新的水稻谷蛋白α—1亚基缺失突变体

从水稻受精卵MNU处理后代中获得4个谷蛋白α－1亚基缺失突变品系。SDS－PAGE和IEF分析表明这些突变体在共同缺失1条pI6.82多肽的同时,或形成新的多肽,或其他多肽表现量增加,这些突变体是由结构基因控制的,IEF分析同时显示2条多肽pI6.82和pI8.58源自同一条谷蛋白前驱体。这4个突变体对于改良水稻谷蛋白品质、研究谷蛋白生物合成遗传调控机制以及揭示谷蛋白基因功能是不可多得的研究材料。     

水稻种子贮藏谷蛋白的改良电泳分析法

利用15％－25％丙烯酰胺梯度凝胶的SDS－PAGE分析法可将水稻种子贮藏谷蛋白分离为3个酸性（α）亚基和3个碱性（β）亚基,通过调节两性电解质比例对现有等用点了和焦电泳分析法进行改良,可将谷蛋白酸性亚基和碱性亚基分别分划为13和14条多肽带,将上述两种方法结合起来的双向电泳分析法可以高清晰度地离析谷蛋白并获得单一多肽,此改良的电泳分析系统有助于确定水稻谷蛋白变异及谷蛋白的生化研究。     

水稻种子贮藏谷蛋白α—2亚基减少突变体

通过筛选水稻（Oryza sativa L.)受精卵的甲基亚硝基脲（MNU）处理后代,获得9个谷蛋白α-2亚基减少（α－2L）突变体。这些突变体依据其SDS－PAGE图谱又可分成3种类型：α－1显增加型（α－1H／α－2L）、β－2减少型（β－2L／α－2L）和α－3显增加型（α3－H／α－2L）。双向电泳分析揭示了产生突变体α-2L的原因在于缺少了一条pI6.71/α－2的多肽;α－1H和α－3H的原因在于分别敢一条新的多肽pI6.50/α-1和pI6.90/α-3;而产生β－2的原因在于缺少一条pI18.74/β－2的多肽。pI6.71/α-2和pI8.74/β-2多肽同时缺失于同一突变体暗示二可能来源于同一前本,为同一基因的产物。这些突变体为水稻谷蛋白遗传规律、生物合成遗传调控机制、基因功能以及蛋白组学研究不可多得的素材。     

水稻谷蛋白突变体的筛选及遗传分析

通过对国内外水稻品种种子的全蛋白分析,筛选到3个谷蛋白突变材料。其中编号W3660种子中37～39kDa与22～23kDa谷蛋白亚基的含量较普通水稻明显降低,而13kDa醇溶蛋白多肽含量则大幅升高;W204和W379种子中37～39kDa与22～23kDa谷蛋白亚基的含量介于普通品种与W3660之间,W379还具超大含量的57kDa多肽,实验证明此多肽属谷蛋白成分。用W3660和普通水稻栽培品种惊人糯(Otorokimochi)构建了杂交群体。后代种子总蛋白SDS-PAGE分析显示,低谷蛋白和高醇溶蛋白性状总是相伴出现;F1种子全部呈现低谷蛋白含量和高醇溶蛋白含量特性;F2种子中呈现低谷蛋白和正常蛋白性状的比例约为3：1;从F3种子分析推断出的F2植株基因型,其低谷蛋白纯合型,杂合型和正常型的分离比例符合1：2：1。表明,W3660的低谷蛋白和高醇溶蛋白性状是由单显性基因控制,而且能稳定地遗传给后代。     

野生大豆(Glycine soja Sieb et ZUCC)球蛋白A_5 A_4 B_3 cDNA的研究(简报)

大豆球蛋白是大豆种子中主要的贮藏蛋白。它们在某些作物中占种子干重20%以上。已经知道大豆球蛋白由6个亚基组成。每个亚基由一或两个酸性多肽(A)和一个碱性多肽(B)组成,多肽之间由二硫键联结。这些亚基是从编码A-B亚基前体的mRNA合成,然后经过转录后加工剪切形成A肽和B肽。至今所有关于球蛋白的基因结构与表达的报道都集中于栽培大豆上。由于我国有丰富的大豆     

湖北省优质杂交稻品种贮藏蛋白的比较研究

研究选取水稻品种共20个,其中包括湖北省近两年审定的优质杂交水稻品种10个(‘鄂早17’、‘鄂早18’、‘两优1193’、‘武香880’、‘岳优26’、‘宜优99’、‘协优96’、‘鄂晚12’、‘鄂中5号’、‘两优277’)以及7个普通杂交水稻(‘G98-202’、‘578’、‘两优637’、‘宜优22’、‘3685’、‘3089’、‘加育948’)和国家审定的优质水稻品种3个(‘嘉育948’、‘两优932’、‘舟903’),采用不连续的SDS-PAGE电泳分析种子贮藏蛋白,结果在谷蛋白中主要分离出57 kD的蛋白前体、37～39 kD的酸性亚基和22～23 kD的碱性亚基。同时,选取品种‘3089’、‘嘉育948’、‘537’、‘舟903’和‘两优932’,利用高效液相色谱法分析了种子贮藏蛋白中的谷蛋白亚基的含量。结果表明,水稻谷蛋白亚基含量的多少可以作为评价品种营养品质优劣的参考依据之一。     

家蚕滞育激素-性信息素合成激活肽基因表达的调节

滞育激素和性信息素合成激活肽是两个重要的昆虫神经肽,这两个神经肽由一个基因编码．利用分子杂交和ＲＴ－ＰＣＲ技术,确定了滞育激素－性信息素合成激活肽基因表达的调节不属于转录后的调节,推定为翻译后形成一个大的前体多肽再剪接为几个成熟的神经肽分子．     

恶性疟原虫杂合多肽抗原融合基因的克隆和表达

合成了５对寡核苷酸片段,分别连接在两种恶性疟原虫杂合多肽（４５肽和５８肽）抗原基因片段（ＨＰＦＧＡ和ＨＰＦＧＢ）的头部和尾部,将这两种片段分别与霍乱毒素Ｂ亚单位（ＣＴ－Ｂ）基因末端融合。两种杂合多肽抗原分别含有数个红内期和红外期有代表性的、并能被Ｔ或Ｂ淋巴细胞识别的保护性抗原表位,ＣＴ－Ｂ基因前端具有促使分泌的信号肽序列。将这两种融合基因的不同重组质粒分别转化大肠杆菌,对转化菌的培养上清检测表明融合蛋白被分泌性表达,既具有恶性疟原虫和ＣＴ－Ｂ抗原性,又保持了ＣＴ－Ｂ与其受体神经节苷脂ＣＭ１结合的生物学活性。     

恶性疟原虫杂合多肽抗原融合基因的克隆和表达

合成了５对寡核苷酸片段，分别连接在两种恶性疟原虫杂合多肽（４５肽和５８肽）抗原基因片段（ＨＰＦＧＡ和ＨＰＦＧＢ）的头部和尾部，将这两种片段分别与霍乱毒素Ｂ亚单位（ＣＴ－Ｂ）基因末端融合。两种杂合多肽抗原分别含有数个红内期和红外期有代表性的、并能被Ｔ或Ｂ淋巴细胞识别的保护性抗原表位，ＣＴ－Ｂ基因前端具有促使分泌的信号肽序列，将这两种融合基因的不同重组质粒分别转化大肠杆菌，对转化菌的培养上清检测表明融     

噬菌体展示技术筛选抗轮状病毒多肽的试验研究

从噬菌体随机展示十五肽文库筛选出4个与轮状病毒粒子特异性结合多肽。经空斑减少抑制实验和MTT法分析表明其中3个多肽对病毒感染培养细胞具有抑制作用,其中序列为QSNPIHIITNTRNHP的C肽具有显著抑制作用,抑制效果达93%,另外2个多肽A和B抑制效果分别为40%与50%。经过多肽序列分析发现这3个十五肽具有2个保守序列,分别是第2至8个氨基酸残基SNPIHII和第12~15个氨基酸残基NIP。胰蛋白酶水解位点分析表明C肽无裂解位点,而A肽和B肽则分别具有3个和4个潜在水解位点。抑制病毒感染液中胰蛋白酶活性,发现A,B两肽也能显著地抑制病毒离体感染。说明所筛选的多肽2个保守序列的完整对抗病毒感染起着重要作用。C肽有望成为一种治疗轮状病毒感染的口服药物。     

Biochemical characterization of rice glutelin

The two major subunits of rice glutelin, the acidic (α) and basic (β) polypeptides were purified by chromatofocusing and cation exchange chromatography, respectively. The molecular weight range of the α polypeptides was 28.5 to 30.8 kilodaltons and the molecular weight range of the β polypeptides was 20.6 to 21.6 kilodaltons. Electrofocusing in polyacrylamide gels showed that the isoelectric points of the α and β polypeptides were 6.5 to 7.5 and 9.4 to 10.3, respectively. At least 12 polypeptides of the α-group and nine polypeptides of the β-group could be separated by electrofocusing. The amino acid compositions of whole glutelin, and the purified α and β subunits were analyzed. The α subunit contained more glutamic acid/glutamine, serine, and glycine, and less alanine, lysine, aspartic acid/asparagine, and isoleucine than the β subunit. A comparison of the amino acid composition of rice glutelin subunits with those of the 11S proteins from eight other plant species indicated that there is more similarity between the β subunits than the α subunits of several diverse plant species.     

水稻谷蛋白是类似于豆球蛋白的蛋白质

水稻是我国的主要粮食作物,它的蛋白质含量为5—14%。在水稻种子蛋白质中,谷蛋白占80%,球蛋白占10%,醇溶蛋白占5%,清蛋白占5%。据报道,水稻种子清蛋白主要由分子量16800的亚基组成,球蛋白由10种不同分子量的亚基通过疏水交互作用相结合,谷蛋白由分子量38000、25000和16000三种亚基通过双硫键相结合:但是,Yamagata(1982)和作者(1983、1984、1986)的研究表明:水稻种子谷蛋白主要由分子量     

The two subfamilies of rice glutelin differ in both primary and higher-order structures

Rice glutelin, which accounts for 70-80% of the total proteins of the seeds, consists of two nutritionally different subfamilies (A and B types). Although the similarity in primary sequences between the two subfamilies is as high as 60%, we established conditions to discriminate the two subfamilies when low amounts of antigen are analyzed by immunoblot methods. The glutelin alpha polypeptides can be resolved into six bands labeled alpha1 to alpha6 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Gel filtration analysis showed that glutelin exists as a polymerized and a smaller molecular weight form. Immunoblot analysis of SDS-PAGE resolved polypeptides showed that alpha2, alpha3, and alpha4 are an A type and that these A types as well as alpha1, a B type, are polymerized. The polymerization tendency clearly differed between the two subfamilies except for alpha1, which may be derived from GluB-4 as suggested by analysis using Escherichia coli expression systems of glutelin cDNA regions corresponding to alpha polypeptides. GluB-4 and all the A type subunits have an extra Cys residue in the hypervariable regions, corresponding to the C-terminal region of alpha polypeptide. Accordingly, the extra Cys residue is hypothesized to be responsible for the polymerization of glutelin.     

Immunological relationships among the major seed proteins of cereals

The structural relationships among the major seed proteins of cereals was evaluated by Western blot analyses using antibodies raised against the wheat gliadin, rice glutelin acidic and basic subunits, and rice prolamine polypeptide. Consitent with the conservation of the primary sequences of these proteins, antibodies to the acidic and basic glutelin subunits cross-reacted with homologous polypeptides from oat as well as pea. The rice glutelin antibodies did not react with the major seed proteins from barley, rye, maize and sorghum. Antibodies raised against the acidic glutelin subunit reacted with the wheat glutenins but antibodies to the basic glutelin subunit did not. A comparison of the published primary sequences of a high molecular weight glutenin and rice glutelin showed little similarity except for a conserved peptide with the motif arg-gln-leu-gln-cys. The possible significance of this conserved element shared by these widely different proteins is discussed. Similar studies with the wheat gliadin antibody showed immunologically related components in plants of the subfamily Festucoideae except for rice. Antibodies raised against the rice prolamine recognized only the rice prolamine, indicating that this polypeptide was structurally distinct from other cereal prolamines. Overall, these results support and help clarify the evolutionary relationship of the cereals.     

Characterization of the DNA-cellulose-binding proteins from Escherichia coli K 12

The various [35S]DNA-binding proteins present in lysates of Escherichia coli K 12 cells have been analyzed by means of two-dimensional SDS-polyacrylamide gel electrophoresis. The proteins were isolated by the DNA-cellulose technique and eluted by increasing concentrations of NaCl (0.15, 0.4, 0.6 and 2 M). Only 2% of the total 35S radioactivity in the lysate became bound to the DNA-cellulose column. A total of 237 polypeptides were detected and the distribution among the salt eluates were 85, 83, 40 and 29 polypeptides, respectively. The 40 major polypeptides with regard to concentrations were also identified from gels stained with a protein-specific reagent. The polypeptides could be divided into two main groups according to pI values, namely, acidic polypeptides (total number, 174) and basic polypeptides (total number, 63). The ratio between acidic and basic polypeptides decreased with increasing salt concentrations in the eluates. The majority of the basic polypeptides had molecular weights in the range 10 000-30 000, whereas the acidic polypeptides had molecular weights from 10 000 to 165 000.     

A rice glutelin and a soybean glycinin have evolved from a common ancestral gene

A cDNA clone covering the entire coding region for a glutelin subunit precursor has been identified from a library of endosperm-developing rice cDNA clones using a mixed oligodeoxynucleotide probe, and then by immunoprecipitation of hybrid-selected translation product with an antiserum against the acidic polypeptides of the glutelin. Analysis of the cDNA insert revealed that rice glutelin is synthesized as precursor polypeptides which undergo post-translational processing to form the nonrandom polypeptide pairs, like glycinin precursors of soybean. By comparing the predicted protein sequence of this precursor from monocots with that of glycinin A1aB1b precursor from dicots, it was found that the overall 32% of the amino acid positions are identical in both proteins. Because regions which show identities are dispersed throughout both molecules, the similarity is not due to convergent evolution, but to divergence evolution from a common ancestral gene.     

Mutants lacking glutelin subunits in rice: mapping and combination of mutated glutelin genes

Nine mutant lines lacking glutelin subunits were selected from M₂ seeds of about 10000 M₁ plants mutagenized with gamma rays or EMS and from 1400 mutant lines selected originally for morphological characters. There were three types of mutants, one line lacking the largest subunit among four minor bands of glutelin acidic subunits (Type 1), five lines lacking the second largest subunit band (Type 2), and three lines lacking the third largest subunit band (Type 3). Mutants lacking the smallest subunit band were not found. Type 1 lacked 2 of the 10 spots of glutelin acidic subunits separated by two-dimensional electrophoresis and 1 of the 11 spots of the 57-kDa glutelin precursor. Type 2 lacked 2 spots of acidic subunits and 1 spot of the 57-kDa glutelin precursor, and had low amounts of 1 of the 8 spots of glutelin basic subunits. Type 3 mutants lacked each of 1 spot of the acidic subunits and glutelin precursor and had low amount of 1 spot of the basic subunits. Genetic analysis of the mutated genes showed that these mutant characters were controlled by single recessive genes named glu-1, glu-2, and glu-3, respectively. Mutated genes of different lines of the same type were found to be at the same locus. RFLP analysis of F₂ plants between the mutant lines and cv `Kasalath' indicated that glu-1 is on chromosome 2, glu-2 on chromosome 10, and glu-3 on chromosome 1. These mutant genes were combined by crossing, and a line lacking the 3 minor bands of the glutelin acidic subunits was developed. However, the total glutelin content of this line was not remarkably reduced, showing a only 13% decrease. Received: 1 April 1996 / Accepted: 14 June 1996