绿色荧光蛋白标记荧光假单胞菌P303及其生存能力检测  

Labeling of Pseudomonas fluorescens P303 strain with green fluorescent protein and monitoring of its survival ability

作　　者：张霞[1] 张杰[1] 李国勋[2] 黄大昉[3] 陈中义[1] 

机构地区：[1]中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室,北京100094 [2]河北农业大学植物保护学院,保定070001 [3]中国农业科学院生物技术研究所,北京100081

出　　处：《植物保护学报》2005年第3期280-286,共7页Journal of Plant Protection

基　　金："973"计划项目(2001CB109005)资助

摘　　要：构建了含有荧光假单胞菌自身启动子PP303的中间质粒载体pGFP.然后利用mini-Tn5转座子,通过接合转移,将两个带有不同启动子的绿色荧光蛋白基因分别整合到荧光假单胞菌P303染色体上,获得了在488nm波长下发光稳定的P303m1和P303m3菌株.PCR鉴定和Southern印迹结果均证明绿色荧光蛋白已随机插入P303染色体.SDS-PAGE结果表明,含有PA1/04/03启动子的P303m3菌株GFP表达量低于含有PP303启动子的P303m1菌株,染色体标记的GFP表达量低于质粒标记.室内平板抑菌试验结果表明,P303m1与出发菌株P303抑菌活性相当,对九种植物病原真菌有较强的拮抗作用.定殖、生存竞争能力研究表明,荧光假单胞菌在自然土壤中和大白菜根际都具有较强的定殖能力.P303和P303m1在自然土壤中第60天的菌量分别为1.63×104和3.3×102cfu/g土(湿重),大白菜根际第50天的菌量分别为3.29×106和4.1×104cfu/g根(湿重).Abstract： The vector pGFP containing PP3o3 promoter was constructed. Then plasmid pJBA28 and pGFP were transformed into E. coli S17-1/λπ respectively, and sequently the conjugation was carried on. S17- 1λπ （pJBA28） and S17-1λπ （pGFP） were donor strains, and P303 was recipient strain. The conjugants P303ml and P303m3 which fluoresced steadily under 488nm were acquired. Both PCR and Southern blotting analysis demonstrated that gfp gene had been inserted into P303 chromosomal DNA randomly. SDS-PAGE assay indicated that the expression of GFP in P303m3 （ carried PAl/04/03 ） was weaker than in P303ml （ carried PP3o3 ） , and the expression of gfp gene located in the chromosome was weaker than in the shuttle plasmid. The growth curve of P303ml certified that it was the same growth speed as the wild strain P303. Genetic stability result showed that the stability of gfp gene remained 100% after 96 h. P303ml remained strong antifungal activity against seven kinds of plant pathogenic fungi as P303. The survival and colonization of P303 and P303ml were investigated by selective culture and PCR identified methods. The results showed that the ability of colonization of P303 and P303ml in the soil and rhizosphere was much stronger than that around phyllosphere. The total population of P303 and P303ml were 1.63×10^4 and 3.30 ×10^2 cfu/g soil （wet weight） in the 60 days after incubation in natural soil, and 3.29×10^6 and 4.1×10^4 cfu/g roots （wet weight） in the 60 days in the rhizosphere of cabbage respectively.

关 键 词：荧光假单胞菌 绿色荧光蛋白 接合转移 SOUTHERN杂交 定殖 绿色荧光蛋白标记 竞争能力 绿色荧光蛋白基因 SOUTHERN 检测 

分 类 号：S476[农业科学—农业昆虫与害虫防治]