Molecular Characteristic of Giant Grouper (Epinephelus Lanceolatus) Vitellogenin

The development of molecular tools has recently opened new direction and facilitated the discovery of the genes involved in these processes and their evolutionary functional significance. Fish oocyte development attracted specific interest in the last century. Morphological investigations were followed by biochemical, physiological, and endocrinological analyses that extended our knowledge of dynamic events that take place during oocyte development and egg formation.


Introduction
The development of molecular tools has recently opened new direction and facilitated the discovery of the genes involved in these processes and their evolutionary functional significance. Fish oocyte development attracted specific interest in the last century. Morphological investigations were followed by biochemical, physiological, and endocrinological analyses that extended our knowledge of dynamic events that take place during oocyte development and egg formation.
Molecular characterization of vitellogenin (Vtg) gene is important because it indirectly leads to the understanding of the role-play in the molecular basis of gonad development in terms of their structure and function [1]. Basically, each gene has its own molecular characteristic that is specific to their action. This includes the Vtg genes, which has certain features that are fundamental and responsible for its actions. The Vtg gene sequence acts as an indicator to the fish reproduction, which can adapt to the environment factor or can influence the gonad development [2]. Study on the molecular levels could permit the understanding of gonad development, gene regulation, structuralfunction relationships, evolution and adaptation to environment.
In teleost fish, as in other oviparous, Vtg is specifically incorporated in the oocyte by receptor-mediated endocytosis through receptors belonging to the low density lipoprotein receptor (LDR) family, which have been named very low density lipoprotein receptors (VLDLRs), Vtg receptors (VtgRs), due to the presence of eight ligand-binding repeats [2]. The other members of the gene family bind various ligands and are involved in lipid metabolism in both vertebrates and invertebrates. Therefore, this function of Vtg component needs to clarify for better understanding in physiology process during oocyte development.
The objective of the present study was to characterize the Giant grouper Vtg gene, and to compare Vtg gene expression between other species as basic information to develop Vtg as biomarker indicator in sex identification of Giant grouper.

Molecular phylogenetic analysis of giant grouper Vtg
The Vtg nucleotide sequence from Giant grouper was characterized using bioinformatics software. A homology search of the deduced amino acid sequence of the obtained Vtg DNAs was carried out using the National Center for Biotechnology Information website (http:// www.ncbi.nlm.nih.gov/). The Vtg Giant grouper sequence ( Figure  1) from previous study [3], was compared with 13 species such as lamprey (Ichthyomyzon unicuspis, GenBank; AAA49327.  The deduced amino acid sequences were aligned using the ClustalW [4] program hosted by the DNA Data Bank of Japan (http://clustalw. ddbj.nig.ac.jp/top-j.html) and subjected to ClustalW analysis to construct a phylogenetic tree using the bootstrapped neighbor-joining method [5]. The sequence obtained was exported to FASTA format in notepad and then, was edited using Bioedit software to remove the unwanted and vector sequences to identify the location of the insert sequence. Multiple alignments from 14 fish peptide sequences of Vtg were conducted using eBiox 5.2.2 program and it were used in the phylogenetic analysis. The phylogenetic analysis was carried out using Molecular Evolutionary Genetic Analysis MEGA version 5.2.2 [6] with Maximum Likelihood and Neighbor Joining algorithms in order to estimate the phylogeny.

Domain
In order to verify the Vtg gene sequences obtained and elucidate structure-function relationship in Vtg, the conserved and essential domains and residues in Vtg and other members of the gene family such as Lipovitellin I (Lv-I) and II (Lv-II), phosvitin (Pv), polyserine track (PT), von Willebrand-factor type-D domain (VWD) were determined by using DELTA BLAST (http://blast.ncbi.nlm.nih.gov/ Blast.cgi.). The molecular characterization of primary structure Giant grouper Vtg gene such as protein domain, families and functional sites were determined by comparing the sequence to other fish.

Three dimensional (3D) structure prediction
Furthermore, in this study the prediction of 3-D structure of Giant grouper with others fishes from different orders were also viewed using protein homology/analogy recognition engine v 2.0 Phyre2 server http://www.sbg.bio.ic.ac.uk/ [7].

Molecular phylogenic analysis
Result of phylogenetic analysis using maximum likelihood (ML) and neighbor joining (NJ) methods showed tree analysis generated two separated tree topology ( Figure 2). In general, showed that Epinephelus lanceolatus Vtg is evolutionary more related to Poecilia latipinna and Kryptolebias marmoratus. It is noted that the distribution of Vtg phylogenetic was significantly different, between freshwater species (Carassius auratus, Catla catla, Danio rerio and Clarias macrochepalus) as one group of Vtg compare to seawater and euryhaline species (Epinephelus lanceolatus, Poecilia latipinna, Kryptobias marmorata, Morone sexatilis, Thunnus thynnus, Fundulus heterocittus, Dicentrachus labrax and Anguilla japonica) for another group. The constructed a phylogenetic tree that places closely related sequences under the same interior node and whose branch lengths closely reproduce the observed distances between sequences.
The results from evolutionary distance estimations are displayed in the distance matrix explorer (Table 1). Results describe the accuracy of pair wise alignment by Clustal under the specific simulation conditions and alignment parameters. Estimation of evolutionary distances between Vtg sequences is important for constructing phylogenetic trees (Figure 1), dating species divergences and understanding the mechanism of evolution of protein. Vtg sequence of giant grouper (Epinephelus lanceolatus) was closed (0.015) with Poecilia latipinna (Genbank: ACV65040.1) and very far from Icthyomyzon unicuspis (Genbank: AAA49327.1 (1.041). Estimating the number of nucleotide or amino acid substitutions needed to compute evolutionary distances is one of the most important subjects in molecular evolutionary genetics and comparative genomics. Estimation of evolutionary distance of Giant grouper Vtg with alignment of 13 other fish homologous sequence, revealed that Giant grouper Vtg was belongs to the marine fishes species rather than freshwater species group.

Domain architecture of Vtg
Study by Babin et al. [8], has proposed the domain architecture  Figure 3. It was clearly seen that α-helix was predominantly present in the Giant grouper Vtg sequence and helix can be grouped into four major groups, which are located in domain region. Analysis indicated that the α-helix, β-sheet and the coil structure configurations have 39.96%, 25.54% and 34.48% respectively. As it can see in Figure 4, the 4-helics can recognize in the different color of domain region.

Three dimensional (3-D) structure prediction
Analysis of the 3-D structure found that E. lanceolatus Vtg gene shows this protein has the typical 4α-helices bundle protein that runs in anti-parallel ( Figure 5A). Based on the color, its can categorized in 4 helix structure, which is Blue, Red, Light green and Green respectively. In the present study, the main structure of Vtg gene in Giant grouper from different species was similar at the 4-helic region (Figures 5B-5F). However, the difference can be seen in Helix-1 (blue) and Helix-4 (red) where the structure was totally different in Lamprey (Icthyomyzon unicuspis) but similar in Catfish (Clarias macrocephalus), Japanese Eel (Anguilla japonica) and European Seabass (Dicentrachus labrax). However, Zebra fish (Danio rerio) 3-D vtg structure was different with giant grouper in the position of reddish color Helix-4.

Discussion
Vitellogenin (Vtg) is an egg yolk precursor expressed in the females of nearly all oviparous species including fish, amphibians, reptiles, birds, most invertebrates, and the platypus. Vtg is the precursor of the lipoprotein and phosphoproteins that make up most of the protein content of yolk. There is potential of Vtg as a biomarker for measuring exposure of oviparous animals to estrogen or estrogen mimics, by using several fish species for which both in vivo and in vitro assays have been developed [9][10][11], Vtg functions as a nutritional source for the developing embryo, rather than as an important functional protein.
Generally, assessment with Blast analysis showed that the Vtg amino acid protein sequences were similar with Vtg gene in the GeneBank database, it is very likely that 14 sequences obtained were Vtg gene fragments. Molecular characteristic of Vtg Giant grouper showed phylogenetic analysis by using maximum likelihood (ML) and neighbor joining (NJ) was generated two separated tree topology. The phylogenetic grouping showed the Giant grouper were closed to Poecilia latipinna and Kryptolebias marmoratus than other group fish at 98-100% similarity in terms of nucleotide and amino acids sequences respectively (Figure 1). It was shown that Giant grouper Vtg gene had the highest homology with Poecilia latipinna. These similarities (0.015 distance matric viewer) were closely related in terms of their Vtg gene sequence although from different environmental and ecological conditions.
Biological similarities are seen between lipoprotein and Vtg from the point of view of binding of hydrophobic molecules, cell specific uptake, and the possibility that these proteins may have a common ancestor [14][15]. In this investigation, the region of Giant grouper Vtg, which   [16] in Zebrafish. It should be noted that an additional DUF 1061 domain of unknown function was identified in the last region of Vtg peptide sequence.
The information from identification of Vtg (such as molecular mass and sequencing) could be useful during preparation of Vtg antibody production. Antibodies production is generated by in vivo or in vitro approaches, their identification relies mainly on screening of hybridoma supernatants or bacterially expressed antibody fragments. The molecular approach can be done on Giant grouper to understand the molecular respond towards fish growth and determine the individual of Giant grouper that has potential to increasing the Vtg production for increase eggs quality.   1  2  3  4  5  6  7  8  9  10  11  12  13  14 ) (