Insects

The oriental fruit fly, Bactrocera dorsalis, was originally collected from Fujian province, People's Republic of China. The adults were reared on an artificial diet consisting of water, yeast powder, honey, and Vitamin C (500:15:15:1) in glass cages. Every 2 days, a banana was put into the cage to collect eggs. After hatching, larvae were reared on banana in plastic basins with sand until pupation. The colonies were kept in a temperature controlled room at 27 ∓ 1 °C, 70 ∓ 5 %RH at 14:10 h L:D throughout development (Shen et al. 2010).

RNA Isolation and cDNA Preparation

Total RNA was isolated from the heads of adult flies (15 mg) using an RNA Isolation Kit (Watson Biotechnologies, Shanghai, China) following the manufacturer's instructions. The total RNA was treated with DNase (TaKaRa, Dalian, China) and dissolved in 40 µL DEPC-treated H₂O and stored at -80 °C until use. The first-strand cDNA was synthesized from 2 µg of DNase-treated RNA by RevertAid™ First Strand cDNA Synthesis Kit (Fermentas, Vilnius, Lithuania) with oligo (dT)18 primers according to the manufacturer's instructions. The reaction mixture was stored at -20 °C for future use.

Amplification of cDNA Fragments

Degenerate primers, Bd1, 5′-ATGAARTTYGGNWSNTGGACNTAYGA-3′ and Bd2, 5′-GCNACCATRAACATDATRCARTTRAA-3′, were designed based on the conserved motifs of nicotinic acetylcholine receptor family to amplify the cDNA fragments (Gao et al. 2007b). Degenerate PCR was conducted using rTaq™ polymerase (TaKaRa, Dalian, China) in 25 µL, including 1 µL cDNA templates, 1 µL each primer (10 µM), 2 µL dNTP (2.5 mM), and 2.5 µL 10× PCR buffer (Mg²⁺ plus). Degenerate PCR amplifications were performed under the following conditions: initial denaturation at 95 °C for 3 min, followed by 34 cycles of denaturation at 95 °C for 30 sec, annealing at 55 °C for 30 sec, and extension at 72 °C for 1 min, with a final extension at 72 °C for 10 min. The PCR products were then separated by 1.5% agarose gel electrophoresis and stained with ethidium bromide (EB). A band of 428 bp cDNA was excised and purified using the Gel Extraction Mini Kit (Watson Biotechnologies, Shanghai).

The purified fragments were cloned into pGEM®-T Easy vector (Promega, Madison, Wisconsin). The ligation reactions were used for transformations with DH5α competent cells (Transgen, Beijing), the DNA inserts of the recombinant clones were identified by PCR with the primers used before, and further confirmed by sequencing in both directions with an ABI-PRISM 3730 sequencer (Invitrogen Life Technologies, Shanghai, China).

Rapid Amplification of cDNA Ends (RACE) and Cloning of the Open Reading Frame (ORF) of Bdorα6

The full-length cDNA of Bdorα6 was obtained by 5′/3′ RACE techniques using a SMARTer™ RACE cDNA Amplification Kit (Clontech, California) according to the manufacturer's instructions. The 3′-RACE-ready cDNA was synthesized from 2 µg of total RNA at 42 °C for 1.5 h with SMARTScribe™ Reverse Transcriptase and the 3′-CDS primer, while the 5′-RACE-ready cDNA was synthesized with SMARTScribe™ Reverse Transcriptase, the 5′-CDS primer, and SMARTer IIA oligo.

Based on the 428 bp cDNA sequence, 4 gene specific primers were designed to amplify the full-length cDNA: 2 forward primers (in the first round reaction, GSP1: 5′-ACAAATGGCGAGTGGTATCTGC-3′ and in the second round reaction, GSP2: 5′-CTTCTATCGCTCACGGTGTTTCTC-3) for 5′-RACE PCR and 2 reverse primers (in the first round reaction, GSP3: 5′-GAGAAACACCGTGAGCGATAGAAG-3′ and in the second round reaction, GSP4: 5′-CGCCGTCGGATTTGTATCGTGAA-3′) for 3′-RACE PCR. Utilizing the gene specific primers, universal primer (UPM, provided by the Clontech kit) and nested universal primer (NUP, provided by the Clontech kit), the 3′-and 5′-RACE were conducted with PrimeSTAR™ HS DNA polymerase (TaKaRa, Dalian, China). Thermal cycling conditions were: pre-denaturation 3 min at 94 °C, 30 cycles of 94 °C for 30 sec, 60 °C -65 °C (according to the annealing temperature of the primer) for 30 sec and 72 °C for 1–1.5 min (according to the size of expected fragment) with a final extension of 10 min at 72 °C. The resulting PCR products of 5′-RACE and 3′-RACE were cloned into pGEM®-T Easy vector (Promega, Madison, Wisconsin) and sequenced as described above.

After the 5′-ends and 3′-RACE sequences were obtained, the contigs were assembled to produce putative full-length sequences by using DNAMAN 5.2.2 (Lynnon BioSoft, Quebec). To verify the full-length cDNA, the primers BdF, 5′-ACATGGACCCGTCGCTGCTAGTC-3′ and BdR, 5′-CATTATCGTGCAATAAGCCTAAA-3′, were designed based on the sequences obtained from 5′-untranslated region (UTR) and 3′-UTR of the gene and used to amplify the ORF of Bdorα6. The PCR products were purified, cloned, and sequenced as described above.

Sequence Analysis

A search for similar sequences was performed using BlastP in the non-redundant protein sequences (nr) database of the NCBI website ( http://www.ncbi.nlm.nih.gov/). Multiple sequence alignment was performed with DNAMAN 5.2.2 (Lynnon BioSoft, Quebec). The signal peptide was predicated by SignalP 3.0 (Bendtsen et al. 2004). Phosphorylation sites and N-linked glycosylation sites were identified by the PROSITE database (Falquet et al. 2002). Transmembrane domains were predicted by the TMHMM program ( http://www.cbs.dtu.dk/services/TMHMM/). The phylogenetic tree construction by MEGA5.04 (Tamura et al. 2011) used the neighbor-joining method. Bootstrap values were calculated on 1000 replications.

Quantitative Real-Time PCR of Bdorα6

The expression levels of Bdorα6 mRNA relative to expression of α-tubulin at different developmental stages and in different tagmata of B. dorsalis were examined using quantitative realtime PCR. Total RNA was isolated from whole bodies at different developmental stages (including egg, first, second and third instar larvae, pupa, and adult) or from heads, thoraces, and abdomens of adult flies, using RNeasy Plus Mini Kit (with gDNA Elimator spin columns, Qiagen, Valencia, California). First strand cDNA was synthesized in a 10 µL reaction mixture using random hexamers by PrimeScript® RT reagent Kit (TaKaRa, Dalian, China). The quantitative realtime PCR was carried out by Mx3000P thermal cycler (Stratagene, La Jolla, California). The PCR amplifications were performed in 25 µL reactions systems containing 2 µL of template cDNA, 12.5 µL iQ™ SYBR® Green Supermix (BIO-RAD, Hercules, CA, USA) and 0.2 mM each of the primers 5′-ATTCGATGGCACGTATCACA-3′ (forward) and 5′-GGGCACCAAGTTAGTCTGGA-3′ (reverse) for Bdorα6, and 5′-CGCATTCATGGTTGATAACG-3′ (forward) and 5′-GGGCACCAAGTTAGTCTGGA-3′ (reverse) for α-tubulin. Amplification conditions were as follows: initial denaturation at 95 °C for 2 min, followed by 40 cycles of denaturation at 95 °C for 15 sec, 60 °C for 30 sec and 72 °C for 30 sec. After reaction, a melting curve analysis from 60 °C to 95 °C was applied to all reactions to ensure consistency and specificity of the amplified product. The real-time PCR analysis was repeated 3 times in independent experiments. The data of different developmental stages and tagmata were normalized according to the expression level of α-tubulin gene (GenBank accession number: GU269902). When we tested the mRNA expression during the different life stages, the pupal stage served as the calibrator sample whose mRNA expression ratio would always equal 1. In addition, when testing mRNA expression in different tagmata, the abdomen served as the calibrator sample.

Statistics Analysis

The PCR efficiencies of Bdorα6 and α-tubulin were calculated by the Mxpro-Mx3000P version 4.01 (Stratagene, La Jolla, California). Data from the quantitative real-time PCR for both the developmental expression and different tagmata expression profiles were subjected to LSD test in analysis of variance (ANOVA) (SPSS 12.0 for Windows).

Cloning and Characterization of Bdorα6

Degenerate primers Bd1 and Bd2 were used to amplify a 428 bp fragment from a cDNA preparation of B. dorsalis. Based on the sequence of the cDNA fragment, we obtained the full-length cDNA through the 5′/3′ RACE technique. It was the closest similarity to other known insect α6-type nAChR subunits at amino acid levels; the gene was named as Bdorα6. The cDNA sequence and deduced amino acid sequence of the Bdorα6 is shown in Fig. 1. The complete cDNA of the Bdorα6 (GenBank accession number: JF974072) consists of 2020 nucleotides with an ORF of 1464 nucleotides encoding 488 amino acids. The cDNA includes a 5′-UTR located 206 bp upstream of the start codon (ATG) and 3′UTR of 350 nucleotides that ended in a poly (A) tail. A possible consensus signal sequence for polyadenylation (AATAAA) is located 27 bp upstream of the poly (A) tail. The theoretical molecular mass of Bdorα6 based on the deduced amino acid sequence was calculated to be 55.6 kDa, with an isoelectric point of 5.41.

We performed a signal peptide scan using the SignalP 3.0 program ( http://www.cbs.dtu.dk/services/SignalP) finding that the protein possesses a predicted 21-residue long signal peptide. The protein has 2 potential N-linked glycosylation sites (N-X-S/T) at positions 44 and 88 in the N-terminal extracellular domain. There are 15 potential phosphorylation sites for protein kinase C, casein kinase II, tyrosine kinase and cAMP- and cGMP- dependent protein kinase, 7 of which are located in the N-terminal extracellular domain, 1 located in the cytoplasmic TM1–TM2 linker, and 7 located in the cytoplasmic TM3–TM4 linker.

Phylogenetic Analysis

To understand the relationships among the Bdorα6 protein with other insect nAChRs, a phylogenetic tree was constructed using the neighbor-joining method (Fig. 2). Phylogenetic analysis indicated that the Bdorα6 was most closely related to Dα6 and Agamα6 with these 3 subunits clustering together, and also showed the relationships with different subunits from other species. Sequence BLASTP revealed that the deduced amino acid sequence of Bdorα6 was highly conserved with alpha6 homolog in other insect species. Compared with proteins in alpha6 subunit from the NCBI database, Bdorα6 shared 94.53% identity with the Dα6 of D. melanogaster (AAM13393), 87.78% identity with the Agamα6 of A. gambiae (AAU12509), 81.08% identity with the Bmα6 of B. mori (ABL67934), and 72.21% identity with Amelα6 of A. mellifera (AAY87894). Therefore, the cloned nAChR subunit gene should belong to α6-type.

Developmental and Tissue-Specific Expression Pattern of Bdorα6

Quantitative real-time PCR was employed to investigate the Bdorα6 gene expression levels during various developmental stages and different adult tagmatas. The result showed that the Bdorα6 gene was expressed in all stages, indicating that it has a role throughout the entire life cycle. The lowest mRNA level was found in pupa stage, and the relative expression levels of Bdorα6 was 23-, 39-, 6-, 2- and 27-fold higher in the egg, first, second and third instar larvae, and adult than in the pupa, respectively. Subsequently, the relative expression level of Bdorα6 in egg, second, third instar larvae, and pupa were significantly lower from that in the first instar larva (P < 0.05) (Fig. 3). There was a descending expression level of Bdorα6 during the developmental period from the first instar larva to the pupa.

The Bdorα6 mRNA was found in different tagmata of adult flies. The relative expression level of Bdorα6 was significantly different (P < 0.05) between different tagmata, and it was 109- and 15-fold higher in the head and thorax relative to the abdomen, respectively (Fig. 4).

Fig. 1.

Nucleotide and deduced amino acid sequence of Bdorα6 cDNA from Bactrocera dorsalis (GenBank accession number: JF974072). The start codon is indicated with bold and the stop codon is indicated both with bold and an asterisk. The predicated signal peptide is double underlined. The Cys-loop domain is underlined with a dotted line. Potential N-linked glycosylation sites are boxed. The position of putative 4 trans-membrane domains (TM1– 4) is shaded. The cysteine doublet (characteristics of nAChR α subunits) is marked with as ‘####’. The potential phosphorylation sites are underlined.