根癌农杆菌介导申克氏孢子丝菌T-DNA插入突变的研究
发布时间：2018-10-15 12:01
【摘要】：申克氏孢子丝菌(Sporothrix schenckii)是一种双相型病原真菌,引起人和动物的孢子丝菌病(Sporotrichosis)。本研究首次成功建立了根癌农杆菌介导的申克氏孢子丝菌JLCC32757遗传转化体系,并探讨了影响转化效率的主要因素。该转化体系效率达600个转化子/10~6个分生孢子,可在短期内获得大量T-DNA(Transfer DNA)插入突变体,且这些突变体有丝分裂稳定。已获得突变菌株2130株,初步建立了小范围的申克氏孢子丝菌T-DNA标签的突变体库,并得到了一些生长、发育、代谢等表型发生改变的突变体,为揭示申克氏孢子丝菌分子机理、探讨致病机制等奠定了坚实的基础。申克氏孢子丝菌T-DNA插入突变体的分子分析表明,T-DNA已插入到申克氏孢子丝菌基因组中,其中87%为T-DNA单拷贝插入,13%为T-DNA多拷贝插入,利用TAIL-PCR即可获得T-DNA插入位点的侧翼序列,表明根癌农杆菌介导的申克氏孢子丝菌遗传转化是一种有效的插入突变策略,是进行申克氏孢子丝菌功能基因组学研究的有力工具。筛选申克氏孢子丝菌T-DNA插入突变体,获得产色素缺陷菌株JLCC32757-M2013。与野生型相比,该菌株失去产色素的能力,其菌丝、分生孢子形态均发生明显改变。分子分析表明,T-DNA以单拷贝插入到申克氏孢子丝菌类泛素结合酶E2基因(SsUBCc),破坏菌体内类泛素结合酶E2基因的正常表达,导致SsUBCc基因和色素合成相关基因在转录水平的表达量降低,菌株毒力下降。以突变株JLCC32757-M2013的SsUBCc基因为参照,PCR扩增模板DNA结果表明,50ng/μl模板DNA稀释1600倍时,仍能获得清晰的PCR扩增产物,含有400个突变体的DNA池的模板核酸浓度足以保证每个突变体PCR扩增的需要。由此以每100个突变体为单位,构建了21个突变体池和DNA池,既保证实验的可靠性又方便从突变体库中筛选靶基因突变的菌株,为充分利用T-DNA插入突变体库进行申克氏孢子丝菌反向遗传学研究提供技术支持。
[Abstract]:(Sporothrix schenckii) is a biphasic pathogenic fungus that causes (Sporotrichosis). In humans and animals. In this study, the JLCC32757 genetic transformation system of Agrobacterium tumefaciens mediated by Agrobacterium tumefaciens was successfully established, and the main factors affecting the transformation efficiency were discussed. The efficiency of the transformation system was 600 transformants / 106 conidia. A large number of T-DNA (Transfer DNA) inserted mutants could be obtained in a short period of time, and these mutants were mitotic and stable. 2130 mutant strains have been obtained, and a small range of mutant library with T-DNA tag of Sporothrix schenckii has been established, and some mutants with phenotypic changes, such as growth, development and metabolism, have been obtained, in order to reveal the molecular mechanism of Sporothrix schenckii. The study of pathogenesis laid a solid foundation. Molecular analysis of T-DNA insertion mutants of Sporothrix schenckii showed that T-DNA had been inserted into the genome of Sporothrix schenckii, in which 87% were T-DNA single copy insertions and 13% were T-DNA multicopy insertions. The flanking sequence of T-DNA insertion site could be obtained by TAIL-PCR. The results showed that Agrobacterium tumefaciens mediated genetic transformation of Sporothrix schenckii was an effective strategy for insertion mutation and a powerful tool for functional genomics study of Sporothrix schenckii. Screening and inserting T-DNA of Sporothrix schenckii into mutants to obtain the pigment-producing strain JLCC32757-M2013. Compared with the wild type, the strain lost the ability to produce pigment, and its hyphal and conidial morphology were obviously changed. Molecular analysis showed that T-DNA was inserted into Schenck's spores with a single copy of ubiquitin binding enzyme E2 gene (SsUBCc), to destroy the normal expression of ubiquitin binding enzyme E2 gene, resulting in a decrease in the expression of SsUBCc gene and pigment synthesis related genes at the transcriptional level. The virulence of the strain was decreased. Using the SsUBCc base of the mutant JLCC32757-M2013 as reference, the results of PCR amplification template DNA showed that when the 50ng/ 渭 l template DNA was diluted 1600 times, a clear PCR amplification product could still be obtained. The concentration of template nucleic acid in the DNA pool containing 400 mutants was sufficient to ensure the PCR amplification of each mutants. Thus, 21 mutants and DNA pools were constructed for every 100 mutants, which not only guaranteed the reliability of the experiment, but also facilitated the screening of target gene mutant strains from the mutants library. To provide technical support for reverse genetics of Sporothrix schenckii by using T-DNA inserted mutants library.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R379
本文编号：2272489
[Abstract]:(Sporothrix schenckii) is a biphasic pathogenic fungus that causes (Sporotrichosis). In humans and animals. In this study, the JLCC32757 genetic transformation system of Agrobacterium tumefaciens mediated by Agrobacterium tumefaciens was successfully established, and the main factors affecting the transformation efficiency were discussed. The efficiency of the transformation system was 600 transformants / 106 conidia. A large number of T-DNA (Transfer DNA) inserted mutants could be obtained in a short period of time, and these mutants were mitotic and stable. 2130 mutant strains have been obtained, and a small range of mutant library with T-DNA tag of Sporothrix schenckii has been established, and some mutants with phenotypic changes, such as growth, development and metabolism, have been obtained, in order to reveal the molecular mechanism of Sporothrix schenckii. The study of pathogenesis laid a solid foundation. Molecular analysis of T-DNA insertion mutants of Sporothrix schenckii showed that T-DNA had been inserted into the genome of Sporothrix schenckii, in which 87% were T-DNA single copy insertions and 13% were T-DNA multicopy insertions. The flanking sequence of T-DNA insertion site could be obtained by TAIL-PCR. The results showed that Agrobacterium tumefaciens mediated genetic transformation of Sporothrix schenckii was an effective strategy for insertion mutation and a powerful tool for functional genomics study of Sporothrix schenckii. Screening and inserting T-DNA of Sporothrix schenckii into mutants to obtain the pigment-producing strain JLCC32757-M2013. Compared with the wild type, the strain lost the ability to produce pigment, and its hyphal and conidial morphology were obviously changed. Molecular analysis showed that T-DNA was inserted into Schenck's spores with a single copy of ubiquitin binding enzyme E2 gene (SsUBCc), to destroy the normal expression of ubiquitin binding enzyme E2 gene, resulting in a decrease in the expression of SsUBCc gene and pigment synthesis related genes at the transcriptional level. The virulence of the strain was decreased. Using the SsUBCc base of the mutant JLCC32757-M2013 as reference, the results of PCR amplification template DNA showed that when the 50ng/ 渭 l template DNA was diluted 1600 times, a clear PCR amplification product could still be obtained. The concentration of template nucleic acid in the DNA pool containing 400 mutants was sufficient to ensure the PCR amplification of each mutants. Thus, 21 mutants and DNA pools were constructed for every 100 mutants, which not only guaranteed the reliability of the experiment, but also facilitated the screening of target gene mutant strains from the mutants library. To provide technical support for reverse genetics of Sporothrix schenckii by using T-DNA inserted mutants library.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2011
【分类号】：R379
【参考文献】
相关期刊论文 前3条
1 胡仕凤;朱木兰;刘力行;唐俊;高必达;陈捷;;康氏木霉REMI突变株的差异表达蛋白分析(简报)[J];分子细胞生物学报;2009年01期
2 冯娟;朱廷恒;崔志峰;汪琨;;农杆菌介导的灰葡萄孢T-DNA插入突变体库构建及插入位点分析[J];微生物学报;2010年02期
3 金波;蒋福升;余美荣;陈铌铍;丁志山;;农杆菌介导几丁质酶及β-1,3-葡聚糖酶基因转化半夏的研究[J];中国中药杂志;2009年14期
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