Studies on Rice Glutelin

The complete amino acid analysis of the whole glutelin preparation from rice endosperm was performed. The recoveries were 101.59% for amino acid residues and 101.68% for nitrogen, and the standard deviations for four determinations on the 22 and 70 hr hydrolyzates were very small. The features of the amino acid composition of the protein were as follows; (1) the high contents of dicarboxylic amino acids, particularly glutamic acid, (2) about 60% of these dicarboxylic amino acids was in the amide form, and (3) the significantly low contents of tryptophan, methionine and half cystine. The amino acid analyses of the two kinds of the subunits of glutelin, the neutral major one and the basic minor one, were also carried out. There were some significant differences between the two subunits, for instance, in the contents of glutamic acid, tryptophan, glycine, half cystine, methionine and lysine. However, the composition of whole glutelin seemed to be settled predominantly by that of the major subunit.     

Studies on Rice Glutelin

In order to isolate and purify glutelin from rice endosperm, Kondo’s method had been widely employed. However, we found that this method was not sufficient to prepare pure glutelin for structural studies, because glutelin prepared by this method would be easily contaminated with some impurities such as carbohydrates, nucleic acids and alkalidenatured albumin and globulin. Then we introduced a new method, which essentially involved the simultaneous extraction of impurities and precipitation of glutelin at pH 10.0 in the presence of sodium chloride. The purified glutelin by this new method had high nitrogen-content, 17.9%, and very low contents of phosphorus, pentose (nucleic acid) and hexose. The N-terminal analysis by FDNB method also revealed that the preparation was chemically pure and glycine was the only one detectable terminus of glutelin.     

Studies on Rice Glutelin

In order to obtain information of the gross-structure of glutelin, chemical and physicochemical properties of S-cyanoethyl glutelin were investigated. Glutelin remained at the origin in polyacrylamide gel electrophoresis, while S-cyanoethyl glutelin migrated in the gel and resolved into two components. The ion-exchange chromatography by carboxymethyl Sephadex C-50 gave further resolution of S-cyanoethyl glutelin into one neutral component corresponding to the anodic component and two basic components corresponding to the cathodic component in polyacrylamide gel electrophoresis at neutral pH. The amino-terminal residue of the neutral component (Component I) could not be detected by the fluorodinitrobenzene method, while both the basic components (Component II and III) had only glycine as the amino-termini. On the basis of dinitrophenyl-glycine found, the minimum molecular weights of Component II and III were calculated at about 35,000 and 43,000 respectively. The relative concentration of these three components was as follows; Component I: Component II: Component III=8:1:1. These facts obviously indicate that glutelin is a very large molecule composed from these three components polymerized by disulfide linkage, Component I being the major subunit.     

Heterogeneity of Rice Glutelin

Three forms of endopolygalacturonase from Saccharomyces fragilis (Kluyveromyces fragilis) were separated by a procedure including adsorption on Amberlite IRC-50, CM Sephadex C-50 column chromatography and repeated preparative disc electrophoresis. Each endo-PG was almost homogenoeus as judged by polyacrylamide gel electrofocusing and disc electrophoresis. The three enzyme were designated as enzymes I, II and III. Enzymes I and II were similar but enzyme HI different from I and II in isoelectric point. The three enzymes resembled one another in eznyme action on pectic acid and other properties. All the three enzymes showed macerating activity toward the potato and carrot tissues.     

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。     

Cell-Free Synthesis of the Rice Glutelin Precursor

Polyadenylated RNA was directly isolated from developing rice(Oryza sativa. L) seeds. The major translation product of theisolated polyadenylated RNA in a rabbit reticulocyte cell-freesystem was about 2 kDa larger than the in vivo labeled 57 kDaglutelin precursor, and was immunoprecipitated by antiserumagainst the glutelinprolamin fraction of mature seeds. (Received January 27, 1986; Accepted June 23, 1986)     

水稻种子贮藏谷蛋白的微细异质性

利用灵敏的等聚焦与SDS－PAGE合的双向电泳分析方法,从水稻（Oryza sativa L.)种子贮藏谷蛋白中至少可以分离为13条酸性和19条碱性多 肽,依据谷蛋白多肽的表现量推测,水稻谷蛋白主要由约6个主效基因控制,肽图谱与N－端氨基酸序列分析可清晰将谷蛋白酸性多肽分为两组,此两组恰好与谷蛋白GluA和GluB两个cDNA克隆组相吻合。     

特种稻谷蛋白亚基的研究

以陕西红香米、绿香米、紫糯、茉莉香米、黑帅、黑宝为实验材料,采用SDS-PAGE方法在pH为8.9,电流为15mA条件下,对其种子的谷蛋白进行了电泳分析,寻找其特征及其之间的差异。结果表明绿香米、黑帅都具有9条特征谱带;茉莉香米具有7条特征谱带;紫糯具有6条特征谱带;黑宝具有5条特征谱带;红香米具有4条特征谱带。分子量较高、蛋白谱带较多的品系为绿香米和黑帅(6～5条);分子量较高、蛋白谱带较少的品系为茉莉香米(2条)。     

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。     

Structural Polypeptides of Simian Virus 40

To determine the number and molecular weights of the structural polypeptides of simian virus 40, we have analyzed purified virus by electrophoresis on 14% polyacrylamide gels containing sodium dodecyl sulfate. Full virus purified by several different methods showed six distinct bands with molecular weights of approximately 43,000 (VP1, containing 70% of virion protein), 32,000 (VP2, 9%), 23,000 (VP3, 10%), 14,000 (VP4, 6%), 12,500 (VP5, 4%), and 11,000 (VP6, 3%) both by analysis of radioactively labeled virions and by visualization of the polypeptide bands after staining. “Empty” virions contain decreased amounts of VP4, 5, and 6. The approximate molecular ratios of the polypeptides were 6.0, 1.0, 1.5, 1.5, 1.1, and 1.0. When virus degraded in an alkaline buffer was analyzed by velocity centrifugation in sucrose gradients, the two larger polypeptides (VP1 and VP2) remained at the top of the gradient, whereas the three smallest polypeptides (VP4, 5, and 6) sedimented as a complex with the viral deoxyribonucleic acid. VP3 was found in association with either VP1 and 2 or VP4, 5, and 6, depending on the conditions of degradation. Presumably, VP1 and VP2, comprising about 80% of the protein, form the capsid of the virus. VP4, 5, and 6 may form a nucleoprotein in the virion, and VP3 may serve as an intermediate structural component.     

水稻谷蛋白GluA-2基因在小麦胚乳中的表达

水稻(Oryza sativa L.)谷蛋白(Glutelin)约占水稻储藏蛋白总量的80%,谷蛋白赖氨酸含量较高并易于被人体消化吸收.为了提高小麦(Triticum aestivum L.)的营养品质,将水稻谷蛋白GluA-2基因的cDNA序列导人小麦栽培品种Bobwhite(T. aestivum cv.Bobwhite).共轰击了600个小麦幼胚,经PCR和Southern杂交鉴定,共获得4棵转GluA-2基因小麦;SDS-PAGE分析表明,GluA-2基因在3棵转基因植株及其后代中表达,在1棵转基因植株中未表达,但其内源的高分子量麦谷蛋白亚基Bx7和By9含量显著降低,并且可遗传至T1代.     

水稻谷蛋白GluA-2基因在小麦胚乳中的表达(英文)

水稻(Oryza sativa L.)谷蛋白(Glutelin)约占水稻储藏蛋白总量的80％,谷蛋白赖氨酸含量较高并易于被人体消化吸收。为了提高小麦(Triticum aestivum L. )的营养品质,将水稻谷蛋白GluA-2基因的cDNA序列导入小麦栽培品种Bobwhite(T. aestivum cv. Bobwhite)。共轰击了600个小麦幼胚,经PCR和Southern杂交鉴定,共获得4棵转GluA-2基因小麦;SDS-PAGE分析表明,GluA-2基因在3棵转基因植株及其后代中表达,在1棵转基因植株中未表达,但其内源的高分子量麦谷蛋白亚基Bx7和By9含量显著降低,并且可遗传至T_代。     

Rice MYB Protein OSMYB5 Specifically Binds to the AACA Motif Conserved among Promoters of Genes for Storage Protein Glutelin

Binding analyses revealed that the AAC--A sequence in glutelingene promoters is the target site of OSMYB5 protein and thatboth the distal and proximal AACA motifs are recognized by thisprotein. These results suggest that the OSMYB5 protein functionsas a trans-acting factor for glutelin gene expression. (Received November 25, 1997; Accepted February 18, 1998)     

Structural Polypeptides of the Granulosis Virus of Plodia interpunctella

Techniques were developed for the isolation and purification of three structural components of Plodia interpunctella granulosis virus: granulin, enveloped nucleocapsids, and nucleocapsids. The polypeptide composition and distribution of protein in each viral component were determined by sodium dodecyl sulfate discontinuous and gradient polyacrylamide slab gel electrophoresis. Enveloped nucleocapsids consisted of 15 structural proteins ranging in molecular weight from 12,600 to 97,300. Five of these proteins, having approximate molecular weights of 17,800, 39,700, 42,400, 48,200, and 97,300, were identified as envelope proteins by surface radioiodination of the enveloped nucleocapsids. Present in purified nucleocapsids were eight polypeptides. The predominant proteins in this structural component had molecular weights of 12,500 and 31,000. Whereas no evidence of polypeptide glycosylation was obtained, six of the viral proteins were observed to be phosphorylated.     

水稻谷蛋白基因GluC启动子的克隆及表达分析

水稻谷蛋白仅在水稻种子胚乳中表达,其启动子是分离胚乳特异性表达启动子的理想材料。本研究克隆了GluC基因启动子pGluC,生物信息学分析表明pGluC内部含有胚乳特异性表达所需要的Skn-1 motif和ACGT-box元件。将pGluC启动子和7个5'端缺失启动子片段构建到pGPTV-GUS载体上,转化水稻愈伤组织,进行组织化学染色和GUS酶活分析。结果表明：全长及截短的-1 911、-1 611、-1 311 bp启动子均能驱动GUS基因在水稻种子胚乳中高效稳定表达。-999、-451、-203、-102 bp启动子失去了胚乳表达特异性,在根、茎或者叶中也检测到GUS表达。该结果为实现外源目的基因在水稻胚乳中特异高效表达提供了理论依据。     

Micro-heterogeneity of Storage Glutelin in Rice Seed (in English)

Nine rice Oryza sativa L.） mutant lines lacking the seed storage glutelin α-2 subunit were obtained from the progenies of fertilized egg cells treated with N-methy-N-nitrosourea (MNU). The mutants could be classified into three types: the α-1 subunit increased type (α-1H/α-2L), decreased the β-2 subunit decreased type (β-2L/α-2L) and the α-3 subunit increased type (α-3H/α-2L) according to their SDS-PAGE profiles. Two-dimensional electrophoresis analysis revealed that all of the mutants lacked a polypeptide of pI 6.71/α-2, while new polypeptides of pI 6.50/α-1 and pI 6.90/α-3 formed in α-1H/α-2L and α-3H/α-2L mutants respectively. Although the β-2L/α-2L mutants did not form new polypeptide, their pI 8.74/β-2 polypeptide was also decreased, suggesting that the two polypeptides decreased in β-2L/α-2L mutants might derive from the same glutelin precursor. These mutant lines are very useful in studying genetic characterisation,the mechanism of genetic regulation on biosynthesis, gene function and proteomics of rice seed storage glutelin.     

水稻低谷蛋白创新种质的选育和鉴定

谷蛋白是水稻种子中含量最高的贮藏蛋白,占种子总蛋白的70%以上,是稻米蛋白中可供人体吸收的主要成分。低谷蛋白稻米可以满足肾脏病人和并发肾脏机能损害的糖尿病患者在蛋白代谢方面的特殊需求,因此培育适合肾脏病人食用的低谷蛋白品种已成为水稻遗传改良的重要目标。本研究通过引进日本低谷蛋白品种LGC-1,于2009年配置其与江苏高产优质粳稻武育粳3号的杂交组合,经过6代自交和农艺性状筛选结合分子标记辅助选择,于2013年选育出3个低谷蛋白新品系。经分子标记检测、种子总蛋白SDS-PAGE分析和谷蛋白含量测定表明,这3个品系谷蛋白含量均降至LGC-1水平,显著低于普通推广品种,且农艺性状表现优异,为水稻低谷蛋白新品种选育提供了优良的新种质。