Yichen Liu

Sequencing and De Novo Analysis of the Hemocytes Transcriptome in Litopenaeus vannamei Response to White Spot Syndrome Virus Infection

Background White spot syndrome virus (WSSV) is a causative pathogen found in most shrimp farming areas of the world and causes large economic losses to the shrimp aquaculture. The mechanism underlying the molecular pathogenesis of the highly virulent WSSV remains unknown. To better understand the virus-host interactions at the molecular level, the transcriptome profiles in hemocytes of unchallenged and WSSV-challenged shrimp (Litopenaeus vannamei) were compared using a short-read deep sequencing method (Illumina). Results RNA-seq analysis generated more than 25.81 million clean pair end (PE) reads, which were assembled into 52,073 unigenes (mean size = 520 bp). Based on sequence similarity searches, 23,568 (45.3%) genes were identified, among which 6,562 and 7,822 unigenes were assigned to gene ontology (GO) categories and clusters of orthologous groups (COG), respectively. Searches in the Kyoto Encyclopedia of Genes and Genomes Pathway database (KEGG) mapped 14,941 (63.4%) unigenes to 240 KEGG pathways. Among all the annotated unigenes, 1,179 were associated with immune-related genes. Digital gene expression (DGE) analysis revealed that the host transcriptome profile was slightly changed in the early infection (5 hours post injection) of the virus, while large transcriptional differences were identified in the late infection (48 hpi) of WSSV. The differentially expressed genes mainly involved in pattern recognition genes and some immune response factors. The results indicated that antiviral immune mechanisms were probably involved in the recognition of pathogen-associated molecular patterns. Conclusions This study provided a global survey of host gene activities against virus infection in a non-model organism, pacific white shrimp. Results can contribute to the in-depth study of candidate genes in white shrimp, and help to improve the current understanding of host-pathogen interactions.

Changes in the organics metabolism in the hepatopancreas induced by eyestalk ablation of the Chinese mitten crab Eriocheir sinensis determined via transcriptome and DGE analysis.

Background To understand the regulation mechanism of eyestalk ablation on the activities of hepatopancreas, Illumina RNA-Seq and digital gene expression (DGE) analyses were performed to investigate the transcriptome of the eyestalk, Y-organ, and hepatopancreas of E. sinensis and to identify the genes associated with the hepatopancreas metabolism that are differentially expressed under eyestalk ablation conditions. Results A total of 58,582 unigenes were constructed from 157,168 contigs with SOAPdenovo. A BlastX search against the NCBI Nr database identified 21,678 unigenes with an E-value higher than 10−5. Using the BLAST2Go and BlastAll software programs, 6,883 unigenes (11.75% of the total) were annotated to the Gene Ontology (GO) database, 7,386 (12.6%) unigenes were classified into 25 Clusters of Orthologous Groups of Proteins (COGs), 16,200 (27.7%) unigenes were assigned to 242 Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways, and1,846 unigenes were matched to “metabolism pathways”. The DGE analysis revealed that 1,416 unigenes were significantly differentially expressed in the hepatopancreas, of which 890 unigenes were up-regulated and 526 unigenes were down-regulated. Of the differentially expressed genes, 382 unigenes were annotated and 63 were classified into metabolism pathways. The results of the real-time polymerase chain reaction (PCR) analysis of four unigenes related to carbohydrate metabolism were consistent with those obtained from the DGE analysis, which demonstrates that the sequencing data were satisfactory for further gene expression analyses. Conclusion This paper reported the transcriptom of the eyestalk, Y-organ, and hepatopancreas from E. sinensis. DGE analysis provided the different expressed genes of the metabolism processes in hepatopancreas that are affected by eyestalk ablation. These findings will facilitate further investigations on the mechanisms of the metabolism of organic substances during development and reproduction in crustaceans.

The protein-protein interaction network of eyestalk, Y-organ and hepatopancreas in Chinese mitten crab Eriocheir sinensis

BackgroundThe protein-protein interaction network (PIN) is an effective information tool for understanding the complex biological processes inside the cell and solving many biological problems such as signaling pathway identification and prediction of protein functions. Eriocheir sinensis is a highly-commercial aquaculture species with an unclear proteome background which hinders the construction and development of PIN for E. sinensis. However, in recent years, the development of next-generation deep-sequencing techniques makes it possible to get high throughput data of E. sinensis tanscriptome and subsequently obtain a systematic overview of the protein-protein interaction system.ResultsIn this work we sequenced the transcriptional RNA of eyestalk, Y-organ and hepatopancreas in E. sinensis and generated a PIN of E. sinensis which included 3,223 proteins and 35,787 interactions. Each protein-protein interaction in the network was scored according to the homology and genetic relationship. The signaling sub-network, representing the signal transduction pathways in E. sinensis, was extracted from the global network, which depicted a global view of the signaling systems in E. sinensis. Seven basic signal transduction pathways were identified in E. sinensis. By investigating the evolution paths of the seven pathways, we found that these pathways got mature in different evolutionary stages. Moreover, the functions of unclassified proteins and unigenes in the PIN of E. sinensis were predicted. Specifically, the functions of 549 unclassified proteins related to 864 unclassified unigenes were assigned, which respectively covered 76% and 73% of all the unclassified proteins and unigenes in the network.ConclusionsThe PIN generated in this work is the first large-scale PIN of aquatic crustacean, thereby providing a paradigmatic blueprint of the aquatic crustacean interactome. Signaling sub-network extracted from the global PIN depicts the interaction of different signaling proteins and the evolutionary paths of the identified signal transduction pathways. Furthermore, the function assignment of unclassified proteins based on the PIN offers a new reference in protein function exploration. More importantly, the construction of the E. sinensis PIN provides necessary experience for the exploration of PINs in other aquatic crustacean species.

Cloning and molecular structure analysis of crustacean hyperglycemic hormone(Ers-CHH)in Eriocheir sinensis

Crustacean hyperglycemic hormone(CHH)is a peptide hormone originally identified in a crustacean neurosecretory complex,the X-organ/sinus gland complex,and located within the eyestalks.CHH is placed in the crustacean hyperglycemic hormone family,which also includes molt-inhibiting hormone(MIH),vitellogenesis-inhibiting hormone(VIH)or gonad-inhibiting hormone(GIH),mandibular organ-inhibiting hormone(MOIH)and insect ion transport peptide(ITP).CHH plays a significant role in the growth,molting and reproduction of crustaceans.By using degenerate primers which were designed according to the N-terminal amino acid sequences of CHH isolated by our lab and the sequences of homologous species CHH,a novel cDNA of crustacean hyperglycemic hormone gene(Ers-CHH,GenBank Accession No:JX485664)was cloned from Chinese mitten crab(Eriocheir sinensis).The full length cDNA consists of 585 bp with a 453 bp open reading frame,encoding 150 amino acids,containing a 30 amino acids signal peptide,and a 42 amino acids CHH precursor-related peptide,a 78 amino acids mature peptide.The mature peptide contains 6 conserved cysteines which formed three disulfide bonds C7-C43、C23-C39、C26-C52.There was an arthropod CHH/MIH/GIH neurohormones family signature in this domain.The three-dimensional structure prediction showed that Ers-CHH mature peptide consisted of 4 α-helixes,and not with the β-sheet structure.5 Prosite patterns,which were analyzed by a Swiss-PdbViewer(v4.0.4),are PS00005:Protein kinase C phosphorylation site(Ser6,Lys8);PS00006:Casein kinase Ⅱ phosphorylation site(Ser17,Glu20);PS00008:N-myristoylation site(Gly38,Cys39,Asn42,Cys43);PS00342:Microbodies C-terminal targeting signal(Ser17,Lys18,Leu19);PS01250:Arthropod CHH/MIH/GIH neurohormones family(Val35,Cys39,Arg40,Asn42,Cys43,Tyr44,Asn46,Phe49,Cys52).The results of the structural model comparison show that the structure of Ers-CHH has high similarity with other CHHs and Ers-CHH.They have no β-sheet structure,except that CHH has 4α-helixes,MIH has 5α-helixes.Although α1 helix of MIH could not be found in CHHs,the position and the amino acid number of α2-α5 helixes in MIH molecular were same as the α1-α4 helixes in CHH.It might be able to interpret the function similarity between CHH and MIH.Multiple alignment results showed that Ers-CHH has the highest identity with Ptychognathus pusillus CHH(92%),and it also shared high identities with Neohelice granulata(86%)and Pachygrapsus marmoratus(72%).However,it showed lower identity with CHH from crayfishes,such as Nephrops norvegicus(49%)and Homarus gammarus(46%).Multiple alignments of Ers-CHH and MIH from other crustaceans showed that only six amino acid residues(Arg13,Asp25,Asn28,Arg31,Arg40,Phe49)in Ers-CHH sequence were same with MIH sequences.It may be helpful for further research on regulation mechanism of CHH in the process of crab growth and carbohydrate metabolism.

Recombinant expression and functional characterization of a Serpin-type serine proteinase inhibitor(Fc-serpin) from the Chinese shrimp(Fenneropenaeus chinensis)

Serine proteinase inhibitors( SPI) play an important role in regulating humoral immunity by controlling the activity of serine proteinase( SP) in the process of protease cascade. Serpin family members participate in numerous biological processes by regulating the activity of serine and cysteine proteinases,such as blood clotting,complement activation,fibrinolysis function,inflammation,tumor suppressor and hormone transfer process. In our previous research,w e cloned a serpin-type serine proteinase inhibitior gene from hemocytes of Chinese shrimp( Fc-serpin,GenBank accession number: DQ318857). The transcript of Fcserpin w as induced in response to the infection of bacteria and w hite spot syndrome virus( WSSV). In this research,recombinant Fc-serpin( rFc-serpin) w as successfully expressed in bacteria E. coli and purified for further research of bioactivity. The concentration of purified target proteins w as 0. 3 g / L. rFc-serpin show ed in vitro antimicrobial activity against main pathogens including Gram-positive,Gram-negative bacteria and fungi. The results suggest that rFc-serpin might play a crucial role in the innate immunity of the shrimp and is expected to be applied in disease control.

Investigation on Electroporation: Mediated Transformation of Chlorella ellipsoidea

Chlorella ellipsoidea is a kind of micro algae biological reactor. It is a suitable eukaryotic expression system for foreign proteins, and the bioactivity of the expressed protein is similar to the natural counterpart. However, introduction of foreign gene into C. ellipsoidea cells is the precondition for its efficient expression. In this paper the electroporation conditions to transform C. ellipsoidea including cultured medium, growth rate of C. ellipsoidea, voltage strength were systematically investigated. BG11 without Mg2+ was selected as the medium for cultivating C. ellipsoidea. The exponential phase cells were harvested and electroporated under different electric voltages. Voltage at the 630 V was identified to be optimal by determining the cells viability after electroporation. Under this condition, the plasmid pSC2 carrying gfp was successfully transformed into C. ellipsoidea. The gfp gene could continuously express for at least 7 days. (This work was financially supported by grants from the National Basic Research Development Program of China (973 programs, 2012CB114405), National High-Tech Research and Development Program of China (863 programs, 2012AA092205 and 2012AA10A401), National Key Technology Research and Development Program (2011BAD13B07 and 2011BAD13B04), Doctoral found of Tianjin normal university: 52X09010 and Open research found for city stage key laboratory of Tianjin normal university).

Recombinant expression and functional characterization of a C-type lectin(Fclectin)from the Chinese shrimp(Fenneropenaeus chinensis)

Chinese shrimp(Fenneropenaeus chinensis)is distributed mainly along Chinese inshore areas,and is one of the most important farmed shrimp in China.The studies on innate immune responses of shrimps,especially on immune defense against the main crustacean pathogens,will provide more knowledge of shrimp immunity to prevent infectious diseases.Invertebrates do not possess an adaptive immune system based on highly specific antibodies and antigen receptors.They must rely on efficient immune defenses capable of protecting them against invading microorganisms.The chief issue of crustacean immunity should concern non-self-recognition mechanisms.Proteins that specifically bind to certain carbohydrate components on the surface of microorganisms play an important role in non-self-recognition and cleaning up of the invading microorganisms.Such proteins are known as pattern recognition receptors(PRRs).Lectins exist in almost all living organisms.Due to their ability of binding to terminal sugars on glycoproteins and glycolipids,lectins are primary candidates for pattern recognition receptors in innate immunity.C type Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean.In the preliminary study,a novel C-type lectin was cloned from hemocytes of Chinese shrimp,(Fenneropenaeus chinensis).It contains two tandem carbohydrate recognition domains(CRDs)/C-type lectin-like domains.Both of the CRDs contain a QPD(Gln-Pro-Asp)motif that has a predicted binding specificity for galactose-type sugar.In this research,two recombinant target proteins(rFclectin-CRD1 and rFclectin-CRD2)were expressed by prokaryotic expression system.The result showed that fusion protein was expressed in the form of inclusion bodies.The LC-ESI-MS analysis showed that two peptide fragments of rFclectin-CRD1 and rFclectin-CRD2 were identical with the corresponding sequence of F.chinensis C-type lectin.Recombinant protein was purified by immobilized-metal affinity chromatography and Ni-NTA technology.The concentrations of purified target proteins were 0.4 g/L.rFclectin-CRD1 and rFclectin-CRD2 had agglutinating and antimicrobial activity against main pathogens in aquaculture in a calcium-dependent manner.The agglutinating activity can be inhibited by multiple carbohydrates,such as galactose,peptidoglycan and lipopolysaccharide.These results suggest that Fclectin,as a Ca2+dependent carbohydrate-recognition protein,is one of the important PRRs.It might play a crucial role in the innate immunity of the shrimp and is expected to be applied in disease control.