植物青枯病原细菌胞外蛋白相关基因的克隆

 用含有转座子Tn5的铜绿假单胞杆菌PAO1826(pMO75∷Tn5,Km^r)诱变我国马铃薯青枯菌小种3号菌株PO41,获得6000多个胞外多糖正常产生的接合子。经SDS-PAGE电泳,筛选出2个胞外蛋白减少9种的突变株PM2644和PM239,其可以引起烟草叶片典型的过敏性反应,致病力比野生型菌株显著降低,皆为原养型菌株。Southern杂交的结果表明,突变株染色体上只有1个转座子插入位点。标记交换证明,9种胞外蛋白缺失与转座子插入相偶联的频率为100%。以聚半乳糖醛酸酶和内葡聚糖酶2种胞外酶为指示蛋白进行定量测定的结果表明,在突变株上清液中和菌体细胞内都没有这2种胞外酶的存在。以突变株PM2644为受体菌,通过基因功能互补的方法,从野生型青枯菌基因文库筛选到2个阳性克隆pPSP1和pPSP2,它们既能恢复2个突变株产生胞外蛋白的能力,也能将2个突变株的致病力恢复到接近野生型菌株的水平。

CLONING OF THE GENE RELATED TO EXTRACELLULAR PROTEINSSECRETED BY Ralstonia solanacearum

More than 6 000 transconjugants producing normal extracellular polysaccharide (EPS) were obtained when a wild type Ralstonia (Pseudomonas) solanacearum (PO41) race 3 was induced by Pseudomonas aeruginosa strain PAO1826 (pMO75::Tn5,Km^r) containing a transductant Tn5. Two mutants, PM2644 and PM239 that were defective in 9 kinds of extracellular proteins (EXPs) were selected by screening the transconjugants on SDS PAGE. These two mutants were prototrophic type and could cause hypersensitive reaction on tobacco leaves. However,their virulence decreased greatly in comparison with wild type. Southern blot hybridization showed that both mutants contained only one insertion site in their chromosomes. Marker exchange mutagenesis indicated that like PM2644 and PM239,100 percent of Km resistant transformants were defective in 9 kinds of EXPs.Activity determination of polygalacturo nase and endoglucanase showed that these two enzymes were absent in culture filtrate and bacteria cells of mutants. Two clones (pPSP1 and pPSP2)restored all deficient EXPs and their lost virulence were screened out from genomic library of wild type strain PO41 by gene function complementation test.