根癌農(nóng)桿菌介導申克氏孢子絲菌T-DNA插入突變的研究
發(fā)布時間：2018-10-15 12:01
【摘要】：申克氏孢子絲菌(Sporothrix schenckii)是一種雙相型病原真菌,引起人和動物的孢子絲菌病(Sporotrichosis)。本研究首次成功建立了根癌農(nóng)桿菌介導的申克氏孢子絲菌JLCC32757遺傳轉(zhuǎn)化體系,并探討了影響轉(zhuǎn)化效率的主要因素。該轉(zhuǎn)化體系效率達600個轉(zhuǎn)化子/10~6個分生孢子,可在短期內(nèi)獲得大量T-DNA(Transfer DNA)插入突變體,且這些突變體有絲分裂穩(wěn)定。已獲得突變菌株2130株,初步建立了小范圍的申克氏孢子絲菌T-DNA標簽的突變體庫,并得到了一些生長、發(fā)育、代謝等表型發(fā)生改變的突變體,為揭示申克氏孢子絲菌分子機理、探討致病機制等奠定了堅實的基礎(chǔ)。申克氏孢子絲菌T-DNA插入突變體的分子分析表明,T-DNA已插入到申克氏孢子絲菌基因組中,其中87%為T-DNA單拷貝插入,13%為T-DNA多拷貝插入,利用TAIL-PCR即可獲得T-DNA插入位點的側(cè)翼序列,表明根癌農(nóng)桿菌介導的申克氏孢子絲菌遺傳轉(zhuǎn)化是一種有效的插入突變策略,是進行申克氏孢子絲菌功能基因組學研究的有力工具。篩選申克氏孢子絲菌T-DNA插入突變體,獲得產(chǎn)色素缺陷菌株JLCC32757-M2013。與野生型相比,該菌株失去產(chǎn)色素的能力,其菌絲、分生孢子形態(tài)均發(fā)生明顯改變。分子分析表明,T-DNA以單拷貝插入到申克氏孢子絲菌類泛素結(jié)合酶E2基因(SsUBCc),破壞菌體內(nèi)類泛素結(jié)合酶E2基因的正常表達,導致SsUBCc基因和色素合成相關(guān)基因在轉(zhuǎn)錄水平的表達量降低,菌株毒力下降。以突變株JLCC32757-M2013的SsUBCc基因為參照,PCR擴增模板DNA結(jié)果表明,50ng/μl模板DNA稀釋1600倍時,仍能獲得清晰的PCR擴增產(chǎn)物,含有400個突變體的DNA池的模板核酸濃度足以保證每個突變體PCR擴增的需要。由此以每100個突變體為單位,構(gòu)建了21個突變體池和DNA池,既保證實驗的可靠性又方便從突變體庫中篩選靶基因突變的菌株,為充分利用T-DNA插入突變體庫進行申克氏孢子絲菌反向遺傳學研究提供技術(shù)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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R379
本文編號：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.
【學位授予單位】：吉林大學
【學位級別】：博士
【學位授予年份】：2011
【分類號】：R379
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相關(guān)期刊論文 前3條
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2 馮娟;朱廷恒;崔志峰;汪琨;;農(nóng)桿菌介導的灰葡萄孢T-DNA插入突變體庫構(gòu)建及插入位點分析[J];微生物學報;2010年02期
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