Application of N-(9-Acridinyl)maleimide as a Fluorescent Detection Reagent of Cysteine and Related Thiols and Disulfide Compounds on Thin-layer Chromatogram

Fluctuations of pungent principles of hot pepper fruits (capsaicinoid), chlorophylls, carotenoid, and fresh fruit weight in Capsicum annuum var. annuum cv. Karayatsubusa at different growth stages after flowering were examined. Capsaicinoid was first detected 20 days after flowering, and reached maximal level around 40 days after flowering, then later decreased gradually. The capsaicinoid composition did not show any appreciable change throughout the stages after flowering. CAP and DC were the major components in all of the stages examined. By using radioisotopic technique, it was found that the main formation and accumulation sites of capsaicinoid are in the placenta of the fruits.     

一个水稻多逆境响应基因OsMsr3克隆与表达分析

为深入探讨水稻对逆境的反应机理并寻找新的植物耐逆基因,采用Affymetrix水稻表达芯片(含51279个转录本)分析了培矮64S全基因组在不同逆境(高温、干旱、低温)胁迫下、不同生育时期叶片和穗中的表达谱,从中筛选出一个受多种逆境诱导表达的基因OsMsr3(Oryza sativa L.multiple stresses responsive gene3,GenBank登陆号为 FJ383169).定量实时PCR分析结果进一步证实了此基因在逆境条件下的诱导表达模式.用 RT-PCR方法扩增获得了包含其完整开放阅读框的cDNA克隆,序列分析表明,其ORF大小为480 bp,编码一个具有160个氨基酸残基的低分子量热激蛋白,推测分子量约为18.0 kD;pI约为6.8.对其编码的蛋白质进行分析,发现其羧基端存在一个HSP20的蛋白保守结构域,与其他植物中的低分子量热激蛋白的相似性介于33.7%～97.5%.对其可能的启动子序列分析,发现6类与逆境反应有关的顺式作用元件.推测该基因在逆境反应中起着重要的作用,进一步的研究正在进行中.     

Biochemical and Crystallographic Characterization of the Starch Branching Enzyme I (BEI) from Oryza sativa L

Starch branching enzyme (SBE) catalyzes the cleavage of α-1.4-linkages and the subsequent transfer of α-1.4 glucan to form an α-1.6 branch point in amylopectin. We overproduced rice branching enzyme I (BEI) in Escherichia coli cells, and the resulting enzyme (rBEI) was characterized with respect to biochemical and crystallographic properties. Specific activities were calculated to be 20.8 units/mg and 2.5 units/mg respectively when amylose and amylopectin were used as substrates. Site-directed mutations of Tyr235, Asp270, His275, Arg342, Asp344, Glu399, and His467 conserved in the α-amylase family enzymes drastically reduced catalytic activity of rBEI. This result suggests that the structures of BEI and the other α-amylase family enzymes are similar and that they share common catalytic mechanisms. Crystals of rBEI were grown under appropriate conditions and the crystals diffracted to a resolution of 3.0 Å on a synchrotron X-ray source.     

Bipolaris eleusines,a potential mycoherbicide candidate for control of barnyardgrass (Echinochloa crus-galli)

An isolate of Bipolaris eleusines was investigated as a potential candidate for biocontrol of barnyardgrass and additional weeds in paddy rice fields and for safety to selected crop species under greenhouse conditions. Barnyardgrass appeared more susceptible at the three-leaf stage than older plants, and disease severity increased as the fungal inoculum increased from 1 × 10⁵ to 1 × 10⁷ conidia/ml when sprayed till run-off. The high application rate caused 73% mortality of barnyardgrass, relative to the non-treated control, but increasing application rate to >1 × 10⁷ conidia/ml did not enhance efficacy (P >.05). This B. eleusines isolate showed no pathogenicity to rice (Oryza sativa spp. indica, O. sativa spp. japonica and an O. sativa hybrid), corn, wheat or any dicot crop species tested while causing slight infection on sorghum and barley. We conclude that B. eleusines, with high efficacy against barnyardgrass and demonstrated safety to rice, is a promising mycoherbicide candidate worthy of further evaluation and development for control of barnyardgrass in paddy rice fields.     

Effects of water stress on plant growth and root proteins in three cultivars of rice (Oryza sativa) with different levels of drought tolerance

Rice ( Oryza sativa L.) is considered a drought-sensitive crop species; however, within this species, there are considerable varietal differences in sensitivity to this environmental stress. In the present work, the effect of water stress on germination, plant growth and root proteins in three rice cultivars (Sinaloa, IR10120 and Chiapas) was analyzed. Seed germination and plant growth were found to be significantly inhibited by polyethylene glycol (PEG)-imposed water deficit in cv. Sinaloa; cvs IR10120 and Chiapas were more tolerant to water stress. Fluorographs of two-dimensional electropherograms of in vivo-labeled polypeptides were analyzed to identify changes in the root protein patterns that resulted when plants were grown in the presence of 10% PEG for 10 days. The treatment induced or increased the synthesis of eight polypeptides or groups of polypeptides in cv. Sinaloa, seven in cv, IR10120 and four in cv. Chiapas. The synthesis of several polypeptides was decreased by the PEG treatment in cv. Sinaloa and cv. IR10120. Most of these PEG-induced changes in the root protein patterns were cultivar-specific and only one 26-kDa protein with a pI of 6.0 was induced by water deficit in the two cultivars Sinaloa and IR10120.     

Polyamines, both common and uncommon, under heat stress in rice (Oryza sativa) callus

Enhanced production and accumulation of free and conjugated polyamines as well as increased activities of their biosynthetic enzymes in plants have been associated with heat stress. Perchloric acid-soluble free, as well as conjugated polyamines, and their metabolic enzymes were studied under 45°C heat stress in callus raised from heat-tolerant and -sensitive rice cultivars. The levels of free and conjugated polyamines, as well as arginine decarboxylase (EC 4.1.1.19) and polyamine oxidase (EC 1.4.34) activities were higher in tolerant than in sensitive callus under non-stressed conditions. Heat stress caused greater accumulation of free and conjugated polyamines in callus of the heat-tolerant cultivar N22 than in that of the heat-sensitive cultivar IR8. In particular, the uncommon polyamines norspermidine and norspermine were detected in cv. N22, which increased appreciably during stress, but they were not detected in callus of cv. IR8. Arginine decarboxylase and polyamine oxidase activities increased to a larger extent in N22 than in IR8 callus during stress, activities that were well correlated with the increased levels of common and uncommon polyamines. Increased levels of transglutaminase activity indicated the high titre of conjugated polyamines.     

Synthesis of a new heterobifunctional linker, N-[4-(aminooxy)butyl]maleimide, for facile access to a thiol-reactive 18F-labeling agent

A new heterobifunctional linker containing an aldehyde-reactive aminooxy group and a thiol-reactive maleimide group, namely N-[4-(aminooxy)butyl]maleimide, was synthesized as a stable HCl salt by O-alkylation of either N-hydroxyphthalimide or N-(4-monomethoxytrityl)hydroxylamine, followed by N-alkylation of maleimide, in an overall yield of 18% (seven steps) or 29% (five steps), respectively. This heterobifunctional linker allowed a simple and efficient synthesis of a maleimide-containing thiol-reactive (18)F-labeling agent. Thus, N-[4-[(4-[(18)F]fluorobenzylidene)aminooxy]butyl]maleimide (specific activity: approximately 3000 Ci/mmol at end of synthesis) was synthesized in two steps involving the preparation of 4-[(18)F]fluorobenzaldehyde, followed by its aminooxy-aldehyde coupling reaction to the heterobifunctional linker, with an overall radiochemical yield of approximately 35% (decay corrected) within approximately 60 min from end of bombardment. Initial (18)F-labeling experiments were carried out using a thiol-containing tripeptide glutathione (GSH) and a 5'-thiol-functionalized oligodeoxynucleotide (5'-S-ODN) in phosphate-buffered saline (PBS, pH 7.5). After standing at room temperature for 10 min, the (18)F-labeled GSH and 5'-S-ODN were obtained in (18)F-labeling yields of approximately 70% and approximately 5% (decay-corrected), respectively. The heterobifunctional linker is easy to synthesize and provides a facile access to the maleimide-containing thiol-reactive (18)F-labeling agent, which could be advantageously employed in the development of (18)F-labeled biomomolecules for use with positron emission tomography.     

Separation and characterization of proteins in rice (Oryza sativa) suspension cultured cells

Proteins extracted from suspension cultured cells of rice were separated by two-dimensional polyacrylamide gel electrophoresis. The separated proteins were electroblotted onto a polyvinylidene difluoride membrane and 103 electroblotted proteins were analyzed. The N-terminal amino-acid sequences of 20 out of 103 proteins were determined in this manner. N-terminal regions of the remaining proteins could not be sequenced and they were inferred to have a blocking group at the N-terminus. Internal amino-acid sequences of 32 proteins were determined by sequence analysis of peptides obtained by Cleveland peptide mapping. The amino-acid sequences determined here were compared with those of known plant and animal proteins. Furthermore, the concanavalin A-peroxidase method was used to determine which of the 103 proteins were glycosylated, and in vitro and in vivo phosphorylation was carried out to identify some of the phosphorylated proteins. Using this experimental approach, we could identify the major proteins involved in growth and development of rice cell suspension cultures and discuss on the physiological function of some of these identified proteins including the calcium binding protein, superoxide dismutase and rice ascorbate peroxidase. This revised version was published online in June 2006 with corrections to the Cover Date.     

Evaluation of resistance of wild rices,Oryza spp., to the whitebacked planthopper,Sogatella furcifera (Horvath) (Homoptera: Delphacidae)

Evaluation of wild rice species for resistance against major insect pests of rice was carried out in both field and greenhouse conditions. A total of 219 wild rice lines were evaluated under field conditions against yellow stem borer, Scirpophaga incertulas (Walker) and leaf folder, Cnaphalocrocis medinalis (Guenee). Of the selected 185 lines tested by “standard seed box screening technique” against whitebacked planthopper, Sogatella furcifera (Horvath), 20 were found to be resistant, 54 moderately resistant, 88 moderately susceptible, and 23 susceptible. Out of the 185 wild rice lines tested against all the three major pests, 17 lines had multiple resistance. Out of these 17 lines, 14 lines were evaluated for mechanisms of resistance against S. furcifera; they showed antixenosis for feeding, as fewer nymphs settled on them compared to the susceptible O. sativa cultivar TN1. However, the eggs were observed to be randomly distributed on them. Antibiosis was exhibited as the nymphal period was prolonged, while there was a decrease in survival and growth index. Thus, besides possessing multiple resistance against all the three major pests during the present investigation, all these 14 wild rice lines belonging to different Oryza spp. exhibited good antixenotic and antibiotic effects.     

The green fluorescent protein (GFP) as a vital screenable marker in rice transformation

 An engineered green fluorescent protein (GFP) from the jellyfish Aequora victoria was used to develop a facile and rapid rice transformation system using particle bombardment of immature embryos. The mgfp4 gene under the control of the 35s Cauliflower Mosaic Virus promoter produced bright-green fluorescence easily detectable and screenable in rice tissue 12–22 days after bombardment. Visual screening of transformed rice tissue, associated with a low level of antibiotic selection, drastically reduced the quantity of tissue to be handled and the time required for the recovery of transformed plants. GFP expression was observed in primary transformed rice plants (T₀) and their progeny (T₁). We describe various techniques to observe GFP in vitro and in vivo. The advantages of this new screenable marker in rice genetic engineering programmes are discussed. Received: 6 October 1997 / Accepted: 9 October 1997     

Novel enzyme immunoassay for thyrotropin-releasing hormone using N-(4-diazophenyl)maleimide as a coupling agent

A novel enzyme immunoassay (EIA) for thyrotropin-releasing hormone (TRH) was developed which used N-(4-diazophenyl)maleimide (DPM) as a new heterobifunctional agent capable of cross-linking TRH to mercaptosuccinyl bovine serum albumin and to beta-D-galactosidase. The resulting conjugates act as the immunogen producing anti-TRH serum in rabbits and the enzyme marker of TRH in the EIA, respectively. This EIA with a double-antibody technique was sensitive and reproducible in measuring TRH at concentrations as low as 50 pg per tube, and monospecific to the hormone showing no cross-reactivity with the hormone analogue L-pGlu-L-His-L-Pro and TRH constituents. Using this assay, the distribution of immunoreactive TRH in the brain was determined easily in rats. The use of DPM should provide a valuable new method for developing EIA hitherto possible for other peptide hormones containing neither a free carboxy nor a free amino group, using imidazole, phenolic, and indole group(s) of the amino acid as a reaction site.     

A Convenient Synthesis of a Mixture of (Z,Z)-3,13-Octadecadienyl Acetate and Its (E,Z)-Isomer,the Attractant for the Cherrytree Borer

A 1:1 mixture of (Z, Z)-3,13-octadecadien-1-ol and its (E, Z)-isomer was synthesized by a Wittig reaction whose stereoselectivity was adjusted to give the products in the required ratio. The corresponding acetate mixture is useful as the attractant for Synanthedon hector.     

Construction of a full bacterial artificial chromosome （BAC） library of Oryza sativa genome

We have constructed a full BAC library for the superior early indica variety of Oryza sativa,Guang Lu Ai 4.The MAX Efficiency DH10B with increased stability of inserts was used as BAC host cells.The potent pBelo BACII with double selection markers was used as cloning vector.The cloning efficiency we have reached was as high as 98%,and the transformation efficiency was raised up to 10^6 transformants/μg of large fragment DNA.The BAC recombinant transformants were picked at random and analyzed for the size of inserts,which turned out to be of 120 kb in length on average.We have obtained more than 20,000 such BAC clones.According to conventional probability equation,they covered the entire rice genome of 420,000 kb in length.The entire length of inserts of the library obtained has the 5-to 6-fold coverage of the genome.To our knowledge,this is the first reported full BAC library for a complex genome.     

以色列野生二粒小麦(Triticum dicoccoides)耐盐性鉴定

采用水培法对从以色列引进的分属8个自然群体的93份野生二粒小麦进行了耐盐性初步鉴定.结果表明:①供试材料中仅有高耐盐材料3份,耐盐材料7份 ,耐盐性较好的材料仅占参试材料总数的10.7%;②8个自然群体的野生二粒小麦的耐盐性存在较大差异,其中3号群体耐盐性明显高于其他群体,说明来自Tabigha地区的野生二粒小麦具有丰富的耐盐基因源;③将经过鉴定的耐盐性较好的野生二粒小麦与普通小麦中的耐盐品种进行比较研究发现,以色列耐盐性较好的野生二粒小麦的耐盐性不如普通小麦中的茶淀红和SW10两个耐盐品种.     

水稻条纹叶枯病抗性位点的检测和效应分析

利用111个家系组成的热研2号（Oryza sativa subsp．japonica‘Reyan2’）／Milyang23（Oryza sativa subsp．indica‘Milyang23’）重组自交系（recombinant inbred lines,RIL）群体（F7）,采用重病区田间自然接种方法,以病情指数作为条纹叶枯病的表型值,鉴定了2个亲本及111个RIL家系对条纹叶枯病的抗性。使用QTL Cartographer软件复合区间作图法,对水稻（Oryza sativa）条纹叶枯病抗性基因进行了QTL分析。结果检测到2个抗水稻条纹叶枯病的QTL,分别位于第2和第11染色体上,其中第11染色体上的QTL贡献率为19．58％,表明这是一个主效的QTL,该QTL及其附近的分子标记,可以用于水稻条纹叶枯病抗性分子标记辅助育种。     

水稻新基因OsRwp34在大肠杆菌中表达的毒害作用

在水稻高温诱导cDNA文库中克隆了一个新的水稻基因O sRwp34,同源性分析表明O sRwp34是一典型的孤儿基因。为了研究O sRwp34的功能,构建了O sRwp34的表达载体pET-28(a)OsRwp34,并转化大肠杆菌菌株BL21(DE3),用IPTG诱导并定时从培养液中取样以检测OD600值和用平板记数法测定活菌数,结果发现在诱导30 m in后宿主菌开始大量死亡,表明O sRwp34的表达产物使宿主菌死亡。随后构建了1个N末端缺失15个氨基酸残基和2个C末端分别缺失12和47个氨基酸残基的O sRwp34缺失体,IPTG诱导后都没有出现毒性作用,推测N和C末端氨基酸残基的同时存在是OsRwp34的毒性作用所必须的。其详细机理有待进一步研究。     

Characterization of Rice Blast Isolates by the Differential System and their Application for Mapping a Resistance Gene,Pi19(t)

To facilitate resistance gene characterization in the present study, the pathogenicities of newly collected blast isolates from rice fields in the Philippines were characterized using international blast differential varieties consisting of 31 monogenic lines that target 24 resistance genes. To classify and designate the blast isolates, we used a new international blast designation system, which has been proposed as a suitable naming system for comparing blast races among different studies. A total of 23 rice blast isolates collected from the Philippines were classified into 16 pathotypes, which showed reaction patterns different from those seen in the standard isolates. Among the blast pathotypes, 11 had differentiating ability for four Pik alleles (Pik, Pik‐m, Pik‐h, and Pik‐p) and Pi1, whereas the standard blast isolates from the Philippines were not able to differentiate these genes. In addition, several blast isolates were avirulent to IRBLt‐K59, IRBL19‐A, and Lijiangxintuanheigu, although the standard differential blast isolates were virulent to these lines. Moreover, two blast isolates were virulent to a monogenic line, IRBL9‐W, which harbours Pi9 and was resistant to all standard differential blast isolates. By using the isolates avirulent to IRBL19‐A, Pi19(t) was successfully mapped in the centromeric region on chromosome 12 with simple sequence repeat markers RM27937 and RM1337. These markers are useful for marker‐assisted Pi19(t) introgression worldwide.     

Synthesis and Hybridization Properties of Oligonucleotides Containing (2 S ,3 R )-9-(2,3,4-Trihydroxybutyl)Adenine

The synthesis and properties of oligonucleotides (ONs) containing 9-(2,3,4-trihydroxybutyl)adenine, A ^C2 and A ^C3, are described. The ON containing A ^C2 involves the 3′ → 4′ and 3′ → 5′ phosphodiester linkages in the strand, whereas that containing A ^C3 possesses the 3′ → 4′ and 2′ → 5′ phosphodiester linkages. It was found that incorporation of the analogs, A ^C2 or A ^C3, into ONs significantly reduces the thermal and thermodynamic stabilities of the ON/DNA duplexes, but does not largely decrease the thermal and thermodynamic stabilities of the ON/RNA duplexes as compared with the case of the ON/DNA duplexes. It was revealed that the base recognition ability of A ^C2 is greater than that of A ^C3 in the ON/RNA duplexes.