Cui-Jie Kang

Molecular cloning and expression patterns of the molt-regulating transcription factor HHR3 from Helicoverpa armigera.

Molt‐regulating transcription factors, hormone receptor 3 (HR3), play important roles in regulating expression of tissue‐specific genes involved in insect molting and metamorphosis. A 1668 bp cDNA encoding a molt‐regulating transcription factor (HHR3) was cloned from Helicoverpa armigera, which encodes a protein made up of 556 amino acids. This 62 kDa protein was found to have an isoelectric point (pI) of 6.52. There was no signal peptide or N‐glycosylation site found in this cDNA. A DNA‐binding region signature of nuclear hormone receptor was found from amino acids 107–133. A possible outside to inside transmembrane helice was found from amino acids 72–90. Northern blots of the larvae revealed five bands of HHR3 named as band 0, 1, 2, 3 and 4 with molecular masses determined as 2.1, 2.6, 3.6, 4.5 and 5.5 kb, respectively. The expression patterns of HHR3 in vivo were variable with developmental stages and tissues. Results showed that band 1–4 of HHR3 was only briefly expressed during molting, which suggested these bands are involved in the regulation of molting cascade, whereas band 0 was expressed in both molting and feeding larvae. Band 1 and 2 of HHR3 could be induced from epidermis of newly molted 6th instar larvae by non‐steroidal ecdysone agonist, RH‐2485.

Characterization of a 2-Cys peroxiredoxin IV in Marsupenaeus japonicus (kuruma shrimp) and its role in the anti-viral immunity

Accumulating evidence suggests that peroxiredoxins (Prx) are key molecules in the pathogenesis of various infectious diseases and are potential therapeutic targets for major diseases such as cancers. In this study, we report a peroxiredoxin IV (Prx IV) in Marsupenaeus japonicus, designated as MjPrx IV, which exhibited peroxidase activity and participated in the anti-white spot syndrome virus (WSSV) immune response. MjPrx IV is a 245-amino acid polypeptide with a predicted 19-amino acid signal peptide, an Ahpc-TSA domain, and a 1-Cys PrxC domain. Phylogenetic analysis revealed that the protein belongs to the Prx IV subfamily. MjPrx IV transcripts were detected in the gills, hepatopancreas, heart, stomach, ovaries, spermary, and intestine tissues, and are upregulated in the gonads, gills and hemocytes of shrimp after WSSV challenge. The mature MjPrx IV peptide was recombinantly expressed in an Escherichia coli system. The protein exhibited peroxidase activity. Furthermore, dsRNA suppression of MjPrx IV increased WSSV replication in shrimp, whereas rMjPrx IV injection into shrimp decreased WSSV replication. These data suggest that MjPrx IV has an important role in shrimp antiviral immunity. To our knowledge, this study is the first to report a shrimp Prx IV that has anti-WSSV activity.

Role of evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) in the antibacterial immunity of Marsupenaeus japonicus

The Toll/Toll-like receptor (TLR) signaling pathway has an important role in the innate immunity of animals. Evolutionarily conserved signaling intermediate in Toll pathways (ECSIT) is a protein that functions as an adaptor protein for the Toll/TLR and bone morphogenetic protein signaling pathways. ECSIT is also a key component in the macrophage bactericidal activity of mammals. However, the function of ECSIT in crustaceans remains unclear. In this study, we cloned and identified a functional ECSIT homologue, MjECSIT 1, from kuruma shrimp Marsupenaeus japonicus. The complementary DNA of MjEcsit 1 is 1442 base pairs long, with an open reading frame of 1221 base pairs that encodes a 407-residue polypeptide. Transcripts of MjEcsit 1 are detected in hemocytes, gills, hepatopancreas, stomach, heart, intestines, testes, and ovaries. Such transcripts are upregulated by Gram-positive bacteria (Staphylococcus aureus) and Gram-negative bacteria (Vibrio anguillarum) injections. The knockdown of MjEcsit 1 by double-stranded RNA injection increases the sensitivity of M. japonicus to S. aureus challenge and weakens the bacterial clearance ability of M. japonicus in vivo. In addition, suppressing MjEcsit 1 restrains the upregulation of two anti-lipopolysaccharide factors by S. aureus injection. The results indicate that MjECSIT 1 is important in the antibacterial immunity of M. japonicus.

Death associated protein 1 (DAP 1) positively regulates virus replication and apoptosis of hemocytes in shrimp Marsupenaeus japonicus

Abstract Death‐associated protein 1 (DAP1) is a small proline‐rich cytoplasmic protein that functions both in the apoptosis and autophage process of mammalian and in the clinical cancer of human. However, little knowledge is known about the homologue gene of DAP1 and its roles in the physiological process of invertebrates. In this paper, we report a novel function of DAP1 in the antivirus immunity of shrimp. A homologue gene of DAP1 was cloned from Marsupenaeus japonicus and named as Mjdap‐1. The full‐length of Mjdap‐1 was 1761 bp with a 309 bp open reading frame that encoded 102 amino acids. Reverse transcription‐PCR results showed that Mjdap‐1 was expressed in all tested tissues, including hemocytes, gills, intestines, stomach, heart, hepatopancreas, testes, and ovaries. In shrimp, Mjdap‐1 transcripts were up‐regulated by white spot syndrome virus (WSSV) infection; Mjdap‐1 knockdown decreased the virus copy in vivo and the mortality of M. japonicus to WSSV challenge. Conversely, injecting the purified recombinant MjDAP1 protein promoted the amplification of virus in shrimp. Flow cytometric assay showed, the virus infection‐induced apoptosis of hemocytes was enhanced by MjDAP1 protein injection and inhibited in MjDAP1 knockdown shrimp. Furthermore, the expression of apoptosis‐inducing factor (AIF) was regulated by Mjdap‐1, but the caspase transcripts were not affected. Our results suggested that MjDAP1 facilitated the amplification of virus in shrimp, which may be attributed to the promotion of hemocyte apoptosis in an AIF‐dependent manner. These results provided a new insight into the function of this protein that may be used for virus disease control. HighlightsDAP1 (Mjdap‐1) is identified in kuruma shrimp Marsupenaeus japonicus.Mjdap‐1 is upregulated in several tissues after white spot syndrome virus challenge.Mjdap‐1 facilitates the amplification of virus in shrimp.Mjdap‐1 promotes the apoptosis of hemocytes in shrimp by affecting the expression of apoptosis inducing factor (AIF)gene.