根癌农杆菌介导水稻基因转化研究进展

简要综述了根癌农杆菌介导水稻基因转化的优点及转化后代的遗传学行为 ,着重分析了影响农杆菌介导水稻基因转化的各种因素和存在问题     

铵态氮/硝态氮对水稻铝吸收的影响及其机制研究

选用两个水稻品种武运粳7号（耐铝品种）和扬稻6号（铝敏感品种）作为实验材料,利用水培试验,通过长期和短期处理相结合的方式研究了NH4+-N和NO3--N对水稻Al吸收的影响及其作用机制。结果表明,与NH4+-N相比,NO3--N促进了水稻对Al的吸收。而且随着NO3--N浓度的提高,水稻根中Al含量显著增加,随着NH4+-N浓度的提高,水稻根中Al含量显著降低。这些结果表明NH4+-N和NO3--N对水稻Al吸收的影响具有浓度效应。当向Al溶液添加pH缓冲剂后,NH4+-N和NO3--N对根尖Al累积的影响变小,表明NH4+-N和NO3--N处理下溶液pH的变化可能是NH4+-N和NO3--N对水稻Al吸收影响不同的一个原因。     

Effects of soil water content on growth,tillering, and manganese uptake of lowland rice grown in the water‐saving ground‐cover rice‐production system (GCRPS)

A 2‐year field experiment and a pot experiment were carried out to compare Mn uptake, tillering, and plant growth of lowland rice grown under different soil water conditions in the ground‐cover rice‐production system (GCRPS) in Beijing, North China. The field experiment was conducted in 2001 and 2002, including two treatments: lowland‐rice variety (Oryza sativa L. spp. japonica) grown under thin (14 μm) plastic‐film soil cover (GCRPS_plastic) at 80%–90% water‐holding capacity (WHC) and traditional lowland rice (paddy control) grown with 3 cm standing‐water table. The pot experiment was conducted in a greenhouse with four treatments: (1) traditional lowland rice: paddy control; (2) GCRPS, water‐saturated soil: GCRPS_saturated; (3) GCRPS at 90% water‐holding capacity (WHC): GCRPS_90%WHC; and (4) GCRPS at 70% WHC: GCRPS_70%WHC. Results of the field experiment showed that dry‐matter production, number of tillers, as well as N and Mn concentrations in rice shoots of GCRPS were significantly lower than in paddy control, while there was no significant difference in shoot Fe, Cu, Zn, and P concentration and nematode populations. In the pot experiment, shoot Mn concentration significantly decreased with decreasing soil water content, while soil redox potential increased. Shoot–dry matter production and tiller number of GCRPS_saturated were significantly higher than in other treatments. Significant correlations were observed between the shoot Mn concentration and tiller number at maximum tillering stage in the field and pot experiment, respectively. We therefore conclude that the limitation of Mn acquisition might contribute to the growth and yield reduction of lowland rice grown in GCRPS. The experiment provides evidence that GCRPS_plastic combined with nearly water‐saturated soil conditions helps saving water and achieving optimum crop development without visual or latent Mn deficiency as observed under more aerobic conditions.     

In vivo staining of reduced iron by 2,2′ bipyridine in rice exposed to iron toxicity

A protocol for a novel method to visualize Fe(II) in rice tissues is proposed. The method is based on the selective formation of a purple‐red color complex of 2,2′ bipyridine and Fe(II). Rice genotypes were exposed to 18 mM Fe(II) in nutrient solution for 2 d. Root systems of intact plants were subsequently placed in 2,2′ bipyridine solutions. The formation of the [Fe(bipy)<$>_3^{2+}<$>] color complex was visualized using bifocal microscopy. The method may improve the selection of genotypes during breeding for Fe‐toxicity resistance of rice.     

Zinc alleviates growth inhibition and oxidative stress caused by cadmium in rice

A hydroponic experiment with two rice cultivars differing in cadmium (Cd) tolerance was conducted to investigate the alleviating effect of zinc (Zn) on growth inhibition and oxidative stress caused by Cd. Treatments consisted of all combinations of two Zn concentrations (0.2 and 1 μM), three Cd concentrations (0, 1, and 5 μM), and two rice cultivars (Bing 97252, Cd‐tolerant; Xiushui 63, Cd‐sensitive). Cd toxicity caused a dramatic reduction in plant height and biomass, chlorophyll concentration and photosynthetic rate, and an increase in Cd concentration in both roots and shoots, malondialdehyde (MDA) concentration, and superoxide dismutase (SOD) and peroxidase (POD) activities in shoots. The response of all these parameters was much larger for Xiushui 63 than for Bing 97252. Addition of Zn to the medium solution alleviated Cd toxicity, which was reflected in a significant increase in plant height, biomass, chlorophyll concentration, and photosynthetic rate, and a marked decrease in MDA concentration and activity of anti‐oxidative enzymes. However, it was noted that Zn increased shoot Cd concentration at higher Cd supply, probably due to the enhancement of Cd translocation from roots to shoots. Therefore, further studies are necessary to determine the effect of Zn supply on Cd translocation from vegetative organs to grains or grain Cd accumulation before Zn fertilizer is applied to Cd‐contaminated soils to alleviate Cd toxicity in rice.     

Arsenic accumulation by rice grown in soil treated with roxarsone

Poultry litter is widely used as a fertilizer for lowland rice in Taiwan and China. However, the organic‐arsenic compound roxarsone (additive of poultry feed) in poultry litter can be absorbed by the plants and the resulting arsenic (As) contamination may pose a serious threat to human health. This study used various amounts of poultry litter contaminated with roxarsone in pot experiments to evaluate the effect of roxarsone on rice agronomic parameters and the bioaccumulation of total and inorganic As in rice‐plant tissues. Rice‐grain yield decreased significantly with increasing As content of the soil, and the critical threshold that killed rice was 200 mg roxarsone (kg soil)^–1. The As concentrations in root, straw, leaf, husk, and grain increased with increasing soil As (p < 1%). At 100 mg roxarsone per kg of soil, the As concentration in the rice grain exceeded the statutory permissible limit of 1.0 mg As (kg dry weight)^–1 and at 25 mg roxarsone (kg soil)^–1, the inorganic As concentrations in grains exceeded the statutory limit of 0.15 mg of inorganic As kg^–1 in China. For all treatments, the As concentrations in various plant tissues at maturity follow the order: root > stem > leaf > husk > grain. Arsenite was the predominant species in root, straw, and grain, while arsenate was the predominant species in leaf and husk. No significant difference existed between the amounts of arsenite and arsenate when various amounts of poultry litter were applied. This result illustrates that large amounts of added roxarsone are not only toxic to rice but also accumulate in grains in the inorganic As forms, potentially posing a threat to human health via the food chain.     

水稻OsHsfA7基因RNA干扰载体的构建及遗传转化研究

为研究水稻热激转录因子基因OsHsfA7的功能及其在水稻耐热育种方面的应用价值,本文通过构建水稻OsHsfA7基因RNA干扰载体获得功能抑制变异材料,从反向遗传学进行基因的功能分析。扩增OsHsfA7c DNA3'编码区470bp片段,分别以反向和正向插入到中间载体pBSK连接的GUS片段两侧,然后将得到的RNA干扰片段克隆到改造的植物表达载体p1301M,构建以CaMV35S启动子驱动表达的水稻OsHsfA7基因RNA干扰表达载体。将该载体转入根癌农杆菌EHA105后,采用农杆菌介导法进行了水稻日本晴品种的遗传转化,获得了23株具有潮霉素抗性的转基因植株,其中12株经GUS染色呈蓝色并且DNA检测10株已插入目的片段,RNA检测其中6株OsHsfA7基因的表达水平下降甚至未检出。结果说明本研究构建的OsHsfA7基因RNA干扰载体对该基因表达沉默是有效的。     

Distinctive expression of a zinc-binding protein in rice callus grown in medium with high zinc concentration

We investigated the growth, contents of water-soluble protein and free amino acids of the callus of japonica rice (Oryza sativa L. cv. Nipponbare) cultured in liquid N6 medium containing a high concentration of zinc. Furthermore, we determined the N-terminal amino acid sequence of a Zn-binding protein expressed in large quantities in the callus. The addition of Zn stimulated the growth of the callus and increased the Zn concentration. The callus subjected to the high-Zn treatment (hereafter referred to as CHZn) contained a larger amount of soluble proteins and a smaller amount of free amino acids than the control callus. Zinc-binding proteins were separated by affinity chromatography. The SDS-PAGE pattern of these proteins showed a distinctive protein band of about 29 kDa. Especially, CHZn contained larger quantities of 29 kDa protein than the control callus. Twenty-seven N-terminal amino acids of the protein were sequenced as DYAPMTLTIVNNCPYPVWPGIQANSGH. Results of homology search to the amino acid sequences from the nr-aa database and the dbEST database suggested that this 29 kDa protein may be a novel zinc-binding protein and that the protein may regulate the concentration of free zinc in the cytoplasm of callus cells through its binding to zinc ions.     

高钾供应加剧了水稻叶片缺镁诱导的氧化胁迫

Magnesium （Mg） deficiency in plant affects photosynthesis and many other metabolic processes. Rice （Oryza sativa L. cv. ＇Wuyunjing 7＇） plants were grown in hydroponics culture at three Mg and two potassium （K） levels under greenhouse conditions to examine the induction of oxidative stress and consequent antioxidant responses in rice leaves due to Mg deficiency. At low Mg （0.2 mmol L 1 Mg supply for two weeks after transplanting） and high K （6 mmol L^-1） for 21 days, the rice plants showed severe Mg deficiency and a significant decreases in the dry matter production. The Mg deficiency in leaves decreased chlorophyll concentrations, photosynthetic activity, and soluble protein, but significantly increased the concentrations of soluble sugars and malondialdchyde （MDA） and the activities of superoxide dismutase （SOLD, EC 1.15.1.1）, catalase （CAT, EC 1.11.1.6） and peroxidase （POD, EC 1.11.1.7）. In addition, Mg concentrations in the leaves and in the shoot biomass were negatively related to the activities of the three antioxidative enzymes and the concentration of MDA in leaves. There were very significant interactive effects between Mg and K supplied in the culture solution on shoot biomass yield, chlorophyll content, photosynthesis rate, the activities of SOD, CAT and POD, and MDA content in the leaves of rice. It is suggested that the high K level in the nutrient solution aggravated the effect of low Mg supply-induced Mg deficiency and created the oxidative damage in rice plants.     

水稻雄蕊导管发育研究

本文以水稻粤泰保持系为材料，采用ATPase定位方法研究了单核至三核花粉期雄蕊导管的发育。单核边位花粉期，分化的导管胞质变浓厚、细胞核染色质浓缩、核转变为新月形并出现凋亡小体；此外，导管内物质也可以大分子形式直接撤离。单核边位晚期，雄蕊导管发育成熟。在二核和三核花粉期，导管内重新出现具有ATPase活性的物质，同样的现象也存在于农垦58s可育花药成熟导管中。以上结果暗示水稻雄蕊导管分化存在程序性死亡及非程序性死亡多种途径，水稻成熟导管内具有ATPase活性物质可能是一种普遍现象。     

Excessive application of farmyard manure reduces rice yield and enhances environmental pollution risk in paddy fields

The present study examined the effect of excessive application of farmyard manure (FM) on rice production and environmental pollution in paddy fields of Japan. A long-term field experiment was conducted during the period 1976–2006 to examine the trends of rice yield and yield components as affected by the excessive FM application (20 Mg ha^?1 year^?1 containing 110 kg N, 180 kg P₂O₅, and 320 kg K₂O). Rice growth, soil fertility, and surface water quality were also assessed in the final year (2006). The results obtained were compared with those of a conventional practice with recommended doses of inorganic fertilizer (IF), i.e. 85 kg N, 68 kg P₂O₅, and 53 kg K₂O ha^?1 year^?1, and an unfertilized control (CR). The excessive FM application resulted in a gradual decrease in grain yield, which was mostly explained by the reduction of grain fertility under the luxuriant rice growth. This reduction may have been due to the higher accumulation of soil nutrients such as N, P, and K. Moreover, the excessive FM application increased chemical oxygen demand, total P, and soluble K concentrations in the paddy surface water and their effluent loads compared to the conventional practice with the recommended IF application.     

Stimulation of phenolic metabolism by silicon contributes to rice resistance to sheath blight

Sheath blight caused by Rhizoctonia solani is a major disease of rice worldwide. Silicon (Si) can enhance rice resistance to sheath blight, but the relation with phenolic metabolism is poorly known. Two rice cultivars with different levels of resistance to R. solani (resistant Teqing and susceptible Ningjing 1) were studied to determine the effects of Si on disease intensity (rated from 0 to 9) and the involvement of phenolic compounds in disease resistance. Variation in the concentrations of phenolics (including total soluble phenolics, flavonoids, and lignin) and in the activities of defense‐related enzymes polyphenoloxidase (PPO) and phenylalanine ammonia‐lyase (PAL) in rice leaf sheaths was investigated. The results show that Si application reduced sheath‐blight disease ratings of Ningjing 1 and Teqing by 2.96 and 0.65, respectively. In uninoculated plants, Si application alone had no significant effects on the concentrations of phenolic compounds or on the activities of PPO and PAL. In inoculated plants, Si application increased phenolics concentrations and PPO and PAL activities only in the susceptible cultivar Ningjing 1. We conclude that Si‐induced enhancement of phenolic metabolism contributed to the improved resistance of rice to sheath blight in the sensitive cultivar.     

Nitric oxide reduces the stress effects of aluminum on the process of germination and early root growth of rice

The present study examined the action of nitric oxide (NO) on the germination process of rice seeds and early root growth under aluminum (Al) stress. Seeds and seedlings of two rice genotypes, with different levels of sensitivity to aluminum stress, were examined after treatment with Al and NO or only with Al. Further, the histochemical localization of Al and NO was performed on the root tissues. In both genotypes, NO was able to neutralize the inhibitory Al effects on germination. In the roots of seedlings, a reduction of Al toxicity as mediated by NO was indicated by an increased root elongation and a reduction of Al accumulation on the root surface in the Al hematoxylin complexation, irrespective of the genotype. The histolocalization of NO in roots using diaminofluorescein diacetate (DAF‐2DA) and confocal microscopy revealed endogenous Al‐induced levels of NO. It is concluded that NO can alleviate Al stress in the seedlings of the studied rice genotypes by improving germination and early root growth and is likely to play a role in a specific stress‐signaling pathway.     

水稻蜡质合成相关基因OsGL1-5的表达模式及逆境响应特征分析

通过生物信息学方法对水稻蜡质合成相关基因OsGLl-5启动子序列特点进行分析;并以水稻中花11不同组织和发育时期的穗子为材料,通过半定量RT-PCR及mRNA原位杂交技术分析OsGLl-5的时空表达特征;同时,分别以200mmol·L-1 NaCl、100μmol· L-1 ABA和1.0％ H2O2处理水稻幼苗,通过半定量RT-PCR分析逆境胁迫下OsGL1-5的表达模式.生物信息学分析表明,OsGL1-5启动子中包括大量与干旱、冷、盐等多种逆境胁迫相关的调控序列.时空表达特征分析表明,OsGL1-5主要在不同发育时期的穗子中表达,且表达部位集中在稃片的毛状体和花药绒毡层中,叶中表达量相对较低,而茎和根中没有检测到表达.NaCl、ABA以及H2O2等逆境胁迫下,OsGL1-5表达量在不同处理时间有所增加.     

Tolerance of rice cultivars to iron toxicity

Iron toxicity is an important growth‐limiting factor for flooded rice production in various parts of the world, including Brazil. Data related to the reaction of rice cultivars to iron concentrations are limited, especially for large numbers of cultivars. Forty rice cultivars were grown in a greenhouse in nutrient solutions containing 0.09, 0.89, and 1.78 mM Fe (5, 50 and 100 ppm Fe). The effects of excess iron were measured on plant height, root length, and root and shoot dry weight. Root and shoot dry weight were found to be more sensitive to excess iron concentration. Based on dry matter yield, reduction of shoots at higher Fe concentrations compared to the optimum or control treatment, rice cultivars were classified as tolerant, moderately tolerant, moderately susceptible or susceptible.

The effect of Fe concentrations on concentrations and contents of other nutrient was also investigated. Higher concentration of Fe in the nutrient solution exerted an inhibiting effect on the concentrations and contents of almost all macro and micronutrients.     

Irrigation and Zn fertilizer management improves Zn phyto‐availability in various rice production systems

Zinc (Zn) is an important micronutrient for rice (Oryza sativa L.) production and its deficiency has been observed in various production systems. High grain Zn concentration is equally important for high rice yield and human health. In this work, the effects of Zn fertilization on seedling growth, grain yield, grain Zn concentration, and their association with root traits were studied under alternate wetting and drying (AWD), aerobic rice (AR), system of rice intensification (SRI), and continuous flooding (CF). Zinc fertilization (15 kg ha^?1) improved nursery seedlings chlorophyll and Zn concentrations, root length, and number of roots with highest values observed in CF. At harvesting, maximum plant height, panicle length, total and panicle bearing tillers, and kernel yield were found with Zn addition in AWD and CF rice systems. Mid season drainage provided at maximum tillering and Zn fertilization increased its concentration in leaves, culms, panicles, and grains under CF and AR at physiological maturity. Most of Zn applied was allocated into culms and panicles, nevertheless, a significant increase in grain Zn concentration was also observed in all production systems. Association of leaf Zn with grain Zn concentration was stronger than with culm and panicle Zn. The results indicate that Zn application after rice nursery transplanting is more important for grain Zn enrichment in all rice systems than for increase in grain yield in all systems except AWD where grain yield was also increased. More grain yield in CF and AWD as compared to SRI and AR can also be attributed to decreased spikelet sterility and to better Zn phyto‐availability in these rice systems at physiological maturity.     

Seeds of doubt: Re‐assessing the impact of grain P concentrations on seedling vigor

Interest in developing crop varieties with low grain phosphorus (P) in order to minimize the removal of P from fields in harvested grain has been limited due to the view that a low‐P grain trait may impair subsequent seedling vigor. This perception is based on relatively few studies, which typically investigated seedling growth on infertile soils, and used seed that may have differed in attributes other than P concentration. To investigate whether these anomalies cast sufficient doubt to warrant renewed research in this field, we compared the growth of rice seedlings from seed low in P obtained from P‐starved plants (P‐starved seed) vs. high‐P seeds (obtained from P‐fertilized plants) in P‐deficient and P‐replete soils. While plants from high‐P seed were superior in the P‐deficient soil, plants grown from P‐starved seed overcame an initial lull in early vigor to obtain similar biomass at maturity as plants grown from high‐P seed. Subsequent experiments were undertaken using high‐P seed vs. seed low in P from a range of rice genotypes that was not obtained from P‐stressed plants (low‐P seed): There was no reduction in seedling vigor or biomass and grain yields at maturity of plants from low‐P seeds in low‐P soil compared to plants from high‐P seed, though responses were genotype‐specific. The results suggest that multiple factors can confound the results of seed P × seedling vigor studies, and that a renewed research effort to define the minimum P levels in seeds required for adequate seedling growth across a range of environments is warranted.     

Subcellular distribution and chemical forms of chromium in rice plants suffering from different levels of chromium toxicity

The subcellular distribution and chemical forms of different heavy metals in rice are correlated with their bio‐toxicity. An experiment was conducted to investigate the subcellular distribution and chemical forms of chromium (Cr) in two rice genotypes (Oryza sativa L. cv. Xiushui 113 and cv. Dan K5) differing in Cr accumulation, to understand the mechanisms of Cr toxicity and resistance in rice plants. The results show that Cr in the roots of rice plants exposed to Cr stress was mainly localized in cell walls, whereas Cr in leaves and stems was mainly present in both cell walls and vacuoles, suggesting that both compartments act as important protective barriers against Cr toxicity in rice plants. Although Cr ions in all plant tissues exist predominantly in the forms extracted by 80% ethanol and distilled water, the amount of Cr in the chemical forms extracted by 2% HAc, 0.6 M HCl, and in residues was significantly increased under the highest Cr level (100 μM Cr) compared to the plants grown under lower Cr levels. These results indicate that excess Cr accumulated in rice plants under Cr stress is bound to undissolved or low‐bioavailable compounds, such as undissolved phosphate and oxalate, being beneficial for rice plants to alleviate Cr toxicity. In addition, under the highest Cr level (100 μM), Dan K5 had a higher percentage of Cr in the chemical forms extracted by 2% HAc, 0.6 M HCl, and in residues compared to Xiushui 113 in both stems and leaves, indicating that more Cr ions in shoots of Dan K5 were bound to undissolved or low‐bioavailable compounds, in comparison with those of Xiushui 113. It is evident that the low bioavailability of Cr in the shoots of Dan K5 is related to a high Cr accumulation.     

Impact of plant genotype and nitrogen level on rice growth response to inoculation with Azospirillum sp. strain B510 under paddy field conditions

Abstract

Twenty rice cultivars, including three genetically-distinct groups (japonica, indica-1, and indica-2), were evaluated for their response to inoculation with Azospirillum sp. strain B510 in paddy fields with standard nitrogen (SN) and low nitrogen (LN) fertilization. In the SN field, the tiller numbers in most indica-2 cultivars, 37?days after transplanting (DAT), were significantly increased by the B510 inoculation, whereas those in 4 japonica cultivars were significantly decreased. A similar growth response was observed in the LN field, although the impacts of the B510 inoculation were more varied than in the SN field. At 58 DAT, the tiller numbers in most cultivars were lower or unaffected by the B510 inoculation under both SN and LN conditions, except that the tiller number of the Nipponbare cultivar, which is classified as japonica, was significantly higher in the LN field only. These results suggest that the effects of inoculation with Azospirillum sp. strain B510 on the growth of rice plants, especially on tiller numbers at the early growth stage, vary depending on the rice genotype, as well as nitrogen level. Therefore, the plant genotypes, growth stages, and fertilization managements must be considered when a plant-associated bacterium is evaluated for beneficial effects under field conditions.