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硬壳蛤壳色差异机制初步研究
胡志
Subtype硕士
Thesis Advisor张涛
2020-05-15
Degree Grantor中国科学院大学
Place of Conferral中国科学院海洋研究所
Degree Discipline生物工程
Keyword硬壳蛤 壳色 色素 分子标记 机制
Abstract

    硬壳蛤(Mercenaria mercenaria)自然栖息于北美大西洋沿岸,是美国重要的渔业资源。1997 年,张福绥院士等人由美国引种硬壳蛤至我国,其耐温耐盐,适合我国大部分池塘养殖,目前已成为一种重要的池塘养殖贝类,养殖面积数十万亩。壳色是双壳贝类一项重要的表型性状,逐渐成为贝类育种关注的热点。在经济贝类中,壳色可影响海产品受消费者的喜爱程度和其市场价格。因此研究硬壳蛤壳色差异的分子机制,对选育壳色新品种具有重要实践意义。

    本研究利用 ELISA 测定贝壳、外套膜类胡萝卜素和黑色素含量,利用 Raman spectra 技术研究贝壳表面着色和未着色区域的微观特征,解释壳色差异的原因;利用 RNA-seq 从壳色发生和壳色沉积阶段研究转录调控特征,筛选出壳色相关的基因和代谢通路;基于转录组数据筛选潜在的壳色分子标记,为将来分子辅助育种提供序列参考。主要研究结果如下:

         1、贝壳和外套膜色素种类和组成

    利用 ELISA 检测硬壳蛤贝壳和外套膜类胡萝卜素和黑色素含量。红色贝壳与杂色贝壳类胡萝卜素、黑色素浓度都显著高于白色贝壳(p<0.05);除 25mm 贝壳类胡萝卜素和黑色素浓度略有下降外,相同壳色不同规格的贝壳类胡萝卜素、黑色素浓度无明显差异;外套膜类胡萝卜素和黑色素的含量与壳色相关,红色个体外套膜类胡萝卜素和黑色素含量显著高于杂色和白色(p<0.05),杂色和白色之间差异不显著(p>0.05)。利用拉曼光谱检测贝壳表面微观物质基础,在色素分布区域可获得多烯-类胡萝卜素的特征信号,也有少数光谱中含有类似于黑色素的特征信号,色素未着色区域未检测到多烯-类胡萝卜素信号峰。

         2、稚贝壳色发生的转录机制

    通过 Illumina 测序对未着色、白色和红色稚贝进行了基因表达研究。黑色素/黑素瘤生成“ABC 转运子类胡萝卜素代谢相关途径卟啉和叶绿素代谢可能参与调节了硬壳蛤稚贝的壳色发生过程。与脂质转运、ABC 转运子以及卟啉和叶绿素代谢相关的基因可能是稚贝壳呈红色的原因。

         3、成贝壳色沉积的转录机制

    通过 Illumina 测序对三种不同壳色硬壳蛤外套膜进行了基因表达研究。色素合成类基因、脂质结合与运输、ATP 合成等基因在红 vs 白中存在显著差异,说明脂质代谢可能影响色素沉积,色素沉积需要消耗能量;信号转导类基因在红 vs杂中存在显著差异,锌离子结合类基因在白 vs 杂中存在显著差异,表明信号转导和锌离子结合可能在杂色壳沉积过程中具有关键作用。

         4、壳色性状候选分子标记的开发

    基于转录组数据,利用转录组比较和 SNP 卡方分析筛选 SSR SNP 标记。筛选出 476 个红色特有 SSR445 个白色特有 SSR481 个杂特有 SSR 标记。获得了 235 个差异显著的 SNP 位点(p<0.05)。

Other Abstract

The hard clam (Mercenaria Mercenaria), which natural habitat is in the Atlantic coast of North America, is a kind of important fishery resources. In 1997, academician Fusui Zhang et al. introduced the hard clams from the United States to China. It is suitable for most of the pond culture in China because of its eurythermal and euryhaline. At present, it has become an important type of pond culture shellfish with a cultured area of hundreds of thousands of mu. Shell color is an important phenotypic trait of bivalve and has become a hotspot in shellfish breeding. Among the economic shellfish, shell color could affect the popularity of seafood by consumers and its market price. Therefore, it is of great practical significance to study the molecular mechanism of shell color difference in hard clam for breeding new varieties of shell color.

In this study, carotenoid and melanin content in the shell and mantle were detected by ELISA kit. Raman spectra was analysis on the surface of shell in the pigmentation region and non-pigmentation region. These explain the reason in different shell color. RNA-seq was used to study the transcriptional regulation characteristics in the stages of shell color generation and shell color pigmentation to screen out shell color-related genes and pathways. Potential shell color molecular markers were screened based on transcriptome data to provide sequence reference for future molecular-assisted breeding. Main results for this study are as follows:

1. Species and composition of pigment in the shell and mantle

The carotenoid and melanin content of shell and mantle were detected by ELISA kit. The carotenoid and melanin concentrations of red shell, and mottled shell were significantly higher than those of white shell (p<0.05). There was no significant difference in the concentration of carotenoids and melanin in the same color shell with different shell lengths, except for a slight decrease in the carotenoids and melanin concentration of 25mm shell. The concentration of carotenoids and melanin in the mantle is associated with the shell color, and the concentration of red individuals is significantly higher than that in the mottled and white (p<0.05), but the difference between the mottled and white is not significant (p>0.05). Raman spectroscopy was used to detect the microscopic material basis of shell surface. The characteristic signal of polyene-carotenoid could be obtained in the pigment distribution area. There is also a small amount of characteristic signal of melanin-like in the spectrum, and no signal peak of polyene-carotenoid can be detected in the white non-pigmentation area.

2. Transcriptional mechanism of shell color generation in the juvenile clam

Illumina sequencing was used to study the gene expression of non-pigmentation juvenile, white and red shell juvenile clams. "Melanin/melanoma formation", "ABC transporter", "carotenoid metabolism related pathway" and "porphyrin and chlorophyll metabolism" might be involved in regulating the pigment generation process of juvenile hard clams. Genes related to lipid transport, ABC transporter and porphyrin and chlorophyll metabolism may account for the red color in the juvenile clams.

3. Transcriptome mechanism of shell color pigmentation in the adult clam

Illumina sequencing was used to study the mantle gene expression of three shell color. There were significant differences in pigment synthesis genes, lipid binding and transport, ATP synthesis in red vs white comparison, indicating that lipid metabolism-related might affect pigmentation, and pigmentation requires energy. Signal transduction genes were significantly different in red vs mottled comparison, while Zinc-binding genes were significantly different in white vs mottled comparison, suggesting that signal transduction and Zinc-binding might play a key role in the pigmentation of mottled shells.

4. Development of candidate molecular markers for shell color trait

SSR and SNP were screened by transcriptome comparison and SNP chi-square analysis based on transcriptome data. 476 red-specific SSRs, 445 white-specific SSRs, and 481 mottled-specific SSRs were developed. 235 SNP with significant differences were obtained (p<0.05).

Subject Area水产学
MOST Discipline Catalogue农学::水产
Language中文
Document Type学位论文
Identifierhttp://ir.qdio.ac.cn/handle/337002/164717
Collection海洋生态与环境科学重点实验室
Recommended Citation
GB/T 7714
胡志. 硬壳蛤壳色差异机制初步研究[D]. 中国科学院海洋研究所. 中国科学院大学,2020.
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