Cloning and expression of glucose regulated protein 78 (GRP78) in<Emphasis Type="Italic"> Fenneropenaeus chinensis</Emphasis>

GRP78 (78 kDa glucose-regulated protein), also known as BiP (immunoglobulin heavy-chain-binding protein), is an essential regulator of endoplasmic reticulum (ER) homeostasis because of its multiple functions in protein folding, ER calcium binding, and controlling of the activation of transmembrane ER stress sensors. In this report, we cloned the full length cDNA of GRP78 (FcGRP78) from Chinese shrimp Fenneropenaeus chinensis. This cDNA revealed a 2,325 bp with 1,968 bp open reading frame encoding 655 amino acids. This is the first reported GRP78 gene in Crustacea. The deduced amino acid sequence of FcGRP78 shared high identity with previously reported insect GRP78s: 86, 87 and 85% identity with GRP78s of Drosophila melanogaster, Aedes aegypti and Bombyx mori, respectively. Northern blot analysis shows that FcGRP78 is ubiquitously expressed in tissues of shrimp. Heat shock at 35°C significantly enhanced the expression of FcGRP78 at the first hour, reached the maximum at 4 h post heat shock, dropped after that and resumed to the normal level until 48 h of post recovery at 25°C. Additionally, differential expression of FcGRP78 was detected in haemocytes, hepatopancreas and lymphoid organ when shrimp were challenged by white spot syndrome virus (WSSV). We inferred that FcGRP78 may play important roles in chaperoning, protein folding and immune function of shrimp.     

Silencing VP28 Gene of White Spot Syndrome Virus of Shrimp by Bacterially Expressed dsRNA

An in vivo expression system to produce large amounts of virus-derived dsRNAs in bacteria to provide a practical control of white spot syndrome virus (WSSV) in shrimp was developed. The bacterially synthesized dsRNA specific to VP28 gene of WSSV promoted gene-specific interference with the WSSV infection in shrimp. Virus infectivity was significantly reduced in WSSV-challenged shrimp injected with VP28-dsRNA and 100% survival was recorded. The inhibition of the expression of WSSV VP28 gene in experimentally challenged animals by VP28-dsRNA was confirmed by RT-PCR and Western blot analyses. Furthermore, we have demonstrated the efficacy of bacterially expressed VP28-dsRNA to silence VP28 gene expression in SISK cell line transfected with eukaryotic expression vector (pcDNA3.1) inserted with VP28 gene of WSSV. The expression level of VP28 gene in SISK cells was determined by fluorescent microscopy and ELISA. The results showed that the expression was significantly reduced in cells transfected with VP28dsRNA, whereas the cells transected with pcDNA-VP28 alone showed higher expression. The in vivo production of dsRNA using prokaryotic expression system could be an alternative to in vitro method for large-scale production of dsRNA corresponding to VP28 gene of WSSV for practical application to control the WSSV in shrimp farming.     

Penaeus monodon chitin-binding protein (PmCBP) is involved in white spot syndrome virus (WSSV) infection

White spot syndrome virus (WSSV) can cause the most serious viral disease of shrimp and has a wide host range among crustaceans. Although researches show a lot about its genome and structure, information concerning the mechanism of how WSSV infects' cells is lacking. In this study, some experiments were applied to confirm the biological meaning of the protein–protein interaction between WSSV envelope protein, VP53A, and Penaeus monodon chitin-binding protein (PmCBP). Immunofluorescent study indicated that PmCBP is located on the cell surface of host cells. PmCBP amounts of about 34 kDa can be detected in both P. monodon and Litopenaeus vannamei tissues by Western blotting. In the in vivo neutralization experiment, both rVP53A and rPmCBP that were produced by Esherichia coli can promote resp. a 40% and 20% survival rate of the shrimp which were challenged by WSSV. Furthermore, a yeast-two-hybrid result revealed that PmCBP could interact with at least 11 WSSV envelope proteins. Those findings suggest that PmCBP may be involved in WSSV infection.     

Differential Gene Expression and Characterization of Tissue-specific cDNA Clones in Oil Palm using mRNA Differential Display

The mRNA differential display method was utilized to study the differential expression and regulation of genes in two species of oil palm, the commercially grown variety Elaeis guineensis, var. tenera and the South American species, Elaeis oleifera. We demonstrated the differential expression of genes in the mesocarp and kernel at the week of active oil synthesis (15 week after anthesis) during fruit development as compare to the roots and leaves and the isolation of tissue-specific and species-specific cDNA clones. A total of eight specific cDNA clones were isolated and their specificities were confirmed by Northern hybridization and classified into three groups. Group one contains four clones (KT3, KT4, KT5 and KT6) that are kernel-specific for E. guineensis, tenera and E. oleifera. The second group represents clone FST1, which is mesocarp and kernel-specific for E. guineensis, tenera and E. oleifera. The third group represents clones MLT1, MLT2 and MLO1 that are mesocarp and leaf-specific. Northern analysis showed that their expressions were developmentally regulated. Nucleotide sequencing and homology search in GenBank data revealed that clones KT3 and KT4 encode for the same maturation protein PM3. While clones MLT1 and MLT2 encode for S-ribonuclease binding protein and fibrillin, respectively. The other clones (KT5, KT6, FST1 and MLO1) did not display any significant homology to any known protein.     

The “Window <Emphasis Type="Italic">t</Emphasis> test”: a simple and powerful approach to detect differentially expressed genes in microarray datasets

This work focuses on differential expression analysis of microarray datasets. One way to improve such statistical analyses is to integrate biological information in the design of these analyses. In this paper, we will use the relationship between the level of gene expression and variability. Using this biological information, we propose to integrate the information from multiple genes to get a better estimate of individual gene variance, when a small number of replicates are available, to increase the power of the statistical analysis. We describe a strategy named the “Window t test” that uses multiple genes which share a similar expression level to compute the variance which is then incorporated a classic t test. The performances of this new method are evaluated by comparison with classic and widely-used methods for differential expression analysis (the classic Student t test, the Regularized t test (reg t test), SAM, Limma, LPE and Shrinkage t). In each case tested, the results obtained were at least equivalent to the best performing method and, in most cases, outperformed it. Moreover, the Window t test relies on a very simple procedure requiring small computing power compared with other methods designed for microarray differential expression analysis. Electronic Supplementary Material  Supplementary material is available for this article at and is accessible for authorized users.     

Cotransformation and differential expression of introduced genes into potato (Solanum tuberosum L.) cv Bintje

Summary The Dutch potato cultivar Bintje has been transformed by Agrobacterium strain LBA1060KG, which contains two plasmids carrying three different DNAs (TL- and TR-DNA on the Agrobacterium rhizogenes plasmid and TKG-DNA on the pBI121 plasmid). Several transformed root clones were obtained after transformation of leaf, stem, and tuber segments, and plants were then regenerated from these root clones. The expression of the various marker genes [rol, opine, -glucuronidase (GUS), and neomycin phosphotransferase (NPTII)] was determined in several root clones and in regenerated plants. The selection of vigorously growing root clones was as efficient as selection for kanamycin resistance. In spite of the location of NPTII and GUS genes on the same T-DNA, 17% of the root clones did not show GUS activity. Nevertheless, Southern blot analysis showed that these root clones contained at least three copies of the GUS gene. Sixty-four per cent of the root clones contained opines. The expression of these genes, however, was negatively correlated with plant regeneration capacity and normal plant development. The differential expression of the marker genes in the transgenic potato tissues is discussed.     

Protection of Penaeus monodon against white spot syndrome virus using a WSSV subunit vaccine

Although invertebrates lack a true adaptive immune response, the potential to vaccinate Penaeus monodon shrimp against white spot syndrome virus (WSSV) using the WSSV envelope proteins VP19 and VP28 was evaluated. Both structural WSSV proteins were N-terminally fused to the maltose binding protein (MBP) and purified after expression in bacteria. Shrimp were vaccinated by intramuscular injection of the purified WSSV proteins and challenged 2 and 25 days after vaccination to assess the onset and duration of protection. As controls, purified MBP- and mock-vaccinated shrimp were included. VP19-vaccinated shrimp showed a significantly better survival (p<0.05) as compared to the MBP-vaccinated control shrimp with a relative percent survival (RPS) of 33% and 57% at 2 and 25 days after vaccination, respectively. Also, the groups vaccinated with VP28 and a mixture of VP19 and VP28 showed a significantly better survival when challenged two days after vaccination (RPS of 44% and 33%, respectively), but not after 25 days. These results show that protection can be generated in shrimp against WSSV using its structural proteins as a subunit vaccine. This suggests that the shrimp immune system is able to specifically recognize and react to proteins. This study further shows that vaccination of shrimp may be possible despite the absence of a true adaptive immune system, opening the way to new strategies to control viral diseases in shrimp and other crustaceans.     

Comparison of Protein Expression Profiles of the Hepatopancreas in Fenneropenaeus chinensis Challenged with Heat-inactivated Vibrio anguillarum and White Spot Syndrome Virus

Fenneropenaeus chinensis (Chinese shrimp) culture industry, like other Penaeidae culture, has been seriously affected by the shrimp diseases caused by bacteria and virus. To better understand the mechanism of immune response of shrimp to different pathogens, proteome research approach was utilized in this study. Firstly, the soluble hepatopancreas protein samples in adult Chinese shrimp among control, heat-inactivated Vibrio-challenged and white spot syndrome virus-infected groups were separated by 2-DE (pH range, 4–7; sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and pH range, 3–10; tricine-SDS-PAGE). Then the differentially expressed protein spots (≥1.5-fold or ≤0.67-fold averagely of controls) were analyzed by LC-ESI-MS/MS. Using Mascot online database searching algorithm and SEQUEST searching program, 48 and 49 differentially expressed protein spots were successfully identified in response to Vibrio and white spot syndrome virus infection, respectively. Based on these results, we discussed the mechanism of immune response of the shrimp and shed light on the differences between immune response of shrimp toward Vibrio and white spot syndrome virus. This study also set a basis for further analyses of some key genes in immune response of Chinese shrimp.     

Spatial and temporal analysis of the local response to wounding

We studied the local response to wounding in Arabidopsis thaliana leaves using a two-step microarray analysis. A microarray containing 3500 cDNA clones was first screened to enrich for genes affected by wounding in the immediate vicinity of the wound (4 h post wounding). 359 non-redundant putative wound responsive genes were then spotted on a smaller wound-response array for detailed analysis of spatial expression (local, adjacent and systemic), timing of expression (0.5, 4, 8, 17 h), and effect of hormone treatments (methyl jasmonate, ethylene and abscisic acid). Our results show that genes that respond early at the site of the wound also respond throughout the plant, with similar kinetics. Early-induced genes which respond systemically encode predominantly signal transduction and regulatory factors (36%), and the expression of many of them is also controlled by methyl jasmonate (about 35% of the 36%). Genes specific to the wound site and the wounded leaf have a slower response to wounding and are mainly metabolic genes. At the wound, many genes of the lignin biosynthesis pathway were induced. In silico analysis of the 5′ promoter regions of genes affected by wounding revealed G-box-related motifs in a significant proportion of the promoters. These results show that the establishment of a systemic response to wounding is a priority for the plant, and that the local response at the wound site is established later. Ethylene and abscisic acid are involved in the local response, regulating repression of photosynthetic genes and expression of drought responsive genes respectively.     

The Role of ABI3 and FUS3 Loci in Arabidopsis thaliana on Phase Transition from Late Embryo Development to Germination

Arabidopsis abi3 and fus3 mutants are defective in late embryo development and their embryos show precocious growth. To understand the function and role of ABI3 and FUS3, we analyzed expression patterns of genes which were normally activated during late embryo development and germination in these mutants. Using the differential display method, both upregulated and downregulated genes were observed in immature siliques of the abi3 fus3 double mutant. Four clones having more abundant expression in the abi3 fus3 double mutant than in wild type were isolated. These genes were activated during wild-type germination, suggesting that some genes that are activated during wild-type germination are precociously activated in the abi3 fus3 mutant during late embryo development. Also, genes that were activated during wild-type germination were isolated and their expression patterns during late embryo development in the wild type and in abi3, fus3, and abi3 fus3 mutants were analyzed. Sixteen such clones were found, and 11 of these showed derepression or precocious activation of gene expression in the mutants. These results indicate that ABI3 and FUS3 negatively regulate a particular set of genes during late embryo development. We also showed that immature fus3 siliques accumulated one-third of the wild-type level of abscisic acid (ABA), but mature fus3 siliques accumulated ABA at a level comparable to that in the wild type. The possible mechanisms of controlling developmental timing in late embryo development as well as collaborative and distinct roles of ABI3 and FUS3 are discussed.     

Monitoring <Emphasis Type="Italic">ALS1</Emphasis> and <Emphasis Type="Italic">ALS3</Emphasis> Gene Expression During In Vitro <Emphasis Type="Italic">Candida albicans</Emphasis> Biofilm Formation Under Continuous Flow Conditions

ALS1 and ALS3 encode cell-surface associated glycoproteins that are considered to be important for Candida albicans biofilm formation. The main goal of the present study was to monitor ALS1 and ALS3 gene expression during C. albicans biofilm formation (on silicone) under continuous flow conditions, using the Centers for Disease Control biofilm reactor (CDC reactor). For ALS1, we found few changes in gene expression until later stages of biofilm formation (72 and 96 h) when this gene appeared to be downregulated relative to the gene expression level in the start culture. We observed an induction of ALS3 gene expression in the initial stages of biofilm formation (0.5, 1, and 6 h), whereas at later stages, this gene was also downregulated relative to the gene expression level in the start culture. We also found that biofilms of an als3/als3 deletion mutant contained less filaments at several time points (1, 6, 24, and 48 h), although filamentation as such was not affected in this strain. Together, our data indicate an important role for ALS3 in the early phases of biofilm formation in the CDC reactor, probably related to adhesion of filaments, while the role of ALS1 is less clear.     

Inhibitory activity of probiotics <Emphasis Type="Italic">Streptococcus phocae</Emphasis> PI80 and <Emphasis Type="Italic">Enterococcus faecium</Emphasis> MC13 against Vibriosis in shrimp <Emphasis Type="Italic">Penaeus monodon</Emphasis>

Occurrence of widespread epizootics among larval and cultured shrimp has put on viable preventive approaches such as application of probiotics on a high priority in aquaculture. In the present study, four probiotics bacteria were isolated from marine fish and shrimp intestine based on the antagonistic activity and nonpathogenic to the host. The isolates of probiotics strains Streptococcus phocae PI80, Enterococcus faecium MC13, Lactococcus garvieae LC149, B49 and one commercial probiotics (ECOFORCE) were fed to post larvae of Penaeus monodon obtained from two different hatcheries to analyze the growth and protection against Vibrio harveyi and V. parahaemolyticus. Growth of P. monodon post larvae fed with probiotic strain S. phocae PI80 was significantly (P < 0.001) higher when compared with control and other three strains in both experiments. The treatment of post larvae with B49 reduced the growth as well as Specific growth rate. Among the three probiotic strains S. phocae PI80 and E. faecium MC13 have effectively inhibited the pathogens. In experiment I high survival (92%) were observed in S. phocae PI80 treated post larvae when challenged with Vibrio harveyi followed by E. faecium MC13 (84%), B49 (76%) and ECOFORCE (68%) but PI80 did not protect the post larvae in the same experiment when they were exposed to V. parahaemolyticus. The probiotic isolate of MC13 has protected the post larvae against V. parahaemolyticus when compared to other probiotics and control. Similarly in the second experiment feeding of S. phocae enhanced the survival of larvae when challenged with V. harveyi. The laboratory studies proved that bacterial probionts S. phocae and E. faecium isolated from shrimp and brackishwater fish has potential applications for controlling pathogenic vibriosis in shrimp culture.