基于Gateway技术的植物表达载体的构建

【目的】为方便基于植物瞬时表达技术的高通量功能基因筛选,利用Gateway技术实现植物表达载体和表达文库的构建,将35S启动子和Gateway技术入门载体pDONR222的同源重组区连接到植物表达载体pCAMBIA0380上,构建Gateway技术兼容的植物表达载体。【方法】分别扩增35S启动子与pDONR222上attP特异识别序列之间的功能区,并将其依次连入根癌农杆菌(Agrobacterium tume faciens)介导的植物表达载体pCAM-BIA0380的多克隆位点区,利用带有attB特异识别区域的GUS基因,对构建的载体功能进行测试。【结果】成功构建了基于Gateway技术的植物表达载体p1104D;载体重组基因的选择性测试结果表明,p1104D对目的基因片段的大小无严格选择性;载体重组效率测试结果表明,p1104D重组平均滴度为5.11×105cfu/mL;GUS报告基因瞬间表达试验结果表明,改造后的载体可以实现目标基因的顺利表达。【结论】基于Gateway技术的植物表达载体p1104D的构建,为实现cDNA文库的高效构建和目标基因的高通量功能筛选提供了可能,有望推动植物-病原互作研究中关键基因的鉴定与克隆。     

Design and validation of a RT‐qPCR procedure for diagnosis and quantification of most types of infectious pancreatic necrosis virus using a single pair of degenerated primers

Infectious pancreatic necrosis virus (IPNV) is an important virus which affects the salmonid aquaculture industry worldwide; therefore, it is important to develop rapid and reliable methods of diagnosis to detect the disease at early stages. Nowadays, RT‐qPCR is replacing other methods because it provides additional information on the viral load, which is important to have a better understanding of the virus replication level and of the stage of the infection and its risk level. The main problem stems from the high diversity of this virus, which can compromise the reliability of the diagnosis. In this study, we have designed an RT‐qPCR procedure for diagnosis and quantification of IPNV based on a single pair of primers targeted to segment B. The procedure has been validated, in vitro and in vivo, testing two different types of standards against seven reference strains and 23 field isolates from different types. The procedure is reliable for the detection of any type, with a detection limit of 31 TCID₅₀ mL^?1, 50 pfu mL^?1 or 66 RNA copies mL^?1, depending on the standard. All the standard curves showed high reliability (R² > 0.95). The results support the high reliability of this new procedure for the diagnosis and quantification of IPNV.     

Soybean molasses as an organic carbon source in the farming of Litopenaeus vannamei (Boone, 1931) in a biofloc system

Soybean molasses was evaluated as a partial replacement for sugarcane molasses as a carbon source for biofloc development in the superintensive culture of Pacific white shrimp (Litopenaeus vannamei). A 50‐day study was conducted with juvenile (3.2 g) shrimp stocked in 16 800 L tanks at a stocking density of 250 shrimp m^?3. Control of total ammonia concentration was performed by the addition of combined mixtures of soybean and sugarcane molasses to the culture water. Three different molasses treatments were evaluated using different soybean‐to‐sugarcane molasses ratios: 15–85%, 38–62% and 60–40% respectively. The control group was treated only with sugarcane molasses. Water quality, chlorophyll a concentration, heterotrophic bacterial load, Vibrio spp. concentration and zootechnical indexes were all evaluated. Total ammonia concentration was controlled by heterotrophic and chemotrophic pathways. Biofloc formation, as quantified by measuring the total suspended solids, was not altered. The Vibrio spp. concentration showed a significant reduction in treatments with soybean‐to‐sugarcane molasses ratios of 38–62% and 60–40%. All combined mixtures of soybean and sugarcane molasses could maintain water quality and productivity in the superintensive culture of L. vannamei using the biofloc system. Thus, the potential use of a residue from agroindustry as a carbon source in a biofloc culture is demonstrated.     

Thermal Biology of Liorhyssus hyalinus (Hemiptera: Rhopalidae) and Nysius simulans (Hemiptera: Lygaeidae), Fed on the Milky Stage of Maize Grains

When quinoa, Chenopodium quinoa Willd., is cultivated in South America outside of its Andean origin, the heteropterans Liorhyssus hyalinus (Fabricius) and Nysius simulans Stål may emerge as important pests. Here we studied the development and reproduction of both species at different constant temperatures in the laboratory. Egg and nymphal development were investigated at 18, 22, 26, 30, 34, and 36°C. For both species, egg incubation time significantly decreased as the temperature increased. Nymphs did not successfully develop at 18°C and the total nymphal time significantly decreased as the temperature increased from 22 to 36°C. Based on a linear day-degree (DD) model, the lower developmental threshold (LDT) temperatures for eggs and nymphs were estimated to be 16.0 and 17.9°C for L. hyalinus, and 16.1 and 19.7°C for N. simulans, respectively. Thermal requirements for egg and nymphal development were 68.6 and 114.8 DD for L. hyalinus, and 77.7 and 190.3 DD for N. simulans, respectively. Reproduction and adult longevity were studied at 22, 26, 30, and 34°C. For both species preoviposition time decreased as temperature increased, and the oviposition period was longest at 26°C. The highest fecundity and egg viability were observed at 30°C, whereas longevities were higher at 22–26°C than at 30–34°C. As the lowest tested temperatures were not suitable to both heteropterans and 30°C was found to be the optimal temperature for development and reproduction, peak densities are expected in warm areas and seasons.     

Identification of helminth species by means of disc electrophoresis

Soluble proteins of 25 helminth species of the classes Trematoda, Cestoidea and Nematoda, were separated by disc electrophoresis using polyacrylamide gel columns. Differences between the species were investigated on the basis of Rm values of the bands. Protein spectra were complemented by the detection of lipoproteins and glycoproteins and by identification of LDH, SHD, peroxidase, esterase and alkaline phosphatase. On the basis of comparison of protein spectra of parasitic worms belonging to three taxonomic classes it was found by means of numerical taxonomy that individual classes are characterized by a certain number of proteins of the same migration properties.     

Physiological Response of Multiple Contrasting Rice (Oryza sativa L.) Cultivars to Suboptimal Temperatures

The physiological response of multiple rice cultivars, eighteen initially and eight cultivars later on, to suboptimal temperatures (ST) conditions was investigated in laboratory and outdoor experimental conditions. Treatment with ST decreased growth in different extents according to the cultivar and affected the PSII performance, determined by chlorophyll fluorescence fast‐transient test, and stomatal conductance, regardless the experimental condition. Two groups of cultivars could be distinguished on the base of their growth and physiological parameters. The group of cultivars presenting higher growths displayed optimal JIP values, and higher instantaneous water use efficiency (WUE_i), due to a lower Gs under ST, unlike cultivars showing lower growth values, which presented worse JIP values and could not adjust their Gs and hence their WUE_i. In this work, we detected at least two cultivars with superior tolerance to ST than the cold tolerant referent Koshihikari. These cultivars could be used as parents or tolerance donors in breeding for new crop varieties. On other hand, positive and significant correlations between data obtained from laboratory and outdoor experiments suggest that laboratory measurements of most of the above mentioned parameters would be useful to predict the response of rice cultivars to ST outdoor.     

Comparative Characterization of Enzymatic Digestion from Fish and Soybean Meal from Simulated Digestive Process of Pacific Bluefin Tuna,Thunnus orientalis

The digestive process of the Pacific bluefin tuna (PBT), Thunnus orientalis, was simulated through two phases of in vitro digestion: acidic digestion with porcine pepsin, followed by alkaline digestion with pancreatic crude extract (PCE) obtained from the PBT to hydrolyze fish meal (FM) and soybean meal (SBM) as protein substrates. The crude protein from FM resulted in a lower degree of hydrolysis (73.3%) compared with SBM (79.2%). However, the resulting digested products showed that FM contained 35% more small peptides, with sizes <6.5 kDa than those from the starting material (>150 kDa). The SBM had an increase of only 1.3% in the similar peptide cut‐offs found after hydrolysis. These results suggested that FM appeared to be a better source of protein according to the amount of low‐molecular weight peptides. In addition, the proteolytic activity of PCE showed that 88.9% of its alkaline proteolytic activity corresponded to trypsin and 2.9% corresponded to chymotrypsin activity. The results shown here demonstrate that peptide sizes are important in identifying suitable protein sources for aquafeed production to reinforce the primary results obtained from the in vitro digestibility using the pH‐Stat system. These results also contribute to a better understanding of the digestibility process in aquatic organisms.     

Associations of fish with various types of littoral habitats in reservoirs

Fish associations with different types of littoral habitats were studied in four canyon‐shaped reservoirs in the Czech Republic in years 2010 and 2011 by gillnets. Two to three habitats per reservoir–beaches (former meadows), stump fields (former forest) and rubble slopes–were defined and sampled along the longitudinal axis of reservoirs. Effects of reservoir, habitat and locality (position along longitudinal axis) on fish biomass, abundance and species structure were tested for juvenile and adult fish separately. Hierarchical analysis of variance revealed that habitats differed significantly in fish biomass and abundance. Redundancy analysis showed that analysed environmental variables had significant influence on fish community structure. Most variability in community structure was explained by reservoir and then by combination of habitat and slope steepness. Locality position had the smallest influence on community structure. For both adult and juvenile fish total abundance and biomass, the most inhabited habitat was beaches; rubble slopes were the least inhabited. Habitat associations differed among species. Among adults, bream Abramis brama, white bream Blicca bjoerkna and roach Rutilus rutilus were associated with beaches and stump fields, whereas perch Perca fluviatilis, ruffe Gymnocephalus cernuus, asp Aspius aspius and pike Esox lucius were associated with rubble slopes. Bream, white bream, bleak Alburnus alburnus, roach, ruffe and pikeperch Sander lucioperca were associated with beaches among juveniles, whereas the only juvenile associated with rubble slopes was perch. We showed that most common species are associated with distinct habitats and also that utilisation of various littoral habitats differs in general.