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。     

黑豆盐溶球蛋白的研究

对经凝胶过滤后的黑豆盐溶球蛋白进行Native-PAGE及双向SDS-PAGE电泳分析,结果表明黑豆7S球蛋白是由3个以非共价键相结合的亚基(MW:84．7kD,72．6kD和49．2 kD)连接而成;黑豆11S球蛋白中则含有以共价键相结合的亚基,同时也可能存在非共价键结合的亚基。其在还原条件下,可分解出5个肽链(MW：38．4kD,35．4kD,34．5kD,21kD和20．6kD),且在电泳图片上明显分为两个区域。     

施氏鲟卵黄蛋白原及其相关蛋白的研究

采用凝胶柱层析、聚丙烯凝胶电泳、免疫印迹（Western blotting）和免疫扩散等方法对施氏鲟卵黄蛋白原（Vi-tellogenin,Vg）及其相关蛋白（Yolk protein,YP）进行了研究。结果表明,施氏鲟血清Vg是一种糖脂磷蛋白,其相对分子量为410kD,由分子量为205kD的两个同源亚基组成。Vg的3种相关蛋白YP1、YP2和YP3。其中YP1相对分子量为370kD,是一种糖脂磷蛋白,由相对分子量为97kD和33kD的两个亚基构成。YP2是一种相对分子量为144kD的磷脂蛋白,由相对分子量为94kD和45kD的2个亚基构成。YP3为相对分子量为66kD的磷蛋白,由相对分子量为30kD的同源亚基构成。     

论文摘要

豌豆种子中含有豆球蛋白和豌豆球蛋白这两类主要的贮存蛋白,后者是可在亚基结构上所免疫特异蛋白加以区别的混合物。两类蛋白在亚基结构上存在微小的差异,并且在结构和数量上受不同的基因型支配。对豆球蛋白的遗传结构和生物合成的研究表明;有一族为许多前体分子编码的基因,又由这些前体分子产生了分子量约为40×10~3(α球     

花生种子发育和萌发过程中贮藏蛋白的合成和降解

以花生品种汕油5 2 3种子为材料,分离纯化花生球蛋白的41 kD和38.5 kD两种主要亚基及伴花生球蛋白的6 0.5 KD亚基并制备抗体.We stern blot分析表明,3种亚基在花生胚组织分化期的胚轴和子叶中就开始合成,其中60.5 kD亚基是最先在胚轴和子叶中大量合成和积累的贮藏蛋白,41 kD和38.5 kD亚基在随后的发育中积累量不断增加;种子萌发时这3种亚基的降解进程不一样,胚轴和子叶中41 kD和38.5kD亚基的降解均先于60.5 kD亚基.     

莲子贮存蛋白的主要亚基及积累模式

红莲(Nelumbo nucifera Gaertn)成熟子叶总蛋白含量达24.35 g/100 g干样品,其贮存蛋白(SP) 的积累模式与豆科种子相似,即随成熟度的提高,SP猛增至占总蛋白含量的86%以上。对莲子不同发育阶段子叶蛋白SDS-PAGE图谱的光密度测定表明莲子叶蛋白有12个主要SP亚基(SP 1～12)。按分子量(MW)大小和积累顺序可分3组A组是98 kD和93 kD的2个亚基,MW最大,积累最晚,但量最多;B组是3个亚基,MW为55～50 kD,大小居中,积累最早,量最少;C组的7个亚基MW最小,在27～14 kD之间,积累较早,量较多。对莲的不同品种、不同器官进行分析后发现,它们都有1个主峰为68 kD的多连峰的代谢蛋白亚基,可能是莲共有的蛋白亚基。     

莲子贮存蛋白的主要亚基及积累模式

红莲（NelumbonuciferaGaertn）成熟于叶总蛋白含量达24．35g／100g干样品,其贮存蛋白（SP）的积累模式与豆科种子相似,即随成熟度的提高,SP猛增至占总蛋白含量的86％以上。对莲子不同发育阶段子叶蛋白SDS-PAGE图谱的光密度测定表明：莲子叶蛋白有12个主要SP亚基（SP1－12）。按分子量（MW）大小和积累顺序可分3组：A组是98kD和93kD的2个亚基,MW最大,积累最晚,但量最多;B组是3个亚基,MW为55－50kD,大小居中,积累最早,量最少;C组的7个亚基MW最小,在27－14kD之间,积累较早,量较多。对莲的不同品种、不同器官进行分析后发现,它们都有1个主峰为68kD的多连峰的代谢蛋白亚基,可能是莲共有的蛋白亚基。     

小麦低分子量麦谷蛋白亚基与面团流变学特性关系的研究

采用十二烷基硫酸钠-聚丙稀酰胺凝胶电泳(SDS-PAGE)分离方法,以牛血清蛋白(67kD)和卵清蛋白(43kD)为分子量标记,对甘肃河西灌区近几年选育的17个小麦品系以及大面积栽培的2个春小麦品种的低分子量麦谷蛋白亚基组成以及不同亚基对面团流变学特性(面团韧性P、延伸性L、面团筋力W)的影响进行分析。19个试验材料中共标记出从35．2～60．5kD的LMW-GS共32条谱带;通过单因素方差分析(ANOVA)和逐步回归分析确定出对面团流变学特性P、L、W值影响显著的7个LMW-GS,分子量由高到低为：52．7kD、52kD、49．3kD、46．7kD、44．8kD、44．2kD、35．2kD。其中35．2kD和46．7kD亚基能显著地增加面团P值,44．8kD亚基能显著地降低面团P值;44．2kD和49．3kD亚基显著增加面团L值,52．7kD亚基降低面团L值;44．8kD、52．7kD亚基能显著降低面团的W值,52．0kD和46．7kD亚基能显著提高面团W值。     

苦瓜种子蛋白及其抑菌作用

SDS-PAGE分析表明,苦瓜种子含有多种蛋白组分,其中57kD和49kD蛋白可能由多个或多种亚基以二硫键结合形成。苦瓜种子蛋白对真菌和细菌有显著抑制效应。     

毛乌素沙地锦鸡种群种子蛋白多样性及其生物学意义

应用多种聚丙烯酰胺凝胶电泳方法对纯化的锦鸡儿种子球蛋白和清蛋白进行分离和分子量测定,占总蛋白７０％左右的球蛋白包括３个不同分子量的蛋白。它们由相同的２１个亚基组成,说明是不同的聚合态,清蛋白在ＰＡＧＥ和ＳＤＳ ＰＡＧＥ中都分出约２０种成分。全蛋白ＰＡＧＥ结果表明最主要的球蛋白和若干清蛋白在种群内和种群间有高水平的多态性,至少有４个可能的位点编码６个蛋白。ＳＤＳ ＰＡＧＥ结果表明全蛋白有约５０个不同     

Molecular heterogeneity of Cowpea (Vigna unguiculata Fabaceae) seed storage proteins

81 wild forms and 110 cultivated cowpea,Vigna unguiculata, accessions from 21 countries of Africa were screened for variability in seed storage proteins. Total seed proteins, albumin and globulin fractions were investigated by means of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and isoelectric focusing (IEF) of nonreduced and/or reduced samples in one- and two-dimensional procedures. The globulin fraction is heterogeneous in molecular weight and contains both legumin-like components and three to six nondisulfide-linked subunits. Three globulin subunits, with molecular weights 110, 76, and 41 kD were found to be composed of disulfide-linked polypeptides. In the nondisulfide-linked fraction, both cultivated and wild forms exhibited patterns of four types (A–D). This fraction contains polypeptide subunits of molecular weights 62, 56, and 52 kD for A type, 62, 56, 54, and 52 kD for B type, 62, 56, 52, and 50 kD for C type, and at least 62, 56, 54, 52, 50, and 49 kD for D type. These subunits present similar multiple charge forms but C and D types possess more basic specific 50 and 49 kD nondisulfide linked components. Major albumin fraction contains subunits of 94, 86, 32, and 24kD. No infraspecific variation was observed in albumin or legumin-like fractions. The discussion is focussed on the relations between genetic variability assessed by storage protein coding genes and phenotypic variability.     

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.     

SSR analysis of genetic diversity and structure of the germplasm of faba bean (Vicia faba L.)

Assessing the diversity and genetic structure of faba bean (Vicia faba L.) germplasm is essential to improve the quality and yield of this economically important crop. In this study, simple sequence repeats (SSRs) were utilized to evaluate the diversity and structure of 35 faba bean genotypes originating from three different geographical regions (Northern Africa, Eastern Africa, and Near East). All 15 SSR loci generated a total of 100 alleles. The allele number per locus varied from 4 to 11, with a mean of 6.67. The expected heterozygosity (H_e) of SSR loci ranged between 0.51 and 0.81, with a mean of 0.63. The PIC value also varied from 0.44 to 0.78, with an average of 0.58. The expected heterozygosity of 22 faba bean genotypes was higher than the observed one. Interestingly, AMOVA analysis showed that much of variability resided within accessions (79.2%). A highly significant difference among regions was also evidenced, and represented 5.3% of the total variation. Moreover, cluster analysis divided the 35 faba bean genotypes into two main clusters. The first main cluster comprised all faba bean genotypes originating from the Near East region, whereas the second main cluster comprised all the genotypes originating from the Northern and Eastern Africa regions, indicating that the Northern and Eastern African faba bean genotypes were more closely related to each other than to the Near East genotypes. Structure analysis also revealed that the 35 faba bean genotypes might be assigned to two populations, in complete accordance with cluster analysis data. In conclusion, this study showed high levels of diversity in the analysed genotypes of faba bean, and could be utilized in future breeding programmes to develop new cultivars of high yield.     

Development of a Simple PCR-based Marker for the Determination of Growth Habit in Vicia faba L. using a Candidate Gene Approach

We have developed the first molecular marker suitable for the selection of determinate growth habit in faba bean using the candidate gene approach. We obtained the sequences of TFL1/CEN like genes from public databases and designed primers on conserved domains. We used three cultivars with determinate growth habit and four accessions (two cultivars and two lines) with indeterminate growth habit. All these genotypes are used in our faba bean breeding program. A single monomorphic PCR fragment was obtained. A set of restriction enzymes was assayed. The enzyme Hind1II produced a clear polymorphism between determinate and indeterminate genotypes. This new cleaved amplified polymorphism (CAP) marker was tested using an F₂ population contrasting for growth habit derived from the cross ‘Verde Bonita’ × 2N52. This marker showed 100% efficiency in discriminating both types of genotypes. Moreover, the codominancy of this marker allows the detection of heterozygous individuals facilitating the breeding process when pyramiding different genes. The perfect cosegregation of the marker with the trait indicates that an orthologue of TFL1/CEN controls the growth habit in faba bean. This marker has been tested in all the genotypes used in our faba bean breeding program as donors of the determinate growth habit. Therefore, it is expected to work well in all the crosses performed with these parental lines as happens in the F₂ tested. The CAPS marker developed in this work will be useful for Marker Assisted Selection programs. In addition, this marker is useful for quality control to determinate the percentage of outsider seeds in commercial seed lots. Moreover, it is a valuable tool to breeders when submitting new faba bean varieties for registration since the method allows guaranteeing that outsider plants remains under the requested limit for registration.     

籽瓜种子蛋白组分和生化特性的研究(简报)

籽瓜是西瓜的变种,其种子板大、味香,是人们喜爱的食品。籽瓜种子蛋白质含量约为38%,其中78%是球蛋白。Blagrove(1980)报道。南瓜、西瓜、甜瓜及黄瓜等瓜类种子球蛋     

蚕豆种子贮藏蛋白质组分的比较研究

以10份蚕豆品种为材料,利用SDS-PAGE方法,对其种子贮藏蛋白亚基组成进行了分析,结果表明:不同品种间的种子贮藏蛋白具有一定的差异,表现出一定的多态性;共分离出23条迁移率不同的亚基条带,13条具有多态性;利用贮藏蛋白亚基条带的信息,分析了品种间的蛋白相似度,相似度指数0.619～0.947,平均0.744;并通过聚类分析,在遗传相似系数0.8260水平上,将供试材料分为了3类,其中第2类具有较丰富的多样性,多样性指数达3.9296;共筛选出7对亲缘关系较远的杂交组合,可为蚕豆品种选育提供一定的科学依据。     

Development of a new diagnostic marker for growth habit selection in faba bean (<Emphasis Type="Italic">Vicia faba</Emphasis> L.) breeding

Faba bean varieties with determinacy of the apical meristem are relevant to green production. A diagnostic CAPS (cleavage amplification polymorphic sequence) marker for determinate growth habit (ti) in faba bean was previously developed by Avila et al. (Mol Breed 17:185–190, 2006) but was effective only on a limited range of cultivars or genotypes. In this study, we studied the reasons for this limited application and developed a new marker useful for most faba bean-breeding programs. By designing a new set of primers, the complete genomic Vf_TFL1 sequences from different genotypes contrasting for the character were obtained and additional base changes associated with the ti phenotype were identified. The comparison among faba bean sequences showed that the previous CAPS marker was based on a SNP (single nucleotide polymorphism) at position 469 in the intron 2–3, a silent mutation. On the contrary, a SNP at position 26 that distinguishes determinate and indeterminate growth habit genotypes lead to an amino acid change (Leu-9 to Arg) in the determinate growth habit genotypes that could account for the ti phenotype. A dCAPS marker based on this SNP that creates a TaqI site in the ti allele was developed. The marker was 100% successful in predicting ti phenotypes in a broad range of faba bean germplasm representing all major cultivars historically grown in Europe. The outcome confirms the utility of the new dCAPS in worldwide marker-assisted selection programs.     

In situ localization of faba bean and oat legumin-type proteins in transgenic tobacco seeds by a highly sensitive immunological tissue print technique

We have used a highly sensitive immunological tissue print technique to study cell- and tissue-specific expression of heterologous genes in transgenic plants. Primary polyclonal antibodies, raised against legumin of faba bean (Vicia faba L.) and 12S globulin of oat (Avena sativa L.) were used to localize these proteins in transgenic tobacco seeds in a streptavidin-alkaline phosphatase assay in combination with biotinylated secondary antibodies producing a higher sensitivity (by several amplification steps) of the assay. Both storage protein genes were found to be expressed in a specific pattern. While legumin is preferentially accumulated in certain parts of the embryo, the oat legumin-type globulin is restricted to the endosperm. The applied technique is highly sensitive with a resolution power down to the singlecell level and allows rapid screening of large numbers of samples.