Complete genome sequence of shrimp hemocyte iridescent virus (SHIV) isolated from white leg shrimp, Litopenaeus vannamei.

Infection with shrimp hemocyte iridescent virus (SHIV), a new virus of the family Iridoviridae isolated in China, results in a high mortality rate in white leg shrimp (Litopenaeus vannamei). The complete genome sequence of SHIV was determined and analyzed in this study. The genomic DNA was 165,809 bp long with 34.6% G+C content and 170 open reading frames (ORFs). Dotplot analysis showed that the longest repetitive region was 320 bp in length, including 11 repetitions of an 18-bp sequence and 3.1 repetitions of a 39-bp sequence. Two phylogenetic trees were constructed based on 27 or 16 concatenated sequences of proteins encoded by genes that are conserved between SHIV homologous and other iridescent viruses. The results of this study, suggest that SHIV should be considered a member of the proposed new genus "Xiairidovirus".

Horizontally transferred genes in the genome of Pacific white shrimp, Litopenaeus vannamei.

BACKGROUND: In recent years, as the development of next-generation sequencing technology, a growing number of genes have been reported as being horizontally transferred from prokaryotes to eukaryotes, most of them involving arthropods. As a member of the phylum Arthropoda, the Pacific white shrimp Litopenaeus vannamei has to adapt to the complex water environments with various symbiotic or parasitic microorganisms, which provide a platform for horizontal gene transfer (HGT). RESULTS: In this study, we analyzed the genome-wide HGT events in L. vannamei. Through homology search and phylogenetic analysis, followed by experimental PCR confirmation, 14 genes with HGT event were identified: 12 of them were transferred from bacteria and two from fungi. Structure analysis of these genes showed that the introns of the two fungi-originated genes were substituted by shrimp DNA fragment, two genes transferred from bacteria had shrimp specific introns inserted in them. Furthermore, around other three bacteria-originated genes, there were three large DNA segments inserted into the shrimp genome. One segment was a transposon that fully transferred, and the other two segments contained only coding regions of bacteria. Functional prediction of these 14 genes showed that 6 of them might be related to energy metabolism, and 4 others related to defense of the organism. CONCLUSIONS: HGT events from bacteria or fungi were happened in the genome of L. vannamei, and these horizontally transferred genes can be transcribed in shrimp. This is the first time to report the existence of horizontally transferred genes in shrimp. Importantly, most of these genes are exposed to a negative selection pressure and appeared to be functional.

Selection of Vibrio harveyi-resistant Litopenaeus vannamei via a three-round challenge selection with a pathogenic strain of V. harveyi.

To obtain Vibrio harveyi-resistant Litopenaeus vannamei shrimp used for study on immune response of shrimp avoid vibriosis, a three-round challenge selection procedure was applied. In this procedure, resistant shrimp were selected gradually via three rounds challenge experiment with a pathogenic strain of V. harveyi at a median and controllable lethal dose of 96-h LD50 (the median lethal dose). After this procedure, the cumulative mortality of selected shrimp during 96 h after injection of V. harveyi at 2.0 × 10(6) cfu shrimp(-1) significantly decreased from 93.3% to 26.7%, the hours of beginning of death and the hours of attaining of the maximum cumulative mortality of shrimp prolonged from 4 h and 10 h to 8 h and 24 h, respectively. The LD50 of 6 h, 12 h, 24 h, 48 h and 96 h of selected shrimp significantly increased to 1.4 ± 0.1 × 10(7) (p < 0.01), 5.5 ± 0.4 × 10(6) (p < 0.01), 3.1 ± 0.2 × 10(6) (p < 0.01), 2.7 ± 0.1 × 10(6) (p < 0.01) and 2.7 ± 0.1 × 10(6) cfu shrimp(-1) (p < 0.01), about 15.9, 15.3, 9.4, 10.0 and 10.4 times of that of normal shrimp, respectively. In conclusion, the resistance of shrimp to Vibrio significantly increased after the three-round challenge selection procedure.

Blocking the large extracellular loop (LEL) domain of FcTetraspanin-3 could inhibit the infection of white spot syndrome virus (WSSV) in Chinese shrimp, Fenneropenaeus chinensis.

Tetraspanins belong to the transmembrane 4 superfamily (TM(4)SF), which span the cell membrane 4 times and act as bridges or connectors. Increasing evidences have shown that tetraspanins play important role in virus infection. The large extracellular loop (LEL) of a tetraspanin is considered as a possible target of some virus. Tetraspanins are widely found in invertebrates, but the functional roles of most invertebrate tetraspanins have remained unknown. Recently, a tetraspanin, called FcTetraspanin-3, was cloned from the cDNA library of Chinese shrimp, Fenneropenaeus chinensis. The FcTetraspanin-3 constitutive expression in all examined tissues and the expression of the gene were highly induced in hepatopancreas, lymphoid organ and intestine by white spot syndrome virus (WSSV) challenge. In this study, we expressed and purified the recombinant peptide containing the LEL domain of FcTetraspanin-3, and produced the anti-LEL polyclone antibody. The expression of FcTetraspanin-3 was observed by real-time PCR and Western blot. Also, the localization of FcTetraspanin-3-positive cells in intestine and hepatopancreas were revealed by immunofluorescence. The results of anti-LEL antibody blocking experiments shown that the antibody can significantly reduce the mortality of shrimp challenged by WSSV. Additionally, dsRNA interference was utilized to examine the functional role of FcTetraspanin-3 in response to WSSV infection, and a sensible decrease of the viral copy number in the tetraspanin knockdown shrimp. These results suggested the blocking of LEL domain of FcTetraspanin-3 could inhibit the infection of WSSV. FcTetraspanin-3 might play an important role in response to WSSV infection, and the LEL domain of FcTetraspanin-3 might mediate the entry of WSSV.

Molecular cloning and expression of NOS in shrimp, Litopenaeus vannamei.

The importance of the nitric oxide synthase (NOS) gene family is demonstrated by many studies in recent years. However, the lack of sequence information and clones of shrimp NOS cDNA limits further study on its characterization and function in this species. In this report, the cDNA of NOS contained full-length ORF was cloned from the Pacific white shrimp, Litopenaeus vannamei. It was of 4680 bp, including a 5'-terminal untranslated region (UTR) of 278 bp, a 3'-terminal UTR of 862 bp, which contained 5 ATTTA repeats, and an open reading frame (ORF) of 3540 bp encoding a polypeptide of 1179 amino acids. It contained a typical NO synthase domain at the N-terminal, next to a flavodoxin 1 domain, a flavin adenine dinucleotide (FAD) binding domain, respectively, and a conservative nicotinamide adenine dinucleotide (NAD) binding domain structure at the C-terminal. Quantitative real-time reverse transcription PCR analysis revealed L. vannamei NOS (LvNOS) to be expressed in most shrimp tissues, with highest expression in the hepatopancreas and weakest expression in skin. The expression of LvNOS after challenge with LPS and poly I:C was tested in hemocytes, hepatopancreas and nerve. The results indicated that the NOS transcript level could be induced in hemocytes by injection with LPS. The highest expression was in the hemocyte, with 8.8 times (at 3 h) as much as that in the control (p < 0.05). However, sharp down-regulation of NOS was found in hepatopancreas and nerve after LPS and poly I:C injection (p < 0.05). These results suggested that NOS might play an important role in shrimp's defense against pathogenic infection.

Arginine kinase from Litopenaeus vannamei: cloning, expression and catalytic properties.

Arginine kinase (AK) is a phosphotransferase that plays a critical role in energy metabolism in invertebrates. In this paper, the full-length cDNA of AK was cloned from shrimp, Litopenaeus vannamei by using RT-PCR and RACE PCR. It was 1446 bp encoding 356 amino acids, and belongs to the conserved phosphagen kinase family. The quantitative real-time reverse transcription PCR analysis revealed a broad expression of AK with the highest expression in the muscle and the lowest in the skin. The expression of AK after challenge with LPS was tested in hemocytes and muscle, which indicated that the two peak values were 6.2 times (at 3 h) and 10.14 times (at 24 h) in the hemocytes compared with the control values, respectively (P < 0.05), while the highest expression of AK was 41 times (at 24 h) in the muscle compared with the control (P < 0.05). In addition, AK was expressed in Escherichia coli by prokaryotic expression plasmid pGEX-4T-2. The recombinant protein was expressed as glutathione s-transferase (GST) arginine kinase (GST-AK) fusion protein, which was purified by affinity chromatography using Glutathione Sepharose 4B. After cleavage from GST by using a site-specific protease, the recombinant protein was identified by ESI-MS and showed AK activity. After treatment with 10 mM ATP, the enzyme activity significantly increased. However, the enzyme activity was inhibited by 10 mM alpha-ketoglutarate, 50 mM glucose and 200 mM ATP. This research suggested that AK might play an important role in the coupling of energy production and utilization and the immune response in shrimps.

[Chromosomal localization and development of SNP markers of a serine protease gene in Farrer's scallop (Chlamys farreri)].

Disease control is a main objective in the culture of Farrer's scallop (Chalmys farreri). Many investigations have focused on the innate immune system of this species. Serine proteases amplify signals in many immune-related pathways and thus play a crucial role in innate immune system. Recently, studies on serine protease in C. farreri were mainly on sequence analysis and expression profile, but no study on gene localization was reported. In this study, a BAC clone (CFB040H03) that contains a serine protease gene of C. farreri was located on the long arms of a pair of homologous chromosomes by BAC-FISH technique. Six SNPs were found inside the serine protease gene using direct sequencing of the PCR products. The chromosomal localization of serine protease gene would provide fundamental support for further research on this gene. The SNPs obtained can be mapped to the genetic linkage map and is helpful to the construction of the integration map of C. farreri.

Molecular cloning, characterization and expression analysis of a putative C-type lectin (Fclectin) gene in Chinese shrimp Fenneropenaeus chinensis.

Lectin is regarded as a potential molecule involved in immune recognition and phagocytosis through opsonization in crustacean. Knowledge on lectin at molecular level would help us to understand its regulation mechanism in crustacean immune system. A novel C-type lectin gene (Fclectin) was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 1482 bp with an 861 bp open reading frame, encoding 287 amino acids. The deduced amino acid sequence contains a putative signal peptide of 19 amino acids. It also contains two carbohydrate recognition domains/C-type lectin-like domains (CRD1 and CRD2), which share 78% identity with each other. CRD1 and CRD2 showed 34% and 30% identity with that of mannose-binding lectin from Japanese lamprey (Lethenteron japonicum), respectively. Both CRD1 and CRD2 of Fclectin have 11 amino acids residues, which are relatively invariant in animals' C-type lectin CRDs. Five residues at Ca2+ binding site 1 are conserved in Fclectin. The potential Ca2+/carbohydrate-binding (site 2) motif QPD, E, NP (Gln-Pro-Asp, Glu, Asn-Pro) presented in the two CRDs of Fclectin may support its ability to bind galactose-type sugars. It could be deduced that Fclectin is a member of C-type lectin superfamily. Transcripts of Fclectin were found only in hemocytes by Northern blotting and RNA in situ hybridization. The variation of mRNA transcription level in hemocytes during artificial infection with bacteria and white spot syndrome virus (WSSV) was quantitated by capillary electrophoresis after RT-PCR. An exploration of mRNA expression variation after LPS stimulation was carried out in primarily cultured hemocytes in vitro. Expression profiles of Fclectin gene were greatly modified after bacteria, LPS or WSSV challenge. The above-stated data can provide us clues to understand the probable role of C-type lectin in innate immunity of shrimp and would be helpful to shrimp disease control.

Identification of a novel DNA methyltransferase 2 from the brine shrimp, Artemia franciscana.

DNA methyltransferase 2 (Dnmt2) is a dual-specificity DNA methyltransferase, which contains a weak DNA methyltransferase and novel tRNA methyltransferase activity. However, its biological function is still enigmatic. To elucidate the expression profiles of Dnmt2 in Artemia franciscana, we isolated the gene encoding a Dnmt2 from A. franciscana and named it as AfDnmt2. The cDNA of AfDnmt2 contained a 1140-bp open reading frame that encoded a putative Dnmt2 protein of 379 amino acids exhibiting 32% approximately 39% identities with other known Dnmt2 homologs. This is the first report of a DNA methyltransferase gene in Crustacean. By using semi-quantitative RT-PCR, AfDnmt2 was found to be expressed through all developmental stages and its expression increased during resumption of diapause cysts development. Southern blot analysis indicated the presence of multiple copies of AfDnmt2 genes in A. franciscana.

Identification and molecular characterization of a peritrophin-like protein from fleshy prawn (Fenneropenaeus chinensis).

Peritrophin, one of the components of the peritrophic matrix, was first isolated from the intestine of insects. It is thought to protect insects from invasion of microorganisms and to stimulate digestion of food. Peritrophin-like proteins have also been found in crustaceans, as a component of the egg layer. In this study, one fragment of the peritrophin-like gene was obtained from fleshy prawn (Chinese shrimp) (Fenneropenaeus chinensis) by panning the T7 phage display library constructed with the shrimp hemocyte cDNA. The total sequence of the peritrophin cDNA was cloned by modified SMART cDNA and LD-PCR methods. The full cDNA is 1048bp and the deduced protein is composed of 274 amino acids, including 21 amino acid signal peptide, and four peritrophin A domains and the latter three forming three chitin-binding domains. Similarity analysis results showed that the peritrophin-like protein from F. chinensis has significant similarities with peritrophin-like and cortical rod proteins from other shrimp. It was inducing expression in hemocytes, heart, stomach, gut, and gills of the infected shrimp, and constitutive expression in the ovaries. No expression signal was detected in the hepatopancreas of either infected or noninfected shrimp. The recombinant peritrophin-like protein has the activity of binding Gram-negative bacteria and strong binding activity to chitin. Therefore, the bacteria and chitin binding activities of the peritrophin-like protein suggest that it may plays a role in immune defense and other physiological resposes.

Characterization of a new member of Iridoviridae, Shrimp hemocyte iridescent virus (SHIV), found in white leg shrimp (Litopenaeus vannamei).

A newly discovered iridescent virus that causes severe disease and high mortality in farmed Litopenaeus vannamei in Zhejiang, China, has been verified and temporarily specified as shrimp hemocyte iridescent virus (SHIV). Histopathological examination revealed basophilic inclusions and pyknosis in hematopoietic tissue and hemocytes in gills, hepatopancreas, periopods and muscle. Using viral metagenomics sequencing, we obtained partial sequences annotated as potential iridoviridae. Phylogenetic analyses using amino acid sequences of major capsid protein (MCP) and ATPase revealed that it is a new iridescent virus but does not belong to the five known genera of Iridoviridae. Transmission electron microscopy showed that the virus exhibited a typical icosahedral structure with a mean diameter of 158.6 ± 12.5 nm (n = 30)(v-v) and 143.6 ± 10.8 nm (n = 30)(f-f), and an 85.8 ± 6.0 nm (n = 30) nucleoid. Challenge tests of L. vannamei via intermuscular injection, per os and reverse gavage all exhibited 100% cumulative mortality rates. The in situ hybridization showed that hemopoietic tissue, gills, and hepatopancreatic sinus were the positively reacting tissues. Additionally, a specific nested PCR assay was developed. PCR results revealed that L. vannamei, Fenneropenaeus chinensis, and Macrobrachium rosenbergii were SHIV-positive, indicating a new threat existing in the shrimp farming industry in China.

Ciona intestinalis as an emerging model organism: its regeneration under controlled conditions and methodology for egg dechorionation.

The ascidian Ciona intestinalis is a model organism of developmental and evolutionary biology and may provide crucial clues concerning two fundamental matters, namely, how chordates originated from the putative deuterostome ancestor and how advanced chordates originated from the simplest chordates. In this paper, a whole-life-span culture of C. intestinalis was conducted. Fed with the diet combination of dry Spirulina, egg yolk, Dicrateria sp., edible yeast and weaning diet for shrimp, C. intestinalis grew up to average 59 mm and matured after 60 d cultivation. This culture process could be repeated using the artificially cultured mature ascidians as material. When the fertilized eggs were maintained under 10, 15, 20, 25 degrees C, they hatched within 30 h, 22 h, 16 h and 12 h 50 min respectively experiencing cleavage, blastulation, gastrulation, neurulation, tailbud stage and tadpole stage. The tadpole larvae were characterized as typical but simplified chordates because of their dorsal nerve cord, notochord and primordial brain. After 8 - 24 h freely swimming, the tadpole larvae settled on the substrates and metamorphosized within 1- 2 d into filter feeding sessile juvenile ascidians. In addition, unfertilized eggs were successfully dechorionated in filtered seawater containing 1% Tripsin, 0.25% EDTA at pH of 10.5 within 40 min. After fertilization, the dechorionated eggs developed well and hatched at normal hatching rate. In conclusion, this paper presented feasible methodology for rearing the tadpole larvae of C. intestinalis into sexual maturity under controlled conditions and detailed observations on the embryogenesis of the laboratory cultured ascidians, which will facilitate developmental and genetic research using this model system.

[The application of microsatellite markers for parentage determination in selective breeding of Pacific white shrimp (Litopenaeus vannamei)].

Used simulations and controlled mating to examine the potential of microsatellite markers in assigning parentage to Pacific white shrimp progeny. Cervus simulations demonstrated that the theoretical expectations for parentage exclusion of 10 microsatellite loci and six most polymorphic of the 10 loci were both 0.99, and the assignment success rate of the 6 most polymorphic microsatellite loci set was nearly to 0.97 with 95% confidence. Based on this information, offspring from 10 crosses where parents were known were genotyped by the 6 microsatellite loci and used for parentage analysis. The result showed that assignment success of the progeny to their 'true' mother and father was 88% and 78% respectively, which were lower than predicted by the Cervus simulations. This could be explained by the existence of null or mutant alleles and by Taq DNA Polymerase slippage in the microsatellite loci.

[A preliminary study on the inheritance of microsatellite in two selective breeding families of shrimp (Litopenaeus vannamei)].

Two full-sib families of Litopenaeus vannamei were used to study the inheritance of allelic variation at 10 microsatellite loci. Of the 20 genotypic ratios observed (10 loci x 2 crosses), 17 ratios conformed to Mendelian segregation. When null alleles were considered, one loci (TUMXLv8.220) confirmed Mendelian expectations in all families. While one loci (TUMXLv3.1) showed deviation from family 06. Three loci (TUMXLv5.66,TUMXLv7.74,TUMXLv8.224)were monomorphic in both controlled crosses; 3 loci were polymorphic and confirmed to Mendelian ratios in all families, can be used for parental analysis and population genetic studies. These results indicated the need to test the inheritance pattern for microsatellite markers in shrimp before using them for population genetic or kinship analysis.

[Cloning and expression of tumor necrosis factor (TNFalpha) cDNA from red seabream pagrus major].

A fragment of TNFalpha cDNA sequence from red seabream was cloned by homology cloning approach with two degenerated primers which were designed based on the conserved regions of other animals' TNF sequences. The sequence was elongated by 3' and 5' RACE to get the full length CDS sequence. This sequence contained 1264 nucleotides that included a 5' UTR of 85 bp, a 3' UTR of 514 bp and an open reading frame (ORF) of 666 bp which could encode 222 amino acids propeptide. In 3' UTR, there were several mRNA instability motifs and three endotoxin-responsive sequences, but the sequence lacked the polyadenylation signal. The deduced peptide had a clear transmembrane domain, a TNFalpha family signature and a TNF2 family profile. The cell attachment sequence and the glycosaminoglycan attachment sites were also found in the sequence. The red seabream TNF sequence shared relatively high similarity with both mammalian TNFalpha and TNFbeta by multiple sequence alignments. Phylogenetic analysis showed that the piscine TNFalpha were located independently in a different branch compared with mammalian TNFalpha and TNFbeta. Based on the primary and secondary structure analysis and gene expression study, we could concluded that the red seabream TNF should be a TNFalpha, not TNFbeta. RT-PCR was used to study TNFalpha transcript expression. 24 h after the red seabream was challenged by Vibrio anguillarum, the RS TNFalpha transcript expression were detected in blood, brain, gill, heart, head kidney, kidney, liver, muscle and spleen. Results showed that TNFalpha mRNA was constitutively expressed in parts of the tissues both in stimulated and unstimulated fish and the expression could be enhanced after the pathogen infection.

[Cloning and analysis of three genes encoding type II CHH family neuropeptides from Fennropenaeus chinensis].

On the basis of sequence similarity, the crustean hyperglycemic hormone (CHH) family peptides have been classified into two types of hormones: type I and type II. Molt-inhibiting hormone (MIH) is a neuropeptide member of type II CHH family. Molting in shrimp is controlled by MIH and ecdysone. By inhibiting the synthesis of ecdysone in the Y-organ, MIH indirectly suppresses the molting activity of shrimp. In this study, we reported the cloning and characterization of 3 gene fragments encoding type II CHH family neuropeptides of the shrimp Fennropenaeus chinensis. According to the complementary DNA sequence of the mult-inhibiting hormone of Fennropenaeus chinensis, 3 primers were designed and synthesized. MP1 and MP2 are sense primers, and MP3 is anti-sense primer. Polymerase chain reaction was performed using genomic DNA of Fennropenaeus chinensis as template. Three PCR products were obtained using primers MP1 and MP3. Their sizes are about 600 bp, 850 bp, 1050 bp, respectively. A 580 bp PCR product was obtained using primers MP2 and MP3. All the 4 PCR products were cloned into pMD18-T vector. The recombinant clones were sequenced using ABI 310 Genetic Analyzer. After sequencing, all the DNA sequences were searched in the GenBank by Blast program to find similar gene sequences. The searching results revealed 3 DNA fragment sequences were of high similarity with CHH family neuropeptide genes from various crustean species. The 3 DNA fragments were named as NP1, NP2, and NP3. Their sizes were 540 bp, 601 bp, and 826 bp, respectively. Using the mRNA sequences with the most similarity to the 3 sequence fragments as reference, the gene structure of the 3 DNA fragment sequences was analyzed. The exons of 3 sequence fragments were aligned with their similar sequences by Clustal W program. Both NP1 and NP2 consisted of 1 intron and 2 exons. NP3 consisted of 2 introns and 3 exons. Sequence analysis suggested that these 3 products belonged to sequence fragments of neuropeptide gene of type II crustacean hyperglycemic hormone family. The exons of NP1, NP2, and NP3 had highest similarity respectively with mRNA of Pem-SCP-C2 and Pem-SGP-C1 both from Penaeus monodon, and FenchMIH from Fennropenaeus chinensis. The identities were 91.5%, 92.8%, 88.9%, respectively. The results suggest NP3 is a fragment of molt-inhibiting hormone gene of Fennropenaeus chinensis NP1 and NP2 are two fragments of neuropeptide genes of type II crustacean hyperglycemic hormone family, which were found in Fennropenaeus chinensis for the first time.

[Molecular cloning and sequence analysis of full length cDNA encoding molt-inhibiting hormone from Fennropenaeus chinensis].

Molt-inhibiting hormone (MIH) is a neuropeptide member belonging to the eyestalk CHH family. Molting in shrimp is controlled by MIH and ecdysone. By inhibiting the synthesis of ecdysone in the Y-organ, MIH indirectly suppress the molting activity of shrimp. A 697 bp full-length encoding molt-inhibiting hormone precursor cDNA, which has been accepted by GenBank (accession number: AF469187), was firstly amplified from the total RNA of eyestalk from Fennropenaeus chinensis by the 3' and 5' rapid amplification of cDNA ends (RACE) method. The 697 bp full-length cDNA encoding MIH precursor was assembled with a 320 bp 3' RACE product and 468 bp 5' RACE product. Results derived from searching by Blast revealed the 697 bp cDNA had high similarity with MIH gene of crustacean. By using Clustal X program, alignment of the amino acid sequence deduced from the 697 bp cDNA with amino acid sequences of 7 MIHs revealed that the deduced amino acid sequence had very high identity with amino acid sequences of MIHs of shrimps. The identities between the deduced amino acid sequence with that of MIH of Marsupenaeus japonicus, Penaeus monodon and Metapenaeus ensis were respectively 95.1%, 83.1% and 79.1%. On the base of all the data, we concluded that the 697 bp full-length cDNA was the cDNA encoding MIH precursor of F. chinensis. Sequence analysis of the 697 bp cDNA revealed a 312 bp open reading frame, and 81 bp 5' untranslated region, and a 302 bp 3' untranslated region. The deduced 103 amino acid polypeptide consisted of a 28 amino acid region of signal peptide and a 75 amino acid region of mature peptide. The six cysteine residues were very conserved in the mature peptide.

Involvement of p90 ribosomal S6 kinase in termination of cell cycle arrest during development of Artemia-encysted embryos.

Artemia has evolved a unique developmental pattern of encysted embryos to cope with various environmental threats. Cell divisions totally cease during the preemergence developmental stage from gastrula to prenauplius. The molecular mechanism of this, however, remains unknown. Our study focuses on the involvement of p90 ribosomal S6 kinase (RSK), a family of serine/threonine kinase-mediating signal transduction downstream of mitogen-activated protein kinase cascades, in the termination of cell cycle arrest during the post-embryonic development of Artemia-encysted gastrula. With immunochemistry, morphology, and cell cycle analysis, the identified Artemia RSK was established to be specifically activated during the post-embryonic and early larval developmental stages when arrested cells of encysted embryos resumed mitoses. In vivo knockdown of RSK activity by RNA interference, kinase inhibition, and antibody neutralization consistently induced defective larvae with distinct gaps between the exoskeleton and internal tissues. In these abnormal individuals, mitoses were detected to be largely inhibited in the affected regions. These results display the requirement of RSK activity during Artemia development and suggest its role in termination of cell cycle (G(2)/M phase) arrest and promotion of mitogenesis. Our findings may, thus, provide insights into the regulation of cell division during Artemia post-embryonic development and reveal further aspects of RSK functions.

[The study of optimal conditions of electroporation in Escherichia coli DH10B strain].

In order to optimize the conditions of construction BAC library, the transformation efficiency of E. coli DH10B was studied in this paper. Our data prove much higher competence of electroporation (reaches 2.19 x 10(10) cfu/microg pUC19 DNA) when harvesting the cells between an OD550 of 0.7 - 0.8. Five different electric field strength (from 9 kV/cm to 25 kV/cm) and three different sized plasmid vector DNAs including pUC19 DNA, pECBAC1 DNA and pCLD04541 DNA, as well as three bacterial artificial chromosomes (BACs) ranging from 40 to 190 kb and their mixture were used to discover the transformation efficiency changes under various conditions. Our data show maximum transformation efficiency and optimal electric field strength of plasmid DNAs drop dramatically with increasing size of the DNA. Molecules of 190 kb transform more than 50-fold less well, on a molar basis, than molecules of 40 kb. And the optimal voltage gradient is strongly dependent on the different sized molecules, for instance, pUC19 reaches the highest transformation efficiency at 21 kV/cm, while the 180 kb BAC DNA gets its best efficiency at 13 kV/cm. This paper demonstrates that conditions may be selected which increase the average size of BAC clones generated by electroporation and could be widely applied in large-insert genome library construction.

A transglutaminase from Chinese shrimp (Fenneropenaeus chinensis), full-length cDNA cloning, tissue localization and expression profile after challenge.

Transglutaminase can catalyze the cross-linking reaction between soluble clotting protein molecules from the plasma for prevention of excess blood loss from a wound and obstructing micro-organisms from invading the wound in crustaceans. A novel transglutaminase (FcTG) gene was cloned from hemocytes of Chinese shrimp Fenneropenaeus chinensis by 3' and 5' rapid amplification of cDNA ends (RACE) PCR. The full-length cDNA consists of 2972bp, encoding 757 amino acids with a calculated molecular mass of 84.96kDa and a theoretical isoelectric point of 5.61. FcTG contains a typical transglutaminase-like homologue (TGc domain: E-value=1.94e-38). Three catalytic sites (Cys-324, His-391 and Asp-414) are present in this domain. The deduced amino acid sequence of FcTG showed high identity with black tiger shrimp TG, kuruma shrimp TG and crayfish TG. Transcripts of FcTG mRNA were mainly detected in gill, lymphoid organ and hemocytes by RT-PCR. RNA in situ hybridization further confirmed that FcTG was constitutively expressed in hemocytes both in the circulatory system and lymphoid organ. The variation of mRNA transcription level in hemocytes and lymphoid organ following injection of killed bacteria or infection with white spot syndrome virus (WSSV) was quantified by RT-PCR. The up-regulated expression of FcTG in shrimp lymphoid organ following injection of bacteria indicates that it is inducible and might be associated with bacterial challenge.