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Cloning and Functional Analysis of Lytic Enzyme Production Related Gene Ctp in Lysobacter Enzymogenes Strain OH11

Author: JiangShuZhen
Tutor: LiuFengQuan
School: Nanjing Agricultural College
Course: Plant Pathology
Keywords: Lysobacter enzymogenes OH11 Regulation of lytic enzyme Gene cloning Functional analysis
CLC: TQ925
Type: Master's thesis
Year: 2010
Downloads: 5
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Abstract


Lysobacter enzymogenes strain OH11 is isolated from cayenne-root soil, is reported for the first time as a bacterial biological control agent in China. The genus Lysobacter belongs to the family Xanthomonadaceae characterized with a high G+C content, gliding motility, has been shown to produce multiple lytic enzymes including chitinase, protease, cellulase andβ-1,3-glucanas. Furthermore, it displays in vitro activity against various plant pathogens, including fungi, oomycetes and nematodes, and has a broad application prospects. Although lytic enzymes (a-lytic protease, chitinase, (3-1,3-glucanase and cellulase) are proved to be the key components agaist various pathogens in L. enzymogenes, however, little is known about the regulatory mechanisms of lytic enzyme production. Thus, the objective of this study is to clone and analyze the function of lytic enzyme-production related genes from L. enzymogenes strain OH11.The mutant library of Lysobacter enzymogenes strain OH 11 was successfully constructed by mating mariner transposon into strain OH11. One mutant D-11, which reduced the activities of several lytic enzymes (a-lytic protease, chitinase, (3-1,3-glucanase and cellulase) was selected from over 5,000 chloramphenicol-resistant (Cmr) mutants. The transposon insertion site was identified as a ctp gene by sub-clone strategy. Sequence analysis showed that the gene was 2214bp with a 69% G+C content. The OH 11 ctp homology analysis indicated that the homoloty between OH 11 and Stenotrophomonas maltophilia strain was 79%, and between OH 11 and Xanthomonas campetris strain was 78%.A ctp deletion mutant Actp was constructed by homologue recombination technology. The following phenotype analysis revealed that, (Ⅰ) the ctp mutant strain Actp have the same growth rate of wild type OH11 in both nutrient-rich medium (2YT) and nutrient-deficient medium (MMX); (Ⅱ) compared to wild-type OH11,△ctp significantly reduced three-type lytic enzyme production (a-lytic protease, (3-1,3-glucanase and cellulase),but had the wild-type ability in chitinase production; (Ⅲ) comparaed to wild-type, the ctp mutant showed a obvious reduction of biofilm formation; (Ⅳ) in both LB ager plates containing carboxymethyl cellulose and laminarin, wild-type OH11 displayed a plumb and smooth colonies, wherease mutant Actp exhibited dry, crimple colonies. However, the ctp mutant displayed wild-type antimicrobial activity against Sclerotinia sclerotiorum, Rhizoctonia solani, and Phytophthora capsici. Meanwhile, all mutant phenotypes were restored in complemented strain Actp (pBBR-PBCTP). In conclusion, this study paved the way for studying the regulatory mechanism in L. enzymogenes.

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