發展快速抽取乳酸菌染色體DNA方法：Xanthogenate-SDS 法，及在選殖Lactobacillus rhamnosus TCELL-1丙胺酸消旋酶基因的應用

Use of the Xanthogenate-SDS to rapid isolate the genomic DNA from lactic acid bacteria and its application for cloning alr gene from Lactobacillus rhamnosus TCELL-1

陳茂全(Mao-Chuan Chen)

指導教授 ： 林志侯

國立清華大學/生命科學院/分子醫學研究所/碩士(2006年)

摘要

乳酸菌為革蘭氏陽性菌，細胞壁有較厚的肽聚醣，增加抽取染色體DNA的困難。傳統使用酵素法抽取乳酸菌染色體DNA，步驟繁瑣而耗時，並產生有毒有機溶劑廢棄物。本實驗發展Xanthogenate- SDS (XS)方法能夠快速抽取乳酸菌染色體DNA而不需要使用機械力或酵素。抽取的DNA能夠進行限制酶降解反應、PCR、基因選殖、含低的蛋白質含量、省時及花費低。但是以XS方法抽取Lactobacillus rhamnosus TCELL-1的染色體DNA時，產量過低；須先經過溶菌酶（終濃度10 μg/μL）反應15-30分鐘再進行XS法抽取(XSL法)後的產量才明顯提高。
以XSL法抽取Lb. rhamnosus TCELL-1染色體DNA後，對丙胺酸消旋酶基因alr進行選殖，得到843bp片段。轉譯成281個胺基酸。與其他乳桿菌胺基酸序列比對，與Lb. casei ATCC334 identity最高，達85.8 %。N端具ALR高度保留區序列AVVKANGYGH特徵，為與cofactor結合的位置。

關鍵字

乳酸菌 ； 鼠李糖乳酸桿菌 ； 快速抽取 ； 染色體 ； 丙胺酸消旋酶 ； 丙胺酸消旋酶基因

並列摘要

Lactic acid bacteria, groups of Gram positive bacteria, have thick peptidoglycan layers in their cell wall, which make the process of cell wall lysis in genomic DNA isolation difficult. A novel method, XS method to isolate genomic DNA from lactobacillus is introduced, which is different from conventional isolation protocol. This method is rapid, requires no enzymatic or mechanical cell disruption, nor multiple organic solvent extractions. Isolated DNA are proven can be used in various molecular biology analysis, such as PCR, restriction enzyme digestion, and cloning. Isolation of genomic DNA from Lactobacillus rhamnosus TCELL-1 using XS method faces the low yield problem, and hence, lysozyme treatment prior to XS treatment (XSL method) is introduced.
DNA isolated from Lb. rhamnosus TCELL-1 via XSL method are used in cloning of alr gene. The partial length of this gene is 843 bp, which can be translated into ALR protein with 281 residues. Comparison of deduced amino acid sequence reveals 85.8 % identity with that of Lb. casei ATCC334. A highly conserved region in N-terminal, AVVKANGYGH, is found.

並列關鍵字

lactic acid bacteria ； Lactobacillus rhamnosus TCELL-1 ； rapid isolation ； genomic DNA ； alanine racemase ； alr gene ； Xanthogenate SDS

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