目录
摘要4
关键词4
Abstract5
Key words5
引言
1 实验材料7
1.1 实验试剂与实验器材7
1.2 菌株和质粒 7
1.3 培养基和菌株生长条件7
1.3.1 培养基的配置7
1.3.2 抗生素8
1.3.3 培养方法及保存条件8
2 实验方法9
2.1 获取Pf5菌株RsmX/Y/Z基因缺失突变体9
2.1.1 引物的设计与合成 9
2.1.2 Pf5菌株基因组的提取 9
2.1.3 PCR扩增反应 10
2.1.4 DNA片段的纯化回收 10
2.1.5 转化与连接 12
2.1.6 三亲交配法转化 14
2.1.7 菌落PCR验证 14
2.2 检测Pf5菌株∆rsmX、∆rsmY和∆rsmZ突变体表现 15
2.2.1 群集运动的检测15
2.2.2 脂肽化合物排油及含量检测15
2.2.3 突变体产生orfamide A对番茄青枯病菌的抑菌作用16
2.2.4 溶血实验16
3 结果与分析16
3.1 三个小RNA突变载体的构建17
3.2 Pf5菌株小RNA突变体的验证17
3．3 Pf5菌株的∆rsmX、∆rsmY和∆rsmZ突变体的群集运动能力的检测17
3.4 Pf5菌株的∆rsmX、∆rsmY和∆rsmZ突变体脂肽类粗提物活性检测18
3.5 sRNA突变体的orfamide A的抑菌作用结果 18
3.6 溶血实验结果19
3.7 Pf5菌株的∆rsmX、∆rsmY和∆rsmZ突变体抗菌脂肽orfamide A的检测.19
4 讨论20
致谢21
参考文献22
小RNA调控生防假单胞菌Pf5菌株抗菌脂肽orfamide A *51今日免费论文网|www.51jrft.com +Q: ^351916072# 
作用研究
摘 要
生防假单胞菌（Pseudomonas spp.）Pf5菌株是研究革兰氏阴性根际促生细菌（plant growth promoting rhizobacteria, PGPR）的典型代表。该生防菌能产生多种抗菌活性物质，还能促进植物生长。在本研究中，我们主要研究该菌株的三个小RNA（rsmX/Y/Z）在合成抗菌脂肽orfamide A中的作用。我们分别构建了这三个小RNA的突变载体，利用自杀性无标记载体pK18mobsacB，构建了∆rsmX、∆rsmY、∆rsmZ突变体。群集运动能力检测结果表明，相比野生型Pf5菌株，突变体∆rsmX群集运动能力显著下降。在该菌株中，群集运动需要抗菌脂肽orfamide A。我们进一步提取了这些菌株的脂肽类物质，检测结果表明，突变体∆rsmX的粗提物几乎没有排油和溶血活性，其对番茄青枯病菌的抑制活性也显著下降。利用HPLC检测结果表明，突变体∆rsmX几乎完全丧失产orfamide A的能力。这说明，在Pf5菌株中，小RNA rsmX具有调控orfamide A合成的功能，具体机理仍需进一步研究。本研究能为提高生防菌Pf5菌株中的活性物质的产品提供理论支持。
Effect of Small RNA on the Regulation of Antibiotic Lipopeptide Orfamide A in Pseudomonas Pf5 Strain
ABSTRACT
Pf5 strain of Pseudomonas SPP. is a typical representative of plant growth promoting rhizosphere bacteria (plant growth promoting rhizobacteria, PGPR). The biocontrol bacteria can produce a variety of antibacterial active substances, but also can promote the growth of plants. In this study, we mainly studied the role of three small rnas (rsmX/Y/Z) of this strain in the synthesis of antimicrobial lipopeptide orfamide A. We constructed mutational vectors for these three small rnas respectively, and constructed ∆rsmX, ∆rsmY, ∆rsmZ mutants using the suicidal unlabeled vector pK18mobsacB. The results of cluster mobility test showed that compared with the wild type pf5 strain, the mutant ∆rsmX cluster mobility was significantly reduced. In this strain, the antibiotic lipopeptide orfamide A was required for clustering. We further extracted the lipid peptides of these strains, and the test results showed that the crude extract of the mutant ∆rsmX had almost no oil discharge and hemolysis activity, and its inhibitory activity against tomato bacterial wilt was also significantly reduced. HPLC results showed that the mutant ∆rsmX almost completely lost its ability to produce orfamide A. This indicates that in pf5 strain, small RNA rsmX has the function of regulating orfamide A synthesis, and the specific mechanism still needs to be further studied. This study can provide theoretical support for the product of improving the active substance in the bioresistant pf5 strain.

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