Isolation and Characterization of Golgi Membranes from Suspension-cultured Cells of Rice (Oryza sativa L.)

The isolation of Golgi membranes from suspension-cultured cellsof rice (Oryza sativa L.) was attempted by linear glycerol densitygradient centrifugation. When "burst" membranes in the pelletobtaind after differential centrifugation at 100,000 ? g weresuspended in 20% (w/w) glycerol in 50 mM malate-NaOH (pH 6.0)and loaded onto a linear density gradient of glycerol, whichextended from 30 to 80% (w/w) in 1 mM EDTA in 50 mM glycylglycine-NaOH(pH 7.5), IDPase, a marker enzyme for Golgi membranes, was separatedfrom other membrane markers on the glycerol gradient. In addition,UDPase and GDPase activities overlapped with the peak fractionof IDPase activity. Furthermore, membrane glycoproteins in eachfraction were characterized by lectin-peroxidase staining. ConcanavalinA and lentil lectin, which have the ability to bind to the high-mannosetype of oligosaccharide, bound to glycoproteins distributedin ER membrane fractions, while wheat germ lectin, castor beanlectin, peanut lectin, and Ulex europaeus lectin-I which recognizethe complex type and/or the mucin type of oligosaccharides interactedwith glycoproteins in the Golgi membrane fractions but not withthose in the ER membrane. These results strongly suggest thatthe oligosaccharide structures of glycoproteins in the ER membraneare of the high-mannose type, whereas glycoproteins in the Golgimembrane have modified N-linked and/or O-linked oligosaccharidechains. (Received November 9, 1988; Accepted October 17, 1989)     

Isolation and Characterization of Matrix Associated Region DNA Fragments in Rice (Oryza sativa L.)

To investigate the interactions between chromosomal DNA andnuclear matrices in higher plants, matrix associated regions(MARs) of rice (Oryza sativa L.) DNAs were cloned. First, weprepared nuclear matrices from isolated nuclei by digestingthem with EcoRl and then extracting with 2 M NaCl. About 6%of the total DNA remained in the nuclear matrices after thisdigestion and extraction. The residual DNA fragments in thenuclear matrices were cloned. Some of the cloned DNA fragmentsshowed binding to certain nuclear proteins. One of the MAR fragmentscontained sequences related to known consensus motifs and ahairpin loop structure. A method is presented for isolationof matrix associated region (MAR) DNAs from plant cells. (Received January 13, 1997; Accepted July 10, 1997)     

Purification and Characterization of Phospholipase D (PLD) from Rice (Oryza sativa L.) and Cloning of cDNA for PLD from Rice and Maize (Zea mays L.)

Phospholipase D (PLD) was purified to high homogeneity fromrice bran (Oryza sativa L.). Two peaks of PLD activity wereresolved by Mono Q anion-exchange chromatography. The molecularmass of PLD in both peaks was 82 kDa on SDS-PAGE and 78 kDain gel filtration. Antibodies raised against the protein inone of the peaks precipitated the enzyme activities in bothpeaks. Enzymatic characteristics of PLD in the two peaks wereidentical except for a difference of 0.1 in the isoelectricpoints. Sequence analysis covering more than 10% of the aminoacids of the proteins and peptide mapping did not detect anydifference in the primary structure of the proteins. A cDNAfor PLD was isolated from rice and it encoded a protein of 812residues. The N-terminal sequences of purified PLDs matchedthe deduced amino acid sequence starting from residue 47. ANorthern blot showed this gene was expressed in leaves, roots,developing seeds and cultured cells, and a Southern blot detecteda single band of rice genomic DNA hybridizing to the cDNA. AcDNA for PLD was also isolated from maize. The similarity ofthe deduced amino acid sequences of PLD was 90% between riceand maize, 73% between the cereals and castor bean. ²Present address: Agribusiness Division, Japan Tobacco Inc.2-1,Toranomon, 2-chome, Minato-ku, Tokyo, 140 Japan     

Cloning and Characterization of Catalases from Rice,Oryza sativa L.

Catalase is the major H₂O₂-scavenging enzyme in all aerobic organisms. From the cDNA sequences of three rice (Oryza sativa L.) genes that encode for predicted catalases (OsCatA, OsCatB, and OsCatC), complete ORFs were subcloned into pET21a and expressed as (His)₆-tagged proteins in Escherichia coli. The recombinant (His)₆-polypeptides were enriched to apparent homogeneity and characterized. With H₂O₂ as substrate, the highest catalase k _cat value (20±1.71×10^?3 min^?1) was found in recombinant OsCatB. The optimum temperatures for catalase activity were 30 °C for OsCatA and OsCatC and 25 °C for OsCatB, while the pH optima were 8.0, 7.5, and 7.0 for OsCatA, OsCatB, and OsCatC respectively. All the catalases were inhibited by sodium azide, β-mercaptoethanol, and potassium cyanide, but only weakly by 3-amino-1,2,4-triazole. The various catalases exhibited different catalase activities in the presence of different salts at different concentrations, OsCatC showing higher salt inhibitory effects than the two other OsCats.     

Fine Mapping of Aroma QTLs in Basmati Rice (Oryza sativa L) on Chromosomes 3, 4 and 8

Aroma is one of the most important quality traits of basmati rice (Oryza sativa L) that leads to high consumer acceptance. Earlier three significant QTLs for aroma, namely aro3-1, aro4-1 and aro8-1, have been mapped on rice chromosomes 3, 4 and 8, respectively, using a population of recombinant inbred lines (RILs) derived from a cross between Pusa 1121 (a basmati quality variety) and Pusa 1342 (a non-aromatic variety). For fine mapping of these QTLs, 184 F₆ RILs were grown in the Kharif season of 2005 at New Delhi and Karnal, India. A total of 115 new SSR markers covering the three QTL intervals were designed and screened for parental polymorphism. Of these, 26 markers were polymorphic between parents, eight for the interval aro3-1, eight for the interval aro4-1 and ten for the interval aro8-1, thus enriching the density of SSR markers in these QTL intervals. Revised genetic maps were constructed by adding 23 of these new markers to the earlier map, by giving physical order of the markers in the pseudomolecules a preference. In the revised maps, the interval for QTL aro4-1 could not be improved further but QTL aro3-1 was narrowed down to an interval of 390 kbp from the earlier reported interval of 8.6 Mbp and similarly the QTL aro8-1 was narrowed down to a physical interval of 430 kbp. The numbers of candidate genes in the aro3-1 and aro8-1 intervals have now been reduced to 51 and 66, respectively. The badh2 gene on chromosome 8 was not associated with the aroma QTL on this chromosome.     

Heterologous Expression and Metal-Binding Characterization of a Type 1 Metallothionein Isoform (OsMTI-1b) from Rice (Oryza sativa)

Metallothioneins (MTs) are ubiquitous, low molecular mass and cysteine-rich proteins that play important roles in maintaining intracellular metal homeostasis, eliminating metal toxification and protecting the cells against oxidative damages. MTs are able to bind metal ions through the thiol groups of their cysteine residues. Plants have several MT isoforms which are classified into four types based on the arrangement of cysteine residues. In the present study, a rice (Oryza sativa) gene encoding type 1 MT isoform, OsMTI-1b, was inserted in vector pET41a and overexpressed in Escherichia coli as carboxy-terminal extensions of glutathione-S-transferase (GST). The recombinant protein GST-OsMTI-1b was purified using affinity chromatography and its ability to bind with Ni²⁺, Cd²⁺, Zn²⁺ and Cu²⁺ ions was analyzed. The results demonstrated that this isoform has ability to bind Ni²⁺, Cd²⁺ and Zn²⁺ ions in vitro, whereas it has no substantial ability to bind Cu²⁺ ions. From competitive reaction with 5,5′-dithiobis(2-nitrobenzoic acid), DTNB, the affinity of metal ions for recombinant form of GST-OsMTI-1b was as follows: Ni²⁺/Cd²⁺ > Zn²⁺ > Cu²⁺     

水稻葡萄糖-6-磷酸脱氢酶cDNA的电子克隆

电子克隆是基因克隆的新策略,以小麦胞质葡萄糖－6－磷酸脱氢酶cDNA(Tagpdl克隆）序列为信息探针,在GenBank水稻nr数据库中找到高度同源的水稻基因组序列,通过人工序列拼接及RT-PCR确认得到了水稻该基因的全长cDNA序列,命名为OsG6PDH,OsG6PDH与小麦Tagpdl克隆的DNA一致率为88％,推导的氨基酸序列与小麦,番茄,烟草的胞质葡萄糖－6－磷酸脱氢酶基因的一致率分别为89％,79％,80％,经RT－PCR表达谱分析,OsG6PDH在水稻幼穗,胚,根,叶中都有表达,在幼穗与根中表达略高,另外,讨论了利用水稻基因组信息的电子克隆方法克隆水稻功能基因的可行性。     

两种水稻GDP解离抑制蛋白基因的分离及特征分析

以Rho家族成员OsRacD为诱饵 ,采用酵母双杂交体系 ,分离到两种与OsRacD互作的水稻RhoGDP解离抑制蛋白的基因 ,分别命名为OsRhoGDI1和OsRhoGDI2 .酵母体内结合和GSTpulldown分析结果显示 ,OsRhoGDI1和OsRhoGDI2与野生型和组成型激活的OsRacD都能结合 ,且不依赖GDI的N端部分序列 ;GDI和Rho的结合具有一定的特异性 .两种GDI在水稻根、地上组织和幼穗等多种组织和器官都有表达 ,但在表达特征上存在明显差异 .研究证实 ,在水稻中存在着调控RhoGTPases的GDP解离抑制蛋白基因家族 ,为Rho蛋白功能及相关信号通路的研究奠定了基础     

逆境诱导水稻谷胱甘肽合成酶基因的表达研究

用RACE方法获得了全长的水稻谷胱甘肽合成酶(GS)基因的cDNA,并命名为OsGS(GeneBankaccession No.:AY453405)。该cDNA全长1 892 bp,编码一个由540个氨基酸组成的多肽,预测其氨基端(N端)含有一段定位叶绿体的信号肽。比较水稻基因组定位结果表明OsGS基因位于水稻12号染色体短臂上,转录区全长6 321 bp,由12个外显子和11个内含子组成。通过RT-PCR对OsGS在水稻正常生长条件和逆境条件下的表达进行了研究。结果表明,在正常生长条件下,OsGS在水稻幼苗的根和叶以及抽穗期水稻的根中表达;但不在抽穗期水稻的叶、茎和幼穗中表达,这显示OsGS在水稻中的表达具有发育和组织特异性。利用抽穗期水稻的叶片为材料,经高温、干旱和重金属逆境处理后,OsGS在抽穗期叶片中的转录被诱导表达;而在盐、低温、伤害逆境下则不被诱导。在轻度和中度干旱胁迫4 h后OsGS基因可被诱导表达。外源ABA处理也能够提高OsGS的转录水平,这显示OsGS可能是依赖ABA信号途径的环境胁迫诱导基因。     

A Cointegrate Ti Plasmid Vector for Agrobacterium tumefaciens - mediated Transformation of indica Rice cv Pusa Basmati 1

An intermediate vector pSSJ1 was constructed by cloning a hph gene and a gus gene with catalase intron in pGV1500. pSSJ1 was cointegrated into a disarmed receptor Ti plasmid pGV2260 harboured in Agrobacterium tumefaciens strain C58C1Rif^R. The resulting A. tumefaciens strain C58C1Rif^R (pGV2260::pSSJ1) stably transformed Oryza sativa L. cv Pusa Basmati 1 scutellum-derived calli at 26% frequency. Introduction of the plasmid pSSJ3 (3′virB, virG and virC of pTiB0542) into A. tumefaciens C58C1Rif^R (pGV2260::pSSJ1) resulted in the elevation of acetosyringone-induced T -strand accumulation. Rice transformation efficiency of the cointegrate plasmid pGV2260::pSSJ1 increased from 26% to 33% in the presence of pSSJ3 and from 26% to 35% in the presence of pToK47 (complete virB, virG and virC). T-DNA integration in To plants was confirmed by Southern hybridization analysis. Inheritance analysis of the T₀ plants with single-copy T-DNA insertions revealed segregation of hygromycin resistance in 3:1 ratio. The feasibility of rice transformation with a cointegrate Ti plasmid vector is clearly established.     

氮磷饥饿诱导的水稻糖转运体基因的cDNA克隆和鉴定

运用快速扣除杂交 (RaSH)方法构建了水稻氮饥饿诱导的cDNA文库。从该文库获得了一个cDNA克隆OsNSI1 (Oryzasativanitrogenstarva tion inducible 1 )。该全长cDNA编码 5 77个氨基酸 ,蛋白分子量为 6 1 .2kD。推测得出的氨基酸序列与其他物种的糖转运体有很高的同源性。水合性分析表明OsNSI1包含有 1 2个跨膜区域和一个中心亲水环。这些数据提示OsNSI1是一个糖转运体蛋白。Southern印迹分析表明OsNSI1是一个单拷贝基因。Northern印迹分析表明OsNSI1主要在叶及根中表达 ,氮、磷饥饿能强烈诱导其表达增强