Evaluation of alternative RNA labeling protocols for transcript profiling with Arabidopsis AGRONOMICS1 tiling arrays

Background

The genus Populus is accepted as a model system for molecular tree biology. To investigate gene functions in Populus spp. trees, generating stable transgenic lines is the common technique for functional genetic studies. However, a limited number of genes have been targeted due to the lengthy transgenic process. Transient transformation assays complementing stable transformation have significant advantages for rapid in vivo assessment of gene function. The aim of this study is to develop a simple and efficient transient transformation for hybrid aspen and to provide its potential applications for functional genomic approaches.

Results

We developed an in planta transient transformation assay for young hybrid aspen cuttings using Agrobacterium-mediated vacuum infiltration. The transformation conditions such as the infiltration medium, the presence of a surfactant, the phase of bacterial growth and bacterial density were optimized to achieve a higher transformation efficiency in young aspen leaves. The Agrobacterium infiltration assay successfully transformed various cell types in leaf tissues. Intracellular localization of four aspen genes was confirmed in homologous Populus spp. using fusion constructs with the green fluorescent protein. Protein-protein interaction was detected in transiently co-transformed cells with bimolecular fluorescence complementation technique. In vivo promoter activity was monitored over a few days in aspen cuttings that were transformed with luciferase reporter gene driven by a circadian clock promoter.

Conclusions

The Agrobacterium infiltration assay developed here is a simple and enhanced throughput method that requires minimum handling and short transgenic process. This method will facilitate functional analyses of Populus genes in a homologous plant system.     

Metabolic profiling of laser microdissected vascular bundles of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Background  

Laser microdissection is a useful tool for collecting tissue-specific samples or even single cells from animal and plant tissue sections. This technique has been successfully employed to study cell type-specific expression at the RNA, and more recently also at the protein level. However, metabolites were not amenable to analysis after laser microdissection, due to the procedures routinely applied for sample preparation. Using standard tissue fixation and embedding protocols to prepare histological sections, metabolites are either efficiently extracted by dehydrating solvents, or washed out by embedding agents.     

3D fluorescent in situ hybridization using Arabidopsis leaf cryosections and isolated nuclei

Background  

Fluorescent hybridization techniques are widely used to study the functional organization of different compartments within the mammalian nucleus. However, few examples of such studies are known in the plant kingdom. Indeed, preservation of nuclei 3D structure, which is required for nuclear organization studies, is difficult to fulfill.     

Development of specific primers for cpSSR analysis in caper,olive and grapevine using consensus chloroplast primer pairs

Chloroplast SSR (cpSSR) markers have demonstrated utility in studying genetic relationships. DNA sequence information of the chloroplast genome is necessary for the development of cpSSR primer pairs. To overcome this limitation, “consensus” primers have been developed to amplify the homologous regions in plants where chloroplast sequences are not available. However, 80% Pinus thunbergi and Nicotiana tabacum developed “consensus” primers tested with grapevine, olive and caper showed multi-locus patterns. The presence of multi-locus patterns requires the use of agarose gel electrophoresis followed from isolation and sequencing of the bands. Herein, a PCR-strategy is proposed to construct specific cpSSR primer pairs without genomic sequence information, giving single-band amplifications that can be directly sequenced. Twelve new specific cpSSR primer pairs were developed for Capparis spinosa L., Olea europea L. and Vitis vinifera L. PCR products were sequenced to confirm the presence of microsatellite sequences, and their transportability was tested on six V. vinifera cultivars. Both single-nucleotide polymorphisms and polymorphic cpSSR were observed in the six grapevine cultivars using the specific cpSSR primers.     

A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction

ABSTRACT: BACKGROUND: We have developed a new analytical approach for isolation and quantification of cytokinins (CK) in minute amounts of fresh plant material, which combines a simple one-step purification with ultra-high pressure liquid chromatography-fast scanning tandem mass spectrometry. RESULTS: Plant tissue samples (1-5 mg FW) were purified by stop-and-go-microextraction (StageTip purification), which previously has only been applied for clean-up and pre-concentration of peptides. We found that a combination of two reverse phases and one cation-exchange phase, was the best tool, giving a total extraction recovery higher than 80%. The process was completed by a single chromatographic analysis of a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) in 24.5 minutes using an analytical column packed with sub-2-microne particles. In multiple reaction monitoring mode, the detection limits ranged from 0.05 to 5 fmol and the linear ranges for most cytokinins were at least five orders of magnitude. The StageTip purification was validated and optimized using samples of Arabidopsis thaliana seedlings, roots and shoots where eighteen cytokinins were successfully determined. CONCLUSIONS: The combination of microextraction with one-step high-throughput purification provides fast, effective and cheap sample preparation prior to qualitative and quantitative measurements. Our procedure can be used after modification also for other phytohormones, depending on selectivity, affinity and capacity of the selected sorbents.     

平菇培养料诱导灭菌工艺比较

为使平菇菌棒生产流程更加顺畅,提高生产效率,对培养料处理流程进行调整试验,结果 A(拌料→装袋→诱导→灭菌→接种)、B(拌料→诱导→灭菌→装袋→接种)、CK(拌料→诱导→装袋→灭菌→接种,为对照)三种处理工艺的单袋产量及生物学效率均没有显著差异,处理A适宜大规模生产,不受季节限制;处理B可用于秋冬季节生产;对照的设备设施要求简单,技术难度小,适宜小规模生产。     

DNA profiling of Capsicum landraces of Manipur

Seven chilli landraces of Manipur belonging to three cultivated species of Capsicum (Capsicum annuum, Capsicum frutescens, and Capsicum chinense) form economically important food crops of the region. The genotypes were characterized using ten random amplified polymorphic DNA (RAPD) markers. The cluster analysis based on Jaccard's similarity coefficient calculated by UPGMA method differentiated the genotypes into two main cluster groups. One cluster represented the C. annuum genotypes while the other cluster represented the C. frutescens and the C. chinense genotypes. C. chinense genotypes were more close to C. frutescens genotypes. Genetic variation between the C. frutescens genotypes was more than among the C. annuum genotypes and the C. chinense genotypes were the least similar ones.     

Scent profiling of Cymbidium ensifolium by electronic nose

The performance of electronic nose (E-nose) for Chinese Cymbidium scent profiling has been evaluated. Changes in scent profiles of two Cymbidium ensifolium cultivars have been monitored at different flowering stages (initial flowering, full flowering, and terminal flowering) and different times combined with two gas collecting devices. Samples were collected by static headspace (SHS) method. How E-nose can be used for pattern recognition and for studying the releasing of flower scent were proposed. Data obtained were subjected to principal component analysis (PCA) and discriminant function analysis (DFA). PCA was performed on the initially instrumental data to explore the structure of each data set and such result showed that the sensory data contained information related to the cultivar and to time spots. DFA was performed to improve the results, leading to clear separations between the sample groups. Gas collecting device did not seriously affect the result of PCA and DFA. Relative aroma intensity (RAI) was proposed as an alternative concept to compare scent intensity between samples on different time points. These results demonstrate the potential application of the E-nose to evaluate the scent profile of flower.     

西瓜砧木专用型育苗基质配方研究

本试验以腐熟中药渣、泥炭、蛭石、珍珠岩为原料,按照不同比例复配成5种育苗基质,选取进口泥炭作为对照(CK),研究不同配比的育苗基质对西瓜砧木(葫芦)苗期生长的影响.结果表明:基质A培育的葫芦幼苗在株高、茎粗、叶柄长以及鲜重上均显著优于其他配比的育苗基质,育苗效果显著提高,可推荐作为西瓜砧木(葫芦)育苗的专用型育苗基质.     

Microarray-based miRNAs profiling in lower limb arteriosclerosis of diabetic rats

AIM: To establish the profiling of microRNAs (miRNAs) in the lower extremity arterial tissue between diabetic rats with lower limb arteriosclerosis (DAS) and diabetic rats with normal lower limb (DN), and to explore the possible molecular mechanisms involved in aberrant miRNA expression in DAS. METHODS: The rat models of DAS and DN were successfully established. The respective lower extremity arterial tissue was isolated. The total miRNAs were purified for a hybridization detection by miRNA microarray. The results of chip scanning and data were analyzed and verified by RT-qPCR. RESULTS: Ten miRNAs related to DAS, including rno-miR-206-3p, rno-miR-133a-5p, rno-miR-133b-3p, rno-miR-133a-3p, rno-miR-325-5p, rno-miR-675-3p, rno-miR-411-5p, rno-miR-329-3p, rno-miR-335 and rno-miR-126a-3p, were determined. All 10 abnormally expressed miRNAs were up-regulated. The validating results of RT-qPCR confirmed 9 of the miRNAs in line with chip expression. Just rno-miR-335 showed the opposite between PCR detection and microarray result. CONCLUSION: A group of miRNAs in diabetic rats suffering from lower limb arteriosclerosis plays an important role in the vascular atherosclerosis process. The abnormal expression of miRNAs is likely to affect the vascular atherosclerosis process.     

Effect of nutrient sources on cucumber production in different substrates

The research was conducted in two successive seasons to compare the effect of nutrient sources, organic manure and inorganic conventional nutrient solution, in cucumber production performed with different local substrates. In fall, the experiment was designed to test three factors, namely cultivar [(a) Armada, (b) Gordion], nutrient source [(a) inorganic nutrient solution, (b) solid organic manure] and substrate [(a) 3 + 1 perlite + clinoptilolite, (b) 1 + 1 perlite + clinoptilolite, (c) 3 + 1 tuff + clinoptilolite, (d) 1 + 1 tuff + clinoptilolite, v/v]. Results showed that organic manuring decrease the total yield by 22.4% in comparison to inorganic nutrient solution. In organic manure treatment, vigorous variety (Armada) gave higher yield than less vigorous variety (Gordion). In the spring season, the tested factors were decreased to two and tested as nutrient source [(a) inorganic nutrient solution, (b) solid organic manure, (c) organic nutrient solution] and substrate. Armada was the only cultivar. Compared to that of the inorganic nutrient solution, total yield was reduced by 10.9% in the organic nutrient solution system and 31.3% in solid organic manure treatment.     

拟南芥RNA 加工因子的开花调控机制

开花调控是植物生长发育中很重要的过程。拟南芥分子遗传学分析表明,MADS-box 转录因
子FLC、RNA 结合蛋白（FCA、FPA 和FLK）和mRNA 3′ 端加工因子（FY）都参与了这一过程。开花因
子通过抑制FLC 表达来促使植物开花;RNA 结合蛋白通过转录后调控来调节FLC 的表达以调控拟南芥开
花。此外,microRNAs 也参与这一过程。本文通过综述上述几个相关因子的调控过程,来阐述RNA 加工
因子参与的拟南芥开花调控机理。     

蛹虫草人工培养基质专利申请情况分析

简介蛹虫草液体发酵、固体栽培和寄主培养等3种人工培养方式,分析各培养方式的基质专利申请数量、申请人分布情况,梳理蛹虫草培养基质研究的发展脉络,指出当前和今后的研究热点和难点问题。     

Water dynamics in two rockwool slab growing substrates of contrasting densities

It is important to determine the dynamics of water and nutrients in the growing substrate used for soil-less cultivation because this allows better time and space management of the water and nutrient supply according to plant needs during each step of the crop cycle. In this study, we focus on the motion of water in rockwool slabs used as growing substrate for a sweet pepper crop. The transport equation is first quoted and root absorption described by a sink term for which the absorption law was experimentally determined. Control volume methods by means of commercial computer fluid dynamics (CFD) software were used to solve the water movement equations numerically in three dimensions. However, as Richards’ equation describing water movements in the substrate is quite different from the Navier–Stokes equations used by the CFD, considerable modifications of the standard transport equations were required. The numerical results were then compared with experimental results and, once validated, the model was reused for a sensitivity analysis with respect to various physical parameters of the substrate. Based on this study, we propose an alternative substrate design more suitable for closed systems.     

Multidimensional fluorescence microscopy of multiple organelles in Arabidopsis seedlings

Background  

The isolation of green fluorescent protein (GFP) and the development of spectral variants over the past decade have begun to reveal the dynamic nature of protein trafficking and organelle motility. In planta analyses of this dynamic process have typically been limited to only two organelles or proteins at a time in only a few cell types.     

Novel applications of motif-directed profiling to identify disease resistance genes in plants

Background

Molecular profiling of gene families is a versatile tool to study diversity between individual genomes in sexual crosses and germplasm. Nucleotide binding site (NBS) profiling, in particular, targets conserved nucleotide binding site-encoding sequences of resistance gene analogs (RGAs), and is widely used to identify molecular markers for disease resistance (R) genes.

Results

In this study, we used NBS profiling to identify genome-wide locations of RGA clusters in the genome of potato clone RH. Positions of RGAs in the potato RH and DM genomes that were generated using profiling and genome sequencing, respectively, were compared. Largely overlapping results, but also interesting discrepancies, were found. Due to the clustering of RGAs, several parts of the genome are overexposed while others remain underexposed using NBS profiling. It is shown how the profiling of other gene families, i.e. protein kinases and different protein domain-coding sequences (i.e., TIR), can be used to achieve a better marker distribution. The power of profiling techniques is further illustrated using RGA cluster-directed profiling in a population of Solanum berthaultii. Multiple different paralogous RGAs within the Rpi-ber cluster could be genetically distinguished. Finally, an adaptation of the profiling protocol was made that allowed the parallel sequencing of profiling fragments using next generation sequencing. The types of RGAs that were tagged in this next-generation profiling approach largely overlapped with classical gel-based profiling. As a potential application of next-generation profiling, we showed how the R gene family associated with late blight resistance in the SH*RH population could be identified using a bulked segregant approach.

Conclusions

In this study, we provide a comprehensive overview of previously described and novel profiling primers and their genomic targets in potato through genetic mapping and comparative genomics. Furthermore, it is shown how genome-wide or fine mapping can be pursued by choosing different sets of profiling primers. A protocol for next-generation profiling is provided and will form the basis for novel applications. Using the current overview of genomic targets, a rational choice can be made for profiling primers to be employed.

     

不同基质对葡萄插条生根和生长的影响

以蛭石、河沙、珍珠岩、炭化稻壳为基质，研究其对葡萄扦插生根的效应。结果表明，以蛭石为扦插基质的插条生根成活率为96．67％；平均根数达29条；根、茎、叶生长量最大，平均株干重达14．07g；愈伤组织出现早，愈伤根和气孔根的根数较多．且生长健壮。