An ordered EST catalogue and gene expression profiles of cassava (<Emphasis Type="Italic">Manihot esculenta</Emphasis>) at key growth stages

A cDNA library was constructed from the root tissues of cassava variety Huanan 124 at the root bulking stage. A total of 9,600 cDNA clones from the library were sequenced with single-pass from the 5′-terminus to establish a catalogue of expressed sequence tags (ESTs). Assembly of the resulting EST sequences resulted in 2,878 putative unigenes. Blastn analysis showed that 62.6% of the unigenes matched with known cassava ESTs and the rest had no ‘hits’ against the cassava database in the integrative PlantGDB database. Blastx analysis showed that 1,715 (59.59%) of the unigenes matched with one or more GenBank protein entries and 1,163 (40.41%) had no ‘hits’. A cDNA microarray with 2,878 unigenes was developed and used to analyze gene expression profiling of Huanan 124 at key growth stages including seedling, formation of root system, root bulking, and starch maturity. Array data analysis revealed that (1) the higher ratio of up-regulated ribosome-related genes was accompanied by a high ratio of up-regulated ubiquitin, proteasome-related and protease genes in cassava roots; (2) starch formation and degradation simultaneously occur at the early stages of root development but starch degradation is declined partially due to decrease in UDP-glucose dehydrogenase activity with root maturity; (3) starch may also be synthesized in situ in roots; (4) starch synthesis, translocation, and accumulation are also associated probably with signaling pathways that parallel Wnt, LAM, TCS and ErbB signaling pathways in animals; (5) constitutive expression of stress-responsive genes may be due to the adaptation of cassava to harsh environments during long-term evolution.     

Cellulosilyticum ruminicola,a Newly Described Rumen Bacterium That Possesses Redundant Fibrolytic-Protein-Encoding Genes and Degrades Lignocellulose with Multiple Carbohydrate- Borne Fibrolytic Enzymes

Cellulosilyticum ruminicola H1 is a newly described bacterium isolated from yak (Bos grunniens) rumen and is characterized by its ability to grow on a variety of hemicelluloses and degrade cellulosic materials. In this study, we performed the whole-genome sequencing of C. ruminicola H1 and observed a comprehensive set of genes encoding the enzymes essential for hydrolyzing plant cell wall. The corresponding enzymatic activities were also determined in strain H1; these included endoglucanases, cellobiohydrolases, xylanases, mannanase, pectinases, and feruloyl esterases and acetyl esterases to break the interbridge cross-link, as well as the enzymes that degrade the glycosidic bonds. This bacterium appears to produce polymer hydrolases that act on both soluble and crystal celluloses. Approximately half of the cellulytic activities, including cellobiohydrolase (50%), feruloyl esterase (45%), and one third of xylanase (31%) and endoglucanase (36%) activities were bound to cellulosic fibers. However, only a minority of mannase (6.78%) and pectinase (1.76%) activities were fiber associated. Strain H1 seems to degrade the plant-derived polysaccharides by producing individual fibrolytic enzymes, whereas the majority of polysaccharide hydrolases contain carbohydrate-binding module. Cellulosome or cellulosomelike protein complex was never isolated from this bacterium. Thus, the fibrolytic enzyme production of strain H1 may represent a different strategy in cellulase organization used by most of other ruminal microbes, but it applies the fungal mode of cellulose production.The ruminant rumens are long believed to be the anaerobic environments efficiently degrading the plant-derived polysaccharides, which is attributed to the inhabited abundant rumen microorganisms. They implement the fibrolytic degradation by the combination of the enzymes comprising of cellulases, hemicellulases, and to a lesser extent pectinases and ligninases (12). The rumen bacteria are outnumbered of the other rumen microbes; however, only a few of cellulolytic bacteria have been isolated from rumens. Ruminococcus flavefaciens, Ruminococcus albus, and Fibrobacter succinogenes are considered to be the most important cellulose-degrading bacteria in the rumen (18), and they produce a set of cellulolytic enzymes, including endoglucanases, exoglucanases (generally cellobiohydrolase), and β-glucosidases, as well as hemicellulases. In addition, the predominant ruminal hemicellulose-digesting bacteria such as Butyrivibrio fibrisolvens and Prevotella ruminicola lack the ability to digest cellulose but degrade xylan and pectin and utilize the degraded soluble sugars as substrates (10, 14). Although the robust cellulolytic species F. succinogenes degrades xylan, it cannot use the pentose product as a carbon source (24). Culture-independent approaches indicate that the three cellulolytic bacterial species represent only ∼2% of the ruminal bacterial 16S rRNA (43). Therefore, many varieties of rumen microbes remain uncultured (2). In recent years, rumen metagenomics studies have revealed the vast diversity of fibrolytic enzymes, multiple domain proteins, and the complexity of microbial composition in the ecosystem (9, 17). Hence, it is likely that the entire microbial community is necessary for the implementation of an efficient fibrolytic process in the rumen, including the uncultured species.In the rumen and other fibrolytic ecosystems, cellulolytic bacteria have to cope with the structural complexity of lignocelluloses and the interspecies competition; thus, not only a variety of plant polymer-degrading enzymes but also a noncatalytic assistant strategy, such as including adhesion of cells to substrates by a variety of anchoring domains, is required (8, 33, 38, 39). The (hemi)cellulolytic enzyme systems have been intensively studied for nonrumen anaerobic bacteria, including Clostridium thermocellum (19, 40), Clostridium cellulolyticum (6), Clostridium cellulovorans (13), and Clostridium stercorarium (47), as well as the rumen species, Rumicoccocus albus (35), Ruminococcus flavefaciens (32), and Fibrobacter succinogenes (4). The results indicate that most of them, except for Fibrobacter succinogenes, produce multiple cellulolytic enzymes integrated in a complex, cellulosome, and free individual proteins.The yak (Bos grunniens) is a large ruminant (∼1,000 kg) in the bovine family that lives mainly on the Qinghai-Tibetan Plateau in China at an altitude of 3,000 m above sea level. It is a local species that lives mainly on the world''s highest plateau. Yaks live in a full-grazing style with grasses, straws, and lichens as their exclusive feed, so the yak rumen can harbor a microbial flora distinct from those of other ruminants due to their fiber-component diet, since diet can be a powerful factor in regulating mammalian gut microbiome (27). A very different prokaryote community structure was revealed for yak rumen in our previous work based on the 16S rRNA diversity, which showed fewer phyla than for cattle but that a higher ratio of sequences was related to uncultured bacteria (2).We previously isolated a novel anaerobic fibrolytic bacterium, Cellulosilyticum ruminicola H1, from the rumen of a domesticated yak (11). Strain H1 grew robustly on natural plant fibers such as corn cob, alfalfa, and ryegrass as the sole carbon and energy sources, as well as on a variety of polysaccharides, including cellulose, xylan, mannan, and pectin, but not monosaccharides such as glucose, which is preferred by most ruminal bacteria. In the present study, using a draft of its genome and enzymatic characterization, we analyzed the enzymatic activities and the structures of the polymer hydrolases of strain H1 that were involved in the hydrolysis of complex polysaccharides.     

Medium optimization and structural characterization of exopolysaccharides from endophytic bacterium Paenibacillus polymyxa EJS-3

In this study, response surface methodology was employed to optimize the medium compositions for the production of exopolysaccharides (EPS) from endophytic bacterium Paenibacillus polymyxa EJS-3. Firstly, fractional factorial design was applied to evaluate the effects of different components in the medium. It was found that sucrose, yeast extract and CaCl₂ influenced significantly the production of EPS. Then, steepest ascent method and central composite design were used to optimize the concentrations of the three variables. As results, the optimal medium compositions were determined as following (g/L): sucrose 188.2, yeast extract 25.8, K₂HPO₄ 5 and CaCl₂ 0.34, with a corresponding yield of 35.26 g/L. In addition, both polysaccharide fractions (EPS-1 and EPS-2) from crude EPS were mainly composed of (2 → 6)-linked β-d-fructofuranosyl residues backbone with (2 → 1)-linked branches based on their structural characterization by FT-IR spectroscopy, methylation analysis and ¹³C NMR spectroscopy.     

Synthesis and characterization of a novel MPEG–chitosan diblock copolymer and self-assembly of nanoparticles

In this paper, a simple and novel method based on free-radical polymerization initiated by potassium persulfate (KPS) was developed to synthesize the MPEG–chitosan diblock copolymer (MPEG–CS). The obtained MPEG–CS diblock copolymer was characterized by Fourier transform infrared (FTIR), ¹H nuclear magnetic resonance (¹H NMR), X-ray diffraction (XRD) and differential scanning calorimetry (DSC), respectively. The MPEG–CS copolymer could self-assemble into nanoparticles in aqueous solution. A typical TEM photography indicated that the well-spherical nanoparticles with diameter at about 200 nm were obtained. In vitro cell culture assay indicated that MPEG–CS nanoparticles are non-toxic and cell-compatible as the polymer concentration was smaller than 0.6 mg/ml. In conclusion, the obtained MPEG–CS nanoparticles might have great potential application in drug-delivery system.     

Association Analysis of the RET Proto-Oncogene with Hirschsprung Disease in the Han Chinese Population of Southeastern China

Hirschsprung disease (HSCR) is a complex congenital disorder characterized by intestinal obstructions caused by the absence of the intestinal ganglion cells of the nerve plexuses in variable lengths of the digestive tract. This study investigated a possible role of the RET proto-oncogene in sporadic HSCR patients in the Han Chinese population. Our results indicated that rs1800858, rs1800860, rs1800863, and rs2075912, located in exons 2, 7, 15, and intron 19 of RET, are strongly associated with the disease (P < 0.01), with rs1800860 and rs1800863 playing a protective role in the pathogenesis of HSCR in the Chinese population. We also showed that the haplotype consisting of four SNPs is significantly associated with HSCR. We did not find a significant difference in the CA-repeat in intron 5 of RET between cases and controls. Our study provided further evidence that the RET gene is involved in the susceptibility to HSCR in the Han Chinese population.     

球孢白僵菌高渗适应性相关基因Bbmpd的克隆与表达分析

【目的】克隆与球孢白僵菌(Beauveria bassiana)的高渗适应性相关基因,并对其功能进行分析,以揭示球孢白僵菌对高渗等逆境适应的分子机理。【方法】利用YADE法克隆T-DNA的侧翼序列并进行基因组步行,获得突变基因的全长及上游序列;利用RT-PCR技术分析突变基因的表达特性以及与Bbhog1的关系;采用同源重组技术敲除Bbmpd基因。【结果】克隆得到插入突变基因及其上、下游序列全长3037bp。该基因与编码球孢白僵菌的1-磷酸甘露醇脱氢酶基因相似性为98%。Bbmpd的表达受高渗环境(0.8mol/L NaCl)的诱导,受Bbhog1信号途径的激活调节,Bbhog1缺失导致Bbmpd表达下调。Bbmpd缺失突变体在高渗胁迫下的生长受到明显抑制。Bbmpd缺失不影响球孢白僵菌在查氏培养基上的生长和产孢。【结论】由T-DNA突变体克隆了编码球孢白僵菌1-磷酸甘露醇脱氢酶基因Bbmpd,该基因的表达受高渗环境的诱导和Bbhog1的调控,与球孢白僵菌高渗适应性相关。     

Static pressure drives proliferation of vascular smooth muscle cells via caveolin-1/ERK1/2 pathway

Intimal hyperplasia plays an important role in various types of vascular remodeling. Mechanical forces derived from blood flow are associated with the proliferation of vascular smooth muscle cells (VSMC). This contributes to many vascular disorders such as hypertension, atherosclerosis and restenosis after percutaneous transluminal angioplasty (PTA). In this study, we show that static pressure induces the proliferation of VSMC and activates its related signal pathway. VSMC from a rat aorta were treated with different pressures (0, 60, 90, 120, 150 and 180 mm Hg) in a custom-made pressure incubator for 24 h. The most active proliferation of VSMC was detected at a pressure of 120 mm Hg. VSMC was also incubated under a static pressure of 120 mm Hg for different time intervals (0, 2, 4, 8, 12 and 24 h). We found that static pressure significantly stimulates VSMC proliferation. Extracellular signal-regulated kinases 1/2 (ERK1/2) activation showed a peak at the pressure of 120 mm Hg at 4-h time point. Moreover, caveolin-1 expression was significantly inhibited by rising static pressure. Downregulation of VSMC proliferation could be found after PD98059 (ERK1/2 phosphorylation inhibitor) treatment. Our data also showed that a siRNA-mediated caveolin-1 knock down increased ERK1/2 phosphorylation and VSMC proliferation. These results demonstrate that static pressure promotes VSMC proliferation via the Caveolin-1/ERK1/2 pathway.     

Trace detection of picloram using an electrochemical immunosensor based on three-dimensional gold nanoclusters

Picloram, a herbicide widely used for broadleaf weed control, is persistent and mobile in soil and water with adverse health and environmental effects. It is important to develop a sensitive method for accurate detection of trace picloram in the environment. In this article, a type of ordered three-dimensional (3D) gold (Au) nanoclusters obtained by two-step electrodeposition using the spatial obstruction/direction of the polycarbonate membrane is reported. Bovine serum albumin (BSA)-picloram was immobilized on the 3D Au nanoclusters by self-assembly, and then competitive immunoreaction with picloram antibody and target picloram was executed. The horseradish peroxidase (HRP)-labeled secondary antibody was applied for enzyme-amplified amperometric measurement. The electrodeposited Au nanoclusters built direct electrical contact and immobilization interface with protein molecules without postmodification and positioning. Under the optimal conditions, the linear range for picloram determination was 0.001-10 μg/ml with a correlation coefficient of 0.996. The detection and quantification limits were 5.0 × 10⁻⁴ and 0.0021 μg/ml, respectively. Picloram concentrations in peach and excess sludge supernatant extracts were tested by the proposed immunosensor, which exhibited good precision, sensitivity, selectivity, and storage stability.     

Mapping of qGL7-2,a grain length QTL on chromosome 7 of rice

A residual heterozygous line(RHL)carrying a heterozygous segment between two SSR loci RM11 and RM134 on the rice chromosome 7 was selected from a set of recombinant inbred lines from the cross D50(javanica)/HB277(indica).The former parent produces much longer grains than the latter.Selfed progenies of this selection were analyzed genotypically(SSRs)and phenotypically(grain length).Grain length was discontinuously variable in the mapping populations,allowing for the placement of this QTL qGL7-2 within a～4.8 cM interval defined by RM351 and RM234.A set of new markers within this region were developed,which narrowed the QTL to a 278 kb region defined by the markers Indel1 and RM21945.This region contains 49 predicted genes.The results also suggest that the novel allele for grain length will be used for the application of marker assisted selection for the improvement of grain length.     

Polymorphisms in PPARD, PPARG and APM1 associated with four types of Traditional Chinese Medicine constitutions

Based on the theory of constitution of Traditional Chinese Medicine (TCM), the human population is divided into nine constitutions including one balanced constitution (Normality) and eight unbalanced constitutions (Yang-deficiency, Yin-deficiency, Phlegm-wetness, Qi-deficiency, Wetness-heat, Blood stasis, Depressed constitution, and Inherited special constitution). Different constitutions have specific metabolic features and different susceptibility to certain diseases. However, whether a genetic basis accounts for such constitution classification is yet to be determined. Here we performed a genetic study to assess the association between genetic variations of metabolic genes including PPARD, PPARG and APM1 and the constitutions. A total of 233 individuals of the Han population in China were classified into four groups, Normality, Yang-deficiency, Yin-deficiency and Phlegm-wetness with whom 23 single nucleotide polymorphisms (SNPs) in the three genes were genotyped using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Biased distribution of PPARD rs2267669 and rs2076167, APM1 rs7627128 and rs1063539 in Yang-deficiency, PPARG Pro12Ala in Yin-deficiency and PPARD rs2076167, APM1 rs266729 and rs7627128 in Phlegm-wetness were observed. The frequencies of Haplotype13 (Hap13) of PPARG in Yin-deficiency, Hap25 of APM1 in Yang-deficiency and Hap2 of PPARD and Hap14 of PPARG in Phlegm-wetness, were significantly different from those in Normality, suggesting those might be group-associated haplotypes. These results suggested that single SNP and haplotypes of PPARD, PPARG and APM1 may underlie the genetic basis of the constitutions classified in TCM.