酵母耐铜基因CUP1改良荧光假单胞菌耐铜性研究初报

利用根际促生菌（PGPR）荧光假单胞菌（Pseudomonas fluorescens）等微生物的生物修复作用可治理土壤重金属污染。酿酒酵母耐铜基因CUP1编码铜金属硫蛋白（Cu-MT），对铜有很高的亲和性，可拮抗重金属铜毒性。为提高荧光假单胞菌的生物修复能力，将酿酒酵母耐铜基因CUP1克隆至荧光假单胞菌。通过引物设计，将SD序列添加至酵母耐铜基因CUP1编码序列前端，PCR扩增得到约200bp的CUP1片段，克隆至测序载体pUC19，经测序证明正确后，胶回收CUP1片段连接到具有广泛宿主范围的柯斯质粒pLAFR3上，经三亲接合转移至荧光假单胞菌AS1.1381，得到重组菌株AS1.1381/pL3CUP1。铜实验表明，重组菌株AS1.1381/pL3CUP1的抗铜能力（6mM Cu2+）显著高于AS1.1381/pLAFR3（4.5mM Cu2+），重组菌株抗铜性得到提高

Preliminary Report on Copper Resistance of Pseudomonas fluorescens Modified with Yeast CUP1 Gene

Bioremediation with the microbe such as Pseudomonas fluorescens,a plant growth-promoting-rhizobacteria(PGPR),could be used to control heavy metal polluted soils.The copper-resistant gene CUP1 of bake yeast encodes copper metallothionein(Cu-MT)which possesses special affinity to copper ions.In this work,in order to improve the ability for bioremediation,the CUP1 gene was cloned into P.fluorescens.The SD sequence mediated by primer was added ahead of the CUP1 gene.The fragments of about 200 bp were amplified and cloned into the sequencing vector pUC19.After confirmed by sequencing,the fragments harboring CUP1 gene was gel-extracted and cloned into the wide-range-host cosmid pLAFR3,and then transformed into P.fluorescens AS1.1381 by tri-parents mating,generated the recombined strain AS1.1381/pL3CUP1.Copper resistance assay showed that the recombined strain AS1.1381/pL3CUP1 had higher copper resistance(6mM Cu2 )than the wide strain ASl.1381/pLAFR3(4.5mM Cu2 ).