实验海洋生物学重点实验室知识库

蜕皮是对虾生长发育和生殖过程中的一个重要事件，其研究具有重要的理论价值和经济意义。然而目前有关对虾蜕皮信号通路的调控网络并不完整，突出的缺口是蜕皮信号传递至EcR/RXR后，蜕皮激素（20-hydroxyecdysone，20E）应答因子如何调控下游分子，进而表现出蜕皮和生长现象，其分子机制并不清楚。本课题以凡纳滨对虾为材料，研究多个蜕皮激素应答基因的结构功能并初步分析了对虾蜕皮信号通路与典型的生长发育信号通路-Wnt通路之间的关系。

通过生物信息学方法分析RNA-Seq数据和基因组数据，我们筛选鉴定和克隆得到了凡纳滨对虾20E重要应答基因LvEcR、LvRXR、LvE75、LvHR3和LvFTZ-F1等，并对以上各基因序列进行基因结构分析，构建系统发生树，分析其表达模式，继而分析各基因的功能。研究表明在本研究中涉及的5个蜕皮激素信号通路关键基因都存在多种可变剪切形式。凡纳滨对虾LvEcR基因存在8种可变剪切体；LvRXR基因存在4种可变剪切体；LvE75基因存在6种可变剪切体；LvHR3和LvFTZ-F1基因分别存在5种和4种可变剪切体。这些可变剪切发生在3’或5’端非编码区，通过外显子选择性剪切，内含子保留等方式产生。我们利用凡纳滨对虾表达谱数据，分析了以上5个基因在胚胎和幼体发育时期、蜕皮过程和不同组织中的表达模式。结果显示，在胚胎和幼体发育时期，5个基因的转录形式最为丰富，而在成虾中，各基因仅存在1-2种转录形式。通过dsRNA干扰功能实验，我们发现LvE75、LvHR3、LvBr-C和LvFTZ-F1分别被干扰后会导致凡纳滨对虾蜕皮率下降，死亡率显著增加。针对死亡或濒死的对虾进行显微观察，我们发现凡纳滨对虾出现头部或尾部不能完成蜕皮，或甲壳不能完全脱落的现象。

为了了解对虾蜕皮和生长之间的关系，我们选择与生长发育密切相关的Wnt基因作为研究对象。在本研究中我们鉴定了凡纳滨对虾Wnt基因家族所有成员，分别命名为LvWnt1、LvWnt2、LvWnt4-LvWnt11、LvWnt16和LvWntA，这些基因都属于单拷贝基因，且发现对虾基因组中存在LvWnt1-LvWnt6保守基因簇。同时结合不同发育时期、不同蜕皮过程和成虾不同组织的RNA-Seq数据初步分析了对虾Wnt基因的表达模式。LvWnts在幼体发育早期开始表达，持续至仔虾时期，但在成虾中只有LvWnt5和LvWntA等少部分LvWnts表达。RT-qPCR验证LvWnts在胚胎和幼体发育时期的表达情况，结果和转录组数据一致。

另外我们利用蜕皮激素20E处理和蜕皮激素信号通路关键基因干扰的样品，采用荧光定量方法，检测多个Wnt基因以及Wnt通路相关的ZW3和Sulf-1基因的表达水平，分析蜕皮信号通路与Wnt通路之间的关联。结果表明，蜕皮激素20E对LvWnt基因有更显著的调控作用，而LvEcR、LvRXR和LvE75基因对所研究的几个LvWnt基因调控不显著。蜕皮激素20E处理后，LvZW3表达水平显著上调，对蜕皮激素比较敏感，而LvSulf-1无明显效果；LvEcR被干扰后，LvZW3表达水平无明显变化，而LvSulf-1表达量显著下调；LvRXR被干扰后，LvZW3表达水平显著下降，LvSulf-1表达量上调；LvFTZ-F1和LvBr-C被干扰后LvZW3和LvSulf-1有一致明显的上调效果。总结以上研究结果，我们推测，对虾的蜕皮和生长是各自不同的信号通路调控，同时两个通路之间具有少量基因相互关联。

Molting plays vital important roles in shrimp development, growth and reproduction, and its research has tremendous theoretical and economic values. However, the regulatory network of molting signal pathway in crustacean is incomplete. What and how the ecdysone response genes to regulate downstream genes to achieve molting and growth is still unclear. In this study, we characterized the structures and functions of ecdysone response genes and analysed the relationship between molting signal pathway and Wnt signal pathway based on transcriptome and genome data from the pacific white shrimp, Litopenaeus vannamei.

We screened and characterized 20E early response genes, LvEcR, LvRXR, LvE75, LvHR3 and LvFTZ-F1.The sequences analysis showed that the five molting related genes had several splicing forms. In L. vannamei, LvEcR had eight alternative spliced isoforms; LvRXR had 4 isoforms; LvE75 had 6, LvHR3 and LvFTZ-F1 has 5 and 4 isoforms, respectively. These isoforms are the results of 3’ and/or 5’ UTR or extron splicing, or intron conserved. Based on L. vannamei transcriptome data, five 20E early response genes showed different expression patterns in different development stages, molting stages and adult different tissues. Together with all results showed that the five genes were transcripted in several isoforms in embryo and larvae, while in adult, every gene was transcripted in one or two isoforms. Silencing of LvE75, LvHR3, LvBr-C and LvFTZ-F1 using double-stranded RNA (dsRNA) caused a significantly decline of the cumulative molting rate and a greatly increase of the cumulative death rate. The dead or dying shrimp were failed to complete molting in head and/or tail of shrimp. These results indicated that LvE75, LvHR3, LvBr-C and LvFTZ-F1 in shrimp played important roles in molting process and survival.

In order to investigate the relationship between molting and growth in shrimp, we focused on Wnt signal pathway that is critically involved in development and growth. We described 12 Wnt genes representing 12 distinct Wnt gene subfamilies in L. vannamei. Based on homolog annotation and phylogenetic analysis, these 12 Wnt genes named as LvWnt1, LvWnt2, LvWnt4-LvWnt11, LvWnt16 and LvWntA. All LvWnts were single-copy genes. Interestingly, LvWnt1 and LvWnt6 were adjacent in the same scaffold in the shrimp genome. All the investigated LvWnt genes were initially expressed at the gastrula or limb bud embryo stages, and last to post larvae. Only parts of them were expressed in adult shrimp, as LvWnt5 and LvWntA. We used RT-qPCR to assess the expression levels in different development stages consisting with transcriptome data.

To further understand the crosstalk between molting and growth, we used qRT-PCR to detect the expression levels of LvWnts, LvZW3 and LvSulf-1 genes in 20E-treated and ecdysone related gene interferenced samples. The results showed that LvWnt genes were significantly regulated by 20E, while LvEcR, LvRXR and LvE75 genes had little influence on LvWnt genes. In addition, LvZW3 and LvSulf-1 were different controlled by 20E and ecdysone response genes. LvZW3 was up-regulated in 20E treated samples; LvSulf-1 was down-regulated in LvEcR interferenced samples; LvZW3 was down-regulated significantly and LvSulf-1 was up-regulated in LvRXR interferenced samples; LvZW3 and LvSulf-1 were up-regulated in LvFTZ-F1 and LvBr-C interferenced samples. In conclusion, we hypothesize that the molting and growth of shrimp are controlled by different signal pathways, and there are crosstalk between two pathways through a few genes.

目  录

摘要........................................................................................................................... I

ABSTRACT.......................................................................................................... III

第一章 文献综述.................................................................................................. 1

1.1 蜕皮信号通路.................................................................................................... 1

1.1.1 蜕皮激素上游调控机制............................................................................. 2

1.1.2 蜕皮激素信号通路下游调控机制............................................................. 4

1.1.3 蜕皮激素信号调控生长的机制研究......................................................... 7

1.2 Wnt信号通路..................................................................................................... 8

1.2.1 Wnt基因的发现.......................................................................................... 8

1.2.2 Wnt基因家族.............................................................................................. 8

1.2.3 Wnt信号通路的作用.................................................................................. 9

1.2.4 蜕皮信号通路与Wnt信号通路的关系.................................................. 10

1.3 本研究的目的及意义...................................................................................... 10

1.4 本研究的主要内容和技术路线...................................................................... 11

第二章 凡纳滨对虾EcR和RXR基因结构、表达模式及功能分析      13

2.1 实验材料与方法.............................................................................................. 13

2.1.1 实验材料................................................................................................... 13

2.1.2 组织取样................................................................................................... 13

2.1.3 数据来源................................................................................................... 13

2.1.4 主要试剂和实验仪器............................................................................... 14

2.1.5 LvEcR和LvRXR基因序列分析............................................................. 14

2.1.6 总RNA提取和cDNA合成.................................................................... 14

2.1.7 LvEcR和LvRCR基因克隆..................................................................... 16

2.1.8 双链RNA（dsRNA）合成及RNA干扰.............................................. 19

2.1.9 荧光定量PCR........................................................................................... 22

2.2 结果和分析...................................................................................................... 24

2.2.1 凡纳滨对虾LvEcR和LvRXR基因的鉴定和isoforms分析............... 24

2.2.2 系统发生分析........................................................................................... 26

2.2.3 基因表达模式........................................................................................... 28

2.2.4 LvEcR和LvRXR基因干扰..................................................................... 31

2.2.5 LvEcR和LvRXR基因被干扰后对下游信号通路基因表达的调控..... 31

2.3 讨论.................................................................................................................. 33

第三章 凡纳滨对虾E75基因可变剪切形式鉴定、表达模式及功能分析 35

3.1 材料与方法...................................................................................................... 35

3.1.1 实验材料及组织取样............................................................................... 35

3.1.2 数据来源................................................................................................... 35

3.1.3 主要试剂及实验仪器............................................................................... 35

3.1.4 序列分析................................................................................................... 35

3.1.5 总RNA的提取及cDNA的合成............................................................ 35

3.1.6 E75基因克隆............................................................................................. 36

3.1.7 荧光定量PCR（qRT-PCR）................................................................... 37

3.1.8 双链RAN合成及最佳干扰剂量摸索..................................................... 38

3.1.9 双链RNA对蜕皮率和死亡率的影响..................................................... 39

3.2 结果和分析...................................................................................................... 39

3.2.1 凡纳滨对虾E75基因的鉴定和isoforms分析....................................... 39

3.2.2 LvE75基因各可变剪切形式在蜕皮周期和不同组织的表达分布........ 44

3.2.3 LvE75基因干扰剂量................................................................................ 45

3.2.4 LvE75基因被干扰后对凡纳滨对虾蜕皮和存活情况的影响................ 45

3.2.5 LvE75基因沉默对蜕皮激素信号通路上下游基因表达的影响............ 46

3.3 讨论.................................................................................................................. 47

第四章 凡纳滨对虾HR3基因和FTZ-F1基因可变剪切鉴定及表达模式分析      49

4.1 材料与方法...................................................................................................... 49

4.1.1 实验材料................................................................................................... 49

4.1.2 数据来源................................................................................................... 49

4.1.3 主要试剂及实验材料............................................................................... 49

4.1.4 总RNA的提取及cDNA的合成............................................................ 49

4.1.5 LvHR3和LvFTZ-F1序列分析................................................................ 49

4.1.6 LvHR3和LvFTZ-F1基因克隆................................................................ 49

4.1.7 荧光定量PCR（qRT-PCR）................................................................... 50

4.1.8 双链RNA（dsRNA）合成及最佳干扰剂量摸索................................. 50

4.1.9 dsRNA干扰对蜕皮率和死亡率的影响................................................... 51

4.2 结果.................................................................................................................. 51

4.2.1 凡纳滨对虾HR3和FTZ-F1基因可变剪切形式鉴定、结构分析....... 51

4.2.2 凡纳滨对虾HR3和FTZ-F1基因在不同发育时期的表达模式........... 54

4.2.3 凡纳滨对虾HR3和FTZ-F1基因在不同组织中的表达模式............... 55

4.2.4 凡纳滨对虾HR3和FTZ-F1基因在蜕皮不同阶段的表达模式........... 56

4.2.5 LvHR3和LvFTZ-F1基因干扰的最佳剂量筛选.................................... 56

4.2.6 LvHR3和LvFTZ-F1基因干扰对蜕皮率和死亡率的影响.................... 57

4.2.7 LvFTZ-F1基因干扰对蜕皮通路其他基因的影响.................................. 58

4.3 讨论.................................................................................................................. 59

第五章 凡纳滨对虾Wnt基因家族鉴定及表达模式分析............... 61

5.1 材料与方法...................................................................................................... 61

5.1.1 实验材料................................................................................................... 61

5.1.2 Wnt基因鉴定............................................................................................ 61

5.1.3 序列分析................................................................................................... 61

5.1.4 进化树分析............................................................................................... 62

5.1.5 LvWnt基因表达模式分析........................................................................ 62

5.1.6 RNA提取和cDNA合成.......................................................................... 62

5.1.7 荧光定量PCR（RT-qPCR）................................................................... 62

5.2 结果.................................................................................................................. 63

5.2.1 LvWnt基因鉴定和结构分析.................................................................... 63

5.2.2 系统发生分析........................................................................................... 69

5.2.3 LvWnt基因表达模式................................................................................ 70

5.2.3.1  LvWnt基因在不同发育时期的表达模式.......................................... 70

5.2.3.2  不同蜕皮时期的表达模式.................................................................. 72

5.2.3.3  不同组织中的表达模式...................................................................... 73

5.3 讨论.................................................................................................................. 74

第六章 凡纳滨对虾蜕皮信号通路与Wnt通路的相互联系.......... 77

6.1 材料和方法...................................................................................................... 77

6.1.1 实验材料................................................................................................... 77

6.1.2 序列分析................................................................................................... 77

6.1.3 荧光定量PCR........................................................................................... 77

6.2 结果与分析...................................................................................................... 78

6.2.1 LvZW3和LvSulf-1基因序列分析.......................................................... 78

6.2.2 20E处理及蜕皮激素应答基因干扰对LvZW3和LvSulf-1基因的影.. 78

6.2.3 20E处理及LvEcR、LvRXR、LvE75基因干扰对Wnt基因的影响.. 79

6.3 讨论.................................................................................................................. 80

结论........................................................................................................................... 83

参考文献................................................................................................................. 85

致  谢...................................................................................................................... 93

个人简历................................................................................................................. 95

硕士期间发表和完成的文章......................................................................... 96