植物乳杆菌中亚硝酸还原酶基因的克隆
关键词：亚硝酸盐；亚硝酸盐还原酶；植物乳杆菌；重组质粒
Cloning of Lactobacillus plantarum nitrite
reductase gene
Abstract: Nitrite is widely present in food, soil and water. Because the nitrite has good bacteriostatic effect, it is permitted as a food additive plays antiseptic effect. And because of its good hair color, nitrite is often used in meat products. Nitrite is an oxidizing agent, it may cause the cells lose the ability of combining with oxygen, and is a potential carcinogen. Therefore, the nitrite content exceed the standard problem has aroused great attention. L. Plantarum is the human probiotic that can produce nitrite reductase. Enzymatic degradation of nitrite is a safe and effective new way. In this subject, we use Lactobacillus plantarum strains that preserved in our laboratory to get nitrite gene sequence. We use TA cloning firstly, and transform it into E.coli DH5α. Through blue-white spot screening and double digests, target fragment would be available. Then construct plasmid pET-21a（+）-NiRs and transform it into E.coli BL21(DE3). The product we obtain could be used in the study of nitrite reductase gene of Lactobacillus plantarum.
Key words: Nitrite；nitrite reductase; Lactobacillus plantarum；Recombinant plasmid
目 录
1 绪论 1
1.1 亚硝酸盐简介 1
1.1.1 亚硝酸盐危害 1
1.1.2亚硝酸盐来源 2
1.2 国内外研究现状 3
1.3研究目的及意义 4
2. 材料 1
2.1 样品和菌种 1
2.2 实验材料 1
2.3主要仪器和设备 2
3．实验方法 3
3.1 查找目的基因碱基序列 4
3.2 培养基的配制 4
3.3 扩菌 5
3.4 接种 6
3.5 中间重组质粒构建 6
3.5.1 植物乳杆菌总DNA提取 6
3.5.2 亚硝酸还原酶基因的PCR扩增 7
3.5.3 TA克隆 8
3.6 DH5α感受态细胞制备 9
3.6.1 CaCl2法 9
3.6.2 快速制备法 9
3.7连接产物转化及蓝白斑筛选 9
3.8 重组质粒的构建 10
3.8.1 PET-21a（+）质粒及TA克隆产物的提取 11
3.8.2 PET-21a（+）质粒及TA克隆产物的双酶切 12
3.8.3酶切产物的纯化、大小片段割胶回收 12
3.8.4 连接反应 13
3.9 E. Coli BL21感受态细胞制备 13
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