Molecular Characterization and Expression Profiling of Nuclear Receptor Gene Families in Oriental Fruit Fly, Bactrocera Dorsalis (Hendel)

Abstract

The oriental fruit fly (Bactrocera dorsalis) is a pest that causes large economic losses in the fruit and vegetable industry, so its control is a major challenge. Nuclear receptors (NRs) are a superfamily of ligand-dependent transcription factors that directly combine with DNA to regulate the expression of downstream target genes. NRs are closely associated with multiple physiological processes such as metabolism, reproduction, and development. Through sequence searches and analysis, we identified 21 B. dorsalis NR genes, all of which contained at least one of the two characteristic binding domains. On the basis of the conserved sequences and phylogenetic relationships, we divided the 21 NR genes into seven subfamilies. All members of the NR0 subfamily and BdHR83, which belonged to the NR2E group, lacked ligand-binding domains. The BdDSF and BdHR51, which also belonged to the NR2Egroup, and BdE78 (which belonged to the NR1E group) all lacked DNA-binding domains. The BdDSF and BdHR83 sequences were incomplete, and were not successfully amplified. Development- and tissue-specific expression profiling demonstrated that the transcript levels of the 19 NR genes varied considerably among eggs, larva, pupae, and adults, as well as among larval and adult male and female tissues. Our results will contribute to a better understanding of NR evolution and expand our knowledge of B. dorsalis physiology.

Keywords: Bactrocera dorsalis; development; spatiotemporal expression; transcription factors.

Figure 1

Domain architectures of 21 NR proteins in B. dorsalis. DNA-binding domain (DBD) (red); ligand-binding domain (LBD) (blue). The deduced amino acid sequences were used to predict the domain architectures in SMART (http://smart.embl-heidelberg.de/) and NCBI Conserved Domains Search (https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi).

Figure 2

Phylogenetic analysis of 90 insect nuclear receptor (NR) proteins from five different species. Bombyx mori (Bm), Ceratitis capitata (Cc), Bactrocera dorsalis (Bd), Drosophila melanogaster (Dm) and Tribolium castaneum (Tc). A bootstrap analysis of 1000 replications was carried out on the trees inferred using the neighbor-joining method, and bootstrap values were shown at each branch of the tree. NRs from B. dorsalis are indicated by red dots.

Figure 3

Developmental mRNA expression profiles of 19 B. dorsalis NR genes (excluding BdHR83 and BdDSF). Relative transcript levels were calculated using α-tubulin (GenBank Accession no.: GU269902) and rps3 (GenBank Accession no.: XM_011212815) as internal references. Three to four independent biological replicates and two technical replicates were performed for each qRT-PCR. E: eggs; L1, L3, L5, L7, and L9, one- to nine-day-old larva on odd days; P1, P3, P5, P7, and P9, one- to nine-day-old pupae on odd days; F: A1, A3, A5, A7, and A9, one- to nine-day-old female adults on odd days; M A1, A3, A5, A7, A9, one- to nine-day-old male adults on odd days. Warm colors (i.e., red) mean high expression levels and cold colors (i.e., blue) mean low expression levels.

Figure 4

Tissue-specific expression of 19 B. dorsalis NR genes (excluding BdHR83 and BdDSF) from four-day-old adults. Relative transcript levels were calculated using α-tubulin (GenBank Accession no.: GU269902) and rps3 (GenBank Accession no.: XM_011212815) as internal references. Three to four independent biological replicates and two technical replicates were performed for each qRT-PCR. Midgut of female/male adults (F/M MG); fat body of female/male adults (F/M FB); Malpighian tubule of female/male adults (F/M MT); ovary of adult females (OV); testis of adult males (TE). Warm colors (i.e., red) indicate high expression levels and cold colors (i.e., blue) indicate low expression levels.

Figure 5

Tissue-specific expression of 19 NR B. dorsalis genes (excluding BdHR83 and BdDSF) from third instar larvae. Relative transcript levels were calculated using α-tubulin (GenBank Accession no.: GU269902) and rps3 (GenBank Accession no.: XM_011212815) as internal references. Three to four independent biological replicates and two technical replicates were performed for each qRT-PCR. Larval midgut (L MG); larval fat body (L FB); larval cuticle (L BP); larval Malpighian tubule (L MT); larval central nervous system (L CNS). Warm colors (i.e., red) indicate high expression levels and cold colors (i.e., blue) indicate low expression levels.

Figure 6

Conserved regions in the NR0 subfamily of B. dorsalis and D. melanogaster NR proteins. Amino acid sequences of the NR0 subfamily catalytic domains were aligned using DNAMAN 6 (https://www.lynnon.com/downloads.html). Identical and highly conserved amino acids are indicated by black (100%), pink (75%), and blue (50%), respectively.

Figure 7

Conserved regions in the NR1 subfamily of B. dorsalis and D. melanogaster NR proteins. Amino acid sequences of the catalytic domains of the NR1 subfamily were aligned using DNAMAN 6 (https://www.lynnon.com/downloads.html). Identical and highly conserved amino acids are indicated by black (100%), pink (75%), and blue (50%), respectively.

Figure 8

Conserved regions in the NR2 subfamily of NR proteins from B. dorsalis (excluding BdHR51, BdDSF, and BdHR83). Amino acid sequences of the catalytic domains of the NR2 subfamily were aligned using DNAMAN 6 (https://www.lynnon.com/downloads.html). Identical and highly conserved amino acids are indicated by black (100%), pink (75%), and blue (50%), respectively.