|关键词||脉红螺 附着变态 转录组 蛋白组 代谢组 Microrna 内参基因 5-ht Receptor Nos|
本研究利用RNA-seq、iTRAQ、GC-MS、Real time PCR等技术对脉红螺幼虫变态过程分子机理展开研究，从转录组水平、蛋白质组水平和代谢组水平揭示了幼虫变态过程调控特征，筛选了脉红螺变态过程中的差异表达的关键转录本/蛋白组/代谢物，并对它们在变态中发挥的潜在生物学功能进行了探讨；开展了脉红螺幼虫变态过程microRNA的响应特征研究，筛选了变态中的差异表达的microRNA并对它们潜在调控的靶基因进行预测，揭示其在变态过程中所发挥的功能；筛选了在脉红螺变态发育过程中和在不同组织中稳定表达的内参基因，为将来进一步研究关键基因在变态过程中的表达水平提供基础；获得关键基因5-HT receptor和NOS的cDNA序列，探讨了其在脉红螺变态过程中表达特点及调控机理。主要研究结果如下：
首次克隆并获得了脉红螺变态信号转导路径中的关键基因（5-HT receptor和NOS）的cDNA 全长，并进行了物种间序列比对和系统进化树的构建。通过qPCR结果发现，5-HT receptor和NOS的表达量均在变态后发生了显著下调。通过免疫组化染色揭示了5-HT receptor在脉红螺幼虫体内的早期发生规律，发现面盘器官上有以5-HT 为信号传导的三条主神经纤维和纤毛基部感受器所连接的复杂神经网络结构，提示其可能与变态密切相关。
The veined rapa whelk (Rapana venosa) is an economically important sea snail in China, and since 1992, there has been interest in its commercial aquaculture. However, sea-ranching efforts have been hampered by difficulties cultivating larvae during the settlement and metamorphosis stages. In countries that do not consume R. venosa, such as the United States, Argentina, and France, this predatory species has become an invasive pest due to unintended worldwide transport and severely disrupts the survival of native bivalves. Because R. venosa population dynamics and spatial expansion are dominated by recruitment and survival rate during metamorphosis, which is a vital process in the species’ biphasic life cycle, understanding the mechanisms behind this process is necessary for both successful aquaculture and invasion control. Moreover, the metamorphosis of R. venosa is unusual compared with other lifelong phytophagous gastropods for exhibiting considerable developmental specificity; the planktonic, pelagic larvae go from filter-feeding on microalgae to carnivorous juveniles that prey on bivalves. This transition occurs rapidly, despite fundamental changes in morphology including velum degeneration and reabsorption, foot reorientation and elongation, as well as secondary-shell growth. Thus, clarifying R. venosa metamorphosis is also of theoretical interest to gastropod researchers.
In our study, we used RNA-seq, iTRAQ sequencing, GC-MS, Real time PCR et al. to study the molecular mechanism of R. venosa metamorphosis from transcriptomic, proteomic, metabonomic insight, the key transcripts/proteins/metabolite during this pelagic to benthic transition were identified and their potential functions were analyzed by bioinformatics methods. We also investigated the microRNA during metamorphosis and the taget gene of differential expressed microRNAs were predicted to reveal their potential function in this process. Besides, the stable reference genes for qPCR were selected to provide a solid base for studying metamorphic key genes. Furthermore, we cloned the cDNAs of key genes (5-HT receptor and NOS) and analyzed their expression during this metamorphic development and we used immunohistochemical method to locate the 5-HT receptor in larvae. Main results are as follows:
1. De novo transcriptome sequencing and analysis of Rapana venosa from six different developmental stages
De novo sequencing was performed to obtain a comprehensive transcriptome profile during early development. A Hi-seq 2500 sequencing run produced 148,737,902 raw reads that were assembled into 212,049 unigenes (average length of 619 nucleotides, of which 70,877 could be annotated). The unigenes were assigned to biological processes and functions after annotation in Gene Ontology, eukaryotic Ortholog Groups and Kyoto Encyclopedia of Genes and Genomes. We also identified 93,196 simple sequence repeats among the unigenes. Six unique sequences associated with neuroendocrine function were analyzed by quantitative real-time PCR. Our data represent the first comprehensive transcriptomic resource for R. venosa. Functional annotation of the unigenes involved in various biological processes could stimulate research on the mechanisms of early development in this species.
2. Transcriptomic analysis of differentially expressed genes during larval metamorphic development by digital gene expression profiling
Fifteen digital gene expression (DGE) libraries from five developmental stages of R. venosa were constructed and sequenced on the IIIumina Hi-Seq 2500 platform. Bioinformatics analysis identified numerous differentially and specifically expressed genes which revealed some genes associated with growth, nervous system, digestive system, immune system and apoptosis participate in important developmental processes. The functional analysis of differentially expressed genes was further implemented by gene ontology and Kyoto encyclopedia of genes and genomes enrichment. DGE profiling provided a general picture of the transcriptomic activities during the early development of R. venosa, which may provide interesting hints for further study.
3. Comprehensive and quantitative proteomic analysis of metamorphosis-related proteins
We analyzed the proteomes of competent R. venosa larvae and post-larvae, resulting in the identification of 5312 proteins, including 470 that were downregulated and 668 that were upregulated after metamorphosis. The differentially expressed proteins reflected multiple processes involved in metamorphosis, including cytoskeleton and cell adhesion, ingestion and digestion, stress response and immunity, as well as specific tissue development.
4. Metabolomic analysis of larval metamorphosis from pelagic to benthic
The present study examined the metabolic profiles of competent larval and post-larval stages of R. venosa using GC−MS. A total of 263 metabolites was detected, 53 of which had different concentrations in larvae and post-larvae: 29 that were apparently higher following metamorphosis and 24 that were lower. Among the metabolites whose concentrations were higher in post-larvae, quinoline-4-carboxylic acid, the dipeptide cysteinylglycine, and anandamide were the most abundant. The metabolites present in higher concentrations in the competent larvae were a suite of oligosaccharides (maltotriose, glucose-6-phosphate, cellobiose, and maltose), L-homoserine, adrenosterone, and sarcosine. Although the roles of these and other metabolites in whelk development are not yet completely known, they provide some clues to the changes in energy metabolism and cell signaling that take place during metamorphosis.
5. Understanding microRNA regulation involved in the metamorphosis
A total of 195 differentially expressed miRNAs was obtained. Sixty-five of these were expressed during the transition from pre-competent- to competent larvae. Thirty-three of these were upregulated and the others were downregulated. Another 123 miRNAs were expressed during the transition from competent- to post-larvae. Ninety-six of these were upregulated and the remaining 27 were downregulated. The expression of miR-276-y, miR-100-x, miR-183-x, and miR-263-x showed an over 100-fold change during development, while the miR-242-x and novel-m0052-3p expression levels changed over 3000-fold. Putative target gene co-expression, gene ontology, and pathway analyses suggest that these miRNAs play important roles in cell proliferation, migration, apoptosis, metabolic regulation, and energy absorption. Twenty miRNAs and their target genes involved in ingestion, digestion, cytoskeleton, cell adhesion, and apoptosis were identified. Nine of them were analysed with real-time PCR, which showed an inverse correlation between the miRNA and their relative expression levels.
6. Selection of housekeeping genes as internal controls for quantitative RT-PCR analysis
We selected 13 candidate genes for suitability as internal controls and measured their expression levels in eight different tissues and twelve larvae developmental stages by qRT-PCR. As a result of further analysis of the expression stability by GeNorm and RefFinder algorithms, we found that EF-1α was the most stable internal control gene in almost all adult tissue samples investigated with RL5 and RL28 as secondary choices. From a developmental perspective, we found that RL28 was the most stable gene in all developmental stages measured, and COX1 and RL5 were appropriate secondary choices.
7. Molecular cloning of key genes cDNAs and their expression analysis during metamorphosis
We obtained full-length cDNAs of 5-HT receptor and NOS in R. venosa for the first time. Analysis by real-time PCR showed the expression level of 5-HT receptor and NOS has a ~2-fold decrease after metamorphosis. The development of serotonin-containing cells in the mollusk R. venosa was also examined using immunohistochemical and histofluorescent methods. The first immunoreactive signal of 5-HT receptor firstly appeared in trochophora stage at the base of the velum and the future eyes position. The 5-HT receptor immunoreactive signals began to appear in the surface of the plate at the bottom of the foot primordial. In competent larvae, the complex nerve network is clearly visible on velum, with three main nerve fibers in the middle of each velum petal and receptors in cilia base. We speculate the 5-HT receptor participate the metamorphic signal transduction.
|学科领域||发育生物学 ; 水产生物学 ; 水域生态学|
|学科门类||理学::生物学 ; 理学::生态学|
|宋浩. 脉红螺幼虫变态过程多组学解析及关键基因的调控作用[D]. 中国科学院海洋研究所. 中国科学院大学,2018.|