aiiA基因原核表达载体的构建与表达

【目的】构建aiiA基因的原核表达载体,并进行诱导表达,检测aiiA蛋白的抗病性,为进一步通过转基因技术培育转aiiA基因植株奠定基础。【方法】从pMDTM19-T Vector+aiiA质粒中酶切获得aiiA基因,将其与pGEX-4T-1表达载体连接构建重组原核表达载体pGEX-4T-1+aiiA,经双酶切及测序鉴定后,进行诱导表达,并对表达产物的功能进行鉴定。【结果】双酶切与PCR检测结果表明,重组原核表达载体pGEX-4T-1+aiiA构建成功。表达条件优化结果表明,在25℃下用0.2mmol/L IPTG诱导9h,aiiA蛋白的表达量最高。aiiA重组蛋白抑菌试验结果表明,其能够降解细菌的AHLs信号分子,猝灭细菌的群体感应,明显减弱病菌的致病力。转aiiA基因尾巨桉抗性检测结果表明,转基因植株抗性明显增强,表现为发病时间延迟,病情指数降低,评价为中等抗病水平。【结论】成功构建了aiiA基因原核表达载体,其诱导表达产物能猝灭细菌的群体感应,明显减弱病菌的致病力。     

高温胁迫下菊花嫁接苗中保护酶活性的变化

【目的】探讨嫁接对菊花耐热性的影响机理及对菊花不同器官耐热性的影响。【方法】比较40℃高温胁迫下菊花嫁接苗和不同生育期扦插苗的叶片、根系和茎段中超氧化物歧化酶(SOD)、过氧化物酶(POD)、过氧化氢酶(CAT)活性的变化与差异。【结果】菊花嫁接苗根茎叶中3种保护酶活性均显著高于扦插苗。与扦插苗相比,嫁接苗叶片、根系和茎段中SOD、POD和CAT活性分别平均提高11.6%,378.0%,260.1%;125.5%,131.3%,203.1%;90.1%,98.0%,90.5%。嫁接苗叶片中以SOD活性最大,均值达375U/g;根系中以POD活性最大,均值达605.87U/g;茎段中3种保护酶活性差异较小,以POD活性略高。【结论】嫁接是提高菊花耐热能力的一项有效措施,嫁接苗根茎叶的耐热性差别显著。     

Digestive enzyme activity during early larval development of the Cuban gar Atractosteus tristoechus

The ontogenesis of digestive enzymes (proteases, amylases, lipases, and phosphatases) in Cuban gar Atractosteus tristoechus was determined in larvae between 5 and 18 days after hatching (DAH). Variations in specific activities of most enzymes were related to the transition from endogenous to exogenous feeding and to the transition from the larval to the juvenile stage. Alkaline protease activity was not detected until 8 DAH in contrast to acid protease activity, which was quantifiable at 5 DAH. Acid protease activity was consistently higher than alkaline protease activity, indicating the presence of a functional stomach in the early stages of larval development. The acid protease activities of larvae and adults were compared by means of zymogram analysis. Four acid protease bands were found in adults (two more than in larvae). This result is the first time that more than one band of acid proteolytic activity has been found in Lepisosteidae. High lipase activity indicated the importance of lipid utilization, particularly during yolk-sac absorption. In contrast to the other enzymes studied, amylase activity was consistently low, probably due to the strictly carnivorous diet of gar larvae and their low capacity for carbohydrate digestion. High activities of aminopeptidase and acid and alkaline phosphatases suggest intestinal absorption. This result, together with the existence of a short gut and a lower proteolytic activity in the intestine than in the stomach, suggest that most of the proteolytic activity takes place in the stomach, while the primary function of the intestine is nutrient uptake.     

Influence of salinity on the energetics of gill and kidney of Atlantic salmon (Salmo salar)

The effect of seawater acclimation and adaptation to various salinities on the energetics of gill and kidney of Atlantic salmon (Salmo salar) was examined. Smolts and non-smolts previously reared in fresh water were exposed to a rapid increase in salinity to 30 ppt. Plasma osmolarity, [Na⁺], [Cl^–], [K⁺] and [Mg⁺⁺] increased in both groups but were significantly lower in smolts than non-smolts. Gill Na⁺, K⁺-ATPase specific activity, initially higher in smolts, increased in both groups after 18 days in seawater. Kidney Na⁺, K⁺-ATPase specific activity was not affected by salinity in either group. Gill and kidney citrate synthase specific activity was not affected by seawater exposure in smolts but decreased in non-smolts. In a second experiment, Atlantic salmon smolts reared in fresh water were acclimated to 0, 10 or 30 ppt seawater for 3 months at a temperature of 13–14°C. Gill Na⁺, K⁺-ATPase was positively correlated with salinity, displaying 2.5- and 5-fold higher specific activity at 10 and 30 ppt, respectively, than at 0 ppt. Kidney Na⁺, K⁺-ATPase specific activity was not significantly affected by environmental salinity. Citrate synthase and cytochrome c oxidase specific activities in gill were slightly (6–13%) lower at 10 ppt than at 0 and 30 ppt, whereas kidney activities were lowest at 30 ppt. Oxygen consumption of isolated gill filaments was significantly higher when incubated in isosmotic saline and at 30 ppt than at 0 ppt, but was not affected by the prior acclimation salinity. The results indicate that although high salinity induces increased gill Na⁺, K⁺-ATPase activity, it does not induce substantial increases in metabolic capacity of gill or kidney.     

Effects of weaning age and diets on ontogeny of digestive activities and structures of pikeperch (<Emphasis Type="Italic">Sander lucioperca</Emphasis>) larvae

Growth and ontogeny of digestive function were studied in pikeperch (Sander lucioperca) larvae weaned on artificial food at different ages. Three weaning treatments initiated respectively on day 9 (W9), day 15 (W15) or day 21 (W21) post-hatching (p.h.) were compared with a control group, fed Artemia nauplii from first feeding until the end of the rearing trial on day 36 p.h. The digestive enzyme activities and the ontogeny of digestive structures were investigated using enzymatic assays and histological methods. Growth of pikeperch larvae was significantly affected by precocious weaning. Pancreatic (trypsin and amylase) and intestinal (leucine-alanine peptidase, leucine aminopeptidase N and alkaline phosphatase) enzyme activities were detected from hatching onwards, increased at the moment of first feeding and then decreased. Pepsin secretion occurred at day 29 p. h. only, concurrently with the stomach development and differentiation of gastric glands. In the early weaning group (W9) the maturation process of intestinal enterocytes seems to be impaired and/or delayed and several signs of malnutrition were recorded. Except for alkaline phosphatase activity, no differences in enzyme activities and development of digestive structures were observed among the control, W21, and W15 groups. Moreover, at the end of the experiment, no differences in proteolytic activities were observed among larvae from the different treatments, indicating that, in surviving individuals, the digestive structures were properly developed and the larvae had acquired an adult mode of digestion. Based on the artificial diet used, our results suggested that pikeperch larvae can be weaned from day 15 p.h. without significant adverse effect on digestive capacities (except for alkaline phosphatase) or development of digestive tract, while earlier weaning impaired the onset of the maturation processes of the digestive system, both in terms of morphological structures and enzymatic activities.     

Micromonospora: An important microbe for biomedicine and potentially for biocontrol and biofuels

Micromonospora species have long been recognized as important sources of antibiotics and also for their unusual spores. However, their involvement in plant-microbe associations is poorly understood although several studies demonstrate that Micromonospora species function in biocontrol, plant growth promotion, root ecology, and in the breakdown of plant cell wall material. Our knowledge of this generally understudied group of actinomycetes has been greatly advanced by the increasing number of reports of their associations with plants, by the deployment of DNA cloning and molecular systematics techniques, and by the recent application of whole genome sequencing. Efforts to annotate the genomes of several Micromonospora species are underway. This information will greatly augment our knowledge of these versatile microorganisms.     

Toxicological effects of TiO2 and ZnO nanoparticles in soil on earthworm Eisenia fetida

Nanoparticles (NPs) of TiO₂ and ZnO are receiving increasing attention due to their widespread applications. To evaluate their toxicities to the earthworm Eisenia fetida (Savigny, 1826) in soil, artificial soil systems containing distilled water, 0.1, 0.5, 1.0 or 5.0 g kg⁻¹ of NPs were prepared and earthworms were exposed for 7 days. Contents of Zn and Ti in earthworm, activities of antioxidant enzymes, DNA damage to earthworm, activity of cellulase and damage to mitochondria of gut cells were investigated after acute toxicity test. The results from response of the antioxidant system combined with DNA damage endpoint (comet assay) indicated that TiO₂ and ZnO NPs could induce significant damage to earthworms when doses were greater than 1.0 g kg⁻¹. We found that Ti and Zn, especially Zn, were bioaccumulated, and that mitochondria were damaged at the highest dose in soil (5.0 g kg⁻¹). The activity of cellulase was significantly inhibited when organisms were exposed to 5.0 g kg⁻¹ of ZnO NPs. Our study demonstrates that both TiO₂ and ZnO NPs exert harmful effects to E. fetida when their levels are higher than 1.0 g kg⁻¹ in soil and that toxicity of ZnO NPs was higher than TiO₂.     

Dwarf mistletoe effects on soil basidiomycete community structure, soil fungal functional diversity, and soil enzyme function: Implications for climate change

We used a combination of molecular, culture and biochemical methods to test the hypothesis that severe infection of pine by dwarf mistletoe (genus Arceuthobium) has significant effects on structure and function of soil fungal communities, and on carbon cycling in soils. PCR and DNA sequencing of the basidiomycete communities in paired blocks of uninfected and infected trees revealed: (1) that the top, organic soil layer in this system is inhabited almost exclusively by ectomycorrhizal fungi; (2) no difference in species richness (6 species core⁻¹ in both) or Shannon-Wiener evenness (0.740 and 0.747 in uninfected and infected blocks respectively), however Shannon-Wiener diversity was significantly greater in infected blocks (1.19 vs 1.94 in uninfected and infected blocks respectively, P < 0.05); (3) significant differences in basidiomycete species composition, with nearly complete absence of two system co-dominant Russula species in infected blocks, and replacement of one co-dominant Piloderma species with another in infected plots, indicating physiological variability within the genus. Soil fungal physiological diversity measured using the Fungilog system was significantly greater in terms of both number of carbon substrates used by culturable soil fungi (both ascomycetes and basidiomycetes) in infected blocks, and the rate at which these substrates were used. Soil enzyme assays revealed greater laccase, peroxidase, and cellulase activities in soils associated with infected trees. Thus, event cascades associated with severe dwarf mistletoe infection not only significantly affected soil fungal species composition and increased species diversity, but also impacted on carbon-related function and functional diversity. Given the geographic range of this pathogen, and forecasts that epidemics of this disease will increase in range in severity with global climate change, these effects have the potential to significantly impact local and global carbon budgets.     

Influence of leaf litter types on microbial functions and nutrient status of soil: Ecological suitability of forest trees for afforestation in tropical laterite wastelands

The impact of forest tree leaf litters on microbial activity and nutrient status of red laterite soil was tested for the ecological suitability of Cassia siamea, Shorea robusta, Acacia auriculiformes and Dalbergia sissoo, which are typically used for afforestation of wastelands in eastern India. The objectives were to compare seasonal variation in soil enzyme activity in 30-years old afforested sites, and to study nutrient status and microbial biomass and function during short-term in-situ incubation of litter in decomposition pits. In afforested soils, enzyme activities significantly varied between litters and seasons. All enzyme activity except invertase dominated in the soils containing Dalbergia and Cassia litters compared to the others. The seasonal effect was enzyme-dependent, with amylase and cellulase reaching peaks during the rainy season but invertase activity showed a reverse trend with lowest values in rainy season, except in Acacia soil, and protease activity was lowest in the soil containing Cassia and Dalbergia during the rainy season. Dehydrogenase activity was negligible in the soils containing Shorea and Acacia, but remained high with respect to Dalbergia and Cassia during all seasons. The decomposition pit study showed significant increase of soil nutrients with respect to litter types and intervals, except with respect to electrical conductivity. Cassia and Dalbergia litters enabled notable increase of soil nutrients than Shorea and Acacia. The soil enzyme activity, in general, increased with duration of litter decay, but microbial biomass C (MBC) decreased over time except in Shorea. Therefore, the enzyme rates normalized to the MBC indicated inverse relations for all enzymes, except in the soil containing Shorea. A positive relationship existed between MBC and soil respiration in Cassia, Acacia and Dalbergia. Analysis of variance revealed main effects of litter types for increasing protease, MBC and CO₂ output, and a main effect of intervals for enhancing enzymes other than cellulase. Rates of soil respiration were greater in soils contain Cassia and Dalbergia, and showed significant differences between litters and between intervals. All enzymes were significantly correlated with electrical conductivity, organic carbon and available phosphorus contents, and all enzymes except invertase were correlated with nitrate concentrations. The acidic soil pH did not affect enzyme activities, and soil nutrients exerted only weak effect on MBC and respiration. Our study showed that leaf litters of Cassia and Dalbergia trees improved the nutrient status and microbial activity in soil more so than Shorea and Acacia litters, and therefore, afforestation using Cassia and Dalbergia trees may be particularly suitable for soil restoration in tropical laterite wastelands.     

Physiological,Biochemical, and Molecular Effects of In Vitro Induced Iron Deficiency in Peach Rootstock Mr.S 2/5

Abstract

On calcareous soils, carbonate induced iron deficiency can have heavy effects on growth and development of several fruit crops. Leaf chlorosis, biochemical, and ultrastructural alterations are the first symptoms causing severe damages to yield and productivity. In this work, some physiological, biochemical, and molecular aspects of iron deficiency stress induced on the peach rootstock Mr.S 2/5 in vitro were studied. The aim was to demonstrate if in vitro culture can be used as a valid and fast method to evoke iron deficiency symptoms and to study plant responses to this kind of abiotic stress. Two different treatments were carried out and compared to a control (MS medium): plantlets grown on MS medium completely free of iron and on MS medium containing 1 mM potassium bicarbonate. After 10 and 20 days from the beginning of the treatments fresh and dry weight, chlorophyll, and carotenoids content were measured. Superoxide dismutase and catalase total activity was measured, and gene expression was analyzed by using etherologous probes (Sod1, Sod3, Sod4, and Cat1) obtained from maize. In the absence of iron or in the presence of bicarbonate Mr.S 2/5 plantlets showed a significant decrease in growth, as quantified by fresh and dry weight. The plantlets showed a severe chlorosis due to a reduction in chlorophyll and carotenoids concentration. Iron deficiency stress caused a reduction of the activity of catalase and superoxide dismutase, while inducing an increase in gene expression.