手性除草剂2甲4氯丙酸对映体在拟南芥中吸收转运及亚细胞分布的差异研究

为探明不同手性构型的2甲4氯丙酸(mecoprop,MCPP)在双子叶植物中的吸收、代谢等差异,以手性14C-MCPP对映体为示踪剂,野生哥伦比亚型拟南芥为受试植物,研究实验室条件下不同手性构型的MCPP在拟南芥中的吸收转运与亚细胞分布特征.结果表明,拟南芥对14C-MCPP 2种对映体的吸收效率分别为90.45％(1...     

Oleosins of Arabidopsis thaliana: expression in Escherichia coli, purification, and functional properties

The interfacial behavior of oleosins, the most abundant proteins from seeds oil bodies, was investigated using the pendant drop method at water/oil interfaces and compared to the behavior of beta-casein and lysozyme, proteins with contrasted emulsifying properties. Recombined high (rS3) and low (rS4) molecular weight oleosins comprising N-terminal histidine tags were purified to electrophoretic homogeneity. rS3 decreased the interfacial tension at the oil/water interface better than rS4, oleosins being more efficient than beta-casein. Oleosins formed aggregates when spread on noncompressed phospholipid (PL) films at the air/water interface as observed using a Langmuir-Blodgett balance equipped with a Brewster angle microscope. Oleosin spread at the surface of a compressed PL monolayer (5-20 mN/m) did not aggregate. Pressure increased immediately and proportionally to the amount of protein spread on the monolayer. The results stress the capacity of oleosins to be inserted in oil and in PL monolayers, which is of particular relevancy to their potential uses as water/oil emulsifiers.     

Rapid, microscale, acetyl bromide-based method for high-throughput determination of lignin content in Arabidopsis thaliana

The acetyl bromide method has been modified to enable the rapid microscale determination of lignin content in Arabidopsis with the goal of determining the genes that control lignin in plants. Modifications include reduction in sample size, use of a microball mill, adoption of a modified rapid method of extraction, use of an ice-bath to stabilize solutions and reduction in the volume of solutions. The microscale method was shown to be rapid, accurate and precise with values in agreement with those determined by the full-scale acetyl bromide method. The extinction coefficient for Arabidopsis lignin, dissolved using acetyl bromide, was determined to be 23.35 g(-1) L cm(-1) at 280 nm. This value is independent of the Arabidopsis accession, environmental growth conditions and is insensitive to lignin structure. The newly developed method can be used to determine lignin content in the inflorescence stems of Arabidopsis for mapping of lignin-related genes.     

The HSP terminator of Arabidopsis thaliana induces a high level of miraculin accumulation in transgenic tomatoes

High-level accumulation of the target recombinant protein is a significant issue in heterologous protein expression using transgenic plants. Miraculin, a taste-modifying protein, was accumulated in transgenic tomatoes using an expression cassette in which the miraculin gene was expressed by the cauliflower mosaic virus (CaMV) 35S promoter and the heat shock protein (HSP) terminator (MIR-HSP). The HSP terminator was derived from heat shock protein 18.2 in Arabidopsis thaliana . Using this HSP-containing cassette, the miraculin concentration in T0 transgenic tomato lines was 1.4-13.9% of the total soluble protein (TSP), and that in the T1 transgenic tomato line homozygous for the miraculin gene reached 17.1% of the TSP. The accumulation level of the target protein was comparable to levels observed with chloroplast transformation. The high-level accumulation of miraculin in T0 transgenic tomato lines achieved by the HSP terminator was maintained in the successive T1 generation, demonstrating the genetic stability of this accumulation system.     

拟南芥Alpha-dioxygenase2(AtDOX2)在毕赤酵母中的表达、纯化及活性鉴定

为了获得具有生物活性的拟南芥(Arabidopsis thaliana)alpha-dioxygenase2(AtDOX2),将其对应基因AtDOX2编码区克隆到酵母表达载体pPIC9k中,获得重组表达载体pPIC9k-AtDOX2,将线性化的重组载体电击转化入毕赤酵母(Pichia pastoris)表达菌株GS115,经G418筛选、PCR鉴定和甲醇诱导时间优化,获得重组AtDOX2的高效表达菌株GS115/pPIC9k-AtDOX2。SDS-PAGE分析结果显示,0.5%甲醇诱导96h重组蛋白表达量最高,其表达量占胞外总蛋白的15%。重组AtDOX2的表观分子量约为70kD,经Ni-NTA柱亲和层析可获得纯度大于80%的重组蛋白。2,2′-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)(ABTS)法测定结果表明重组蛋白具有过氧化物酶活性,且其活性受Ca2+和Mg2+激活,受EDTA、咪唑和Mn2+抑制;2,4-二硝基苯肼(2,4-DNP)法测定结果显示,重组AtDOX2具有双加氧酶活性,Ca2+对其双加氧酶活性也有激活作用。结果说明利用酵母表达系统获得...     

Analysis of amino acid uptake and translocation in Arabidopsis with a low‐cost hydroponic system

In soils, amino acids may be an important source of nitrogen for plants, at least in those where organic matter is not quickly degraded. The physiology of uptake of amino acids by roots was mainly studied in the 70's and 80's, before genes encoding amino acid importers were cloned in the 90's. While two families of amino acid transporters have been identified, yielding a total of about 100 genes, the role of each member is yet to be elucidated. As a tool for studying the role of amino acid transporters from Arabidopsis we set up a new hydroponic system suitable for radioisotope use. This system enables reproducible amino acid uptake by roots and estimation of the transport to the shoots of the amino acid taken up. We show that the rates of glutamine (Gln) uptake by wild‐type roots and transfer to the shoots were linear, and that other tested amino acids were translocated to the shoots with lower efficiency than Gln. A T‐DNA insertion mutant for a Gln exporter was compared to the wild‐type plants. Gln uptake and transfer were similar in both genotypes, showing that the suppression of the exporter did not affect uptake or transfer of amino acids to the shoots. The main advantage of the hydroponic system presented here is that all the materials used to grow Arabidopsis are virtually free and can therefore be discarded, a useful feature when working with radioactivity.     

拟南芥低温诱导cor15a基因的启动子克隆及在转基因马铃薯中的表达

利用PCR方法从拟南芥(Arabidopsis thaliana)品种Columbia的基因组中扩增出低温诱导基因cor15a的启动子片段并插入克隆载体。序列分析表明，克隆的启动子长900bp，与目前已经发表的启动子序列完全一致。将此启动子插入pBI121的gus基因及NOS终止子上游构成表达载体pLB，通过直接法导入根癌农杆菌(Agrobacterium tumefaciens)LBA4404，并转化马铃薯(Solarium tuberosum)获得转化再生植株。再生植株PCR检测和PCR-Southern鉴定结果证明，cor15a基因启动子已经成功地转入到马铃薯植株基因组中。GUS组织染色证明拟南芥cor15a启动子在马铃薯中也同样具有低温诱导表达的能力。     

Localization of MT2a Gene promoter expression is different from the site(s) of copper accumulation in the roots of transgenic Arabidopsis thaliana

Accumulation of mRNA for MT2a, a metallothionein of Arabidopsis thaliana, is known to be upregulated by excess copper (Cu). We compared the localization of the promoter activity of the MT2a gene and the accumulation site(s) of CU in the roots of transgenic A. thaliana. Open reading frame of the β-glucuronidase (GUS) gene was fused with the MT2a gene promoter and introduced into A. thaliana. Strong GUS activity was observed in the region near or within the stele in the presence of CU at a high concentration (high-CU condition). Electron probe X-ray micro-analysis demonstrated that under high-CU conditions, CU accumulated most abundantly in the cortex, where GUS activity was not significantly induced. These results indicate that the sites showing the MT2a promoter activity and CU accumulation did not coincide with each other, suggesting that CU is not a direct activator of the MT2a promoter.     

Efficiency of mannose-binding plant lectins in controlling a homopteran insect,the red cotton bug

Yield losses of different crops due to the attack of various classes of insects are a worldwide problem. Sucking type homopteran pests causing damage to many crop species are not controlled by commonly known insecticidal proteins, namely, Bacillus thuringiensis delta-endotoxin (Bt). This study describes the purification of mannose-binding lectins from three different monocotyledonous plants (Allium sativum, Colocasia esculenta, and Diffenbachia sequina) and their effects on a homopteran insect, the red cotton bug. All of them had a detrimental effect on the growth and development of the insect, where A. sativum bulb lectin showed the highest mortality of all, in particular. The same bulb lectin not only affected the growth and fecundity of the insect but also imparted drastic changes in the color, weight, and size, even on the second generation of the insects which have been reared on artificial diet supplemented with a sublethal dose of the lectin. Thus, this finding opens up a possibility of using this lectin as an important component in crop management.     

Rapid, low cost thin-layer chromatographic screening method for the detection of ochratoxin A in green coffee at a control level of 10 microg/kg.

A thin-layer chromatographic (TLC) screening method was developed for the detection of ochratoxin A (OTA) in green coffee at a control level of 10 microg/kg. The method is based on extraction of OTA with a mixture of phosphoric acid and dichloromethane, purification by liquid-liquid partition into sodium hydrogen carbonate, separation by normal-phase TLC, and detection by visual estimation of fluorescence intensity under a UV lamp at 366 nm. The method was validated by performing replicate analyses of uncontaminated green coffee material spiked at 3 different levels of OTA (5, 10, and 20 microg/kg), and also by comparing results obtained on a series of test trial green coffees naturally contaminated with OTA (range 0.2 to 136.7 microg/kg) with those measured by a quantitative immunoaffinity/HPLC method. The agreement between the two methods was excellent, and neither false positive nor false negative results were recorded. This screening method is rapid, simple, robust, and very cheap, which makes it particularly well adapted for implementation in coffee-producing countries.     

Sources of resistance in primary synthetic hexaploid wheat (Triticum aestivum L.) to insect pests: Hessian fly, Russian wheat aphid and Sunn pest in the fertile crescent

Hessian fly, Mayetiola destructor (Say), Russian wheat aphid (RWA), Diuraphis noxia (Kurdjumov), and Sunn pest, Eurygaster integriceps (Puton), are among the most important insect pests of wheat in North Africa, West and Central Asia. Host plant resistance is the most economical, environmentally friendly and practical means of controlling insect pests. Through field and greenhouse screening, several sources of resistance to Hessian fly, RWA and Sunn pest have been identified in wheat and its wild relatives. To further broaden the genetic base of resistance to these pests, 914 fixed lines of synthetic hexaploid wheat (SHW) commonly designated as primary synthetic wheat were evaluated for resistance to Hessian fly, RWA and Sunn pest. The initial screenings for RWA and Sunn pest were carried out in the field at Tel Hadya, Aleppo, Syria, and for Hessian fly in the greenhouse at Tel Hadya during the 2009 and 2010 seasons. Promising accessions from the initial screening for Hessian fly, RWA and Sunn pest were evaluated for confirmation in replicated trials in the greenhouse and field. Fifteen SHWs showed high levels of resistance to Hessian fly and four showed moderate resistance. A wheat line derived from the cross (Triticum turgidum/T. dicoccoides) also showed a high level of resistance to Hessian fly. The level of resistance to RWA in SHW was considerably lower; only one SHW and one durum wheat ‘Altar 84’ exhibited a high level of resistance, while four SHW were moderately resistant. There were 21 SHW genotypes and one durum wheat ‘Langdon’ found resistant to Sunn pest feeding at the vegetative stage. Crosses between these potentially novel resistance sources and elite bread wheat were initiated. Genetic and genomic studies using these accessions are ongoing to identify and characterize the resistance genes and reveal potentially new resistance genes, which will be useful in breeding programs to develop wheat germplasm with multiple resistances to these pests.     

Biosafety, antioxidant status, and metabolites in urine after consumption of dried cranberry juice in healthy women: a pilot double-blind placebo-controlled trial

This study assessed the effect of an 8 week consumption of dried cranberry juice (DCJ) on 65 healthy young women. Basic biochemical and hematological parameters, antioxidant status, presence of metabolites in urine, and urine ex vivo antiadherence activity were determined throughout the trial. A 400 mg amount of DCJ/day had no influence on any parameter tested. A 1200 mg amount of DCJ/day resulted in a statistically significant decrease in serum levels of advanced oxidation protein products. This specific protective effect against oxidative damage of proteins is described here for the first time. Urine samples had an inhibitory effect on the adhesion of uropathogenic Escherichia coli strains, but no increase in urine acidity was noted. Hippuric acid, isomers of salicyluric and dihydroxybenzoic acids, and quercetin glucuronide were identified as the main metabolites. In conclusion, cranberry fruits are effective not only in the prevention of urinary tract infection but also for the prevention of oxidative stress.     

Inoculation with indigenous rhizobial isolates enhanced nodulation,growth, yield and protein content of soybean (Glycine max L.) at different agro-climatic regions in Ethiopia

Abstract

Soybean cultivation in Ethiopia is dominated by smallholder farmers who use little or no inputs, often resulting in low yields. The use of effective rhizobia strains was considered as an ecologically and environmentally sound approach for soybean production. Field experiments were conducted during 2015/16 cropping seasons at two different agro-climatic regions in Ethiopia to investigate the effectiveness of local soybean isolates for improving nodulation, growth, yield and quality of soybean. Ten treatments comprising of seven indigenous rhizobia isolates, one exotic strain, nitrogen fertilized treatment and uninoculated control were arranged in randomized complete block design in three replications. Results of the experiment revealed that nodule number and nodule dry weight significantly increased from nil in the uninoculated control to 14–34 and 110–521?mg plant^?1, respectively due to inoculation with different isolates. Furthermore, inoculation significantly increased shoot dry weight by 24–46%, shoot nitrogen concentration by 20–30%, shoot N content by 29–49%, plant height by 14–41%, pods per plant by 12–38%, seeds per pod by 7–19%, thousand seed weight by 15–24%, grain yield by 22–115% and protein content by 7–39% compared with the uninoculated control. Generally, isolates Jm-1-Bo, As-5-Aw, Bk-3-Aw, Cw-6-Aw and MAR 1495 performed better than the others in most yield parameters at both locations of which Jm-1-Bo and As-5-Aw were the local isolates performing best irrespective of location, and were superior to the effective exotic standard strain. Therefore, isolates Jm-1-Bo, As-5-Aw and Bk-3-Aw could be utilized as candidates for inoculant production at large scale in areas with similar agroecology.     

Identification of genetic loci in a rhizosphere-inhabiting,species of Pseudomonas involved in expression of a phytoparasitic nematode ovicidal factor

Pseudomonas sp. BG33R was isolated from peach orchard soil suppressive to the phytoparasitic ring nematode, Mesocriconema xenoplax. Tn5 transposon mutagenesis was utilized to generate five BG33R mutants 246, 1122, 1233, 1397, and 1917, which lacked ovicidal activity and also exhibited altered fluorescent siderophore and protease production. Southern blot analysis revealed that each of the five mutants contained a single unique Tn5 insertion. Mobilization of selected BG33R genomic cosmid clones into mutants 246 and 1917 restored egg-kill activity and restored normal protease and siderophore production. Furthermore, a genomic cosmid clone hybridizing to the Tn5 insertion site in 1122 also complemented mutant 1917. Site-directed mutagenesis of BG33R using the cloned Tn5 insertion site from mutant 1397 resulted in the loss of ovicidal activity and restoration of the hyperfluorescence phenotype. Sequence analysis was performed on the genetic regions flanking the Tn5 insertion site in all five egg-kill-negative mutants. Open reading frame analysis and amino acid comparative database searches of the Tn5 insertion sites in the five mutants revealed a high degree of homology to the vgrG gene, the two-component sensor kinase protein, rtpA (gacS analog), a UDP-glucose 4-epimerase gene, a siderophore receptor protein, and the 50S ribosomal protein, L21. The involvement of these genes in the production of the egg-kill factor is discussed.     

Micromorphology of a Mousterian paleosol in aeolianites at the site Habonim, Israel

A paleosol with Mousterian tools in aeolianites at the Habonim site (Carmel Coastal plain, Israel) is a complex pedosedimentary sequence which records a succession of soil-forming episodes and stages of coastal dune instability. We identified three strong soils and one accretionary soil within Mousterian pedocomplex via micromorphological techniques, coupled with FTIR and SEM/EDAX. In the lowermost, red sandy soil (hamra) in Unit IV, decalcification led to the formation of micritic coatings, disrupted by faunal churning, while rubefication resulted in red ferric segregations. Illuviation of ferruginious clay in the form of clay coatings is lacking, probably due to its strong incorporation into the matrix. Redox features were intensified in the pseudogley of the next stage (Unit III). Polyphase calcitic features here indicate that illuviation of clay pre-dates illuviation of micrite and its later recrystallization into sparitic calcite. Further deterioration of drainage conditions and increased accumulation of fine particles led to the formation of vertisol (Unit II). Paleovertisol is characterized by a maximum of clay, stress-originated microfabric, abundant Mn precipitates and calcitic pseudomorphs. The peak of landscape instability occurred later and is recorded in the upper Unit I, in which micromorphological signs of colluvial and aeolian processes are juxtaposed with various ferric accumulations, associated with gleying. The final episode occurred at a time of climatic instability, devegetation, and probably, a simultaneous groundwater rise in the littoral zone.