蚕豆种质资源清蛋白遗传多样性分析?

利用SDS-PAGE对101份蚕豆种质资源进行了清蛋白遗传多样性分析,共检测出蛋白带2625条。除共有带外,迁移率不同的谱带类型36种,其中相对分子量为92kD、75kD、62kD、40kD、34kD、17kD、13kD的谱带在检测的种质材料中出现的频率最高,分别为92.08%、90.10%、99.01%、95.05%、95.05%、98.02%、95.05%。其余29种谱带类型具有较强的多态性,多态性谱带平均为16.09条,多态性比例为44.69%。每份种质材料的清蛋白谱带数介于21～31条之间,平均25.99条;供试种质间遗传相似系数0.6111～0.9722,平均0.7122。3个地理类群内多样性指数0.9879,类群间多样性指数0.0121,表明蚕豆清蛋白的变异主要来源于类群内。聚类分析将参试种质分为6类,与以往蚕豆种质分类研究结果类似,表明清蛋白能在一定程度上反映种质间的亲缘关系。研究结果对于蚕豆蛋白质品质育种具有一定的参考价值。     

乌塌菜种质资源遗传多样性的ISSR分析

利用ISSR技术对48份乌塌菜种质资源进行遗传多样性分析。从60条随机引物中筛选出稳定性强、条带清晰且多态性丰富的9条引物进行PCR扩增,共扩增出103条谱带,平均每个引物扩增出11.4条带,其中多态性带85条,多态性位点百分率为82.68%。不同乌塌菜种质间遗传相似系数变幅为0.59~0.97,说明ISSR标记能够揭示材料间较高的遗传多样性。利用UPGMA聚类分析,ISSR标记能将48份乌塌菜品种完全区分开,48份乌塌菜种质被划分为4个类群,聚类结果与叶片颜色相关,为乌塌菜品种资源的研究利用提供参考。     

47份水稻品种资源的ISSR遗传多样性分析

为研究广东省惠州市种植的常规水稻品种的遗传多样性,本实验利用ISSR标记对47份水稻品种资源进行遗传多样性检测.从49条引物中筛选出5条重复性好,条带清晰的引物进行PCR扩增,共扩增出53条带,每个引物可以扩增出9～13条带,平均为10.6条,其中47条具有多态性,比率为88.7%.不同水稻品种间遗传相似系数变幅为0.319～0.936,平均达0.691,说明ISSR标记能够揭示材料间较高的遗传多样性.通过聚类,从分子水平对水稻品种资源的遗传关系进行分析,并对47份水稻品种资源进行分类,ISSR标记能将47份水稻品种完全区分开,为水稻品种资源的研究利用提供参考.     

蝴蝶兰种质资源遗传多样性的ISSR分析

利用ISSR分子标记对24个蝴蝶兰(Phalaenopsis)栽培品种进行了遗传多样性分析.筛选出的12个ISSR引物共扩增出301条清晰的谱带,其中多态性条带268条,多态性比率为89.1%.品种间的遗传相似系数在0.37～0.93之间,表明品种间具有较高的遗传多样性.UPGMA聚类分析可将供试材料分为3组,分类结果与材料来源及花器官表型具有密切的关系.     

RAPD和ISSR分子标记对果蔗种质资源的遗传多样性研究

利用RAPD与ISSR分子标记技术对40份不同地方果蔗种质的遗传多样性进行分析。从供试材料中筛选到具有多态性的RAPD引物23条,ISSR引物28条。23条RAPD引物共扩增出250条带,多态性条带比率为70%,相似系数变化范围在0.68-1.00之间;28条ISSR引物共扩增出301条带,多态性条带比率为77.1%,相似系数变化范围在0.66-1.00之间。根据两种标记的结果,用UPGMA法对40份果蔗种质材料进行聚类分析,结果表明,RAPD和ISSR均将40份果蔗种质分为4类:第Ⅰ类为32份地方果蔗品种,包括福建、江西、浙江、广西、云南等地的品种;第Ⅱ类为外引黑皮果蔗Badila和丰城紫皮果蔗;第Ⅲ类为杂交种白鳝、歪干担、肚度、温岭果蔗以及人工杂交选育的果蔗品种474;第Ⅳ类只有广东的黄皮果蔗。这两种标记的聚类结果相关分析表明,它们存在呈极显著相关(r=0.9746)。但ISSR标记比RAPD标记可检测到更大的遗传变异。     

利用RAPD标记分析大麦种质资源的遗传多样性

利用RAPD标记对19份西藏近缘野生大麦材料、33份我国不同省市的地方品种以及8份国外引进大麦品种共60份大麦种质资源的遗传多样性进行检测.结果表明材料间遗传差异明显.32个RAPD引物中,有25个引物(占78.13%)可扩增出清晰且具多态性的条带,另外7个引物能扩增出1～3条清晰但无多态性的条带.每个引物可扩增出1～8条多态性带,平均为3.72条.32个引物共产生119条DNA片段,其中87条具有多态性,多态性比率(PPB)为73.11%,平均多态信息量(PIC)为0.434;每个位点平均有效等位基因数(Ne)为2.304;材料间遗传相似系数GS变化范围为0.757～0.981,平均值为0.871.19份来源于西藏的近缘野生大麦材料间GS值变幅为0.818～0.969,平均为0.892;33份我国栽培大麦地方品种间的GS值变化范围为0.783～0.981,平均为0.879;8份分别来自8个国家的栽培大麦品种间的GS值变幅为0.820～0.956,平均为0.882.根据RAPD标记分析的结果,对60份大麦种质资源进行聚类分析,在平均GS值0.871水平上60份大麦材料可聚为5类,聚类结果能在一定程度上反应材料的地理分布关系,但某些相同地理来源的材料也较分散地分布在整个聚类树中.本研究从分子水平上进一步证明了我国栽培大麦丰富的遗传多样性,是世界栽培大麦的遗传多样性中心之一.     

鹅观草种质资源醇溶蛋白遗传多样性研究

以8个地理类群的90份鹅观草野生种质材料为研究对象,采用A-PAGE(酸性聚丙烯酰胺)凝胶电泳技术进行蛋白质水平遗传多样性检测.研究结果表明,来源于不同居群的鹅观草共分离出26条谱带,每个材料可以分离出5～26条迁移率不同的谱带, 平均谱带数为16.39条,其中平均多态性谱带为12.65条,多态性比例为77.22%;基于供试材料醇溶蛋白每个位点谱带出现的频率,分别计算了地理类群内多样性指数(0.345)和总多样性指数(0.471),类群间的遗传分化系数为26.8%,表明鹅观草变异的73.2% 来源于类群内;90份供试材料醇溶蛋白的Jaccard遗传相似系数变异范围为0.133 3～1.000,平均遗传相似系数为0.395 7;利用种子醇溶蛋白可将90份材料分为12类,鹅观草种质资源之间的亲缘关系呈现出一定的地域性规律;不同地理类群间的遗传多样性指数从高到低的排列顺序依次为云南＞四川＞内蒙古＞新疆＞山西＞甘肃＞宁夏＞河北.因此在进行鹅观草种质资源收集和原地保护时,建议对云南和四川地区的鹅观草种质资源应给予极大关注.     

节瓜自交系遗传多样性的ISSR分析

利用ISSR分子标记技术对36份节瓜自交系进行遗传多样性分析。从100条ISSR引物中筛选出14条多态性明显、条带清晰、反应稳定的引物,对36份节瓜材料基因组DNA进行扩增,共扩增出76条清晰稳定的条带,其中多态性条带45条,多态性比例为59.21%。36份材料间遗传相似系数在0.57~0.96之间,表明材料间遗传多样性较为狭窄。聚类分析结果显示,以遗传相似系数0.76为阈值时,可将36份节瓜自交系材料聚为3类,分类结果与供试材料的地理来源较为吻合。基于聚类分析结果,可为今后节瓜的新品种选育、遗传改良以及分子遗传连锁图谱构建的杂交亲本选择提供科学依据。     

猕猴桃属16个雄性材料遗传多样性的ISSR分析

利用ISSR分子标记对雄性猕猴桃16个材料进行遗传多样性分析。从100条引物中筛选出10条引物用于ISSR扩增,共扩增出172条带,其中多态性条带140条,多态性百分率为81.4%;经POPGENE 1.32软件分析结果显示,16个雄性猕猴桃材料的遗传距离在0.1503~0.5128之间,平均Nei's基因多样性指数(H)为0.2416,平均Shannon信息指数(I)为0.4048;聚类分析结果显示,在遗传相似系数为0.64处可将供试材料分成4类,第Ⅰ类为中华和美味猕猴桃,第Ⅱ类为阔叶、毛花猕猴桃,第Ⅲ类为魁绿猕猴桃,第Ⅳ类为四萼猕猴桃。结果表明ISSR可用于雄性猕猴桃遗传多样性研究,该研究结果可为猕猴桃种质资源的进一步开发利用提供重要信息。     

37份龙眼种质资源亲缘关系的ISSR分析

本研究利用ISSR技术对37份龙眼种质资源进行遗传多样性检测.研究结果表明,从100条ISSR引物中筛选出7条重复性好,条带清晰的引物对37份龙眼品种基因组DNA进行扩增,共扩增出54条带,其中43条具有多态性,比率为79.6%.不同龙眼品种间遗传相似系数变幅为0.69～0.97,平均达0.83,说明ISSR标记能够揭示材料间较高的遗传多样性.UPGMA聚类结果表明,ISSR标记能将37份龙眼品种完全区分开,并能将来源于中国、越南和泰国的37份龙眼品种分别聚类到中国、越南和泰国三大品种群,说明龙眼品种资源的亲缘关系与地理因素有关,三个国家的龙眼品种之间存在较大的遗传差异.本研究结果将为为龙眼品种资源的研究利用提供参考.     

OCCURRENCE OF LOW MOLECULAR WEIGHT AND HIGH CYSTEINE CONTAINING ALBUMIN STORAGE PROTEINS IN OILSEEDS OF DIVERSE SPECIES

The proteins in the oilseeds of species from 11 families, including sunflower, mustard, linseed, almond, lupin, peanut, cucumber, Brazil nut, hazelnut, yucca, castor bean, and cottonseed were studied. Sucrose gradient centrifugation showed that a substantial proportion of the total seed protein from each species migrated with a 2S sedimentation coefficient. The 2S proteins, being water-soluble and thus termed albumins, comprised 20–60% of the total seed proteins, while faster migrating globulins comprised the rest. The amino acid compositions of the 2S proteins were characterisitic of storage proteins by having a high amide content. However, the 2S proteins are different from the classical globulin storage proteins in having a high content of cysteine. It is proposed that 2S albumins are seed storage proteins with a wide distribution and with chemical properties distinct from those of the globulin storage proteins. They play an additional and unique role of providing sulfur reserve for germination.     

Mobilization of storage proteins in soybean seed (Glycine max L.) during germination and seedling growth

During germination and early growth of the seedling, storage proteins are degraded by proteases. Currently, limited information is available on the degradation of storage proteins in the soybean during germination. In this study, a combined two-dimensional gel electrophoresis and mass spectrometry approach was utilized to determine the proteome profile of soybean seeds (Glycine max L.; Eunhakong). Comparative analysis showed that the temporal profiles of protein expression are dramatically changed during the seed germination and seedling growth. More than 80% of the proteins identified were subunits of glycinin and β-conglycinin, two major storage proteins. Most subunits of these proteins were degraded almost completely at a different rate by 120h, and the degradation products were accumulated or degraded further. Interestingly, the acidic subunits of glycinin were rapidly degraded, but no obvious change in the basic chains. Of the five acidic subunits, the degradation of G2 subunit was not apparently affected by at least 96h but the levels decreased rapidly after that, while no newly appearing intermediate was detected upon the degradation of G4 subunit. On the other hand, the degradation of β-conglycinin during storage protein mobilization appeared to be similar to that of glycinin but at a faster rate. Both α and α' subunits of β-conglycinin largely disappeared by 96h, while the β subunits degraded at the slowest rate. These results suggest that mobilization of subunits of the storage proteins is differentially regulated for seed germination and seedling growth. The present proteomic analysis will facilitate future studies addressing the complex biochemical events taking place during soybean seed germination.     

The Infection Court of Faba Bean Seedlings for Oospores of Peronospora viciae f.sp. fabae in Soil

The infection court of Faba bean seedlings for oospores of Peronospora viciae f.sp. fabae in soil was determined. Soil naturally infested with oospores was placed as 3-cm thick layers at four different depths relative to Faba bean seeds. Seedlings with downy mildew were obtained only from seeds sown in the middle of a 3-cm layer of oosporeinfested soil. No infection was obtained from oosporeinfested soil placed more than 1.5 cm above or below seeds. Histological observations showed that the hypocotyl and first part of the main root were the most probable sites of infection.     

Isolation and Partial Characterization of a Seed Lectin from Tepary Bean that Delays Bruchid Beetle Development

Four isolectin forms of a seed lectin from mature seed of tepary bean (Phaseolus acutifolius) were isolated using solubility fractionation, affinity chromatography, and high performance liquid chromatography. The subunits are polypeptides with an apparent molecular mass of 30,000 daltons. The 30 kilodalton subunits are produced starting approximately 13 days after flowering and subsequently comprise a major fraction of the proteins found in the mature seed. The amino terminus of each isolectin fraction was determined to be highly homologous with that of the subunits of common bean (Phaseolus vulgaris L.) phytohemagglutinin (PHA). The tepary isolectin cross-reacts with both erythroagglutinating and leucoagglutinating subunits of PHA antibodies, although differential cross-reactivity was noted. A seed protein fraction enriched in tepary bean lectin was found to be toxic to bean bruchid beetles (Acanthoscelides obtectus), when incorporated into their diets at incremental concentrations from (1-5% w/w) above that of PHA concentrations in mature seeds of the susceptible common bean variety “Red Kidney.”     

In vitro and in silico analyses of Vicia faba L. on Peroxisome proliferator–activated receptor gamma

The agonists of peroxisome proliferator–activated receptor gamma (PPARγ) from natural victual products were used as antidiabetic agents. Faba bean (Vicia faba L.) is a consequential legume that was known to possess potential antidiabetic activity, whose mechanism of action was unknown. The current study was focused to ascertain gene expression of the nuclear receptor PPARγ by Faba bean pod extract in rat cell lines (RINm5F).The real‐time polymerase chain reaction analysis demonstrated that Faba bean pod extract in concentrations of 160 µg/mL have shown 4.97‐fold stimulation compared with control. The cells treated with 320 µg/mL has shown 5.89‐fold upregulation, respectively. Furthermore, in silico docking analysis was carried out against PPARγ, using the bioactive compounds identified from Faba bean pod extracts, which were known reported compounds from the literature. The results suggest that gene expression of PPARγ was inhibited by the constituents in Faba bean. In silico analysis prognosticates, butein has a high binding energy (?8.6 kcal/mol) with an atomic contact energy of ?214.10, followed by Apigenin and Quercetin against PPARγ. Similarly, the percentage of interaction was high for butein, followed by Apigenin and Quercetin than other compounds comparatively. Hence, the results conclude inhibition of PPARγ by the bioactive compounds from Faba bean, which may provide insights into developing future therapeutic molecules for diabetes mellitus.     

A comparative study of the role of the major proteinases of germinated common bean (Phaseolus vulgaris L.) and soybean (Glycine max (L.) Merrill) seeds in the degradation of their storage proteins

Two types of cysteine proteases, low-specificity enzymes from the papain family and Asn-specific from the legumain family are generally considered to be the major endopeptidases responsible for the degradation of seed storage proteins during early seedling growth. The action of the corresponding enzymes (CPPh1 and LLP, respectively) from common bean (Phaseolus vulgaris L.) on phaseolin (the common bean storage protein), and on the homologous soybean (Glycine max (L.) Merrill) storage protein, beta-conglycinin, was studied. Under the action of LLP, proteolysis of phaseolin was limited to cleavage of its interdomain linker. No cleavage of the interdomain linker occurred in beta-conglycinin with LLP. LLP action was restricted to splitting off the disordered N-terminal extensions of alpha and alpha' subunits. No extensive hydrolysis (degradation to short TCA-soluble peptides) of either protein occurred under the action of LLP. CPPh1 cleaved the phaseolin subunits into roughly half-sized fragments at the onset of proteolysis. The cleavage was accompanied by a small (8-10%) decrease of protein. No decrease of protein occurred with further incubation. Thus the two most active proteinases detected in common bean seedlings individually were incapable of the extensive degradation of phaseolin. Extensive hydrolysis of phaseolin was only achieved by the consecutive action of LLP and CPPh1. Similar cleavages occurred during the action of CPPh1 on beta-conglycinin. However, by contrast with phaseolin, CPPh1 by itself accomplished the extensive hydrolysis of beta-conglycinin. The differences in the course of proteolysis of the proteins studied were determined by their structural peculiarities.     

Storage globulins pass through the Golgi apparatus and multivesicular bodies in the absence of dense vesicle formation during early stages of cotyledon development in mung bean

During seed development and maturation, large amounts of storage proteins are synthesized and deposited in protein storage vacuoles (PSVs). Multiple mechanisms have been proposed to be responsible for transporting storage proteins to PSVs in developing seeds. In this study, a specific antibody was raised against the mung bean (Vigna radiata) seed storage protein 8S globulin and its deposition was followed via immunogold electron microscopy in developing mung bean cotyledons. It is demonstrated that non-aggregated 8S globulins are present in multivesicular bodies (MVBs) in early stages of cotyledon development where neither dense vesicles (DVs) nor a PSV were recognizable. However, at later stages of cotyledon development, condensed globulins were visible in both DVs and distinct MVBs with a novel form of partitioning, with the internal vesicles being pushed to one sector of this organelle. These distinct MVBs were no longer sensitive to wortmannin. This study thus indicates a possible role for MVBs in transporting storage proteins to PSVs during the early stage of seed development prior to the involvement of DVs. In addition, wortmannin treatment is shown to induce DVs to form aggregates and to fuse with the plasma membrane.     

Advances in the molecular biology of plant seed storage proteins

Plant seed storage proteins were among the first proteins to be isolated (20); however, only recently, as a result of using molecular biology techniques, have the amino acid sequences of many of these proteins been determined. With the accumulation of amino acid sequence data for many vicilin-type storage proteins much has been learned concerning the location of conserved amino acid regions and other regions which can tolerate amino acid sequence variation. Combining this knowledge with recent advances in plant gene transfer technologies will allow molecular biologists to correct (by using amino acid replacement mutations) the sulfur amino acid deficiency inherent to bean seed storage proteins. The development of more nutritious soybean and common bean seeds will be of benefit to programs involving human and animal nutrition.     

Hydrolysis of Crystalloid Storage Protein in Castor Bean Seed Endosperm During and Following Germination

Hydrolysis of the insoluble crystalloid storage proteins ofcastor bean endosperm during germination released buffer-solublepolypeptides with molecular weights in the presence of sodiumdodecyl sulphate of 30000–40000. These polypeptides appearto be dimers since the addition of 2-mercaptoethanol decreasestheir molecular weights to 15000–22000. Hydrolysis ofthe crystalloid proteins was detected 12–18 h after seedimbibition (HAI), which is before the completion of germination;maximum rates were attained at 30 HAI. During this period, parallelincreases in free amino acids were observed. Hydrolysis of thecrystalloid proteins during early germination was insensitiveto cycloheximide treatment and therefore did not require newlysynthesized proteases. Hydrolysis was effected by proteaseswhich were made in an inactive form during seed developmentand activated upon seed imbibition. Key words: Castor bean, crystalloid storage protein hydrolysis, seed germination, endosperm     

Characterization of Blue-Green Fluorescence in the Mesophyll of Sugar Beet (Beta vulgaris L.) Leaves Affected by Iron Deficiency

Protease C1, an enzyme from soybean (Glycine max [L.] Merrill cv Amsoy 71) seedling cotyledons, was previously determined to be the enzyme responsible for the initial degradation of the alpha' and alpha subunits, but not the beta subunit, of beta-conglycinin storage protein. The sizes of the proteolytic products generated by the action of protease C1 suggest that the cleavage sites on the alpha' and alpha subunits of beta-conglycinin may be located in their N-terminal domain, which is not found in the beta subunit of beta-conglycinin. To check this hypothesis, storage proteins from other plant species that are homologous to either the alpha'/alpha or the beta subunit of beta-conglycinin were tested as substrates. As expected, the convicilin from pea (Pisum sativum), a protein homologous to the alpha' and alpha subunits of beta-conglycinin, was digested by protease C1. The vicilins from pea as well as vicilins from adzuki bean (Vigna angularis), garden bean (Phaseolus vulgaris), black-eyed pea (Vigna unguiculata), and mung bean (Vigna radiata), storage proteins that are homologous to the beta subunit of soybean beta-conglycinin, were not degraded by protease C1. Degradation of soybean beta-conglycinin involves a sequential attack of the alpha subunit at multiple sites, culminating in the formation of a stable intermediate of 53.5 kD and a final product of 48.0 kD. The cleavage sites resulting in this formation of the intermediates and final product were determined by N-terminal analysis. These were compared to the known amino acid sequences of the three beta-conglycinin subunits. Results showed these two polypeptides to be generated by proteolysis of the alpha subunit at regions bearing long strings of acidic amino acid residues.