摘 要

解淀粉芽孢杆菌（Bacillus amyloliquefaciens）FZB42是一株背景研究较为深入的革兰氏阳性植物根际友好细菌菌株，该菌对植物的生长具很好的有促进作用。在其自身的生长过程中可以产生一系列的代谢产物。这些代谢产物能够具有广泛地抑制真菌和细菌的活性。因此，解淀粉芽孢杆菌同时具有生物除污、植物促生及生物防治等特性。已有的研究表明解淀粉芽孢杆菌sRNA Bas01的功能可能涉及到细菌的生物膜形成和趋化性，而这两种作用跟细菌在植物根部的定殖能力具有密切关系。 因此研究sRNA Bas01基因对解淀粉芽孢杆菌生物膜形成和趋化性的影响，对于揭示“细菌—植物”相互作用的分子机制，具有重要意义。

本文通过研究解淀粉芽孢杆菌（B. amyloliquefaciens）FZB42及其Bas01突变株生物膜形成区别，探讨Bas01基因对FZB42游动性影响，主要研究内容和结果如下：

1、将初始菌株FZB42分别接种至不同琼脂浓度(分别为0.5%、 0.75%、1%.0、1.25%和1.5%）以确定解淀粉芽孢杆菌生物膜形成最适培养基。培养基测定结果表明，不同浓度的培养基对解淀粉芽孢杆菌生物膜群移有一定的影响，在一定浓度范围内，随着浓度的提高，菌落长得越小。0.5%琼脂浓度的培养基细菌长得最好，故可使用0.5%的培养基浓度进行测试sRNA基因对解淀粉芽孢杆菌生物膜群移的影响。

2、通过将菌株sRNA突变株、sRNA超表达菌株及初始菌株FZB42（作为阳性对照菌株）接种至确定的最适培养基上，诱导基因表达并观察生物膜形成情况，测定结果表明，sRNA基因对FZB42群移现象有一定的影响，有sRNA基因的解淀粉芽孢杆菌群移现象明显，而无sRNA基因的解淀粉芽孢杆菌无明显群移现象。说明Bas01对解淀粉芽孢杆菌FZB42游动性及在形成菌膜过程中起重要作用。

关键词：解淀粉芽孢杆菌；sRNA Bas01；生物膜；群移

Effects of Bas01 sRNA on biofilm formation of Bacillus amyloliquefaciens

Abstract

Bacillus amyloliquefaciens FZB42 is a depth study of the background of the gram positive PGPR strains, which have a good effect on the growth of plants. A series of metabolites can be produced in the process of its own growth. These metabolites make it widely used to inhibit the activity of fungi and bacteria. Therefore, Bacillus amyloliquefaciens with bioremediation, biological control and plant growth promoting properties. Previous studies have indicated that the function of sigD in sRNA solution may be related to the biofilm formation and chemotaxis of bacteria, and these two kinds of effects have close relationship with the colonization ability of bacteria in the plant roots. Therefore, it is important to study the effect of Bas01 sRNA gene on the formation and chemotaxis of the biofilm formation and chemotaxis of Bacillus subtilis, which is important to reveal the molecular mechanism of the interaction between bacteria and plants.

The contents and results of this paper are as follows:

1. The initial strain FZB42 line to the solid LB tablet, overnight culture 8h. The shaking flask culture of LB liquid 10mL medium was inoculated to different agar concentration (1.5%, 0.75%, 1%.0, 1.25% and 0.5%) respectively, until OD=5.0. Added to the diluted to OD=1 in PBS buffer each treatment into 5 culture in the center of the base position 0, injected with bacteria liquid, air drying for 10 minutes, add insulation box each 1 hour observation sRNA expression induced changes in membrane material and bacillus, continuous observation of 8 hours.The results showed that the different concentrations of the culture medium had certain effect on the solution of the biofilm group. In a certain concentration range, with the increase of concentration, the colony grew smaller. 0.5% agar concentration of the culture medium bacteria grow best, it can be used to test the concentration of 0.5% of the sRNA gene to test the effect of gene on the biological membrane of Bacillus subtilis.

2. Through the strain sRNA mutant strain (denoted as FBS48), sRNA super expression strain (denoted as FBS49) and initial strain FZB42 (as positive control strain, denoted as FBS01) crossed to the solid LB tablet, overnight culture 8h. And then inoculated into 10mL liquid LB culture medium shake flask culture, until OD=5.0. Added to the diluted to OD=1 in PBS buffer each treatment into 5 culture in the center of the base position 0.1ml bacilli, air drying for 10 minutes, add insulation box each 1 hour observation sRNA expression induced changes in membrane material and bacillus, continuous observation for 8 hours.The medium determination results show that sRNA genes of FZB42 group shift phenomenon has certain influence, sRNA genes of Bacillus amyloliquefaciens group shift phenomenon is obvious, and no sRNA genes in the solution of Bacillus amyloliquefaciens, no significant group shift phenomenon.

Key words： Bacillus subtilis，Bas01 sRNA， biofilm ，group shift

目录

绪 论 1

第一章 文献综述 2

1 解淀粉芽孢杆菌 2

1.1解淀粉芽孢杆菌的一般特性 2

1.3解淀粉芽孢杆菌的应用 3

1.4解淀粉芽孢杆菌的sRNA 4

1.5群移 4

第二章 培养基对解淀粉芽孢杆菌生物膜群移的影响 6

2.1材料 6

2.1.1供试菌株 6

2.1.2试剂 6

2.2方法 7

2.2.1.菌液的制备 7

2.2.2群移的培养 7

2.3结果与讨论 7

2.3.1结果 7

2.3.2讨论 9

第三章 Bas01基因对解淀粉芽孢杆菌生物膜群移的影响 10

3.1材料 10

3.2方法 10

3.2.1菌液的制备 10

3.2.2群移的培养 10

3.3结果 10

3.3.1 Bas01基因对FZB42细胞膜形成的影响 10