蚕豆叶片气孔保卫细胞中微管的分布

本实验用考马斯亮蓝染色及间接免疫荧光等细胞化学方法对蚕豆叶片气孔保卫细胞中微管的形态分布作了初步观察研究。考马斯亮蓝染色后光学显微镜下观察，发现蚕豆叶片下表皮气孔保卫细胞中有丝状物存在，其分布情况为：由核向细胞的背壁、腹壁呈辐射状排列，或呈一种交错网络结构。经微管解聚剂甲氨膦（ＡＰＭ）处理的保卫细胞，无明显丝状物存在。在间接免疫荧光定位中，经荧光显微镜及共焦激光扫描显微镜观察，其保卫细胞中微管的分     

衣藻肌动蛋白-绿色荧光蛋白融合基因在酵母细胞中的表达*

将构建的含有衣藻(Chlamydomonas reinhardtii)动蛋白(actin)和绿色荧光蛋(green fluorescent protein,GFP)融合基因的酵母表达载体引入裂殖酵母(Schizosaccharomyces pombe)，衣藻肌动蛋白和荧光蛋白的融合基因得到了表达。表达actin-GFP融合蛋白的酵母细胞在蓝光激发下可以观察到绿色荧光。在EMM培养基上actin-gfp强烈表达，但在硫胺素(thiamine)存在时只进行微弱表达。在融合基因强烈表达时，actin-gfp表达产物在酵母细胞中以聚集形式存在，细胞破碎后发现表达产物主要分布于沉淀中。     

绿色荧光蛋白与黄瓜花叶病毒运动蛋白融合基因的构建及在大肠杆菌中的表达

采用重组－PCR方法，扩增获得绿色荧光蛋白（GFP）与黄瓜花叶病毒运动蛋白（CMV MP）融合基因，将其克隆到pKEN上，构建了该融合基因的原核表达载体pKENG-M。SDS－PAGE及免疫印迹分析显示，57kD的融合蛋白基因在大肠杆菌DH5α中正确表达，激光共聚焦显微镜分析表明，在488nm光激发下，菌体呈亮绿色，表明融合蛋白保持绿色荧光特性。实验结果为进一步研究CMV MP的功能及其与胞间连丝和其他细胞成分的作用关系奠定了基础。     

亚非马蜂溶血肽基因与增强型绿色荧光蛋白基因融合在昆虫细胞中的表达

构建亚非马蜂(Polisteshebraeus)溶血肽基因重组转移载体pBacHT-GFPTPhMT,转化含穿梭载体Bacmid的受体菌Escherichiacoli(DH10Bac)中,得重组穿梭载体Bacmid-GFPTPhM,再用Lipofectin介导其DNA转染粉纹夜蛾(Trichoplusiani)细胞系Tn。SDS-PAGE分析表明,感染Bacmid-GFPTPhM的Tn-5B1-4细胞的表达产物在约为34kD处出现特异性条带,表达量约占细胞总蛋白的3%。Westernblot显示表达产物具有良好的免疫活性。感染Bacmid-GFPTPhM的细胞表达时相动态分析进一步表明,与增强型绿色荧光基因融合的亚非马蜂溶血肽基因的重组病毒已在昆虫细胞中进行了成功地表达,感染后72h表达量可达最高水平。     

绿色荧光蛋白标记可移动载体质粒的构建及其在嗜水气单胞菌的表达

以多寄主可移动载体质粒ｐＳＵＰ１０１１为基础载体,构建了绿色荧光蛋白（ＧＦＰ）标记的重组质粒,通过细菌接合,将重组质粒转移到嗜水气单胞菌Ｊ－１中。经紫外光检测和ＳＤＳ－ＰＡＧＥ电泳分析,证实重组粒在该 可表达ＧＦＰ。在无选择压力的培养条件下,２４小时后,重组质粒的稳定率在７２．２％,３６小时后,稳定率在５０％。显示ＧＦＰ作为报告基因在嗜水气单胞菌致病机理研究的应用前景。     

红色荧光标记载体pBacA4DsRed的构建及其在蚕卵中的瞬时表达

红色荧光蛋白(RFP)是从海葵(Discosoma)中分离的一种新荧光蛋白基因,可以在紫外线激发下发出红色荧光,最大发射波长为583nm,在细胞中荧光转换效率高,是目前发现     

拟南芥质膜水通道蛋白RD28保守区段在大肠杆菌中的表达及其抗体的制备

实验利用ＰＣＲ技术,从拟南芥原膜水旧白ＲＤ２８的ｃＤＮＡ中扩增了所有植物细胞质膜水通道蛋白保守区段（４２０ｂｐ）,并将其构入ｐＧＥＸ－ＫＧ。酶切、测序分析表明重组质粒ｐＧＥＸ－ＲＤ２８结构正确。０．１ｍｍｏｌ／Ｌ的ＩＰＴＧ可诱导表达分子量约４０ｋＤ融合蛋白ＧＳＴ－ＲＤ２８,该蛋白主要以非包涵体的形式存在。诱导表达后的菌体超声裂解液经谷胱甘肽活化的Ｓｅｐｈａｒｏｓｅ ４Ｂ亲和层析柱纯化得到了高纯度Ｇ     

猪Ⅱ型圆环病毒ORF2基因在Hela细胞中的表达及表达蛋白的定位

猪圆环病毒（Porcine circovims，PCV）属于环状病毒属，该病毒无囊膜，基因组为单链环状DNA，大小约为1．7kb，编码2个主要的开放阅读框架：ORF1和ORF2，ORF2编码病毒的核衣壳蛋白（Nawagitgul et al，2000），可能在PCV2病毒致病方面具有重要的作用。PCV2可引起断奶仔猪多系统衰竭综合症（PMWS）．给养猪业带来了巨大的经济损失。分别用ORF2基因的亚单位疫苗免疫猪、核酸疫苗免疫小白鼠，均具有良好的效果（Blanchard et al，2003；Kamstrup et al，2004）。关于PCV2疫苗的研究在我国尚未处报道。本实验在异源启动子CMV下实现了ORF2在真核细胞中的表达。     

铝诱导蚕豆气孔保卫细胞凋亡研究

植物叶表面的气孔保卫细胞是研究信号转导的模式实验系统,对环境变化反应灵敏而准确,采用蚕豆叶面气孔保卫细胞,研究了铝（AlCl3）对细胞的毒性效应。结果表明,在1～10 mmol.L^-1范围内,AlCl3可使气孔保卫细胞活性降低,部分细胞死亡,且随着浓度的增高细胞死亡率增高;死细胞呈现核固缩、核降解、凋亡小体等典型凋亡特征。凋亡抑制剂Z-Asp-CH2-DCB或TLCK与AlCl3共同作用时,保卫细胞死亡率显著降低;一定浓度的抗坏血酸（AsA）或过氧化氢酶（CAT）以及Ca2＋螯合剂乙二醇四乙酸酯（EGTA）或Ca2＋通道抑制剂LaCl3与AlCl3共同作用时,细胞死亡率降低。研究结果表明,铝诱导的蚕豆保卫细胞死亡可能是一种细胞凋亡过程,由胁迫诱发的活性氧介导,通过激活质膜钙通道,引起胞内Ca2＋水平改变,进而介导细胞凋亡。     

Autofertility in a pure line faba bean (Vicia faba L.) germplasm collection

Summary A pure line faba bean germplasm collection (BPL) was derived by a process of cyclic single plant progenies starting in 1979 through 1985. In 1985/86, 840 of these pure line accessions from 677 original populations from 41 countries were evaluated for autofertility by comparing the mean of five plants where all flowers were tripped vs. five plants with no tripped flowers for number of seeds produced (seed index). In a replicated trial grown at the same time, seed yield, 100 seed weight, number of pods per plant, seeds per pod, and number of flowers per inflorescence were also evaluated. A high number of BPL accessions failed to set seeds in the screen houses without tripping, the mean seed index measure of autofertility was 0.51 with a C.V. of 74.2%. There were marked differences among countries of origin for autofertility, with Egypt having the highest (SI = 0.94) and the USSR the lowest (SI = 0.26) autofertility, however, most countries expressed a large range, suggesting that breeders should first look in their own material for this trait rather than searching in exotic germplasm. In general, there was a higher level of autofertility in Middle Eastern countries that may be due to natural selection for non-dependency on pollinators for high yield. Contrary to the findings of others, large seeded accessions had a high autofertility (SI = 0.704) compared to small seeded accessions (SI = 0.384), however, both had similar ranges. In general, there was no significant correlations among autofertility and yield and its components. The variability found in this large pure line collection for autofertility is of potential use for developing cultivars not dependent on insect populations for high yield and for developing self-pollinated faba bean.     

Collection, evaluation and classification of Greek populations of faba bean (Vicia faba L.)

Fifty-five Greek Vicia faba L. populations, collected from diverse areas, were planted at two dry and low fertility sites for evaluation and classification. Yield evaluation, which was carried out by Principal Component Analysis (PCA) on the basis of seven yield traits, showed the number of pods per plant, number of ovules and seeds per pod, and branching from the basal nodes to be the most important traits for population evaluation regarding yield. For population classification, four dissimilarity coefficients (Manhattan, Average Taxonomic Distance, Euclidean distance and squared Euclidean distance) and four multivariate methods (PCA, UPGMA, Neighbor-joining and Principal Coordinate Analysis) were evaluated using fifteen morphological and seven yield traits. Neighbor-joining was chosen as the most suitable multivariate method. This method combined with PCA for the seven yield traits, placed the populations into six groups. As revealed by the application of PCA on all twenty-two traits the grouping was based mainly on pod characteristics, stem thickness, plant height, 1000 seed weight and branching from basal nodes. Based on the results of the present study, a model is proposed for conserving cross-pollinated species, such as faba bean.     

Comparative investigation on the susceptibility of faba bean (Vicia faba L.) and sunflower (Helianthus annuus L.) to iron chlorosis

Comparative physiological studies on iron (Fe) chlorosis of Vicia faba L. and Helianthus annuus L. were carried out. High internal Fe contents in Vicia cotyledons (16–37 μg) were completely used for plant growth and Fe chlorosis was not inducible by the application of nitrate (with or without bicarbonate). In Helianthus, low quantities of Fe in the seeds (4 μg) were insufficient for normal growth and without Fe in the nutrient solution, Fe chlorosis was obtained in all treatments. This chlorosis was an absolute Fe deficiency. Also, the treatment with 1 μM Fe in the nutrient solution and nitrate (with or without bicarbonate) led to severe chlorotic symptoms associated with low leaf Fe concentrations and high Fe concentrations in the roots. In contrast, Helianthus grown with NH₄NO₃ and 1 μM Fe had green leaves and high leaf Fe concentrations. However, with NO₃ supply (with or without bicarbonate), Fe translocation from the roots to the upper plant parts was restricted and leaves were chlorotic. Chlorotic and green sunflower leaves may have the same Fe concentrations, leaf Fe concentration being dependent on Fe translocation into the leaf at the various pH levels in the nutrient solution. At low external pH levels (controlled conditions) more Fe was translocated into the leaf leading to similar leaf Fe concentrations with higher chlorophyll concentrations (NH₄NO₃) and with lower chlorophyll concentrations (NO₃). This indicates a lower utilization of leaf Fe of NO₃ grown sunflower plants. Utilization of Fe in faba bean leaves is presumably higher than in sunflower leaves. In Vicia xylem sap pH was not affected by nitrate. In contrast, the xylem sap pH in Helianthus was permanently increased by about 0.4 pH units when fed with nitrate (with or without bicarbonate) compared with NH₄NO₃ nutrition. The xylem sap pH is indicative of leaf apoplast pH. From our earlier work (Mengel et al., 1994; Kosegarten und Englisch, 1994) we therefore suppose that in Helianthus, Fe immobilization occurs in the leaf apoplast due to high pH levels when grown with nitrate (with or without bicarbonate).     

Phenolics, phytic acid, and phytase in Canadian-grown low-tannin faba bean (Vicia faba L.) genotypes

Thirteen low-tannin faba bean genotypes grown at two locations in north central Alberta in 2009 were evaluated to investigate the variation in seed characteristics, phenolic and phytate contents, and phytase and antioxidant activities and to elucidate the relationship of these components as a breeding strategy. Seed characteristics including color were predominantly genotype dependent. The faba bean genotypes with total phenolic content ranging from 5.5 to 41.8 mg of catechin equiv/g of sample was linearly related to tannin content and the best predictor of antioxidant activity. Phytic acid content and phytase activity varied significantly among genotypes and between locations, ranging from 5.9 to 15.1 g/kg and from 1606 to 2154 FTU/kg sample, respectively. Multivariate data analysis performed on 19 components analyzed in this study using principal component analysis (PCA) and cluster analysis demonstrate that differences in seed characteristics, phenolic components, phytic acid, and phytase are major factors in segregating faba bean genotypes. The relatively low phytic acid content and high phytase activity of these low-tannin faba bean genotypes are beneficial/essential traits for their use in human and animal nutrition.     

Genetic variability for protein content and S-aminoacids in broad-beans (Vicia faba L.)

Summary Approximately 600 different samples of the broad bean collection have been evaluated for crude protein content of the seeds.111 samples were also evaluated for S-aminoacids. The values ranged from 19.5 to 34.0 for protein content and 0.97 to 1.45 for half-cystine and 0.62 to 0.99 for methionine. Protein content was negatively correlated with seed weights, highest values been found in the small seeded samples of Ethiopia and Afghanistan. Mediterranean samples exibited quite large variability. S-aminoacid per protein unit were negatively correlated with the protein content and determination coefficients had intermediate values. Sulphur aminoacids expressed as % of dry seed weight were positively correlated with protein content, but determination values were rather low. Analyses of protein and sulphur aminoacids in seeds from a 10×10 diallel cross scheme gave information on genetic mechanisms involved in the character's control; additive and non additive components of variance were significant, indicating difficulties in practical breeding programmes.

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Genetische Variabilität des Gehalts an Rohprotein und schwefelhaltigen Aminosäuren bei Ackerbohnen (Vicia faba L.)

Zusammenfassung Fast 600 Sortimentsnummern der Ackerbohnenkollektion wurden auf den Rohproteingehalt der Samen untersucht, 111 auch auf den auf Gehalt an schwefelhaltigen Aminosäuren. Der Rohproteingehalt schwankt von 19,5 bis 34,0%, der Gehalt an Cystein von 0,97 bis 1,45% und der an Methionin von 0,62 bis 0,99%. Der Rohproteingehalt war negativ mit dem Samengewicht korreliert, die höchsten Werte wurden bei den kleinsamigen Sippen aus Äthiopien und Afghanistan gefunden. Die Sippen aus dem Mittelmeergebiet zeigen eine sehr große Variabilität. Der relative Gehalt an S-haltigen Aminosäuren war negativ mit dem Rohproteingehalt korreliert und das Bestimmtheitsmaß hatte mittlere Werte. Der absolute Gehalt an S-haltigen Aminosäuren war positiv mit dem Rohproteingehalt korreliert, aber das Bestimmtheitsmaß war niedriger.Die Analysen des Rohproteingehaltes sowie des Gehaltes an schwefelhaltigen Aminosäuren der Samen (von F₁-Pflanzen) aus einem 10×10 Diallel-Kreuzungsschema ergaben Informationen über genetische Mechanismen in der Kontrolle der Merkmale; die additive und die nicht additive Varianzkomponente waren signifikant und offenbaren die Schwierigkeiten in den praktischen Züchtungsprogrammen.

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, , (Vicia faba)

600 . 111 , (-). 19,5 34,0%; — 0,97 1,45%, — 0,62 0,99%. ; . . - , . - , . - f₁ ( 10× 10) , ; , .

     

Root turnover of faba beans (Vicia faba L.) and its interaction with P and K supply

An increased root turnover can be a mechanism of improved nutrient‐uptake efficiency. The objectives of this study were to investigate P and K efficiency of faba beans (Vicia faba L.), to determine their root growth and root turnover, and to assess the relevance of root turnover on P and K uptake at limited supply. Faba beans were grown as part of a long‐term fertilization experiment on fertilized plots (control) and plots that had not received any P or K fertilizer for 16 years (P0, K0). Although the unfertilized soils were low and very low in their P‐ and K‐supply level, respectively, no differences in shoot‐dry‐matter production occurred compared to the control. However, relative K concentration in dry matter of the K0 plants (control plants = 100) decreased during the experiment and was only 60% of the control at the final harvest. This indicated a high K‐utilization efficiency of faba bean. Relative phosphorus concentration increased in the P0 treatment and was not different from the control at the last harvest, indicating an improvement in P‐uptake efficiency with time. The size of the standing root system determined by sequential auger sampling (net development) was not influenced by P and K supply. Total root production as measured by the ingrowth‐core method was about 6 times higher than the average size of the standing root system and even increased under low‐K conditions. This indicated a fast root turnover. Modeling soil nutrient transport and uptake revealed that calculated uptake of the control was up to 48% higher when root turnover was taken into account, compared to calculations based on the net development of the root systems. This is due to a better soil exploitation. Under K shortage, root turnover resulted in a 117% higher calculated uptake, which was close to measured K uptake. Root turnover was also of benefit for P uptake, but calculated P uptake was significantly less than measured, indicating that root turnover was of little importance for P uptake of faba beans.     

Carbon costs of nitrate reduction in broad bean (Vicia faba L.) and pea (Pisum sativum L.) plants

The present study aimed at the assessment of carbon (C) costs for nitrate reduction by measuring the additional CO₂ amounts released from roots of nitrate‐fed plants in comparison with urea‐fed plants. Only roots were suitable for these determinations, because nitrate reduction in illuminated shoots is fed nearly exclusively by reducing equivalents coming directly from photosynthetic processes. Therefore, in a first experiment, the sites of nitrate reduction were determined in nodule‐free broad bean (Vicia faba L.) and pea (Pisum sativum L.) plants grown in pots filled with quartz sand and supplied with KNO₃. The extent of nitrate reduction in the various plant organs was determined by measuring in vitro nitrate reductase activity and in situ ¹⁵NO reduction. Only between 9% and 16% of nitrate were reduced in roots of German pea cultivars, whilst 52% to 65% were reduced in broad bean roots. Therefore, C costs of the process could be determined only in broad bean, using an additional pot experiment. The C costs amounted to about 4.76 mol C (mol N)^–1 or 4 mg C (mg N)^–1, similar to those measured earlier for N₂ fixation. The high proportion of nitrate reduction in shoots of pea plants implies that only very little C is required for this nitrate fraction. This can explain the better root growth of nitrate‐nourished pea plants in comparison with N₂‐fixing organisms, which need C compounds for N₂ reduction in roots. Moreover, a different availability of photosynthates in roots of plant genotypes could explain physiologically the occurrence of “shoot and root reducers” in nature.     

Effectiveness of native Rhizobium on nodulation and yield of faba bean (Vicia faba L.) in Eastern Ethiopia

This study was initiated to evaluate the effect of locally isolated Rhizobium on nodulation and yield of faba bean at Haramaya, Ethiopia for three consecutive years. Ten treatments comprising of eight effective isolates of rhizobia, uninoculated, and N-fertilized (20 kg N ha^?1) were laid out in a randomized complete block design with three replications. The result of the experiment indicated that all inoculation treatments increased nodule number and dry weight over the control check in all cropping seasons. The result, however, showed the non-significant effect of Rhizobium inoculation on shoot length, number of tiller per plant and 100 seed weight in all cropping season. Inoculating Haramaya University Faba Bean Rhizobium (HUFBR)-15 in 2011 and National Soil Faba Bean Rhizobium (NSFBR)-30 in 2012 and 2013 gave the highest grain yields (4330, 5267 and 4608 kg ha^?1), respectively. These records were 75%, 48%, and 5% over the uninoculated treatment of respective years. Over the season, NSCBR-30 inoculation resulted in the highest nodulation and grain yield production as compared to the other treatments. In general, isolates from central Ethiopia were better than those isolated from eastern Ethiopia and Tropical Agricultural Legume (TAL)-1035 in enhancing faba bean production at Haramaya site. Therefore, NSFBR-30 is recommended as a candidate isolate for faba bean biofertilizer production in eastern Ethiopia soils.     

Characterization of Greek populations of faba bean (Vicia faba L.) and their evaluation using a new parameter

Using data from four environments of a two-year experiment, a collection of 55 Greek faba bean populations wascharacterized on the basis of ten morphological and ten agronomical traits. The collection can be described ashaving flowers with medium intensity of streaks on standard petal, two to three flowers per inflorescence, leaveswith six leaflets per leaf, sub-elliptic leaflet shape and small or medium leaflet size. The stems had weak ormedium pigmentation at flowering time, were of low or medium thickness, had medium height and mediumbranching from basal nodes and high resistance to lodging. Pods were mainly basal with pendent attitude, flattenedin shape, had dark colour at maturity and were small with few ovules and seeds. Most seeds had testa of light greencolour. This characterization makes the present collection similar to other South European faba bean populations.Apart from characterization, collections should also be evaluated from the breeding point of view. Since themost desirable populations within a collection are those with valuable but 'low frequency' levels of traits, theparameter R (rareness) was proposed. For each trait, a partial rareness (R ) parameter is calculated using only ithose trait levels that can be considered as having 'low frequency'. The parameter R is the sum of the 'partialrarenesses'. Populations with high frequencies of the 'lowest frequency' levels of traits in the collection arecharacterized by high values of R and can, therefore, be more easily identified.