黑曲霉缺失突变体ΔsodC的构建及其基因功能研究

发布时间：2018-03-25 02:15

本文选题：黑曲霉　切入点：基因敲除　出处：《合肥工业大学》2017年硕士论文

【摘要】：黑曲霉(Aspergillus niger),曲霉属真菌中的一个常见种,既是重要的工业发酵菌种,但同时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)是生物体内重要的抗氧化损伤的金属酶类,其主要功能是清除生物体内产生的超氧自由基。在黑曲霉中主要有Cu/Zn型的sodC和Mn型的sodB。为探究超氧化物歧化酶sodC基因对黑曲霉在生理代谢方面所起的作用,本文利用同源重组的技术原理,对黑曲霉A.niger MA70.15中的sodC基因进行敲除。首先,以黑曲霉基因组DNA为模板,扩增获得sodC目的基因的上下游侧翼片段,利用重叠序列对上下游片段进行PCR连接。将该片段与质粒pC3进行连接,并将其转入DH5α感受态细胞,通过蓝白斑筛选及PCR鉴定,得到已构建成功的重组质粒。通过CaCl_2/PEG转化法将重组质粒转入黑曲霉原生质体。通过多步骤的筛选及基因组PCR鉴定,得到sodC基因敲除突变体ΔsodC。进一步以黑曲霉A.niger MA70.15和ΔsodC为实验材料,对其进行后续研究。通过观察发现黑曲霉A.niger MA70.15和Δsod C在正常的培养条件下,在生长量,菌斑直径等方面均无差异,表明在体外正常的培养条件下,黑曲霉中sodC基因的缺失对其生长并无影响。通过对两者体内SOD活性进行检测,发现基因sodC的缺失导致黑曲霉中整体的SOD水平的降低,表明sodC基因对菌体内整体的SOD活性有贡献。此外我们通过观察黑曲霉A.niger MA70.15和ΔsodC在高浓度的金属离子如,Cu2+,Zn2+,Al3+,Cd2+,H_2O_2及超氧阴离子诱导剂甲萘醌等多种胁迫条件下菌丝体的生长及孢子的萌发情况,发现与野生型相比,甲萘醌能显著抑制突变体体内的菌丝体生长及孢子萌发。同时,甲萘醌处理显著提高了ΔsodC突变体体内ROS及MDA含量。我们发现经抗坏血酸预处理的野生型及突变体的菌丝体比直接在甲萘醌条件下生长的要快。上述结果表明sodC基因在黑曲霉抵抗外界氧化胁迫的过程中起到重要的抗氧化的作用。此外,与野生型相比,在侵染水果时随着损害的加深,缺失突变体?sodC对水果的侵染能力明显延缓。对侵染果实中的ROS及MDA含量进行检测,发现被突变体所侵染的水果组织内的ROS及MDA含量都要低于野生型的。进一步对在侵染条件下的菌体进行RNA提取并进行荧光定量PCR,发现在侵染条件下突变体体内的SOD编码基因(主要有sod B、An04g04870、An08g03890、An16g05520)的表达量要明显高于野生型的。表明在果实侵染过程中,黑曲霉体内的sodC基因对其全毒力有贡献。上述结果表明sodC基因在黑曲霉中起到的重要的抗氧化作用,并且它是参与果实侵染的致病基因。过氧化氢酶(Catalases,CAT,EC 1.11.1.6.),作为黑曲霉体内抗氧化酶系统中的重要成员。利用黑曲霉缺失突变体ΔcatA,通过与野生型菌株的表型进行比较,探究其在黑曲霉生长代谢过程中所起的作用。通过观察发现野生型与突变体ΔcatA在正常的培养条件下,生长量和菌斑直径等方面均无差异,这表明在正常的培养条件下,catA基因的缺失对黑曲霉生长并无影响。对两者体内的CAT酶活性进行测定,发现基因catA的缺失导致黑曲霉中整体的CAT酶活水平的降低。并发现H_2O_2处理对ΔcatA突变体菌丝体的生长有明显的抑制作用。同时,与野生型相比,在侵染水果的过程中,由缺失突变体ΔcatA所造成的损伤直径要明显小于由野生型所造成的。这些结果都初步证明了catA基因在黑曲霉的生长过程中可能起到的重要的抗氧化作用,而且catA参与了黑曲霉对果实的侵染过程。
[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.

【学位授予单位】：合肥工业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：Q933

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