植物植酸酶及其在饲料中的应用前景

植物植酸酶不但能分解内源植酸磷 ,对外源植酸磷同样有明显的降解作用。在饲粮中添加植酸酶活性高的植物性饲料 ,可提高猪和家禽对植酸磷的利用率 ,降低粪便中磷的排泄量 ,提高生产性能。麦类籽实中具有较高的天然植酸酶活性 ,发芽能显著提高种子中植酸酶的活性 ,因而有希望通过发芽提高麦类籽实中的植酸酶活性 ,经提纯浓缩后可达到在实际生产中应用的水平 ,从而减少在饲料中添加无机磷或价格昂贵的微生物植酸酶。     

转枯草芽孢杆菌植酸酶基因烟草对不同介质中植酸磷的吸收利用

利用转入枯草芽孢杆菌植酸酶基因的不同烟草株系,分别在无菌培养基、砂培和土培试验中研究了转植酸酶基因烟草对植酸磷的吸收和利用.结果表明,在无菌培养基试验中,所有转植酸酶基因烟草对植酸磷的吸收利用能力均显著高于野生型,其生物量比野生型提高了3.6～10.7倍,总磷吸收量提高了2.2～4.6倍;在沙培和土培中,转植酸酶基因烟草对植酸磷的吸收利用与野生型相比,生物量和总磷吸收量差异不显著.这说明转植酸酶基因在无菌条件下可以提高植物吸收利用植酸磷的能力,但是在自然条件下,由于微生物分解或矿物固定等原因,其作用不稳定,需要进一步研究克服土壤中的限制因素,才能使转基因植物充分发挥作用.     

磷肥对土壤中镉的植物有效性影响及其机理

为寻求保障镉污染农田稻米质量安全的有效措施,采用盆栽方法研究了低镉磷肥(Cd＜0.2 mg·kg^-1)及不同施磷量(0.10、0.20 g P₂O₅·kg^-1)对污染稻田土壤中(潮泥田)镉的植物有效性影响,并探讨了相关机理.结果表明： 在0.10 g·kg^-1磷剂量水平下,与对照(无磷肥)相比,钙镁磷和磷酸二氢钾处理显著提高了土壤pH和降低了土壤镉活性,钙镁磷和过磷酸钙处理显著降低了水稻对镉的吸收累积;当施磷量增至0.20 g·kg^-1时,磷酸氢钙处理显著提高了土壤pH和降低了土壤镉活性,钙镁磷、磷酸二氢钾和磷酸氢钙处理下DTPA提取态镉含量降低11.8%、9.8%和11.8%,NH₄OAc提取态镉含量降低9.5%、7.1%和7.1%;5种磷肥处理均显著降低了水稻茎叶中镉含量(降幅24.9%～50.8%),除磷酸氢钙处理外,糙米镉含量的降幅均达到显著水平,钙镁磷和过磷酸钙处理下糙米镉含量接近国家粮食卫生标准(GB 2715-2005).5种供试磷肥中,能提高土壤pH的磷肥(钙镁磷、磷酸二氢钾和磷酸氢钙)降低土壤镉有效性的效果显著,含钙磷肥(钙镁磷和过磷酸钙)降低水稻镉积累的效果较好.磷肥化学性质的差异可能是影响其效果的主要原因,选择碱性含钙磷肥对控制污染农田中作物吸收累积镉更有效.     

磷肥对土壤中镉的植物有效性影响及其机理

为寻求保障镉污染农田稻米质量安全的有效措施,采用盆栽方法研究了低镉磷肥(Cd＜0.2 mg·kg-1)及不同施磷量(0.10、0.20 g P2O5·kg-1)对污染稻田土壤中(潮泥田)镉的植物有效性影响,并探讨了相关机理.结果表明:在0.10 g·kg-1磷剂量水平下,与对照(无磷肥)相比,钙镁磷和磷酸二氢钾处理显著提高了土壤pH和降低了土壤镉活性,钙镁磷和过磷酸钙处理显著降低了水稻对镉的吸收累积;当施磷量增至0.20 g·kg-1时,磷酸氢钙处理显著提高了土壤pH和降低了土壤镉活性,钙镁磷、磷酸二氢钾和磷酸氢钙处理下DTPA提取态镉含量降低11.8％、9.8％和11.8％,NH4OAc提取态镉含量降低9.5％、7.1％和7.1％;5种磷肥处理均显著降低了水稻茎叶中镉含量(降幅24.9％ ～ 50.8％),除磷酸氢钙处理外,糙米镉含量的降幅均达到显著水平,钙镁磷和过磷酸钙处理下糙米镉含量接近国家粮食卫生标准(GB 2715-2005).5种供试磷肥中,能提高土壤pH的磷肥(钙镁磷、磷酸二氢钾和磷酸氢钙)降低土壤镉有效性的效果显著,含钙磷肥(钙镁磷和过磷酸钙)降低水稻镉积累的效果较好.磷肥化学性质的差异可能是影响其效果的主要原因,选择碱性含钙磷肥对控制污染农田中作物吸收累积镉更有效.     

模拟增温对中亚热带杉木人工林土壤磷有效性的影响

气候变暖改变与土壤磷循环相关的生物地球化学过程,对陆地生态系统磷循环产生直接或间接影响。为研究亚热带地区杉木人工林土壤磷有效性对增温的响应,开展了模拟增温实验。实验设置对照组及增温组(5℃),经过1.5a的短期增温,对杉木人工林的土壤全磷、有机磷、微生物量磷、有效磷、酸性磷酸酶活性及相关土壤理化性质进行测定,结果表明:增温处理下,土壤酸性磷酸酶活性提高约1.5倍,土壤全磷、微生物量磷以及有机磷含量分别减少了6%、34%和12%,土壤有效磷含量增加25%。可见,短期增温通过提高土壤磷酸酶活性进而促进土壤有机磷矿化和降低土壤微生物固磷量,从而增加土壤磷有效性,但是增温导致潜在可利用的土壤微生物量磷大幅度的降低,将有可能加剧亚热带杉木人工林土壤磷限制。     

植酸酶研究进展及土壤植酸酶应用展望

磷元素是农作物的主要营养限制因子之一,开发利用土壤中的磷资源对解决作物的磷素限制意义深远。植酸是土壤有机磷的主要形态,其矿化分解并释放有效磷的过程是一个酶促反应,植酸酶是这一过程的关键酶。植酸酶在土壤改良及农业的可持续发展领域具有较强的应用前景,高通量测序技术的出现也为土壤植酸酶研究提供了全新的思路和策略。综述了植酸酶的多样性、获取技术、提高产率的策略及在农业领域的应用现状,分析了土壤植酸酶研究存在的问题和未来发展的趋势,并对其应用前景进行了展望。     

菌根真菌磷酸酶活性对红三叶草生境中土壤有机磷亏缺的影响

利用PVC分室培养装置研究了菌根际和菌丝际磷酸酶活性变化与土壤有机磷亏缺间的关系,结果表明,施用有机磷（植酸钠）能促进菌根根系侵染、提高土壤磷酸酶尤其是酸性磷酸酶的活性,使菌丝际范围变宽。菌丝际的存在使土壤有机磷亏缺范围加大,与非菌根植物相比,由于菌根真菌的作用,植物能更容易地从有机磷中获得磷营养以满足植物生长的需要,从而使其干物重和磷吸收量更高。     

亚热带毛竹林土壤磷组分和微生物对施氮的响应

磷(P)是植物和微生物生长的重要营养元素,亚热带地区土壤P有效性较低,且长期高氮(N)沉降可能会造成土壤P有效性进一步降低。本试验开展于戴云山毛竹林,分析了施N 3年对土壤的基本理化性质、P组分、微生物生物量和酸性磷酸单酯酶活性的影响。结果表明: 施N显著增加了土壤NO₃^--N含量,提高了土壤N有效性,但显著降低了易分解态有机磷占全磷的比例,且总有机碳与总有机P的比例>200。土壤微生物生物量碳、微生物生物量磷、酸性磷酸单酯酶活性、微生物生物量碳/微生物生物量磷和微生物生物量氮/微生物生物量磷随施N量的增加而增加。此外,易分解态有机磷占总磷比例与微生物生物量磷呈显著负相关关系。因此,施N加剧了土壤P有效性限制,提高了微生物对P的需求。     

转基因植物表达植酸酶研究进展

植酸是植物体内磷的主要存在形式,其绝大部分不能被单胃动物消化吸收,而随粪便排出体外造成环境污染;同时,植酸又是一种抗营养因子,它通过络合植物体内的一些营养成分而降低植物的营养价值。通过植物转基因方法使植物自身表达足量的植酸酶,以减小植酸带来的不利影响,是提高植物性饲料营养价值和控制环境磷污染的一种经济有效的措施。就转基因植物植酸酶的优势、研究现状、存在的问题及其发展前景进行了综述。     

植酸酶高产菌株的诱变选育

无花果曲霉是一种可以产植酸酶的菌株,其代谢产物植酸酶可以将有机植酸磷降解为无机磷。以此菌株为出发菌株,确定了它的最适pH值为1.3~1.4,最适温度为55~60℃。同时,为获得高酶活的突变株,进行亚硝基胍和紫外线处理,经初筛得到99株高效突变株,再经复筛和传代试验,得到1株植酸酶活性是出发菌株2.47倍的突变株NTG-23。     

Fate and effects of phosphorus additions in soils under N2-fixing red alder

Soil phosphorus (P) dynamics are controlled by the interaction of geochemical, biochemical and biological processes. Changes in species composition or management could alter the relative importance of these processes. We examined soil P dynamics in two plantations of N₂-fixing red alder (Alnus rubra) by determining the fate and effects of added fertilizer P. History of the plantations varied such that sites were previously occupied by 60-yr-old stands of alder or non-fixing Douglas-fir (Pseudotsuga menziesii). Without fertilization, the soil with a longer period of alder influence had more organic P (P_o) and less sorbed inorganic P (Hydroxide- and Bicarb-extractable P_i). Fertilization increased soil total P, and 88% of the fertilizer was accounted for in the surface mineral soil (0–15 cm). Sorbed P_i was the major sink for fertilizer P (55–60%), independent of site history. Although P_o was 35–70% of soil P in unfertilized plots, added P did not accumulate as P_o. Neither site history nor P addition influenced phosphatase activity. Fertilization increased decomposition during incubation of the organic horizon, suggesting that late-stage decomposition is P-limited in these N-rich soils. On the time-scale of a few years, geochemical sorption and desorption of inorganic P were the most important processes controlling the distribution of added P. Organic P accumulation is expected to occur over a longer time frame, linked to the production and turnover of organic matter.     

Expression of a fungal phytase gene in Nicotiana tabacum improves phosphorus nutrition of plants grown in amended soils

Transgenic Nicotiana tabacum plants expressing a chimeric phytase gene (ex::phyA) from the soil fungus Aspergillus niger were generated. Three independently transformed lines showed increased extracellular phytase activity compared with a vector control and wild-type plants, both of which had no detectable extracellular phytase. Transgenic N. tabacum plants grown in sterile agar supplied with phosphorus (P) as phytate accumulated 3.7-fold more P than vector control plants. Despite this, the expression of ex::phyA in plants did not lead to an improved accumulation of P from two unamended P-deficient soils. However, when soils were amended with either phytate or phosphate and lime, transgenic plants accumulated up to 52% more P than controls. Positive responses by transgenic plants were, in some instances, coincident with a putative increase in soil phytate. We conclude that the development of plants that exude phytase to the soil may not ensure improved plant P nutrition, as the availability of phytate in the soil also appears to be critical. Nevertheless, if plants that express ex::phyA are combined with soil amendments that promote the availability of phytate, there is the potential to enhance the P nutrition of crop plants and to improve the efficiency of P fertilizer use in agricultural systems.     

Uptake of phosphate from soils and fertilizers as affected by soil P availability and solubility of phosphorus fertilizers

Fertilizers labelled with ³²P were used to measure amounts of phosphorus, P_s and P_F, taken up by Lolium perenne from available soil P and from P fertilizer respectively, when applied at a rate of 66 mg P·(kg soil^–1) in greenhouse experiments. The quantity P_s of phosphorus taken up from soil in the presence of P fertilizer was compared to the quantity P_o taken up from soil without P fertilizer. The quantity (P_s–P_o) is positive for low P_o values, i.e. in soils poor in available phosphorus, but is negative for high P_o values indicating that an input of P fertilizer can induce a decrease in the utilization of available soil phosphorus. Moreover, for a given soil, the quantity (P_s–P_o) depends on the chemical form of the fertilizer. The standard method of evaluation of P fertilizer efficiency is based on the assumption that P_s=P_o, but P_s can differ from P_o. This result can explain the contradictory data published from field experiments about the efficiency of the various P fertilizers.     

Novel Glucose-1-Phosphatase with High Phytase Activity and Unusual Metal Ion Activation from Soil Bacterium Pantoea sp. Strain 3.5.1

Phosphorus is an important macronutrient, but its availability in soil is limited. Many soil microorganisms improve the bioavailability of phosphate by releasing it from various organic compounds, including phytate. To investigate the diversity of phytate-hydrolyzing bacteria in soil, we sampled soils of various ecological habitats, including forest, private homesteads, large agricultural complexes, and urban landscapes. Bacterial isolate Pantoea sp. strain 3.5.1 with the highest level of phytase activity was isolated from forest soil and investigated further. The Pantoea sp. 3.5.1 agpP gene encoding a novel glucose-1-phosphatase with high phytase activity was identified, and the corresponding protein was purified to apparent homogeneity, sequenced by mass spectroscopy, and biochemically characterized. The AgpP enzyme exhibits maximum activity and stability at pH 4.5 and at 37°C. The enzyme belongs to a group of histidine acid phosphatases and has the lowest K_m values toward phytate, glucose-6-phosphate, and glucose-1-phosphate. Unexpectedly, stimulation of enzymatic activity by several divalent metal ions was observed for the AgpP enzyme. High-performance liquid chromatography (HPLC) and high-performance ion chromatography (HPIC) analyses of phytate hydrolysis products identify dl-myo-inositol 1,2,4,5,6-pentakisphosphate as the final product of the reaction, indicating that the Pantoea sp. AgpP glucose-1-phosphatase can be classified as a 3-phytase. The identification of the Pantoea sp. AgpP phytase and its unusual regulation by metal ions highlight the remarkable diversity of phosphorus metabolism regulation in soil bacteria. Furthermore, our data indicate that natural forest soils harbor rich reservoirs of novel phytate-hydrolyzing enzymes with unique biochemical features.     

Phosphorus Transformations in an Oxisol under contrasting land-use systems: The role of the soil microbial biomass

It is generally assumed that phosphorus (P) availability for plant growth on highly weathered and P-deficient tropical soils may depend more on biologically mediated organic P (P_o) turnover processes than on the release of adsorbed inorganic P (P_i). However, experimental evidence showing the linkages between P_o, microbial activity, P cycling and soil P availability is scarce. To test whether land-use systems with higher soil P_o are characterized by greater soil biological activity and increased P mineralization, we analyzed the partitioning of P among various organic and inorganic P fractions in soils of contrasting agricultural land-use systems and related it to biological soil properties. Isotopic labeling was used to obtain information on the turnover of P held in the microbial biomass. Soil samples were taken from grass–legume pasture (GL), continuous rice (CR) and native savanna (SAV) which served as reference. In agreement with estimated P budgets (+277, +70 and 0 kg P ha^–1 for CR, GL and SAV, respectively), available P estimated using Bray-2 and resin extraction declined in the order CR > GL > SAV. Increases in Bray-2 and resin P_i were greater in CR than GL relative to total soil P increase. Organic P fractions were significantly less affected by P inputs than inorganic fractions, but were a more important sink in GL than CR soils. Extractable microbial P (P_chl) was slightly higher in GL (6.6 mg P kg^–1) than SAV soils (5.4 mg P kg^–1), and significantly lowest in CR (2.6 mg P kg^–1). Two days after labeling the soil with carrier free ³³P, 25, 10 and 2% of the added ³³P were found in P_chl in GL, SAV and CR soils, respectively, suggesting a high and rapid microbial P turnover that was highest in GL soils. Indicators of P mineralization were higher in GL than CR soils, suggesting a greater transformation potential to render P_o available. Legume-based pastures (GL) can be considered as an important land-use option as they stimulate P cycling. However, it remains to be investigated whether crops planted in pasture–crop rotations could benefit from the enhanced P_o cycling in grass–legume soils. Furthermore, there is need to develop and test a direct method to quantify P_o mineralization in these systems.     

Extracellular secretion of Aspergillus phytase from Arabidopsis roots enables plants to obtain phosphorus from phytate

Phosphorus (P) deficiency in soil is a major constraint for agricultural production worldwide. Despite this, most soils contain significant amounts of total soil P that occurs in inorganic and organic fractions and accumulates with phosphorus fertilization. A major component of soil organic phosphorus occurs as phytate. We show that when grown in agar under sterile conditions, Arabidopsis thaliana plants are able to obtain phosphorus from a range of organic phosphorus substrates that would be expected to occur in soil, but have only limited ability to obtain phosphorus directly from phytate. In wild-type plants, phytase constituted less than 0.8% of the total acid phosphomonoesterase activity of root extracts and was not detectable as an extracellular enzyme. By comparison, the growth and phosphorus nutrition of Arabidopsis plants supplied with phytate was improved significantly when the phytase gene (phyA) from Aspergillus niger was introduced. The Aspergillus phytase was only effective when secreted as an extracellular enzyme by inclusion of the signal peptide sequence from the carrot extensin (ex) gene. A 20-fold increase in total root phytase activity in transgenic lines expressing ex::phyA resulted in improved phosphorus nutrition, such that the growth and phosphorus content of the plants was equivalent to control plants supplied with inorganic phosphate. These results show that extracellular phytase activity of plant roots is a significant factor in the utilization of phosphorus from phytate and indicate that opportunity exists for using gene technology to improve the ability of plants to utilize accumulated forms of soil organic phosphorus.     

Assimilation of Phytate-phosphorus by the Extracellular Phytase Activity of Tobacco (Nicotiana tabacum) is Affected by the Availability of Soluble Phytate

Phytate, the major organic phosphorus in soil, is not readily available to plants as a source of phosphorus (P). It is either complexed with cations or adsorbed to various soil components. The present study was carried out to investigate the extracellular phytase activities of tobacco (Nicotiana tabacum variety GeXin No.1) and its ability to assimilate external phytate-P. Whereas phytase activities in roots, shoots and growth media of P_i-fed 14-day-old seedlings were only 1.3–4.9% of total acid phosphatase (APase) activities, P starvation triggered an increase in phytase secretion up to 914.9 mU mg⁻¹ protein, equivalent to 18.2% of total APase activities. Much of the extracellular phytase activities were found to be root-associated than root-released. The plants were not able to utilize phytate adsorbed to sand, except when insoluble phytate salts were preformed with Mg²⁺ and Ca²⁺ ions for supplementation. Tobacco grew better in sand supplemented with Mg-phytate salts (31.9 mg dry weight plant⁻¹; 0.68% w/w P concentration) than that with Ca-phytate salts (9.5 mg plant⁻¹; 0.42%), presumably due to its higher solubility. We conclude that insolubility of soil phytate is the major constrain for its assimilation. Improving solubility of soil phytate, for example, by enhancement of citrate secretion, may be a feasible approach to improve soil phytate assimilation.     

Characterization of transgenic Trifolium subterraneum L. which expresses phyA and releases extracellular phytase: growth and P nutrition in laboratory media and soil

Transgenic Trifolium subterraneum expressing a phytase gene (phyA) from Aspergillus niger were generated. Five independently transformed lines showed an average 77‐fold increase in exuded phytase activity in comparison with null segregant and wild‐type controls. Unlike other phosphatases, exuded phytase activity was unaffected by P supply, verifying the constitutive expression of phyA. Transgenic T. subterraneum grown in agar with P supplied as phytate, took up 1.3‐ to 3.6‐fold more P than controls and had equivalent P uptake to plants supplied with orthophosphate. This unique phenotype was compromised when the plants were grown in soil. None of the five lines showed increased shoot biomass or total P uptake in an unfertilized, low‐P soil taken from under permanent pasture. With addition of P, one of the five transgenic lines had consistently greater P nutrition compared with control plants. Despite variable growth and P nutrition responses, P uptake per root length was on average greater for transgenic lines. Exudation of phytase by transgenic T. subterraneum allowed utilization of P from phytate in non‐sorbing, sterile laboratory media, but was less effective when plants were grown in soil. Release of extracellular phytase is therefore not the only requirement for the acquisition of P from endogenous soil phytate by plants.     

Inorganic and organic P in soil solutions from three upland soils

Soil solutions from three P-deficient Cambisols were analyzed for inorganic orthophosphate (P_i), organically combined phosphorus (P_o), total phosphorus (P_t) and residual phosphorus (P_r=P_t–(P_o+P_i)). The solutions were obtained by centrifugation of soil samples wetted-up to 90% field capacity. Increasing the centrifugal force from 750 to 1400×g (for 60 minutes) increased the volume of soil solution obtained by 17–35%. Increasing the centrifugation period from 30 to 90 minutes (at 1000×g) increased the volume by 2–12%. The effect of the different centrifugation conditions on the P composition of soil solutions were not critical and had little effect on either P_t concentration or on the distribution of P between P_i, P_o and P_r fractions. Soil solutions were also obtained on a seasonal basis over a 2-year period. The soils, fresh from the field, were wetted-up to 90% field capacity and centrifuged at 1000×g for 60 minutes to isolate the soil solution. Although the soils were derived from contrasting parent rock, and had different Fe and Al sesquioxide contents, the P_t concentrations of the soil solutions and the distribution between the fractions were similar. Annual average P_t concentrations for the 3 soils ranged from 93 to 114 and 63 to 89 g dm^-3 during the first and second year, respectively. Seasonal changes were of a similar order as those resulting from differences in soil type. During May, June, August and October soil solutions had average P_t concentrations ranging from 82 to 111 and 51 to 119 g P dm^-3 in 1989 and 1990, respectively. P_o was a major P component in soil solution and exceeded the amount of P_i by about 5–20 times.     

Effect of particle size and microbial phytase on phytate degradation in incubated maize and soybean meal

The objective of the study was to evaluate the effect of screen size (1, 2 and 3 mm) and microbial phytase (0 and 1000 FTU/kg as-fed) on phytate degradation in maize (100% maize), soybean meal (100% SBM) and maize–SBM (75% maize and 25% SBM) incubated in water for 0, 2, 4, 8 and 24 h at 38°C. Samples were analysed for pH, dry matter and phytate phosphorus (P). Particle size distribution (PSD) and average particle size (APS) of samples were measured by the Laser Diffraction and Bygholm method. PSD differed between the two methods, whereas APS was similar. Decreasing screen size from 3 to 1 mm reduced APS by 48% in maize, 30% in SBM and 26% in maize–SBM. No interaction between screen size and microbial phytase on phytate degradation was observed, but the interaction between microbial phytase and incubation time was significant (P<0.001). This was because microbial phytase reduced phytate P by 88% in maize, 84% in maize–SBM and 75% in SBM after 2 h of incubation (P<0.05), whereas the reduction of phytate P was limited (<50%) in the feeds, even after 24 h when no microbial phytase was added. The exponential decay model was fitted to the feeds with microbial phytase to analyse the effect of screen size and feed on microbial phytase efficacy on phytate degradation. The interaction between screen size and feed affected the relative phytate degradation rate (R_d) of microbial phytase as well as the time to decrease 50% of the phytate P (t1/2) (P<0.001). Thus, changing from 3 to 1 mm screen size increased R_d by 22 and 10%/h and shortened t1/2 by 0.4 and 0.2 h in maize and maize–SBM, respectively (P<0.05), but not in SBM. Moreover, the screen size effect was more pronounced in maize and maize–SBM compared with SBM as a higher phytate degradation rate constant (K_d) and R_d, and a shorter t1/2 was observed in maize compared with SBM in all screen sizes (P<0.05). However, a higher amount of degraded phytate was achieved in SBM than in maize because of the higher initial phytate P content in SBM. In conclusion, reducing screen size from 3 to 1 mm increased K_d and R_d and decreased t1/2 in maize and maize–SBM with microbial phytase. The positive effect of grinding on improving microbial phytase efficacy, which was expressed as K_d, R_d and t1/2, was greater in maize than in SBM.