Cloning of Chitinase Genes and Generation of DNA Fragments for the Specific Detection From Streptomyces saraceticus and S. parvulus

• Main Authors: Lih-Ren Kong, 宮力仁
• Other Authors: Chang-Hsien Yang
• Format: Others
• Language: zh-TW
• Published: 2000
• Online Access: http://ndltd.ncl.edu.tw/handle/92636732748580796984

碩士 === 國立中興大學 === 農業生物科技學研究所 === 88 === Saprophytic gram-positive bacteria in genus of Streptomyces have been shown to have characteristics useful for application as biocontrol agents to fight against soil-borne plant fungal pathogens. These characteristics include the production of enzymes such as chitinase to degrade fungal cell wall directly. Strain of S. saraceticus N45 and S. parvulus has been shown to harbor the high chitinase activity. For their potential use as biological control agent in the future, the cloning of chitinase genes and the development of a method to quickly and precisely detect their appearance become necessary. The cloning of chitinase genes from S. saraceticus N45 and S. parvulus was performed using PCR-based strategy. DNA fragments amplified from both species using specific degenerate primers showed significant homology to S. plicatus chi63 and S. lividans ChiC genes. Southern and PCR analysis indicated that these chitinase genes were located in the plasmid rather than in the main chromosome. These partial DNA sequences can be used to further clone the full-length sequence for these chitinase genes from both S. saraceticus N45 and S. parvulus. The cloned chitinase genes will be used in biological control in the future. For the detection of S. saraceticus N45, RAPD-PCR was used to amplify random DNA fragments from S. saraceticus N45 genome. Three DNA fragments, 417, 523 and 655 bp in length, were further isolated and sequenced. Southern analysis indicates that these three DNA fragments were specifically present in S. saraceticus N45 genome in a single or low copy manner and can be used to specifically identify S. saraceticus N45. Nest primers were further designed from both ends of these three fragments and used for PCR reactions. A single specific band was produced for each nest primer pairs amplified from genomic DNA of S. saraceticus N45. These three single bands are S. saraceticus N45 specific and could not be amplified from other species of Streptomyces or other bacteria such as Ralstonia solanacearum, Agrobacterium tumefaciens and E.coli. By the detection of the coexistence of these three fragments in various conditions, the appearance of S. saraceticus N45 can be ensured greatly. For the detection of S. parvulus, RAPD-PCR was used to amplify random DNA fragments from S. parvulus genome. One DNA fragment, 516 bp in length, was further isolated and sequenced. Southern analysis indicates that this DNA fragment was specifically present in S. parvulus genome in a single copy manner. Nest primer was further designed from both ends of this fragment and used for PCR reaction. A single specific band was amplified from genomic DNA of S. parvulus and could not be amplified from other species of Streptomyces or other bacteria such as Ralstonia solanacearum, Agrobacterium tumefaciens and E.coli.