Isolation and characterization of insertion sequences (IS) from Xanthomonas campestris pv. campestris

• Main Authors: Chin-Hong Wang, 王志宏
• Other Authors: Jiann-Hwa Chen
• Format: Others
• Language: zh-TW
• Published: 1994
• Online Access: http://ndltd.ncl.edu.tw/handle/34919823674274058538

碩士 === 國立中興大學 === 分子生物研究所 === 82 === Xanthomonas campestris pv. campestris (Xc) 為一革蘭氏陰性菌,可引 起十字花科蔬菜黑腐病, 本研究首先藉由質體pUCD800 長度變化來分離 Xc 內的轉位子 (Insertion sequence,以下簡稱 IS),並藉由南方雜配法 來探討 IS 在不同菌系內分布情形及與致病 (或抗病)機制間的關係.將攜 帶有質體 pUCD800 之 Xc 二品系 XC11 及 Xc17, 培養於 sucrose 培養 基中, 由於 pUCD800 帶有 sacB/R 基因, 而它的基因產物造成菌體在含 sucrose 的環境下 lysis. 因此, 可藉由在 5% sucrose培養基中篩選到 質體 pUCD800 上 sacB/R 發生變化的存活變異株. 以 Xc11(pUCD800), Xc17(pUCD800) 分別進行三次獨立的 independent culture 培養,經由限 制■圖譜 (restricrion enzemes mapping)方法, 總共可將插入的 DNA 片段分類為十二種,由於在分析過程中有六種插入片段其質體發生 DNA rearrangment, 故此處暫不予分析. 而分析其它六種插入片段, 發現均有 典型的相反重覆序列(inverted repeat) 及 target duplication. 目前 可根據其相反 重覆序列分為 typeI, typeII, 和 type III 三種.以這六 種 IS 為探針,分別與 Xc11, Xc17, 和一個 Xc11 自變性無病源性突變 種 Xc11A genomic DNA 進行南方雜配. 若以 typeI 或 type II 為 probe, 則 Xc17, Xc11 經 EcoRI, Hind III-digested 的 genomic DNA hybridization pattern顯示 type I 或 type II 與致病 (或抗病) 基因可能具有 linkage 的現象. 如以 type III IS 做 probe, 所得到 Xc11, Xc11A, Xc17 的 hybridization pattern 沒有差別. 顯示 type III 與致病 (或抗病) 基因可能不具有 linkage 的現象. Xanthomonas campestris pv. campestris (Xc) is a Gram-negtive bacterium that causes black rot of crucifers. It can also produce the exopolysaccharide xantham gum that is utilized broadly in the industry. In this study, we first isolate the insertion sequences (ISs) in Xc, and then localize the pathogenecity-related gene(s) to vicinity of certain ISs. The plasmid pUCD800 has been transfered into two strains of X. campestris, Xc11 and Xc17,and the surviving mutants were selected on 5% sucrose-containing medium. For each strain, we have collected three independent cultures. Based on the size and the restriction sites cantained in the inserted fragments, totally insertion fragments have been isolated and can be grouped into 12 groups. These six insertion fragment can be furture grouped into three types according to their terminal inverted repeat identical. Southern blot analysis using 32-p- lableled DNA fragments containing different types of IS as probes indicated that HindIII- and EcoRI-digested genomic DNA of Xc11, XC11A, an avirulent strain of Xc11, and Xc17. It appears that typeI and/or type II IS, but not type III, are linked to the pathogencity-related genes in Xc.