应用随机PCR方法鉴定一株真养产碱杆菌

采用随机引物PCR技术从新建细胞培养室空气中获得一段长414bp的片段,通过克隆测序及序列分析,结果表明所测序列与真养产碱杆菌主要参考菌株的同源性分别高达79%-83%,由其推导的氨基酸序列与真养产碱杆菌主要参考菌株的同源性高达86.4%-89.1%,从而确定所分离菌株为真养产碱杆菌。     

Genetic and transgenic perturbations of carbon reserve production in Arabidopsis seeds reveal metabolic interactions of biochemical pathways

The biosynthesis of seed oil and starch both depend on the supply of carbon from the maternal plant. The biochemical interactions between these two pathways are not fully understood. In the Arabidopsis mutant shrunken seed 1 (sse1)/pex16, a reduced rate of fatty acid synthesis leads to starch accumulation. To further understand the metabolic impact of the decrease in oil synthesis, we compared soluble metabolites in sse1 and wild type (WT) seeds. Sugars, sugar phosphates, alcohols, pyruvate, and many other organic acids accumulated in sse1 seeds as a likely consequence of the reduced carbon demand for lipid synthesis. The enlarged pool size of hexose-P, the metabolites at the crossroad of sugar metabolism, glycolysis, and starch synthesis, was likely a direct cause of the increased flow into starch. Downstream of glycolysis, more carbon entered the TCA cycle as an alternative to the fatty acid pathway, causing the total amount of TCA cycle intermediates to rise while moving the steady state of the cycle away from fumarate. To convert the excess carbon metabolites into starch, we introduced the Escherichia coli starch synthetic enzyme ADP-glucose pyrophosphorylase (AGPase) into sse1 seeds. Expression of AGPase enhanced net starch biosynthesis in the mutant, resulting in starch levels that reached 37% of seed weight. However, further increases above this level were not achieved and most of the carbon intermediates remained high in comparison with the WT, indicating that additional mechanisms limit starch deposition in Arabidopsis seeds.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

扶桑绵粉蚧组织蛋白酶B基因的克隆、原核表达和不同发育阶段表达分析

昆虫组织蛋白酶B在昆虫代谢过程中发挥重要作用。本研究利用RACE技术克隆了扶桑绵粉蚧Phenacoccus solenopsis Tinsley组织蛋白酶B基因的开放阅读框（ORF）序列, 命名为PsCb （GenBank登录号: JQ727999）。生物信息学分析表明, 该基因的开放阅读框包含927 bp的片段, 编码308个氨基酸。多序列比对表明, 该基因编码的蛋白在N端变异较大, 在C端保守性高。组织蛋白酶B基因的系统进化树结果表明扶桑绵粉蚧组织蛋白酶独自成为一支。原核表达电泳检测到一条大约35 kDa的目的条带, 与预测的蛋白分子量相符。组织蛋白酶B基因在扶桑绵粉蚧各个虫态均有表达, 卵期表达量相对较低, 2龄若虫期达到最高峰, 然后下降。本研究为进一步研究该基因的功能并开发出组织蛋白酶抑制剂, 从而研制出扶桑绵粉蚧杀卵剂和胚胎发育抑制剂等提供理论依据。     

PA2800 Plays an Important Role in Both Antibiotic Susceptibility and Virulence in Pseudomonas aeruginosa

Pseudomonas aeruginosa is an important human pathogen which causes a variety of infections. P. aeruginosa infections are often difficult to treat due to the pathogen’s resistance to many antibiotics. Previously, it has been reported that a transposon insertion mutant in gene PA2800 of P. aeruginosa PAO1 was more sensitive to tetracycline and ciprofloxacin. Further characterization of this gene, a vacJ homolog, in this study indicated that this gene plays an important role in both antibiotic susceptibility and virulence in P. aeruginosa. The role of PA2800 in antibiotic susceptibility probably signifies its involvement in maintaining outer membrane stability, similar to the role of vacJ in E. coli and Shigella flexneri. However, in contrast to vacJ in other bacteria, PA2800 also affects antibiotic susceptibility by affecting the expression of oprH in P. aeruginosa. As shown by in vivo studies using a Drosophila melanogaster infection model, significantly increased virulence was observed in the PA2800 mutant when compared to the wild type, and such a difference is likely a result of disrupted outer membrane stability and altered expression of znuA in the mutant. The role of PA2800 or vacJ in antibiotic susceptibility and pathogenicity seems to be unique in P. aeruginosa in which it affects both outer membrane stability as well as gene expression.     

GB Virus C Envelope Protein E2 Inhibits TCR-Induced IL-2 Production and Alters IL-2-Signaling Pathways

GB virus type C (GBV-C) viremia is associated with reduced CD4(+) T cell expansion following IL-2 therapy and with a reduction in T cell activation in HIV-infected individuals. The mechanism(s) by which GBV-C might alter T cell activation or IL-2 signaling have not been studied. In this study, we assess IL-2 release, IL-2R expression, IL-2 signaling, and cell proliferation in tet-off Jurkat cells expressing the GBV-C envelope glycoprotein (E2) following activation through the TCR. TCR activation was induced by incubation in anti-CD3/CD28 Abs. IL-2 release was measured by ELISA, STAT5 phosphorylation was assessed by immunoblot, and IL-2Rα (CD25) expression and cell proliferation were determined by flow cytometry. IL-2 and IL-2Rα steady-state mRNA levels were measured by real-time PCR. GBV-C E2 expression significantly inhibited IL-2 release, CD25 expression, STAT5 phosphorylation, and cellular proliferation in Jurkat cells following activation through the TCR compared with control cell lines. Reducing E2 expression by doxycycline reversed the inhibitory effects observed in the E2-expressing cells. The N-terminal 219 aa of E2 was sufficient to inhibit IL-2 signaling. Addition of purified recombinant GBV-C E2 protein to primary human CD4(+) and CD8(+) T cells inhibited TCR activation-induced IL-2 release and upregulation of IL-2Rα expression. These data provide evidence that the GBV-C E2 protein may contribute to the block in CD4(+) T cell expansion following IL-2 therapy in HIV-infected individuals. Furthermore, the effects of GBV-C on IL-2 and IL-2-signaling pathways may contribute to the reduction in chronic immune activation observed in GBV-C/HIV-coinfected individuals.     

Variability and adaptability of Miscanthus species evaluated for energy crop domestication

A growing body of evidence indicates that second‐generation energy crops can play an important role in the development of renewable energy and the mitigation of climate change. However, dedicated energy crops have yet to be domesticated in order to fully realize their productive potential under unfavorable soil and climatic conditions. To explore the possibility of domesticating Miscanthus crops in northern China where marginal and degraded land is abundant, we conducted common garden experiments at multiple locations to evaluate variation and adaptation of three Miscanthus species that are likely to serve as the wild progenitors of the energy crops. A total of 93 populations of Miscanthus sinensis, Miscanthus sacchariflorus, and Miscanthus lutarioriparius were collected across their natural distributional ranges in China and grown in three locations that represent temperate grassland with cold winter, the semiarid Loess Plateau, and relatively warm and wet central China. Evaluated with growth traits such as plant height, tiller number, tiller diameter, and flowering time, the Miscanthus species showed high levels of genetic variation within and between species. There were significant site × population interactions for almost all traits of M. sacchariflorus and M. sinensis, but not M. lutarioriparius. The northern populations of M. sacchariflorus had the highest establishment rates at the most northern site owing to their strong cold tolerance. An endemic species in central China, M. lutarioriparius, produced not only the highest biomass of the three species but also higher biomass at the Loess Plateau than the southern site near its native habitats. These results demonstrated that the wild species harbored a high level of genetic variation underlying traits important for crop establishment and production at sites that are colder and drier than their native habitats. The natural variation and adaptive plasticity found in the Miscanthus species indicated that they could provide valuable resources for the development of second‐generation energy crops.     

Engineering a homobutanol fermentation pathway in Escherichia coli EG03

A homobutanol fermentation pathway was engineered in a derivative of Escherichia coli B (glucose [glycolysis] => 2 pyruvate + 2 NADH; pyruvate [pyruvate dehydrogenase] => acetyl-CoA + NADH; 2 acetyl-CoA [butanol pathway enzymes] + 4 NADH => butanol; summary stoichiometry: glucose => butanol). Initially, the native fermentation pathways were eliminated from E. coli B by deleting the genes encoding for lactate dehydrogenase (ldhA), acetate kinase (ackA), fumarate reductase (frdABCD), pyruvate formate lyase (pflB), and alcohol dehydrogenase (adhE), and the pyruvate dehydrogenase complex (aceEF-lpd) was anaerobically expressed through promoter replacement. The resulting strain, E. coli EG03 (ΔfrdABCD ΔldhA ΔackA ΔpflB Δ adhE ΔpdhR ::pflBp6-aceEF-lpd ΔmgsA), could generate 4 NADH for every glucose oxidized to two acetyl-CoA through glycolysis and the pyruvate dehydrogenase complex. However, EG03 lost its ability for anaerobic growth due to the lack of NADH oxidation pathways. When the butanol pathway genes that encode for acetyl-CoA acetyltransferase (thiL), 3-hydroxybutyryl-CoA dehydrogenase (hbd), crotonase (crt), butyryl-CoA dehydrogenase (bcd, etfA, etfB), and butyraldehyde dehydrogenase (adheII) were cloned from Clostridium acetobutylicum ATCC 824, and expressed in E. coli EG03, a balanced NADH oxidation pathway was established for homobutanol fermentation (glucose => 4 NADH + 2 acetyl-CoA => butanol). This strain was able to convert glucose to butanol (1,254 mg l(-1)) under anaerobic condition.     

FgVELB is associated with vegetative differentiation, secondary metabolism and virulence in Fusarium graminearum

The velvet complex containing VeA, VelB and LaeA has been showed to play critical roles in the regulation of secondary metabolism and diverse cellular processes in Aspergillus spp. In this study, we identified FgVelB, a homolog of Aspergillus nidulans VelB, from Fusarium graminearum using the BLASTP program. Disruption of FgVELB gene led to several phenotypic defects, including suppression of aerial hyphae formation, reduced hyphal hydrophobicity and highly increased conidiation. The mutant showed increased resistance to osmotic stress and cell wall-damaging agents, which may be related to a high level of glycerol accumulation in the mutant. Additionally, the mutant exhibited increased sensitivity to the phenylpyrrole fungicide fludioxonil. Ultrastructural and histochemical analyses revealed that conidia of FgVELB deletion mutant contained numerous lipid droplets. Pathogenicity assays showed FgVELB deletion mutant was impaired in virulence on flowering wheat head, which is consistent with the observation that FgVelB is involved in the regulation of deoxynivalenol biosynthesis in F. graminearum. All of the defects were restored by genetic complementation of the mutant with wild-type FgVELB gene. Yeast two hybrid assays showed that FgVelB does not interact with FgVeA. Taken together, results of this study indicated that FgVelB plays a critical role in the regulation of various cellular processes in F. graminearum.