霍乱弧菌基因组中非编码DNA功能的初步研究
关键词：霍乱弧菌；非编码DNA；毒力基因
The Basic Research of Non-coding DNA in Vibrio cholera
Abstract： Cholera is a acute intestinal infection deadly infectious disease caused by Vibrio cholerae , which attack rapidly and spread quickly。 Study found that a large fraction of the sequence of non-encoded proteins in the genome of Vibrio cholerae accounted for more than 15% percent of the total genome of Vibrio cholerae。 Therefore, we focus on the size of more than 1000kb, somehow conservative non-coding DNA sequences, try to figure out whether these non-coding sequence have some unknown and important role。 Junker(http://pranag。physics。iisc。ernet。in/junker/) is a professional website to find the interval sequences in the genome。 We used the website to successfully construct four knockout strains, named ∆VC0388。5、∆VC0548。5、∆VCA0390。5、∆VCA0884。5。Through the related physiological experiment, we finally found that the lack of VC0388。5 can lead to obvious growth defects of Vibrio cholerae, the lack of VC0548。5 can lead to the large expression of main virulence genes tcpA。
Key words: Vibrio cholerae; non-encoded DNA; virulence genes
目 录
摘要 3
关键词 3
Abstract。 3
Key words 3
绪论 4
1 材料 5
1。1 供试菌株与质粒 5
1。2 培养基与培养条件 6
1。2。1 培养基配方 6
1。2。2 抗生素与浓度 6
1。3 实验用酶及试剂 6
2 方法 6
2。1 霍乱弧菌及大肠杆菌高效电转化感受态细胞的制备（以大肠杆菌为例） 6
2。2 重组载体及酶连产物的电转化 6
2。3 二亲接合 6
2。4 ∆VC0388。5、∆VC0548。5、∆VCA0390。5、∆VCA0884。5敲除菌株的构建 7
2。4。1 总基因以及质粒的提取 7
2。4。2 利用PCR及over-lapping 得到间隔序列同源臂片段 7
2。4。3 重组质粒的构建 7
2。4。4 ∆VC0388。5、∆VC0548。5、∆VCA0390。5、∆VCA0884。5敲除菌株的筛选 7
2。5 构建霍乱弧菌主要毒力基因tcpA 的冷光表达菌株 7
2。6 生长曲线的测定 7
2。7 敲除株对过氧化氢（H2O2）敏感性检测 7
2。8 生物膜实验 8