皱纹盘鲍不同选系对弧菌刺激的差异响应及其机制研究

IOCAS-IR > 实验海洋生物学重点实验室

	皱纹盘鲍不同选系对弧菌刺激的差异响应及其机制研究
	钟兆山
学位类型	硕士
导师	刘晓
	2017-05-27
学位授予单位	中国科学院大学
学位授予地点	北京
学位专业	水产养殖
关键词	皱纹盘鲍弧菌选择育种免疫相关基因抗弧菌筛选
摘要	皱纹盘鲍是我国重要的海水养殖贝类，也是人们喜爱的海产品之一，通过增养殖工作者的努力以及各种新式养殖模式的普及，中国已成为世界上首屈一指的养鲍大国，虽然我国鲍养殖品种较少，主要为皱纹盘鲍和南方沿海少数的杂色鲍，但是仅凭皱纹盘鲍一种的产量已经超过世界上其他国家各种鲍养殖产量的总和。现在鲍养殖产量的逐年提高离不开优质鲍品种的推广、充足饵料的投喂、良好的养殖水质、精心的养殖管理以及重要的疾病防控手段。皱纹盘鲍是软体动物门中普通的一员，与其他海洋无脊椎动物一样，没有进化出以抗体为主的特异性免疫系统，仅拥有非特异性免疫系统或者说先天免疫系统。鲍的先天免疫在复杂多变的海洋环境中起到重要作用，在与环境相互选择进化的过程中，不断完善的先天免疫系统使这种古老的物种在地球上生活了上亿年。有历史记载人类在7万5千年前已经开始利用及食用鲍。在我国，鲍自古以来就被当作海产珍品，涉及到鲍的美食有很多，且鲍全身是宝，鲍壳也是名贵中药，在我国鲍的大规模养殖与此也颇有关系。世界上鲍品种虽多，但是各种鲍在养殖过程中都有疾病爆发的情况，特别在大规模集约化的养殖条件下，伴随环境污染、海洋酸化及全球变暖的加剧，鲍养殖的海洋环境也日渐恶化，疾病频发，这其中弧菌导致的疾病占绝大多数。弧菌疾病是海洋生物的常见致病菌，对海洋生物及人类危害严重。本研究从观察到皱纹盘鲍不同选育群体在受到弧菌侵染后有不同的死亡率后，进行了如下研究：皱纹盘鲍不同品系对弧菌耐受性差异的检测；皱纹盘鲍抗弧菌品系的筛选及筛选效果的检测；抗弧菌筛选后的群体与一般群体对弧菌刺激时的差异响应。现将本阶段实验结果总结如下： 1 皱纹盘鲍不同品系对弧菌耐受差异的检测皱纹盘鲍“97”、“98”选育群体均用三种弧菌（溶藻弧菌、哈维氏弧菌和鳗弧菌）进行攻毒实验，实验周期为7天，在不同的弧菌浓度下，皱纹盘鲍两选育群体都随注射弧菌菌液的浓度升高而死亡率增加。并且可以发现皱纹盘鲍“97”和“98”选育群体对溶藻弧菌（bb3）的耐受性类似，而皱纹盘鲍“98”选育群体对哈维氏弧菌（bb4）和鳗弧菌（vm）的耐受性高于“97”选育群体。 2皱纹盘鲍抗弧菌品系的筛选及筛选效果的检测利用检测皱纹盘鲍不同选育群体敏感性存活下的个体，混合一起命名为R1群体；再用较高浓度三种弧菌混合菌液注射4059只健康未经筛选过的皱纹盘鲍“97”选育群体G6个体，其中bb3、bb4、vm三种弧菌的终浓度为5×10⁶，1×10⁵，5×10⁶ cfu/mL待死亡率稳定时统计，发现总共存活鲍321只，累积死亡率达到92.09%，筛选强度为8%，将存活下的皱纹盘鲍群体定义为R2群体，对应的所有未经过筛选的群体定义为CK群体。皱纹盘鲍R1群体和CK群体在同时注射三种弧菌混合菌液挑战后，R1群体死亡率为36.67±4.49%，CK群体死亡率为84.00±6.37%，两者死亡率差异显著（P＜0.01）；对六个月后筛选的群体R2及未筛选过的群体CK同时注射单一弧菌bb3、bb4及vm，浓度分别为1×10⁶cfu/mL、1×10⁶cfu/mL、3×10⁷cfu/mL发现待两组死亡率稳定后，其中注射bb3后R2群体死亡率为13.33±4.71%，CK群体为53.33±9.43%，两者死亡率差异显著（P＜0.01），注射bb4后，R2群体死亡率为23.33±4.71%，CK群体为83.33±12.47%，两者死亡率差异显著（P＜0.01），注射vm后，R2群体死亡率为0%，CK群体为36.67±4.71%，两者死亡率差异显著（P＜0.01），利用弧菌的混合再次注射及单独再次注射同时说明了抗弧菌筛选的效果明显。 3抗弧菌筛选后的群体R2与一般群体CK对弧菌刺激时的差异响应本研究挑选了5个与皱纹盘鲍免疫防御相关的基因，包括皱纹盘鲍防御素基因（Defensin）、溶菌酶基因（Lysozyme）、热激蛋白70基因（HSP70），一个与细胞凋亡相关基因一氧化氮合成酶基因（NOS）和一个与功能多样的，且研究逐渐深入的血蓝蛋白基因（Hemocyanin）；通过研究相同弧菌注射刺激后，这些基因的表达量变化，来反映皱纹盘鲍两群体受到弧菌刺激时的差异响应。
其他摘要	Haliotis discus hannai Ino is one of the most important aquaculture products in China and it is also one of the favorite seafoods in China. Through the proliferation of breeding and breeding workers’ hard work also with the popularization of various new farming modes, China has become the first abalone breeding country in the world. There are two mainly breeding species in china, one is Haliotis discus hannai another is Haliotis diversicolor, but the yield of Haliotis discus hannai has exceeded that of other countries’ abalone in the world. Now the high annual production of abalone cannot be separated from the promotion of high quality abalone germplasm resource, adequate feeding of food, good quality of cultural water, careful management of the breeding and important means of disease prevention and control. Abalone is an ordinary member of mollusk. Like other marine invertebrates, there is no evolutionary antibody-specific immune system, only the non-specific immune system or the innate immune system. Abalone’s innate immunity plays an important role in the complex and volatile marine environment, and the continuous improvement of innate immunity in the process of evolution with the environment makes this ancient species alive for hundreds of millions years on Earth. There are historical records of human beings has begun to use abalone before 75,000 years ago. In our country, since the ancient treasure has been used as delicious seafood, there are a lot of food related to abalone, for another thing the shell of abalone is also expensive Chinese medicine, and large-scale farming may has a relationship with this. Although there are many varieties of abalone in the world, but all kinds of abalone in the breeding process have the outbreak of some diseases, especially in large-scale intensive farming conditions, as well as environmental pollution, ocean acidification and global worming in the case of abalone breeding the marine environment is also deteriorating. Which vibrio caused by the disease accounted for the vast majority. Vibrio disease is a common pathogen of marine life, which is harmful to marine life and human. Since the observation of the mortality were different after infection Vibrio, the following studies were carried out: The different tolerance of two selection lines of Haliotis discus hannai to vibrio challenge; selecting the tolerance line of abalone, and the select effect of vibrio disease resistance breeding; the different response between vibrio resistance selection line and general population of Haliotis discus hannai. Now the results of this stage are summarized as follows: 1 The detection of vibrio tolerance in different strains of Haliotis discus hannai. The experimental system was tested with 3 kinds of vibrio, and the experimental period was 7 days. At different Vibrio concentrations, the mortality rate increased with the concentration of the vibrio. The tolerability of Vibrio alginolytica (bb3), “97” selection line was similar to "98" selection line, but for the tolerability of V. harveyi (bb4) and V. anguillarum (vm), 98 selection line was higher than that of "97". 2 Vibrio resistance line’s selection and the test of screening effect R1 population was the survial abalones which used for detection of vibrio tolerance in first chapter.The final mixed vibrio concentration of bb3, bb4 and vm was 5×10⁶ cfu/mL,1×10⁵ cfu/mL, and 5×10⁶ cfu/mL to inject to 4059 health abalones. When the mortality was stable, we make a statistics that found a total survival of 321, and the cumulative mortality rate was 92.09%, in line with the general selection intensity, and its selection intensity was 8%. The selected population is defined as R2 population, and all the unselected population is defined as CK population. The mortality rate of R1 and CK population was 36.67±4.49% and 84.00±6.37% especially after the challenge of three kinds of Vibrio mixed bacteria, and the difference was significant. We injected the same dose of bb3 , bb4 or vm vibrio bacteria to R2 and CK population, which the vibrio dose were 1×10⁶、1×10⁶ 、3×10⁷ cfu/mL.For bb3 injection, we found that the moratility rate of R2 population was 13.33±4.71% and CK population was 53.33±9.43%; for bb4 injection, we found that the moratility rate of R2 population was 23.33±4.71% and CK population was 83.33±12.47%; for vm injection, we found that the moratility rate of R2 population was 0 and CK population was 36.67±4.71%; These three tests also proved we had a successful selection. 3 Different response in gene expression level between population R2 and population CK against vibrio stimulation In this study, we selected five genes that related to Haliotis discus hannai immune defense. It is Defensin, Lysozyme, HSP70, NOS and Hemocyanin. We can easy to distinguish CK and R2 population for the different expression of these genes.
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/136685
专题	实验海洋生物学重点实验室
推荐引用方式 GB/T 7714	钟兆山. 皱纹盘鲍不同选系对弧菌刺激的差异响应及其机制研究[D]. 北京. 中国科学院大学,2017.

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