高,低蛋白含量的大豆种子贮藏蛋白积累的比较研究

利用ＳＤＳ－ＰＡＧＥ及电子显微镜等技术比较分析和观察了两个大豆品种（Ｇｌｙｃｉｎｅ ｍ ａｘ（Ｌ．） Ｍｅｒｒ．）种子贮藏蛋白积累的差异。结果表明,在子叶发育过程中,蛋白含量高（４５％ ）的“公交８０５９－３”比蛋白含量低（３５％ ）的“ＧＤ－１５１５”蛋白含量高,且增长速率较快;蛋白质及７Ｓ和１１Ｓ亚基积累的时期较早,尤其是７Ｓ的β亚基。电镜观察表明“公交８０５９－３”蛋白质在液泡中的积累,前期与“ＧＤ１５１５”相似,中期以后的液泡中,蛋白体占整个细胞的比例均高于“ＧＤ１５１５”。这表明：大豆蛋白含量的差异有其生化及结构上的基础     

植物种子贮藏蛋白质及其细胞内转运与加工

高等植物种子成熟过程中贮存大量的贮藏蛋白质作为种子发芽和初期生长的重要营养来源。根据溶解性不同,种子贮藏蛋白质可分为白蛋白、球蛋白、醇溶蛋白和谷蛋白4类。在种子胚发育过程中,醇溶蛋白在粗面内质网合成后形成蛋白质聚集体,直接出芽形成蛋白体并贮存其中。白蛋白、球蛋白和谷蛋白在粗面内质网以分子量较大的前体形式合成后,根据各自的分选信号进入特定的运输囊泡,经由受体依赖型运输/聚集体形式运输转运至蛋白质贮藏型液泡中,然后经过液泡加工酶等的剪切转换为成熟型贮藏蛋白质并贮存其中。蛋白质的合成、分选、转运和加工等过程影响种子蛋白质的品质及含量。该文对种子贮藏蛋白质的分类和运输、加工以及这些过程对种子蛋白质品质和含量的影响进行了概述。     

植物种子贮藏蛋白质及其细胞内转运与加工

高等植物种子成熟过程中贮存大量的贮藏蛋白质作为种子发芽和初期生长的重要营养来源。根据溶解性不同, 种子贮藏蛋白质可分为白蛋白、球蛋白、醇溶蛋白和谷蛋白4类。在种子胚发育过程中, 醇溶蛋白在粗面内质网合成后形成蛋白质聚集体, 直接出芽形成蛋白体并贮存其中。白蛋白、球蛋白和谷蛋白在粗面内质网以分子量较大的前体形式合成后, 根据各自的分选信号进入特定的运输囊泡, 经由受体依赖型运输/聚集体形式运输转运至蛋白质贮藏型液泡中, 然后经过液泡加工酶等的剪切转换为成熟型贮藏蛋白质并贮存其中。蛋白质的合成、分选、转运和加工等过程影响种子蛋白质的品质及含量。该文对种子贮藏蛋白质的分类和运输、加工以及这些过程对种子蛋白质品质和含量的影响进行了概述。     

槐种子发育过程中子叶细胞显微结构的变化和过氧化物酶同工酶变…

槐（Ｓｏｐｈｏｒａ ｊａｐｏｎｉｃａ Ｌ．）种子发育过程中,子叶细胞大量合成和积累储藏物质,光镜和电镜下用组织化学观察到开花后３０ｄ,叶肉细胞高度液泡化,其中分而着大量的质体、粗面内质网、一些线粒体的小脂体。淀粉开始积累。开花后６０ｄ,淀粉和脂体不断长大,数量明显增多,液泡中蛋白体开始形成。从花后９０ｄ开始,液泡中有大量蛋白体形成且不断长大,有些蛋白体变得不规则形。至种子成熟期,叶肉细胞几乎完全被     

槐种子发育过程中子叶细胞显微结构的变化和过氧化物酶同工酶变化的动态

槐（SophorajaponicaL．）种子发育过程中,子叶细胞大量合成和积累储藏物质,光镜和电镜下用组织化学方法观察到开花后30d,叶肉细胞高度液泡化,其中分布着大量的质体、粗面内质网、一些线粒体和小脂体。淀粉粒开始积累。开花后60d,淀粉粒和脂体不断长大,数量明显增多,液泡中蛋白体开始形成。从花后90d开始,液泡中有大量蛋白体形成且不断长大,有些蛋白体变得不规则形。至种子成熟期,叶肉细胞几乎完全被蛋白体及脂体所填充。淀粉粒及大多数细胞器消失。基于上述观察,还对种子发育过程中子叶细胞过氧化物酶活力及同工酶港的变化进行了观察和分析。     

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。     

羧甲基壳聚糖对玉米籽粒氮代谢关键酶和种子贮藏蛋白含量的影响

以羧甲基壳聚糖（ＮＣＭＣ） 处理玉米开花期果穗,发育中籽粒的谷氨酰胺合成酶（ＧＳ） 、谷氨酸脱氢酶（ＧＤＨ） 及谷丙转氨酶（ＧＰＴ） 活性均明显增强,而蛋白水解酶活性下降。羧甲基壳聚糖处理离体玉米籽粒酶提取液,酶液中ＧＤＨ 活性明显提高。羧甲基壳聚糖处理玉米果穗,其发育籽粒中可溶性蛋白和成熟种子中贮藏蛋白含量明显提高。这都表明,羧甲基壳聚糖对玉米氮代谢、蛋白质合成与积累具有明显的生理调节作用。     

羧甲基壳聚糖对玉米籽粒氮代谢关键酶和种子贮藏蛋白含量的影响

以羧甲基壳聚糖（ＮＣＭＣ）处理玉米开花期果穗,发育中籽粒的谷氨酰胺合成酶（ＧＳ）,谷氨酸脱氢酶民（ＧＤＨ）及谷丙转氨酶（ＧＰＴ）活性均明显增强,而蛋白水解酶活性下降。羧甲基壳聚糖处理离体玉米籽粒酶提取液,酶液中ＧＤＨ活性明显提高。羧甲基壳聚糖处理玉米果穗,其发育籽粒中可溶性蛋白和成熟种子中贮藏蛋白含量明显提高,这都表明,羧甲基壳聚糖对玉米氮代谢、蛋白质合成与积累具有明显的生理调节作用。     

赤豆种子萌发中幼苗酰脲含量及尿囊素酶活力变化动态

赤豆种子萌发过程中,幼苗迅速合成酰脲,在酰脲含量达到最大值前,尿囊酸含量高于尿囊素含量,子叶合成酰脲最迅速;酰脲含量达到最大值后,茎带叶的酰脲含量最高。幼苗累积酰脲在品种间相似。种子萌发过程中,幼苗尿囊素酶活力迅速呈线性增加,且与酰脲含量变化趋势一致。幼苗尿囊素酶较耐热。     

温度对大豆(Glycine max)种子发育过程中蛋白质、脂肪和淀粉积累过程的影响

目前人们仍不清楚温度是如何影响发育中的大豆（Glycine max L．）种子蛋白质和脂肪积累过程以及基因型不同的大豆是否对温度具有相同的反应。研究拟通过对3个基因型大豆在不同温度处理下,种子发育过程中的蛋白质和脂肪的积累模式研究,以了解温度对种子组分的调节机理。3个基因型大豆品种（Evans,PI132．217,和Proto）种子盆栽在温度为27／20％（中温）的生长箱中生长到开花。在开花后第10天,将其中的一个生长箱的温度调节到35／27℃（高温）;另一个调到20／12℃（低温）。生长在高温和中温条件下的大豆,在开花的第21天开始收集豆荚,每3d取1次样。生长在低温条件下的大豆,在开花的第25天开始收集豆荚,每5d取1次样。结果表明,3个基因型大豆种子均在高温下生长快,成熟早,在中温下生长速率最大,低温下生长速率低但种子生长期延长。当种子获得60％-70％总干重时种子脂肪含量达到最大（中温）,高温使其提前出现,低温则被推后。在低温下,种子中蛋白质和脂肪两者积累模式相同,但蛋白质积累速率低。在高温和中温条件下,种子蛋白质和脂肪的积累模式不同。在种子获得60％～70％的总干重之前,蛋白质和脂肪积累模式相同,但在种子获得60％～70％的总干重之后,蛋白质积累呈上升趋势,而脂肪积累停止或下降。同时在种子发育的晚期伴随着蛋白质含量增加,淀粉和蔗糖含量快速下降。虽然3个基因型大豆种子的蛋白质和脂肪积累模式均明显受温度影响,但在不同温度条件下和不同生长阶段中高蛋白质品种Proto和PI132．217（蛋白质稳定型）蛋白质含量总是高于低蛋白质品种Evans,而且两者差异显著。这一研究表明温度不能改变品种在蛋白质和脂肪合成上的遗传特性。遗传育种在提高大豆种子蛋白质含量上仍起决定作用,但是合理的播种时期在提高大豆种子蛋白质和脂肪含量上也是不可忽视的问题。     

Leaf urea metabolism in potato. Urease activity profile and patterns of recovery and distribution of (15)N after foliar urea application in wild-type and urease-antisense transgenics

The influence of urease activity on N distribution and losses after foliar urea application was investigated using wild-type and transgenic potato (Solanum tuberosum cv Désirée) plants in which urease activity was down-regulated. A good correlation between urease activity and (15)N urea metabolism (NH(3) accumulation) was found. The general accumulation of ammonium in leaves treated with urea indicated that urease activity is not rate limiting, at least initially, for the assimilation of urea N by the plant. It is surprising that there was no effect of urease activity on either N losses or (15)N distribution in the plants after foliar urea application. Experiments with wild-type plants in the field using foliar-applied (15)N urea demonstrated an initial rapid export of N from urea-treated leaves to the tubers within 48 h, followed by a more gradual redistribution during the subsequent days. Only 10% to 18% of urea N applied was lost (presumably because of NH(3) volatilization) in contrast to far greater losses reported in several other studies. The pattern of urease activity in the canopy was investigated during plant development. The activity per unit protein increased up to 10-fold with leaf and plant age, suggesting a correlation with increased N recycling in senescing tissues. Whereas several reports have claimed that plant urease is inducible by urea, no evidence for urease induction could be found in potato.     

Molybdenum accumulation and storage in Klebsiella pneumoniae and Azotobacter vinelandii.

In Klebsiella pneumoniae, Mo accumulation appeared to be coregulated with nitrogenase synthesis. O2 and NH+4, which repressed nitrogenase synthesis, also prevented Mo accumulation. In Azotobacter vinelandii, Mo accumulation did not appear to be regulated Mo was accumulated to levels much higher than those seen in K. pneumoniae even when nitrogenase synthesis was repressed. Accumulated Mo was bound mainly to a Mo storage protein, and it could act as a supply for the Mo needed in component I synthesis when extracellular Mo had been exhausted. When A. vinelandii was grown in the presence of WO2-(4) rather than MoO2-(4), it synthesized a W-containing analog of the Mo storage protein. The Mo storage protein was purified from both NH+4 and N2-grown cells of A. vinelandii and found to be a tetramer of two pairs of different subunits binding a minimum of 15 atoms of Mo per tetramer.     

AAP1 regulates import of amino acids into developing Arabidopsis embryos

The embryo of Arabidopsis seeds is symplasmically isolated from the surrounding seed coat and endosperm, and uptake of nutrients from the seed apoplast is required for embryo growth and storage reserve accumulation. With the aim of understanding the importance of nitrogen (N) uptake into developing embryos, we analysed two mutants of AAP1 (At1g58360), an amino acid transporter that was localized to Arabidopsis embryos. In mature and desiccated aap1 seeds the total N and carbon content was reduced while the total free amino acid levels were strongly increased. Separately analysed embryos and seed coats/endosperm of mature seeds showed that the elevated amounts in amino acids were caused by an accumulation in the seed coat/endosperm, demonstrating that a decrease in uptake of amino acids by the aap1 embryo affects the N pool in the seed coat/endosperm. Also, the number of protein bodies was increased in the aap1 endosperm, suggesting that the accumulation of free amino acids triggered protein synthesis. Analysis of seed storage compounds revealed that the total fatty acid content was unchanged in aap1 seeds, but storage protein levels were decreased. Expression analysis of genes of seed N transport, metabolism and storage was in agreement with the biochemical data. In addition, seed weight, as well as total silique and seed number, was reduced in the mutants. Together, these results demonstrate that seed protein synthesis and seed weight is dependent on N availability and that AAP1-mediated uptake of amino acids by the embryo is important for storage protein synthesis and seed yield.     

Incorporation of N from intravenously administered 15N labelled urea into the bacterial protein in the sheep.

The experiment carried out on two wethers demonstrated that nitrogen of intravenously injected urea, labelled with 15N was incorporated into total and bacterial nitrogen fraction of the digesta flowing through the rumen and duodenum. The amount of 15N in the bacterial fraction flowing throught the rumen and duodenum was relatively low in comparison with the amount of 15N in the total nitrogen (14,8% and 8,1% in the rumen and 6,6% and 7,9% in the duodenum. The ratio of the amount of bacterial-N to total-N in the rumen content (12,7 and 7,5%) was only slightly lower than the ratio of bacterial 15N to total 15N. In the duodenum this ratio was a little higher (8,7 and 10,0%). Blood urea nitrogen was utilized only partly in biosynthesis of bacterial protein. The results showed that only a small amount of blood urea nitrogen retained in the organism was utilized for microbial protein synthesis and the majority in some different way.     

花生胚离体发育及渗调物质的影响

在无外源激素培养基上花生胚能继续发育．渗调物质如甘露醇可抑制胚早萌,维持胚性发育,促进贮藏蛋白质合成和累积．渗调物质对胚离体发育的调控与其提高胚内源ＡＢＡ含量有关．     

Kinetic improvement of an algal diacylglycerol acyltransferase 1 via fusion with an acyl‐CoA binding protein

Microalgal oils in the form of triacylglycerols (TAGs) are broadly used as nutritional supplements and biofuels. Diacylglycerol acyltransferase (DGAT) catalyzes the final step of acyl‐CoA‐dependent biosynthesis of TAG, and is considered a key target for manipulating oil production. Although a growing number of DGAT1s have been identified and over‐expressed in some algal species, the detailed structure?function relationship, as well as the improvement of DGAT1 performance via protein engineering, remain largely untapped. Here, we explored the structure?function features of the hydrophilic N‐terminal domain of DGAT1 from the green microalga Chromochloris zofingiensis (CzDGAT1). The results indicated that the N‐terminal domain of CzDGAT1 was less disordered than those of the higher eukaryotic enzymes and its partial truncation or complete removal could substantially decrease enzyme activity, suggesting its possible role in maintaining enzyme performance. Although the N‐terminal domains of animal and plant DGAT1s were previously found to bind acyl‐CoAs, replacement of CzDGAT1 N‐terminus by an acyl‐CoA binding protein (ACBP) could not restore enzyme activity. Interestingly, the fusion of ACBP to the N‐terminus of the full‐length CzDGAT1 could enhance the enzyme affinity for acyl‐CoAs and augment protein accumulation levels, which ultimately drove oil accumulation in yeast cells and tobacco leaves to higher levels than the full‐length CzDGAT1. Overall, our findings unravel the distinct features of the N‐terminus of algal DGAT1 and provide a strategy to engineer enhanced performance in DGAT1 via protein fusion, which may open a vista in generating improved membrane‐bound acyl‐CoA‐dependent enzymes and boosting oil biosynthesis in plants and oleaginous microorganisms.     

Effectiveness of Sesbania rostrata and Phaseolus calcaratus as green manure for upland rice grown in acidic soil

Two field experiments on green manuring were conducted under upland acidic soil (pH = 4.35) conditions with the following objectives: (1) to determine the influence of inoculation site, P fertilization, and liming on the biomass production, N content, N accumulation, and N availability of S. rostrata grown in an acidic soil, (2) to compare the effectiveness of S. rostrata, P. calcaratus and urea as N sources for upland rice as affected by liming and N source-sowing time combination, and (3) to assess the effect of liming and N source-sowing time combination on % Ndff (N derived from the fertilizer), % Ndfs (N derived from soil), % FNU (fertilizer N utilization), and FNY or fertilizer N yield (kg N ha^–1) of upland rice grown in acidic soil. At 2 weeks after incorporating S. rostrata (95 days after lime application), liming significantly increased N availability by more than 2-fold suggesting that the decomposition of S. rostrata by soil microflora was stimulated by lime. Liming, phosphorus application, and inoculation site improved significantly the dry biomass production, N content and N accumulation of S. rostrata; thus, enhancing its green manuring potential. Regardless of liming, S. rostrata whether applied at 0 week or 2 weeks before sowing was superior to urea in improving grain and straw yields. P. calcaratus when applied at 2 weeks before sowing also produced higher grain yield than urea. Immediate sowing of upland rice after green manure incorporation did not affect negatively the growth and development of upland rice; hence, farmers could save at least 2 weeks in their cropping calendar. N source-sowing time combination had a highly significant influence on % Ndff, % Ndfs, % FNU, N uptake, and fertilizer N yield of upland rice. However, only N uptake was influenced significantly by liming. The rice plant obtained significantly higher % Ndfs from the soils treated with green manure than those treated with urea regardless of liming. The % FNU and % Ndff from the green manures were 11-37% and 9-25%, respectively. These values are much lower than those obtained under continuously flooded soil conditions possibly because of the differences in the organic matter decomposer populations and N loss mechanisms between sloping upland conditions and continuously flooded conditions.     

Antisense inhibition of the plastidial glucose-6-phosphate/phosphate translocator in Vicia seeds shifts cellular differentiation and promotes protein storage

The glucose-6-phosphate/phosphate translocator (GPT) acts as an importer of carbon into the plastid. Despite the potential importance of GPT for storage in crop seeds, its regulatory role in biosynthetic pathways that are active during seed development is poorly understood. We have isolated GPT1 from Vicia narbonensis and studied its role in seed development using a transgenic approach based on the seed-specific legumin promoter LeB4. GPT1 is highly expressed in vegetative sink tissues, flowers and young seeds. In the embryo, localized upregulation of GPT1 at the onset of storage coincides with the onset of starch accumulation. Embryos of transgenic plants expressing antisense GPT1 showed a significant reduction (up to 55%) in the specific transport rate of glucose-6-phosphate as determined using proteoliposomes prepared from embryos. Furthermore, amyloplasts developed later and were smaller in size, while the expression of genes encoding plastid-specific translocators and proteins involved in starch biosynthesis was decreased. Metabolite analysis and stable isotope labelling demonstrated that starch biosynthesis was also reduced, although storage protein biosynthesis increased. This metabolic shift was characterized by upregulation of genes related to nitrogen uptake and protein storage, morphological variation of the protein-storing vacuoles, and a crude protein content of mature seeds of transgenics that was up to 30% higher than in wild-type. These findings provide evidence that (1) the prevailing level of GPT1 abundance/activity is rate-limiting for the synthesis of starch in developing seeds, (2) GPT1 exerts a controlling function on assimilate partitioning into storage protein, and (3) GPT1 is essential for the differentiation of embryonic plastids and seed maturation.     

Comparative Study on the Accumulation of Storage Protein in the Soybean Seeds with High and Low Protein Contents

The difference in storage protein accumulation between two soybean was compared by SDS-PAGE and electron microscopy. The results showed that the “Gongjiao 8059-3” (protein content 45 %) was more rapid in protein accumulation rate and higher in protein content than that in “GD 1515” (protein content 35 %) during cotyledon development. The subunits (7S and l1S) of the storage protein accumulated earlier in “Gongjiao 8059-3” than in “GD 1515”. Under electron microscope, the protein accumulated in vacuoles was similar between the two cultivars, but the protein density was higher and the volume of protein bodies was larger in cells after mid developing stage in “Gongjiao 8059-3”. These results showed that difference in protein contents may be based on biochemical and structural alterations.