Abstract:  【Aim】 This study aims to clone the transcript variant X3 of Pangolin isoforms A/H/I/S (Pangolin X3), a key gene downstream of the Wnt signaling pathway in the silkworm, Bombyx mori, and analyze its sequence and expression characteristics. 【Methods】 Pangolin X3 of B. mori was retrieved from the NCBI database, and the primers were designed according to its coding sequence (CDS). Pangolin X3 was cloned from the midgut and hemolymph of B. mori larvae using PCR, and then verified by sequencing. The sequence characteristics of Pangolin X3 were analyzed using SilkDB 3.0, SMART, multiple sequence alignment and phylogenetic tree. The relative expression levels of Pangolin X3 in different tissues (head, hemolymph, integument, gonads, midgut, anterior silk gland, middle silk gland, posterior silk gland, fat body and Malpighian tubules) of the day-3 5th instar larvae of B. mori were analyzed using qRT-PCR. 【Results】 The CDS of Pangolin X3 (GenBank accession no.: XM_038020921) was cloned from the midgut and hemolymph of B. mori larvae. It has an open reading frame of 1 560 bp in length, encoding 519 amino acid residues with the predicted molecular weight of 55.86 kD and the isoelectric point of 7.53. Pangolin X3 contains a conserved β-catenin binding site and an HMG domain, its amino acid sequence is relatively conserved among different insects, especially the HMG domain that binds to DNA, but some amino acid residues in the N-terminal CTNNB1 domain that binds to β-catenin are variable. Tissue expression profile revealed that the relative expression levels of Pangolin X3 in the midgut, hemolymph and gonads of the day-3 5th instar larvae of B. mori were high, while those in the head, integument, anterior silk gland, middle silk gland, posterior silk gland, fat body and Malpighian tubules were low. 【Conclusion】 In this study, we cloned Pangolin X3 of B. mori and analyzed its sequence and expression characteristics, providing a basis for an in-depth study of the biological function of Pangolin in B. mori.

Key words:  Bombyx mori, Wnt; Pangolin X3, gene cloning, sequence analysis, expression characteristics