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对虾抗弧菌性状分子育种技术研发和抗性机制探究
张倩
学位类型博士
导师李富花
2020-05-20
学位授予单位中国科学院大学
学位授予地点中国科学院海洋研究所
学位名称理学博士
关键词凡纳滨对虾 副溶血弧菌 分子育种 抗性机制
摘要

   对虾是世界重要的海水养殖动物,其良种选育是产业发展的基石。在过去三十年,基于数量遗传学的选育方法,对虾重要经济性状的遗传改良取得显著成效。前期的选育研究较多集中在生长性状,对于遗传力较低的抗性性状研究较少。近年来,世界对虾产业因急性肝胰腺坏死综合征(Acute hepatopancreas necrosis diseaseAHPND)的爆发遭受了严重的打击。AHPND主要是由携带pVA1质粒的副溶血弧菌(Vibrio parahaemolyticus)感染引起,选育抗副溶血弧菌的对虾种质被认为是解决AHPND的根本途径。由于对抗病性状的遗传评估准确率较低以及表型难以度量等问题,利用传统育种技术进行抗病性状的选育效率较低。随着基因分型技术的发展,分子育种技术日渐成熟,将传统选育技术与分子育种技术结合,对加快对虾抗弧菌性状的遗传改良具有重要意义。本论文以凡纳滨对虾(Litopenaeus vannamei)为实验对象,分别从标准化抗弧菌性状测试方法的建立、对虾抗弧菌表型组研究、抗性机理解析和抗性相关分子标记筛选等方面开展了系统研究,为对虾的抗病育种和病害防治提供了重要理论依据和技术支撑。研究取得的主要进展如下:

1.建立了对虾抗副溶血弧菌标准化测试方法。研究了不同培养条件下副溶血弧菌的生长规律和生长特点,建立了用于对虾抗性测试的副溶血弧菌规模化培养方法;比较了处于不同生长时期的副溶血弧菌对凡纳滨对虾幼虾的感染能力,证实处于平台期前期的副溶血弧菌更适合感染实验。通过改进副溶血弧菌培养方式,优化感染方法,使对虾抗弧菌性状测试标准化。

2.开展了对虾抗副溶血弧菌感染的表型组研究。针对凡纳滨对虾的AHPND病理特征,研究了副溶血弧菌感染对虾的主要靶组织——肝胰腺的颜色变化与病原菌载量的关系,并将弧菌感染对虾的进程划分为四个等级。肝胰腺颜色变化和副溶血弧菌载量可被开发为对虾对弧菌抗性的表型参数,为家系或群体的抗病能力测定提供新的表型指标。

3.建立了对虾体内副溶血弧菌载量的定量检测方法,比较了对虾抗性和敏感家系在弧菌感染过程中体内副溶血弧菌的动态变化规律。首先根据副溶血弧菌毒力因子序列设计引物和探针,经过引物筛选、体系优化,建立了用于检测带毒素质粒的副溶血弧菌的TaqMan探针荧光定量PCR技术,使检测的灵敏度和准确性明显提高。比较了对虾的抗性家系和敏感家系在弧菌浸泡感染后体内副溶血弧菌的动态变化特征,结果表明抗性家系体内副溶血弧菌的载量显著低于敏感家系,提示抗性家系可能通过抑制副溶血弧菌在体内的增殖从而减少对机体的影响,这为进一步解析抗性机制奠定了基础。

4.开展对虾抗性家系和敏感家系的比较转录组研究。以课题组长期选育的材料为基础,结合近两年副溶血弧菌感染测试的家系数据和系谱关系,对抗性家系和敏感家系进行了转录组测序,筛选获得392个差异表达基因。其中,表皮蛋白(Flexible cuticle protein)和肌球蛋白(Myosin)基因在抗性家系中的表达量较高,而与代谢和免疫相关的基因在敏感家系中表达量较高。这些差异基因的表达水平可能与对虾家系的抗病能力强弱有关,可为对虾抗性家系的选育提供重要参考。

5.通过关联分析技术筛选鉴定了与对虾抗副溶血弧菌性状相关的SNP标记和候选基因。通过对对虾的敏感材料和抗性材料进行目标区域测序,分析了1,566SNPs的不同等位基因在易感材料和抗性材料中的频率,通过关联分析获得30个与对虾抗弧菌性状相关的SNPs,并利用候选基因关联分析方法,在LvALF6基因上筛选获得6个与抗弧菌性状相关的SNPs,为对虾抗病育种提供了重要的分子标记和候选基因。

 

其他摘要

    Shrimp is the main marine cultivated species in the world and selective breeding is the foundation for shrimp aquaculture industry. In the past 30 years, remarkable achievements have been achieved in genetic improvement for important economic traits of shrimp based on quantitative genetics. Previous genetic breeding works have mainly focused on the growth trait, while relatively few work focused on resistance trait with low heritability. Since the outbreak of acute hepatopancreas necrosis disease (AHPND) in recent years, the shrimp aquaculture industry has suffered severe damage. AHPND is mainly caused by infection of Vibrio parahaemolyticus carrying a plasmid (pVA1). Therefore, selective breeding for broodstocks resistant against V. parahaemolyticus is regarded as a key approach to solve this disease problem. However, the progress for breeding shrimp with disease resistance trait by traditional selective breeding techniques is very slow due to the low accuracy for genetic evaluation and high difficulty for measuring the phenotypes. With the development of genotyping technology, molecular breeding techniques are becoming more powerful. Combining traditional selection breeding with molecular breeding technologies is of great significance for accelerating the genetic improvement of disease resistance against V. parahaemolyticus. In this paper, we developed a series of studies in molecular breeding of Pacific white shrimp (Litopenaeus vannamei) in disease resistance against Vibrio, including the establishment of standardized test method for shrimp resistance test, phenotypes for shrimp resistant to Vibrio, analysis of resistance mechanism, and the identification of molecular markers related to disease resistance. These data will provide important instructions and information for disease resistant shrimp breeding and disease control in aquaculture industry. The main results are as follows:

1. A standardized method for testing shrimp resistance against V. parahaemolyticus was established. By analyzing the growth characteristics of V. parahaemolyticus under different culture modes, a method for large-scale culture of pathogenic bacteria in field was established. By comparing the infection ability of V. parahaemolyticus at different growth stages, we found that the bacteria at the pre-platform phase were more suitable for infection experiments. In summary, a standard method for testing the shrimp resistance against Vibrio was established through improving the culture mode for Vibrio and optimizing the infection approach in field. 

2. Studies on the phenotypes of shrimp after infection of V. parahaemolyticus. Based on the pathological characteristics of AHPND in shrimp, the relationship between the color variations and bacterial load in the hepatopancreas during the infection process was analyzed, and the infection process could be divided into four grades, which have apparent correlation with the color of hepatopancreas. Therefore, the color of the hepatopancreas in shrimp and bacterial load are possible to be used as phenotypic parameters of shrimp during V. parahaemolyticus infection, which will provide new indicators for evaluating the disease resistance of families or populations of shrimp.

3. A quantitative method for V. parahaemolyticus was established, which was used to study the dynamics of Vibrio in disease resistance families and disease susceptible families. Based on the sequences of PirAVp and PirBVp in V. parahaemolyticus, specific primers and probes were designed, and a real time PCR method with TaqMan probe was established with high sensitivity and accuracy to detect the load of V. parahaemolyticus in shrimp. Through detecting the dynamic changes of pathogenic bacteria between resistant and susceptible families during V. parahaemolyticus infection, we found that shrimp from disease resistant families showed much lower bacterial load than shrimp from disease susceptible families. These data suggested that shrimp with disease resistance might prevent the occurrence of disease through inhibiting the replication of the pathogen in shrimp, which provided some information for explaining the resistance mechanism of shrimp against Vibrio.

4. A comparative transcriptome study between distease resistant and susceptible families was carried out to explore the molecular mechanism of the resistance. Among the shrimp families after long time breeding by our group, resistant family and susceptible family were used for transcriptome analysis after analyzing the pedigree of families and their performance during Vibrio infection. A total of 392 differentially expressed genes (DEGs) were screened between disease resistant shrimp and disease susceptible shrimp. Flexible cuticle protein and myosin showed higher expression levels in disease resistant shrimp, while some metabolism and immunity related genes showed higher expression level in disease susceptible shrimp. These DEGs might reflect the resistance of shrimp to disease and could provide index for the selection of disease resistant family.

5. SNP markers and candidate genes related to the resistance of shrimp to V. parahaemolyticus were identified using association analysis. By sequencing the target regions of resistant and susceptible materials, 1,566 SNPs and their frequency of alleles in susceptible and resistant groups were obtained. A total of 30 SNP markers with significant difference were screened. Besides, other 6 SNPs with significant difference which were located at LvALF6 were screened through candidate gene association analysis. All these SNPs could provide useful genetic markers and candidate genes for the breeding of shrimp with disease resistance.

学科领域生物学 ; 遗传学
学科门类理学 ; 理学::生物学
页数150
语种中文
目录第1章 绪论 1 1.1对虾产业发展现状 1 1.1.1对虾产业发展现状概述 1 1.1.2我国对虾产业面临的问题 2 1.2对虾疾病与研究现状 4 1.2.1对虾主要的疾病 4 1.2.2急性肝胰腺坏死综合征的致病机理 6 1.2.3急性肝胰腺坏死综合征的防治措施 8 1.3水产动物育种技术 10 1.3.1水产动物选择育种的常用方法 10 1.3.2现代动物育种技术在水产动物中的应用 14 1.3.3对虾的选育进展 18 1.3.4对虾抗病性状的选育研究进展 21 1.4研究目的和意义 23 第2章 对虾抗弧菌性状标准化测试方法建立 25 2.1引言 25 2.2材料与方法 25 2.2.1病原菌在摇床和静置两种培养条件下的生长规律与纯度检测 26 2.2.2不同生长时期的副溶血弧菌感染能力比较 29 2.2.3 TSB培养基的加入对感染实验的影响分析 29 2.2.4梯度浸泡和单次浸泡感染方法比较 31 2.3结果 31 2.3.1病原菌在摇床和静置两种培养条件下的生长曲线与纯度 31 2.3.2不同生长时期的病原菌浸泡感染对虾的效果比较 34 2.3.3加入TSB培养基的浓度对感染实验的影响分析 38 2.3.4梯度浸泡和单次浸泡感染方法的比较 41 2.4讨论 42 第3章 对虾抗副溶血弧菌性状表型组指标研究 45 3.1引言 45 3.2材料与方法 46 3.2.1 VpAHPND浸泡感染对虾 46 3.2.2 VpAHPND感染对虾后肝胰腺的颜色与病原菌载量检测 46 3.3结果 47 3.3.1 VpAHPND感染对虾的累积死亡率 47 3.3.2 VpAHPND感染对虾后肝胰腺的颜色变化 47 3.3.3对虾肝胰腺颜色与病原菌载量的关系 54 3.4讨论 58 第4章 对虾感染后病原菌载量与抗病性能关系研究 60 4.1引言 60 4.2材料与方法 60 4.2.1 TaqMan荧光定量PCR引物和探针设计 60 4.2.2重组质粒标准品的制备 62 4.2.3对虾抗性家系和敏感家系的筛选 64 4.2.4家系样品的基因组提取 66 4.3结果 67 4.3.1 Taqman探针实时荧光定量PCR方法的建立 67 4.3.2抗性家系4345和敏感家系4383体内病原菌的增殖规律 68 4.3.3抗性家系4461和敏感家系4430体内病原菌的增殖规律 72 4.3.4抗性家系4410和敏感家系4340体内病原菌的增殖规律 72 4.4讨论 73 第5章 对虾抗弧菌家系和敏感家系的比较转录组分析 76 5.1引言 76 5.2材料与方法 77 5.2.1凡纳滨对虾抗性和敏感家系的筛选 77 5.2.2制备转录组材料 77 5.2.3转录组测序文库构建与高通量测序 80 5.2.4转录组数据的分析 80 5.2.5实时荧光定量PCR验证转录组结果 82 5.3结果 85 5.3.1数据质控和组装统计 85 5.3.2抗性家系和敏感家系之间差异基因统计与转录组质量验证 86 5.3.3抗性家系和敏感家系之间差异基因的功能分析 88 5.4讨论 95 第6章 对虾抗副溶血弧菌相关分子标记筛选 97 6.1引言 97 6.2材料与方法 97 6.2.1凡纳滨对虾抗性和敏感材料的制备 97 6.2.2对虾在注射感染后体内病原菌载量检测 99 6.2.3基于目标区段测序的SNP分型 99 6.2.4与抗VpAHPND相关的SNPs筛选 100 6.2.5 混池测序结果验证 100 6.2.6 VpAHPND感染对虾后候选基因的时序表达谱 102 6.2.7 LvALF6和LvPI3K在不同基因型的个体中表达差异分析 102 6.2.8 LvALF6的候选基因关联分析 103 6.3结果 103 6.3.1注射VpAHPND感染凡纳滨对虾的累积死亡率 103 6.3.2凡纳滨对虾在注射感染后不同组织中的病原菌载量检测 104 6.3.3抗VpAHPND相关SNPs筛选 104 6.3.4 混池测序验证结果 110 6.3.5抗VpAHPND相关的候选基因 111 6.3.6 LvALF6和LvPI3K在不同基因型个体中的时序表达谱 116 6.3.7 LvALF6的候选基因关联分析 117 6.4讨论 118 结论与展望 122 参考文献 124 附录I主要试剂 145 附录II主要仪器 146 致谢 147 作者简历及攻读学位期间发表的学术论文与研究成果 149
文献类型学位论文
条目标识符http://ir.qdio.ac.cn/handle/337002/164647
专题实验海洋生物学重点实验室
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张倩. 对虾抗弧菌性状分子育种技术研发和抗性机制探究[D]. 中国科学院海洋研究所. 中国科学院大学,2020.
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