Dinoflagellates are microscopic unicellular microalgae found in aquatic environments worldwide. One common feature of dinoflagellates is the presence of two unequal flagella: The longitudinal flagellum is round while the transverse flagellum is ribbon-shaped. Currently little is known about the intracellular ultrastructure of the dinoflagellate flagella apparatus and molecular aspects of the flagella assembly are unknown.
In this study a combination of ultrastructure studies using scanning (SEM) and transmission electron microscopy (TEM), and molecular analysis of transcriptomics data was used to gain understanding on the flagella apparatus of the toxic marine dinoflagellate Alexandrium minutum. Samples for SEM and TEM observations were prepared using standard protocols. For transcriptomics analysis, total RNA was harvested from exponential phase cultures during the light and dark culture periods. The transcriptome was sequenced using HiSeq sequencing, assembled and annotated. The assembled sequences were searched for putative genes involved in flagella synthesis, assembly and functioning based on annotations and reverse Blast analysis.
The putative genes were used in phylogenetic and in silico protein analysis. Transcriptome sequencing produced 399,169 unigenes, of which 412 were related to flagella biosynthesis and function. Structural flagella genes found were α, β and γ dynein; α, β and γ tubulin; radial spoke, basal body, central pair wd, central pair pf20, flagellar protofilament ribbon protein, calcium binding, sodium–type flagellar motor component and nexin. There were also several intraflagellar transport (IFT) genes namely 20, 27, 52, 57, 74, 80, 81, 88, 122 and 172.
The other flagella related genes found were genes for kinesin II and cytoplasmic dynein 2. Ultrastructure observation by TEM showed that the Cells had the typical dinoflagellate organelles. The chloroplast contained two or three apprised thylakoids, and was surrounded by three membranes, the eyespot is located dorsally and composed of one or two layers of globules situated within the chloroplast, In the periphery of the cell, trichocysts, mucocysts, and unidentified multilayered material were visible This study provided for the first time a large volume of gene data that can be used to better understand the molecular basis of flagella biosynthesis and function in dinoflagellates.
Table of Contents
CHAPTER I INTRODUCTION
1.1 RESEARCH BACKGROUND
1.2 PROBLEM STATEMENT
1.3 OBJECTIVES OF RESEARCH AND SCOPE OF WORKS
CHAPTER II LITERATURE REVIEW
2.1 Dinoflagellates
2.1.1 General characteristics of dinoflagellate
2.1.2 Genetic characteristics
2.1.3 Morphology and taxonomy
2.1.4 Life cycles
2.1.5 Molecular phylogenies
2.1.6 Harmful algal blooms
2.1.7 Ecology
2.1.8 Alexandrium minutum
2.2 Eukaryotic flagella apparatus
2.2.1 Structure of eukaryotic flagella
2.2.2 flagellar assembly
2.3 Scanning and transmission electron microscopy in dinoflagellate
2.3.1 Scanning electron microscopy (SEM)
2.3.2 Transmission electron microscopy (TEM)
CHAPTER III Material and methods
3.1 Cell culture
3.1.1 Preparation sterile seawater
3.1.2 Preparation medium culture
3.1.3 Culturing
3.2 Cell harvesting and total RNA extraction
3.2.1 Cell harvesting
3.2.2 Total RNA extraction
3.3 EST Database Analysis and Managemen
3.3.1 Pipeline of Experiments
3.3.2 Output Statistics
3.3.3 Phylogenetic constructions
3.4 Microscopy
3.4.1 Light microscopy
3.4.2 Scanning electron microscopy
3.4.3 Transmission electron microscopy
CHAPTER IV result and discussion
4.1 microscopy
4.1.1 Light microscop
4.1.2 Scanning electron microscopy
4.1.3 Transmission electron microscopy
4.2 molecular analyses
4.2.1 Overview of transcriptome sequencing results
4.3 Flagellar unigens
4.3.1 Known structural flagellar genes
4.3.2 Intraflagellar transport components
CHAPTER V CONCLUSION AND FUTURE WORKS
5.1 CONCLUSION
5.2 SUGGESTIONS FOR FUTURE WORK
Research Objectives and Thematic Focus
This thesis aims to characterize the ultrastructure of the toxic marine dinoflagellate Alexandrium minutum and to elucidate the molecular aspects of flagellar assembly through transcriptome sequencing and analysis, providing new genomic data to understand the underlying mechanisms in dinoflagellate biology.
- Ultrastructural examination of Alexandrium minutum using electron microscopy (SEM and TEM).
- Transcriptome sequencing and de novo assembly to create a comprehensive gene database.
- Identification and phylogenetic analysis of structural flagellar genes and their roles/functions.
- Investigation of intraflagellar transport (IFT) genes and their function in flagellar assembly.
Excerpt from the Book
2.2 EUKARYOTIC FLAGELLA APPARATUS
Motile cilia and flagella, presumably first discovered by Dutch microscopist Antonius Von Leeuwenhoek, have long attracted the interest of cell biologists. Present on nearly in most organisms and every vertebrate cell, flagella is a complex organelle that has function in diverse biological roles such as motility, transport liquid substance, and sensory conveyancing (reviewed by Pazour & Witman, 2003; Davenport & Yoder 2005; Pan et al. 2005; Marshall & Nonaka, 2006; Christensen et al. 2007). Flagella is microtubule based structures that project from the cell surface and act as cellular antennae (Pazour & Witman, 2003; Marshall & Nonaka, 2006; Christensen et al. 2007), Some group of organisims such as advanced fungi, cellular slime moldes, red algae, conifers and angiosperms lost the susceptibility of assembling of flagella within adaptation to life(Raven et al. 1999).
2.2.1 Structure of eukaryotic flagella
Eukaryotic cilia and flagella are microtubule-based organelles protruding from modified centrioles or basal bodies and have essentially identical structures. Most motile cilia and flagella assign a highly conserved 9+2 arrangement of microtubules in the axonemal core structure (Hyams et al. 1975; Hyams et al. 1978); The ciliary membrane surrounds the axoneme and is continuous with the plasma membrane of the cell body, but has a unique composition of lipids and membrane proteins essential for sensory functions (Emmer et al. 2010; Rohatgi & Snell, 2010). The axoneme is assembled onto the ‘basal body’, a modified centriole consisting of 9 microtubule triplets, two of which are extended during the formation of the axoneme (Ishikawa and Marshall, 2011). Nine doublet microtubules (DMTs) leaguer two central singlet microtubules of the central pair complex (CPC) and radial spokes connect them together (Jianfeng & Lin 2012).
Summary of Chapters
CHAPTER I INTRODUCTION: Summarizes the background on dinoflagellates, their significance in marine ecosystems, and the research objectives regarding flagellar apparatus studies.
CHAPTER II LITERATURE REVIEW: Provides a technical overview of dinoflagellate biology, eukaryotic flagella structures, and current microscopy techniques used in this research field.
CHAPTER III Material and methods: Details the experimental protocols used, including cell culture, RNA extraction, transcriptome sequencing, and downstream bioinformatics analyses.
CHAPTER IV result and discussion: Presents the findings regarding ultrastructure and molecular analyses, specifically focusing on structural flagellar genes and IFT components.
CHAPTER V CONCLUSION AND FUTURE WORKS: Synthesizes the major project outcomes and offers perspectives on future research challenges in dinoflagellate genomics.
Keywords
Alexandrium minutum, dinoflagellates, flagella, ultrastructure, transcriptome, gene assembly, intraflagellar transport, IFT, dynein, tubulin, axoneme, microscopy, marine biotechnology, genomics, phylogeny.
Frequently Asked Questions
What is the core focus of this research?
The research is dedicated to understanding the ultrastructure and molecular basis of flagellar assembly in the toxigenic marine dinoflagellate Alexandrium minutum.
What are the primary fields of study involved?
The study integrates marine biotechnology, microbiology, molecular biology, and bioinformatic analysis, specifically focusing on transcriptomics and electron microscopy.
What is the main research question or objective?
The objective is to identify and characterize the structural genes of flagella and putative intraflagellar transport (IFT) genes to bridge the current knowledge gap regarding flagellar biosynthesis and regulation in dinoflagellates.
Which scientific methods were employed?
The study utilized a combination of Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM) for structural analysis, alongside Illumina HiSeq transcriptome sequencing and in silico phylogenetic/protein analysis.
What is treated in the main part of the thesis?
The main section covers the experimental methodology, the presentation of transcriptomic data, and detailed discussions on identified structural flagellar genes (like dynein and tubulin) and intraflagellar transport components.
Which keywords best describe this study?
Keywords include Alexandrium minutum, flagella, ultrastructure, transcriptome, intraflagellar transport (IFT), dynein, and bioinformatics.
How does the genome of Alexandrium minutum compare to other eukaryotes?
The thesis notes that Alexandrium minutum, like other dinoflagellates, exhibits enormous and complex genomes that contain diverse, often uncharacterized gene sequences, standing in contrast to the genome sizes of most other eukaryotic algae.
Why are IFT genes relevant to this research?
IFT genes are essential for the bidirectional transport of proteins required for flagellar assembly and maintenance; their identification provides insights into the molecular regulation of the dinoflagellate flagellar apparatus.
- Quote paper
- Nahid Khalili (Author), 2013, Ultrastructural and Molecular Aspects of Flagella Assembly in the Marine Dinoflagellate Alexandrium Minutum, Munich, GRIN Verlag, https://www.grin.com/document/1416320
