ChPDIA3 targeted by miR-126-x and miR-21-y responds to Vibrio harveyi infection in Crassostrea hongkongensis

Abstract

Introduction: The Hong Kong oyster (Crassostrea hongkongensis), as the main marine aquaculture shellfish in the South China Sea, not only has high economic and ecological value, but also is an ideal model for conducting research on pathogen-host interactions. In the cultivation process of C. hongkongensis, there is a challenge posed by vibrios. To improve the antibacterial strains of C. hongkongensis, we have studied the gene associated with immunity, PDIA3.

Methods and findings: In this study, we cloned the PDIA3 sequence of the C. hongkongensis, using the RACE technique. It has a total of 2081 bp and contains a 5'-UTR of 55 bp and a 3'-UTR of 547 bp. The ChPDIA3 gene sequence has an ORF frame that is 1479 bp in length and encodes 492 amino acids. Analysis of the phylogenetic tree constructed by Neighbor Joining method showed that ChPDIA3 clustered with other shellfishes into a single unit, which was consistent with the law of species evolution.

Discussion: The highest expression of ChPDIA3 was detected in gill tissues of the C. hongkongensis using RT-qPCR, and significantly higher expression in V. harveyi and LPS infection than Poly(I:C) (P<0.05). This may indicate that ChPDIA3 is primarily involved in the immune response against bacterial infections in the C. hongkongensis. The binding sites of miR-126-x, miR-21-y and ChPDIA3 were detected using dual luciferase experiments, respectively. The results showed that both miR-126-x and miR-21-y inhibited the 3'-UTR region of ChPDIA3. This suggested that both miR-126-x and miR-21-y inhibited ChPDIA3 expression. This study will help to further understand the function of ChPDIA3 in response to pathogen infection, thus providing new ideas for understanding the resistance and adaptation of the C. hongkongensis to Vibrio infection.

Keywords: Crassostrea hongkongensis; PDIA3; Vibrio harveyi; immune response; miRNA regulation.

Figure 1

A PDIA3 gene in C. hongkongensis and phylogenetic tree of the ChPDIA3 protein. (A) Schematic presentation of ChPDIA3. Black line, structure of the gene in the genome. Dark red and dark green boxes, ChPDIA3 transcript and its coding region. (B) Phylogenetic tree and structural information of selected PDIA3 protein. GenBank accession number (NCBI): Homarus americanus (XP_042223300.1), Penaeus vannamei (XP_027212336.1), Penaeus monodon (XP_037788996.1), Cyprinus carpio (XP_018979324.2), Poecilia formosa (XP_007566541.1), Takifugu rubripes (XP_029700442.1), Mus musculus (NP_031978.2), Homo sapiens (NP_005304.3), Puma concolor (XP_025774328.1), Canis lupus familiaris (XP_535453.3), Sepia pharaonic(CAE1328800.1), Octopus bimaculoides (XP_014785522.1), Octopus sinensis (XP_029643538.1), Mytilus galloprovincialis (VDI57500.1), Ostrea edulis (XP_048735080.2), Crassostrea gigas (XP_011453191.2). (C) Alignment of partial PDIA3 domain containing active conserved site Thioredoxin_CS.

Figure 2

Expression of ChPDIA3 in different tissues of the C. hongkongensis. Different lowercase letters indicate significant differences between treatment groups (P<0.05).

Figure 3

Expression pattern of ChPDIA3 in different treatment groups. Different lowercase letters indicate significant differences between treatment groups (P<0.05).

Figure 4

Expression patterns of genes after RNA interference. (A) shows the expression of the ChPDIA3 gene at different time points (12h, 24h, 48h, 72h) in the RNA interference group, the Vibrio group (injected with Vibrio harveyi), and the control group (injected with PBS) after RNA interference. (B–D) display the expression trends of downstream genes of ChPDIA3 (such as CALR, LMAN1, and LMAN2) in the endoplasmic reticulum protein processing pathway at different time points when ChPDIA3 is silenced by dsRNA. Different lowercase letters indicate significant differences between treatment groups (P<0.05).

Figure 5

Dual luciferase detection of miR-126-x and miR-21-y regulation of ChPDIA3. (A) represents the miR - 126 - x mimics group, showing the luciferase activity of HEK293T cell lines co - transfected with miR - 126 - x mimics and pmirGLO - PDIA3 - 3’ - UTR. (B) represents the miR - 21 - y mimics group, indicating the luciferase activity of HEK293T cell lines co - transfected with miR - 21 - y mimics and pmirGLO - PDIA3 - 3’ - UTR. “*” indicates an extremely significant difference between groups (P < 0.01).