油菜种子发育早期的油体发生与调控

以甘蓝型油菜（Brassica napus L.）品种‘Westar’和‘Topas’为材料,通过超微结构观察和荧光定量PCR技术对油菜胚胎发育早期油体的发生、油体蛋白及脂肪酸合成转录因子基因的表达情况进行分析。结果显示：油体出现在油菜胚胎发育早期,在授粉9~11 d后（球形胚时期）的胚体和胚柄中均存在直径小于0.5 μm的油体;荧光定量实验结果表明,除BnCLO3的表达量在整个胚胎发育阶段无明显变化外,其他油体蛋白基因Oleosins、Steroleosins和BnCLO1的表达量在心形胚时期就明显增多并持续增长;脂肪酸合成转录因子BnLEC1、BnL1L、BnWRI1和BnFUS3在胚胎发育阶段,基因表达规律均呈先上升再下降的趋势,但达到最高值的时间存在差异,其中BnLEC1最早,BnL1L其次,BnWRI1和BnFUS3较晚。研究结果表明甘蓝型油菜在球形胚时期出现油体,其结构蛋白和转录调控因子基因的表达自心形胚开始明显增多。     

甘蓝型油菜油体数量及面积之和与含油量的相关性

利用荧光染料尼罗红染色和激光扫描共聚焦显微观察技术,建立了油菜油体观察或生物体内中性脂类物质定性鉴定的研究体系。对高油品种宁油14号、宁油18号、ZH-088和低油品种ZL-366、NjY008、Westar共6个甘蓝型油菜品种子叶贮藏细胞内的油体进行了观察。研究发现：油菜种子成熟过程中,油体从着色不明显的小颗粒,逐渐发育形成着色清晰的球状大油体。种子成熟干燥后,油体间很少发生聚合。在成熟干燥的种子中,油体集中分布于子叶贮藏细胞中央,呈椭圆形或不规则形状,较少为圆形。通过研究种子内油体与含油量的关系,发现高油品种组与低油品种组之间在单个子叶贮藏细胞内油体数量和截面积之和存在明显差异,而在高油品种组内或低油品种组内的差异不明显。结果显示,油菜种子细胞中油体的数量和总面积与含油量之间存在正相关,可作为高油分材料的选择依据。     

甘蓝型油菜油体数量及面积之和与含油量的相关性

利用荧光染料尼罗红染色和激光扫描共聚焦显微观察技术, 建立了油菜油体观察或生物体内中性脂类物质定性鉴定的研究体系。对高油品种宁油14号、宁油18号、ZH-088和低油品种ZL-366、NjY008、Westar共6个甘蓝型油菜品种子叶 贮藏细胞内的油体进行了观察。研究发现: 油菜种子成熟过程中, 油体从着色不明显的小颗粒, 逐渐发育形成着色清晰的球状大油体。种子成熟干燥后, 油体间很少发生聚合。在成熟干燥的种子中, 油体集中分布于子叶贮藏细胞中央, 呈椭圆形或不规则形状, 较少为圆形。通过研究种子内油体与含油量的关系, 发现高油品种组与低油品种组之间在单个子叶贮藏细胞内油体数量和截面积之和存在明显差异, 而在高油品种组内或低油品种组内的差异不明显。结果显示, 油菜种子细胞中油体的数量和总面积与含油量之间存在正相关, 可作为高油分材料的选择依据。     

不同品种油甘子果实营养成分评价

为系统分析评价不同品种油甘子(Phyllanthus emblica)果实的营养品质,以‘赤皮’‘玻璃油甘’‘东坑甜’‘饼甜’‘六月白’和‘马蹄甜’6个品种油甘子果实为材料,测定7项营养成分含量,运用主成分分析法对果实营养成分含量进行分析评价。结果表明,各品种油甘子维生素C和还原糖含量均较高;维生素C与蛋白质呈显著正相关,还原糖与可溶性固形物呈显著正相关。通过主成分分析提取出3个主成分,认为‘赤皮’和‘玻璃油甘’2个品种可作为油甘子丰产林建设的优良栽培品种。     

强休眠玉米种子休眠前后的蛋白差异表达

以强休眠玉米自交系08-641为试验材料,分别对处于休眠状态下的新鲜收获种子和经过10 d后熟作用破除休眠的种子进行了蛋白质组学差异表达分析。结果表明,通过双向电泳技术在3次重复试验下休眠状态的08-641鲜种子蛋白2-DE图谱上共检测到约600个蛋白质点,在经过10 d后熟作用破除休眠的08-641种子蛋白2-DE图谱上共检测到约620个蛋白质点,其中下调表达蛋白质点4个,上调表达蛋白质点4个,新增蛋白质点8个,缺失表达蛋白质点7个。经过质谱鉴定的差异表达蛋白质主要涉及球蛋白、胚胎晚期丰富蛋白、豆球蛋白等贮藏物蛋白质;蛋白酶体、山梨醇脱氢酶等参与物质代谢的蛋白质;热激蛋白等参与蛋白质结构、细胞功能调控的蛋白质。推测08-641种子休眠是由于种子内休眠相关蛋白的过量表达或缺失抑制了种子的正常萌发。     

油菜种子发育早期的油体发生与调控

以甘蓝型油菜( Brassica napus L.)品种‘Westar’和‘Topas’为材料,通过超微结构观察和荧光定量PCR技术对油菜胚胎发育早期油体的发生、油体蛋白及脂肪酸合成转录因子基因的表达情况进行分析。结果显示:油体出现在油菜胚胎发育早期,在授粉9 ~ 11 d后(球形胚时期)的胚体和胚柄中均存在直径小于0. 5 μm的油体;荧光定量实验结果表明,除 BnCLO3 的表达量在整个胚胎发育阶段无明显变化外,其他油体蛋白基因 Oleosins 、 Steroleosins 和 BnCLO1 的表达量在心形胚时期就明显增多并持续增长;脂肪酸合成转录因子 BnLEC1 、 BnL1L 、 BnWRI1 和 BnFUS3 在胚胎发育阶段,基因表达规律均呈先上升再下降的趋势,但达到最高值的时间存在差异,其中 BnLEC1 最早, BnL1L 其次, BnWRI1 和 BnFUS3 较晚。研究结果表明甘蓝型油菜在球形胚时期出现油体,其结构蛋白和转录调控因子基因的表达自心形胚开始明显增多。     

‘哲引3号’ב北京杨’杂交子代悬浮细胞系的建立和植株再生

采用“固—液—液—固”培养方法,分别以［‘哲引3号杨’（Populus pseudo-simonii×P.nigra ‘Zhenyin3#’）ב北京杨’（P.×beijingensis）］杂交子代的种子、子叶和下胚轴为材料,开展了多个杂交子代悬浮细胞系建立和培养研究,结果显示：（1）不同基因型的种子愈伤诱导率差异显著,不同基因型的子叶、下胚轴愈伤诱导率差异不显著;不同基因型的初始悬浮细胞系密实体积差异均显著。（2）采用静置分层和细胞筛双层过滤结合的方法能把游离胚性细胞从初始悬浮细胞系中分离出来,不同基因型来源的游离胚性细胞数量差异不显著。（3）种子和子叶来源的胚性悬浮细胞系能在不含NH+4的液体培养基中诱导出球形愈伤,这些愈伤薄片能在含有CPPU的分化培养基上实现植株再生,最后共获得了20个基因型的来自种子球形愈伤的再生植株和另外20个基因型的来自子叶球形愈伤的再生植株。     

盐胁迫对不同品种小白菜种子萌发特性的影响

采用5个小白菜(Brassica chinensis)品种(‘七宝青’、‘夏冬青’、‘四月慢’、‘南京中杆’和‘605’),用100 mmol/L NaCl对种子发芽作盐胁迫处理,从发芽势、发芽率、发芽指数、盐害指数、胚长和胚鲜重等指标比较了5个小白菜品种的抗盐性。结果表明,‘七宝青’种子的耐盐性优于其它品种。     

油菜叶片总蛋白质双向电泳样品制备方法的改进

以甘蓝型油菜"扬油6号"的叶片为试验材料,分别采用传统的TCA/Acetone(三氯乙酸/丙酮沉淀法)和改进的PEG(polyethylene glycol)分步提取法提取叶片可溶性总蛋白,并利用条件一致的蛋白质双向电泳体系进行比较。TCA/Acetone法提取的蛋白质双向电泳图谱背景中由于高丰度"housekeeping"结构蛋白的存在,特别是叶片中参与光合作用的Rubisco蛋白的干扰,图谱中低丰度调控蛋白受到了高度覆盖和遮蔽现象,影响双向电泳图谱的质量。而PEG分步提取法提取的蛋白质样品,可以剔除Rubisco蛋白,使获得的双向电泳图谱清晰,无斑点间的遮蔽现象,为油菜叶片蛋白质组定量和定性分析提供了丰富的信息。     

干旱胁迫下不同抗旱水平陆地棉的叶片蛋白质组学比较研究

为了探讨陆地棉品种抗旱机理,以陆地棉抗旱品种‘中H177’和不抗旱品种‘中S9612’为材料,运用双向电泳结合质谱技术,分析干旱胁迫下不同陆地棉三叶期叶片蛋白质组分差异变化。结果表明:干旱胁迫下,不同陆地棉叶片蛋白表达差异较大;‘中H177’出现30个差异表达蛋白质点,‘中S9612’出现47个差异表达蛋白质点,只在‘中H177’表达差异的蛋白点11个,只在‘中S9612’表达差异的蛋白点28个,差异表达一致蛋白点8个,表达不一致蛋白点11个。质谱共鉴定出43个差异表达蛋白;功能分类分析表明,干旱胁迫蛋白参与光合作用、物质与能量代谢、抗逆相关蛋白、物质运输和活性氧清除;Rubisco活化酶和能量代谢相关蛋白ATP合成酶类表达差异最大。研究结果可以初步为陆地棉抗旱机理的探讨提供一定的理论基础。     

Fusion of oil bodies in endosperm of oat grains

Few microscopical studies have been made on lipid storage in oat grains, with variable results as to the extent of lipid accumulation in the starchy endosperm. Grains of medium- and high-lipid oat (Avena sativa L.) were studied at two developmental stages and at maturity, by light microscopy using different staining methods, and by scanning and transmission electron microscopy. Discrete oil bodies occurred in the aleurone layer, scutellum and embryo. In contrast, oil bodies in the starchy endosperm often had diffuse boundaries and fused with each other and with protein vacuoles during grain development, forming a continuous oil matrix between the protein and starch components. The different microscopical methods were confirmative to each other regarding the coalescence of oil bodies, a phenomenon probably correlated with the reduced amount of oil-body associated proteins in the endosperm. This was supported experimentally by SDS-PAGE separation of oil-body proteins and immunoblotting and immunolocalization with antibodies against a 16 kD oil-body protein. Much more oil-body proteins per amount of oil occurred in the embryo and scutellum than in the endosperm. Immunolocalization of 14 and 16 kD oil-body associated proteins on sectioned grains resulted in more heavy labeling of the embryo, scutellum and aleurone layer than the rest of the endosperm. Observations on the appearance of oil bodies at an early stage of development pertain to the prevailing hypotheses of oil-body biogenesis.     

子莲新品种‘武植子莲1号’和‘武植子莲2号’产量与营养品质分析

对子莲（Nelumbo nucifera Gaertn.）新品种‘武植子莲1号’和‘武植子莲2号’与其他12个主栽子莲品种的莲子产量和品质性状进行分析,并通过隶属函数分析法对他们的营养品质性状进行综合评价。结果显示：不同子莲品种的产量和营养品质性状差异显著,同一品种在不同发育时期莲子的可溶性糖和淀粉含量等营养指标差异较大;鲜莲子的可溶性糖含量显著高于成熟莲子,而蛋白质和淀粉含量显著低于成熟莲子。与12个主栽子莲品种相比,‘武植子莲1号’在产量上较为突出,成熟莲子的淀粉含量达52.15%,是生产天然淀粉的优良品种;‘武植子莲2号’鲜莲子的直链淀粉和支链淀粉含量低,可溶性糖含量达23.43%,适合鲜食。隶属函数分析结果表明,‘武植子莲1号’和‘武植子莲2号’的综合营养品质较好,具有很高的应用价值。     

Size and stability of reconstituted sesame oil bodies

Oil bodies of sesame seeds comprise a triacylglycerol matrix, which is surrounded by a monolayer of phospholipids embedded with unique proteins, mainly structural proteins termed oleosins. Artificial oil bodies were successfully reconstituted with various compositions of triacylglycerols, phospholipids, and oil-body proteins. The sizes of reconstituted oil bodies displayed a normal distribution with an average size proportional to the ratio of triacylglycerols to oil-body proteins. Both thermostability and structural stability of reconstituted oil bodies decreased as their sizes increased, and vice versa. Proteinase K digestion indicated that oleosins anchored both native and reconstituted oil bodies via their central hydrophobic domains. The stability of reconstituted oil bodies, as well as the purified ones from sesame seeds, could be substantially enhanced after their surface proteins were cross-linked by glutaraldehyde or genipin.     

Oleosins prevent oil-body coalescence during seed imbibition as suggested by a low-temperature scanning electron microscope study of desiccation-tolerant and -sensitive oilseeds

In order to clarify further the physiological role of oleosins in seed development, we characterized the oil-body proteins of several oilseeds exhibiting a range of desiccation sensitivities from the recalcitrant (Theobroma cacao L., Quercus rubra L.), intermediate (Coffea arabica L., Azadirachta indica A. Juss.) and orthodox categories (Sterculia setigera Del., Brassica napus L.). The estimated ratio of putative oleosins to lipid in oil bodies of Q. rubra was less than 5% of the equivalent values for rapeseed oil bodies. No oleosin was detected in T. cacao oil bodies. In A. indica cotyledons, oil bodies contained very low amounts of putative oleosins. Oil bodies both from C. arabica and S. setigera exhibited a similar ratio of putative oleosins to lipid as found in rapeseed. In C. arabica seeds, the central domain of an oleosin was partially sequenced. Using a low temperature field-emission scanning electron microscope, the structural stability of oil bodies was investigated in seeds after drying, storage in cold conditions and rehydration. Despite the absence or relative dearth of oleosins in desiccation-sensitive, recalcitrant oilseeds, oil bodies remained relatively stable after slow or fast drying. In A. indica seeds exposed to a lethal cold storage treatment, no significant change in oil-body sizes was observed. In contrast, during imbibition of artificially dried seeds containing low amounts of putative oleosins, the oil bodies fused to form large droplets, resulting in the loss of cellular integrity. No damage to the oil bodies occurred in imbibed seeds of Q. rubra, C. arabica and S. setigera. Thus the rehydration phase appears to be detrimental to the stability of oil bodies when these are present in large amounts and are lacking oleosins. We therefore suggest that one of the functions of oleosins in oilseed development may be to stabilize oil bodies during seed imbibition prior to germination. Received: 22 April 1997 / Accepted: 5 June 1997     

Oleosin genes in maize kernels having diverse oil contents are constitutively expressed independent of oil contents

In seeds, the subcellular storage oil bodies have a matrix of oils (triacylglycerols) surrounded by a layer of phospholipids embedded with abundant structural proteins called oleosins. We used two maize (Zea mays L.) strains having diverse kernel (seed) oil contents to study the effects of varying the oil and oleosin contents on the structure of the oil bodies. Illinois High Oils (IHO, 15% w/w oils) and Illinois Low Oils (ILO, 0.5%) maize kernels were the products of breeding for diverse oil contents for about 100 generations. In both maize strains, although the genes for oil synthesis had apparently been modified drastically, the genes encoding oleosins appeared to be unaltered, as revealed by Southern blot analyses of the three oleosin genes and sodium dodecyl sulfate-polyacrylamide gel electrophoresis with immunoblotting of the oleosins. In addition, both strains contained the same three oleosin isoforms of a defined proportion, and both accumulated oils and oleosins coordinately. Oleosins in both strains were restricted to the oil bodies, as shown by analyses of the various subcellular fractions separated by sucrosedensity-gradient centrifugation. Electron microscopy of the embryos and the isolated organelles revealed that the oil bodies in IHO were larger and had a spherical shape, whereas those in ILO were smaller and had irregular shapes. We conclude that in seeds, oleosin genes are expressed independent of the oil contents, and the size and shape of the oil bodies are dictated by the ratio of oils to oleosins synthesized during seed maturation. The extensive breeding for diverse oil contents has not altered the apparent mechanism of oil-body synthesis and the occurrence of hetero-dimer or -multimer of oleosin isoforms on the oil bodies.Abbreviations IHO Illinois High Oils - ILO Illinois Low Oils This work was supported by a USDA NRICGP grant. We thank Dr. J.W. Dudley of the University of Illinois for the IHO and ILO maize kernels, and Dr. W. Thomson for discussion on the stereological method.     

Photosynthetic characteristics in different peanut cultivars under conditions of drought and re-watering at seedling stage

Aims In China, peanut (Arachis hypogaea) is mainly cultivated in the semi-arid and rain-fed areas, and drought is the most prominent environmental stress to its growth. However, studies on the physiological responses of different peanut cultivars to drought and re-watering are lacking. Our objectives were to investigate the relationship between photosynthetic characteristics and drought tolerance, and to explore the ability to recover from drought damage in different peanut cultivars.
Methods A pot experiment was conducted with artificial water stress treatment, and the photosynthetic characteristics were determined in twelve peanut cultivars under the conditions of drought stress and re-watering at the seedling stage. The drought tolerance was assessed by drought resistance coefficient of biomass in seedling. The recovery capacity was assessed by compensatory growth of plant.
Important findings Five cultivars, including ‘Shanhua 11’, ‘Rugaoxiyangsheng’, ‘A596’, ‘Shanhua 9’, and ‘Nongda 818’, showed over-compensatory growth after re-watering, and their capacity of compensatory growth had significant positive correlation with drought tolerance (p < 0.01). The net photosynthetic rate (P_n), stomatal conductance (G_s), intercellular CO₂ concentration (C_i), maximum photochemical efficiency (F_v/F_m), PSII actual quantum yield (ΦPSII), and photochemical quenching coefficient (q_P) all decreased over the course of drought stress, and then increased following re-watering, with the amplitude of changes being smaller in the more drought tolerant cultivars. Seven days of drought did not result in significant differences in the photosynthetic characteristics among majority of the peanut cultivars tested (p > 0.05). After 14 days of drought, the values of photosynthetic variables differed significantly among the peanut cultivars with different drought tolerance (p < 0.05). The values of P_n, G_s, Φ_PSII, F_v/F_m, and q_P in the cultivars ‘Shanhua 11’, ‘Rugaoxiyangsheng’, ‘A596’, and ‘Shanhua 9’fully recovered five days after re-watering, while those in the cultivars ‘79266’, ‘ICG6848’, ‘Baisha 1016’, and ‘Hua 17’ did not fully recover even after 10 days of re-watering; the values of those photosynthetic variables were significantly greater (p < 0.05) in the more drought tolerant cultivars following re-watering. Correlation analysis showed that the drought tolerance was significantly and positively correlated with P_n, Φ_PSII, F_v/F_m, and q_P after 14 days of drought stress and after five days of re-watering, respectively (p < 0.01). Therefore, under drought stress at 40% of relative water content (RWC) for 14 days and after five days of re-watering at the seedling stage, the P_n, Φ_PSII, F_v/F_m, and q_P could be used for identifying the level of damage and recovery capacity of peanut cultivars. The cultivar ‘Shanhua 11’ can be used as a reference for drought adaptability identification in peanut.     

Protein and lipid composition analysis of oil bodies from two Brassica napus cultivars

Oil bodies were purified from mature seed of two Brassica napus crop cultivars, Reston and Westar. Purified oil body proteins were subjected to both 2-DE followed by LC-MS/MS and multidimensional protein identification technology. Besides previously known oil body proteins oleosin, putative embryo specific protein ATS1, (similar to caleosin), and 11-beta-hydroxysteroid dehydrogenase-like protein (steroleosin), several new proteins were identified in this study. One of the identified proteins, a short chain dehydrogenase/reductase, is similar to a triacylglycerol-associated factor from narrow-leafed lupin while the other, a protein annotated as a myrosinase associated protein, shows high similarity to the lipase/hydrolase family of enzymes with GDSL-motifs. These similarities suggest these two proteins could be involved in oil body degradation. Detailed analysis of the two other oil body components, polar lipids (lipid monolayer) and neutral lipids (triacylglycerol matrix) was also performed. Major differences were observed in the fatty acid composition of polar lipid fractions between the two B. napus cultivars. Neutral lipid composition confirmed erucic acid and oleic acid accumulation in Reston and Westar seed oil, respectively.     

Synthesis of the major oil-body membrane protein in developing rapeseed (Brassica napus) embryos. Integration with storage-lipid and storage-protein synthesis and implications for the mechanism of oil-body formation.

The synthesis of the major protein and lipid storage reserves during embryogenesis in oilseed rape (Brassica napus L., cv. Mikado) has been examined by biochemical, immunological and immunocytochemical techniques. The mature seeds contained about 45% (w/w) storage oil and 25% (w/w) protein. There were three major seed protein components, i.e. about 40-50% total protein was cruciferin, 20% was napin and 20% was a 18 kDa hydrophobic polypeptide associated with the proteinaceous membrane surrounding the storage oil bodies. Embryogenesis was divided into four overlapping stages with regard to the synthesis of these storage components: (1) for the first 3 weeks after flowering, little, if any, synthesis of storage components was observed; (2) storage-oil synthesis began at about week 3, and maximal rates were from weeks 4 to 7; (3) synthesis of the soluble storage proteins cruciferin and napin started at week 6 and rates were maximal between weeks 8 and 11; (4) the final stage was the synthesis of the 19 kDa oil-body polypeptide, which started at weeks 8-10 and was at a maximal rate between weeks 10 and 12. The synthesis of the 19 kDa oil-body protein therefore occurred independently of the synthesis of the soluble seed storage proteins. This former synthesis did not occur until shortly before the insertion of the 19 kDa polypeptide into the oil-body membrane. No evidence was found, either from sucrose-density-gradient-centrifugation experiments or from immunogold-labelling studies, for its prior accumulation in the endoplasmic reticulum. Conventional and immunogold-electron-microscopic studies showed that oil bodies were synthesized in the early to middle stages of seed development without a strongly electron-dense membrane. Such a membrane was only found at later stages of seed development, concomitantly with the synthesis of the 19 kDa protein. It is proposed that, in rapeseed embryos, oil bodies are initially formed with no proteinaceous membrane. Such a membrane is formed later in development after insertion by ribosomes of the hydrophobic 19 kDa polypeptide directly into the oil bodies.     

Oleate desaturation and acyl turnover in sunflower (Helianthus annuus L.) seed lipids during rapid temperature adaptation

In-vivo experiments with developing sunflower (Helianthus annuus L.) seeds demonstrated that oleate desaturase activity was stimulated by low temperature (10 °C), repressed by high temperature (30 °C) and rapidly restored by returning the seeds to low temperature. Within time periods of 2–4 h, in which the de-novo fatty acid synthesis was negligible, the percentages of oleate (18:1) and linoleate (18:2) were modified in the seed lipids as a consequence of temperature adaptation. When the seeds were transferred to low temperature, the 18:2 content increased in all lipids from both microsomal membranes and oil bodies. After shifting to high temperature, the overall 18:2 content remained constant, but the 18:2 content decreased in diacylglycerols, phosphatidylcholine (PC) and other polar lipids of the two fractions and also in triacylglycerols (TAGs) of the microsomes but increased in TAGs of the oil bodies. The results indicate that the mechanism for the rapid adaptation of sunflower seeds to temperature changes involves (i) the synthesis or activation of oleate desaturase at low temperature and the reversible inhibition of this enzyme at high temperature and (ii) the exchange of 18:1 and 18:2 between TAGs and PC. Under both low and high temperature, 18:1 is transferred from reserve TAGs to PC and 18:2 is transferred from PC to reserve TAGs. At low temperature, 18:1 is desaturated to 18:2 thus allowing the enrichment of membrane lipids with 18:2, the excess being stored in reserve TAGs. At high temperature, however, and provided that oleate desaturase is repressed, the membrane lipids become enriched in 18:1 and the oil-body TAGs become enriched in 18:2. Received: 11 August 1997 / Accepted: 10 November 1997