Efficient expression of green fluorescent protein （GFP） mediated by a chimeric promoter in Chlamydomonas reinhardtii

To improve the expression efficiency of exogenous genes in Chlamydomonas reinhardtii, a high efficient expression vector was constructed. Green fluorescent protein （GFP） was expressed in C. reinhardtii under the control of promoters： RBCS2 and HSP70A-RBCS2. Efficiency of transformation and expression were compared between two transgenic algae： RBCS2 mediated strain Tran-Ⅰ and HSP70A-RBCS2 mediated strain Tran-Ⅱ. Results show that HSP70A-RBCS2 could improve greatly the transformation efficiency by approximately eightfold of RBCS2, and the expression efficiency of GFP in Tran-Ⅱ was at least double of that in Tran-Ⅰ. In addition, a threefold increase of GFP in Tran-Ⅱ was induced by heat shock at 40℃. All of the results demonstrated that HSP70A-RBCS2 was more efficient than RBCS2 in expressing exogenous gene in C. reinhardtii.     

Vitreoscilla hemoglobin gene (vgb) improves lutein production in Chlorella vulgaris

Vitreoscilla hemoglobin is an oxygen-binding protein that promotes oxygen delivery and reduces oxygen consumption under low oxygen conditions to increase the efficiency of cell respiration and metabolism. In this study, we introduced a Vitreoscilla hemoglobin gene （vgb） into Chlorella vulgaris by Agrobacterium tumefaciens-mediated transformation （ATMT）. PCR analysis confirmed that the vgb gene was successfully integrated into the Chlorella vulgaris genome. Analysis of biomass obtained in shake flasks revealed transformant biomass concentrations as high as 3.28 g/L, which was 38.81% higher than that of the wild-type strain. Lutein content of transformants also increased slightly. Further experiments recovered a maximum lutein yield of 2.91 mg/L from the transformants, which was 36.77% higher than that of the wild-type strain. The above results suggest that integrated expression of the vgb gene may improve cell growth and lutein yield in Chlorella vulgaris, with applications to lutein production from Chlorella during fermentation.     

Heat-shock protein 70 expression in shrimp Fenneropenaeus chinensis during thermal and immune-challenged stress

Using western immunoblotting, we obtained heat-shock protein 70 (HSP70) induction data and distribution in different tissues from shrimp Fenneropenaeus chinensis during thermal and immune-challenged stresses. This is probably the first report of the effects of various stressors on the expression of HSP70 in shrimp.HSP70 was prominently induced in hepatopancreas and gills, but not in muscle, eyestalk and hemolymph, when the shrimp were exposed to heat shock and Vibrio anguillavium-challenged stresses. Cold shock and WSSV treatment had no significant effects on the levels of HSP70 expression in all tissues examined. HSP70 induction was greatest after 2 h exposure to heat shock stress, which was elevated after acute heat shock exposure of 10℃ above ambient temperature.     

Low temperature stress on the hematological parameters and HSP gene expression in the turbot Scophthalmus maximus

To study the effect of low temperature stress on hematological parameters and HSP gene expression in the turbot( Scophthalmus maximus),water temperature was lowered rapidly from 18 to 1°C.During the cooling process,three individuals were removed from culture tanks at 18,13,8,5,3,and 1°C.Blood samples and tissues were taken from each individual,hematological indices and HSP gene expression in tissues were measured.The red blood cell count,white blood cell count,and hemoglobin concentration decreased significantly( P 0.05) as temperature decreased.Enzyme activities of plasma alanine transaminase and creatine kinase increased as temperature decreased,whereas aspartic transaminase and γ-glutamyl transpeptidase activities displayed no obvious changes above 1°C and lactate dehydrogenase activity increased first and then decreased.Blood urea nitrogen and uric acid levels were highest at 8°C,and creatinine concentration was highest at 3°C.The concentrations of plasma cortisol,cholesterol,and triglyceride all increased significantly( P 0.05) as temperature decreased.The serum glucose concentration increased first and then decreased to the initial level.The HSP70 m RNA expression showed various patterns in different tissues,whereas HSP90 m RNA expression showed the same tendency in all tissues.Overall,these results indicate that temperature decreases in the range of 8 to 5°C may induce a stress response in S.maximus and that temperature should be kept above 8°C in the aquaculture setting to avoid damage to the fish.     

Genetic Transformation of Nannochloropsis oculata with a Bacterial Phleomycin Resistance Gene as Dominant Selective Marker

The gene ble from Streptoalloteichus hindustanus is widely used as a selective antibiotic marker.It can control the phleomycin resistance,and significantly increase the tolerance of hosts to zeocin.The unicellular marine microalga Nannochloropsis oculata is extremely sensitive to zeocin.We selected ble as the selective marker for the genetic transformation of N.oculata.After the algal cells at a density of 2×10~7 cells mL~(-1) was digested with 4% hemicellulase and 2% driselase for 1 h,the protoplasts accounted for 90% of the total.The ble was placed at the downstream of promoter HSP70A-RUBS2 isolated from Chlamydomonas reinhardtii,yielding a recombinant expression construct p MS188.The construct was transferred into the protoplasts through electroporation(1kV,15 μS).The transformed protoplasts were cultured in fresh f/2 liquid medium,and selected on solid f/2 medium supplemented with 500 ngmL~(-1) zeocin.The PCR result proved that ble existed in the transformants.Three transformants had been cultured for at least 5 generations without losing ble.Southern blotting analysis showed that the ble has been integrated into the genome of N.oculata.The ble will serve as a new dominant selective marker in genetic engineering N.oculata.     

Physicochemical effects on sulfite transformation in a lipid-rich Chlorella sp. strain

SO2 is very rapidly hydrated to sulfurous acid in water solution at pH value above 6.0,whereby sulfite is yielded from the disassociation of protons.We aimed to improve the sulfite transformation efficiency and provide a basis for the direct utilization of SO2 from flue gas by a microalgal suspension.Chlorella sp.XQ-20044 was cultured in a medium with 20 mmol/L sodium sulfite under different physicochemical conditions.Under light conditions,sulfite concentration in the algal suspension reduced linearly over time,and was completely converted into sulfate within 8 h.The highest sulfite transformation rate(3.25 mmol/(L·h)) was obtained under the following conditions:35°C,light intensity of 300μmol/(m2·s),NaHCO3 concentration of 6 g/L,initial cell density(OD540) of 0.8 and pH of 9-10.There was a positive correlation between sulfite transformation rate and the growth of Chlorella,with the conditions favorable to algal growth giving better sulfite transformation.Although oxygen in the air plays a role in the transformation of SO2-3 to SO2-4,the transformation is mainly dependent on the metabolic activity of algal cells.Chlorella sp.XQ-20044 is capable of tolerating high sulfite concentration,and can utilize sulfite as the sole sulfur source for maintaining healthy growth.We found that sulfite 20 mmol/L had no obvious effect on the total lipid content and fatty acid profiles of the algae.Thus,the results suggest it is feasible to use flue gas for the mass production of feedstock for biodiesel using Chlorella sp.XQ-20044,without preliminary removal of SO2,assuming there is adequate control of the pH.     

Insertal Orientation Has No Influence on the Expression of gfp Gene and the Growth of the Host Synechococcus sp. PCC7942

In transgenic process, a foreign gene can be integrated in the host genome in two directions, which may influence its expression. In order to study the effects of insertal orientation, the gfp reporter gene was inserted in the isiAB locus of Synechococcus sp.PCC7942 in different directions, and the GFP expression levels and the growth of the transgenic algae were compared. It was showed that the gfp gene could express in each direction, and no significant difference was detected on algal growth and GFP expression levels between the two recombinant algae.     

Genetic transformation of Platymonas (Tetraselmis) subcordiformis (Prasinophyceae, Chlorophyta) using particle bombardment and glass-bead agitation

Platymonas (Tetraselmis) subcordiformis is a unicellular marine green alga. It was found to be very sensitive to the herbicide Basta through a sensitivity test indicating it could be employed as a selective agent. The bar gene is a practicable and selectable marker gene. The vector containing the expression cassette of the bar gene was transferred to P. subcordiformis by both particle bombardment and glass-bead agitation and transformants were then selected using Basta. Finally, Southern blotting analysis indicated that the bar gene had been successfully integrated into the nuclear genome of P. subcordiformis using both of the transgenic techniques, with the transformation efficiency of the glass-bead method being slightly higher than that of particle bombardment. This is the first report on stable transformation of P. subcordiformis, and will improve fundamental research and enlarge application of this alga.     

Heat-shock protein 70 expression in shrimp Fenneropenaeus chinensis during thermal and immune-challenged stress

Using western immunoblotting we obtained heat-shock protein 70 (HSP70) induction data and distribution in different tissues from shrimpFenneropenaeus chinensis during thermal and immune-challenged stresses. This is probably the first report of the effects of various stressors on the expression of HSP70 in shrimp. HSP70 was prominently induced in hepatopancreas and gills, but not in muscle, eyestalk and hemolymph, when the shrimp were exposed to heat shock andVibrio anguillavium-challenged stresses. Cold shock and WSSV treatment had no significant effects on the levels of HSP70 expression in all tissues examined. HSP70 induction was greatest after 2 h exposure to heat shock stress, which was elevated after acute heat shock exposure of 10°C above ambient temperature. Supported by the National Key Fundamental Research Program “Study of the Major Diseases and Resistance Mechanism of Mariculture Organisms”, NSFC, No. 30140017, and the international cooperative program “Immunaqua” by the European Commission, No. ICA4-CT-2001-10023.     

Effects of Salinity Fluctuation Frequency on the Osmolarity, Na^＋-K^＋ -ATPase Activity and HSP70 Expression in Juvenile Chinese Shrimp, Fenneropenaeus chinensis

Experiments were conducted to examine the effects of salinity fluctuation frequency on the osmolarity, Na+-K+-ATPase activity and HSP70 of Chinese shrimp Fenneropenaeus chinensis with initial wet body weight of 1.460 g ± 0.091 g. The salinity in the control group (D0) was 28 throughout the experiment, whereas treatments D2, D4, D6 and D8 were subjected to different salinity fluctuation frequencies of 2, 4, 6 and 8 d, respectively. The salinity in treatments D2, D4, D6 and D8 was kept at 28 for 2, 4, 6 and 8...     

Low temperature stress on the hematological parameters and <Emphasis Type="Italic">HSP</Emphasis> gene expression in the turbot <Emphasis Type="Italic">Scophthalmus maximus</Emphasis>

To study the effect of low temperature stress on hematological parameters and HSP gene expression in the turbot (Scophthalmus maximus), water temperature was lowered rapidly from 18 to 1°C. During the cooling process, three individuals were removed from culture tanks at 18, 13, 8, 5, 3, and 1°C. Blood samples and tissues were taken from each individual, hematological indices and HSP gene expression in tissues were measured. The red blood cell count, white blood cell count, and hemoglobin concentration decreased significantly (P < 0.05) as temperature decreased. Enzyme activities of plasma alanine transaminase and creatine kinase increased as temperature decreased, whereas aspartic transaminase and γ-glutamyl transpeptidase activities displayed no obvious changes above 1°C and lactate dehydrogenase activity increased first and then decreased. Blood urea nitrogen and uric acid levels were highest at 8°C, and creatinine concentration was highest at 3°C. The concentrations of plasma cortisol, cholesterol, and triglyceride all increased significantly (P < 0.05) as temperature decreased. The serum glucose concentration increased first and then decreased to the initial level. The HSP70 mRNA expression showed various patterns in different tissues, whereas HSP90 mRNA expression showed the same tendency in all tissues. Overall, these results indicate that temperature decreases in the range of 8 to 5°C may induce a stress response in S. maximus and that temperature should be kept above 8°C in the aquaculture setting to avoid damage to the fish.     

Effects of salinity fluctuation frequency on the osmolarity, Na+-K+-ATPase activity and HSP70 expression in juvenile chinese shrimp, Fenneropenaeus chinensis

Experiments were conducted to examine the effects of salinity fluctuation frequency on the osmolarity, Na⁺-K⁺-ATPase activity and HSP70 of Chinese shrimp Fenneropenaeus chinensis with initial wet body weight of 1.460 g ± 0.091 g. The salinity in the control group (D0) was 28 throughout the experiment, whereas treatments D2, D4, D6 and D8 were subjected to different salinity fluctuation frequencies of 2, 4, 6 and 8 d, respectively. The salinity in treatments D2, D4, D6 and D8 was kept at 28 for 2, 4, 6 and 8 d, respectively, decreased abruptly to salinity 24, lasted for another 2 d, and then was raised to its initial value 28. This was a complete salinity fluctuation cycle that afterwards repeated itself. After 32 days, the osmolarity in treatments D2, D4, D6 and D8 was significantly lower than that in treatment D0 (P<0.05). There were significant differences in both muscle and eyestalks HSP70 expression among groups. The HSP70 expressions in muscle and eyestalks in group D4 were 61.4% and 57.0% higher, respectively, than that in the control group D0 (P<0.05). There were, however, no significant differences in gill or hepatopancreas Na⁺-K⁺-ATPase activity between the treatments and the control.     

Effects of rapid temperature changes on HK,PK and HSP70 of <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis> in different seasons

Activities of hexokinase(HK), pyrnvate kinase(PK)and levels of HSPT0 were measured to evaluate the response of Litopenaeus vannamei to rapid temperature changes under controlled laboratory conditions. Shrimps were subjected to a quick temperature change from 27℃ to 17℃ for the summer case(Cold temperature treatment), or from 17℃ to 27℃ for the winter case(Warm temperature treatment). After 0. 5, 1, 3, 6, 12, 24, 48, and 72 h of exposure time, shrimps were sampled and prepared for further analysis. The results showed that the effect of acute temperature changes on activities of HK was significant. Patterns of variations of the two glycolytic enzymes suggested that enzymes in the glycolysis cycle could adjust their activities to meet the acute temperature change. The HSP70 level increased in both cold and warm temperature treatments, suggesting that the rapid temperature changes activated the process of body's self-protection. But the difference in expression peak of HSP70 might be related to the different body size and the higher thermal sensitivity to temperature increase than to temperature decrease of L. Vannamei.