Fig 1.
Genomic, phylogenetic and conservation analysis of Amys in 29 species.
A: The number of Amy genes in 29 species across different taxa (Deuterostomia, Lophotrochozoa, and Ecdysozoa) with genomic data. B: Phylogenetic analysis of Amy genes from the 29 species using neighbor-joining method with 1000 bootstrap replicates. C: Sequence logo showing amino acid conservation patterns in Amy proteins, where letter height indicates relative residue frequency and amino acids are color-coded by physicochemical properties.
Fig 2.
Domain organization and transcriptomic expression patterns of the Amy family in L. vannamei.
A: The structural domain organization of Amy proteins in L. vannamei annotated by SMART. B: Transcriptomic expression in different tissues (n = 3 biological replicates). Tissues: brain (Br), thoracic nerve (TN), abdominal nerve (AN), eyestalk (Es), gill (Gi), hepatopancreas (Hp), stomach (St), intestine (In), heart (Ht), muscle (Ms), ovary (Ov), testis (Ts). C: Transcriptomic expression in different ontogenetic development and molting stages (n = 3 biological replicates). Ontogenetic development stages (a): early fertilized egg (pre-E), late fertilized egg (post-E), nauplius stages (N1-N6), zoea stages (Z1-Z3), mysis stages (M1-M3) and postlarvae (Pl). Molting stages (b): intermolt (C), early to late pre-molt (D0-D4), post-molt (P1-P2).
Fig 3.
Full-length cDNA sequence and 3D structural modeling of Lv-Amy.
A: Full-length cDNA sequence and deduced amino acid sequence of Lv-Amy. The translational start codon (atg) and stop codon (taa) are shown in red, the signal peptide, Aamy and Aamy_C are shown in the boxes with different colors, and the active site, Ca² ⁺ -binding site and catalytic site are indicated with different symbols. B: 3D structural model of Lv-Amy showing domain organization with the signal peptide, Aamy domain and Aamy_C domain distinguished by colors.
Fig 4.
Tissue-specific expression and localization of Lv-Amy.
A: Tissue-specific expression profiles of Lv-Amy detected by qPCR, with transcript levels normalized to the reference gene and expressed as mean ± SD (n = 3). Tissues follow abbreviations in Fig 2B. B: Histological and FISH analysis of Lv-Amy in digestive tissues of L. vannamei. H/E stain indicates sections stained with hematoxylin and eosin. In the FISH, yellow signals represent positive hybridization and blue signals indicate DAPI-stained nuclei. Tissues: hepatopancreas (Hp), stomach–midgut junction (SMJ), midgut (MG), hindgut (HG) and stomach (St). The hepatopancreas contains three major cell types: blister-like cells (B), resorptive cells (R) and embryonic cells (E). The intestine includes epithelium (ep), muscle (mus) and lumen (lum). The stomach contains lower ampullary setae (las), food particles (fp) and setules (st).
Fig 5.
Expression, purification and refolding of recombinant Lv-Amy protein.
A: SDS-PAGE analysis of whole-cell proteins, soluble fraction and insoluble fraction from IPTG-induced E. coli expressing Lv-Amy. B: SDS-PAGE of inclusion body proteins purified under denaturing conditions by Ni² ⁺ -IMAC. C: SDS-PAGE of refolded protein. M: Protein molecular weight markers, 1: control, 2: whole-cell proteins after IPTG induction, 3: soluble fraction after cell lysis, 4: insoluble fraction after cell lysis, P: target protein (red arrow).
Fig 6.
Effect of temperature and pH on the activity of recombinant Lv-Amy protein.
A: Effect of temperature on the activity of Lv-Amy. B: Effect of pH on the activity of Lv-Amy. Values are shown as mean ± SD (n = 3 technical replicates per condition), error bars indicate SD.