iTRAQ-based proteomic analysis of the hepatopancreas from Litopenaeus vannamei after trans-vp28 gene Synechocystis sp. PCC6803 immunization.

Litopenaeus vannamei (Pacific white shrimp) is one of the most commercially important varieties of shrimp cultivated in the world. Shrimp farming is a high-risk, capital-intensive industry that is susceptible to periodic outbreaks of diseases caused by viral and bacterial pathogens. Thus, there is a need to develop economically viable methods of disease control. The hepatopancreas of crustaceans are known to have an important role in their innate immune response. In this study, we have explored the immune response of the hepatopancreas from L. vannamei fed with trans-vp28 gene Synechocystis sp. PCC6803 using iTRAQ-based proteomics. A total of 214 differentially expressed proteins (DEPs) were identified, of which 143 were up-regulated and 71 were down-regulated. These proteins have diverse roles in the cell cytoskeleton and cell phagocytosis, antioxidant defense process and the response of immune related proteins. Among these proteins, the immunity associated with the functional annotation of L. vannamei was further analysed. In addition, 4 DEPs (act1, N/A, H and C7M84_013542) were analysed using parallel reaction monitoring (PRM). This is the first report of proteomics in the hepatopancreas of L. vannamei immunized with trans-vp28 gene Synechocystis sp. PCC6803.

[Advances in the research and applications of orange fluorescent protein].

Fluorescent proteins can be used as probes to investigate intercellular molecular interactions and trace the pathway of specific metabolites, thus providing a detailed and accurate description of various metabolic processes and cellular pathways in living cells. Nowadays, the existing fluorescent proteins cover almost all spectral bands from ultraviolet to far-red. These fluorescent proteins have been applied in many fields of bioscience with the help of high-resolution microscopy, making great contributions to the development of biology. It is generally agreed that orange fluorescent proteins refer to the fluorescent proteins at the spectral range of 540-570 nm. In recent years, researches on orange fluorescent proteins have made great progress, and they have been widely applied in the field of biology and medicine as reporter protein and fluorescence resonance energy transfer as fluorescent receptor. This paper reviews the studies in the field of orange fluorescent proteins over the last 15 years, with the special focus on the development and application of orange fluorescent proteins to provide the basis for the future studies.

Effects of Synechococcus sp. PCC 7942 harboring vp19, vp28, and vp (19 + 28) on the survival and immune response of Litopenaeus vannamei infected WSSV.

This study aimed to assess the effect of oral administration of Synechococcus sp. PCC 7942 harboring vp19, vp28, and vp(19 + 28)against infection by white spot syndrome virus (WSSV) on juveniles of Litopenaeus vannamei. L. vannamei was orally administrated by feeding with different mutants of Synechococcus for 10 days, and then challenged with WSSV. The cumulative mortality of vp19, vp28, vp (19 + 28) groups was lower than that of the positive control group (57.8%, 62.2%, 71.1%, respectively); vp (19 + 28) group had a better protection rate than vp19 and vp28 groups. The analysis of shrimp immunological parameters showed that, after WSSV injection, the activity of superoxide dismutase, phenol oxidase, catalase, and lysozyme in the hepatopancreas of vp19, vp28, and vp (19 + 28) groups was higher than in the positive group; at the same time, growth performances of L. vannamei of experimental groups were better than control groups. Results showed that the Synechococcus mutants harboring vp19, vp28, and vp (19 + 28) could be used both as drug and feed to also enhance the defensive ability of juvenile shrimp against WSSV infection by increasing the activity of immune related enzymes.

[Pathogenicity of white-spot syndrome virus in Macrobrachium nipponensis via different infection routes].

Macrobrachium nipponensis is delicious and has high economic value, but its susceptibility to white-spot syndrome virus (WSSV) is unknown. Susceptibility, morbidity, and multiplication of WSSV in M. nipponense were studied by epidemiological survey, infection experiment and qPCR. M. nipponense was the natural host of WSSV, and the natural carrying rate was about 8.33%. M. nipponense could be infected with WSSV via oral administration, muscle injection and immersion, and the cumulative infection rate of 10 d exposure was 100%, and the cumulative mortality rates were 100%, 75% and 0%, respectively. The infection of WSSV is fast by muscle injection. The virus content after 5 day's injection is 1 000 times higher than that of the first day of infection, and the mortality rate reached 100% after 8 days. The median lethal dose (LD50) measured as the mortality of infected M. nipponense via injection indicated the LD50 in the concentration of WSSV of 2.71×105 virions/µL. In shrimp farming, M. nipponense can be infected by ingesting WSSV infected shrimp or dead shrimp, and also by soaking in WSSV-containing water and thus become a vector, consequently affecting the spread and pathogenicity of WSSV.

[Effects of light quality on cell growth and psbA promoter of engineered Synechococcus sp. PCC7002].

Light quality can regulate both psbA genes and vector promoter psbA of the engineered Synechococcus. Through light regulation, we tried to improve yield of the recombinant protein for vp28 gene-expressed Synechococcus sp. PCC7002. To drive photon-capturing efficiently, three limiting factors (irradiance, temperature and pH) were optimized by measuring net photosynthesis. High cell density cultures were performed with variant ratios of white, red and blue light in a 5-L photo-bioreactor. Yields of biomass, expressions of vp28 and transcription levels of psbA were compared. High ratio blue light-induced vp28 transcription had tripled and the relative accumulation of VP28 protein was doubled. The relative expressions of psbAII and psbAIII had positive correlations with higher ratio of blue light, not the red light. With high ratio red light inducing, dry biomass reached 1.5 g/L in three days. Therefore, we speculated that red light accelerated biomass accumulation of the transgenic strain and blue light promoted transcription for PpsbA and psbA. These results provided useful information for mass production of cyanobacteria and its secondary metabolites.

Regulation of pepc gene expression in Anabaena sp. PCC 7120 and its effects on cyclic electron flow around photosystem I and tolerances to environmental stresses.

Since pepc gene encoding phosphoenolpyruvate carboxylase (PEPCase) has been cloned from Anabaena sp. PCC 7120 and other cyanobacteria, the effects of pepc gene expression on photosynthesis have not been reported yet. In this study, we constructed mutants containing either upregulated (forward) or downregulated (reverse) pepc gene in Anabaena sp. PCC 7120. Results from real-time quantitative polymerase chain reaction (RT-qPCR), Western blot and enzymatic analysis showed that PEPCase activity was significantly reduced in the reverse mutant compared with the wild type, and that of the forward mutant was obviously increased. Interestingly, the net photosynthesis in both the reverse mutant and the forward mutant were higher than that of the wild type, but dark respiration was decreased only in the reverse mutant. The absorbance changes of P700 upon saturation pulse showed the photosystem I (PSI) activity was inhibited, as reflected by Y(I), and Y(NA) was elevated, and dark reduction of P700(+) was stimulated, indicating enhanced cyclic electron flow (CEF) around PSI in the reverse mutant. Additionally, the reverse mutant photosynthesis was higher than that of the wild type in low temperature, low and high pH, and high salinity, and this implies increased tolerance in the reverse mutant through downregulated pepc gene.

Recombinant expression and functional analysis of a Chlamydomonas reinhardtii bacterial-type phosphoenolpyruvate carboxylase gene fragment.

To investigate the function of a bacterial-type phosphoenolpyruvate carboxylase (PEPC2) derived from photosynthetically-grown Chlamydomonas reinhardtii, a fragment of the pepc2 gene was cloned and expressed in Escherichia coli. After optimal induction for 6 h, PEPC activity in the reverse mutant was lower than wild type (0.9 vs. 1.7 U/mg protein), and soluble protein was also lower than wild type (119 vs. 186 mg/g dry wt). In contrast, the total lipid content was increased from 56 (in wild type) to 71 mg/g dry wt, despite the growth rate being slightly diminished. The changes in PEPC activity, soluble protein and total lipid in the forward mutant were the opposite (2.4 U/mg, 230 mg/g, and 44 mg/g dry wt, respectively). Together, these data indicate that PEPC may function as a metabolic pivot in the regulation of protein and lipid accumulation in this alga.

Homologous comparisons of photosynthetic system I genes among cyanobacteria and chloroplasts.

It has now believed that chloroplasts arose from cyanobacteria, however, during endosymbiosis, the photosynthetic genes in chloroplasts have been reduced. How these changes occurred during plant evolution was the focus of the present study. Beginning with photosystem I (PSI) genes, a homologous comparison of amino acid sequences of 18 subunits of PSI from 10 species of cyanobacteria, chloroplasts in 12 species of eucaryotic algae, and 28 species of plants (including bryophytes, pteridophytes, gymnospermae, dicotyledon and monocotyledon) was undertaken. The data showed that 18 genes of PSI can be divided into two groups: Part I including seven genes (psaA, psaB, psaC, psaI, psaJ, ycf3 and ycf4) shared both by cyanobacteria and plant chloroplasts; Part II containing another 11 genes (psaD, psaE, psaF, psaK, psaL, psaM, btpA, ycf37, psaG, psaH and psaN) appeared to have diversified in different plant groups. Among Part I genes, psaC, psaA and psaB had higher homology in all species of cyanobacteria and chloroplasts. Among Part II genes, only psaG, psaH and psaN emerged in seed plants.

[Exploitation and utilization of rich lipids-microalgae, as new lipids feedstock for biodiesel production--a review].

As a renewable energy sources to replace conventional fossil fuels, biodiesel fuels have been becoming increasingly requirements to global fuels market. Biodiesel derived from oil crops cannot realistically satisfy even more fraction of the raw material existing costs and soil competitive demand for its growth. Microalgae appear to be the advantage of costs that is capable of higher photosynthetic efficiency, larger biomass, faster growth compared to those of oil crops. Lipid content of many microalgae is usually 80% of its dry weight. Genetic microalgae with high-oil productivity by genetic manipulations are capable of making microalgal biodiesel economically competitive with petrodiesel through large-scale production of genetic microalgal biomass. As demonstrated here, the use of biodiesel fuels in home and abroad are currently introduced, and the cost advantage of microalgae as the raw material is analyzed; And moreover, the progress of microalgal genetic engineering in regulation of lipid metabolism and the problems in the construct of genetic microalgae strains as well as approaches for making microalgal biodiesel appear to be an important source of renewable fuel that has the potential to completely displace fossil diesel are discussed in this review.

[Studies on relationship between the expression of hTNF-alpha gene and photosynthesis in Anabaena sp. IB02].

The effects of illumination on growth of Anabaena sp. IB02 and hTNF-alpha expression were studied. Photosynthetic activity, PS I and PS II activity of Anabaena sp. IB02 were assayed. Illumination enhanced the growth of Anabaena sp. IB02 and hTNF-a expression. Some relations were observed between hTNF-alpha expression and ture photosynthesis activity, PS I, PS II activity of Anabaena sp. IB02. Significant differences of the photosynthetic activity of host were detected simultaneously when hTNF-a expressed: the respiration rate increased (-68%), the light saturation point descended (+66%), all these suggested that the metabolic charge of host were increased and grow faster than wild type under low illumination.

Interactions between organic and inorganic carbon sources during mixotrophic cultivation of Synechococcus sp.

Glucose at 3 g l(-1) markedly accelerated growth of Synechococcus sp. PCC 7002. The net photosynthesis rate was 263 micromol O2 (mg Chl a h)(-1) for mixotrophic culture and 146 micromol O2 (mg Chl a h)(-1) for photoautotrophic culture. Additing 1 g NaHCO3 l(-1) to the glucose-supplemented culture enhanced the photosynthetic rate by 18%, and the total carbon consumption rate was raised to 2.5 mg l(-1) (mg chl a h)(-1) from a previously negative value. An interaction between organic and inorganic carbon metabolism was established.

[Bioinformatics studies on photosynthetic system genes in cyanobacteria and chloroplasts].

This study compared homology of base sequences in genes encoding photosynthetic system proteins of cyanobacteria (Synechocystics sp. PCC6803, Nostoc sp. PCC7120) with these of chloroplasts (from Marchantia Polymorpha, Nicotiana tobacum, Oryza sativ, Euglena gracilis, Pinus thunbergii, Zea mays, Odentella sinesis, Cyanophora paradoxa, Porphyra purpurea and Arabidopsis thaliana) by BLAST method. While the gene sequence of Synechocystics sp. PCC6803 was considered as the criterion (100%) the homology of others were compared with it. Among the genes for photosystem I, psaC homology was the highest (90.14%) and the lowest was psaJ (52.24%). The highest ones were psbD (83.71%) for photosystem II, atpB (79.58%) for ATP synthase and petB (81.66%) for cytochrome b6/f complex. The lowest ones were psbN (49.70%) for photosystem II, atpF (26.69%) for ATP synthase and petA (55.27%) for cytochrome b6/f complex. Also, this paper discussed why the homology of gene sequences was the highest or the lowest. No report has been published and this bioinformatics research may provide some evidences for the origin and evolution of chloroplasts.

[Spectroscopic properties of Heterosigma akashiwo under iron limitation].

Room-temperature absorption spectra of H. akashiwo cells under iron limitation showed a chlorophyll c absorption peak at 630 nm, 2 nm blue-shifted from its normal position of 632 nm. Moreover, because of the increase in the relative carotenoid abundance compared to Chl a, there was an extra shoulder peak at 480 nm for the iron-limited cells. Their fluorescence spectra (77 K) have one prominent chlorophyll emission peak at 685 nm. By comparison with Fe-replete cells (10 mumol.L-1), the fluorescence yield from 685 nm band increased by about 2 times in Fe deplete cells (5 nmol.L-1), and about 1.4 times in low iron cells (100 nmol.L-1), respectively. 48 h after Fe addition, the height of 685 nm peak was considerably decreased from that observed in low iron (100 nmol.L-1) and iron deficient (ID) cells before Fe addition, which indicated that there was a significantly higher energy dissipation, and thus, a less effective photosynthesis under the lack of Fe.

[Induction of biochemical composition in Heterosigma akashiwo under Fe stress].

The iron-stress mediated effects on biochemical constituents of red tide alga H. akashiwo were examined in Fe-replete and iron deficiency, and low iron batch cultures. The content of all pigments decreased under iron limitation, and the cellular chlorophyll a concentration decreased by more than 2-fold compared to that under iron replete condition. This change in Chl a was accompanied by a corresponding decrease in Chl c per cell, resulting in no trend in the ratio of Chl c to Chl a. The cellular carotenoids content decreased by more than 1.5-fold compared to that under iron replete condition. As a consequence, carotenoids/chlorophyll a ratio increased in Fe-deficient cells. Carbohydrate content was reduced under iron stress, and total proteins decreased with the decrease of iron concentration. A crude fractionation of the soluble proteins demonstrated that 17 kD and 55 kDa proteins in soluble fraction were induced.

Cloning and expression of metallothionein mutant alpha-KKS-alpha in Anabaena sp. PCC 7120.

The mouse metallothionein (mMT) mutant alpha-KKS-alpha has a higher capacity for binding heavy metals than wild type mMT. The mMT mutant alpha-KKS-alpha gene was placed under the control of the strong promoter PpbsA to generate the intermediate vector pRL-alpha-KKS-alpha. pRLalpha-KKS-alpha was then linked with the plasmid pDC-08 to construct shuttle expression vector pDC-alphaKKS-alpha. This expression vector was transformed into Anabaena sp. PCC 7120 using triparental conjugative transfer. After antibiotic selection (ampicillin and kanamycin), transgenic Anabaena was identified by PCR and Western blotting. The expression level of the mMT mutation alpha-KKS-alpha reached 7.4 mg/g dry cells weight, as detected by ELISA, and heavy metal resistance of the transgenic Anabaena was significantly improved.

Co-expression of Triosephosphate Isomerase, Fructose-1, 6-bisphosphate Aldolase and Fructose-1, 6-bisphosphatase in E.coli.

To establish a way to control or to decrease the daily increasing concentration of atmospheric CO(2), metabolically engineering Cyanobacteria was taken for the improvement of its efficiency of photosynthetic CO(2) fixation. As a preliminary stage of this study, three genes coding for three important Calvin cycle enzymes, i.e. triosephosphate isomerase (TPI), fructose-1, 6-bisphosphate aldolase(FBP aldolase),and fructose-1, 6-bisphosphatase(FBPase), respectively, have been cloned into one plasmid, pTrcFAT, which is controlled by promoter trc. Successful co-transcriptional expression of these three genes resulted inhigh yields of these enzymes under the induction of 0.25 mmol/L IPTG. Bioassay showed that the expressed enzymes from one liter of culture could directly catalyze DHAP conversion into 700 &mgr;mol of fructose-6-phosphate (F-6-P) per one minute. Furthermore, in order to introduce the three genes co-expression system into Cyanobacteria, a shuttle plasmid between E.coli and Cyanobacteria was constructed using plasmid pTrcFAT and a shuttle vector pDC-8, forming ashuttle plasmid pDCFAT-2 containing a dimer of the three genes co-expression operator. Successful co-expression in E.coli of pDCFAT-2 with higher full activity has been obtained. This shuttle was used to transform of Cyanobacteria Synechococcus sp. PCC 7942, and a few positive colonies were obtained.