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.     

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)。     

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。     

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.     

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。     

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。     

Structural Relationship among the Rice Glutelin Polypeptides

When the glutelin protein fraction of rice (Oryza sativa L.) seeds was fractionated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, three size classes of proteins, 51 kilodaltons (kD), 34 to 37 kD, and 21 to 22 kD, as well as a contaminating prolamine polypeptide of 14 kD were detected. Antibodies were raised against these proteins and employed in studies to determine whether a precursor-product relationship existed among the glutelin components. Antibodies of the 34 to 37 kD and 21 to 22 kD polypeptides strongly reacted with the 51 kD protein, and conversely, anti-51 kD protein cross reacted with both of the putative subunits. Immunoprecipitation of in vitro translated products resulted in the synthesis of only the precursor form, indicating that the α and β subunits are proteolytic products of the 51 kD precursor protein. The poly(A)⁺ RNA directed in vitro translated product was about 2000 daltons larger than both the authentic glutelin precursor and the in vitro translated product from polysome run-off synthesis. Western blot analysis of the 34 to 37 kD and 21 to 22 kD polypeptides partially digested with Staphylococcus aureus V8 protease revealed distinct patterns indicating that these proteins are structurally unrelated. As observed for the glutelins, the rice prolamines are also synthesized as a precursor of 16 kD, 2000 daltons larger than the mature polypeptide. Addition of dog pancreatic microsomal membranes to a wheat germ protein translation system resulted in the processing of the prolamine preprotein but not the preproglutelin to the mature form.     

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。     

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。     

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.     

偃麦草属植物醇溶蛋白和谷蛋白多态性及系统学研究

运用酸性聚丙烯酰胺凝胶电泳(A-PAGE)和十二烷基硫酸钠聚丙烯酰胺凝胶电泳(SDS-PAGE)对偃麦草属(Elytrigia)24个物种的醇溶蛋白和谷蛋白进行了研究。以中国春为参照,按醇溶蛋白和谷蛋白电泳图谱条带迁移距离大小和条带多态性对供试材料进行聚类分析。结果显示,24份供试材料均呈现不同的醇溶蛋白和谷蛋白电泳图谱,共分离出醇溶蛋白带纹83条和谷蛋白带纹53条,多态性均达100%,并且在相同染色体组组成的物种中,染色体数目越多,其蛋白带纹越多;偃麦草属24个物种的醇溶蛋白和谷蛋白图谱均有明显差异,蛋白图谱可作为鉴定偃麦草属物种的指纹图谱。聚类分析结果显示,在材料间的醇溶蛋白遗传相似性系数为0.66时,24份材料被划分为6个主要类群,含E和St基因组的物种具有较近的亲缘关系;在谷蛋白遗传相似性系数为0.62时,24份材料被聚为4大类。聚类结果表明,染色体组成未知的物种Et.pachynera可能为异源多倍体物种。     

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。     

The Processing of a 57-kDa Precursor Peptide to Subunits of Rice Glutelin

The processing of a 57-kDa peptide into 37- and 22-kDa subunitsof glutelin, a major storage protein of rice, was confirmedby the immunological compatibility between the precursor andglutelin subunits. The 57-kDa peptide reacted with the antiseraraised against purified 37-kDa and 22-kDa subunits of glutelin.The processing was further confirmed by alteration of an invivo protein synthesis by monensin, a sodium ionophore whichinhibits the intracellular transport of secretory and membraneproteins. Infusion of monensin into developing rice grains resultedin suppressed formation of mature glutelin subunits with concomitantaccumulation of the 57-kDa peptide. The present results indicatethat both subunits of rice glutelin were produced by post-translationalcleavage of the 57-kDa peptide. (Received July 9, 1986; Accepted October 1, 1986)     

水稻谷蛋白GluA-2基因5′上游序列控制下的UidA基因在转基因水稻胚乳中的表达模式

为了研究谷蛋白胚乳特异性表达启动子在我国栽培稻品种中的表达模式,将UidA基因分别置于水稻谷蛋白GluA—2基因750bp和2．3kb上游序列下游,利用农杆菌转化法导人栽培稻品种中花8号并获得转基因植株。Southern blot检测表明,UidA基因已经整合到水稻基因组当中并以单拷贝存在。Northern blot检测表明,开花后13～15d和11～13d,UidA基因和水稻内源的GluA—2基因的表达量分别达到最高,随后逐渐降低。对转基因植株种子的GUS染色表明,UidA基因仅在胚乳中表达,在糊粉层中GUS表达量最高。测定了2．3kb和750bp转基因植株种子的GUS活性,结果表明前者的GUS活性是后者的2～3倍。序列分析表明,位于GluA—2基因转录启始位点上游2170bD的G-box可能是一个与表达量相关的顺式调控元件。