Lihua Qiu

Characterization of skin ulceration syndrome associated microRNAs in sea cucumber Apostichopus japonicus by deep sequencing.

MicroRNAs (miRNAs) constitute a family of small RNA species which have been demonstrated to be one of key effectors in mediating host-pathogen interaction. In this study, two haemocytes miRNA libraries were constructed with deep sequenced by illumina Hiseq2000 from healthy (L1) and skin ulceration syndrome Apostichopus japonicus (L2). The high throughput solexa sequencing resulted in 9,579,038 and 7,742,558 clean data from L1 and L2, respectively. Sequences analysis revealed that 40 conserved miRNAs were found in both libraries, in which let-7 and mir-125 were speculated to be clustered together and expressed accordingly. Eighty-six miRNA candidates were also identified by reference genome search and stem-loop structure prediction. Importantly, mir-31 and mir-2008 displayed significant differential expression between the two libraries according to FPKM model, which might be considered as promising targets for elucidating the intrinsic mechanism of skin ulceration syndrome outbreak in the species.

An immune responsive multidomain galectin from bay scallop Argopectens irradians

Galectins are a family of beta-galactoside-binding lectins which play crucial roles in innate immunity of vertebrates and invertebrates. In the present study, the cDNA of a galectin with multiple carbohydrate-recognition domains (CRDs) was cloned from bay scallop Argopectens irradians (designated AiGal1) by expressed sequence tag (EST) and rapid amplification of cDNA ends (RACE) techniques. The full-length cDNA of AiGal1 was of 2235 nucleotides, encoding a polypeptide of 549 amino acids. SMART program analysis revealed that AiGal1 contained four galectin CRDs, and all the CRDs contained the two consensus motifs essential for ligand-binding. Quantitative real-time PCR was employed to investigate the tissue distribution of AiGal1 mRNA and temporal expression in haemocytes of scallops challenged with Vibrio anguillarum, Micrococcus luteus and Pichia pastoris. The AiGal1 mRNA could be detected in all tested tissues with the highest expression level in hepatopancreas. After challenged by V. anguillarum and M. luteus, the expression level of AiGal1 mRNA was both up-regulated and reached the maximum level at 9 h (1.52 fold, P < 0.05) and 18 h (2.89 fold, P < 0.01) post challenge, respectively. However, there was no significant difference in the mRNA expression of AiGal1 in haemocytes after P. pastoris challenge (P > 0.05). These results collectively indicated that AiGal1 was a new member of the galectin family and involved in the immune responses against bacterial infection.

Characterization and function analysis of Hsp60 and Hsp10 under different acute stresses in black tiger shrimp, Penaeus monodon

Heat shock proteins (Hsps) are a class of highly conserved proteins produced in virtually all living organisms from bacteria to humans. Hsp60 and Hsp10, the most important mitochondrial chaperones, participate in environmental stress responses. In this study, the full-length complementary DNAs (cDNAs) of Hsp60 (PmHsp60) and Hsp10 (PmHsp10) were cloned from Penaeus monodon. Sequence analysis showed that PmHsp60 and PmHsp10 encoded polypeptides of 578 and 102 amino acids, respectively. The expression profiles of PmHsp60 and PmHsp10 were detected in the gills and hepatopancreas of the shrimps under pH challenge, osmotic stress, and heavy metal exposure, and results suggested that PmHsp60 and PmHsp10 were involved in the responses to these stimuli. ATPase and chaperone activity assay indicated that PmHsp60 could slow down protein denaturation and that Hsp60/Hsp10 may be combined to produce a chaperone complex with effective chaperone and ATPase activities. Overall, this study provides useful information to help further understand the functional mechanisms of the environmental stress responses of Hsp60 and Hsp10 in shrimp.

RNA-Seq analysis of immune-relevant genes in Lateolabrax japonicus during Vibrio anguillarum infection

Lateolabrax japonicus is one of the main marine aquatic fish species, and is popularly cultured in East Asia due to its highly commercial value. In recent years, because of large-scale and intensive farming and seawater pollution, fish diseases keep breaking out. However, systematic study on L. japonicus immunogenetics is limited due to the deficiency of deep sequencing technologies and genome backgrounds. In this study, the widely analysis at the transcriptome level for L. japonicus that infected with Vibrio anguillarum was performed. In total, 334,388,688 high quality reads were obtained in six libraries (HK-VA, HK-PBS, LI-VA, LI-PBS, SP-VA and SP-PBS) and de novo assembled into 101,860 Unigenes with an average unigene length of 879 bp. Based on sequence similarity 30,142 unigenes (29.59%) were annotated in the public databases. Comparative analysis revealed, 1,202, 3034 and 3519 differentially expressed genes (DEGs) were identified in three comparisons (HK-PBS VS HK-VA, LI-PBS VS LI-VA and SP-PBS VS SP-VA). Enrichment and pathway analysis of the DEGs was also carried out to excavate the candidate genes related to immunity. In conclusion, this study identifies and evaluates dozen of potential immune related pathways and candidate genes, which are indispensable for padding genomic resources of L. japonicus, and would lay the foundation for further studying and illuminating the mechanism of host-pathogen interactions.

Characterization and expression analysis of a chitinase gene (PmChi-4) from black tiger shrimp (Penaeus monodon) under pathogen infection and ambient ammonia nitrogen stress.

ABSTRACT Chitinase is a multi‐gene family, which play important physiological roles in crustaceans, involved in several biological processes, including digestion, molting and defense against viruses. In the present study, a chitinase‐4 gene (PmChi‐4) was cloned from Penaeus monodon by rapid amplification of cDNA ends (RACE). The full length of PmChi‐4 cDNA was 2178 bp, including an 1815 bp open reading frame (ORF) which encoded 604 amino acid residues. The predicted PmChi‐4 protein was 67.7 kDa and shared 61%–88% identity with the type of Chi‐4s from other crustaceans. Quantitative real‐time (qRT‐PCR) analysis indicated that PmChi‐4 was expressed ubiquitously with the high expression level in hepatopancreas. PmChi‐4 was expressed throughout the whole larvae stages, and the highest level of PmChi‐4 transcripts was detected at Mysis3 stage, which indicated that PmChi‐4 may be involved in larval metamorphosis. In order to know whether PmChi‐4 was related to the immune response of shrimp, Streptococcus agalactiae and Vibrio harveyi were chosen to challenge the shrimp, PmChi‐4 transcripts were significantly increased and reached to the maximum at 6 h in hepatopancreas and at 12 h in gill, respectively. The results suggested that PmChi‐4 participated in the immune defenses to pathogen infection. Besides, the ammonia nitrogen stress treatment was also carried out, PmChi‐4 transcripts were significantly decreased in hepatopancreas and gill and the result showed that PmChi‐4 may be involved in ammonia nitrogen stress in P. monodon. Overall, our present study lay a foundation for further research into the biological function and regulation of chitinase in P. monodon. HighlightsA chitinase‐4 gene was cloned from P. monodon by RACE method.The full length of PmChi‐4 cDNA was 2178 bp, including an 1815 bp ORF.qRT‐PCR was performed to study the PmChi‐4 expression pattern in different tissues and larvae stages.PmChi‐4 expression level increased after S. agalactiae and V. harveyi infection.PmChi‐4 expression level declined after ammonia nitrogen stress treatment.

Characterization of complement 1q binding protein of tiger shrimp, Penaeus monodon, and its C1q binding activity.

The receptor for the globular heads of C1q, C1qBP/gC1qR/p33, is a multicompartmental, multifunctional cellular protein with an important role in infection and in inflammation. In the present study, we identified and characterized the complement component 1q subcomponent binding protein (C1qBP) from the tiger shrimp Penaeus monodon (designated as PmC1qBP). The open reading frame of PmC1qBP encodes 262 amino acid residues with a conserved MAM33 domain, an arginine-glycine-aspartate cell adhesion motif, and a mitochondrial targeting sequence in the first 53 amino acids. PmC1qBP shares 32%-81% similarity with known C1qBPs and clusters with lobster gC1qR under phylogenetic analysis. The temporal PmC1qBP mRNA expression in the hepatopancreas was significantly enhanced at 9 h after Vibrio vulnificus challenge. The native PmC1qBP was expressed in the gills, hepatopancreas, ovaries, and intestines as a precursor (38 kDa) and the active peptide (35 kDa). The recombinant PmC1qBP protein was expressed in Escherichia coli BL21, and was purified using nickel-nitrilotriacetic acid agarose. A complement 1q binding assay indicated that the rC1qBP protein competitively binds to C1q in mouse serum. The data reveal that PmC1qBP is not only involved in shrimp immune responses to pathogenic infections, but also cross-binding to the mouse C1q.

The immune response of the C-Jun in the black tiger shrimp (Penaeus monodon) after bacterial infection

Abstract The transcription factor C‐Jun widely exists in vertebrates and invertebrates and plays an important role in various kinds of stimulus response. In this study, PmC‐jun gene was first cloned from Penaeus monodon. The full‐length cDNA of PmC‐jun was 1857 bp in length and included an 879 bp open reading frame (ORF), which encoded 293 amino acids. qRT‐PCR analysis results showed that PmC‐jun mRNAs were ubiquitously expressed in all the examined tissues. The highest expression level was observed in gill, followed by hepatopancreas. The expression patterns of PmC‐jun after Vibrio harveyi and Streptococcus agalactiae injections were studied by qRT‐PCR experiment. PmC‐jun increased obviously in the gill and hepatopancreas. The expression pattern of PmC‐jun in the hepatopancreas was further studied using in situ hybridization (ISH) method. The mRNA expression level of PmC‐jun significantly increased in the hepatopancreas after bacterial infection. The expression sites of PmC‐jun were almost unchanged. PmC‐jun played a regulatory role in pathogen invasion. HighlightsC‐Jun gene was identified and characterised in Penaeus monodon.qRT‐PCR indicated that PmC‐Jun mRNAs were widely expressed and the peak level was observed in the gills and hepatopancreas.PmC‐Jun was up‐regulation after Vibro harveyi and Streptococus agalactiae infection.PmC‐Jun plays an important role in Penaeus monodon immune response.

TLR3 gene in Japanese sea perch (Lateolabrax japonicus): Molecular cloning, characterization and expression analysis after bacterial infection

&NA; Toll‐like receptors (TLRs) play important roles in fish innate immune and are involved in the defense process of bacteria invasion. In the present study, the full‐length cDNA of TLR3 from the sea perch, Lateolabrax japonicus, was cloned and characterized. The full length of LjTLR3 cDNA was 3265 bp including an open reading frame of 2679 bp encoding a peptide of 922 amino acids. Tissues distribution analysis indicated that LjTLR3 showed a tissue‐specific variation with high expression in spleen, head‐kidney and liver. In order to investigate LjTLR3 functions against bacteria infection, the expression patterns of LjTLR3 after Vibrio harveyi and Streptococcus agalactiae challenge were detected by qRT‐PCR, and the results showed that LjTLR3 was significant up‐regulated after both bacteria stimulation in head‐kidney, spleen and liver in a time‐depended manner. Furthermore, the results by in situ hybridization experiments showed that positive signals of LjTLR3 mRNA in infected spleen and head‐kidney were more numerous than that in the control group. In addition, intracellular localization revealed that LjTLR3 is distributed in the cytoplasm. In summary, these findings suggest that LjTLR3 was involved in the immune process under bacteria infection. This study would benefit to further clarify the roles of fish TLRs in the immune process and contribute to further study on enhancing disease resistance of L. japonicus. HighlightsTLR3 was cloned and characterized from the sea perch, Lateolabrax japonicus.LjTLR3 protein is located in the cytoplasm.TLR3 was highly expressed in the spleen, head‐kidney and liver.TLR3 was up‐regulated after V. harveyi and S. agalactiae infection by qRT‐PCR.The both bacterial infection enhanced the expression of TLR3 by in situ hybridization.

Genomic structure, expression pattern, and functional characterization of transcription factor E2F-2 from black tiger shrimp (Penaeus monodon)

Transcription factor E2F-2 is a regulator of cell cycle. Researchers identified E2F-2 genes from yeasts to humans, but few reports investigated E2F-2 gene from black tiger shrimp. In the present study, we cloned E2F-2 gene from black tiger shrimp (Penaeus monodon). Full-length PmE2F-2 complementary DNA sequence measures 3,189 bp with an open reading frame of 1,371 bp. Complete PmE2F-2 genomic sequence (17,305 bp) of P. monodon contains nine exons, which are separated by eight introns. Quantitative real-time polymerase chain reaction (qRT-PCR) analysis indicated that PmE2F-2 is highly expressed in hepatopancreas and ovaries of P. monodon. Highest PmE2F-2 expression levels were observed in stage III ovarian development of P. monodon. PmE2F-2 expression levels were significantly augmented in ovaries of P. monodon after 5-hydroxytryptamine injection and eyestalk ablation. RNA interference experiments were conducted to examine PmE2F-2, PmCDK2, and PmCyclin E expression profiles. PmE2F-2 was successfully knocked down in ovaries and hepatopancreas via double-stranded RNA (dsRNA)–E2F-2 injection. In the same organs, PmE2F-2 expression localization and level were investigated through in situ hybridization, which revealed consistent results with those of qRT-PCR. After dsRNA—E2F-2 injection, gonadosomatic index of shrimp was significantly lower than those following dsRNA—GFP and phosphate-buffered solution injections. Therefore, PmE2F-2 may be involved in ovarian maturation in P. monodon.

Molecular cloning, expression and functional analysis of three subunits of protein phosphatase 2A (PP2A) from black tiger shrimps (Penaeus monodon)

Protein phosphatase 2A (PP2A) is a cellular serine-threonine (Ser/Thr) phosphatase that plays a crucial role in regulating most cellular functions. In the present study, the full-length cDNAs of three subunits of PmPP2A (PmPP2A-A, PP2A-B and PP2A-C) were cloned from Penaeus monodon, which are the first available for shrimps. Sequence analysis showed that PmPP2A-A, PmPP2A-B and PmPP2A-C encoded polypeptides of 591, 443, and 324 amino acids, respectively. The mRNAs of three subunits of PmPP2A were expressed constitutively in all tissues examined, and predominantly in the ovaries. In ovarian maturation stages, the three subunits of PmPP2A were continuously but differentially expressed. Dopamine and 5-hydroxytryptamine injection experiments were conducted to study the expression profile of three subunits of PmPP2A, and the results indicated that PmPP2A played a negative regulatory role in the process of ovarian maturation. In addition, the recombinant proteins of three subunits of PmPP2A were successfully obtained, and the phosphatase activity of PmPP2A was tested in vitro. The results of this study will advance our understanding about the molecular mechanisms of PmPP2A in Penaeus monodon.