| Eucalyptus robusta, one of key economic tree species in China, due to a great deal of chemical fertilizer had to be applied to meet the nutrition requirements in the life cycle, its industry had exerted substantial negative impact on the environment.Owing to the ability of forming associative nitrogen fixation system with various plants, Diazotroph was considered a very promising bacterial flora.By sequencing the variable region of 16S rRNA gene after PCR (accession number was KM460926.1 in NCBI database) as well as combining the Biolog microbial identification system with the conventional methods, a free-living nitrogen fixing bacteria (isolated formthe soil of Eucalyptus plantations and provisionally nominated as KO108) was classified as Klebsilla oxytoca, a strain in the genus of Klebsilla which was belong to Enterbateriaceae,with the nature of Gram-negative and acid-producing.Drug resistance of KO108 was tested with the disc agar diffusion method. The results showed that it was sensitive to nitrofurantoin, streptomycin, ceftriaxone, kanamycin, ofloxacin, doxycycline, ciprofloxacin, chloromycetin; and moderately sensitive to cotrimoxazole but resistant to polymyxin, penicillin G, tetracycline, erythromyc. In particular, the diameter of inhibition zone of penicillin G against it was zero.In this study, the mutants of KO108 were obtained by random insertion with transposon mTn5gusA-pgfp21, and basing on the screening of mutants with ability to colonize on the surface of root of E.robusta, a mutually beneficial growth-promoting system of woody-plant-nitrogen-fixing-bacteria was tried to establish.The mutant library of KO108 was constructed by bi-parent conjugation of KO108 with Escherichia coli S17.1, and by this method, transposon mTn5gusA-pgfp21 in E. coli S17.1 was introduced into KO108 then randomly inserted in to the genetic code, therefore, mutants tagged with Kan resistance, GUS and GFP activity was obtained. The quality of the KO108 mutant library was assessed by Southern blot with the gfp (750 bp) as a probe against any mutant in the library and the results showed that insertion of transposon mTn5gusA-gfp21 in the K. oxytoca genomes was single, this verified good randomness for the mutant library.Mutants with E. robusta root-colonizing property were screened by co-culturing of E. robusta and mutants, and only two mutants, named as MA and MD respectively, meet the requirements, however, the parent of this tow mutants, wild KO108, did not.The flanking sequence of mTn5gusA-pgfp21 integration site in the genome of MA and MD were verified by inverse PCR, with primers designed from the terminal part of GUS1 and IS50R in mTn5gusA-pgfp21 and templates derived from MA and MD genome after digesting with Sau3A I then self-ligating.Two fragments of 671 bp and 195 bp were obtained by the above mentioned methods and they were further analyzed using basic local alignment search tool(BLAST).The results showed that, at the nucleotide level, fragment of MA had 87% was identity with NADP(H) quinone oxidoreductase gene and that of MD was 99% with the gene cluster of lipopolysaccharide synthesis in K.oxytoca.The 1329 clones genomic library of KO108 was constructed by Epicentre pWEBTM Cosmid Cloning Kit. Its coverage and randomness were confirmed by the digestion profiles with EcoR I of 20 plasmids in 20 clones randomly selected from the library. The results showed that all tested plasmids contained the 8.179 bp cosmid fragment and an exogenous one, but the later was diverse in size, with an average of about 40 kb, the maximum 50 kb and minimum 25 kb, which suggested that the total capacity of this library was about 52 Mb,5 times of KO108 genome, 99.8% theoretical possibility to encompass any gene in this strain.By dot blotting,3 clones containing NADP(H) quinone oxidoreductase gene was screened from genomic library of KO108. This created a foundation for finding out the relationship between the NADP(H) quinone oxidoreductase gene and the specific of KO108 colonizing on the root surface of E. Robusta. |
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