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A transferrin gene associated with development and 2-tridecanone tolerance in Helicoverpa armigera - PubMed

A transferrin gene associated with development and 2-tridecanone tolerance in Helicoverpa armigera

L Zhang et al. Insect Mol Biol. 2015 Apr.

Abstract

The full-length cDNA (2320 bp) encoding a putative iron-binding transferrin protein from Helicoverpa armigera was cloned and named HaTrf. The putative HaTrf sequence included 670 amino acids with a molecular mass of approximately 76 kDa. Quantitative PCR results demonstrated that the transcriptional level of HaTrf was significantly higher in the sixth instar and pupa stages as compared with other developmental stages. HaTrf transcripts were more abundant in fat bodies and in the epidermis than in malpighian tubules. Compared with the control, the expression of HaTrf increased dramatically 24 h after treatment with 2-tridecanone. Apparent growth inhibition with a dramatic body weight decrease was observed in larvae fed with HaTrf double-stranded RNA (dsRNA), as compared with those fed with green fluorescent protein dsRNA. RNA interference of HaTrf also significantly increased the susceptibility of larvae to 2-tridecanone. These results indicate the possible involvement of HaTrf in tolerance to plant secondary chemicals.

Keywords: 2-tridecanone; Helicoverpa armigera; RNA interference; transferrin.

© 2014 The Authors. Insect Molecular Biology published by John Wiley & Sons Ltd on behalf of The Royal Entomological Society.

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Figures

Figure 1
Figure 1

Multiple alignment of transferrin amino acid sequences from various insects: Spodoptera litura transferrin (DQ470073.1), Bombyx mori transferrin (NM 001043549.1), Manduca sexta transferrin (M62802.1), Chilo suppressalis transferrin (AB158473.1), Plutella xylostella transferrin (AB282638.1), Choristoneura fumiferana transferrin (AY563106.1), Anagasta kuehniella transferrin (HM026347.1), Papilio xuthus transferrin 1 (AK402830.1). Positions of potential iron-binding residues are indicated by rectangles, conserved cysteine residues by ‘*’ and conserved lysine residues by ‘&’. ‘<’ and ‘>’ show the N-terminal lobe and C-terminal lobe, respectively. The 20 amino acid signal peptide sequence (MVSKIKYIYLLVLACVCVQA) is underlined. The similar and identical residues are shaded in gray and black.

Figure 2
Figure 2

Phylogenetic relationships of transferrin amongst Helicoverpa armigera and other species. ‘Δ’ indicates the Lepidoptera. Full species names and GenBank accession numbers: Spodoptera litura transferrin (DQ470073.1), Bombyx mori transferrin (NM 001043549.1), Manduca sexta transferrin (M62802.1), Chilo suppressalis transferrin (AB158473.1), Plutella xylostella transferrin (AB282638.1), Anagasta kuehniella transferrin (HM026347.1), Riptortus clavatus transferrin (AF046126.1), Apis mellifera transferrin (AY336528.1), Solenopsis invicta transferrin (AY940116.1), Apriona germari transferrin (AY894342.1), Romalea microptera transferrin (AY362358.2), Periplaneta americana transferrin (JQ257002.1), Blattella germanica transferrin (L05340.1), Drosophila melanogaster transferrin 1 (NM 078677.2), D. melanogaster transferrin 2 (NM 079320.2), D. melanogaster transferrin 3 (NM 079035.2), Musca domestica transferrin (HQ645966.1), Sarcophaga peregrina transferrin (D28940.1), Culex pipiens pallens transferrin (HM370559.1), Culex quinquefasciatus transferrin (XM001863377.1). Numbers on nodes indicate bootstrap values from 10 000 replicates.

Figure 3
Figure 3

(A) Tissue-specific expression patterns of Helicoverpa armigera transferrin (HaTrf) in larvae. The relative expression of HaTrf in different tissues (haemolymph, head, midgut, fat body, epidermis and malpighian tubule) of sixth-instar larvae. (B) Developmental expression profiles of HaTrf. The expression of HaTrf in different development stages: egg, first-instar larvae (1st), second-instar larvae (2nd), third-instar larvae (3rd), fourth-instar larvae (4th), fifth-instar larvae (5th), sixth-instar larvae (6th), pupa, adult female, adult male. (C) Expression profiles of HaTrf under 2-tridecanone induction. Comparison of the untreated group at each time point (0, 12, 24 and 48 h) with the transcriptional profile of HaTrf in the sixth-instar larvae under 2-tridecanone (the concentration of 2-tridecanone in the diet was 10 mg/g, w : w). Bars with different letters for each group are significantly different at P < 0.05.

Figure 4
Figure 4

The double-stranded RNA (dsRNA)-mediated depletion of Helicoverpa armigera transferrin (HaTrf) transcripts in larvae fed with HaTrf dsRNA. Second-instar larvae were fed on a diet containing dsRNA at 5 or 35 μg/g (w : w), and some individuals from each sample were collected at 12, 24, 36 and 48 h after feeding. PCR bands and the histogram show the quantitative PCR results of HaTrf transcription. Green fluorescnt protein (GFP) dsRNA was used as a control using the same concentrations. In each diagram, bars sharing the same letter for each group are not significantly different at P > 0.05.

Figure 5
Figure 5

Systemic RNA interference effect in Helicoverpa armigera observed using fluorescent-labelled double-stranded RNA (dsRNA). Fifteen first-instar larvae were fed on a diet containing fluorescent-labelled dsRNA at 35 μg/g (w : w), and samples were collected at 6, 12, 24 and 48 h after feeding for fluorescence signal scanning. The left panels were observed using a stereo microscope (SM) and the right panels were observed at 492 nm (FAM) on an Olympus SZX86.

Figure 6
Figure 6

Effect of double-stranded RNA (dsRNA)-mediated depletion of Helicoverpa armigera transferrin (HaTrf) transcripts on larval development. (A) Photograph of the HaTrf dsRNA-mediated growth inhibition phenotype, as compared with the green fluorescent protein (GFP) dsRNA control. (B) HaTrf dsRNA-mediated body weight changes at 0 days (0 d), 1 day (1 d), 3 days (3 d) and 5 days (5 d) compared with the GFP dsRNA control, bars sharing the same letter for each group are not significantly different at P > 0.05.

Figure 7
Figure 7

Survival rates of larvae fed on Helicoverpa armigera transferrin (HaTrf) double-stranded RNA (dsRNA) containing 2-tridecanone. Survival rates were evaluated by feeding second-instar larvae with HaTrf dsRNA (35 μg/g) containing 2-tridecanone (0.1 mg/g) for 1 day (1 d), 3 days (3 d) and 5 days (5 d), bars sharing the same letter for each group are not significantly different at P > 0.05. GFP, green fluorescent protein.

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