R-ISSR as a new tool for genomic fingerprinting, mapping, and gene tagging

In the present study we propose and test the concept of R-ISSR, a new tool for genomic fingerprinting, mapping, and gene tagging. The concept is based on the fact that primers for inter-simple sequence repeat (ISSR) and random-amplified polymorphic DNA (RAPD) analysis elicit different genomic information, and the combined use of these 2 kinds of primers in the same polymerase chain reaction (PCR) reactions might reveal new genomic loci that could not be detected with either technique alone. The feasibility of this tool was first electronically simulated with sequence analysis software andArabidopsis chromosome sequence. Next, different combinations of ISSR and RAPD primers were applied in real PCR reactions to detect new genomic loci in 2 maize lines (Q319 and 1145). Sequencing gels were used to separate PCR products and showed good resolving ability in comparison with agarose gels. RAPD primers could be successfully used with ISSR primers for the detection of new genomic loci and applied in a new way for genomic mapping, fingerprinting, and gene tagging.     

Necrostatin-1 protects against glutamate-induced glutathione depletion and caspase-independent cell death in HT-22 cells

Glutamate, a major excitatory neurotransmitter in the CNS, plays a critical role in neurological disorders such as stroke and Parkinson's disease. Recent studies have suggested that glutamate excess can result in a form of cell death called glutamate-induced oxytosis. In this study, we explore the protective effects of necrostatin-1 (Nec-1), an inhibitor of necroptosis, on glutamate-induced oxytosis. We show that Nec-1 inhibits glutamate-induced oxytosis in HT-22 cells through a mechanism that involves an increase in cellular glutathione (GSH) levels as well as a reduction in reactive oxygen species production. However, Nec-1 had no protective effect on free radical-induced cell death caused by hydrogen peroxide or menadione, which suggests that Nec-1 has no antioxidant effects. Interestingly, the protective effect of Nec-1 was still observed when cellular GSH was depleted by buthionine sulfoximine, a specific and irreversible inhibitor of glutamylcysteine synthetase. Our study further demonstrates that Nec-1 significantly blocks the nuclear translocation of apoptosis-inducing factor (a marker of caspase-independent programmed cell death ) and inhibits the integration of Bcl-2/adenovirus E1B 19 kDa-interacting protein 3 (a pro-death member of the Bcl-2 family) into the mitochondrial membrane. Taken together, these results demonstrate for the first time that Nec-1 prevents glutamate-induced oxytosis in HT-22 cells through GSH related as well as apoptosis-inducing factor and Bcl-2/adenovirus E1B 19 kDa-interacting protein 3-related pathways.     

Influence of supports on catalytic behavior of nickel catalysts in carbon dioxide reforming of toluene as a model compound of tar from biomass gasification

A series of supported Ni catalysts including Ni/MgO, Ni/γ-Al2O3, Ni/α-Al2O3, Ni/SiO2 and Ni/ZrO2 was tested in CO2 reforming of toluene as a model compound of tar from biomass gasification in a fluidized bed reactor, and characterized by the means of temperature programmed reduction with hydrogen (H2-TPR), XRD, TEM and temperature programmed oxidation (TPO). Combining the characterization results with the performance tests, the activity of catalyst greatly depended on Ni particles size, and the stability was affected by the coke composition. Both of them (Ni particle size and coke composition) were closely related to the interaction between nickel and support which would determine the chemical environment where Ni inhabited. The best catalytic performance was observed on Ni/MgO due to the strong interaction between NiO and MgO via the formation of Ni-Mg-O solid solution, and the highest dispersion of Ni particle in the basic environment.     

Enrichment of a common wheat genetic map and QTL mapping for fatty acid content in grain

DArT and SSR markers were used to saturate and improve a previous genetic map of RILs derived from the cross Chuan35050 × Shannong483. The new map comprised 719 loci, 561 of which were located on specific chromosomes, giving a total map length of 4008.4 cM; the rest 158 loci were mapped to the most likely intervals. The average chromosome length was 190.9 cM and the marker density was 7.15 cM per marker interval. Among the 719 loci, the majority of marker loci were DArTs (361); the rest included 170 SSRs, 100 EST-SSRs, and 88 other molecular and biochemical loci. QTL mapping for fatty acid content in wheat grain was conducted in this study. Forty QTLs were detected in different environments, with single QTL explaining 3.6-58.1% of the phenotypic variations. These QTLs were distributed on 16 chromosomes. Twenty-two QTLs showed positive additive effects, with Chuan35050 increasing the QTL effects, whereas 18 QTLs were negative with increasing effects from Shannong483. Six sets of co-located QTLs for different traits occurred on chromosomes 1B, 1D, 2D, 5D, and 6B.     

Molecular variation and evolution of the tyrosine kinase domains of insulin receptor <Emphasis Type="Italic">IRa</Emphasis> and <Emphasis Type="Italic">IRb</Emphasis> genes in Cyprinidae

The insulin receptor (IR) gene plays an important role in regulating cell growth, differentiation and development. In the present study, DNA sequences of insulin receptor genes, IRa and IRb, were amplified and sequenced from 37 representative species of the Cyprinidae and from five outgroup species from non-cyprinid Cypriniformes. Based on coding sequences (CDS) of tyrosine kinase regions of IRa and IRb, molecular evolution and phylogenetic relationships were analyzed to better understand the characteristics of IR gene divergence in the family Cyprinidae. IRa and IRb were clustered into one lineage in the gene tree of the IR gene family, reconstructed using the unweighted pair group method with arithmetic mean (UPGMA). IRa and IRb have evolved into distinct genes after IR gene duplication in Cyprinidae. For each gene, molecular evolution analyses showed that there was no significant difference among different groups in the reconstructed maximum parsimony (MP) tree of Cyprinidae; IRa and IRb have been subjected to similar evolutionary pressure among different lineages. Although the amino acid sequences of IRa and IRb tyrosine kinase regions were highly conserved, our analyses showed that there were clear sequence variations between the tyrosine kinase regions of IRa and IRb proteins. This indicates that IRa and IRb proteins might play different roles in the insulin signaling pathway.     

Molecular tagging and mapping of the erect panicle gene in rice

Erect panicle (EP) is one of the more important traits of the proposed ideotype of high-yielding rice. Several rice cultivars with the EP phenotype, which has been reported to be controlled by a dominant gene, have been successfully developed and released for commercial production in North China. To analyze the inheritance of the EP trait, we generated segregating F₂ and BC₁F₁ populations by crossing an EP-type variety, Liaojing 5, and a curved-panicle-type variety, Fengjin. Our results confirmed that a dominant gene controls the EP trait. Simple-sequence repeat (SSR) and bulked segregant analyses of the F₂ population revealed that the EP gene is located on chromosome 9, between two newly developed SSR markers, RM5833-11 and RM5686-23, at a genetic distance of 1.5 and 0.9 cM, respectively. Markers closer to the EP gene were developed by amplified fragment length polymorphism (AFLP) analysis with 128 AFLP primer combinations. Three AFLP markers were found to be linked to the EP gene, and the nearest marker, E-TA/M-CTC₂₀₀, was mapped to the same location as SSR marker RM5686-23, 1.5 cM from the EP gene. A local map around the EP gene comprising nine SSR and one AFLP marker was constructed. These markers will be useful for marker-assisted selection (MAS) for the EP trait in rice breeding programs.     

Orally active and brain permeable proline amides as highly selective 5HT2c agonists for the treatment of obesity

Brain-penetrable proline amides were developed as 5HT2c agonists with more than 1000-fold binding selectivity against 5HT2b receptor. After medicinal chemistry optimization and SAR studies, orally active proline amides with robust efficacy in a rodent food intake inhibition model were uncovered.     

Precise mapping of a quantitative trait locus interval for spike length and grain weight in bread wheat (Triticum aestivum L.)

The spike characteristics length, spikelet density and fertile floret number are related yield components and are important in cereal improvement. QSpl.nau-2D is a major quantitative trait locus controlling spike length (SPL) detected in the recombinant inbred line population developed by crossing wheat (Triticum aestivum) cultivars Nanda2419 with Wangshuibai. In this study, to validate its genetic effect and determine its precise location, QSpl.nau-2D’s near-isogenic line (NIL) was developed using Mianyang99-323 as the recurrent parent through marker-assisted selection. Field trials showed that the NIL not only had significantly longer spikes on average than the recurrent parent but also had significantly higher grain weight, but did not differ in spikelet number and kernel number per spike. In the F2 population derived from a cross of the NIL with Mianyang99-323, QSpl.nau-2D functioned like a single gene and conditioned the SPL in a partially dominant manner, and was thus designated as HL1 (for head length). To precisely map HL1, 89 recombinants, consisting of 11 genotypes, were identified in the NIL-derived F2 population of 674 plants by using markers in the Xwmc25–Xgpw4080 interval. Phenotyping these lines showed that the introduction of a 0.9-cM interval flanked by Xcfd53 and DG371 in Nanda2419 resulted in longer spikes and a higher grain weight in the NIL. The availability of markers closely linked to HL1 could facilitate its use in breeding programs.     

Monitoring of Gene Expression Profiles and Isolation of Candidate Genes Involved in Pollination and Fertilization in Rice (Oryza Sativa L.) with a 10K cDNA Microarray

To monitor gene expression profiles during pollination and fertilization in rice at a genome scale, we generated 73,424 high-quality expressed sequence tags (ESTs) derived from the green/etiolated shoot and pistil (0-5 h after pollination, 5hP) of rice, which were subsequently used to construct a cDNA microarray containing ca. 10 000 unique rice genes. This microarray was used to analyze gene expression in pistil unpollinated (UP), 5hP and 5DAP(5 days after pollination), anther, shoot, root, 10-day-old embryo (10EM) and 10-day-old endosperm (10EN). Clustering analysis revealed that the anther has a gene-expression profile more similar to root than to pistil and most pistil-preferentially expressed genes respond to pollination and/or fertilization. There are 253 ESTs exhibiting differential expression (e +/- 2-fold changes) during pollination and fertilization, and about 70% of them can be assigned a putative function. We also recovered 20 genes similar to pollination-related and/or fertility-related genes previously identified as well as genes that were not implicated previously. Microarray and real-time PCR analyses showed that the array sensitivity was estimated at 1-5 copies of mRNA per cell, and the differentially expressed genes showed a high correlation between the two methods. Our results indicated that this cDNA microarray constructed here is reliable and can be used for monitoring gene expression profiles in rice. In addition, the genes that differentially expressed during pollination represent candidate genes for dissecting molecular mechanism of this important biological process in rice.