中国科学院海洋研究所机构知识库
Advanced  
IOCAS-IR  > 海洋生物技术研发中心  > 学位论文
题名: 温度及细菌间作用对杀鲑气单胞菌致病性的影响
作者: 孟令杰
学位类别: 硕士
答辩日期: 2017-06
授予单位: 中国科学院大学
授予地点: 北京
导师: 刘鹰
关键词: 生态因子,杀鲑气单胞菌,群体感应,温度,毒力因子
学位专业: 环境工程
中文摘要: 大西洋鲑作为一种高经济价值的养殖鱼种,因其体型较大、肉质良好、口感鲜美、营养丰富等优点,深受全世界消费者的喜爱。目前我国已有养殖公司从北欧、南美等地引入良种,采用循环水系统工厂化养殖洄游型大西洋鲑。然而,近年来大西洋鲑养殖业的发展受到了杀鲑气单胞菌(Aeromonas salmonicida)病害问题的严重制约。杀鲑气单胞菌是一类条件致病菌,只有当环境适合的情况下才能导致鱼体发病。因此,探究环境因素对于杀鲑气单胞菌致病性的影响显得尤为重要,但目前对此仍缺乏足够的研究。本论文通过生产调查与实验验证进行分析,对温度和细菌种间相互作用这两个环境中重要的生态因子做了初步探究,阐述其对于杀鲑气单胞菌致病性的影响,得到的主要结论如下:
1、杀鲑气单胞菌病害爆发时,大西洋鲑死亡率变化与温度、亚硝酸盐浓度变化呈相关性
在调研时间段内,生产车间水质总体控制良好。除亚硝酸盐浓度在大西洋鲑死亡率较高时期波动到1 mg/L以上,其余水质参数基本符合大西洋鲑养殖规范。各水质指标间变化趋势存在相关性,较为普遍的是水温同亚硝酸盐浓度的变化呈显著负相关(Pearson相关系数-0.505,P<0.01),氨氮浓度同亚硝酸盐浓度变化呈显著正相关(Pearson相关系数0.587,P<0.01)。在各水质指标中,水温及亚硝酸盐浓度同大西洋鲑死亡率变化显著相关。其中,水温与死亡率之间的相关性普遍存在于被调查的各个系统中,呈显著负相关(P<0.01)。随后,通过回归分析预测大西洋鲑死亡率M=19.573-1.042T+0.155 NO2-,方程极显著(P<0.01),调整后R2=0.494,表明该方程可以在一定程度上解释杀鲑气单胞菌爆发时大西洋鲑死亡率的变化。
2、温度影响杀鲑气单胞菌生长、代谢及毒力基因转录
温度是影响杀鲑气单胞菌病害的重要生态因子。采用8 ℃、10 ℃、13 ℃、15 ℃、18 ℃和28 ℃的温度梯度分别对杀鲑气单胞菌培养,收集胞外产物,进行SDS-PAGE凝胶电泳分析。结果发现,28 ℃下杀鲑气单胞菌胞外蛋白成分最复杂,该温度下胞外蛋白成分多于低温培养,但其缺失分子量为40 kDa、100 kDa的蛋白。根据胞外产物类型,选取10℃、18℃、28℃温度梯度组进行研究。不同温度培养下杀鲑气单胞菌的生长速率、AI-2信号分子活性、形态均具有较大差异。随后,对三个温度培养的杀鲑气单胞菌进行转录组分析,发现不同组间分别存在843、1212和521个表达差异基因,主要集中在催化、结合等生物学过程中,并多参与细胞组分、膜构成等功能。KEGG富集分析发现114个通路中存在差异表达基因,如典型的Ⅲ型分泌系统各蛋白编码基因,在28℃培养时表达量显著升高。这些结果表明杀鲑气单胞菌中基因表达响应温度改变,在转录水平发生变化。
3、杀鲑气单胞菌能够合成并分泌具有活性的AI-2信号分子
AI-2信号分子参与的Ⅱ型群体感应是细菌种间的重要交流方式,其他细菌有可能通过群体感应影响杀鲑气单胞菌的致病能力,并且实验表明杀鲑气单胞菌AI-2信号活性受到温度调节。首先,采用哈维氏弧菌BB170生物检测系统,检测到6株来源不同的杀鲑气单胞菌菌株均能产生AI-2信号分子活性。后续实验表明,菌株C4 AI-2信号分子活性在不同生长阶段具有差异,同时受到不同生长环境影响。基因克隆与测序验证了杀鲑气单胞菌基因组中Ⅱ型群体感应相关基因luxSpfs,通过生物信息学分析,表明杀鲑气单胞菌中LuxS蛋白具备群体感应所需关键氨基酸位点组氨酸His54、组氨酸His58、半胱氨酸Cys128和甘氨酸Gly93,模型构建则证明LuxS具有功能性二级结构。以上结果表明杀鲑气单胞菌中LuxS蛋白具有合成AI-2信号分子功能。经系统发生树分析,杀鲑气单胞菌中LuxS与同属其他细菌相似性最高,其次是同为水产致病菌的迟缓爱德华氏菌。
4、Ⅱ型群体感应影响杀鲑气单胞菌多种表型及侵染大西洋鲑能力
本研究利用同源重组方法构建杀鲑气单胞菌luxS基因缺失菌株C4ΔluxS,经基因型及表型验证正确。生长实验表明,杀鲑气单胞菌C4ΔluxS菌株较野生株具有一定生长优势。缺失LuxS后,选取的代表性毒力基因表达量多没有降低趋势,仅表面A蛋白编码基因vapA表达量显著降低(P<0.01),表现为表面疏水性降低3.59倍,无法观察到细胞自凝集现象。通过扫描电镜观察发现不同基因型细菌大小、形态也略有不同,验证以上实验结果。攻毒实验表明,受到杀鲑气单胞菌C4ΔluxS菌株侵染的大西洋鲑较受到野生型菌株侵染的大西洋鲑,更早表达天然免疫因子TLR5S、IFNα、SOD及POD。研究结果表明杀鲑气单胞菌中表面A蛋白受LuxS调控,LuxS可以间接帮助杀鲑气单胞菌提高自我保护能力,延长在宿主中的存活时间。
英文摘要: For the characteristics of big individual, good meat quality, great taste and rich in unsaturated fatty acid etc, Atlantic Salmon(Salmo salar L)was popular in the world, as a kind of cultured fish with high economic value. Now there is domestic farm, which has introduced superior varieties of Atlantic Salmon from northern Europe and south America to China, and cultured fish by using Recirculating Aquaculture System. However, disease caused by Aeromonas salmonicida has seriously restricted the development of the aquaculture industry of Atlantic Salmon. A.salmonicida is an opportunistic pathogen and only cause disease under appropriate environment. Therefore, it is very important to explore the impact of ecological factors on the pathogenicity of A.salmonicida, but relative reports are few. This thesis combined the production survey and a series of experiments, and selected the interaction among bacteria and water temperature as two important ecological factors, to verify the regulatory function of these factors on the bacterial virulence. Main conclusions are carried out as follows:
In production, water temperature is related to the mortality of Atlantic salmon with the infection of A.salmonicida
During the period of survey, water quality was controlled well in the workshop generally. In addition to the concentration of nitrite fluctuating over 1 mg/L, other water parameters satisfied with Atlantic salmon culture practices. The variation trend of water parameters correlate with each other, especially, water temperature is negative correlated with the change of nitrite concentration (Pearson correlation coefficient -0.505, P<0.01), and ammonia is significantly positive correlated nitrite concentration (Pearson correlation coefficient 0.587, P<0.01). There is significant correlation between water temperature/nitrite concentration and mortality of Atlantic salmon, especially water temperature is negatively correlated with mortality (P<0.01). By regression analysis, mortality of Atlantic salmon M=19.573-1.042T+0.155 NO2-,P<0.01, adjusted R2 0.494, indicating this quation can be used to predict the mortality of Atlantic salmon with water parameters when A.salmonicida outbreaking.
Temperature affects virulence of A.salmonicida
ECPs of A.salmonicida which cultured under 8℃, 10℃, 13℃, 15℃, 18℃ and 28℃ are different with each other, in which bacteria from 28℃ could secret more proteins than other from temperature, but protein of 40 kDa and 100 kDa are lost art 28℃. Growth rate, AI-2 activity and morphology are different for bacteria cultured under 10℃, 18℃, 28℃. Among these three groups, 843, 1212 and 521 genes differentialy express, which concentrate on the reaction such as catalize and bingding. Differential expressed genes distribute in 114 KEGG pathways, for example, expression of type three secretion system related coding genes are negatively regulated at 28℃. Results demonstrate that temperature could affect the virulence of A.salmonicida.
AI-2 quorum sensing exists in A.salmonicida
AI-2 activity could be detected from 6 different A.salmonicida strains. In C4, AI-2 activity changes in different growth phase, and is influenced of different culture condition. Gene cloning and sequencing demonstrate AI-2 quorum sensing related luxS and pfs genes integrally exist in A.salmonicida. Bioinformation shows that LuxS in A.salmonicida has key amino acid sites His54, His 58, Cys128 and Gly93 for AI-2 production, and functional protein secondary structure. The result shows that LuxS has function to synthesis AI-2 signals. Phylogenetic tree indicates that LuxS of A.salmonicida mostly homology with other Aeromonas. Bacteria, then Edwardsiella tarda.
AI-2 quorum sensing affects pathogenicity of A.salmonicida
This study constructed a A.salmonicida luxS deficiency strain by homologous recombination, and verified the right recombination by both genetic and phenotype. In same culture condition, C4ΔluxS mutant grows better than wild type, and expression level of most virulence genes do not decrease. However, expression of A-layer protein coding gene vapA decreases significantly in mutant strain (P<0.01), cell hydrophobicity decreases 3.59-fold and auto-aggregation disappears. Results above are verified by SEM observation, and cell size, morphology also change with the lost of LuxS. In challenge experiment, Atlantic salmon infected with C4ΔluxS express innate immune factors TLR5S、IFNα、SOD and POD earlier. Study data indicates that A-layer protein is regulated by LuxS, so LuxS could assist A.salmonicida self-protect, and prolong the survival time in host.
语种: 英语
内容类型: 学位论文
URI标识: http://ir.qdio.ac.cn/handle/337002/136682
Appears in Collections:海洋生物技术研发中心_学位论文

Files in This Item:
File Name/ File Size Content Type Version Access License
孟令杰毕业论文0613.pdf(1582KB)学位论文--延迟开放2018-6-13后可获取 联系

作者单位: 1.中国科学院海洋研究所
2.中国科学院大学

Recommended Citation:
孟令杰. 温度及细菌间作用对杀鲑气单胞菌致病性的影响[D]. 北京. 中国科学院大学. 2017.
Service
Recommend this item
Sava as my favorate item
Show this item's statistics
Export Endnote File
Google Scholar
Similar articles in Google Scholar
[孟令杰]'s Articles
CSDL cross search
Similar articles in CSDL Cross Search
[孟令杰]‘s Articles
Related Copyright Policies
Null
Social Bookmarking
Add to CiteULike Add to Connotea Add to Del.icio.us Add to Digg Add to Reddit
所有评论 (0)
暂无评论
 
评注功能仅针对注册用户开放,请您登录
您对该条目有什么异议,请填写以下表单,管理员会尽快联系您。
内 容:
Email:  *
单位:
验证码:   刷新
您在IR的使用过程中有什么好的想法或者建议可以反馈给我们。
标 题:
 *
内 容:
Email:  *
验证码:   刷新

Items in IR are protected by copyright, with all rights reserved, unless otherwise indicated.

 

 

Valid XHTML 1.0!
Copyright © 2007-2017  中国科学院海洋研究所 - Feedback
Powered by CSpace