【作者】 王伟；

【导师】 赵斌；

【作者基本信息】 华中农业大学 ， 微生物学， 2008， 硕士

【摘要】 基因cadA1编码的是P型ATP酶，它是一种能够对细胞内重金属离子起到平衡调节作用的运输子。但是在恶臭假单胞菌（Pseudomonas putida）中，cadA1在抗重金属方面的功能还知道的很少。本研究的目的就是为了确定cadA1是否与P.putida CD2的重金属抗性相关。本研究构建了P.putida CD2的cadA1中断突变株，然后通过测定该突变株对不同重金属的抗性能力来研究cadA1的功能，将中断突变株对Cd²⁺、Zn²⁺、Co²⁺、Pb²⁺、Cu²⁺和Mn²⁺6种重金属的最小抑制浓度（MIC）进行了测定。结果显示，与野生型相比，MIC值并未有所变化。这表明了cadA1中断突变株与野生型对这6种重金属的抗性能力不存在差异。为了确定cadA1是否与重金属的抗性有关，也将cadA1在对重金属敏感的P.putida CD2突变株中进行了过量表达，观察细菌对重金属的抗性水平是否有提高。结果显示cadA1的过量表达能够提高P.putida CD2的czcCBA1突变株对Zn²⁺的抗性能力。这说明了CadA1能够提供对Zn²⁺的抗性，但这样一个结论却没有在cadA1的中断实验中得到证实。这给我们的启示是：cadA1在P.putida CD2中确实是有抗重金属的功能，但是它可能受到了细胞内某些因子的调控从而不能施展其自身的能力。本研究还利用绿色荧光蛋白测定的方法来初步研究了cadA1的表达调控。通过构建基因报告质粒pEGP-PCAD，然后利用它来监测恶臭假单胞菌中（野生型菌株，czcCBA1突变株，cadA2突变株）重金属对cadA1启动子的表达量的影响。结果显示cadA1启动子的表达只能够在P.putida CD2-2（也就是czcCBA1突变株）中被诱导，而且只被Zn²⁺诱导。这些也就说明了在P.putida CD2中，cadA1的表达只能被Zn²⁺诱导，而且CzcCBA1的存在会抑制这个诱导作用。结合上述三个方面的结果，最后确定了cadA1的功能：cadA1能够提供对Zn²⁺的抗性，但是它只是在P.putida CD2中，当czcCBA1发生了突变时才会大量合成蛋白CadA1，以此来作为细胞中的一个补救途径抵抗Zn²⁺。在一般情况下，由于CzcCBA1的存在，CadA1的量非常之小以至于它在细胞对重金属的解毒中根本起不到任何作用。据我们了解，本研究是第一次证明了在P.putida CD2中P型ATP酶基因cadA1与Zn²⁺的抗性有关。更多还原

【Abstract】 The cadA1 gene encodes a P-type ATPase in P. putida. The P-type ATPases are a family of transporters that mediate the uptake and extrusion of heavy metal ions. But little is known about the function of cadA1 in heavy metal resistance. The aim of the present work is to determine if the cadA1 gene is involved in tolerance to heavy metal ions in P. putida CD2.In this study, a mutant strain of P. putida CD2 was constructed by disrupting cadA1. The function of cadA1 was studied by determining the resistance of the cadA1 disrupted strain to different heavy metal ions. But no difference in the MIC was observed between P. putida CD2-1 and CD2. It was shown that the cadA1 disrupted strain did not change the ability in resistance to Cd²⁺、Zn²⁺、Co²⁺、Pb²⁺、Cu²⁺ and Mn²⁺ compared with the wild type.For determining the effect of CadA1 on heavy metal resistance, the cadA1 gene was A1so overexpressed in heavy metal-sensitive mutants of P. putida CD2. The overexpression of cadA1 increased resistance to Zn²⁺ in the czcCBA1 disrupted mutant of P. putida CD2. It indicates that the cadA1 gene confers Zn²⁺ resistance which has not been proven by disrupting cadA1, suggesting that the cadA1 gene is functional, but may be regulated by some unknow factors in P. putida CD2 that it cannot exert its function in heavy metal resistance.Further more, the regulation of cadA1 expression was studied by GFP fluorescence assay. A reporter plasmid pEGP-PCAD was constructed and used to monitor the expression of PcadA1 in response to heavy metal ions in P. putida CD2、CD2-2 and CD2-3. It showed that PcadA1 was specific, responding at a significant level to Zn²⁺ in P. putida CD2-2. But there was A1most no expression of PcadA1 in P. putida CD2 or CD2-3 when induced by heavy metal ions. It reveals that expression of cadA1 is induced by Zn²⁺ and this induction is inhibited by the presence of CzcCBA1 in P. putida CD2.In combination of the results obtained, it was demonstrated that cadA1 can confer resistance to Zn²⁺, but the level of CadA1 in P. putida CD2 is so low because of the presence of CzcCBAl that it contributes little to Zn²⁺ resistance. However, expression of cadA1 would increase to serve as a salvage pathway for Zn²⁺ resistance when czcCBA1 disrupted. To the best of our knowledge, this work has provided the first evidence that the cadA1 gene confers Zn²⁺ resistance in P. putida CD2.更多还原