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刺参体色发生生理特征与调控机制的基础研究
邢丽丽
Subtype博士
Thesis Advisor杨红生
2019-05-13
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Name理学博士
Keyword仿刺参 体色 色素沉积 Mitf基因 遗传育种
Abstract

从无脊椎动物到哺乳动物都存在着色素沉积过程。动物的色素沉积除了实现稀有和美丽的体色外,还具有诸多功能,包括伪装、体温调节、交配选择、个体交流、抗干燥、盐度适应以及免疫等,这些作用使得体色成为生物生存及繁殖相关的重要表型特征。此外,色素沉积过程也为理解进化生物学中的其他关键问题,如适应、物种形成及平衡多态性的维持等,提供了与之相关并且具体可见的表型特征。

棘皮动物体色多样,甚至在物种内体色也有巨大差异,而有关棘皮动物色素及色素沉积的研究125多年前就开始了,至今依然是研究热点之一。另外,棘皮动物属于后口动物,其临近分类物种包括脊索动物,因此,相对于其他无脊椎动物,棘皮动物许多参与发育、再生和色素形成的基因与哺乳动物的关系更密切。刺参作为棘皮动物之一,具有多种体色和重要的营养、医药价值,在亚洲许多国家和地区被认为是最具价值的海洋商业物种。目前青刺参较为普遍,紫刺参和白刺参数量很少且更受消费者青睐。然而,迄今为止,对于刺参体色形成的分子和遗传机制知之甚少,甚至在棘皮动物中亦是如此。本研究通过Illumina转录组、IBT蛋白组、LC-QTOF-MS代谢组、ChIP-seqReal time PCR、原位杂交、免疫组化和遗传育种等技术来揭示刺参体色形成的分子和遗传机制。主要研究结果如下:

1. 刺参色素种类、含量及黑色素细胞比较研究

在本项研究中,对白刺参、浅青刺参、深青刺参及紫刺参四种色型刺参体壁进行了色素种类、含量及黑色素细胞比较研究。色素种类及含量比较实验结果表明:白刺参的色素种类远远少于其他颜色刺参;除白刺参外,浅青、深青、紫刺参中都存在黑色素、虾青素、β-胡萝卜素和叶黄素这些生物色;黑色素对于刺参体色的形成起到最主要作用;结构色鸟嘌呤在所有色型刺参中都是最高的。TEM(透射电子显微镜)黑色素细胞比较分析实验结果表明:白刺参中黑色素细胞数量最少且不存在成熟的黑色素小体;随着刺参色型颜色加深,其黑色素颗粒数量越多;深青刺参和紫刺参中除了存在成熟黑色素小体,还有黑色素颗粒分泌到细胞外。

2. 刺参体色发生各阶段生长生理特性比较研究

在本项研究中,从生长、组织、细胞三个层面系统描述了不同色型刺参不同体色发生阶段的具体差异。在生长生理方面,无论是体色发生率还是生长率都是紫刺参高于青刺参高于白刺参。组织结构方面,不同色型刺参体色发生三阶段的体壁结构比较研究表明,体壁表皮层厚度和黑色素细胞密度皆为紫刺参高于青刺参高于白刺参。色素细胞方面,TEM实验研究表明,紫刺参成熟黑色素小体数量最多,体色发生后阶段出现黑色素颗粒分泌。表皮层厚度和黑色素含量可能是导致三种色型刺参之间生长和存活率差异的新发现因素。

3. 刺参体色发生三阶段转录组分析

在本项研究中,通过Illumina测序,对白、青、紫刺参体色发生的三阶段进行了基因表达动态变化趋势分析,并筛选出与白化、色素沉积、体色多态性相关的差异表达基因,为系统解析刺参体色发生及白化的分子调控机制奠定了基础。但令人意外的是,无论是本项研究中获得的刺参转录组数据还是刺参基因组数据,以及海胆基因组、转录组中,都未发现酪氨酸酶基因,而酪氨酸酶是黑色素形成通路中最为关键的酶,它将酪氨酸转化为DOPA。因此推测刺参乃至棘皮动物黑色素合成路径中的酪氨酸酶功能可能由其他基因编码的酶取代,或者酪氨酸酶基因序列在某些分类单元中是高度不保守的,同时也不排除黑色素有外源摄取的可能。

4. 紫刺参体色发生三阶段蛋白组分析

在本项研究中,首次使用IBT蛋白质组测序技术来揭示紫刺参体壁色素沉积的分子机制。通过对紫刺参体色发生前、体色发生时、体色发生后三个阶段进行蛋白质组分析,共鉴定出5580个蛋白,其中,紫刺参体色发生时阶段相对于体色发生前阶段有1099个差异蛋白,体色发生后阶段相对于体色发生时阶段中有339个差异蛋白。GOKEGG分析表明,“黑色素合成”, “黑色素小体”,“黑素瘤”, “色素生物合成过程”, “表皮发育”, Ras信号通路”, Wnt信号通路”的相关差异蛋白可能涉及到紫刺参色素合成和调控。该项研究鉴定出的差异蛋白对于揭示刺参色素沉积机制具有重要作用,并且为今后研究刺参抗黑素瘤、白化、UV损伤及其他疾病奠定了基础。

5. 刺参体色发生阶段代谢组分析

在本项研究中,利用UHPLC-QTOF/MS技术对处于体色发生时阶段的白刺参、青刺参、紫刺参进行了代谢组分析,共鉴定出2633种代谢物,筛选出了参与色素沉积、体色形成过程的潜在差异显著代谢物,为阐明刺参代谢过程和体色发生的相关机制奠定了基础。

6. MITF基因在白、青、紫刺参中的差异研究

MITF基因是黑色素细胞发育以及黑色素合成通路中最关键的基因之一。本项研究涵盖原位杂交、免疫组化、ChIP-seqreal time PCRWestern Blot、以及多态性等实验。其中,real time PCR实验表明,MITF mRNA的表达量在紫刺参最高,白刺参最低;Western Blot实验表明,MITF蛋白的表达量在紫刺参最高,白刺参最低;免疫组化实验表明,MITF蛋白主要在表皮层表达;原位杂交实验表明,MITF 基因主要在表皮层表达;多态性实验表明,1-9号外显子中,共存在20SNP位点,其中两个位点是非同义突变,其余的是同义突变。ChIP-seq实验筛选出了MITF可能结合的DNA序列。该项研究全面阐释了MITF基因在不同颜色刺参中的表达情况,并筛选出了与体色相关的SNP,后续将在良种选育中进行应用。

7. 刺参杂交、自交家系子代分析

在本项研究中,采用完全双列杂交设计,构建了九个家系。研究结果表明,除白刺参外,自交家系子代与亲代体色一致,杂交子代体色多数与亲代母本体色一致,杂交子代相对于自交子代生长更为快速。根据本研究结果推断,野生白刺参体色遗传不稳定,需要不断选育;紫刺参体色遗传稳定且个体间体色差异极小,是研究刺参体色遗传的理想材料;刺参生长可能存在杂交优势,可通过不同杂交家系获得优势组合;刺参体色遗传受母系性状影响。

Other Abstract

Pigmentation processes occur from invertebrates to mammals. Body wall pigmentation in animals serves many functions, in addition to achieving rare and beautiful coloration. Body color and coloration patterns are important phenotypic traits associated with the survival and reproductive activities in many organisms, including camouflage, thermoregulation, mating selection, social interactions, desiccation resistance, salinity adaptation, and immunity. Furthermore, Pigmentation processes provide a traceable and relevant trait for understanding key issues in evolutionary biology such as adaptation, speciation and the maintenance of balanced polymorphisms.

Echinoderms have been a rich source of pigment and pigmentation research for over 125 years. These animals are enormously diverse in color with rich differences even within a species. In addition, they are also basal branching deuterostomes whose sister taxa include chordates. Therefore, many of the genes involved in development, in regeneration, and in pigment formation are more closely related to mammals. Here, we consider pigment formation in Apostichopus japonicus. The sea cucumber Apostichopus japonicus, which has nutritive and medical properties, is considered the most valuable commercial species in many parts of Asia. Compared with the green morph, the purple and white morph are rare and have great appeal to consumers. However, little is currently known about the molecular and genetic mechanism of body color formation in A. japonicus, even in echinoderm. In this study, we used Illumina sequencing, IBT sequencing, LC-QTOF-MS, ChIP-seq, real time PCR, in-situ hybridization, immunohistochemistry, genetic breeding, et al. to reveal the molecular and genetic mechanism of body color formation in A. japonicus. Main results are as follows:

1. Comparative study on the pigment type, pigment content and melanocytes of A. japonicus

In this study, we conducted research on pigment composition and melanin content among white, light-green, dark-green, and purple morphs of sea cucumber, A. japonicus. The results of the comparison experiments of pigment types and contents showed that there were fewer types of pigments in white sea cucumber than in the other color morphs; the biological color pigments melanin, astaxanthin, β-carotene, and lutein were detected in light-green, dark-green, and purple sea cucumbers; melanin played the most important role in the formation of body color; the concentration of guanine which belonged to structural color is highest in all color morphs. Transmission electron microscopy analyses revealed that white sea cucumbers had the fewest epidermal melanocytes in the body wall, and their melanocytes contained fewer melanosomes as well as non-pigmented pre-melanosomes. Sea cucumbers with deeper body colors contained more melanin granules. In the body wall of dark-green and purple sea cucumbers, melanin granules were secreted out of the cell.

2. Growth, histology and ultrastructure differences in three pigmentation stages of green, white and purple morphs of A. japonicus

In this study, the growth, histology and ultrastructure of two newly discovered white and purple color morphs were investigated and compared with the common green morph, in the same breeding environment. At 50 days old, the growth and survival rates and the extent of pigmentation were the highest in purple, then green color morphs, and were lowest in white morphs. The white morph had fewer, and less developed, epidermal melanocytes compared with the green and purple color morphs. The results suggested that growth and survival rates were highest in purple sea cucumber and lowest in white sea cucumber. Epidermis thickness and melanin content may be the newfound factors contributed to the differences of growth and survival rate among three color morphs.

3. Transcriptome analysis of three sea cucumber color morphs in three pigmentation stages

In this study, Illumina sequencing was performed to analyze the gene expression dynamic trend of white, green and purple sea cucumbers in three pigmentation stages, and to screen the differential expressed genes related to albinism, pigmentation and body color polymorphism. These results may provide base for systematically analyzing the molecular regulation mechanism of body color formation and albinism of sea cucumbers. Surprisingly, the major enzyme responsible for melanin synthesis and converting tyrosine to DOPA, tyrosinasea copper containing phenol oxidaseis not detectable yet, neither in the transcriptome data obtained in this study nor the genome data of sea cucumbers. Perhaps this enzyme activity is accomplished by various different genes or is highly variable in sequence in certain taxa. There is also the possibility of exogenous intake of melanin.

4. IBT-based quantitative proteomics analysis of purple sea cucumber in three pigmentation stages

In this study, isobaric tags (IBT) were first used to reveal the molecular mechanism of pigmentation in the body wall of the purple sea cucumber. We analyzed the proteomes of purple sea cucumber in early pigmentation stage (Pa), mid pigmentation stage (Pb) and late pigmentation stage (Pc), resulting in the identification of 5580 proteins, including 1099 differentially expressed proteins in Pb: Pa and 339 differentially expressed proteins in Pc: Pb. GO and KEGG analyses revealed possible differentially expressed proteins, including “melanogenesis”, “melanosome”, “melanoma”, “pigment-biosynthetic process”, “Epidermis development”, “Ras-signaling pathway”, “Wnt-signaling pathway”, involved in pigment synthesis and regulation in purple sea cucumbers. The large number of differentially expressed proteins identified here should be highly useful in further elucidating the mechanisms underlying pigmentation in sea cucumbers. Furthermore, these results may also provide the base for further identification of proteins involved in resistance mechanisms against melanoma, albinism, UV damage, and other diseases in sea cucumbers.

5. Metabolome analysis of three sea cucumber color morphs in mid-pigmentation stage

In this study, UHPLC-QTOF/MS technique was performed to analyze the metabolome of white sea cucumber, green sea cucumber and purple sea cucumber in the mid-pigmentation stage. A total of 2633 metabolites were identified. And the differential metabolites potentially related to pigmentation and body color formation were screened out, which lays a foundation for elucidating the related mechanisms of the metabolic process and body color formation mechanism of sea cucumbers.

6. Differences of MITF gene in white, green and purple sea cucumbers

The MITF gene is one of the most critical genes in melanocyte development and melanin synthesis pathways. In this study, we conducted in situ hybridization, immunohistochemistry, ChIP-seq, real time PCR, Western Blot, and polymorphism analysis experiments. Among them, real time PCR experiment showed that the expression level of MITF mRNA was the highest in purple sea cucumber and the lowest in white sea cucumber; Western Blot experiment showed that the expression level of MITF protein was the highest in purple sea cucumber and the lowest in white sea cucumber; immunohistochemistry experiment showed that the MITF protein was mainly expressed in the epidermis. The in situ hybridization experiment showed that the MITF gene was mainly expressed in the epidermis. Gene polymorphism experiment showed that there were 20 SNPs in exon 1-9, two of which were non-synonymous mutations, and the rest were synonymous mutations. The ChIP-seq experiment screened out DNA sequences that might be bound by MITF. This study comprehensively explained the expression of MITF gene in different color morphs, and screened out the SNP related to body color, which will be applied in the breeding.

7. Self-crossing and Hybridization family construction of sea cucumbers

In this study, nine families were constructed using a complete double-column hybrid design. The results showed that, the body color of wild white sea cucumber is not stable and requires continuous breeding. Purple sea cucumber is an ideal material for studying the color inheritance of sea cucumber. There may be hybridization advantages in the growth of sea cucumber, and the superior combinations can be obtained through different hybrid families. The genetic mechanism of sea cucumber body color is affected by maternal traits.

Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/156906
Collection海洋生态与环境科学重点实验室
Recommended Citation
GB/T 7714
邢丽丽. 刺参体色发生生理特征与调控机制的基础研究[D]. 中国科学院海洋研究所. 中国科学院大学,2019.
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