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KMID : 0903519950380040313
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Journal of the Korean Society of Agricultural Chemistry and Biotechnology
1995 Volume.38 No. 4 p.313 ~ p.319
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Clonig of CM - cellulase Gene of Rhizobium meliloti TAL1372 in Escherichia coli
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Abstract
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The involvement of the cell-wall degrading enzymes in Rhizobium has long been an unsolved question about the infection process in the formation of root nodule. To assess the contribution of the cellulase to the nodulation of rhzobia, here we report the production of cellulase from R. meliloti TAL1372 which degrade carboxymethylcellulose (CMC) model substrate with CMC-plate method. We constructed a genomic library by cloning Sau3A-digested genomic DNA from R meliloti TAL1372 into the BamHI site of the cosmid vector pLAFR3. Out of more than one thousand transductants of E. coli, one clone (pRCB-71) had CM-cellulose activity and contained pLAFR3 cosmid with 30 kb insert of R. meliloti DNA. The product of CM-cellulose gene was analyzed by native PAGE. About 45 kD protein was considered to be a product of the gene. Tn5 mutagenesis reveals that the structural gene located in a ca 3kb KpnI fragment. The cellulose-minus mutants of R. meliloti TAL1372 were obtained by Tn5 mutagenesis of pRC8-71 and marker exchange techniques. Analyses of the nodulation ability of these Tn5 mutants showed that the CM-cellulose gene of R. meliloti TAL1372 may be involved in early nodulation development on alfalfa.
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