Expression of a Soybean (Glycine max [L.] Merr.) Seed Storage Protein Gene in Transgenic Arabidopsis thaliana and Its Response to Nutritional Stress and to Abscisic Acid Mutations

Among the three subunits of [beta]-conglycinin, the 7S seed storage protein of soybean (Glycine max [L.] Merr.), expression of the [beta] subunit gene is unique. Accumulation of the [beta] subunit is enhanced in sulfate-deficient soybean plants, and its mRNA levels increase when abscisic acid (ABA) is added to the in vitro cotyledon culture medium. Transgenic Arabidopsis thaliana lines carrying a gene encoding the [beta] subunit was constructed and grown under sulfate deficiency. Accumulation of both [beta] subunit mRNA and protein were enhanced in developing A. thaliana seeds. Accumulation of one of the A. thaliana seed storage protein mRNAs was also enhanced by sulfate deficiency, although the response was weaker than that observed for the soybean [beta] subunit mRNA. When the aba1-1 or abi3-1 mutations were crossed into the transgenic A. thaliana line, accumulation of the [beta] subunit was significantly reduced, whereas accumulation of the A. thaliana seed storage protein was not greatly affected. These results indicate that soybean and A. thaliana share a common mechanism for response to sulfate deficiency and to ABA, although the sensitivity is different between the species. The transgenic A. thaliana carrying the [beta] subunit gene of [beta]-conglycinin will be a good system to analyze these responses.     

Promoters of Rice Seed Storage Protein Genes Direct Endosperm-Specific Gene Expression in Transgenic Rice

The promoters of genes encoding rice seed storage proteins (glutelin,prolamin, globulin and albumin) were analyzed for their abilityto direct rß-gIucuronidase (GUS) gene expression intransgenic rice plants. All promoters tested could direct endosperm-specificexpression of the GUS reporter gene irrespective of variableactivities and patterns in the endosperm. (Received February 27, 1998; Accepted May 16, 1998)     

Ectopic Expression of Riboflavin-binding Protein Gene TsRfBP Paradoxically Enhances Both Plant Growth and Drought Tolerance in Transgenic Arabidopsis thaliana

Riboflavin is the precursor of the coenzymes flavin monophosphate (FMN) and flavin adenine dinucleotide (FAD), which serve as indispensable redox cofactors in all plants. Numerous data indicate that riboflavin is involved in pathogen resistance but less data are available on abiotic stress tolerance. In this experiment, the overexpression of the riboflavin-binding protein resulted in an enhancement of vegetative growth and net photosynthetic rate, and an acceleration of floral transition in transgenic Arabidopsis thaliana REAT11 (containing less than half the normal levels of free riboflavin, FMN, and FAD) compared to wild-type Col-0 under nonstressed conditions. The effect of drought stress on the antioxidant response of Col-0 and REAT11 was compared, where 20- and 40-day-old grown plants were subjected to 10 % PEG 6000 treatment for 2 days. Stress conditions caused a significant increase in H₂O₂ accumulation, lipid peroxidation, and membrane permeability in Col-0 over that in REAT11. Greater activity levels of superoxide dismutase, ascorbate peroxidase, and glutathione reductase were observed in the leaves of REAT11 compared to those of Col-0. Significant increases in total ascorbate and glutathione content and higher ratios of ASC/DHA: (ASC and DHA are reduced and oxidized ascorbate, respectively) and GSH/GSSG: (GSH and GSSG are reduced and oxidized glutathione, respectively) were observed in the leaves of REAT11 compared to those in Col-0 under drought conditions. In addition, enhancement of free proline and soluble sugar accumulation was observed in REAT11 compared to Col-0 under stress. Our results suggest that a slight deficiency in free riboflavin can paradoxically induce both a higher vegetative growth rate and an enhanced tolerance to drought in transgenic plants. The “stress escape” hypothesis is proposed here to explain this interesting phenomenon.     

Expression of 2S Seed Storage Protein Gene of Brassica juncea in Transgenic Tobacco Plants under Constitutive and Seed-specific Promoters

The coding and upstream promoter regions of Brassica juncea 2S seed storage protein gene were amplified by polymerase chain reaction. The plant expression cassettes containing 2S seed storage protein gene under the control of either a constitutive Caulimovirus 35S promoter or a seed specific 2S protein promoter and the polyadenylation signal of a pea rbcS gene were used for Agrobacterium — mediated transformation of tobacco (Nicotiana tabacum cv Petit Havana). Integration of 2S gene was confirmed by Southern blot and PCR analysis of plant genomic DNA. Expression of introduced 2S protein gene was monitored by slot blot analysis of total RNA using 2S protein sequence as hybridization probe and also by immunodot blot analysis using specific antiserum of 2S protein. Expression was either constitutive with CaMV 35S promoter or highly seed-specific with Brassica 2S promoter.     

Maternal Effects of mto1 Mutation, That Causes Overaccumulation of Soluble Methionine, on the Expression of a Soybean {beta}Conglycinin Gene Promoter-GUS Fusion in Transgenic Arabidopsis thaliana

ß-Conglycinin, the 7S seed storage protein of soybean(Glycine max [L.] Merr.), is comprised mainly of three subunits,designated , ' and ß. Expression of the gene encodingthe ß subunit is unique because its expression hasbeen shown to be down-regulated by exogenously applied L-methioninein immature soybean cotyledon cultures in vitro. Arabidopsisthaliana strain carrying a mto1-1 mutation overaccumulates solublemethionine. By using this mutant, we analyzed the effects ofmethionine on expression of the ß subunit gene invivo. Reciprocal crosses were made between the mto1-1 mutantand a transgenic A. thaliana strain, designated SNTß3,which carries a ß-glucuronidase (GUS) reporter geneunder the control of the promoter region of the ßsubunit gene. Analysis of GUS activity in F₁ seeds indicatedthat the GUS activity was dramatically repressed when the mto1-1mutant plants were used as female parents. We constructed astrain which carries both the transgene and mto1-1 mutationin the homozygous state. Analyses of the GUS activity in seedsof this double homozygous strain indicated that the GUS activitywas repressed to 2.5% of control by introduction of the mto1-1mutation. These results indicate that the ß subunitgene promoter activity in seeds is down-regulated by maternalgenotype and suggest that soluble methionine, or its mobilemetabolite, is translocated from mother plants to repress ßsubunit gene expression in seeds. ⁵Present address: Division of Biological Sciences, GraduateSchool of Science, Hokkaido University, Kita-ku, Sapporo, 060Japan ⁶Present address: Department of Biotechnology, Faculty of Agriculture,The University of Tokyo, Bunkyo-ku, Tokyo, 113 Japan     

An Auxin-Regulated Gene of Arabidopsis thaliana Encodes a DNA-Binding Protein

We have isolated a single-copy gene from the plant Arabidopsis thaliana, called dbp, which encodes a lysine-rich, DNA-binding protein. The Dbp protein has a molecular weight and a composition resembling histone H1. When the dbp gene was expressed in bacteria, the protein product bound DNA nonspecifically. The dbp gene is expressed constitutively in all parts of the plant but is induced five times above this basal level in apical zones. In vitro hormone-depletion experiments showed that the expression in the shoot apex could be induced by exogenous auxin. In situ hybridizations in the root apex indicated that the expression of dbp is enhanced in the region of cell division.     

反义trxs基因对转基因小麦种子内源trxh基因表达的影响

以转反义硫氧还蛋白基因(anti-trxs)株系01TY70-1-17-5及其对照小麦品种‘豫麦70’为试验材料,以小麦中稳定表达的肌动蛋白基因actin为内标,用半定量反转录聚合酶链式反应(semi-QRT-PCR)方法,对转基因株系及其对照种子中trxh基因时空表达情况进行了检测。检测结果表明,花后15~30d转基因株系trxh基因转录量平均比对照下降了20.1%,花后25d显著低于对照(P<0.05);胚乳trxh基因转录量最低,平均比对照低19.4%;种子吸涨24h时间内,转基因株系trxh基因转录量较对照均略低,但差异不显著。表明,外源trxs基因的导入直接干扰了内源基因的表达。     

拟南芥春化相关基因VRN2的克隆及其植物表达载体的构建

本文利用特异性引物,从拟南芥RNA中提取春化相关基因VRN2 cDNA序列,GenBank登录号AY063047。该基因序列大小为1 354 bp,编码区为1 323 bp,编码氨基酸441个。将克隆片段插入中间载体pBPFΩ7,经PstI酶切回收带有P35S启动子和nos终止子片段,连接pBI121载体,构建VRN2基因的植物表达载体。     

Floral-dip法大豆GmDREB转录因子转拟南芥研究

DREB（dehydration responsive element binding）转录因子通过调控下游多个抗逆相关基因的表达,能有效提高植物的抗逆性。将构建的植物高效表达载体GmDREB：：pCAMBIA1304,借助优化的floral-dip法,转入模式植物拟南芥,并经潮霉素Hygromycine（40-50mg&#183;L^-1）抗性筛选得到22棵抗性植株。对抗性植株再进行PCR和GUS检测获得19颗阳性苗,阳性率为86．3％。对T1代种子进行抗性分离比例统计,有4个株系的分离比例接近3：1,符合孟德尔遗传定律,说明外源基因GmDREB在这些株系的染色体中可能是单拷贝插入。继续对上述4个株系的后代进行抗性筛选．现已得到2个纯合的转基因株系。导入的报告基因GUS组织染色检测表明,转入大豆DREB基因在拟南芥的根系和子叶中均有大量表达,并在叶脉中表达。     

The Partial Characterization of the Major Seed Storage Proteins in Arabidopsis thaliana L.

This study was aimed at the characterization of the major storage proteins in Arabidopsis thaliana. Two major protein fractions, i.e., the fraction Ⅰ and Ⅱ proteins, were isolated from the extract of mature seeds of this plant by molecular seive gel filtration chromatography. Various polyacrylarnide gel electrophoretic techniques were used to study the properties and polypeptide compositions of these two protein fractions. In was shown that during the SDS gel electrophoresis, fraction Ⅰ protein was separated into 6 major bands with the mol. was. of 34, 31, 29, 28 and 19-20 kD, respectively, whereas Fraction Ⅱ protein migrated as 3 low mol. wt. bands (10-12 kD) on the same gel. Non-denaturing native gel electrophoresis revealed that fraction Ⅰ was a neutral protein and Fraction Ⅱ was a positively charged basic protein with an isoelectric point (pI) higher than 8.8. Fraction I protein was further separated into at least 16 polypeptides in isoelectric focusing/SDS two-dimensional gel electrophoresis, i.e. each SDS band contained 3-4 polypeptides with the same mol. wt. but different pis. This suggested a more complex polypeptide composition of this protein. The properties of fraction Ⅰ and Ⅱ proteins were in good accordance with that of the 12s and 1.7s storage globulins in seeds of many other dicotyledonous plants, and therefore had been characterized as the two major seed storage proteins in this species. These two storage globulins were shown to be accumulated within a defined period during the late stage of seed development (12-14 DAF) and became predominant protein components in mature seeds. In the mean time, a few points in relation to the polypeptide composition and subunit molecular configuration of the 12s globulin were noted.     

拟南芥SPX1蛋白原核表达及纯化分析

包含SPX结构域的蛋白在高等真核生物中广泛存在,这类蛋白的功能多数还不太清晰,但发现有些与磷信号相关,有些与铁信号相关。拟南芥中含有SPX结构域的蛋白可分为4个家族,本研究中的拟南芥SPX1(At SPX1)属于一个只含有SPX结构域的蛋白组成的家族,其它家族成员还包含额外的基因序列。进化树分析表明,At SPX1编码的氨基酸序列与双子叶植物具有较高的一致性,与单子叶植物进化距离较远。为了揭示At SPX1蛋白的结构形态与其生物学功能之间的联系,开展了At SPX1蛋白质体外可溶性表达实验,构建了原核体外表达载体,在大肠杆菌(E.coli)细胞中获得了该蛋白可溶性高表达。表达的蛋白包含有His标签方便了蛋白纯化,插入的SUMO融合蛋白标签可以通过蛋白酶切除,而目标蛋白通过硫酸铵沉淀实现了纯化。进一步分子筛层析分析表明At SPX1以单体形式存在。实验结果提供了一套表达纯化At SPX1蛋白的有效方案。     

Storage Protein Composition of Soybean Cotyledons Grown In Vitro in Media of Various Sulfate Concentrations in the Presence and Absence of Exogenous l-Methionine

Supplemental methionine in a complete culture medium increased the methionine content of the protein fraction of cultured soybean (Glycine max L. Merrill) cotyledons (Thompson, Madison, Muenster 1981 Phytochemistry 20: 941-945). To explain the observed increase in protein methionine, we have measured the amounts and subunit compositions of 7S and 11S storage proteins and determined the amino acid compositions of the three major protein fractions (2-5S, 7S, 11S) of seeds developed on plants and of cultured cotyledons grown in the presence or absence of supplemental l-methionine. Development of cultured cotyledons was representative of development of seeds on plants. The ratios of 11S to 7S proteins, the subunit contents, and amino acid compositions of their storage protein fractions were similar, but not identical. Supplemental methionine increased the mole percent methionine in each of the three protein fractions of cultured cotyledons and changed the amounts of several other amino acids. Supplemental methionine inhibited expression of the 7S β-subunit gene. Concomitant with the absence of the β-subunit, which contains no methionine, was an increase in the ratio of 11S to 7S proteins, and an increase in the methionine content of the subunits composing these fractions. Inhibition of β-subunit gene expression by methionine in cultured cotyledons provides a reproducible, easily controlled system for the study of eucaryotic gene expression.