Directional and direct cloning strategy for high-throughput generation of recombinant baculoviruses

The baculovirus expression vector system (BEVS) has become one of the most widely used systems for routine protein expression. We have developed an improved strategy to clone foreign genes directionally and directly into the baculovirus genome vector via a one-step procedure to generate recombinant viruses in a week. In this work, we constructed a host strain Escherichia coli DH10BacHB1.1, which contains the modified baculovirus shuttle genome vector pHBMBacmid1.1 for the cloning vector. The treated PCR products of foreign genes were ligated with the Bsu36I-digested vector. Then Spodoptera frugiperda (Sf9) cells were transfected directly with the ligation mixture. Using this method, the DsRed fluorescence protein and mannanase genes have been cloned in the baculovirus genome and expressed in the Sf9 cells. This strategy not only provides a means for high-throughput construction of recombinant baculoviruses, but also offers an idea of constructing other large plasmids and DNA virus-based expression vectors.     

A rice glutamate receptor-like gene is critical for the division and survival of individual cells in the root apical meristem

Glu receptors are known to function as Glu-activated ion channels that mediate mostly excitatory neurotransmission in animals. Glu receptor-like genes have also been reported in higher plants, although their function is largely unknown. We have identified a rice (Oryza sativa) Glu receptor-like gene, designated GLR3.1, in which mutation by T-DNA insertion caused a short-root mutant phenotype. Histology and DNA synthesis analyses revealed that the mutant root meristematic activity is distorted and is accompanied by enhanced programmed cell death. Our results supply genetic evidence that a plant Glu receptor-like gene, rice GLR3.1, is essential for the maintenance of cell division and individual cell survival in the root apical meristem at the early seedling stage.     

HCC-associated protein HCAP1, a variant of GEMIN4, interacts with zinc-finger proteins

The gene HCAP1 (HCC-associated Protein 1), one variant of GEMIN4, has been mapped in a minimum LOH region on chromosome 17p13.3 and encodes a 1047-amino acid protein. Function predictions based on the amino acid sequence of protein HCAP1 revealed it to contain one helix-loop-helix motif and one leucine zipper domain. Using yeast two-hybrid screening, five zinc-finger proteins were identified as HCAP1-interacting proteins. Among them, NDP52 (nuclear dot protein 52) appeared most frequently in positive clones and was the most strongly interacting protein. Then, the interaction between HCAP1 and NDP52 was confirmed by GST pull-down assay and a coimmunoprecipitation experiment. Moreover, an immunofluorescent staining assay indicated that NDP52 colocalizes with HCAP1 in the cytoplasm. By deletion analysis, the leucine zipper domain of HCAP1 and the zinc finger domain of NDP52 were identified as important regions responsible for the interaction.     

A new human gene hNTKL-BP1 interacts with hPirh2

NTKL (N-terminal kinase-like protein) encodes an evolutionarily conserved kinase-like protein and is mapped around chromosomal breakpoints found in several carcinomas, suggesting that NTKL dysfunction may be involved in carcinogenesis. Recently, we identified a novel mouse gene, mNTKL-BP1 (NTKL-binding protein 1), encoding a protein interacting with NTKL. For further study, a new human gene, hNTKL-BP1, which is highly homologous with mNTKL-BP1, was used as bait in yeast two-hybrid system. hPirh2 (human p53-induced RING-H2 protein) was identified as hNTKL-BP1 interacting protein. The specific interaction of two proteins was confirmed by pull-down assay in vitro and co-immunoprecipitation in vivo. Moreover, an immunofluorescent staining assay showed that hNTKL-BP1 colocalizes with hPirh2 in SMMC 7721 cells. It will stimulate further investigation into whether hNTKL-BP1 is the substrate of hPirh2 or interaction of hNTKL-BP1 with hPirh2 enhances or represses the ubiquitin-protein ligase activity of hPirh2.     

Enhancement of 1,3-propanediol Production by <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis> with Fumarate Addition

Addition of 5 mm fumarate to cultures of Klebsiella pneumoniae enhanced the rate of glycerol consumption and the production of 1,3-propanediol (PDO). Compared to the control, the activity of glycerol dehydrogenase increased by 35, 33 and 46%, the activity of glycerol dehydratase increased by 160, 210 and 115%, and the activity of 1,3-propanediol oxidoreductase increased by 25, 39 and 85% when, respectively, 5, 15 and 25 mm fumarate were provided. At the same time, the ratio of NAD⁺ to NADH decreased by 20, 23 and 29%. Using a 5 l bioreactor with 5 mM fumarate addition, the specific rate of glycerol consumption and the productivity of PDO was 30 mmol/l h and 17 mmol/l h, respectively, both increased by 35% over the control. Revisions requested 15 July 2005; Revisions received 30 August 2005     

Brittle Culm1, a COBRA-Like Protein,Functions in Cellulose Assembly through Binding Cellulose Microfibrils

Cellulose represents the most abundant biopolymer in nature and has great economic importance. Cellulose chains pack laterally into crystalline forms, stacking into a complicated crystallographic structure. However, the mechanism of cellulose crystallization is poorly understood. Here, via functional characterization, we report that Brittle Culm1 (BC1), a COBRA-like protein in rice, modifies cellulose crystallinity. BC1 was demonstrated to be a glycosylphosphatidylinositol (GPI) anchored protein and can be released into cell walls by removal of the GPI anchor. BC1 possesses a carbohydrate-binding module (CBM) at its N-terminus. In vitro binding assays showed that this CBM interacts specifically with crystalline cellulose, and several aromatic residues in this domain are essential for binding. It was further demonstrated that cell wall-localized BC1 via the CBM and GPI anchor is one functional form of BC1. X-ray diffraction (XRD) assays revealed that mutations in BC1 and knockdown of BC1 expression decrease the crystallite width of cellulose; overexpression of BC1 and the CBM-mutated BC1s caused varied crystallinity with results that were consistent with the in vitro binding assay. Moreover, interaction between the CBM and cellulose microfibrils was largely repressed when the cell wall residues were pre-stained with two cellulose dyes. Treating wild-type and bc1 seedlings with the dyes resulted in insensitive root growth responses in bc1 plants. Combined with the evidence that BC1 and three secondary wall cellulose synthases (CESAs) function in different steps of cellulose production as revealed by genetic analysis, we conclude that BC1 modulates cellulose assembly by interacting with cellulose and affecting microfibril crystallinity.     

Effect of soil microorganisms and labile C availability on soil respiration in response to litter inputs in forest ecosystems: A meta‐analysis

Litter inputs can influence soil respiration directly through labile C availability and, indirectly, through the activity of soil microorganisms and modifications in soil microclimate; however, their relative contributions and the magnitude of any effect remain poorly understood. We synthesized 66 recently published papers on forest ecosystems using a meta‐analysis approach to investigate the effect of litter inputs on soil respiration and the underlying mechanisms involved. Our results showed that litter inputs had a strong positive impact on soil respiration, labile C availability, and the abundance of soil microorganisms, with less of an impact related to soil moisture and temperature. Overall, soil respiration was increased by 36% and 55%, respectively, in response to natural and doubled litter inputs. The increase in soil respiration induced by litter inputs showed a tendency for coniferous forests (50.7%)> broad‐leaved forests (41.3%)> mixed forests (31.9%). This stimulation effect also depended on stand age with 30‐ to 100‐year‐old forests (53.3%) and ≥100‐year‐old forests (50.2%) both 1.5 times larger than ≤30‐year‐old forests (34.5%). Soil microbial biomass carbon and soil dissolved organic carbon increased by 21.0%‐33.6% and 60.3%‐87.7%, respectively, in response to natural and doubled litter inputs, while soil respiration increased linearly with corresponding increases in soil microbial biomass carbon and soil dissolved organic carbon. Natural and doubled litter inputs increased the total phospholipid fatty acid (PLFA) content by 6.6% and 19.7%, respectively, but decreased the fungal/bacterial PLFA ratio by 26.9% and 18.7%, respectively. Soil respiration also increased linearly with increases in total PLFA and decreased linearly with decreases in the fungal/bacterial PLFA ratio. The contribution of litter inputs to an increase in soil respiration showed a trend of total PLFA > fungal/bacterial PLFA ratio > soil dissolved organic carbon > soil microbial biomass carbon. Therefore, in addition to forest type and stand age, labile C availability and soil microorganisms are also important factors that influence soil respiration in response to litter inputs, with soil microorganisms being more important than labile C availability.     

Neighboring trees regulate the root‐associated pathogenic fungi on the host plant in a subtropical forest

Root‐associated fungi and host‐specific pathogens are major determinants of species coexistence in forests. Phylogenetically related neighboring trees can strongly affect the fungal community structure of the host plant, which, in turn, will affect the ecological processes. Unfortunately, our understanding of the factors influencing fungal community composition in forests is still limited. In particular, investigation of the relationship between the phytopathogenic fungal community and neighboring trees is incomplete. In the current study, we tested the host specificity of members of the root‐associated fungal community collected from seven tree species and determined the influence of neighboring trees and habitat variation on the composition of the phytopathogenic fungal community of the focal plant in a subtropical evergreen forest. Using high‐throughput sequencing data with respect to the internal transcribed spacer (ITS) region, we characterized the community composition of the root‐associated fungi and found significant differences with respect to fungal groups among the seven tree species. The density of conspecific neighboring trees had a significantly positive influence on the relative abundance of phytopathogens, especially host‐specific pathogens, while the heterospecific neighbor density had a significant negative impact on the species richness of host‐specific pathogens, as well as phytopathogens. Our work provides evidence that the root‐associated phytopathogenic fungi of a host plant depend greatly on the tree neighbors of the host plant.