Construction of EGFP-Labeling System for Visualizing the Infection Process of Xanthomonas axonopodis pv. citri in planta

Xanthomonas axonopodis pv. citri (Xac) is the causal agent of citrus bacterial canker, an economically important disease to world citrus industry. To monitor the infection process of Xac in different citrus plants, the enhanced green florescent protein (EGFP) visualizing system was constructed to visualize the propagation and localization in planta. First, the wild-type Xac was isolated from the diseased leaves of susceptible 'Bingtang' sweet orange, and then the isolated Xac was labeled with EGFP by triparental mating. After PCR identification, the growth kinetics and pathogenicity of the transformants were analyzed in comparison with the wild-type Xac. The EGFP-labeled bacteria were inoculated by spraying on the surface and infiltration in the mesophyll of 'Bingtang' sweet orange leaves. The bacterial cell multiplication and diffusion processes were observed directly under confocal laser scanning microscope at different intervals after inoculation. The results indicated that the EGFP-labeled Xac releasing clear green fluorescence light under fluorescent microscope showed the infection process and had the same pathogenicity as the wild type to citrus. Consequently, the labeled Xac demonstrated the ability as an efficient tool to monitor the pathogen infection.     

Identification of quantitative trait loci for rice grain element composition on an arsenic impacted soil: Influence of flowering time on genetic loci

Elements in grain crops such as iron, zinc and selenium are essential in the human diet, whereas elements such as arsenic are potentially toxic to humans. This study aims to identify quantitative trait loci (QTLs) for trace elements in rice grain. A field experiment was conducted in an arsenic enriched field site in Qiyang, China using the Bala × Azucena mapping population grown under standard field conditions. Grains were subjected to elemental analysis by inductively coupled plasma mass spectroscopy. QTLs were detected for the elemental composition within the rice grains, including for iron and selenium, which have previously been detected in this population grown at another location, indicating the stability of these QTLs. A correlation was observed between flowering time and a number of the element concentrations in grains, which was also revealed as co‐localisation between flowering time QTLs and grain element QTLs. Unravelling the environmental conditions that influence the grain ionome appears to be complex, but from the results in this study one of the major factors which controls the accumulation of elements within the grain is flowering time.     

Sequence variability in three mitochondrial DNA regions of Spirometra erinaceieuropaei spargana of human and animal health significance

Sequence variability in three mitochondrial DNA (mtDNA) regions, namely cytochrome c oxidase subunit 3 (cox3), NADH dehydrogenase subunits 1 and 4 (nad1 and nad4) in Spirometra erinaceieuropaei spargana from different geographical regions in China was examined. A portion of each of the cox3 (pcox3), nad1 (pnad1) and nad4 genes (pnad4) were amplified separately from individual S. erinaceieuropaei spargana by polymerase chain reaction (PCR). Representative amplicons were subjected to sequencing in order to estimate sequence variability. The sequences of pcox3, pnad1 and pnad4 were 541, 607 and 847?bp in length, respectively. The A+T contents of the sequences were 68.39-68.76% (pcox3), 63.76-64.91% (pnad1) and 67.18-67.77% (pnad4), respectively, while the intra-specific sequence variations within each of the S. erinaceieuropaei spargana were 0-1.5% for pcox3, 0-2.8% for pnad1 and 0-2.7% for pnad4. Phylogenetic analysis using neighbour joining (NJ), maximum likelihood (ML) and maximum parsimony (MP) methods, indicated that all the spargana isolates in Hunan Province represented S. erinaceieuropaei. These findings demonstrated clearly the usefulness of the three mtDNA sequences for population genetics studies of S. erinaceieuropaei spargana of human and animal health significance.     

<Emphasis Type="Italic">Ptcorp</Emphasis> gene induced by cold stress was identified by proteomic analysis in leaves of <Emphasis Type="Italic">Poncirus trifoliata</Emphasis> (L.) Raf.

A proteomic approach was employed to investigate the cold stress-responsive proteins in trifoliate orange (Poncirus trifoliata (L.) Raf.), which is a well-known cold tolerant citrus relative and widely used as rootstock in China. Two-year-old potted seedlings were exposed to freezing temperature (−6°C) for 50 min (nonlethal) and 80 min (lethal), and the total proteins were isolated from leaves of the treated plants. Nine differentially accumulated proteins over 2-fold changes in abundance were identified by two-dimensional gel electrophoresis and mass spectrometry. Among these proteins, a resistance protein induced by the nonlethal cold treatment (protein spot #2 from P. trifoliata) was selected as target sequence for degenerated primer design. By using the designed primers, a PCR product of about 700 bp size was amplified from P. trifoliata genomic DNA, which was further cloned and sequenced. A nucleotide sequence of 676 bp was obtained and named Ptcorp. Blast retrieval showed that Ptcorp shared 88% homology with an EST of cold acclimated Bluecrop (Vaccinium corymbosum) library (Accession number: CF811080), indicating that Ptcorp had association with cold acclimation. Semiquantitative RT-PCR analysis demonstrated that Ptcorp gene was up-regulated by cold stress which was consistent with the former result of protein expression profile. As the resistance protein (NBS-LRR disease resistance protein family) gene was up-regulated by cold stress in trifoliate orange and satsuma mandarin, it may imply that NBS-LRR genes might be associated with cold resistance in citrus.     

Systematic investigation of Amphioxus (Branchiostoma floridae) microRNAs

Ascaris lumbricoides and Ascaris suum are parasitic nematodes living in the small intestine of humans and pigs, and can cause the disease ascariasis. For long, there has been controversy as to whether the two ascaridoid taxa represent the same species due to their significant resemblances in morphology. However, the complete mitochondrial (mt) genome data have been lacking for A. lumbricoides in spite of human and animal health significance and socio-economic impact globally of these parasites. In the present study, we sequenced the complete mt genomes of A. lumbricoides and A. suum (China isolate), which was 14,303 bp and 14,311 bp in size, respectively. The identity of the mt genomes was 98.1% between A. lumbricoides and A. suum (China isolate), and 98.5% between A. suum (China isolate) and A. suum (USA isolate). Both genomes are circular, and consist of 36 genes, including 12 genes for proteins, 2 genes for rRNA and 22 genes for tRNA, which are consistent with that of all other species of ascaridoid studied to date. All genes are transcribed in the same direction and have a nucleotide composition high in A and T (71.7% for A. lumbricoides and 71.8% for A. suum). The AT bias had a significant effect on both the codon usage pattern and amino acid composition of proteins. Phylogenetic analyses of A. lumbricoides and A. suum using concatenated amino acid sequences of 12 protein-coding genes, with three different computational algorithms (Bayesian analysis, maximum likelihood and maximum parsimony) all clustered in a clade with high statistical support, indicating that A. lumbricoides and A. suum was very closely related. These mt genome data and the results provide some additional genetic evidence that A. lumbricoides and A. suum may represent the same species. The mt genome data presented in this study are also useful novel markers for studying the molecular epidemiology and population genetics of Ascaris.     

Effect of a DNA vaccine harboring two copies of inhibin α (1-32) fragments on immune response, hormone concentrations and reproductive performance in rats

The objective was to investigate the effects of a novel DNA vaccine (pcISI) harboring two copies of inhibin α (1-32) fragments on immune response, hormone concentrations and reproductive performance in rats. Female Wistar rats (n = 18 per group) were immunized (twice, 4 wk apart) with 10, 50, or 100 μg (T₁, T₂ and T₃, respectively), of the pcISI plasmid. At 4 wk after the second immunization, plasma antibody titers were higher (P < 0.05) in T₃ than in either T₁ or T₂ (0.341 ± 0.123, 0.236 ± 0.068, and 0.251 ± 0.077, respectively, mean ± SD). Concurrrently, plasma concentrations of FSH and estradiol were highest (P < 0.05) in T₃, and were higher (P < 0.05) in T₁ and T₂ than in control groups. For antibody-positive rats, there was a correlation (P < 0.01) between antibody titer and FSH concentrations after two pcISI immunizations. The number of mature follicles in the T₃ group (46.00 ± 4.65) was higher (P < 0.05) than in two control groups (29.25 ± 3.72 and 27.92 ± 3.48), and also higher (P < 0.05) than in T₁ and T₂ (37.17 ± 4.99 and 38.75 ± 7.09). Antibody-positive rats had more mature ovarian follicles than negative rats (46.75 ± 4.23 vs. 35.60 ± 3.38, P < 0.05). Moreover, litter size and number of placentas were increased (P < 0.05) in the pcISI immunization groups, except for the T₁ group, compared to the control groups. In conclusion, the pcISI DNA vaccine successfully induced a humoral immune response, improved reproductive hormone concentrations, stimulated follicular development, and increased number of placentas and litter size. Furthermore, 100 μg yielded the best immune response.     

Development of novel chloroplast microsatellite markers for Miscanthus species (Poaceae)

? Premise of the study: A set of novel chloroplast microsatellite markers (cpSSRs) was developed for the bioenergy crop Miscanthus, and their utility in cross-species amplification was evaluated. ? Methods and Results: Twenty-eight novel primers flanking cpSSR loci were designed from a complete chloroplast genome sequence of Saccharum officinarum, a species closely related to Miscanthus. These primers were then tested on eight Miscanthus species, among which 16 cpSSR loci were found to be polymorphic. The number of alleles per polymorphic locus ranged from two to seven, with an average 3.94 alleles. ? Conclusions: These cpSSR markers can be applied to all Miscanthus species and will be useful for studying Miscanthus population structure, diversity, and phylogeography.     

Melamine causes apoptosis of rat kidney epithelial cell line (NRK-52e cells) via excessive intracellular ROS (reactive oxygen species) and the activation of p38 MAPK pathway

There was an outbreak of urinary stones associated with consumption of melamine-tainted milk products in 2008 in China, leading to serious illness of many infants and even death. We have recently demonstrated that melamine causes oxidative damage on the NRK (normal rat kidney)-52e cells. The objective of this study was to explore the cellular signalling pathway that mediates the cell apoptosis induced by melamine in the NRK-52e cells. Fluorescence microscope showed that melamine enhanced intracellular ROS (reactive oxygen species) levels of the NRK-52e cells. AO/EB (acridine orange/ethidium bromide) staining and flow cytometry revealed that melamine increased apoptotic and necrotic percentages of the NRK-52e cells in a dose-dependent manner. Notably, MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide] assays and flow cytometry displayed that SB203580, an inhibitor for p38 MAPK (mitogen-activated protein kinase) pathway, increased the proliferation of the NRK-52e cells and reduced the apoptotic and necrotic percentages of the NRK-52e cells. Western blots further demonstrated that p38 phosphorylation was activated by melamine in the NRK-52e cells and inhibitor SB203580 blocked the increase of p38 phosphorylation induced by melamine. Together, these results suggested that melamine causes apoptosis of the NRK-52e cells via excessive intracellular ROS and the activation of p38 MAPK pathway. This study thus offers a novel insight into molecular mechanisms by which melamine has adverse cytotoxicity on renal tubular epithelial cells.     

马铃薯遗传转化体系的优化及玉米淀粉分支酶基因SBEⅡb的导入

以马铃薯脱毒试管苗茎段为转化受体材料,建立并优化了农杆菌介导的马铃薯遗传转化体系。通过农杆菌介导法将玉米淀粉分支酶基因(Starch branching enzyme b,SBEⅡb)的过表达载体转化马铃薯,接种762个茎段,共获得35株抗性植株。经PCR检测获得了4株转基因阳性植株;对转基因植株进一步进行GUS活性组织化学染色,发现转基因植株的茎段与试管薯均被染上蓝色,表明外源SBEⅡb基因已整合到马铃薯基因组,且正常表达。     

太湖新银鱼移植对(鳖)早期摄食和生长的影响

研究选择长江中游西洞庭湖水系太湖新银鱼移植水体(黄石水库)和未移植水体(蒙泉水库),研究太湖新银鱼(Neosalanx taihuensis Chen)移植对浮游动物食性鱼类(Hemiculter leucisculus Basilewsky)早期生长和摄食的影响。2009年7月下旬和8月中旬共采集稚鱼157尾,其中7月下旬采集稚鱼在14—23日龄之间,两水体间生长差异不显著;8月中旬采集稚鱼在20—49日龄之间,黄石水库生长率显著小于蒙泉水库。对样品耳石日轮分析发现25日龄之前两水体稚鱼生长率相似,之后黄石水库稚鱼较蒙泉水库生长慢。食性分析发现25日龄前两水体稚鱼食物组成相似,主要摄食轮虫、小型枝角类和桡足类;25日龄后黄石水库稚鱼食性没有显著变化,而蒙泉水库稚鱼则转食大型枝角类、昆虫及鱼卵和仔鱼。两水体气候条件、营养状况、鱼类区系组成上基本相同,是否有太湖新银鱼移植是两水体间的主要差别。太湖新银鱼春群在1—5月间繁殖,而的繁殖在6月下旬之后。因此在早期生活史阶段与太湖新银鱼的食物竞争会主要发生在转食大型浮游动物之后。太湖新银鱼摄食使黄石水库大型浮游动物饵料资源短缺,稚鱼在25日龄后不能转食,是导致黄石水库幼鱼在25日龄后生长减慢的重要因素。