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1、摘要目的：解淀粉芽孢杆菌在自然环境中广泛分布，易于分离和培养，对人类和人类无毒无害。其代谢物丰富，具有广谱抗菌活性，抗逆性强，生长快，稳定性好，在植物病害的防治中具有多种有益作用，非常适合生物的大规模生产和应用。本研究基于实验室中先前的实验并使用现有的质粒pKSV7，本研究尝试选择合适的反向筛选标记以使无痕基因操作成为可能，并为解淀粉芽孢杆菌C10的遗传修饰提供强大的工具。方法：相关研究显示，在菌体内体外胸腺嘧啶类似物5-FU可以生成有毒物质5-dUMP，利用这一原理可以敲除解淀粉芽孢杆菌C10内的upp基因，之后获得C10upp这一突变菌株，基于这一底盘细胞可以进行自杀型基因敲除载体pKSU
2、-amyA的构建，此载体内含有upp基因，并成功敲除了C10中的amyA基因片段。结果：1. 构建的pKSV7-upp载体经酶切以及测序确认载体构建成功。在探索解淀粉芽孢杆菌C10的电转化条件后，确定通过LB培养基过夜培养的解淀粉芽孢杆菌 C10在LBS培养基（每升水0.5 M山梨糖醇，10克氯化钠，10克胰蛋白胨和5克酵母提取物）中转接，同时添加0.64%甘氨酸溶液，0.05%Tween 80（w/v）溶液，1.02%DL -苏氨酸溶液，32 振荡培养至 OD600=0.9。重悬感受态细胞时用MSG缓冲液（0.5 M山梨糖醇，0.5 M甘露糖醇和10甘油）。电转化时预设电压为2000V，电击
3、时间为5ms,电击后加900 uL复苏培养基（LB培养基里加0.5 M山梨醇，0.38M甘露醇），180rpm 32复苏3h，涂布含Cm的LB平板。2. 经过对upp基因敲除菌株C10upp菌株和原始菌株 C10进行了生长曲线测定，发现upp基因的缺失对菌株生长速率没有影响。以此为依据可以在解淀粉芽孢杆菌的遗传学改造过程中将upp基因作为菌株的反向筛选标记基因。3. 在pKSV7载体中移入upp基因，再一次将含有upp表达盒的反向筛选质粒 pKSU通过接合转移导入解淀粉芽孢杆菌C10upp。将重新导入upp基因的突变菌株C10upp（pKSU）和突变菌株 C10upp在 0.35mM的5-FU
4、的平板上涂布，在此平板上含有upp基因的突变菌株C10upp（pKSU）出现生长异常，这表明含有upp基因的菌株对5-FU具有较高的敏感度，这一现象也从侧面表明了在对解淀粉芽孢杆菌进行遗传学改造时，upp基因可以作为一种反向标记基因。4. 构建敲除载体pKSU-amyA，经过电转化进入突变菌株C10upp，经过氯霉素与5-FU敏感性检测后，得到突变菌株C10uppamyA。同时对突变菌株C10uppamyA进行了淀粉酶水解实验。实验结果显示，突变菌株C10uppamyA由于-淀粉酶基因的缺失，不能形成水解圈。实验的成功证实了解淀粉芽孢杆菌C10中基因敲除体系的成功建立，为后续在解淀粉芽孢杆菌C
5、10中的遗传操作奠定了基础。关键词：解淀粉芽孢杆菌；载体构建；upp；基因敲除AbstractObjective: Bacillus amyloliquefaciens is widely distributed in the natural environment, easy to isolate and culture, non-toxic and harmless to humans and humans. It is rich in metabolites, has broad-spectrum antibacterial activity, strong stress resista
6、nce, fast growth, and good stability. It has a variety of beneficial effects in the control of plant diseases and is very suitable for large-scale production and application of organisms. This study is based on previous experiments in the laboratory and uses the existing plasmid pKSV7. This study at
7、tempts to select suitable reverse screening markers to enable traceless gene manipulation and provides a powerful tool for genetic modification of Bacillus amyloliquefaciens C10 .Methods: Using the principle of 5-FU, a thymine analog in vitro, to produce 5-dUMP, which is toxic to bacteria, the upp g
8、ene in Bacillus amyloliquefaciens C10 was knocked out to obtain a mutant strain C10upp. Based on this chassis cell, The suicide gene knockout vector pKSU-amyA with the upp gene was successfully knocked out of the amyA gene fragment in C10.Results:1. The constructed pKSV7-upp vector was successfully
9、digested with enzyme digestion and sequencing. After exploring the electrotransformation conditions of Bacillus amyloliquefaciens C10, it was determined that Bacillus amyloliquefaciens C10 cultured overnight in LB medium in LBS medium (0.5 M sorbitol per liter of water, 10 g sodium chloride, 10 g tr
10、yptone and 5g yeast extract), while adding 0.64% glycine solution, 0.05% Tween 80 (w / v) solution, 1.02% DL-threonine solution, shake culture at 32 until OD600 = 0.9. Resuspend competent cells with MSG buffer (0.5 M sorbitol, 0.5 M mannitol and 10% glycerol). The preset voltage during electrotransf
11、ormation is 2000V, and the electric shock time is 5ms. After the electric shock, 900 uL recovery medium (0.5 M sorbitol and 0.38M mannitol in LB medium) is added, and the cells are recovered at 180 rpm and 32 C for 3 hours. flat.2.After determining the growth curve of the upp gene knockout strain C1
12、0upp and the original strain C10, it was found that the deletion of the upp gene has no effect on the growth rate of the strain. Therefore, the upp gene can be used as a reverse screening marker gene to genetically modify Bacillus amyloliquefaciens.3.By constructing the pKSV7 vector containing the u
13、pp gene, the reverse selection plasmid pKSU containing the upp expression cassette was once again introduced into Bacillus amyloliquefaciens C10upp by conjugation transfer. The mutant strain C10upp (pKSU) and mutant strain C10upp that reintroduced the upp gene were coated on a 0.35 mM 5-FU plate, an
14、d C10upp (pKSU) could not grow normally on a 5-FU plate, indicating that the upp gene replenished strain was recovered The sensitivity to 5-FU was proved, and the upp gene was proved to be the feasibility of reverse marker gene in the genetic modification of Bacillus amyloliquefaciens.4.Construction
15、 of the knockout vector pKSU-amyA, which was transformed into the mutant strain C10upp by electrotransformation. After the sensitivity detection of chloramphenicol and 5-FU, the mutant strain C10uppamyA was obtained. At the same time, amylase hydrolysis experiments were performed on the mutant strai
16、n C10uppamyA. The experimental results showed that the mutant strain C10uppamyA could not form a hydrolysis circle due to the deletion of the -amylase gene. The success of the experiment confirmed the understanding of the successful establishment of the gene knockout system in Bacillus amyloliquefaciens C10, which laid the foundation for subsequent genetic manipulation in Bacillus amyloliquefaciens
