摘 要

本次试验使用一种称作LFH-PCR的方法使解淀粉芽胞杆菌FZB42中的Hfq基因发生突变。LFH-PCR通过把从质粒上获得的氯霉素抗性基因（Cat基因）与从菌株FZB42的基因组上获得的Hfq上下游两段同源序列连接起来而起作用的。第一步是Hfq上下游两段同源序列的扩增，而其中能与氯霉素抗性基因（Cat基因）有二十几个互补配对的碱基是通过引物添加上去的。第二步是，以氯霉素抗性基因（Cat基因）作为模版，含有与Cat基因互补配对的碱基的Hfq上下游两段同源序列作为长引物进行扩增从而获得连接产物。最后通过把LFH-PCR产物转化入FZB42中，通过同源重组，使标记基因Cat基因插入到Hfq基因中从而使Hfq基因发生突变。

关键词：LFH-PCR ；Hfq基因；同源重组

Using LFH-PCR to synthetize the Cat Marker Cassettes with Long Flanking Homology Regions to knock out the Hfq Gene of Bacillus amyloliquefaciens

ABSTRACT

This experiment is to knock out the Hfq gene of Bacillus amyloliquefaciens FZB42,using a PCR-method called LFH-PCR.This method works through linking two PCR fragments from genomic DNA of FZB42 to Cat gene,a resistance marker making FZB42 resistant to chloramphenicol.The first step was to amplify two long flanking homology DNA fragments of Hfq gene.And about 20 base pairs extensions homologous to the Cat marker were added to these two long flanking homology DNA fragment by special-designed primers.The second step was to get the joining PCR product,using the Cat marker as template and those two long molecules as two long primers.Finally,the Cat marker was to be added into the Hfq gene through homology recombination after the transformation of LFH-PCR-generated disruption cassette to FZB42.Thus,the Hfq gene was to be broken.

Key Words: LFH-PCR; Hfq(Host Factor Qbeta) gene; Homology Recombination

目 录

1综述.............................................................................1

1.1引言.........................................................................1

1.2 Hfq在细胞层面的功能.........................................................1

1.3 Hfq在sRNA调控中作用........................................................2

1.4 Hfq功能的结构基础...........................................................3

1.5 Hfq行使功能的机制...........................................................4

1.6 本实验的目的及意义...........................................................5

2材料与方法.......................................................................5

2.1 CTAB法提取FZB42基因组......................................................5

2.1.1试剂.....................................................................5

2.1.2操作步骤.................................................................5

2.2 LFH-PCR引物设计.............................................................6

2.3 连接产物的扩增...............................................................7

2.4 连接PCR.....................................................................9

2.5 转化........................................................................10

2.5.1 10*MC 试剂配制..........................................................11

2.5.2 操作步骤................................................................11

2.6 使用菌落PCR筛菌............................................................12

3 实验结果与分析..................................................................12

3.1 连接产物的扩增结果..........................................................13

3.2 连接PCR扩增的结果..........................................................15

3.3 菌落PCR结果................................................................16

4 致谢............................................................................16

5 参考文献........................................................................16

1综述

1.1引言

RNA伴性分子Hfq最初是于1968在大肠杆菌E.coli中被发现的[1]。最初它被认为是噬菌体QB进行有效复制而必需的一种当家因子（host factor），它的名字也是由此得来[2,3]。Hfq是RNA结合蛋白质家族的一员，而这种蛋白质家族在真核生物，原核生物和古细菌中都有发现[4]。因此在真核生物中存在Hfq的类似物也是意料之中的。举例来说，Hfq被认为与在真核生物中参与mRNA剪切与降解活动的Sm蛋白质与Sm-like蛋白质同源[5]。这类蛋白质家族的特征是能形成结合RNA的环状复合物。真核细胞中的Sm蛋白质一般形成异聚环，而细菌中的hfq到目前为止，都被发现是形成同聚环[3]。近期研究发现Hfq在细菌的生命活动中发挥着重要的作用，下面首先从细胞层面介绍Hfq的功能。