【作者】 唐华英；

【导师】 李有志；

【作者基本信息】 广西大学 ， 微生物学， 2006， 硕士

【摘要】 重金属是生物体必需的元素，然而过高的浓度会对有机体产生毒性。在真核和细菌中通过各种机制达到对重金属的抗性。铜转运蛋白P型ATP酶在真核生物中广泛存在，在胞内起着转运铜的作用，以达到对铜的抗性。 根据已报道的与铜转运P型ATP酶同源的氨基酸序列，设计简并引物，在丝状真菌Penicillium janthinillum GXCR中扩增得到与铜转运P型ATP酶基因同源的部分序列。构建了该基因RNA干扰表达载体pCPXBn-ihp1，采用PEG/Cacl₂介导的方法转化GXCR原生质体，经过PCR检测、Southern杂交分析证实获得三个干扰突变体，对它们进行实时荧光定量PCR分析表明铜转运P型ATP酶基因表达水平降低，分别是野生型的24.92％、40.23％和32.89％。对突变体进行抗铜水平检测发现抗Cu²⁺能力与野生型对照有下降，表明铜转运P型ATP酶基因在丝状真菌微紫青霉菌Penicillium janthinillum GXCR的抗铜能力上起一定作用。更多还原

【Abstract】 Although many heavy metals are essential, high heavy metal concentrations are toxic to organism. Resistance to high heavy metal concentrations is achieved by a variety of mechanisms both in bacteria and in fungi. Copper-transporting P-type ATPases are found in eukaryotes as well, where they play a major role in intracellular copper transport.DNA sequence which was partial homogeneous with Copper-transporting P-type ATPase gene was amplified from filaceous fungi Penicillium janthinillum GXCR by designed degenerate primers based on the reported amino acid sequence. A RNAi expression vector pCPXBn-ihp1 was constructed aimed at interfering the copper-transporting P-type ATPase gene of Penicillium janthinillum GXCR and then transfered into protoplasts of GXCR with PEG/CaCl₂ method. Three mutants were identified by PCR and Southern hybriding blot. The expression level of Copper-transporting P-type ATPase genes of the three mutants decreased by 24.92%、 40.23% and 32.89%, respectively, measured with real-time fluorescence quantitative PCR （Q-PCR） . Compared to the wild type, the mutants’ resistant capacity to Cu²⁺ declined and indicates that the Copper-transporting P-type ATPase gene plays an important role in resistance to heavy metal ion such as Cu²⁺ in filaceous fungi Penicillium janthinillum GXCR更多还原