表达血管内皮生长因子-siRNA及双自杀基因yCDglyTK的联合基因载体构建及其应用研究

构建了新型联合基因载体pcDNA3.1(-)VEGF-siRNA/yCDglyTK,研究其在人胃癌细胞系SGC7901细胞中的表达和杀伤作用.构建靶向血管内皮生长因子(VEGF)的干扰质粒pGenesil-VEGF-siRNA,采用PCR法从中扩增siRNA表达框(含U6启动子),亚克隆至双自杀基因载体pcDNA3.1(-)CV-yCDglyTK,构建联合基因质粒pcDNA3.1(-)VEGF-siRNA/yCDglyTK;通过酶切、测序等鉴定重组质粒;以磷酸钙纳米颗粒为载体,将干扰质粒、双自杀基因质粒及联合基因质粒转染SGC7901细胞,RT-PCR、Western-blot验证目的基因表达;MTT法检测转染细胞对5-氟胞嘧啶(5-FC)的敏感性.结果表明:酶切及测序证实联合基因载体pcDNA3.1(-)VEGF-siRNA/yCDglyTK构建成功;SGC7901细胞转染联合基因质粒后,RT-PCR、Western-blot证实融合自杀基因表达,而VEGF基因表达下调;在前体药物5-FC作用下,转染联合基因组细胞存活率最低,与其他组比较有统计学差异.成功构建联合基因载体pcDNA3.1(-)VEGF-si...     

Introduction to ion trap mass spectrometry: Application to the structural characterization of plant oligosaccharides

This review will focus on ion trap mass spectrometry (ITMS) and the application of this technique to the structural analysis of carbohydrates. The basic principles of operation of the electrostatic ion traps are briefly described and the applicability of the technique to the structural characterization of carbohydrates is illustrated with the analysis of arabinoxylan oligosaccharides by ion trap mass spectrometry.     

Global functional analyses of rice promoters by genomics approaches

Promoters play key roles in conferring temporal, spatial, chemical, developmental, or environmental regulation of gene expression. Promoters that are subject to specific regulations are useful for manipulating foreign gene expression in plant cells, tissues, or organs with desirable patterns and under controlled conditions, and have been important for both basic research and applications in agriculture biotechnology. Recent advances in genomics technologies have greatly facilitated identification and study of promoters in a genome scale with high efficiency. Previously we have generated a large T-DNA tagged rice mutant library (TRIM), in which the T-DNA was designed with a gene/promoter trap system, by placing a promoter-less GUS gene next to the right border of T-DNA. GUS activity screens of this library offer in situ and in planta identifications and analyses of promoter activities in their native configurations in the rice genome. In the present study, we systematically performed GUS activity screens of the rice mutant library for genes/promoters constitutively, differentially, or specifically active in vegetative and reproductive tissues. More than 8,200 lines have been screened, and 11% and 22% of them displayed GUS staining in vegetative tissues and in flowers, respectively. Among the vegetative tissue active promoters, the ratio of leaf active versus root active is about 1.6. Interestingly, all the flower active promoters are anther active, but with varied activities in different flower tissues. To identify tissue specific ABA/stress up-regulated promoters, we compared microarray data of ABA/stress induced genes with those of tissue-specific expression determined by promoter trap GUS staining. Following this approach, we showed that the peroxidase 1 gene promoter was ABA up-regulated by 4 fold within 1 day of exposure to ABA and its expression is lateral root specific. We suggest that this be an easy bioinformatics approach in identifying tissue/cell type specific promoters that are up-regulated by hormones or other factors. Su-May Yu and Swee-Suak Ko equally contributed to this work.     

Analysis of gene-trap Ds rice populations in Korea

Insertional mutagen-mediated gene tagging populations have been essential resources for analyzing the function of plant genes. In rice, maize transposable elements have been successfully utilized to produce transposant populations. However, many generations and substantial field space are required to obtain a sufficiently sized transposant population. In rice, the japonica and indica subspecies are phenotypically and genetically divergent. Here, callus cultures with seeds carrying Ac and Ds were used to produce 89,700 lines of Dongjin, a japonica cultivar, and 6,200 lines of MGRI079, whose genome is composed of a mixture of the genetic backgrounds of japonica and indica. Of the more than 3,000 lines examined, 67% had Ds elements. Among the Ds-carrying lines, 81% of Dongjin and 63% of MGRI079 contained transposed Ds, with an average of around 2.0 copies. By examining more than 15,000 lines, it was found that 12% expressed the reporter gene GUS during the early-seedling stage. GUS was expressed in root hairs and crown root initials at estimated frequencies of 0.78% and 0.34%, respectively. The 5,271 analyzed Ds loci were found to be randomly distributed over all of the rice chromosomes. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Sung Han Park, Nam Soo Jun, Chul Min Kim are contributed equally to this paper     

Metabolic profiling and systematic identification of flavonoids and isoflavonoids in roots and cell suspension cultures of Medicago truncatula using HPLC-UV-ESI-MS and GC-MS

An integrated approach utilizing HPLC-UV-ESI-MS and GC-MS was used for the large-scale and systematic identification of polyphenols in Medicago truncatula root and cell culture. Under optimized conditions, we were able to simultaneously quantify and identify 35 polyphenols including 26 isoflavones, 3 flavones, 2 flavanones, 2 aurones and a chalcone. All identifications were based upon UV spectra, mass spectral characteristics of protonated molecules, tandem mass spectral data, mass measurements obtained using a quadrupole time-of-flight mass spectrometer (QtofMS), and confirmed through the co-characterization of authentic compounds. In specific instances where the stereochemistry of sugar conjugates was uncertain, subsequent enzymatic hydrolysis of the conjugate followed by GC-MS was used to assign the sugar stereochemical configuration. Comparative metabolic profiling of Medicago truncatula root and cell cultures was then performed and revealed significant differences in the isoflavonoid composition of these two tissues.     

OsRRM, a Spen-like rice gene expressed specifically in the endosperm

We used the promoter trap technique to identify a rice plant, named 107^#, in which the β-glucuronidase （GUS） reporter gene was expressed specifically in the endosperm. A single copy of the T-DNA was inserted into the plant genome, and a candidate gene OsRRM was identified by the insertion. The OsRRM promoter directed GUS expression specifically in rice endosperm, analogous to the GUS expression pattern observed in 107^#. OsRRMis a single-copy gene in rice and encodes a nuclear protein containing 1 005 amino-acid residues with two RNA recognition motifs and one Spen paralog and ortholog C-terminal domain. Westem blot analysis confirmed that the OsRRM protein was specifically expressed in rice endosperm. Ectopic expression of OsRRM in transgenic plants led to abnormalities, such as short stature, retarded growth and low fructification rates. Our data, in conjunction with the reported function of Spen genes, implicated OsRRM in the regulation of cell development in rice endosperm.     

Synthesis and ESR studies of a novel cyclic nitrone spin trap attached to a phosphonium group-a suitable trap for mitochondria-generated ROS?

In this study we report the synthesis and biological application of a novel cyclic nitrone spin trap containing a phosphonium cation. This new spin trap ([4-(2-methyl-1-oxy-3, 4-dihydro-2H-pyrrole-2-carbonyloxy)-butyl]-triphenyl-phosphonium bromide, MitoBMPOBr) is a derivative of the cyclic nitrone, 5-tert-butoxycarbonyl 5-methyl-1-pyrroline N-oxide (BMPO). MitoBMPOBr forms radical adducts upon trapping of superoxide and hydroxyl radicals that exhibit highly distinct and characteristic EPR spectra. The stability of these adducts is comparable to those of BMPO. Because of the presence of a positively-charged phosphonium moiety, MitoBMPOBr may be suitable for trapping reactive oxygen species (ROS) in the mitochondria.     

两种苹果蠹蛾性引诱剂诱捕器诱捕效率比较及地面植被的影响

在甘肃省高台县通过对三角胶粘式和水盆式这2种类型苹果蠹蛾Cydia pomonella(L.)诱捕器的田间比较研究。结果显示,二者在诱捕效率上存在的明显差异。在苹果蠹蛾密度较低的条件下,三角胶粘式诱捕器平均日诱捕量为2.50只,比水盆式诱捕器高出近2.84倍,最早监测到成虫的时间也比水盆式诱捕器提前3~4d,因此,三角胶粘式诱捕器具有更高的监测效率。结果还显示地面植被的遮盖极大地降低诱捕器的诱捕效果,严重时会使诱捕器对密度较低的苹果蠹蛾种群的监测功能丧失。     

Virus population extinction via ecological traps

Populations are at risk of extinction when unsuitable or when sink habitat exceeds a threshold frequency in the environment. Sinks that present cues associated with high-quality habitats, termed ecological traps, have especially detrimental effects on net population growth at metapopulation scales. Ecological traps for viruses arise naturally, or can be engineered, via the expression of viral-binding sites on cells that preclude viral reproduction. We present a model for virus population growth in a heterogeneous host community, parameterized with data from populations of the RNA bacteriophage Φ6 presented with mixtures of suitable host bacteria and either neutral or trap cells. We demonstrate that viruses can sustain high rates of population growth in the presence of neutral non-hosts as long as some host cells are present, whereas trap cells dramatically reduce viral fitness. In addition, we demonstrate that the efficacy of traps for viral elimination is frequency dependent in spatially structured environments such that population viability is a nonlinear function of habitat loss in dispersal-limited virus populations. We conclude that the ecological concepts applied to species conservation in altered landscapes can also contribute to the development of trap cell therapies for infectious human viruses.