青霉菌Penicillium sp.GXCR1有毒重金属盐的抗性及其遗传转化体系的研究
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摘要
从矿山排水沟污泥中分离到一株高抗铜的同时对苯菌灵(Benomyl)敏感的青霉菌(Penicillium sp.)GXCR1。该菌的ITS核苷酸序列与102株青霉菌的ITS核苷酸序列有95％以上的同源性。其最适生长温度为32℃，最佳生长pH值为5。
在含Cu~(2+)的PDA固体培养基上：抗Cu~(2+)的水平达200mM，在5mM的Mn~(2+)存在下，其抗Cu~(2+)水平达800mM。
在含重金属盐的PDB液体培养基中抑制该菌生长的Cu~(2+)、Zn~(2+)、Al~(3+)、Cr~(3+)，Cr~(6+)、Cd~(2+)、Pb~(2+)、Ni~(2+)最小浓度分别为＞200mM、＞1200mM、＞500mM、＞60mM、10mM、10mM、10mM和3mM；能在含40mM Cu~(2+)、200mM Zn~(2+)和1mM Cd~(2+)组合，40mM Cu~(2+)、100mM Zn~(2+)和3mM Pb~(2+)组合，或者200mM Zn~(2+)、3mM Cd~(2+)和3mM Pb~(2+)组合的复合重金属盐的PDB液体培养基中生长。
在含重金属盐的水溶液中自然菌体表面对Cu~(2+)、Zn~(2+)吸附量分别达到26.19和258.3mg／g干菌体，而且吸附量远大于二者分别在菌体内的蓄积量。碱预处理的菌体对Cu~(2+)的吸附量达76.9mg／g干菌体。在Cu~(2+)、Zn~(2+)离子共存时，对Cu~(2+)的吸附量大于对Zn~(2+)的吸附量。
制备该菌原生质体的最佳菌龄为从孢子接种起培养24h。原生质体制备得率3.2×10~7个／ml，再生率达31％，原生质体的质粒转化效率为0.3个转化子／μgDNA。
用紫外诱变法获得了GXCR1的铜抗性水平明显降低的一株突变体MGXCR1。
A strain of Penicillium sp. GXCR1 was isolated from waste sludge out of the effluent channel in the mining area. The GXCR1 has very high copper resistance, and was susceptible to the Benomyl. The nucleotide sequences of ITS DNA of the GXCR1 shared more than 95% homology with 102 strains of P.spp. Its optimum growth temperature was at 32 , and the optimum growth pH was at pH 5.
On the PDA medium, the level of Cu2+resistance of the GXCR1 was 200mM, and even up to 800mM if in the presence of 5mM Mn2+.
In the liquid medium PDB with the added individual metal salt, the minimal concentration of the individual heavy metal inhibiting the GXCR1 growth was > 200mM Cu2+, >1200mM Zn2+,>500mM Al34 and 60mM Cr3+, 10mM Cd2+ ,10mM Pb2+ , 10mM Ni2+ and 3mM Cr6+ respectively; It could growth well in liquid medium PDB with one of the following heavy metal combinations: 40mM Cu2+, 200mM Zn2+ and 1mM Cd2+, 40mM Cu2+, 100mM Zn2+and 3mM Pb2+, or 200mM Zn2+, 3mM Cd2+ and 3mM Pb2+.
The biosorption capacity of heavy metals of the GXCR1 was up to 26.19mg Cu2+/g DW and 258.3mgZn2+/g DW respectively. The quantity of to Cu2+ and Zn2+ biossorption of the mycelium surfaces was much larger than that of both accumulation inside the cells of mycelia. The biosorption of mycelia pretreated with 0.2M NaOH was up to 76.9mg Cu2+/g DW. The Cu2+ biosorption quantity of mycelia was much larger than that of Zn2+ biosorption when under coexsistance of both metal salts.
The optimum mycelial age for preparation of protoplast was at 24h time-point after inoculation and culture of conidia. The rate of the prepared protoplast was 3.2 107/ml. The reproduction rate of the prepared protoplast was up to 31%. Efficiency of protoplast transformation with the plasmid was 0.3 transformants/ug DNA.
A strain of the mutant MGXCR1 with a much lower level of Cu resistance was selected by ultraviolet mutagenesis from the wild type strain GXCR1.
在含Cu~(2+)的PDA固体培养基上：抗Cu~(2+)的水平达200mM，在5mM的Mn~(2+)存在下，其抗Cu~(2+)水平达800mM。
在含重金属盐的PDB液体培养基中抑制该菌生长的Cu~(2+)、Zn~(2+)、Al~(3+)、Cr~(3+)，Cr~(6+)、Cd~(2+)、Pb~(2+)、Ni~(2+)最小浓度分别为＞200mM、＞1200mM、＞500mM、＞60mM、10mM、10mM、10mM和3mM；能在含40mM Cu~(2+)、200mM Zn~(2+)和1mM Cd~(2+)组合，40mM Cu~(2+)、100mM Zn~(2+)和3mM Pb~(2+)组合，或者200mM Zn~(2+)、3mM Cd~(2+)和3mM Pb~(2+)组合的复合重金属盐的PDB液体培养基中生长。
在含重金属盐的水溶液中自然菌体表面对Cu~(2+)、Zn~(2+)吸附量分别达到26.19和258.3mg／g干菌体，而且吸附量远大于二者分别在菌体内的蓄积量。碱预处理的菌体对Cu~(2+)的吸附量达76.9mg／g干菌体。在Cu~(2+)、Zn~(2+)离子共存时，对Cu~(2+)的吸附量大于对Zn~(2+)的吸附量。
制备该菌原生质体的最佳菌龄为从孢子接种起培养24h。原生质体制备得率3.2×10~7个／ml，再生率达31％，原生质体的质粒转化效率为0.3个转化子／μgDNA。
用紫外诱变法获得了GXCR1的铜抗性水平明显降低的一株突变体MGXCR1。
A strain of Penicillium sp. GXCR1 was isolated from waste sludge out of the effluent channel in the mining area. The GXCR1 has very high copper resistance, and was susceptible to the Benomyl. The nucleotide sequences of ITS DNA of the GXCR1 shared more than 95% homology with 102 strains of P.spp. Its optimum growth temperature was at 32 , and the optimum growth pH was at pH 5.
On the PDA medium, the level of Cu2+resistance of the GXCR1 was 200mM, and even up to 800mM if in the presence of 5mM Mn2+.
In the liquid medium PDB with the added individual metal salt, the minimal concentration of the individual heavy metal inhibiting the GXCR1 growth was > 200mM Cu2+, >1200mM Zn2+,>500mM Al34 and 60mM Cr3+, 10mM Cd2+ ,10mM Pb2+ , 10mM Ni2+ and 3mM Cr6+ respectively; It could growth well in liquid medium PDB with one of the following heavy metal combinations: 40mM Cu2+, 200mM Zn2+ and 1mM Cd2+, 40mM Cu2+, 100mM Zn2+and 3mM Pb2+, or 200mM Zn2+, 3mM Cd2+ and 3mM Pb2+.
The biosorption capacity of heavy metals of the GXCR1 was up to 26.19mg Cu2+/g DW and 258.3mgZn2+/g DW respectively. The quantity of to Cu2+ and Zn2+ biossorption of the mycelium surfaces was much larger than that of both accumulation inside the cells of mycelia. The biosorption of mycelia pretreated with 0.2M NaOH was up to 76.9mg Cu2+/g DW. The Cu2+ biosorption quantity of mycelia was much larger than that of Zn2+ biosorption when under coexsistance of both metal salts.
The optimum mycelial age for preparation of protoplast was at 24h time-point after inoculation and culture of conidia. The rate of the prepared protoplast was 3.2 107/ml. The reproduction rate of the prepared protoplast was up to 31%. Efficiency of protoplast transformation with the plasmid was 0.3 transformants/ug DNA.
A strain of the mutant MGXCR1 with a much lower level of Cu resistance was selected by ultraviolet mutagenesis from the wild type strain GXCR1.
引文
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116．中华人民共和国国家标准：污水综合排放标准(GB 8978-1996)
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