实验海洋生物学重点实验室知识库

肌肉是由肌细胞经历发育分化、融合为成熟的肌纤维及肌纤维生长等过程发育而来，这些过程受到多种基因的调控。Shisa蛋白家族是近年来发现的一类含有N端半胱氨酸结构域和C端脯氨酸区的单跨膜衔接蛋白家族，在脊椎动物的发育、癌症的产生和细胞凋亡中发挥重要的作用。shisa-2的表达在鱼类胚胎中具有体节特异性，同时体节是形成肌肉的基础，然而shisa-2在鱼类肌肉生长发育中的功能尚不清楚。

本研究以具有重要经济价值的海水养殖鱼类牙鲆（Paralichthys olivaceus）和模式生物斑马鱼（Danio rerio）为研究对象，克隆获得了牙鲆和斑马鱼的shisa-2基因，并对其所编码的氨基酸序列进行了分析；通过原位杂交和RT-PCR分析了基因在牙鲆和斑马鱼胚胎期的时空表达特点，同时利用qPCR分析了基因在牙鲆二、三倍体胚胎中的表达差异；通过细胞转染的方法，分析了蛋白的亚细胞定位；通过在牙鲆和斑马鱼胚胎中注射mRNA的方法进行基因的瞬时过表达研究，分析了其对肌肉发育标志因子myod表达的影响；构建了肌肉特异启动子驱动的表达载体，期望获得持续过表达的转基因斑马鱼，从而分析过表达shisa-2对肌肉发育的影响；在牙鲆和斑马鱼中进行了基因编辑实验，期望获得shisa-2突变体并分析基因的缺失对肌肉发育的影响。相关研究结果将丰富鱼类肌肉发育和调控机制的理论，为牙鲆等经济鱼类养殖提供参考。

1.  从牙鲆和斑马鱼中克隆得到shisa-2序列，对氨基酸序列进行分析发现，牙鲆和斑马鱼Shisa-2 都具有一个N端的信号肽区域、富含半胱氨酸结构域、跨膜区和脯氨酸区域。通过构建系统发育进化树发现，牙鲆Shisa-2与罗非鱼、金头鲷的聚在一起，而斑马鱼Shisa-2与鲤鱼的聚在一起。  

2.  RT-PCR分析表明，牙鲆shisa-2在未受精卵中有微弱表达，在原肠期之后，随着肌节的形成，shisa-2的表达量逐渐增加；原位杂交显示shisa-2 表达在体节中，同时表达在早期脑泡、视泡、耳囊、鳃弓中。在斑马鱼胚胎中，在原肠期以后，其在逐渐发育形成的前体节中胚层中表达量较高，在早期脑泡、视泡、耳囊中也表达。这些结果表明shisa-2在牙鲆和斑马鱼中有着体节特异性的表达特征，可能参与了肌肉发育过程的调控。

3.  在牙鲆和斑马鱼中对shisa-2的功能做了初步探究。将构建的重组表达载体pEGFP- N1- shisa-2转染到Hela细胞中，结果显示Shisa-2 蛋白定位在细胞内质网上。在瞬时过表达shisa-2的牙鲆和斑马鱼胚胎中，通过原位杂交发现肌肉特异因子myod的表达受到影响。通过CRISPR/Cas9技术对牙鲆和斑马鱼的shisa-2 进行敲除，免疫组化分析牙鲆F0代杂合突变体肌纤维融合及体节形态的情况，结果显示其体节形态出现异常。在斑马鱼中筛选获得了shisa-2 敲除的F1代杂合突变体。同时，将构建的肌肉特异性启动子驱动表达shisa-2的载体与Tol2转座酶共注射到斑马鱼胚胎中，经筛选得到了F1代转基因shisa-2斑马鱼。

Myoblast experience a process of differentiation, development, and maturation fusing into muscle fibers. Muscle fibers contine to grow and develop becoming mature muscles. These processes are controlled by a variety of genes. The Shisa protein family, which has been discovered in recent years, is a family of single transmembrane adaptor proteins containing an N-terminal signal peptide, cysteine ​​domain and a C-terminal proline region. Some researches have shown the Shisa plays an important role in the development of vertebrates, the generation of cancer, and apoptosis. The expression of shisa-2, one member of this family genes, shows somite specific in fish embryos, while the somite will development into muscle. But the function of shisa-2 in fish muscle growth and development is still unclear.

In this study, the marine aquaculture fish olive flounder（Paralichthys olivaceus）which has important economic value, and the model organism zebrafish (Danio rerio) were used as the research objects.Flounder and zebrafish shisa-2 were cloned, and the predicted encoded amino-acids were analyzed. The spatiotemporal expression characteristics of the genes in the embryonic stage of olive flounder and zebrafish were analyzed through in situ hybridization and RT-PCR; at the same time, the differences of expression during embryoic development between diploid and triploid flounder embryos were analyzed by qPCR. The subcellular localization of the protein was analyzed by cell transfection. Transient overexpression was performed by injecting shisa-2 mRNA into olive flounder and zebrafish embryos, and its effect on the expression of muscle development marker gene myod was analyzed. An expression vector driven by a muscle-specific promoter was constructed, and it was hoped to obtain continuously overexpressed transgenic zebrafish for analyzing the effect of overexpression on muscle development. Gene editing experiments were carried out in olive flounder and zebrafish, and it was hoped that mutants would be obtained and genes analyzed. The results will enrich the theory of fish muscle development and regulation, providing reference for commercial fish farming.

1. The shisa-2 was cloned from olive flounder and zebrafish, and the amino-acids sequences analysis found that both of olive flounder and zebrafish Shisa-2 contained N-terminal signal peptide, Cystein rich domain, transmembrance region and Proline rich region. By constructing a phylogenetic tree, it was found that flounder Shisa-2 clustered with that of tilapia and gilthead seabream, and zebrafish Shisa-2 clustered with that of carp.

2. RT-PCR results showed that shisa-2 was weakly expressed in unfertilized eggs in olive flounder embryos, and after the gastrulation stage, the expression of shisa-2 increased with the formation of somite. In situ hybridization shows that shisa-2 was expressed in the pre-somite mesoderm, and also in the early brain vesicles, optic vesicles, and ear vesicles. In zebrafish embryos, shisa-2 was highly expressed in the developing somites after the gastrulation stage and expressed in the early brain vesicles, optic vesicles, and ear vesicles, too. These results indicate that shisa-2 has somites specific expression characterization and it might be involved in muscle development.

3. The function of shisa-2 was investigated in olive flounder and zebrafish. The recombinant expression vector pEGFP-N1-shisa-2 was constructed and transfected into Hela cells, and the results showed that Shisa-2 protein was located in the endoplasmic reticulum. In the olive flounder and zebrafish embryos transiently overexpressing of shisa-2, in situ hybridizion results showed that the expression of the muscle-specific factor myod was changed. The shisa-2 of olive flounder and zebrafish were knocked out through CRISPR/Cas9 methods. In F0 heterozygous mutant olive flounder, the fusion of muscle fibers and the morphology of the somite were analyzed through immunhischemistry, and the results showed that the morphology of the somite of the heterozygous mutant was abnormal. F1 generation mutants were also screened in zebrafish. At the same time, the shisa-2 expression vector driven by muscle-specific promoter was constructed and microinjected into zebrafish embryos with Tol2 tanspatase, and the F1 transgenic shisa-2 zebrafish was got through screening.

第1章  引言... 1

1.1  肌肉的生长发育及功能... 1

1.1.1  骨骼肌的结构与功能... 1

1.1.2  鱼类肌肉的生长方式... 2

1.1.3  相关基因及信号通路在肌肉发育中的作用... 2

1.1.4  鱼类二、三倍体肌肉相关基因比较的研究... 4

1.2  内质网在肌肉发育中的作用... 4

1.2.1  内质网的结构与功能... 4

1.2.2  内质网在肌肉发育中的作用... 5

1.3  Shisa家族基因... 7

1.3.1  Shisa蛋白的结构... 7

1.3.2  shisa 基因的功能... 7

1.3.3  Shisa蛋白的作用机制... 8

1.3.4  shisa-2 研究进展... 9

1.4  立项依据和目的意义... 9

第2章 牙鲆和斑马鱼shisa-2基因的克隆及表达分析... 11

2.1  实验材料与方法... 11

2.1.1  实验材料与样品的获取... 11

2.1.2  主要试剂... 12

2.1.3  主要仪器设备... 12

2.1.4  实验方法... 13

2.1.4.1  总RNA 的提取... 13

2.1.4.2  DNase 消化总RNA.. 13

2.1.4.3  cDNA 第一条链的合成... 14

2.1.4.4  shisa-2 基因的克隆... 14

2.1.4.5  RT-PCR.. 16

2.1.4.6  整胚原位杂交... 17

2.1.4.7  绝对定量表达分析... 20

2.1.4.8  shisa-2系统发育进化分析... 21

2.2  结果        22

2.2.1  牙鲆和斑马鱼shisa-2 基因的克隆与序列分析... 22

2.2.2  牙鲆shisa-2 基因在胚胎发育各时期的时空表达... 25

2.2.3  斑马鱼shisa-2 基因在胚胎发育各时期的时空表达... 27

2.2.4  二、三倍体牙鲆中shisa-2 的表达量差异... 29

2.3  讨论        31

2.3.1  牙鲆和斑马鱼shisa-2 基因序列特征分析... 31

2.3.2  牙鲆和斑马鱼shisa-2 基因在胚胎发育过程中的表达... 31

2.3.3  shisa-2在二、三倍体牙鲆中的表达差异分析... 33

第3章 牙鲆和斑马鱼shisa-2 基因功能的初步分析... 35

3.1  实验材料及方法... 35

3.1.1  实验材料... 35

3.1.2  主要试剂... 35

3.1.3  主要仪器设备... 35

3.1.4  实验方法... 36

3.1.4.1  Shisa-2 蛋白的亚细胞定位... 36

3.1.4.2  shisa-2 mRNA过表达实验... 40

3.1.4.3  牙鲆shisa-2基因敲除实验... 43

3.1.4.4  转基因shisa-2斑马鱼的构建... 49

3.2  实验结果... 53

3.2.1  基因组验证... 53

3.2.2  Shisa-2 蛋白的亚细胞定位... 54

3.2.3  牙鲆胚胎中过表达shisa-2 对myod表达的影响... 55

3.2.4  斑马鱼胚胎中过表达shisa-2 对myod表达的影响... 56

3.2.5  牙鲆shisa-2 突变体的筛选... 57

3.2.6  斑马鱼shisa-2 突变体的筛选... 60

3.2.7  牙鲆shisa-2杂合突变个体肌纤维形态... 63

3.2.8  转基因shisa-2斑马鱼的筛选... 66

3.3 讨论        67

3.3.1  Shisa 蛋白的亚细胞定位... 67

3.3.2  shisa-2 基因对体节及肌肉发育的影响... 68

总 结... 70

参考文献... 71

致  谢... 71

作者简历及攻读学位期间发表的学术论文与研究成果    80