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。     

甘蓝型油菜种子中油体的超微结构及蛋白质组分析

以甘蓝型油菜（Brassica napus L.）含油量较高的品种‘ZS11’、含油量中等的品种‘Westar’和‘Topas’以及含油量较低的品种‘ZS10’为实验材料,通过超微结构观察和统计,比较分析不同品种种子中油体形态、大小和数量的差异。研究结果显示,品种‘ZS11’种子子叶细胞油体排列致密,形态较小,大部分油体的直径低于1 μm;而在含油量中等或较低的品种中,种子子叶细胞油体排列均显疏松,其中‘Westar’和‘Topas’的油体较大,而‘ZS10’的油体大小不一。本研究还通过双向电泳分析进一步检测了‘Westar’和‘ZS11’种子中总蛋白和油体蛋白的差异表达情况。结果显示,‘Westar’和‘ZS11’种子总蛋白双向电泳图谱中,表达量具有2倍以上差异的蛋白质点共有57个;其中在‘Westar’中特异表达的种子总蛋白质点有24个,在‘ZS11’中有23个。在上述2个品种油体蛋白双向电泳图谱中,表达量具有2倍以上差异的蛋白质点共有52个,在品种‘Westar’中特异表达的有2个,‘ZS11’中有13个。表明不同含油量的油菜品种种子在油体的结构和蛋白组份上均存在差异。     

拟南芥菜(Arabidopsis thaliana)子叶中蛋白体形成的超微结构和免疫电镜定位研究

本文对拟南芥菜(Arabidopsis thaliana)种子发育过程中贮藏蛋白的积累和蛋白体的形成进行了超微结构和免疫电镜定位的研究。常规超薄切片的电镜观察表明,在开花后第10天(10 DAF),高电子密度的蛋白质物质开始在子叶细胞的液泡中沉积。这一过程一直延续到种子接近成熟(14 DAF),这时液泡中充满了蛋白质物质,转变成为大的蛋白体。利用了该种植物主要种子贮藏蛋白之一的12 s球蛋白的单克隆抗体作为免疫探针,以蛋白质A-胶体金电镜技术对12 s种子蛋白进行了细胞内定位,证实了在液泡中积累的物质为种子贮藏蛋白。实验结果表明在拟南芥菜中,子叶细胞中的液泡是蛋白体的前体,肯定了蛋白体的发生起源于液泡的观点。本文还对应用胶体金电镜技术进行细胞内定位的某些问题作了初步探讨。     

西瓜种子发育和萌发过程中子叶细胞超微结构的变化

西瓜种子子叶内贮存物质开始积累时,细胞质内有大量核糖体、质体、线粒体,内质网片段和囊泡,种子脱水期至成熟期,细胞器的数量减少,成熟种子子叶细胞的细胞壁不连续,几乎观察不到细胞器的存在,种子萌发过程中内质网,线粒体,质体的数目逐渐增多,叶肉细胞的质体发育成叶绿体,种子形成过程中,在子叶细胞大液泡分隔的同时,膨胀的内质网囊泡内积累蛋白质（直径0.1-0.4μm),这些小的蛋白质球体最终进入液泡形成大的蛋白体（直径1-3μm);萌发种子贮存蛋白质被水解的同时,一些脂体进入液泡并被分解,同时液泡融合;脂类物质开始积累的时间早于蛋白质,积累的量较蛋白质多,但在萌发种子中被彻底水解的时间晚于蛋白质,淀粉粒的数量在种子形成时减少,种子萌发时在表皮细胞和叶肉细胞内都重新合成。     

莲种子萌发和幼苗生长时期营养物质的代谢变化

莲子叶细胞中储存了丰富的营养物质, 主要为蛋白质、淀粉和淀粉质体DNA。这些贮藏物质为种子萌发和幼苗的生长提供必需的能量和养料。通过组织化学和显微镜观察, 研究莲从种子萌发到植株生长至具有4个节时, 子叶中贮藏物质消耗的全过程。在此过程中, 子叶中的贮藏物质不断降解,营养物质发生转运。蛋白体首先发生降解, 其大量降解主要发生在幼苗三叶期。淀粉质体降解时会聚 集成团, 之后体积逐渐减小, 最后完全降解。种子萌发后65天是子叶贮藏物质消耗末期, 淀粉质体DNA的含量比萌发后20天的三叶期明显减少。细胞壁的形态结构发生多种形式的变化, 细胞壁发生的这些变化与子叶细胞间物质的运输有关。含多糖的球形颗粒通过维管束在子叶中运输。     

莲种子萌发和幼苗生长时期营养物质的代谢变化

莲子叶细胞中储存了丰富的营养物质,主要为蛋白质、淀粉和淀粉质体DNA.这些贮藏物质为种子萌发和幼苗的生长提供必需的能量和养料.通过组织化学和显微镜观察,研究莲从种子萌发到植株生长至具有4个节时,子叶中贮藏物质消耗的全过程.在此过程中,子叶中的贮藏物质不断降解,营养物质发生转运.蛋白体首先发生降解,其大量降解主要发生在幼苗三叶期.淀粉质体降解时会聚集成团,之后体积逐渐减小,最后完全降解.种子萌发后65天是子叶贮藏物质消耗末期,淀粉质体DNA的含量比萌发后20天的三叶期明显减少.细胞壁的形态结构发生多种形式的变化,细胞壁发生的这些变化与子叶细胞间物质的运输有关.含多糖的球形颗粒通过维管束在子叶中运输.     

马占相思子叶离体培养中细胞早期变化的研究

用光学和电子显微镜观察马占相思子叶在离体培养中的细胞早期变化,结果表明,未培养的子叶薄壁细胞内充满大量贮藏物质,在脱分化过程中,贮藏物质逐渐减少,细胞核体积增大,液泡蛋白体出现,质体转化为原质体,线粒体,内质网等细胞器数量增加,马脱分化启动后的细胞分裂方式进行了讨论。     

春秋花生种子细胞化学的比较研究

采用泉花10号,汕油71的春秋花生种子为材料,利用细胞化学方法,在环氧树脂厚切片中分别显示出下胚轴和子叶中的多糖、蛋白质和脂类,并对其数量、形态及分布进行比较.结果表明,春花生种子下胚轴细胞较大,且内部结构清晰,排列规则,发育充分,而秋花生种子成熟度较低.对于子叶细胞,在春花生中,其贮藏物质丰富含大量的脂体、蛋白体,排列紧密;而秋花生细胞内部结构疏松,液泡中尚未充满蛋白质,但淀粉粒似较多.这就从细胞学的角度解释了生产上采用春花生种子的优越性..另外,在方法上对环氧树脂厚切片中多糖、蛋白质、脂类的细胞化学染色方法和染色保存进行了讨论.     

油菜高含油量基因工程研究

油菜是世界上重要油料作物之一,是世界食用植物油的重要来源。近十年来,随着其种植面积的不断扩大,目前已成为世界第二大植物油来源,因此提高油菜种子含油量具有重大的经济利用价值。近年来,基因工程技术的飞速发展带来了优化油菜品种资源的新方法。三酰甘油对种子油脂的形成十分重要,它是油菜种子最主要的储藏脂类。将三酰甘油合成代谢途径中的关键酶基因及一些转录因子转入到油菜组基因中,一方面增加种子中关键酶基因的表达;另一方面增加转录因子表达以增强糖酵解和三酰甘油形成的相关基因表达,增加底物浓度和三酰甘油合成的速度,期待获得高含油量的转基因油菜。本文综述了国内外关于油菜油酯代谢关键酶基因及调控基因的研究进展,并展望了未来提高油菜含油量的发展思路。     

两种品质类型花生品质形成与子叶细胞超微结构的关系

在大田条件下研究了两种品质类型花生(Arachis hypogaea)品质形成的动态差异及其子叶细胞超微结构的差异。结果表明, 高蛋白品种‘XB023’的蛋白质含量在籽仁发育前期较高油品种‘鲁花9号’低, 后期显著高于‘鲁花9号’, 且成熟期籽仁8种必需氨基酸组分含量均高于‘鲁花9号’, 其中谷氨酸、赖氨酸和亮氨酸含量差异极显著; ‘XB023’脂肪含量在籽仁发育期一直低于‘鲁花9号’。‘XB023’各时期的籽仁可溶性糖含量和油酸/亚油酸(O/L)值均显著低于‘鲁花9号’。两品种在果针入土10天时子叶细胞即形成淀粉粒、脂体和蛋白体, 随后脂体、蛋白体的数量不断增加, 淀粉粒先增大后逐渐缩小解体。‘XB023’的脂体达到最大的时间早于‘鲁花9号’, 而‘鲁花9号’的脂体快速积累的时间比‘XB023’长。两品种蛋白体大小都在果针入土40天时达到最大值, ‘XB023’的蛋白体在籽仁发育后期数量增加较快。高蛋白品种较高的蛋白质含量由其子叶细胞中较大蛋白体的大小和较多的蛋白体数量决定, 而高油品种较高的脂肪含量是由其较多的脂体数量决定。     

Seed Structure Characteristics to Form Ultrahigh Oil Content in Rapeseed

Background

Rapeseed (Brassica napus L.) is an important oil crop in the world, and increasing its oil content is a major breeding goal. The studies on seed structure and characteristics of different oil content rapeseed could help us to understand the biological mechanism of lipid accumulation, and be helpful for rapeseed breeding.

Methodology/Principal Findings

Here we report on the seed ultrastructure of an ultrahigh oil content rapeseed line YN171, whose oil content is 64.8%, and compared with other high and low oil content rapeseed lines. The results indicated that the cytoplasms of cotyledon, radicle, and aleuronic cells were completely filled with oil and protein bodies, and YN171 had a high oil body organelle to cell area ratio for all cell types. In the cotyledon cells, oil body organelles comprised 81% of the total cell area in YN171, but only 53 to 58% in three high oil content lines and 33 to 38% in three low oil content lines. The high oil body organelle to cotyledon cell area ratio and the cotyledon ratio in seed were the main reasons for the ultrahigh oil content of YN171. The correlation analysis indicated that oil content is significantly negatively correlated with protein content, but is not correlated with fatty acid composition.

Conclusions/Significance

Our results indicate that the oil content of YN171 could be enhanced by increasing the oil body organelle to cell ratio for some cell types. The oil body organelle to seed ratio significantly highly positively correlates with oil content, and could be used to predict seed oil content. Based on the structural analysis of different oil content rapeseed lines, we estimate the maximum of rapeseed oil content could reach 75%. Our results will help us to screen and identify high oil content lines in rapeseed breeding.     

Influence of microgravity on ultrastructure and storage reserves in seeds of Brassica rapa L.

Successful plant reproduction under spaceflight conditions has been problematic in the past. During a 122 d opportunity on the Mir space station, full life cycles of Brassica rapa L. were completed in microgravity in a series of three experiments in the Svet greenhouse. Ultrastructural and cytochemical analyses of storage reserves in mature dry seeds produced in these experiments were compared with those of seeds produced during a high-fidelity ground control. Additional analyses were performed on developing Brassica embryos, 15 d post pollination, which were produced during a separate experiment on the Shuttle (STS-87). Seeds produced on Mir had less than 20% of the cotyledon cell number found in seeds harvested from the ground control. Cytochemical localization of storage reserves in mature cotyledons showed that starch was retained in the spaceflight material, whereas protein and lipid were the primary storage reserves in ground control seeds. Protein bodies in mature cotyledons produced in space were 44% smaller than those in the ground control seeds. Fifteen days after pollination, cotyledon cells from mature embryos formed in space had large numbers of starch grains, and protein bodies were absent, while in developing ground control seeds at the same stage, protein bodies had already formed and fewer starch grains were evident. These data suggest that both the late stage of seed development and maturation are changed in Brassica by growth in a microgravity environment. While gravity is not absolutely required for any step in the plant life cycle, seed quality in Brassica is compromised by development in microgravity.     

Design of New Genome- and Gene-Sourced Primers and Identification of QTL for Seed Oil Content in a Specially High-Oil Brassica napus Cultivar

Rapeseed (Brassica napus L.) is one of most important oilseed crops in the world. There are now various rapeseed cultivars in nature that differ in their seed oil content because they vary in oil-content alleles and there are high-oil alleles among the high-oil rapeseed cultivars. For these experiments, we generated doubled haploid (DH) lines derived from the cross between the specially high-oil cultivar zy036 whose seed oil content is approximately 50% and the specially low-oil cultivar 51070 whose seed oil content is approximately 36%. First, to address the deficiency in polymorphic markers, we designed 5944 pairs of newly developed genome-sourced primers and 443 pairs of newly developed primers related to oil-content genes to complement the 2244 pairs of publicly available primers. Second, we constructed a new DH genetic linkage map using 527 molecular markers, consisting of 181 publicly available markers, 298 newly developed genome-sourced markers and 48 newly developed markers related to oil-content genes. The map contained 19 linkage groups, covering a total length of 2,265.54 cM with an average distance between markers of 4.30 cM. Third, we identified quantitative trait loci (QTL) for seed oil content using field data collected at three sites over 3 years, and found a total of 12 QTL. Of the 12 QTL associated with seed oil content identified, 9 were high-oil QTL which derived from the specially high-oil cultivar zy036. Two high-oil QTL on chromosomes A2 and C9 co-localized in two out of three trials. By QTL mapping for seed oil content, we found four candidate genes for seed oil content related to four gene markers: GSNP39, GSSR161, GIFLP106 and GIFLP046. This information will be useful for cloning functional genes correlated with seed oil content in the future.     

植物油体在生物技术中的应用研究进展

油体是植物种子尤其是油料植物种子的重要贮脂细胞器,具有较强的物化稳定性,而且易于通过离心法分离提取。研究表明,油体是由外层的磷脂和油体结合蛋白以及包裹在内部的液态基质（主要为三酰甘油）形成的弹性球体或椭球体。目前,在植物中共发现三类油体结合蛋白,它们主要存在于油体表面。鉴于油体和油体结合蛋白的结构特殊性,二者在生物技术领域得到了广泛应用。本文重点综述了油体在表达纯化外源蛋白方面的优势、策略以及在生产药用蛋白、制备固定化酶、捕捉抗体和药用酶、生产营养素和提高植物抗性等多个领域的研究进展,并介绍了人工油体和油体乳化剂方面的开发应用情况。     

Differential,temporal and spatial expression of genes involved in storage oil and oleosin accumulation in developing rapeseed embryos: implications for the role of oleosins and the mechanisms of oil-body formation

The temporal and spatial expression of oleosin and ₉-stearoyl-ACP desaturase genes and their products has been examined in developing embryos of rapeseed, Brassica napus L. var. Topas. Expression of oleosin and stearate desaturase genes was measured by in situ hybridisation at five different stages of development ranging from the torpedo stage to a mature-desiccating embryo. The temporal pattern of gene expression varied dramatically between the two classes of gene. Stearate desaturase gene expression was relatively high, even at the torpedo stage, whereas oleosin gene expression was barely detectable at this stage. By the stage of maximum embryo fresh weight, stearate desaturase gene expression had declined considerably while oleosin gene expression was at its height.In contrast to their differential temporal expression, the in situ labelling of both classes of embryo-specific gene showed similar, relatively uniform patterns of spatial expression throughout the embryo sections. Immunogold labelling of ultra-thin sections from radicle tissue with anti-oleosin antibodies showed similar patterns to sections from cotyledon tissue. However, whereas at least three oleosin isoforms were detectable on western blots of homogenates from cotyledons, only one isoform was found in radicles. This suggests that some of the oleosin isoforms may be expressed differentially in the various types of embryo tissue. The differential timing of stearate desaturase and oleosin gene expression was mirrored by similar differences in the timing of the accumulation of their ultimate products, i.e. storage oil and oleosin proteins. Oil-body fractions prepared from young (2.5 mg) embryos contained very little oleosin protein, as examined by SDS-PAGE and western blotting, whereas identically prepared fractions from dry seeds contained over 10% (w/w) oleosin. Dehydration of oil bodies from young embryos resulted in their breakdown and coalescence into large clumps of oil which could not be re-emulsified, even after rehydration. In contrast, the oleosin-rich oil bodies from mature embryos were stable to dehydration and subsequent rehydration. It is suggested that, in developing rapeseed embryos, the accumulation of storage oil and oleosins is not concomitant but that the eventual deposition of oleosins onto the surfaces of storage oil bodies is essential for their stability during seed desiccation.Abbreviations ABA abscisic acid - ACP acyl carrier protein - GLC gas-liquid chromatography - PBS phosphate-buffered saline     

黄连木果实中油体的发育

黄连木（Pistacia chinensis）是一种重要的木本油料植物,其果实中贮存着大量的油脂,这些油脂分子主要存在于果皮、种皮和胚的油体中。在光学显微镜下观察发现,果皮中油的积累开始于果实发育晚期,果皮开始变红时;种皮中油体的发育开始于果实发育早期;胚中油体的发育开始于球形胚时期。透射电子显微镜观察结果显示,种皮和胚中的油体形成于内质网,而果皮中的油体则分别由内质网、质体和液泡形成。尼罗红荧光标记显示,内质网形成的油体始终以独立单元的形式存在。种皮和胚中也贮藏蛋白体,但发育晚于油体。果皮、种皮和子叶中都贮存少量的淀粉粒。     

Expression and subcellular targeting of a soybean oleosin in transgenic rapeseed. Implications for the mechanism of oil-body formation in seeds

Two genomic clones, encoding isoforms A and B of the 24 kDa soybean oleosin and containing 5 kbp and 1 kbp, respectively, of promoter sequence, were inserted separately into rapeseed plants. T₂ seeds from five independent transgenic lines, three expressing isoform A and two expressing isoform B, each containing one or two copies of the transgene, were analysed in detail. In all five lines, the soybean transgenes exhibited the same patterns of mRNA and protein accumulation as the resident rapeseed oleosins, i.e. their expression was absolutely seed-specific and peaked at the mid-late stages of cotyledon development. The 24 kDa soybean oleosin was targeted to and stably integrated into oil bodies, despite the absence of a soybean partner isoform. The soybean protein accumulated in young embryos mainly as a 23 kDa polypeptide, whereas a 24 kDa protein predominated later in development. The ratio of rapeseed:soybean oleosin in the transgenic plants was about 5:1 to 6:1, as determined by SDS-PAGE and densitometry. Accumulation of these relatively high levels of soybean oleosin protein did not affect the amount of endogenous rapeseed oleosin. Immunoblotting studies showed that about 95% of the recombinant soybean 24 kDa oleosin (and the endogenous 19 kDa rapeseed oleosin) was targeted to oil bodies, with the remainder associated with the microsomal fraction. Sucrose density-gradient centrifugation showed that the oleosins were associated with a membrane fraction of buoyant density 1.10–1.14 g ml^?1, which partially overlapped with several endoplasmic reticulum (ER) markers. Unlike oleosins associated with oil bodies, none of the membrane-associated oleosins could be immunoprecipitated in the presence of protein A-Sepharose, indicating a possible conformational difference between the two pools of oleosin. Complementary electron microscopy-immunocytochemical studies of transgenic rapeseed revealed that all oil bodies examined could be labelled with both the soybean or rapeseed anti-oleosin antibodies, indicating that each oil body contained a mixed population of soybean and rapeseed oleosins. A small but significant proportion of both soybean and rapeseed oleosins was located on ER membranes in the vicinity of oil bodies, but none were detected on the bulk ER cisternae. This is the first report of apparent targeting of oleosins via ER to oil bodies in vivo and of possible associated conformational/ processing changes in the protein. Although oil-body formation per se can occur independently of oleosins, it is proposed that the relative net amounts of oleosin and oil accumulated during the course of seed development are a major determinant of oil-body size in desiccation-tolerant seeds.     

Analysis of QTLs for erucic acid and oil content in seeds on A8 chromosome and the linkage drag between the alleles for the two traits in Brassica napus

The history of canola breeding began with the discovery of germplasm with low erucic acid content in seeds of spring forage cultivar in tbe 1950's.FAEI,mutations led to a dramatic decrease of the seed erucic acid content in Arabidopsis thaliana.The products of the two FAEI loci.BnA8.FAEI and BnC3.FAEI,showed additive effects to the level of erucic acid content in oilseed rape.Previous research believed that the pleiotropy of FAEI was responsible for the decrease in seed oil content along with the reduction of seed erucic acid content in the modern cultivars.TN DH population was developed from a canola cultivar Tapidor and a Chinese traditional cultivar Ningyou7.The population had been tested in 10 and 11 environments to map QTLs for the erucic acid content and oil content in seeds.As the map resolution increased,a novel QTL for seed erucic acid content was revealed,after Meta-analysis,7 cM away from the most significant seed erucic acid content QTL where BnA8.FAEI is located.Seven independent QTLs for seed oil content(qOC) were detected around the two seed erucic acid content QTLs(qEA)across 39.20 cM on linkage group A8.Two of the qOCs co-localized with the two qEAs,respectively,and were detected in a single environment.The otherfive qOCs were detected in 10 of ll environments independent of qEAs.Alleles from Tapidor in all the QTLs at the 0-39.20 cM region contributed negative effects to either erucic acid content or oil content in seeds.Parallel,genocontent source.Through rounds of crossbreeding with oil-cropped cultivars and intensive selection for multi generations,Tapidor still had the controlled by the five qEA-independent qOCs,with low seed erucic acid content.Ninety cultivars of B.napus from 8 countries were used to analyze the genetic drag with 9 molecular markers located in the QTL confidence intervals (24.04cM) on linkage group A8.It was noticed that more than 46% of the cultivars with low seed erucic acid content trait remained the genotype of low seed oil content at least in one locus.Backcross and marker-assisted selection could break the genetic drag between the low oil content and erucic acid in seeds in the process for breeding modern high seed oil content canola cultivars.     

Oil Body Biogenesis during Brassica napus Embryogenesis

Although the oil body is known to be an important membrane enclosed compartment for oil storage in seeds, we have little understanding about its biogenesis during embryogenesis. In the present study we investigated the oil body emergence and variations in Brassica napus cv. Topas. The results demonstrate that the oil bodies could be detected already at the heart stage, at the same time as the embryos began to turn green, and the starch grains accumulated in the chloroplast stroma. In comparison, we have studied the development of oil bodies between Arabidopsis thaliana wild type (Col) and the low-seed-oil mutant wrinkled1–3 . We observed that the oil body development in the embryos of Col is similar to that of B. napus cv. Topas, and that the size of the oil bodies was obviously smaller in the embryos of wrinkled1–3 . Our results suggest that the oil body biogenesis might be coupled with the embryo chloroplast.