根癌农杆菌LBA4404介导的微紫青霉菌菌株GXCR遗传转化体系的建立
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摘要
从基因水平来研究丝状真菌与重金属间的相互作用具有重要的意义，近年来发展的根癌农杆菌介导的遗传转化(ATMT)与传统的遗传转化方法相比较，ATMT具有高效、稳定、高频单拷贝随机插入、适用范围广等优点，是目前研究丝状真菌遗传转化的主要手段之一。
基于本实验室工作曾分离得到一株高抗多种重金属盐并对苯菌灵敏感的微紫青霉菌(Penicillium janthinellum)菌株GXCR的良好基础，研究将建立由根癌农杆菌LBA4404介导的GXCR菌株的遗传转化体系，为从基因水平上研究该菌株对重金属盐抗性的可能机制提供基础。本研究在质粒pGSA1252基础上构建了T-DNA区含有苯菌灵抗性基因(Benomylresistance)的双元载体pG-Bn，通过三亲转化法导入到根癌农杆菌LBA4404中，与稀释至10~7个／ml的GXCR孢子共培养两天后筛选得到具有苯菌灵抗性的转化株367株，对随机挑取的40株转化株总DNA进行PCR验证，有98％的转化株均能扩增出目的片段，转化效率达到了3．7个转化株／10个小菌块。随机挑选30个能扩增出目的片段的转化株中，有13个转化株不能在CuSO_4 PDA平板上生长，9个转化株的生长明显受CuSO_4抑制。同时对表型明显发生变化的转化株总DNA进行Southern杂交分析，结果显示62．5％的转化株为T-DNA单拷贝整合插入，易于利用插入突变来分析相关基因的功能，为对微紫青霉菌菌株GXCR的抗重金属遗传机制的研究提供了宝贵的材料。
It is beneficial to study the interaction between microorganisms and heavy mental in gene level. Agrobacterium tumefaciens-mediated transformation (ATMT) is an efficient tool for filamentous fungi because of high frequencies of transformation, stable DNA integration, random insertion and wide application that were difficult to transform with traditional methods.
We previously isolated a Penicillium janthinellum strain GXCR with high resistance to many kinds of heavy mental salts and sensitive to benomyl. In this study, the genetic transformantion of GXCR mediated by Agrobacterium tumefaciens was successfully constructed. Make researching about its mechanism of resistance in gene level is possible. We constructed a binary vector named pG-Bn whose T-DNA harboring Benomyl resistance gene on the base of vector pGSA1252. Then, we transfer pG-Bn to LBA4404 by triparental cross and co-cultured with spores of GXCR. Now, we have obtained 367 transformants from that genetic system, PCR tests shows that 38 of 40 transformants can amplificate benomyl gene, the transformation efficiency is approximate 3.7 mutants /10 colonys. We choose 30 of 40 tranformations mentioned above at random, and we found that thirteen of them can't growth on the PDA which with CuSO_4 , and nine of them have obviously different in the concentration of CuSO_4 to wild-type, the rest are no visible different to wild-type. Southern blot analysis of transformants, the results show that 62.5 % of the mutants were T-DNA single copy intergration, that make the factional analysis of related genes via insertion mutation more easier. And also provided precious materials for the study of the genetic mechanism about GXCR resist to heavy mental.
基于本实验室工作曾分离得到一株高抗多种重金属盐并对苯菌灵敏感的微紫青霉菌(Penicillium janthinellum)菌株GXCR的良好基础，研究将建立由根癌农杆菌LBA4404介导的GXCR菌株的遗传转化体系，为从基因水平上研究该菌株对重金属盐抗性的可能机制提供基础。本研究在质粒pGSA1252基础上构建了T-DNA区含有苯菌灵抗性基因(Benomylresistance)的双元载体pG-Bn，通过三亲转化法导入到根癌农杆菌LBA4404中，与稀释至10~7个／ml的GXCR孢子共培养两天后筛选得到具有苯菌灵抗性的转化株367株，对随机挑取的40株转化株总DNA进行PCR验证，有98％的转化株均能扩增出目的片段，转化效率达到了3．7个转化株／10个小菌块。随机挑选30个能扩增出目的片段的转化株中，有13个转化株不能在CuSO_4 PDA平板上生长，9个转化株的生长明显受CuSO_4抑制。同时对表型明显发生变化的转化株总DNA进行Southern杂交分析，结果显示62．5％的转化株为T-DNA单拷贝整合插入，易于利用插入突变来分析相关基因的功能，为对微紫青霉菌菌株GXCR的抗重金属遗传机制的研究提供了宝贵的材料。
It is beneficial to study the interaction between microorganisms and heavy mental in gene level. Agrobacterium tumefaciens-mediated transformation (ATMT) is an efficient tool for filamentous fungi because of high frequencies of transformation, stable DNA integration, random insertion and wide application that were difficult to transform with traditional methods.
We previously isolated a Penicillium janthinellum strain GXCR with high resistance to many kinds of heavy mental salts and sensitive to benomyl. In this study, the genetic transformantion of GXCR mediated by Agrobacterium tumefaciens was successfully constructed. Make researching about its mechanism of resistance in gene level is possible. We constructed a binary vector named pG-Bn whose T-DNA harboring Benomyl resistance gene on the base of vector pGSA1252. Then, we transfer pG-Bn to LBA4404 by triparental cross and co-cultured with spores of GXCR. Now, we have obtained 367 transformants from that genetic system, PCR tests shows that 38 of 40 transformants can amplificate benomyl gene, the transformation efficiency is approximate 3.7 mutants /10 colonys. We choose 30 of 40 tranformations mentioned above at random, and we found that thirteen of them can't growth on the PDA which with CuSO_4 , and nine of them have obviously different in the concentration of CuSO_4 to wild-type, the rest are no visible different to wild-type. Southern blot analysis of transformants, the results show that 62.5 % of the mutants were T-DNA single copy intergration, that make the factional analysis of related genes via insertion mutation more easier. And also provided precious materials for the study of the genetic mechanism about GXCR resist to heavy mental.
引文
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