Cloning and in silico analysis of the 5′ putative regulatory regions of four vitellogenin genes from Cx. Tarsalis

The transcription factor binding sites (GATA, C/EBP, EcRE) which are required for tissue- and stage-specific vitellogenin expression in mosquito vitellogenin genes are usually located approximately 2 kb 5′ upstream of the initial vitellogenin transcriptional start site [1]. Four vitellogenin genes from Cx. tarsalis were isolated previously [24]–[25]. In this study, we cloned the putative 5′ regulatory regions for the four distinct vitellogenin genes using a genome walking approach [24]. Isolated sequences were 3159-bp, 1976-bp, 2950-bp and 2133-bp for Vg1a, Vg1b, Vg2a and Vg2b, respectively. Sequences were deposited into GenBank under accession numbers KF271781 - KF271784. Analysis showed that the sequences of the two Vg1 promoters were highly conserved in the region from translation start codon “ATG” to 123-bp upstream, then increased in divergence. Likewise, sequences of the two Vg2 promoters were highly-conserved in the region from “ATG” to 166-bp upstream, then increased in divergence. Sequences between the Vg1 and Vg2 regulatory regions were highly divergent.

Despite this divergence, rtPCR indicated that all four vitellogenin promoters had similar expression profiles and were active both pre- and post-bloodmeal [25], suggesting that they may share similar cis-regulatory elements. We analyzed these sequences to identify putative responsive elements. Even with highly divergence sequences, most of the transcription factor binding sites (GATA, TATA, C/EBP, EcRE), which are required for tissue- and stage- specific vitellogenin expression in the Ae. aegypti Vg1 gene could be found in all four Cx. tarsalis vitellogenin promoters. Differences between each vitellogenin promoter were also identified (Figure 1).

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Figure 1. Schematic illustration of putative of regulatory elements in the promoter regions of the four Culex tarsalis vitellogenin genes.

https://doi.org/10.1371/journal.pone.0088994.g001

Activity analysis of four vitellogenin promoters in transgenic D. melanogaster

Germline transformation of Cx. tarsalis has not yet been achieved, so we used transgenic D. melanogaster to investigate the function of the Cx. tarsalis vitellogenin promoters in vivo. We designed 4 piggyBac constructs that each had the general organization: pBac[3XP3-DsRed-CxVg-promoter-EGFP] and generated transgenic Drosophila by embryonic microinjection with pBac helper plasmid. Three lines for each promoter were randomly selected for reporter gene expression analysis.

To monitor the expression level of GFP gene, RT-PCR and qRT-PCR analysis were employed. The results showed that GFP expression in D. melanogaster fat bodies was observed when driven by all Cx. tarsalis promoters, but expression levels were highly variable between different vitellogenin promoters and between different independent fly lines of the same promoter (Figures 2 and 3). In general, GFP expression driven by Vg2a and Vg2b promoters was higher than when driven by Vg1a and Vg1b promoters. The highest expression was observed in line Vg2bL3, which had 315-fold higher GFP expression compared to line Vg1aL3, the line with the lowest detectable GFP expression (no expression was detectable in line Vg1aL1). These results show that different lines transformed with the same promoter sequence had highly variable reporter gene expression, indicating position effects due to the integration site of the transposon in the Drosophila genome.

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Figure 2. Expression of GFP gene in transgenic Drosophila by RT-PCR.

Three independent lines for each promoter were assayed. The constitutive ribosomal protein 49 gene (rp49) in D. melanogaster was examined as an endogenous control. N  =  negative control.

https://doi.org/10.1371/journal.pone.0088994.g002

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Figure 3. Variation in reporter gene (GFP) expression among transgenic Drosophila melanogaster lines.

Relative expression levels of GFP were normalized to the expression of ribosomal protein 49 (rp49). Expression folds are calculated by comparison to line Vg1aL3, which had the lowest detectable GFP expression. Error bars represent standard errors. Letters represent statistical differences.

https://doi.org/10.1371/journal.pone.0088994.g003

Ethics statement

Mosquitoes were bloodfed on commercially obtained chicken blood using a membrane feeder.

Mosquitoes

Cx. tarsalis used in this study were from the KNWR colony, which was collected in the Kern National Wildlife Refuge (KNWR) in Kern County, California [25]. Larvae were reared at a standard density of 200 larvae/pan and fed a 1∶2∶2 blend of fish food, rabbit pellets and bovine liver extract. Adults were maintained on 10% sucrose solution at 27±1°C and 80±5% relative humidity with a 14 h/10 h light/dark cycle.

Genomic DNA extraction and partial coding sequence isolation

Genomic DNA was isolated from female adults using the procedure previously described [24]. Based on the available An. gambiae, Ae. aegypti and Cx. pipiens vitellogenin sequences from GenBank, primers were designed to PCR-amplify the partial coding sequence at the 5′ end of the genes (Table 1). PCR products were cloned using the TOPO TA cloning kit for sequencing (Invitrogen).

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Table 1. PCR primers list.

https://doi.org/10.1371/journal.pone.0088994.t001

Genomic DNA extraction, 5′ promoter region isolation and bioinformatics analysis of putative transcriptional factors binding site

Isolation of the 5′-flanking regions of vitellogenin promoter was performed by genome walking as previously described [24]. Sequence analyses were performed using BLAST (http://www.ncbi.nlm.nih.gov/blast/) and protein sequence alignments conducted using Vector NTI Advance 10 (Invitrogen). Putative transcription factor binding sites were identified using the web-based Conreal server (http://conreal.niob.knaw.nl/).

Plasmid construction and transgenic Drosophila melanogaster

For each vitellogenin gene, a pair of primers was designed to amplify the region 2–3 kb upstream from the start codon (Table 1). PCR reactions proceeded with incubation at 94°C for 2 min, followed by 35 cycles consisting of 94°C for 30 s, 58°C for 30 s and 72°C for 2 min.

Each amplified putative promoter fragment was cloned upstream of the GFP coding sequence in pGlow (Invitrogen). Each promoter, together with GFP and the BGH polyadenylation sequence from pGlow was PCR-amplified and digested using Asc I and Fse I (for Vg1 promoters) or Fse I (for Vg2 promoters) and inserted into the Fse I site in pBac[3XP3-DsRed] to generate four constructs which each had the general organization: pBac[3XP3-DsRed-CxVg-promoter-GFP-BGH]. Each construct was injected into D. melanogaster embryos (w¹¹¹⁸) with the helper plasmid phsp-pBac [31]. Transgenic D. melanogaster were identified by red eye fluorescence using a Leica MZ95 microscope with DsRed filter (exciter HQ545/30x; emitter HQ620/60m) and lines established.

RT-PCR gel analysis of GFP transcripts in transgenic D. melanogaster

Drosophila fatbodies were dissected from adult female transgenic flies and total RNA extracted using the SV Total RNA Isolation system (Promega). 10–15 transgenic female adults (3 to 5 days old) for each sample were processed for RNA extraction. To remove genomic DNA contamination, the DNA-free™ kit (Ambion) was employed following the manufacturer's protocol. Two micrograms of total RNA was used as template for first strand cDNA synthesis using M-MLV Reverse Transcriptase (Promega). A pair of GFP specific primers (Table 1) was used for PCR. The PCR reaction was 2 min at 94°C, followed by 35 cycles consisting of 94°C for 30 s, 50°C for 30 s, and 72°C for 4 min 30 sec, and a final 72°C extension for 10 min. No-RT controls were included with every reaction.

Quantitative RT-PCR assays

To quantify expression levels of GFP driven by Cx. tarsalis vitellogenin promoters in transgenic D. melanogaster, we conducted quantitative real-time PCR using GFP-specific and the rp49-specific (single-copy nuclear gene) primer sets (Table 1). Quantitative PCR (qPCR) was performed using the Rotor-Gene Q Real-Time PCR System (QIAGEN) using the Rotor-Gene SYBR Green PCR Kit (QIAGEN). qPCR for both GFP and rp49 proceeded with incubation at 95°C for 5 min, followed by 40 PCR cycles consisting of 95°C denaturation for 5 s, 60°C annealing for 5 s and 72°C extension for 5 s and a final melting curve analysis of the PCR product to verify the specificity and identity, with ramp from 60°C to 95°C, rising by 1°C each step and 90 s for the pre-melt conditioning on first step, 5 s for each step afterwards. We tested 5 samples per treatment and each sample was replicated 3 times. Threshold cycle (Ct) values of the target gene GFP and rp49 were calculated for each replicate. Expression folds of GFP between different constructs of vitellogenin promoters or lines were calculated using the comparative CT method of relative quantization [32]. No-RT controls were included with every reaction. Expression values between lines were statistically compared by Kruskal-Wallis test with the Conover-Inman method for multiple comparisons using StatsDirect software (statsdirect.com).