实验海洋生物学重点实验室知识库

病害问题仍然是对虾养殖业的瓶颈，严重困扰着产业的健康发展。对虾属于无脊椎动物，主要依赖先天免疫体系抵御病原的感染。然而值得注意的是，对虾等甲壳动物中陆续被报道的“免疫致敏”或“训练免疫”现象表明，对虾表现出类似于脊椎动物适应性免疫的特征。目前，虽然部分报道针对该现象背后的机制开展了研究，但是其分子机制仍不清晰。富亮氨酸重复序列（Leucine-rich repeat, LRR）是一保守性较高的氨基酸序列，具有重要的免疫学功能，特别是在无颌类脊椎动物中报道的适应性免疫分子可变淋巴细胞受体（Variable lymphocyte receptor, VLR），其主要功能域由Leucine-rich repeat（LRR）组成。本研究以凡纳滨对虾为材料，首先基于基因组和转录组数据，鉴定了对虾中含LRR结构域的蛋白（LRR domain-containing proteins, 亦称LRR proteins）编码基因，并对其中两条在推导氨基酸序列上与VLR分子特征类似的LRR蛋白编码基因以及一条编码含LRR和Ig结构域（LRRIGs）的基因开展了深入分析和功能研究，阐明了它们在对虾中的免疫学作用，并初步揭示了它们是否与对虾“免疫致敏”现象相关。本论文的主要研究进展如下：

1）凡纳滨对虾LRR蛋白编码基因的鉴定。我们基于目前已报道的含LRR结构域的蛋白序列信息在凡纳滨对虾组学数据中对其LRR蛋白家族编码基因进行了筛选鉴定，获得52条编码框完整的LRR蛋白编码基因序列。对筛选的可信度比较高的LRR蛋白编码基因进行基因和推导氨基酸序列结构分析，根据分析结果和注释信息将对虾中LRR蛋白分为12类。除LRR结构域外，许多预测的LRR蛋白还包含其他已报道与生长发育、免疫、代谢等生理过程相关的功能域。进一步对凡纳滨对虾LRR蛋白序列进行分析，发现了部分与无颌类脊椎动物可变淋巴细胞受体蛋白序列特征类似的编码基因（LvVLR-likes），说明凡纳滨对虾基因组可能编码与无颌类脊椎动物VLR类似的“雏形分子”。

2）凡纳滨对虾LvVLRA-like基因的序列分析及免疫学功能研究。序列分析结果显示，凡纳滨对虾LvVLRA-like蛋白与无颌类脊椎动物VLRA有类似的结构组成，除LRR结构域外还包含LRRNT、LRRCT、信号肽和跨膜结构域；进化分析显示，与TLR相比，LvVLRA-like与VLR具有更高的亲缘关系；亚细胞定位实验结果显示该蛋白主要分布在细胞膜上，说明该蛋白可能是一种膜蛋白；组织分布实验结果显示该基因在凡纳滨对虾各免疫组织中呈泛表达模式，且其转录表达水平在副溶血弧菌感染初期的多个免疫相关组织中其转录表达水平显著上调；LvVLRA-like基因被敲降后，副溶血弧菌感染的宿主体内病原数量约是对照组（注射dsEGFP后进行副溶血弧菌感染）对虾中的9倍，且各个时间点宿主累积死亡率也显著高于对照组，说明该基因可能参与凡纳滨对虾抗副溶血弧菌感染的过程；随后，为了进一步确定该基因发挥作用的调控途径，我们检测了该基因被干扰36h小时后凡纳滨对虾体内抗菌先天免疫信号通路相关基因的表达变化，发现两个NF-κB转录因子编码基因（LvDorsal和LvRelish）以及四条抗菌肽编码基因（LvALF1、LvALF2、LvCrustin1和LvPenaeidin1）都呈显著下调表达趋势，说明凡纳滨对虾LvVLRA-like参与抑制副溶血弧菌增殖是通过调控NF-κB先天免疫信号通路实现的；在病原二次感染后的对虾中，该基因的表达水平与首次感染后没有明显差异，表明该基因可能与对虾“免疫致敏”现象无关。

3）凡纳滨对虾LvVLRB-like基因的序列分析及免疫学功能研究。序列分析结果显示，LvVLRB-like蛋白只包含LRR结构域和信号肽，属典型的分泌蛋白；组织表达谱分析结果显示LvVLRB-like基因主要在凡纳滨对虾肝胰腺中表达，在其它组织中水平较低；免疫响应分析结果显示，LvVLRB-like基因在革兰氏阳性菌表皮葡萄球菌感染6h、12h显著上调表达，在革兰氏阴性菌副溶血弧菌感染6h显著上调表达；原核重组表达的LvVLRB-like蛋白对副溶血弧菌有一定的凝集活性，表明该蛋白对病原有一定的识别和结合能力；该基因敲降后，副溶血弧菌感染24h后对虾肝胰腺中病原菌数量明显高于对照组，约为3.84倍，各个时间点的宿主累计死亡率也显著高于对照组；同时，该基因敲降显著降低了LvALF1和LvPenaeidin1两种抗菌肽编码基因的表达水平，但重组LvVLRB-like蛋白对体外培养血细胞的吞噬活性没有显著影响，表明该蛋白在体内与病原结合后可能启动体液免疫信号通路；病原二次感染后的对虾中，该基因的表达水平与首次感染后没有明显差异，表明该基因可能与对虾“免疫致敏”现象无关。

4）凡纳滨对虾含LvLRRIG 基因的序列分析及免疫学功能研究。序列分析结果表明，凡纳滨对虾LvLRRIG基因编码一种膜受体蛋白，序列包含一个信号肽（SP），一个LRRNT，12个LRR基序, 三个Ig结构域以及一个跨膜结构域（TM）；进化分析结果表明，凡纳滨对虾LvLRRIG蛋白与甲壳动物及昆虫中类似蛋白的亲缘关系较近；副溶血弧菌感染后，LvLRRIG基因在四种被检测免疫相关组织包括肠、鳃、血细胞和Oka中均发生上调表达，特别是在病原感染早期上调表达更为显著；LvLRRIG基因被敲降后，经副溶血弧菌感染的对虾肝胰腺中细菌数量显著增加，约为对照组的10倍；同时，LvLRRIG基因敲降引起了先天免疫信号通路基因包括核转录因子（NF-κB）和四种抗菌肽编码基因（LvRelish、 LvALF1、 LvALF2、 LvPenaeidin1、LvCrustin1）表达量的显著下降，表明凡纳滨对虾LvLRRIG能够通过调控先天免疫信号通路参与宿主的抗病原感染过程；另外，病原二次感染后的对虾中，该基因的表达水平与首次感染后没有明显差异，表明该基因可能与对虾“免疫致敏”现象无关。

本论文主要对凡纳滨对虾LRR蛋白家族编码基因进行了筛选鉴定，并在此基础上对其中两种VLR-like基因及一种LRRIG基因进行了深入分析及免疫学功能探究，初步揭示了它们在病原感染过程中的免疫学功能及潜在的作用机制，为无脊椎动物含LRR蛋白的免疫学功能解析提供了新的认识；同时，研究结果也为VLR基因的分子起源提供了参考。

The disease problem is still the bottleneck of the shrimp aquaculture, which seriously disturbs the healthy development of aquaculture industry. Like other invertebrates, shrimps primarily rely on innate immune system to fight pathogens infection. However, it is worth noting that shrimps may exist characteristics similar to vertebrate adaptive immunity based on "immunosensitization" or "training immunity" phenomena reported in crustaceans. Although the studies related to mechanisms behind the phenomena have been performed, the molecular mechanism is still unclear. Leucine-rich repeat (LRR) is a highly conserved amino acid sequence with important immunological functions. A typical example is the variable lymphocyte receptor (VLR) in jawless vertebrates, whose main functional domain consists of LRR motifs. In the present study, we identified genes encoding LRR domain-containing proteins (also called LRR proteins) in the shrimp Litopenaeus vannamei based on the genomic and transcriptomic data. Among them, two genes encoding LRR proteins exhibiting similar amino acid sequence features to that of VLRs and one gene encoding LRR and Ig domains containing protein (LRRIGs) were further analyzad functionally studied. Their immunological roles in L. vannamei were clarified and their relationship with the "immunosensitization" phenomena was revealed. The main research progresses were as follows.

1) Identification of the genes encoding LRR proteins in L. vannamei. The LRR domain-containing proteins in L. vannamei were screened in the genome and transcriptome database using reported LRR sequences. A total of 52 LRR proteins encoding gene sequences with complete coding frames were identified. The gene and protein structure prediction analysis of the identified LRR proteins with high screening reliability was performed and they were classified into 12 categories according to the analysis results and annotation. In addition to the LRR domain, many LRR proteins also contain other functional domains which have been reported to be involved in growth, development, immunity, metabolism and other physiological processes. Several VLR-like genes (LvVLR-likes), encoding protein sequences with similar domain composition to VLRs in jawless fishes, were identified through further sequence analysis. The data indicated that the shrimp genome might encode the ancestors of VLR genes in jawless invertebrates.

2) Sequence characterization and functional study of LvVLRA-like gene in L. vannamei. Sequence characterization showed that LvVLRA-like protein exhibited similar domain composition to VLRA in jawless invertebrates. In addition to LRR domain, LvVLRA-like protein also contained signal peptide, LRRNT, LRRCT, and the transmembrane region. Evolutionary analysis shows that LvVLRA-like has a higher relationship with VLR than TLR. Subcellular localization analysis showed that LvVLRA-like protein was mainly detected on the cell membrane, indicating that the protein might be a membrane-bound receptor protein. Tissue distribution analysis showed that LvVLRA-like transcripts wildely existed in various immune tissues of L. vannamei. The transcriptional expression level of LvVLRA-like was significantly up-regulated in main immune related tissues at the early stage after pathogen infection. After knockdown of LvVLRA-like, the total number of pathogenic bacteria in the hepatopancreas of shrimp infected with V. parahaemolyticus was 9-fold higher than that in shrimp injected with dsEGFP and infected with V. parahaemolyticus. The cumulative mortality rates of shrimp in LvVLRA-like knockdown group were also significantly higher than those in the control group at each time point. The data suggested that LvVLRA-like gene might be involved in the immune defense of shrimp against V. parahaemolyticus infection. In order to know how LvVLRA-like gene plays functions, we further detected the expression changes of genes in the antibacterial signaling pathways in shrimp after the gene was knocked down. The data showed that two NF-κB transcription factors encoding genes (LvDorsal and LvRelish) and four antimicrobial peptide encoding genes (LvALF1, LvALF2, LvCrustin1 and LvPenaeidin1) were significantly down-regulated in shrimp at 36 h after LvVLRA-like knockdown. This indicated that LvVLRA-like might play immune functions through regulating NF-κB innate immune signaling pathways. The expression level of LvVLRA-like gene in shrimp receiving a secondary infection was not apparently different from that in shrimp after first infection, suggesting that the gene might not be related to the "immunosensitization" phenomena in L. vannamei.

3) Sequence characterization and functional study of LvVLRB-like gene in L. vannamei. Sequence analysis showed that LvVLRB-like protein consisted of the LRR domain and a signal peptide, which was a typical secreted protein. Tissue distribution analysis showed that LvVLRB-like gene mainly expressed in hepatopancreas as well as in other tissues of shrimp with a low level. The expression levels LvVLRB-like gene in hepatopancreas of shrimp were significantlt up-regulated at 6 h and 12 h after injected with the Gram-positive bacteria S. epidermidis, while it was significantly up-regulated at 6 h after injected with the Gram-negative bacteria V. parahaemolyticus. The recombinant LvVLRB-like protein exhibited apparent bacterial agglutination activity against V. parahaemolyticus, suggesting that the protein could recognize and bind to the pathogenic bacteria. After knockdown of LvVLRB-like, the total number of pathogenic bacteria in the hepatopancreas of shrimp infected with V. parahaemolyticus was 3.84-fold higher than that in shrimp injected with dsEGFP and infected with V. parahaemolyticus. The cumulative mortality rates of shrimp in LvVLRB-like knockdown group were also significantly higher than those in the control group at each time point. Meanwhile, the expression levels of two antimicrobial peptide encoding genes (LvALF1 and LvPenaeidin1) were down-regulated in shrimp after LvVLRB-like knockdown. However, the phagocytic activity of in vitro cultured hemocytes to V. parahaemolyticus was not affected after adding recombinant LvVLRB-like protein. These data suggested that LvVLRB-like gene participated in host immune defense against V. parahaemolyticus infection probably through regulating antibacterial immune signaling pathways of L. vannamei. The expression level of LvVLRB-like gene in shrimp receiving a secondary infection was not apparently different from that in shrimp after first infection, suggesting that the gene might not be related to the "immunosensitization" phenomena in L. vannamei.

4) Sequence characterization and functional study of LvLRRIG gene in L. vannamei. Sequence analysis showed that LvLRRIG encoded a protein with a signal peptide (SP), an LRRNT, 12 LRR motifs, three LRR Ig domains, and a transmembrane motif (TM). Phylogenetic analysis showed that LvLRRIG is closely related to LRRIG protein from other crustaceans and insects. After V. parahaemolyticus infection, the expression levels of LvLRRIG gene were significantly up-regulated in intestine, gills, hemocytes and Oka, especially at the early infection stage. After knockdown of LvLRRIG, the total number of pathogenic bacteria in the hepatopancreas of shrimp infected with V. parahaemolyticus was 10-fold higher than that in shrimp injected with dsEGFP and infected with V. parahaemolyticus. Meanwhile, the expression levels of the NF-κB encoding gene LvRelish and four antibacterial peptide encoding genes (LvALF1, LvALF2, LvPenaeidin1 and LvCrustin1) were significantly down-regulated in shrimp after LvLRRIG knockdown, indicating that LvLRRIG participated in the host immune defense by regulating the innate immune signaling pathway. The expression level of LvLRRIG gene in shrimp receiving a secondary infection was not apparently different from that in shrimp after first infection, suggesting that the gene might not be related to the "immunosensitization" phenomena in L. vannamei.

The present study identified LRR proteins encoded by L. vannamei genome. In further, two VLR-likes genes and one LRRIG gene was studied and their immunological functions and potential regulatory mechanisms were explored in shrimp. The present data provided new insights into the immunological functions of invertebrate LRR proteins as well as references for the study of molecular origin of VLRs.

第一章 绪论... 1

1.1 无脊椎动物“免疫致敏”现象及相关免疫分子... 1

1.2 脊椎动物适应性免疫机制... 3

1.3 无颌类脊椎动物中的可变淋巴细胞受体（VLR）... 4

1.4 LRR蛋白结构特征及功能研究现状... 8

1.5 无脊椎动物LRR蛋白研究现状... 10

1.6 本研究的目的与意义... 11

第二章 凡纳滨对虾LRR蛋白家族成员的筛选鉴定... 12

2.1 研究方法... 12

2.1.1 凡纳滨对虾LRR蛋白序列提取... 12

2.1.2 凡纳滨对虾LRR蛋白功能域聚类分析... 13

2.1.3 凡纳滨对虾LvVLR-like基因鉴定与序列分析... 13

2.2 结果... 14

2.2.1 凡纳滨对虾LRR蛋白聚类分析... 14

2.2.2 凡纳滨对虾LRR蛋白家族成员基因结构分析... 19

2.2.3 LvVLR-like基因及蛋白结构特征... 21

2.2.4 凡纳滨对虾LvVLR-like基因可变剪切形式... 22

2.3 讨论... 22

第三章 凡纳滨对虾LvVLRA-like基因免疫学功能研究... 25

3.1实验材料与方法... 25

3.1.1实验用虾及组织取样... 25

3.1.2实验所用菌株和试剂... 26

3.1.3实验所用引物... 26

3.1.4样品总RNA提取及cDNA的合成... 30

3.1.5 LvVLRA-like基因克隆... 32

3.1.6 LvVLRA-like基因序列分析... 36

3.1.7 实时荧光定量PCR实验（qRT-PCR）... 36

3.1.8 双链RNA合成及最佳干扰剂量筛选... 37

3.1.9 RNA干扰实验优势菌株16S测序鉴定... 41

3.1.10 LvVLRA-like亚细胞定位实验... 42

3.1.11 病原二次感染及基因表达检测... 46

3.2实验结果... 48

3.2.1 LvVLRA-like序列特征和结构分析... 48

3.2.2 LvVLRA-like 三维结构模式分析... 50

3.2.3 LvVLRA-like进化分析... 51

3.2.4 LvVLRA-like抑制副溶血弧菌的增殖... 53

3.2.5 LvVLRA-like参与的先天性免疫应答信号途径... 57

3.2.6 LvVLRA-like基因与“免疫致敏”. 58

3.3讨论... 59

第四章 凡纳滨对虾LvVLRB-like基因的分析及免疫学功能探究... 61

4.1实验材料与研究方法... 61

4.1.1实验用虾及组织取样... 61

4.1.2实验所用菌株和试剂... 62

4.1.3实验所用引物... 62

4.1.4总RNA提取及第一链cDNA的合成... 65

4.1.5 LvVLRB-like基因克隆... 66

4.1.6 LvVLRB-like基因序列分析... 70

4.1.7 LvVLRB-like基因干扰实验... 71

4.1.8 RNA干扰实验优势菌株16S测序鉴定... 74

4.1.9实时荧光定量PCR（qRT-PCR）... 74

4.1.10原核重组蛋白的表达与纯化... 75

4.1.11细菌凝集实验... 78

4.1.12血细胞吞噬活性检测... 78

4.1.13病原二次感染及基因表达检测... 79

4.2实验结果... 80

4.2.1 LvVLRB-like基因结构及氨基酸序列比对... 80

4.2.2 LvVLRB-like抑制副溶血弧菌的增殖... 83

4.2.3 LvVLRB-like细菌凝集活性检测... 86

4.2.4 LvVLRB-like与凡纳滨对虾先天性免疫应答... 88

4.2.5 凡纳滨对虾LvVLRB-like基因与“免疫致敏”. 89

4.3小结与讨论... 90

第五章 凡纳滨对虾LvLRRIG基因免疫学功能研究... 92

5.1实验材料与方法... 92

5.1.1实验用虾及组织取样... 92

5.1.2实验所用菌株和试剂... 93

5.1.3实验所用引物... 93

5.1.4样品总RNA提取及cDNA的合成... 97

5.1.5 LvLRRIG基因克隆... 97

5.1.6 LvLRRIG基因序列分析... 97

5.1.7 实时荧光定量PCR实验（qRT-PCR）... 97

5.1.8 双链RNA合成及最佳干扰剂量的筛选... 98

5.1.9 RNA干扰实验优势菌16S测序鉴定... 99

5.1.10病原二次感染及基因表达检测... 99

5.2实验结果... 99

5.2.1 LvLRRIG基因序列特征... 99

5.2.2 LvLRRIG蛋白氨基酸多序列比对及进化树分析... 100

5.2.3 LvLRRIG分子抑制副溶血弧菌增殖... 103

5.2.4 LvLRRIG蛋白参与的先天性免疫应答信号途径... 105

5.2.5 凡纳滨对虾LvLRRIG基因与“免疫致敏”. 106

5.3小结与讨论... 107

第六章 总结与展望... 109

6.1全文总结... 109

6.1.1 凡纳滨对虾LRR蛋白的鉴定和序列分析... 109

6.1.2 凡纳滨对虾VLR类似基因免疫学功能研究... 109

6.1.3 凡纳滨对虾LvLRRIG基因免疫学功能探究... 109

6.2本文的创新点... 110

6.3未来工作展望... 110

参考文献... 111

附录... 125

附录A：实验所用部分试剂配方... 125

附录B：论文相关学术名词（缩写）... 126

致 谢... 129

作者简历及攻读学位期间发表的学术论文... 129

作者简历... 129

在读期间学术论文发表情况... 129