Simultaneous and Systemic Knock-down of Big Defensin 1 and 2 gene Expression in the Pacific Oyster Crassostrea gigas using Long Double-stranded RNA-mediated RNA Interference
- DOI : 10.5657/FAS.2014.0377
- Author: Jee Bo Young, Kim Min Sun, Cho Mi Young, Lee Soon Jeong, Park Myung Ae, Kim Jin Woo, Choi Seung Hyuk, Jeong Hyun Do, Kim Ki Hong
- Organization: Jee Bo Young; Kim Min Sun; Cho Mi Young; Lee Soon Jeong; Park Myung Ae; Kim Jin Woo; Choi Seung Hyuk; Jeong Hyun Do; Kim Ki Hong
- Publish: Fisheries and aquatic sciences Volume 17, Issue3, p377~380, 30 Sep 2014
-
ABSTRACT
RNA interference (RNAi)-mediated transcriptional knock-down of
Crassostrea gigas big defensin 1 and 2 genes (Cg -BigDef1 andCg -BigDef2) was investigated. The cDNA sequences ofCg -BigDef1 andCg -BigDef2 were identical, excluding an additional fragment of 20 nucleotides inCg -BigDef1; thus, a long double-stranded RNA (dsRNA) targeting the mRNA ofCg -BigDef2 effectively downregulated bothCg -BigDef2 andCg -BigDef1. In addition, long dsRNA targeting green fluorescent protein (GFP) did not affect transcription of the two big defensin genes. These results suggest that the transcriptional downregulation ofCg -BigDef1 andCg -BigDef2 was mediated by sequence-specific RNA interference (RNAi). Despite injection of long dsRNA targetingCg -BigDef2 into only the adductor muscle, knock-down ofCg -BigDef1 andCg -BigDef2 was observed in the adductor muscle, hemocytes, mantle, and gills, suggestive of systemic spread of RNAi inC. gigas . Furthermore, the inhibitory effect of dsRNA persisted until 72 h post-injection, indicative of a long-lasting RNAi-mediated knock-down of target genes.
-
KEYWORD
Crassostrea gigas , Long double-stranded RNA , RNA interference , Big defensins
-
RNA interference (RNAi), a highly conserved cellular mechanism in eukaryotic organisms, is a form of post-transcriptional homologous gene-silencing induced by double-stranded RNA (dsRNA) (Fire et al., 1998; Fire, 1999). DsRNA in cells is cleaved into small dsRNA fragments called the short interfering RNA (siRNA) by an RNase III-like enzyme, Dicer. These fragments are then incorporated into the RNA-induced silencing complex (RISC), which can degrade target mRNA in a sequence-specific manner, resulting in knock-down of the expression of the target gene (Bernstein et al., 2001; Sharp, 2001; Zamore, 2001; Hannon, 2002). Although RNAi-mediated knockdown of specific genes has been demonstrated in some species of gastropods, such as
Aplysia (Lee et al., 2001),Lymnaea stagnalis (Korneev et al., 2002), andBiomphalaria glabrata (Jiang et al., 2006), and in bivalves such as the Pacific oysterCrassostrea gigas (Fabioux et al., 2009; Huvet et al., 2012) and Zhikong scallopChlamys farreri (Wang et al., 2011), RNAi has not commonly been applied in mollusks.Antimicrobial peptides (AMPs) are small amphipathic peptides that play a role in the innate defense of hosts against microbial infections (Hancock and Scott, 2000; Bulet et al., 2004; Otero-González et al., 2010). Big defensin, initially identified in the horseshoe crab
Tachypleus tridentatus (Saito et al., 1995), is a type of AMP with two structurally and functionally different domains. The N-terminal domain is highly hydrophobic and more active against Gram-positive bacteria, while the C-terminal domain is cysteine-rich, cationic, and more active against Gram-negative bacteria (Saito et al., 1995). In bivalve mollusks, several big defensins have been reported. Zhao et al. (2007) identified the first one from bay scallop (Argopecten irradians ) and showed that it had microbicidal activity against Gram-positive and Gram-negative bacteria and fungi. In the hard clam (Mercenaria mercenaria ), expression of a big defensin gene significantly increased after challenge with a protistan parasite called quahog parasite unknown (Perrigault et al., 2009). A big defensin identified from clam (Venerupis philippinarum ) showed increased transcription afterVibrio anguillarum challenge, and exhibited bactericidal activity against both Gram-positive and Gram-negative bacteria (Zhao et al., 2010). Recently, Gerdol et al. (2012) reported eight big defensins from the Mediterranean mussel (Mytilus galloprovincialis ). In the Pacific oyster (Crassostrea gigas ), six AMPs including three defensins (Cg -Def,Cg -Defh1,Cg -Defh2) and three big defensins (Cg -BigDef1,Cg -BigDef2,Cg -BigDef3) have been reported (Gueguen et al., 2006; Gonzalez et al., 2007; Rosa et al., 2011). Among the three big defensins,Cg -BigDef1 andCg -BigDef2 were transcriptionally upregulated after injection with heat-killed or live bacteria (Rosa et al., 2011).Although various methods can be used to analyze gene function, RNAi-mediated knock-down of gene expression is a convenient method for
in vivo analysis of gene function. However, forin vivo experiments, it is important to understand whether the RNAi effect is systemic and how long the RNAi effect will last. In the present study, we investigated whether focal injection of a long dsRNA targetingCg -BigDef2 could systemically induce RNAi-mediated transcriptional knock-down ofC. gigas big defensin 1 and 2 genes over time.> Long double-stranded RNA (dsRNA) preparation
Double-stranded RNAs (dsRNAs) specific to oyster big defensin 2 (
Cg -BigDef2) and to green fluorescent protein (GFP) were synthesizedin vitro using the Megascript RNAi kit (Ambion, Austin, TX, USA) according to the manufacturer’s instructions. Briefly, DNA fragments partially encoding cDNA ofCg -BigDef2 and GFP genes were amplified via polymerase chain reaction (PCR) with gene-specific primers (Table 1) and cloned into the pGEM T-easy vector (Promega, Madison, WI, USA). After confirming the sequences, plasmids containingCg -BigDef2 and GFP were digested withEco RI/Nco I andEco RI/Hind III, respectively. Then these genes were subcloned into the LITMUS 28i vector (New England Biolabs, Ipswich, 378MA, USA) and named pdT7-BigDef2 and pdT7-GFP, respectively. For single-stranded RNA (ssRNA) production, each vector was linearized withEco RI andin vitro transcription was performed. Each antisense strand was synthesized after linearizing the vector withNco I (Cg -BigDef2) orHind III (GFP). Equal amounts of sense and antisense RNA were annealed to produce dsRNA. Digestion of the template DNA and any ssRNA remaining in the dsRNA, as well as purification of dsRNA, were performed using reagents in the Megascript kit (Ambion).> Oysters and dsRNA administration
Pacific oysters (
C. gigas ; shell length 6–8 cm) were obtained from a commercial oyster farm in Korea and maintained in filtered seawater at 20°C. For the experiment, 36 oysters were randomly selected, divided into 3 groups of 12 oysters, put into a 250-L tank, and acclimated for 1 week without changing the water. After starting the injection experiment, cultured water in the tank was completely replaced once a day with newly filtered seawater. Oysters in each group were injected with dsRNA targetingCg -BigDef2 or GFP into the adductor muscle at a dose of 50 μg per oyster. Oysters in the control group were injected with phosphate buffered saline (PBS) alone. At 12, 24, 48 and 72 h post-injection, three oysters in each group were randomly removed to analyze knock-down of target gene expression.> Effect of long dsRNAs on the expression of
Cg -BigDef1 and two genes inC. gigas Hemolymph withdrawn from the adductor muscle was immediately centrifuged at 1,000 g for 20 min at 4°C to obtain hemocytes. A part of the adductor muscle, as well as the mantle and gill, were excised from each oyster. Total RNA was extracted using RNAiso plus reagent (Takara, Shiga, Japan). Prior to synthesizing first-strand cDNA, 1 μg total RNA was treated with RNase-free DNaseI (Promega). To synthesize first-strand cDNA, 1 μg total RNA treated with DNase was incubated with 0.5 μL random primer (0.5 μg/mL, Promega) at 80°C for 5 min and further incubated at 42°C for 60 min in reaction mixture containing 2 μL each of 10 mM dNTP mix (Takara), 0.5 μL M-MLV reverse transcriptase (Promega), and 0.25 μL RNase inhibitor (Promega) in a final reaction volume of 10 μL. PCR was performed with 2×Prime Taq Premix (Genet Bio, Daejeon, Korea), 1 μL 10-1 diluted cDNA template, and oligonucleotide primer pairs for amplification of the each target BigDef gene (
Cg -BigDef 1, 2-F andCg -BigDef 1-R orCg -BigDef 2-R) and the control elongation factor (EF) gene (EF-F and EF-R) (Table 1). Thermal cycling conditions were 1 cycle of 3 min at 95°C (initial denaturation) followed by 35 cycles (forCg -BigDef1), 27 cycles (forCg -BigDef2), or 25 cycles (for EF) of 30 s at 95°C, 30 s at 60°C, and 30 s at 72°C, with a final extension step of 7 min at 72°C. PCR samples to be compared were electrophoresed on 1% agarose gels and stained with ethidium bromide (EtBr).Transcription of the
Cg -BigDef2 gene was clearly downregulated by injection of long dsRNA targeting theCg -BigDef2 gene (Fig. 1). Transcription of theCg -BigDef1 gene also decreased after injection of long dsRNA targeting theCg -Big- Def2 gene. However, expression of theCg -BigDef2 gene in oysters was not affected by injection of long dsRNA targeting the GFP gene.Injection of long dsRNA targeting mRNA of the
Cg -BigDef2 gene into the adductor muscle of oyster induced knockdown of the two BigDef genes in that muscle, as well as in the mantle, gill, and hemocytes (Fig. 1). Transcriptional knockdown ofCg -BigDef1 andCg -BigDef2 genes in oysters injected with long dsRNA targeting theCg -BigDef2 gene persisted until 72 h post-injection (Fig. 1).Long dsRNA targeting mRNA of
Cg -BigDef2 transcriptionally downregulated bothCg -BigDef1 andCg -BigDef2. Because cDNA sequences ofCg -BigDef1 andCg -BigDef2 are identical excluding an additional fragment of 20 nucleotides inCg -BigDef1 (Rosa et al., 2011), transcription of bothCg -BigDef1 andCg -BigDef2 may be suppressed by siRNAs originating from long dsRNA targetingCg -BigDef2. Furthermore, we did not observe a decrease in expression of the two big defensin genes after injection of long dsRNA targeting GFP, suggesting that the knock-down ofCg -BigDef1 andCg - BigDef2 was mediated by a sequence-specific RNAi phenomenon. Although Rosa et al. (2011) reported that hemocytes are the only tissues that expressCg -BigDefs, we found mRNAs ofCg -BigDef1 and 2 in hemocytes as well as other tissues, including the adductor muscle, mantle, and gill, which may be the result of hemocyte infiltration. Rosa et al. (2011) also indicated that other cell types can expressCg -BigDef genes.In some organisms including
Drosophila and vertebrates, dsRNA taken up by a cell is not transmitted to other cells (Roignant et al., 2003; Bitko et al., 2005), whereas in cells ofCaenorhabditis elegans and some other invertebrates, dsRNA can spread to other cells, resulting in a systemic RNAi response (Winston et al., 2002; Dong and Friedrich, 2005). Fabioux et al. (2009) and Huvet et al. (2012) reported dsRNAmediated knock-down of target genes in oysters. However, the injection site and expression analysis in their experiments were restricted to the gonad, and little information is available on the use of systemic RNAi in oysters. In the present study, despite the injection of BD2-dsRNA into only the adductor muscle, knock-down ofCg -BigDef1 and 2 was observed not only in the muscle but also in hemocytes, the mantle, and gills, suggestive of systemic spread of RNAi inC. gigas .The antimicrobial activity of molluscan big defensins has been demonstrated by analyzing
in vitro bactericidal activity using recombinant big defensins or shown indirectly based on transcriptional upregulation of big defensin genes in response to bacterial infection (Zhao et al., 2007, 2010; Perrigault et al., 2009; Rosa et al., 2011). In the present study, we systemically downregulated two big defensin genes inC. gigas using RNAi, which can be used in future studies to analyze the functions of big defensins in oyster.-
10. Gueguen Y, Herpin A, Aumelas A, Garnier J, Fievet J, Escoubas JM, Bulet P, Gonzalez M, Lelong C, Favrel P, Bachere E 2006 Characterization of a defensin from the oyster Crassostrea gigas. Recombinant production, folding, solution structure, antimicrobial activities, and gene expression [J Biol Chem] Vol.281 P.313-323
-
[Fig. 1.] RNA interference of big defensin 1 and 2 expression in oysters Crassostrea gigas by long double-stranded RNA (dsRNA). Transcriptional expression of elongation factor (control), big defensin 1 and big defensin 2 genes in adductor muscle (AM ), mantle (M), gill (G), and hemocytes (H) in oysters Crassostrea gigas that were injected with long dsRNA targeting mRNA of big defensin 2 (dsDef ), green fluorescent protein (dsGFP) or phosphate buffered saline (PBS) into adductor muscle. At each 12, 24, 48 and 72 h post-injection, 3 oysters in each group were randomly sampled and transcription of the genes was analyzed by RT-PCR. Bands represent transcripts of each individual oyster (n = 3) in each group.