Actin gene from blood cockle Anadara granosa as a potential housekeeping gene for gene expression analysis

Actin gene has been used to control gene expression in several species. However, there is a lack of information on actin gene in the family Arcidae, including Anadara granosa. The objective of this research was to explore actin gene expression and characteristic from A. granosa induced by mercury. Blood cockle samples were acclimatized for two days prior to HgCl2 exposure. Concentrations of HgCl2 used were 0, 1, 2, and 10 mg/l. Samples were subjected to HgCl2 for 0-48 h. Following induction, samples were dissected and isolated to collect RNA samples. Intact RNA samples were reverse transcibed and amplified using PCR method. PCR products were then sequenced. The expression of actin gene was constant despite variation in mercury concentration. A 353 bp fragment of AgACT cDNA was successfully amplified. As compared to other actin gene sequences from other molluscs, distinct sequences were observed. Hence, the actin gene from A. granosa is specific and can be utilized as housekeeping gene in future gene expression studies.


Introduction
Blood cockle Anadara granosa (also known as Tegillarca granosa) is a commercial bivalve inhabiting sandy-mud substrate in intertidal and subtidal ecosytems.These ecosystems are continuously exposed to low and high tides, and connect terestrial and marine ecosystems.This environment presents a challenge to organisms as they must cope with fluctuating water condition and antropogenic impact.Changing environmental conditions, such as temperature, salinity, mercury concentration, parasite, and ultraviolet (UV) radiation, stimulates stress on bivalve and other organism (Farcy et al., 2009;Kravets et al., 2012).
Stresses usually cause protein dysfunction, such as protein denaturation and aggregation.To protect from this deleterious impact, genes that have potential for stabilizing protein and assist protein refolding under stress conditions are required.The expression of the target gene indicates the level of stress.However, the existence of stress gene in an organism is not necessarily constitutive.According to Morga et al. (2010), the relative expression of the target gene requires an internal control or a housekeeping gene.The internal control is an endogenous control that allows correction of experimental variations.
Actin gene has been used as a housekeeping gene to normalize gene expression in human (Goidin et al., 2001), sheep (Garcia-Crespo et al., 2005), mice (Ikegami et al., 2002), and bivalve Crassostrea gigas (Farcy et al., 2009).It encompasses constant expression levels and needs no induction factor to be expressed.Given this character, it may be utilized as a housekeeping constitutive gene.The character of housekeeping constitutive gene is functional as an internal control in normalization for mRNA expression level.Normalization is necessarily for the correction for identifying differences in gene expression which are usually due to sample condition and treatment, as well as induction material (Yperman et al., 2004).
Actin is a highly conserve protein-encoding gene and one of the major components of cytoskeleton that plays an important role in most eukaryotic cells (Cooper and Crain, 1982).All eukaryote cells express actin which constitutes as much as 50% of the total cellular protein.It serves a number of functions and seems to be involved in such vital cellular processes as cell motility, contractil, mitosis and cytokinesis, intracellular transport, and cell secretion.On the other hand, actin plays a role in gene transcription (Zheng et al., 2009).
Actin gene plays an important role as a housekeeping gene to standardize quantification in gene expression studies.Lack of information on actin gene may result in a less acurate quantification of gene expression.There is yet no information on actin gene of the family Arcidae, especially A. granosa.Therefore, this research as a sequential research aimed at exploring stress gene expression in response to mercury induction.The objective was to explore actin gene expression and characteristic from A. granosa induced by mercuric chloride at various concentrations.Hence, partial characterization is applied on a relatively conserve sequence of A. granosa.The sequence of A. granosa actin gene obtained from this research may be adopted for other bivalves in the Arcidae family.

Sampling
Blood cockles were sampled using Indonesian traditional gear and manual without gear, in November 2012 from two coastal waters of Banten Province, i.e., Banten Bay, Bojonegara and Lada Bay, Panimbang, Indonesia (Figure 1).The cockles were transported to the laboratory and then placed on separate aquarium filled with fresh sea water.They were acclimatized for 48 h prior to exposure to mercury chloride (HgCl 2 ).

HgCl 2 Exposure
Blood cockles were exposed to three different concentrations of HgCl 2 (1, 2, and 10 mg/l).The period of exposure was 24 and 48 h.Control cockles were left without mercury treatment.There were three animals on each mercury treatment; therefore, there were 60 animals used in this research.The cockle size was 2.753 ± 0.427 cm.At the end of the mercury treatment, gills were dissected to be used in analyses on RNA isolation.

RNA isolation
Gills were grinded to isolate RNA using GeneJet RNA Purification Kit (Thermo Scientific Inc.).The proedure followed manufacturer's instruction.RNA integrity was obtained by loading the sample onto 1.2% agarose gel and run in electroforesis chamber.The RNA samples were then monitored underneath UV transluminator.RNA purity was measured with spectrophotometer.

cDNA synthesis
Reverse transcription for cDNA synthesis was accomplished using RevertAid Transcriptase (Thermo Scientific Inc.).RT-PCR procedure followed manufacturer's instruction.cDNA samples were used as templates in amplification of housekeeping gene (β-actin).
PCR mix composition for cDNA AgACT (A. granosa β-actin) gene was 3 µl cDNA template added with PCR mix from Platinum Taq Polymerase (Invitrogen) according to the manufacturer's instruction.PCR was carried out using AB Verity-PCR machine.The PCR condition was 94°C for 3 minutes pre denaturation, and 35 cycles on such subsequent steps as denaturation at 94°C for 45 seconds, primer annealing temperature at 55°C for 1 minute, elongation at 72°C for 1 minutes.Finally, post PCR step was conducted at 72°C for 7 minutes, and followed by cooling at 15°C for 10 minutes.PCR product integrity was observed using 1.5% agarose gel underneath UV transluminator.

DNA sequencing and sequence analysis
DNA sequencing was carried out using sequencer machine.It was completed in both forward and reverse primers at First Base Laboratory, Singapore.Alignment for β-actin gene was performed using MEGA4 program (Tamura et al., 2007).Genetic distance between A. granosa samples and human sequence was computed, based on p-distance value of nucleotide and amino acid pairwise.Phylogenetic tree was then constructed based on Neighbor Joining (NJ) (Tamura et al., 2007).

Total mRNA isolation
Total mRNA from blood cockle A. granosa was successfully isolated using RNAzol reagent.Prior to isolation, cockles were exposed to mercury chloride at the levels of 1, 2, and 10 mg/l for the time intervals of 24, 48, and 72 h.To investigate the integrity, the mRNA samples were run on electrophoresis at 85 volt for 30 minutes. Figure 2 indicates mRNA bands with two ribosomal RNA bands.
RNA purity measured using spectrophotometer ranged between 1.582 and 1.902.Given the significant integrity and purity of the total mRNA, samples then deserved to be proceeded as templates for total cDNA syntehsis.cDNA amplification from Anadara granosa βactin (AgACT) gene A 353 bp fragment of AgACT cDNA was succesfully amplified using human β-actin primers (Figure 3).PCR products were checked for size and yield on 1.5% agarose gel prior to sequencing.
All of the bands of the AgACT PCR product show consistent expressions indicated by thickness of the PCR band.According to Thellin et al. (1999), housekeeping gene performed constant levels for many samples of the same species may be used as an internal control for any other genes of interest.Therefore, the experiment was proceeded to next analysis such sequencing analysis.
Comparisons of p-distance among AgACT sequences revealed nucleotide and amino acid substitution values in the range of 0.000-0.013(Table 1) and 0.000-0.032(Table 2), respectively.On the other hand, comparisons between AgACT and other molluscan sequences signified nucleotide and amino acid substitution values in the range of 0.225 to 0.251 (Table 1) and 0.210 and 0.226 (Table 2), respectively.
Phylogenetic analysis indicates that AgACT nucleotide and amino acid sequences were different from other molluscs (Figure 4 and 5).Accordingly, there were two distinctive groups among AgACT individuals represented specific haplotypes.The first haplotype group represented low levels of mercury concentration, i.e., control (0 mg/l), individual induced by mercury of 1 mg/l/24 h, and 1 mg/l/48 h.On the other hand, iindividuals exposed to mercuric chloride at the level of 2 and 10 mg/l formed a second haplotype group.
Actin is highly conserved and ubiquitous in eukaryotes.In animals, actin is primarily involved in muscle contraction in differentiated muscle tissue.In nonmuscle animal cells, actin is involved in a variety of processes including cytoskeletal structure, cellular motility, cell-surface mobility, intracellular transport, and possibly, chromosomal condensation and mitosis.Given to its characteristic, actin gene is commonly used as housekeeping gene (Morga et al., 2010).As a molecular agent, housekeeeping gene, such as βactin is applied for internal standard in many RNA analyses.The synthesis of the molecules is often considered as being very few fluctuating in comparison to that of others (Thellin et al., 1999).
This research revealed the thickness level of PCR bands of AgACTs resulting in a constant expression without considering the treatment levels and exposure times.In contrast, numerous studies have shown that actin gene exhibited inconstant expressions.Morga et al. (2010) found inconsistency of β-actin expression of Ostrea edulis infected by parasite Bonamia ostreae.Parasite Bonamia ostreae seemed to affect the expression of actin.Actin is involved in the cytoskeleton structure which plays a pivotal role in the phagocytosis and encapsulation.Expression variabilities of β-actin were also discovered on fish Ictalurus punctatus related to food depredation and low water stress.Physiological state of the fish might affect reference gene expression in a tissue specific manner (Small et al., 2008).Inconsistent expressions of actin gene were found as well in ovine heart valves (Yperman et al., 2004), heatinduced chicken (Banerji et al., 1986), and human asthmatic airways (Glare et al., 2002).Hence, those former studies failed to use actin gene as an internal control.
According to Morga et al. (2010), a gene with stable expression may be applied as an internal control in order to normalize the gene expression.As a result, this research suggested that A. granosa actin (AgACT) gene, providing similar response to induced factor such as mercuric chloride, may be used as standard to normalize the gene of interest.
In spite of its conserve character, this research revealed that β-actin gene in respect to nucleotide and amino acid sequences from samples of A. granosa denoted variety of the sequences among individuals.
Comparison of nucleotide sequences from A. granosa and other molluscs concerns with number of singleton and parsimony sites.The singleton sites explain that there is only one nucleotide difference among the sequences and those are specific identification for A. granosa.While parsimony sites explain that there are at least two nucleotide differences among the sequences.This research recorded that number of singleton and parsimony sites is slightly comparable.
This research revealed a dissimilarity sequence of both nucleotide and amino acid between AgACT and molluscan β-actin sequences.Although there were some conserved sites in nucleotide and amino acid sequences, ancestral relationship is distant.It is as a consequence of the notion that the greater the genetic distance, the more variation of nucleotides.Therefore, AgACT isolated from A. granosa may be considered as the candidate for β-actin gene specific for A. granosa.not yet been isolated from Arcidae bivalve, moreover A. granosa.Therefore, the gene will be very important as a positive control in analysis of gene expression on such bivalve.

Conclusions
Expression of β-actin gene in blood cockle A. granosa induced by mercury was constant in spite of variation in mercury concentration.A. granosa β-actin gene was successfully sequenced producing 353 bp nucleotide partial fragment.This gene was specific; therefore, it can be used as an internal control to normalize gene expression for the bivalve family Arcidae.

Figure 2 .
Figure 2. Electrophoretic result of RNA isolated from blood cockle A. granosa.Two solid bands of RNA indicate RNA ribosomal 28S and 18S.

Table 1 .
Nucleotide dissimilarities between β-actin gene of Anadara granosa and other mollusc sequences were aligned by CLUSTAL IV and computed by p-Distance comparison.Alignment and computation were constructed with MEGA4.02software.

Table 2 .
Amino acid dissimilarities between β-actin gene of Anadara granosa and other mollusc sequences were aligned by CLUSTAL IV and computed by p-Distance comparison.Alignment and computation were constructed with MEGA4.02software.
To date, β-actin gene has