实验海洋生物学重点实验室知识库

  抗菌肽作为先天免疫应答产生的一类重要的效应分子，在宿主抗感染免疫防御中发挥重要作用。Crustin是甲壳动物广泛存在的一类抗菌肽，目前已在甲壳动物中报道了I、II、III和IV四种类型，它们在体外具有抗微生物、抑制蛋白酶等多种活性。然而，目前对于这类蛋白的体内功能和作用机制了解甚少。本论文以凡纳滨对虾为研究对象，以已有的对虾组学数据为基础，系统分析了crustin（LvCrustin）基因的序列结构特征和功能多样性，并对其中占多数类型的三条代表性crustin基因的体内作用机制进行了深入研究，研究结果不仅丰富了对虾先天免疫的基础理论，也为对虾的病害防治提供了重要理论指导。论文主要进展如下：

1. 对虾crustin基因的序列多样性和特征分析：在转录组水平上鉴定了34条具有完整开放阅读框（ORF）的crustin基因LvCrustins，基于氨基酸序列特征，将其分为6种类型（Type I、II、III、IV、VI和VII型），其中Type I型和II型包含的基因数量较多，TypeI型Crustin包含三个亚类（Type Ia,、Ib和Ic）, Type II 型Crustin包含两个亚类（Type IIa和IIb）。这些类型中的Type VI和VII两种类型和Type Ib和Ic两个亚类为本研究新发现；此外还发现了3条具有非典型乳清酸蛋白（WAP）结构域的crustins。表达模式分析结果显示：大多数LvCrustins主要在表皮、胃、鳃、肠或血细胞等中的两种或以上组织高表达；有些在表皮、胃或鳃中呈现高表达的LvCrustins，在对虾蜕皮的不同阶段，其表达量表现出明显不同：如在对虾蜕皮后其表达量显著高于其他蜕皮时期；其中发现一条在卵巢中呈现高表达的LvCrustin，其转录本主要存在于囊胚期之前的胚胎期，而肢芽期之后几乎不表达。经进一步qPCR检测发现，其中有15条在转录组中具有较高表达量的LvCrustins均在副溶血弧菌或WSSV感染后呈现明显的上调表达，提示其在对虾应对病原感染的过程中起着重要作用。综上所述，对虾中的crustin基因在结构和功能上均显示出明显的多样性特征。

2. Ⅰa型crustin代表基因—LvCrustin Ⅰa-1的功能研究：LvCrustin Ⅰa-1具有典型的WAP结构域，主要在血细胞、肠道、鳃、表皮、胃等组织中表达。LvCrustin Ⅰa-1被敲降后，凡纳滨对虾消化道微生物群落结构发生显著变化，且感染弧菌后体内可培养细菌数目显著高于对照组，表明LvCrustin通过调控体内微生物群落平衡的方法抵御病原感染。菌群功能预测分析结果显示，LvCrustin Ⅰa-1被敲降后对虾消化道菌群的运动能力、感知和响应外界环境的能力下降，与宿主及其他细菌的相互作用减弱，菌群增殖、能量代谢等生理功能受到影响。LvCrustin Ⅰa-1敲降后对虾肠道转录组分析结果显示，对虾肠道中胞外基质组分、细胞连接、细胞骨架、细胞凋亡、免疫信号通路及效应因子相关基因的表达显著下调，表明对虾肠道上皮的物理和化学屏障受到破坏。上述结果表明，LvCrustin Ⅰa-1可通过调控对虾消化道菌群组成及调节对虾肠道免疫水平等方式发挥其免疫学功能。

3. 具有非典型WAP结构域的代表性基因LvCrustin Ⅰa-2的功能研究：通过与具有典型WAP结构域的 Ⅰa 型crustin基因进行多重序列比对，发现LvCrustin Ⅰa-2的WAP结构域中第2和7位上的两个半胱氨酸残基发生突变。LvCrustin Ⅰa-2主要在胃、肠道、鳃以及表皮等与外界环境直接接触的组织中表达。通过双链RNA敲降LvCrustin Ⅰa-2，导致凡纳滨对虾在感染副溶血弧菌后体内可培养细菌数量显著高于对照组，表明LvCrustin Ⅰa-2参与了副溶血弧菌的感染过程。然而LvCrustin Ⅰa-2重组蛋白对对虾致病菌没有显示出明显的抑制活性，仅具有一定的菌结合活性。此外，LvCrustin Ⅰa-2敲降后对虾肠道微生物群落结构发生显著变化，其中脱甲基菌（Demequina）、丙酸菌（Propionibacterium）、厌氧孢菌（Anaerospora）以及小海员菌（Nautella）等潜在致病菌比例显著增加，弧菌（Vibrio）、拟杆菌（Bacteroidia）等细菌比例显著下降。菌群功能预测分析结果显示，LvCrustin Ⅰa-2被敲降后，对虾肠道菌群中两相系统、细菌趋化性、细菌分泌系统等通路丰度显著降低，表明菌群的定植能力、响应外界环境的能力以及细菌间的相互作用均减弱。上述结果表明LvCrustin Ⅰa-2可能不是通过直接杀灭致病菌的方式抵御病原感染，而是通过调控体内微生物群落平衡的方式发挥其免疫学功能。

4. 鳃高表达 Ⅱa 型crustin—LvCrustin Ⅱa-1的功能研究：LvCrustin Ⅱa-1主要在对虾鳃中高表达。副溶血弧菌浸泡对虾后，LvCrustin Ⅱa-1的表达量显著上调。LvCrustin Ⅱa-1基因沉默的对虾，浸泡副溶血弧菌后，体内可培养细菌数目显著高于对照组，表明LvCrustin Ⅱa-1具有抵御环境中致病菌感染的功能。外周海水和对虾鳃的微生物组比较分析结果显示，对虾鳃中环境微生物的比例显著低于外周海水，而有益共生菌的比例明显高于外周海水，表明对虾鳃中微生物的定植受到宿主的明显调控。LvCrustin Ⅱa-1被敲降后对虾鳃中潜在致病菌和环境微生物的比例发生显著变化，表明LvCrustin Ⅱa-1在调控对虾鳃内的微生物群落中具有重要作用。

  通过以上研究，明确了对虾crustin基因的结构和功能的多样性，揭示了crustin基因通过调控体内微生物群落结构及机体免疫水平等体内作用机制，发挥抵御病原感染的功能。研究结果不但完善了甲壳动物抗菌肽作用机理等免疫学认知，而且也为对虾病害防治策略提供了新的视角。

        Antimicrobial peptides (AMPs) are a kind of immune effectors in the immune response, which play important roles in the immune defense of the host against pathogen infection. Crustins are one kind of AMPs which are widely distributed in crustaceans. Four types of crustin, including type I, II, III, and IV, have been reported in crustaceans with multiple functions in vitro, including antimicrobial activity, protease inhibition, and so on. However, knowledge about their in vivo functions and functioning mechanism is still very limited. In the present study, we analyzed the sequence diversity and studied the functions of crustin in the Pacific white shrimp Litopenaeus vannamei base on the omics data. Three representative crustins were studied further to uncover their in vivo function in shrimp. The present study will not only enrich the basic theory of shrimp innate immunity, but also provide important instructions for developing disease control stragies in shrimp. The main progresses were as follows.

1. Analyses on the sequence diversities and characterization of crustins in shrimp. A total of 34 full-length crustin encoding genes were identified in Litopenaeus vannamei. Sequence analysis of their deduced mature peptides revealed that these crustins could be categoried into six types, including Type I, II, III, IV, VI, and VII. Type I and Type II crustins accounted for the majority of the identified crustins. Type I crustins contained three sub-types, including Type Ia, Ib and Ic, and Type II crustins contained two sub-types, Type IIa and IIb. Among all identified crustins, two types (Type VI and VII) and two subtypes (Type Ib and Ic) are the newly identified types or subtypes of crustins in the present study. Furthermore, three LvCrustin genes with atypical WAP domain, were identified. Expression analysis showed that most LvCrustins were mainly distributed in two or more shrimp tissues among epidermis, stomach, intestine, gill, and hemocytes. Several LvCrustins, mainly distributed in epidermis, stomach and gill, showed the highest expression levels in post-molt stage during the molting cycle of shrimp, , which indicating that they might participate in the immune protection to shrimp during molting. One identified crustin, which was predominantly distributed in the ovary of shrimp, showed expression only in embryo developmental stages before gastrula, which suggested that this crustin might be a maternal immune effector. Quantitative PCR analysis on 15 selected crustins with relatively high expression level in shrimp showed that most of these genes were apparently up-regulated after Vibrio parahemolyticus or WSSV infection. These data indicated that these crustins might play important roles in shrimp againt pathogen infection. To summarize, the crustin genes in Litopenaeus vannamei presented high diversities in structures and functions.

2. Functional study of the representative crustin of Type Ia, LvCrustin Ⅰa-1. LvCrustin Ⅰa-1 is a type I crustin with a typical WAP domain. It was mainly distributed in hemocytes, intestine, gill, epidermis and stomach of the shrimp. After knockdown of LvCrustin Ⅰa-1 with dsRNA, the intestinal microbiota were changed apparently, and the bacteria number in hepatopancreas of shrimp upon V. parahaemolyticus infection dramatically increased, indicating that the pathogen defense function of LvCrustin might be performed through modulation on the intestinal microbiota homeostasis. Functional prediction on the intestinal microbiota showed that the locomotivity, sensory ability, interspecific competition, reproduction ability, and energy metabolism of microbiota declined significantly after LvCrustin Ⅰa-1 was knocked down. The result of transcriptome analysis on shrimp intestine after LvCrustin Ⅰa-1 knockdown suggested the extracellular matrix component, cell junction, cytoskeleton, apoptosis, immune pathway and effector relevant genes were down-regulated, indicating the intestinal epithelial barrier of the LvCrustin Ⅰa-1-silensed shrimp was broken. These data suggested that LvCrustin Ⅰa-1 not only had microbiota modulation function but also played immunoregulation functions in intestine.

3. Functional study of the representative crustin with an atypical WAP domain, LvCrustin Ⅰa-2. Analysis on the WAP of LvCrustin Ⅰa-2 showed that the cysteins located on position 2 and 7 were changed to other amino acids. This gene was mainly distributed in the tissues which are directly contact with the environment, such as intestine, gill, epidermis and stomach in the shrimp. Knockdown of LvCrustin Ⅰa-2 with dsRNA resulted in a significant increase of the bacteria number in hepatopancreas of shrimp upon V. parahaemolyticus infection, showing that LvCrustin Ⅰa-2 participated in pathogen infection process. Recombinant LvCrustin Ⅰa-2 protein showed some microorganism-binding activity rather than antibacterial activity against tested bacteria. Furthermore, significant difference existed between the intestinal microbiota in shrimp before and after LvCrustin Ⅰa-2 knockdown based on the result of alpha and NMDS analysis. Knockdown of LvCrustin Ⅰa-2 increased the proportion of Demequina, Nautella, Propionibacterium, Anaerospora and decreased the proportion of Bacteroidia and Vibrio. The functional prediction of intestinal microbiota showed that the sensory ability, colonial ability, and interspecific competition of microbiota declined after LvCrustin Ⅰa-2 was knocked down. These data suggested that LvCrustin Ⅰa-2 might perform its immunological function through modulation to the intestinal microbiota homeostasis rather than direct inhibition on the bacterial growth in shrimp.

4. Functional study of the representative crustin of type Ⅱa crustin, LvCrustin Ⅱa-1 which is highly expressed in the gills of shrimp. The expression level of LvCrustin Ⅱa-1 was significantly up-regulated at different time points after Vibrio parahaemolyticus immersion. When LvCrustin Ⅱa-1 was silenced by dsRNA interference, the counts of bacteria colonies in the hepatopancreas of shrimp increased significantly after V. parahaemolyticus immersion, which indicated that the infection progress of pathogenic bacteria was accelerated after the expression of LvCrustin Ⅱa-1 decreased. Compared with the microbiota in seawater outside shrimp, the lower proportion of environmental bacteria and higher proportion of symbiont in the gill microbiota of shrimp indicated that the bacterial colonization was modulated by the host. Knockdown of LvCrustin Ⅱa-1 changed the proportion of some potential pathogens and environmental bacteria, which supported the idea that the new identified crustin in the gill played important roles in modulation of the microbiota community in in the gills of the shrimp.

        In summary, the present study revealed diversity of crustins in structures and functions in shrimp. Functional studies suggested that LvCrustins denfended pathogen infection probably through modulating the microbiota and immune level of the host. The results of the study will increase the knowledge about the immune mechanism of crustacean AMPs, and provide new insights into the disease control strategy in shrimp aquaculture.

目  录

第1章  绪论... 1

1.1  对虾常见病害及免疫系统概述... 1

1.1.1  对虾常见病害... 1

1.1.2  对虾免疫系统概述... 2

1.2  抗菌肽... 5

1.2.1  抗菌肽的分类... 5

1.2.2  抗菌肽的抗微生物活性及其作用机制... 6

1.2.3  抗菌肽功能的多样性... 7

1.2.4  抗菌肽在调控微生物群落结构中的作用... 8

1.3  甲壳动物抗菌肽研究进展... 9

1.3.1  甲壳动物抗菌肽的多样性... 9

1.3.2  对虾抗菌肽及其在抵御病原感染中的作用... 10

1.3.3  甲壳动物抗菌肽调控体内微生物群落的研究进展... 11

1.3.4  Crustin研究进展... 12

1.4  本研究的目的与意义... 14

第2章  凡纳滨对虾Crustin基因的多样性... 15

2.1  引言... 15

2.2  材料与方法... 15

2.2.1  序列鉴定及分析... 15

2.2.2  实验动物... 16

2.2.3  病原感染及组织取样... 16

2.2.4  总RNA的提取... 17

2.2.5  cDNA的合成... 18

2.2.6  荧光定量PCR.. 18

2.3  结果... 20

2.3.1  序列鉴定及分类分析... 20

2.3.2  LvCrustins的组织分布分析... 24

2.3.3  LvCrustins在对虾蜕皮过程中的免疫防护作用... 25

2.3.4  LvCrustin在母源免疫中的作用... 26

2.3.5  病原刺激后LvCrustins的表达量变化... 27

2.4  讨论... 28

第3章  LvCrustin Ⅰa-1在对虾体内微生物群落调控及免疫调节中的作用研究... 33

3.1  前言... 33

3.2  材料与方法... 33

3.2.1  实验动物及组织取样... 33

3.2.2  LvCrustin Ⅰa-1基因的组织分布... 33

3.2.3  LvCrustin Ⅰa-1基因的克隆... 33

3.2.4  LvCrustin Ⅰa-1的序列分析... 37

3.2.5  双链RNA合成及干扰剂量的摸索... 37

3.2.6  LvCrustin Ⅰa-1沉默对虾的细菌感染实验... 40

3.2.7  LvCrustin Ⅰa-1沉默对虾肠道微生物的16s扩增子分析... 41

3.2.8  LvCrustin Ⅰa-1沉默对虾肠道转录组分析... 43

3.2.9  统计分析... 45

3.3  实验结果... 45

3.3.1  LvCrustin Ⅰa-1基因的序列分析... 46

3.3.2  LvCrustin Ⅰa-1基因的组织分布... 46

3.3.3  LvCrustin Ⅰa-1的体内抗菌作用... 47

3.3.4  LvCrustin Ⅰa-1对对虾消化道内微生物的调控作用... 49

3.3.5  LvCrustin Ⅰa-1基因的沉默对对虾肠道基因转录表达的影响... 61

3.4  讨论... 77

第4章  具有非典型WAP结构域的LvCrustin Ⅰa-2在对虾抵御病原感染中的作用机制研究... 87

4.1  引言... 87

4.2  材料与方法... 87

4.2.1  LvCrustin Ⅰa-2基因的组织分布... 87

4.2.2  LvCrustin Ⅰa-2基因的克隆及序列分析... 88

4.2.3  LvCrustin Ⅰa-2双链RNA的合成及干扰剂量的摸索... 89

4.2.4  LvCrustin Ⅰa-2沉默对虾的细菌感染实验... 89

4.2.5  LvCrustin Ⅰa-2蛋白的原核表达... 90

4.2.6  LvCrustin Ⅰa-2蛋白的纯化... 92

4.2.7  LvCrustin Ⅰa-2重组蛋白对致病菌的最小抑菌浓度实验... 93

4.2.8  LvCrustin Ⅰa-2蛋白的菌结合活性检测... 95

4.2.9  LvCrustin Ⅰa-2沉默对虾消化道微生物组分析... 96

4.2.10  统计分析... 97

4.3  实验结果... 97

4.3.1  LvCrustin Ⅰa-2的序列结构特征分析... 97

4.3.2  LvCrustin Ⅰa-2的组织分布... 99

4.3.3  对虾LvCrustin Ⅰa-2基因的敲降对副溶血弧菌感染进程的影响... 100

4.3.4  LvCrustin Ⅰa-2的原核表达、抑菌活性及菌结合活性... 102

4.3.5  LvCrustin Ⅰa-2对肠道微生物的调控作用... 104

4.4  讨论... 109

第5章  对虾鳃中高表达Crustin Ⅱa-1在调控鳃微生物群落中的作用研究    113

5.1  前言... 113

5.2  材料与方法... 114

5.2.1  LvCrustin Ⅱa-1基因的组织分布检测... 114

5.2.2  LvCrustin Ⅱa-1基因的克隆... 114

5.2.3  LvCrustin Ⅱa-1的序列分析... 115

5.2.4  凡纳滨对虾在弧菌浸泡感染过程中LvCrustin Ⅱa-1的表达量检测... 115

5.2.5  LvCrustin Ⅱa-1双链RNA的合成及干扰剂量的摸索... 116

5.2.6  LvCrustin Ⅱa-1沉默对虾的浸泡感染实验... 117

5.2.7  LvCrustin Ⅱa-1沉默的对虾中鳃的微生物组分析... 117

5.2.8  统计分析... 119

5.3  实验结果... 119

5.3.1  LvCrustin Ⅱa-1的序列分析... 119

5.3.2  LvCrustin Ⅱa-1的组织分布... 121

5.3.3  副溶血弧菌浸泡感染对虾后LvCrustin Ⅱa-1基因的表达量变化... 122

5.3.4  LvCrustin Ⅱa-1体内抗菌作用... 122

5.3.5  LvCrustin Ⅱa-1对对虾鳃中微生物群落结构的调控作用... 123

5.4  讨论... 131

结论与展望... 135

参考文献... 137

致  谢... 163

作者简历及攻读学位期间发表的学术论文与研究成果... 165