的作用,本文利用同源重組的技術原理,對黑曲霉A.niger MA70.15中的sodC基因進行敲除。首先,以黑曲霉基因組DNA為模板,擴增獲得sodC目的基因的上下游側翼片段,利用重疊序列對上下游片段進行PCR連接。將該片段與質粒pC3進行連接,并將其轉入DH5α感受態(tài)細胞,通過藍白斑篩選及PCR鑒定,得到已構建成功的重組質粒。通過CaCl_2/PEG轉化法將重組質粒轉入黑曲霉原生質體。通過多步驟的篩選及基因組PCR鑒定,得到sodC基因敲除突變體ΔsodC。進一步以黑曲霉A.niger MA70.15和ΔsodC為實驗材料,對其進行后續(xù)研究。通過觀察發(fā)現(xiàn)黑曲霉A.niger MA70.15和Δsod C在正常的培養(yǎng)條件下,在生長量,菌斑直徑等方面均無差異,表明在體外正常的培養(yǎng)條件下,黑曲霉中sodC基因的缺失對其生長并無影響。通過對兩者體內SOD活性進行檢測,發(fā)現(xiàn)基因sodC的缺失導致黑曲霉中整體的SOD水平的降低,表明sodC基因對菌體內整體的SOD活性有貢獻。此外我們通過觀察黑曲霉A.niger MA70.15和ΔsodC在高濃度的金屬離子如,Cu2+,Zn2+,Al3+,Cd2+,H_2O_2及超氧陰離子誘導劑甲萘醌等多種脅迫條件下菌絲體的生長及孢子的萌發(fā)情況,發(fā)現(xiàn)與野生型相比,甲萘醌能顯著抑制突變體體內的菌絲體生長及孢子萌發(fā)。同時,甲萘醌處理顯著提高了ΔsodC突變體體內ROS及MDA含量。我們發(fā)現(xiàn)經抗壞血酸預處理的野生型及突變體的菌絲體比直接在甲萘醌條件下生長的要快。上述結果表明sodC基因在黑曲霉抵抗外界氧化脅迫的過程中起到重要的抗氧化的作用。此外,與野生型相比,在侵染水果時隨著損害的加深,缺失突變體?sodC對水果的侵染能力明顯延緩。對侵染果實中的ROS及MDA含量進行檢測,發(fā)現(xiàn)被突變體所侵染的水果組織內的ROS及MDA含量都要低于野生型的。進一步對在侵染條件下的菌體進行RNA提取并進行熒光定量PCR,發(fā)現(xiàn)在侵染條件下突變體體內的SOD編碼基因(主要有sod B、An04g04870、An08g03890、An16g05520)的表達量要明顯高于野生型的。表明在果實侵染過程中,黑曲霉體內的sodC基因對其全毒力有貢獻。上述結果表明sodC基因在黑曲霉中起到的重要的抗氧化作用,并且它是參與果實侵染的致病基因。過氧化氫酶(Catalases,CAT,EC 1.11.1.6.),作為黑曲霉體內抗氧化酶系統(tǒng)中的重要成員。利用黑曲霉缺失突變體ΔcatA,通過與野生型菌株的表型進行比較,探究其在黑曲霉生長代謝過程中所起的作用。通過觀察發(fā)現(xiàn)野生型與突變體ΔcatA在正常的培養(yǎng)條件下,生長量和菌斑直徑等方面均無差異,這表明在正常的培養(yǎng)條件下,catA基因的缺失對黑曲霉生長并無影響。對兩者體內的CAT酶活性進行測定,發(fā)現(xiàn)基因catA的缺失導致黑曲霉中整體的CAT酶活水平的降低。并發(fā)現(xiàn)H_2O_2處理對ΔcatA突變體菌絲體的生長有明顯的抑制作用。同時,與野生型相比,在侵染水果的過程中,由缺失突變體ΔcatA所造成的損傷直徑要明顯小于由野生型所造成的。這些結果都初步證明了catA基因在黑曲霉的生長過程中可能起到的重要的抗氧化作用,而且catA參與了黑曲霉對果實的侵染過程。
[Abstract]:Niger (Aspergillus Niger), Aspergillus in a common species, industrial fermentation is an important pathogenic fungi, but also has the infection ability. Superoxide dismutase (Superoxide dismutase, SOD EC, 1.15.1.1) is an important anti oxidative damage of metal enzymes, its main function is to the scavenging of superoxide radical in vivo. Produced in Aspergillus Niger are mainly Cu/Zn type sodC and type Mn sodB. for exploring the role of superoxide dismutase sodC gene of Aspergillus niger in physiological metabolism, the principle of homologous recombination, sodC gene of Aspergillus niger A.niger in MA70.15 knock in addition to. First, Aspergillus niger genomic DNA as template, amplified sodC gene flanking fragments of downstream fragments were connected by overlap PCR sequence. The fragment was connected with plasmid pC3, And put it into a DH5 alpha competent cell, through the blue white screening and PCR identification, the recombinant plasmid has been successfully transformed by CaCl_2/PEG. The recombinant plasmid was transferred into protoplasts of Aspergillus niger. Through screening and identification of genomic PCR of multiple steps, sodC gene knockout mutant of Aspergillus niger A.niger Delta sodC. further to MA70.15 and sodC as experimental materials, further studies on it. Through the observation that Niger A.niger MA70.15 and SOD C in normal culture condition, the growth rate, there were no differences in plaque diameter, suggesting that in vitro normal culture conditions, the deletion of sodC gene in Aspergillus Niger had no effect on the growth of. The detection of both SOD activity in vivo, found missing the sodC gene leads to the decrease of the overall level of SOD in Aspergillus niger, suggesting that the sodC gene contributes to the activity of SOD bacteria in the whole. Besides I Through the observation of Aspergillus niger A.niger MA70.15 and sodC in high concentration of metal ions such as Cu2+, Zn2+, Al3+, Cd2+, germination and growth of mycelium and spores of H_2O_2 induced by superoxide anion agent menadione and other stress conditions, compared with wild type, menadione can significantly inhibit mutant in vivo the mycelium growth and spore germination. At the same time, menadione treatment significantly improved sodC mutants in vivo ROS and MDA content. We found that the mycelium of wild type and mutant ascorbic acid pretreatment of the body than in menadione faster growth conditions. The results showed that the sodC gene plays an important antioxidant resistance the role of oxidative stress in the process of the outside world in Niger. In addition, compared with the wild type, the infection of fruit with damage deepened, the ability of sodC to infect mutants? Fruit was significantly delayed infection in fruit. ROS and MDA were detected by ROS and the content of MDA, found that mutants infected fruit tissues were lower than that in wild type. Further RNA extraction of bacteria in the infection condition and fluorescence quantitative PCR, found in the infection condition in the mutant gene encoding SOD (mainly SOD B, An04g04870, An08g03890, An16g05520) expression was significantly higher than that of wild type. The fruit showed in the infection process, sodC gene of Aspergillus niger in vivo contribute to its full virulence. These results indicated that the important role of anti oxidation to sodC gene in Aspergillus niger, and it is a causative gene involved in fruit infection. Hydrogen peroxide enzyme (Catalases, CAT, EC, 1.11.1.6.), as an important member in Niger antioxidase system. Using Aspergillus niger mutant catA, the phenotype with the wild type strain were compared, to explore its The growth of Aspergillus niger by metabolic process. Through the observation that the wild type and mutant catA in normal culture condition, there were no differences in growth rate and plaque diameter etc., which shows that in normal culture condition, the deletion of catA gene on the growth of Aspergillus niger and have no effect on the enzymatic activity of CAT. Both in vivo were found missing the catA gene leads to the decrease of Aspergillus niger CAT enzyme activity level and overall. The results showed that H_2O_2 had obvious inhibitory effect on catA mutant mycelial growth. At the same time, compared with the wild type fruit during the infection, caused by a catA deletion mutant the damage diameter was smaller than that caused by the wild type. These results demonstrated that antioxidant effects of catA gene may play in the process of growth of Aspergillus niger in important, and catA is involved in the invasion of the fruit of Aspergillus niger Dyeing process.

【學位授予單位】：合肥工業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：Q933

【參考文獻】

相關期刊論文 前5條

1 錢程;馬晨;湯永嬌;李建國;;果蔬中食源性致病微生物的研究進展[J];熱帶農業(yè)科學;2014年12期

2 李荔;張克勤;楊金奎;;過氧化物酶體與病原真菌的致病性[J];微生物學通報;2011年06期

3 郭艷梅;鄭平;孫際賓;;黑曲霉作為細胞工廠:知識準備與技術基礎[J];生物工程學報;2010年10期

4 于慧敏;馬玉超;;工業(yè)微生物代謝途徑調控的基因敲除策略[J];生物工程學報;2010年09期

5 許楊;涂追;;絲狀真菌基因敲除技術研究進展[J];食品與生物技術學報;2007年01期

，

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