实验海洋生物学重点实验室知识库

       类胡萝卜素是一类广泛存在于动物、植物和微生物的萜烯类物质，包括番茄红素（lycopene）、β胡萝卜素（β-carotene）、虾青素（astaxanthin）、叶黄质（lutein）等，它是人类重要的营养物质，通常具有抗氧化、保护视力、增强免疫力等作用。在这些类胡萝卜素中，虾青素的抗氧化能力最强，是维生素C的6000倍，被称为“抗氧化之王”。虾青素广泛存在于虾蟹等甲壳动物中，但是甲壳动物自身并不能从头合成虾青素，只能从食物中摄取或经过代谢反应获得。目前关于动物体内虾青素的吸收、转运、代谢、利用等过程的了解还很有限。前期研究中，我们实验室成功培育出了能够显著富集虾青素的脊尾白虾新品种“科苏红1号”，它被认为是一种富含虾青素的突变体，外观呈红色。该突变型和野生型脊尾白虾相比，其体内的虾青素含量提高了10倍以上，解析其虾青素富集的分子机理，对于深入了解动物对虾青素的吸收代谢过程，改良甲壳动物品质性状具有重要意义。

        本论文以“科苏红1号”新品种的虾青素富集性状为目标性状开展遗传解析，通过转录组测序分析筛选了突变型红虾和野生型脊尾白虾之间的差异表达基因及通路，并针对类胡萝卜素代谢通路上游的类胡萝卜素加氧酶基因进行了功能研究；通过BSR（Bulked Segregant RNA-Seq）定位技术挖掘了脊尾白虾虾青素富集性状相关单核苷酸多态性（SNP），定位了虾青素富集性状的决定区段；鉴定获得一个与虾青素富集性状密切相关的候选基因，并对该基因的相关功能进行了验证。研究结果为解析脊尾白虾虾青素富集性状的分子机制提供了重要信息，也可为开展甲壳动物品质性状的遗传选育提供重要的理论指导。论文的主要进展如下：

1. 突变型和野生型脊尾白虾的比较转录组分析：为揭示突变型脊尾白虾虾青素富集的潜在分子机制，我们对“科苏红1号”和野生型脊尾白虾的头胸部组织进行了转录组测序和差异比较分析，测序共获得了78224条unigenes，其中差异表达基因（DEG）1863个。在这些差异基因中，902个在突变型红虾中相对高表达，961个相对低表达。差异基因的GO和KEGG富集分析结果显示溶酶体通路是富集最多差异基因的通路，而代谢过程相关的大部分通路在突变型中表现出相对下调的趋势。实时荧光定量PCR（qPCR）验证结果也显示，结合、转运相关基因包括甲壳蓝蛋白、载脂蛋白、羧酸酯酶、表皮蛋白等均在突变型红虾与野生型白虾中呈现差异表达。以上结果说明突变型红虾在虾青素结合、转运和代谢相关的生物学过程中发生了变化，而对这些过程的研究有助于我们进一步揭示脊尾白虾进行虾青素富集的机制。

2. 脊尾白虾中类胡萝卜素分解相关酶的鉴定与功能分析：类胡萝卜素加氧酶通常能够促进类胡萝卜素的裂解，从而影响动物体内类胡萝卜素的累积。通过分析转录组数据，我们在脊尾白虾体内鉴定到7条类胡萝卜素加氧酶相关基因（EcBCO-like）和2条类胡萝卜素异构加氧酶基因（carotenoid isomerooxygenase-like），对这些基因进行分析显示7条类胡萝卜素加氧酶聚为一支，而与类胡萝卜素异构酶的亲缘关系较远。组织表达分析显示EcBCO-like基因主要在肝胰腺或胃中高表达，而carotenoid isomerooxygenase-like基因在眼中高表达，说明这两类基因发挥的作用不同。进一步对EcBCO-like1、EcBCO-like3和EcBCO-like6进行功能研究，发现EcBCO-like1被敲降后脊尾白虾死亡率升高，说明该基因可能在脊尾白虾的基础代谢方面发挥着重要作用，EcBCO-like3的干扰没有引起脊尾白虾发生明显的变化，而EcBCO-like6被敲降后肝胰腺中类胡萝卜素含量发生了显著变化，证实EcBCO-like6在类胡萝卜素代谢中发挥着重要作用。

3. 脊尾白虾中虾青素富集性状相关分子标记和基因的定位及分析：我们通过BSR技术挖掘了虾青素富集性状相关的SNPs，并在性状分离的回交家系和F2代家系材料中进行验证，最终鉴定获得了与虾青素富集性状紧密连锁的SNP标记559个，共分布在347个基因上。通过进一步验证获得3个候选基因Unigene0053862、Unigene0016297和Unigene0027594，基因组比对显示这3个基因位于同一scaffold上。此外，候选基因的表达量分析显示Unigene0053862和Unigene0016297在突变型和野生型脊尾白虾之间无差异，而Unigene0027594在突变型和野生型白虾中表达量差异显著，因此Unigene0027594被确定为脊尾白虾虾青素富集性状相关的候选基因。

4. 脊尾白虾中虾青素富集性状候选基因的鉴定及功能分析：首先对Unigene0027594的cDNA序列在突变型红虾和野生型白虾中进行分析发现存在1个SNP差异，该SNP并未引起编码蛋白的改变；不同组织的表达分析结果显示该基因在野生型白虾的各个组织均有表达，但在突变型红虾的各个组织表达量极低，且不同发育时期的表达结果显示Unigene0027594在野生型白虾中自无节幼体期开始高表达，但在红虾各个发育时期始终呈现低表达，亚细胞定位结果显示该基因定位于细胞膜。利用RNA干扰技术（RNAi）敲降野生型白虾中Unigene0027594的表达，发现表达量显著降低但未发现明显的表型变化。为进一步探讨其在突变型和野生型脊尾白虾中差异表达的原因，我们针对该基因的启动子区序列进行了分析，结果显示突变型红虾中ATG起始密码子前约1 kb的位置处插入了一段约5 kb的大片段，启动子的活性分析显示Unigene27594的启动子位于5 kb插入片段的后面，且大片段的插入会导致启动子活性的大幅降低。以上结果说明Unigene0027594启动子的大片段插入很可能是突变型红虾发生虾青素富集的关键因素。

Carotenoids are tetraterpene pigments which exist in microorganisms, plants and animals, which include lycopene, β-carotene, astaxanthin, lutein and so on. They are important nutrients for human beings, acting as antioxidants, visual pigments, and even with the essential effects on immune defense. Among these carotenoids, astaxanthin shows the strongest antioxidant capacity with 6,000 times higher than vitamin C, which is known as the "king of antioxidants". It is widely found in crustaceans such as shrimps and crabs, however, astaxanthin cannot be synthesized de novo in animals, which are usually accumulated from diets or modified through metabolic reactions. There are limited studies on the absorption, transport, metabolism and utilization of astaxanthin in animals. In recent years, we found a mutant of the ridgetail white prawn Exopalaemon carinicauda, which have been reared and identified as a new variety “Kesuhong No.1” with high level of astaxanthin. The concentration of total astaxanthin in the orange-red body variant prawns is 10 folds higher than that in the wild type prawns. Therefore, revealing the molecular mechanism of astaxanthin accumulation in the ridgetail white prawn is of great significance for understanding the metabolic process of astaxanthin in animals and improving the quality traits of crustaceans.

In this research, the genetic analysis of the astaxanthin enrichment trait of the new variety "Kesuhong No.1" was carried out. The differentially expressed genes and pathways between the new variety and the wild type prawns were identified based on the comparative transcriptome analysis. Additionally, characterization and function analysis of the carotene oxygenase, which contributed to the metabolic processes of carotenoids, were also performed. Those SNPs associated with astaxanthin enrichment trait, which showing the significant genotype differences between the wild type prawns and the orange-red variant, were mined by BSR-Seq. And the trait determining region related to astaxanthin enrichment was analyzed on the basis of the correlation analysis and genetic linkage analysis. The identification and function analysis of candidate gene underlining the astaxanthin enrichment in E. carinicauda was also conducted. These results provide important information for understanding the molecular mechanism of astaxanthin accumulation in E. carinicauda, and essential reference for the breeding strategies in crustaceans. The main research progresses were as follows.

1. Comparative transcriptome analysis in the mutant and wild type prawns. To reveal the potential molecular mechanism of astaxanthin enrichment in E. carinicauda, transcriptome analysis was performed between the mutant and the wild type prawns. A total of 78224 unigenes were obtained and 1863 were identified as DEGs, in which 902 unigenes showed higher expression levels, while 961 unigenes presented lower expression levels in the mutant. Based on the GO analysis and KEGG analysis, “Lysosome” was the most significantly enriched pathway, and most pathways in Metabolism were down-regulated in the mutant. The results of qPCR detection also showed that genes related to binding and transport, including crustacyanin, Apolipoprotein D (ApoD), carboxylesterase and cuticle proteins, showed significantly differential expression between the two types of prawns. These data indicated that the biological processes related to astaxanthin transport, metabolism and binding showed significant expression differences between the mutant and the wild type prawns, and these differences may help us find out the mechanism of astaxanthin enrichment in E. carinicauda.

2. Identification and functional analysis of enzymes related to carotenoid cleavage in E. carinicauda. Carotene oxygenases can contribute to the degradation of carotenoids, and some researches have shown that they have important effects on carotenoid accumulation in animals. In our study, nine homologous genes were identified in the transcriptome database of ridgetail white prawn, including seven EcBCO-like genes and two carotenoid isomerooxygenase-like genes. Phylogenetic analysis showed that these two carotenoid isomerooxygenase-like genes completely separated from EcBCO-like genes, and EcBCO-like genes except EcBCO-like1 were clustered together. Additionally, tissue distribution analysis showed that EcBCO-like genes were highly expressed in tissues like hepatopancreas or stomach, while carotenoid isomerooxygenase-like genes were highly expressed in the eyestalk. This indicated that these two types of genes might play different roles. We then conducted the RNA interference on EcBCO-like1, EcBCO-like3 and EcBCO-like6 for functional research, and results showed that the mortality rate of these prawns increased rapidly after knockdown of EcBCO-like1. This indicated that EcBCO-like1 might be of importance in fundamental metabolism process. No phenotype change was observed after EcBCO-like3 knockdown, while prawns injected with dsEcBCO-like6 showed color changes in their hepatopancreas and the content of carotenoid in their hepatopancreas was much higher than that in the control prawns. This result suggested that EcBCO-like6 may play an important role in carotenoid metabolism.

3. Mapping and genetic analysis of genes related to astaxanthin enrichment in E. carinicauda. Polymorphic SNPs were identified by using the BSR-Seq data and verified in the mapping population F1, F2. A total of 559 SNP markers associated with astaxanthin enrichment were identified, and they were distributed on 347 genes. Then three genes including Unigene0053862, Unigene0016297 and Unigene0027594 were found tightly linked to the trait and located on the same scaffold. In addition, the expression analysis of candidate genes showed that Unigene0053862 and Unigene0016297 were with no difference in the expression level between the new variety and the wild type prawns, while Unigene0027594 was differentially expressed in these two populations. Therefore, Unigene0027594 was identified as the candidate key gene that was tightly linked to the mutant.

4. Identification and functional analysis of candidate gene related to astaxanthin enrichment in E. carinicauda. There was only one polymorphic SNP in the cDNA sequence of Unigene0027594 between the new variety and wild type prawns, and the SNP was located in the non-coding region. Expression analysis showed that this gene was expressed in all tissues of wild type prawns, but the expression level in the new variety was extremely low. Through analyzing its expression at different developmental stages, we found that Unigene0027594 was highly expressed in the wild type prawns since the nauplius stage. However, the expression level of Unigene0027594 was always significantly lower than that in the wild type prawns at different developmental stages. In addition, the expression of GFP-Unigene0027594 fusion protein revealed its localization on the cell membrane. After knockdown of Unigene0027594 in the wild type prawns, the expression level of this gene was lower but there was no apparent phenotypic change. In order to understand the reasons for its differential expression in the new variety and the wild type prawns, the sequence of the promoter region of Unigene0027594 was analyzed, and the result showed that a fragment about 5 kb was inserted at the position about 1 kb before the initiation codon in the new variety. In addition, the promoter activity was determined by dual-luciferase reporter assay, and the analysis showed that the promoter of Unigene0027594 was located behind the inserted fragment, and the insertion of the fragment will result in a substantial decrease in promoter activity. These results suggested that the inserted fragment in the promoter region of Unigene0027594 may be the key factor of astaxanthin enrichment in E. carinicauda.

第一章 绪论............................................................................................................1
1.1 类胡萝卜素的概述...........................................................................................1
1.1.1 类胡萝卜素的结构与种类........................................................................1
1.1.2 类胡萝卜素的功能....................................................................................3
1.2 动植物中类胡萝卜素的合成与代谢机制...................................................4
1.2.1 植物中类胡萝卜素的合成........................................................................4
1.2.2 动物对类胡萝卜素的吸收与转运............................................................6
1.2.3 动物对类胡萝卜素的结合与沉积............................................................7
1.2.4 动物对类胡萝卜素的分解........................................................................7
1.2.5 虾青素的合成与代谢................................................................................8
1.3 动植物品质性状与类胡萝卜素的关系..........................................................11
1.3.1 生物常见品质性状...................................................................................12
1.3.2 类胡萝卜素对品质性状的影响...............................................................13
1.3.3 植物中类胡萝卜素富集品质性状的遗传解析.......................................14
1.3.4 动物中类胡萝卜素富集品质性状的遗传解析.......................................15
1.3.5 品质性状的遗传解析策略.......................................................................17
1.4 甲壳动物类胡萝卜素研究进展......................................................................19
1.4.1 甲壳动物类胡萝卜素的种类...................................................................19
1.4.2 类胡萝卜素对甲壳动物及其品质性状的影响.......................................20
1.4.3 甲壳动物中类胡萝卜素富集性状的形成机制.......................................21
1.5 本研究的目的与意义......................................................................................22
第二章 基于 RNA-seq 技术筛选虾青素富集性状相关差异基因......................25
2.1 引言..................................................................................................................25
2.2 材料及方法......................................................................................................25
2.2.1 实验材料及取样.......................................................................................25
2.2.2 实验所用试剂及菌株...............................................................................26
2.2.3 实验所用引物...........................................................................................26
2.2.4 样品总 RNA 的提取 ................................................................................27
2.2.5 测序文库构建...........................................................................................28
2.2.6 转录组的组装及注释...............................................................................29
2.2.7 差异表达基因的分析...............................................................................29
2.2.8 第一链 cDNA 的合成 .............................................................................29
2.2.9 实时荧光定量 PCR .................................................................................30
2.3 结果..................................................................................................................31
2.3.1 转录组测序基本信息...............................................................................31
2.3.2 Unigene 基本注释....................................................................................32
2.3.3 差异表达基因的分析...............................................................................35
2.3.4 差异表达基因的验证...............................................................................42
2.4 讨论..................................................................................................................43
第三章 脊尾白虾 β-Carotene Oxygenase-like 基因的鉴定与功能分析
................................................................................................................................47
3.1 引言..................................................................................................................47
3.2 材料及方法......................................................................................................47
3.2.1 实验材料及取样.......................................................................................47
3.2.2 实验所用试剂及菌株...............................................................................48
3.2.3 实验所用引物...........................................................................................48
3.2.4 样品总 RNA 的提取 ...............................................................................49
3.2.5 第一链 cDNA 的合成 .............................................................................49
3.2.6 EcBCO-like 基因的克隆..........................................................................50
3.2.7 EcBCO-like 基因序列分析......................................................................53
3.2.8 EcBCO-like 基因的组织分布..................................................................56
3.2.9 EcBCO-like 基因的双链合成及干扰实验..............................................56
3.2.10 类胡萝卜素投喂实验.............................................................................60
3.3 实验结果..........................................................................................................61
3.3.1 脊尾白虾中 EcBCO-like 基因的多样性.................................................61
3.3.2 EcBCO-like 基因的进化分析..................................................................62
3.3.3 EcBCO-like 基因的组织分布..................................................................63
3.3.4 EcBCO-like 基因的干扰实验..................................................................65
3.3.5 EcBCO-like 基因敲降后的投喂实验......................................................65
3.4 讨论..................................................................................................................67
第四章 脊尾白虾中虾青素富集性状的 BSR 分析.............................................71
4.1 引言..................................................................................................................71
4.2 材料及方法......................................................................................................71
4.2.1 实验材料及取样.......................................................................................71
4.2.2 实验所用试剂及菌株...............................................................................71
4.2.3 实验所用引物...........................................................................................71
4.2.4 脊尾白虾基因组的提取...........................................................................72
4.2.5 性状相关 SNP 的筛选 ............................................................................73
4.2.6 性状相关 SNP 的验证 ............................................................................73
4.3 实验结果..........................................................................................................74
4.3.1 BSR-Seq 测序结果...................................................................................74
4.3.2 SNP 验证家系材料的准备 .....................................................................74
4.3.3 性状相关 SNP 位点和基因的发掘 ........................................................75
4.3.4 SNP 及候选基因验证 .............................................................................76
4.4 讨论..................................................................................................................80
第五章 脊尾白虾中虾青素富集性状候选基因的表达分析及功能验
证............................................................................................................................83
5.1 引言..................................................................................................................83
5.2 材料及方法......................................................................................................83
5.2.1 实验材料及取样.......................................................................................83
5.2.2 实验所用试剂及菌株...............................................................................84
5.2.3 RNA 的提取 ............................................................................................84
5.2.4 第一链 cDNA 的合成 .............................................................................85
5.2.5 目的基因的克隆.......................................................................................85
5.2.6 Unigene0027594 基因的序列分析..........................................................86
5.2.7 不同组织和发育时期 Unigene0027594 的表达量检测.........................86
5.2.8 Unigene0027594 的双链合成及干扰实验..............................................87
5.2.9 亚细胞定位...............................................................................................89
5.2.10 候选基因启动子的克隆.........................................................................94
5.2.11 双荧光素酶实验.....................................................................................94
5.3 实验结果..........................................................................................................98
5.3.1 Unigene0027594 的克隆与序列分析......................................................98
5.3.2 Unigene0027594 的表达分析..................................................................99
5.3.3 Unigene0027594 的干扰实验................................................................100
5.3.4 Unigene0027594 的亚细胞定位............................................................101
5.3.5 Unigene0027594 的启动子区分析........................................................102
5.3.6 Unigene0027594 启动子活性................................................................103
5.4 讨论................................................................................................................104
结论与展望..........................................................................................................107
参考文献..............................................................................................................111
附录......................................................................................................................131
致谢......................................................................................................................133
作者简历及攻读学位期间发表的学术论文与研究成果..................................135