Molecular Characterization and Function Analysis of the Vitellogenin Receptor from the Cotton Bollworm, Helicoverpa armigera (Hübner) (Lepidoptera, Noctuidae)

Abstract

Developing oocytes accumulate plentiful yolk protein during oogenesis through receptor-mediated endocytosis. The vitellogenin receptor (VgR), belonging to the low-density lipoprotein receptor (LDLR) family, regulates the absorption of yolk protein. In this work, the full-length vitellogenin receptor (HaVgR) in the cotton bollworm Helicoverpa armigera was identified, encoding a 1817 residue protein. Sequence alignment revealed that the sequence of HaVgR contained all of the conservative structural motifs of LDLR family members, and phylogenetic analysis indicated that HaVgR had a high identity among Lepidoptera and was distinct from that of other insects. Consistent with other insects, HaVgR was specifically expressed in ovarian tissue. The developmental expression pattern showed that HaVgR was first transcribed in the newly metamorphosed female adults, reached a peak in 2-day-old adults and then declined. Western blot analysis also revealed an ovarian-specific and developing expression pattern, which was consistent with the HaVgR mRNA transcription. Moreover, RNAi-mediated HaVgR knockdown strongly reduced the VgR expression in both the mRNA and protein levels, which inhibited the yolk protein deposition in the ovaries, led to the dramatic accumulation of vitellogenin and the up-regulation of HaVg expression in hemolymph, and eventually resulted in a declined fecundity. Together, all of these findings demonstrate that HaVgR is a specific receptor in uptake and transportation of yolk protein for the maturation of oocytes and that it plays a critical role in female reproduction.

Fig 1

Diagrammatic comparison of primary structure of HaVgR with other insects (A) and the molecular phylogenetic tree constructed based on the sequence of vitellogenin receptor (VgR) and low density lipoprotein receptors (B). EGFc indicate non-calcium binding. The YWXD containing repeats that form b-propeller domains are labeled LY. The percentages on the left indicate overall identity of each protein compared to HaVgR. SP, signal peptide; LBD, lipid binding domain; O, potential O-linked sugar domain; TM, transmembrane domain; C, cytoplasmic domain. The protein names and accession numbers used in this analysis are listed in S2 Table.

Fig 2. qRT-PCR and western blot analysis of sex-, tissue-specific and development expression patterns of HaVgR.

A: Sex-specific expression of HaVgR. The total RNA samples were extracted from abdomens of female and male adults. B: Tissue-specific expression of HaVgR. The total RNA samples were extracted from various of female tissues and segment: ovary, epidermis, midgut, fat body, malpighian tubules and head. C: Development expression patterns of HaVgR. Samples were extracted from abdomens of the 10-days-old pupae (P), 0-day-old adults (0dA), 1-day-old adults (1dA), 2-days-old adults (2dA), 3-days-old adults (3dA), 4-days-old adults (4dA), 5-days-old adults (5dA), 6-days-old adults (6dA)), 7-days-old (7dA), 8-days-old (8dA). The bars represent the average (±SE) of biological repeats. Different letters indicate significant difference (P <0.05). ** indicate significant (P <0.01) differences between two groups.

Fig 3. The ovarian development of H. armigera.

The ovarian images of pupae, 0-day-old female to 8-day-old female (A-J) were observed. Bar: A-J = 2 mm.

Fig 4. Detection of mRNA and protein level in RNA-interference-treated insects.

(A): The expressions levels of HaVgR were determine by qPCR and western blot after injection of dsVgR 24, 48 and 72 h. (B): The expression levels of HaVg were analyzed by qPCR and western blot after injection of dsVgR 48 h. The dsGFP treatment group was used as a negative control and DEPC water was used as a blank control. The bars represent the average (±SE) of biological repeats. Different letters indicate significant difference (P <0.05).

Fig 5. Ovary development was evaluated after RNAi-mediated knockdown of HaVgR.

Ovaries were dissected and photographed under dissection microscopy at days 4 post-injection, the dsGFP was injected as a negative control and DEPC water was injected as the blank control. DEPC water (A-A2), dsGFP (B-B2) and dsVgR (C-C2).