胚芽乳酸桿菌無性狀質體pLP31-8之特性分析與應用

Characterization and Application of a Cryptic Plasmid pLP31-8 from Lactobacillus plantarum

黃文正(Wen-Zheng Huang) ； 陳正文(Zeng-Weng Chen) ； 王泓智(Hung-Chih Wang) ； 林俊宏(Jiunn-Horng Lin) ； 宣詩玲(Shih-Ling Hsuan) ； 王志鵬(Jyh-Perng Wang)

《臺灣農業化學與食品科學》 57卷5&6期 (2019/12) Pp. 240-251

https://doi.org/10.6578/TJACFS.201912_57(5_6).0005

摘要

胚芽乳酸桿菌（Lactobacillus plantarum）為一種兼性異質發酵之乳酸菌，常見於發酵蔬菜中，亦可分離自人類糞便、牛奶及乳酪。過去之研究指出，多數胚芽乳酸桿菌菌株帶有單個或多個不同大小之質體。本研究主要針對味增分離菌株L. plantarum ATIT-031之最小無性狀質體pLP31-8進行定序與特性分析。另亦進一步利用此質體建構新型穿梭載體，並分析穿梭載體之宿主範圍與其於宿主中之複製數。研究中，先針對L. plantarum ATIT-031中之最小質體pLP31-8進行分離與選殖。DNA定序結果顯示，pLP31-8之大小為1,753 bp，其中包含單股複製起始點（sso）、雙股複製起始點（dso）、複製蛋白質基因（rep）及複製數調控蛋白質基因（cop）序列，推測此質體為滾輪型複製質體。即時定量聚合酶連鎖反應測定結果顯示，pLP31-8於L. plantarum ATIT-031之複製數為1。之後，將EcoRI切位經突變之pLP31-8與大腸桿菌選殖載體pBRCM-MCS進行結合，建構成穿梭載體pBRLP31-8。電轉形之結果顯示，pBRLP31-8可轉形至胚芽乳酸桿菌（Lactobacillus plantarum）ATIT-018、鼠李糖乳酸桿菌（Lactobacillus rhamnosus）BCRC 16000、食竇魏斯氏菌（Weissella cibaria）ATIT-044及枯草桿菌（Bacillus subtilis）DB430中。即時定量聚合酶連鎖反應測定結果顯示，穿梭載體pBRLP31-8於ATIT-018、BCRC 16000、ATIT-044及DB430中之複製數分別為5、5、65及22。未來將進一步利用此穿梭載體進行上述宿主之表現系統開發。

關鍵字

胚芽乳酸桿菌 ； 無性狀質體 ； 穿梭載體

並列摘要

Lactobacillus plantarum is a facultative heterofermentative lactic acid bacterium, which is commonly found in fermented vegetables. L. plantarum can also be isolated from human feces, milk, and cheese. Previous studies have confirmed that most L. plantarum strains contain single or multiple plasmids of various sizes. In this study, the smallest cryptic plasmid of L. plantarum ATIT-031 isolated from miso, designated as pLP31-8, was sequenced and characterized. In addition, the plasmid was used to construct a new shuttle vector; then, the host range of the shuttle vector and its copy number in the host were analyzed. First, the smallest plasmid pLP31-8 of L. plantarum ATIT-031 was separated and cloned. The DNA sequencing result revealed that the size of pLP31-8 is 1,753 bp, encompassing single-strand origin (sso), double-strand origin (dso), replication-associated protein gene (rep), and copy number regulatory protein gene (cop). It is suggested that pLP31-8 replicates by the rolling-circle mode. The result of real-time quantitative polymerase chain reaction revealed that the copy number of pLP31-8 in L. plantarum ATIT-031 is one. Subsequently, the pLP31-8 with mutated EcoRI site was ligated with an E. coli cloning vector pBRCM-MCS to generate the shuttle vector pBRLP31-8. The results of electrotransformation revealed that pBRLP31-8 could be transformed into L. plantarum ATIT- 018, Lactobacillus rhamnosus BCRC 16000, Weissella cibaria ATIT-044, and Bacillus subtilis DB430. The results of real-time quantitative polymerase chain reaction revealed that the shuttle vector pBRLP31-8 had copy numbers of 5, 5, 65, and 22 in ATIT-018, BCRC 16000, ATIT-044, and DB430, respectively. The shuttle vector will be further used to develop expression systems for the aforementioned hosts.

並列關鍵字

Lactobacillus plantarum ； cryptic plasmid ； shuttle vector