Molecular characteristic of CcBurs-α and CcBurs-β in C. chinensis.
A: Multiple alignments of the amino acid sequences of CcBurs-α with homologs from five other insect species. Black represents 100% identity, red represents 75% identity, and blue represents <75% identity. CcBurs-α (C. chinensis, OR488624), DcBurs-α (Diaphorina citri, XP_008468249.2), ApBurs-α (Acyrthosiphon pisum, XP_001946341.1), MpBurs-α (Myzus persicae, XP_022171710.1), HvBurs-α (Homalodisca vitripennis, XP_046670477.1), DmBurs-α (Drosophila melanogaster, CAH74223.1). The corresponding GenBank accession numbers are as follows. B: Multiple alignments of the amino acid sequences of CcBurs-β with homologs from five other insect species. Black represents 100% identity, red represents 75% identity, and blue represents <75% identity. CcBurs-β (C. chinensis, OR488625), DcBurs-β (D. citri, AWT50591.1), HvBurs-β (H. vitripennis, XP_046671521.1), NvBurs-β (Nezara viridula, AZC86173.1), LsBurs-β (Laodelphax striatellus, AXF48186.1), DmBurs-β (D. melanogaster, CAH74224.1). The corresponding GenBank accession numbers are as follows. C: Predicted protein tertiary structures of CcBurs-α and CcBurs-β. D: Western blot analysis of Bursicon proteins using anti-His-Tag antibody with non-reduced and reduced SDS-PAGE. The left numbers indicate the positions of pre-stained protein markers. Lanes of α, β, and α+β represent separate expression of CcBurs-α, CcBurs-β, or co-expressed of α+β. Monomers were not present under non-reduced conditions. E-F: Relative mRNA expression of CcBurs-α and CcBurs-β after 25 °C or 10 °C treatments at 3, 6, 9, 12, and 15 d (n=3). G-H: Effect of temperature receptor CcTRPM knockdown on the mRNA expression of CcBurs-α and CcBurs-β at 3, 6, and 10 d under 10°C condition (n=3). Data in 1E-1H are shown as the mean ± SE with three independent biological replications, with at least 50 nymphs for each biological replication. Statistically significant differences were determined using pair-wise Student’s t-test in SPSS 26.0 software, and significance levels were denoted by *** (p < 0.001).