Metabolic regulation of alpha-amylase gene expression in transgenic cell cultures of rice (Oryza sativa L.)

Expression of two genes in the alpha-amylase gene family is controlled by metabolic regulation in rice cultured cells. The levels of RAmy3D and RAmy3E mRNAs in rice cultured cells are inversely related to the concentration of sugar in the culture medium. Other genes in the rice alpha-amylase gene family have little or no expression in cultured cells; these expression levels are not controlled by metabolic regulation. A RAmy3D promoter/GUS gene fusion was metabolically regulated in the transgenic rice cell line 3DG, just as the endogenous RAmy3D gene is regulated. An assay of GUS enzyme activity in 3DG cells demonstrated that RAmy3D/GUS expression is repressed when sugar is present in the culture medium and induced when sugar is removed from the medium. The 942 bp fragment of the RAmy3D promoter that was linked to the coding region of the GUS reporter gene thus contains all of the regulatory sequences necessary for metabolic regulation of the gene.     

Isolation of a novel auxin receptor from soluble fractions of rice (Oryza sativa L.) shoots

We report a single stranded conformational polymorphism (SSCP) analysis of the coding region of the dopamine D1 receptor (DRD1) in Tourette's syndrome (n = 50) and control (n = 50) subjects. Tourette's syndrome populations with comorbidity for attention deficit-hyperactivity disorder (AD-HD) (n = 35) and obsessive compulsive disorder (OCD) (n = 30) were also screened. As a related study, we also screened patients diagnosed with alcohol dependence (n = 72). The present study discovered no DRD1 coding region mutations in any of the Tourette's syndrome or alcohol dependent patients. One silent mutation, a C for a T at Ile49, was discovered in one control subject. The non-polymorphic structure of the DRD1 gene among the Tourette's syndrome, Tourette's syndrome comorbid with AD-HD and OCD and the alcohol dependent populations screened by SSCP suggests that coding region mutations of the DRD1 gene are unlikely to contribute to the inheritance of these disorders.     

水稻染色体片段代换系群体的构建及应用研究进展

定位和克隆水稻重要农艺性状QTL,是水稻功能基因组学研究的重要方向,是分子标记辅助选择选育高产、优质、多抗水稻新品种的重要基础.染色体片段代换系是进行QTL分析的理想材料.介绍了水稻染色体片段代换系群体的构建原理,综述了其构建及应用研究进展,并对其研究方向进行了展望.     

Reversible and Irreversible Drought-Induced Changes in the Anther Proteome of Rice(Oryza sativa L.)Genotypes IR64 and Moroberekan

Crop yield is most sensitive to water deficit during the reproductive stage.For rice,the most sensitive yield component is spikelet fertility and the most sensitive stage is immediately before heading.Here,we examined the effect of drought on the anther proteome of two rice genotypes:Moroberekan and IR64.Water was withheld for 3 d before heading(3DBH)in well watered controls for 5 d until the flag leaf relative water content(RWC)had declined to 45-50%.Plants were then re-watered and heading occurred 2-3 d later,representing a delay of 4-5 d relative to controls.The anther proteins were separated at 3 DBH,at the end of the stress period,and at heading in stressed/re-watered plants and controls by two-dimensional(2-D)gel electrophoresis,and 93 protein spots were affected reproducibly in abundance by drought during the experiment across two rice genotypes.After drought stress,upon re-watering,expressions of 24 protein spots were irreversible in both genotypes,60 protein spots were irreversible in IR64 but reversible in Moroberekan,only nine protein spots were irreversible in Moroberekan while reversible in IR64.Among them,there were 14 newly drought-induced protein spots in IR64;none of them was reversible on re-watering.However,there were 13 newly drought-induced protein spots in Moroberekan,10 of them were reversible on re-watering,including six drought-induced protein spots that were not reversed in IR64.Taken together,our proteomics data reveal that drought-tolerant genotype Moroberekan possessed better recovery capability following drought and re-watering at the anther proteome level than the drought-sensitive genotype IR64.The disruptions of drought to rice anther development and pollen cell functions are also discussed in the paper.     

DNA fingerprints of rice (Oryza sativa) obtained from hypervariable chloroplast simple sequence repeats

The aim of this research was to develop a convenient polymerase chain reaction-based assay that would allow intraspecific chloroplast variability to be detected. Our approach is based on the detection of length polymorphism within chloroplast mononucleotide microsatellite loci. Information from the fully sequenced rice chloroplast genome was used to identify 12 regions with a minimum of ten uninterrupted mononucleotide repeats. Primers flanking these repeats were used in conjunction with polymerase chain reaction to examine levels of polymorphism in six wild and 14 cultivated rice accessions. A total of six of the primer pairs revealed length polymorphism with between two and five size variants being detected. Diversity indices varied between 0.07 and 0.72. The length variation detected at multiple, physically linked sites was used to identify 15 unique haplotypes with an overall diversity index of 0.90. This level of polymorphism is sufficiently high to allow chloroplast variability to be studied at the intraspecific level. An additional 47 Oryza sativa accessions were also assayed with 31 unique chloroplast haplotypes being detected. The distribution of these haplotypes is described in relation to isozyme groupings and subspecies differentiation. The relevance and implications of these results for plant population genetics and the management of germplasm collections is discussed.     

Construction of a DNA library from chromosome 4 of rice (Oryza sativa) by microdissection

To evaluate the role of excitatory amino acids in secondary injury occurring after spinal cord trauma, several experimental studies focusing on the the changes of amino acid levels in the spinal cord have been performed to date. However, because of technical limitations, it has not been possible to correlate the local changes of excitatory amino acids with the total tissue levels of excitatory amino acids. To investigate the connection between the spread of injury and the excitatory amino acids, we assessed, the local changes of aspartate through novel experimental approaches like immunoreactivity via fluorescence microphotometry and histopathology while also analyzing the total tissue levels of amino acids via HPLC. These studies were performed using a model of incomplete cervical spinal cord injury in rats. Through this approach, we found that the levels of excitatory amino acids, such as glutamate and aspartate, began to decrease immediately after injury. No significant decrease was observed in the other amino acids. Similarly, local changes in aspartate in the spinal cord were observed using fluorescence microphotometry. The decrease in the anterior and posterior horns was rapid up to 15 min after injury, but, slowed thereafter, suggesting that a release of excitatory amino acids occurred at the site of primary injury almost immediately following injury. At 15-min post-injury large neurons within the injured cord appeared intact on histopathological analysis demonstrating that the alteration of excitatory amino acids occurs prior to histopathological change. Histopathological change in the white matter occurred more slowly than in the anterior and posterior horns, suggesting the spread of the lesion by secondary damage due to an autoclastic mechanism.     

Comparative RFLP mapping of a wild rice, Oryza officinalis, and cultivated rice, O. sativa

A comparative RFLP map was constructed in a wild rice, Oryza officinalis, by using 139 genomic and cDNA probes that had been used previously to map RFLPs in O. sativa. Nine of the 12 chromosomes of O. officinalis were highly homosequential to those of O. sativa. A major rearrangement of gene order was detected in chromosome 1 and small inversions were found in chromosomes 3 and 11. Fourteen translocated RFLP markers were found, and chromosome 11 contained a high frequency of such translocated segments. Results were consistent with meiotic and trisomic analysis, which suggested that the genomes of O. officinalis and O. sativa were similar. Applications of comparative maps in plant breeding and gene cloning are discussed.     

Superoxide dismutase enhances tolerance of freezing stress in transgenic alfalfa (Medicago sativa L.)

Activated oxygen or oxygen free radicals have been implicated in a number of physiological disorders in plants including freezing injury. Superoxide dismutase (SOD) catalyzes the dismutation of superoxide into O2 and H2O2 and thereby reduces the titer of activated oxygen molecules in the cell. To further examine the relationship between oxidative and freezing stresses, the expression of SOD was modified in transgenic alfalfa (Medicago sativa L.). The Mn-SOD cDNA from Nicotiana plumbaginifolia under the control of the cauliflower mosaic virus 35S promoter was introduced into alfalfa using Agrobacterium tumefaciens-mediated transformation. Two plasmid vectors, pMitSOD and pChlSOD, contained a chimeric Mn-SOD construct with a transit peptide for targeting to the mitochondria or one for targeting to the chloroplast, respectively. The putatively transgenic plants were selected for resistance to kanamycin and screened for neomycin phosphotransferase activity and the presence of an additional Mn-SOD isozyme. Detailed analysis of a set of four selected transformants indicated that some had enhanced SOD activity, increased tolerance to the diphenyl ether herbicide, acifluorfen, and increased regrowth after freezing stress. The F1 progeny of one line, RA3-ChlSOD-30, were analyzed by SOD isozyme activity, by polymerase chain reaction for the Mn-SOD gene, and by polymerase chain reaction for the neo gene. RA3-ChlSOD-30 had three sites of insertion of pChlSOD, but only one gave a functional Mn-SOD isozyme; the other two were apparently partial insertions. The progeny with a functional Mn-SOD transgene had more rapid regrowth following freezing stress than those progeny lacking the functional Mn-SOD transgene, suggesting that Mn-SOD serves a protective role by minimizing oxygen free radical production after freezing stress.     

水稻硫转运蛋白基因OsST的亚细胞定位

[目的]构建水稻硫酸根转运基因OsST与YFP黄色荧光蛋白融合表达载体,对OsST蛋白进行亚细胞定位.[方法]从水稻叶片的cDNA中克隆OsST基因ORF全长,测序验证后连入pA7-YFP表达载体,通过基因枪将融合载体转入洋葱上表皮细胞,在激光共聚焦显微镜下观察细胞中荧光发光部位.[结果]OSST蛋白定位于细胞膜和核膜上.[结论]为进一步研究硫转运蛋白的功能及硫酸根运输的机理奠定了基础.     

Rice (Oryza sativa) centromeric regions consist of complex DNA

Rice bacterial artificial chromosome clones containing centromeric DNA were isolated by using a DNA sequence (pSau3A9) that is present in the centromeres of Gramineae species. Seven distinct repetitive DNA elements were isolated from a 75-kilobase rice bacterial artificial chromosome clone. All seven DNA elements are present in every rice centromere as demonstrated by fluorescence in situ hybridization. Six of the elements are middle repetitive, and their copy numbers range from approximately 50 to approximately 300 in the rice genome. Five of these six middle repetitive DNA elements are present in all of the Gramineae species, and the other element is detected only in species within the Bambusoideae subfamily of Gramineae. All six middle repetitive DNA elements are dispersed in the centromeric regions. The seventh element, the RCS2 family, is a tandem repeat of a 168-bp sequence that is represented approximately 6,000 times in the rice genome and is detected only in Oryza species. Fiber-fluorescence in situ hybridization analysis revealed that the RCS2 family is organized into long uninterrupted arrays and resembles previously reported tandem repeats located in the centromeres of human and Arabidopsis thaliana chromosomes. We characterized a large DNA fragment derived from a plant centromere and demonstrated that rice centromeres consist of complex DNA, including both highly and middle repetitive DNA sequences.     

Enrichment process of lanthanum as a nonessential trace element in leaf cells of lettuce (Lactuca sativa L.)

Rare earth elements (REEs) as nonessential trace elements are enriched in living organisms and threaten their health. To early detect and reduce REE enrichment in living organisms, scientists are focused on clarifying the enrichment process of REEs in living organisms and its risks. However, the enrichment process of REEs in edible plant cells has remained unclear. Herein, by using interdisciplinary methods and techniques, the enrichment process of lanthanum (La(III)) in the leaf cells of lettuce (Lactuca sativa L.) was investigated. (1) When La(III) exposure dose is 0.5–5 μmol/L, La(III) is enriched outside the plasma membrane (PM). In this zone, La(III) is bound to vitronectin-like protein (VN) to form La–VN complexes; (2) When La(III) exposure dose is 5–20 μmol/L, besides the zone outside the PM, La(III) is also enriched on the PM and bound to arabinogalactan proteins (AGPs) to form La–AGPs complexes; (3) When La(III) exposure dose is 20–140 μmol/L, besides the zone outside and on the PM, La(III) is enriched inside the PM; (4) When La(III) exposure dose is 60–140 μmol/L, malondialdehyde content (an important indicator of invisible damage) significantly increases. Thus, as La(III) exposure dose increases, La(III) gradually migrates from outside the PM to the PM and inside the PM, enriching in these zones in turn. The enriched La(III) will cause invisible damage to lettuce leaf cells and even enter human bodies along food chains. These results provide references for investigating the enrichment process of REEs in plants and its environmental risks, and finding strategies to early detect and reduce REE enrichment in plants.     

紫花苜蓿菌核病抗病基因ISSR标记初步研究

[目的]研究紫花苜蓿菌核病抗病基因的ISSR标记技术.[方法]运用ISSR分子标记技术,结合集群分离分析法时5株抗病植株和7株感病植株进行抗菌核病基因连锁的分子标记筛选;用叶片离体接种法对高抗83号×高感4号杂交F<,1>代的94个植株进行抗性验证.[结果]在93个ISSR引物中,有35个引物能够产生清晰稳定的扩增条带,其中6个引物能在抗痛、感病DNA池间产生9个特异性条带.抗性验证试验结果显示:825-1400、831-1480、850-1800、858-1600、866-1900、888-1400可以作为苜蓿菌核病抗性基因的ISSR分子标记.[结论]研究结果为紫花苜蓿菌核痛抗病基因的定位、克隆、转基因等深入研究奠定了基础.     

Molecular dissection of B cell antigen receptor signaling (review)

Metchnikowin is a recently discovered proline-rich peptide from Drosophila with antibacterial and antifungal properties. Like most other antimicrobial peptides from insects, its expression is immune-inducible. Here we present evidence that induction of metchnikowin gene expression can be mediated either by the TOLL pathway or by the imd gene product. We show that the gene remains inducible in Toll-deficient mutants, in which the antifungal response is blocked, as well as in imd mutants, which fail to mount an antibacterial response. However, in Toll-deficient;imd double mutants, metchnikowin gene expression can no longer be detected after immune challenge. Our results suggest that expression of this peptide with dual activity can be triggered by signals generated by either bacterial or fungal infection. Cloning of the metchnikowin gene revealed the presence in the 5' flanking region of several putative cis-regulatory motifs characterized in the promoters of insect immune genes: namely, Rel sites, GATA motifs, interferon consensus response elements and NF-IL6 response elements. Establishment of transgenic fly lines in which the GFP reporter gene was placed under the control of 1.5 kb of metchnikowin gene upstream sequences indicates that this fragment is able to confer full immune inducibility and tissue specificity of expression on the transgene.     

Molecular cloning and characterization of a cDNA encoding asparagine synthetase from soybean (Glycine max L.) cell cultures

A cDNA encoding glutamine-dependent asparagine synthetase was isolated from dark-adapted Glycine max cell culture. The deduced amino acid sequence showed 76-89% identity with other plant sequences. The gene for asparagine synthetase is expressed predominantly in shoots as compared to roots of etiolated plants and the level of expression decreases following light treatment, suggesting that the gene expression is down-regulated by light.     

Characterization of the gene for delta1-pyrroline-5-carboxylate synthetase and correlation between the expression of the gene and salt tolerance in Oryza sativa L

A cDNA for delta1-pyrroline-5-carboxylate (P5C) synthetase (cOsP5CS), an enzyme involved in the biosynthesis of proline, was isolated and characterized from a cDNA library prepared from 14-day-old seedlings of Oryza sativa cv. Akibare. The deduced amino acid sequence of the P5CS protein (OsP5CS) from O. sativa exhibited 74.2% and 75.5% homology to that of the P5CS from Arabidopsis thaliana and Vigna aconitifolia, respectively. Northern blot analysis revealed that the gene for P5CS (OsP5CS) was induced by high salt, dehydration, treatment of ABA and cold treatment, while it was not induced by heat treatment. Simultaneously, accumulation of proline was observed as a result of high salt treatment in O. sativa. Moreover, the levels of expression of OsP5CS mRNA and content of proline under salt stress condition were compared between a salt-tolerant cultivar, Dee-gee-woo-gen (DGWG) and a salt-sensitive breeding line, IR28. It was observed that the expression of the P5CS gene and the accumulation of proline in DGWG steadily increased, whereas those in IR28 increased slightly.