Mapping of genetic modifiers of <Emphasis Type="Italic">Plcd1</Emphasis> in scant hair mice (<Emphasis Type="Italic">snthr</Emphasis><Superscript><Emphasis Type="Italic">1Bao</Emphasis></Superscript>)

The scant hair mutant mouse (locus symbol: snthr ^1Bao ) is a recessive mutation that originated in an ethylnitrosourea chemical carcinogenesis study using the DBA/2J inbred strain. The gene responsible for the mutation was previously determined to be phospholipase C, delta 1 (Plcd1; mutant allele symbol Plcd1 ^snthr1Bao ). To map the modifiers of Plcd1, an intercross (DBA/2J-snthr ^1Bao /snthr ^1Bao × C57BL/6J+/+) was conducted. The F2 mutant progeny exhibited a variety of alopecia phenotypes; all F2 mutants (n=507) were classified into 3 groups (mild, moderate, and severe alopecia) and genotyped based on 96 microsatellites. A major QTL was identified on mouse chromosome (mChr) 15 at 12 cM with an LOD score greater than 7 (P < 0.0001). Three minor QTLs were detected on mChr 2, 5, and 7 at 40, 84 and 48 cM, respectively. The QTLs on mChr 7 and 15 were associated with minor alopecia while the QTLs on mChr 2 and 5 were associated with moderate to severe alopecia. No antagonistic or synergistic effects among or between the 4 QTLs were found. Integrating the functions of the 4 potential regulatory QTLs and mutant Plcd1 ^snthr1Bao , we found that these QTLs might contribute to variations of scant hair severity by altering the Ca²⁺ signal pathways in mouse skin.     

Mechanism of <Emphasis Type="Italic">Aulacophora femoralis chinensis</Emphasis> Weise feeding behavior and chemical response of host <Emphasis Type="Italic">Cucumis sativus</Emphasis> L.

One of the frontiers in ecological study is the inter-action between plant and insect mediated by secondary metabolites[1]. Recent studies on the chemical interactions in the food chain of crop-pest-natural enemy showed that insects have olfactory response to the volatile al-lelochemicals released from plants[2,3]. Insects could in-teract with plants through their vision, olfaction and taste, and taste could be the most direct response mechanism of their herbivory. Some beetles (Acalymma, Aul…     

Preparation of high selectivity silicalite-1 membranes by two-step <Emphasis Type="Italic">in situ</Emphasis> hydrothermal synthesis

High selectivity silicalite-1 membranes were synthesized on silica tubes by in situ hydrothermal synthesis. Using a two-step synthesis, a membrane with a separation factor of 99 was prepared for separating an ethanol/water mixture at 60°C. The average (n = 4) flux and separation factor of the membranes were 0.47 kg m-2 h-1 and 89, respectively. The membranes exhibited high reproducibility, and the relative standard deviation of the average (n = 4) flux and separation factor were only 5.3% and 9.2%, respectively. These results suggest that silica is a suitable support for synthesis of high-performance silicalite-1 membranes.     

Comparative proteomics analysis of <Emphasis Type="Italic">OsNAS1</Emphasis> transgenic <Emphasis Type="Italic">Brassica napus</Emphasis> under salt stress

Salt stress is one of the major abiotic stresses in agricultural plants worldwide. We used proteomics to analyze the differential expression of proteins in transgenic OsNAS1 and non-transformant Brassica napus treated with 20 mmol/L Na₂CO₃. Total protein from the leaves was extracted and separated through a high-resolution and highly repetitive two-dimensional electrophoresis (2-DE) technology system. Twelve protein spots were reproducibly observed to be upregulated by more than 2-fold between transgenic and non-transformant B. napus. These 12 spots were digested in-gel with trypsin and characterized by matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) to obtain the peptide mass fingerprints. Protein database searching revealed that 5 of these proteins are involved in salt tolerance: dehydrogenase, glutathione S-transferase, peroxidase, 20S proteasome beta subunit, and ribulose-1,5-bisphosphate carboxylase/oxygenase. The potential functions of these identified proteins in substance and energy metabolism, stress tolerance, protein degradation, and cell defense are discussed. The salt tolerance of the transgenic rapeseed was significantly improved by the introduction of the OsNAS1 gene from Brazilian upland rice of Oryza sativa (cv. IAPAR 9).     

<Emphasis Type="Italic">In situ</Emphasis> FTIR investigation on phase transformations in BN nanoparticles

Fourier transformation infrared spectrum （FTIR） was employed to investigate phase transformation of nanoparticle BN, containing eBN, rBN, wBN and cBN, in which eBN and rBN are the main components. We found that part of eBN was converted into rBN at 200--350℃. At and above 400℃, eBN transformed into wBN, and some of wBN were converted into cBN simultaneously. This conclusion is supported by the results from high- resolution transmission electron microscopic （HRTEM） and Raman spectroscopic measurements. In this work, the converting processes between different BN phases were continuously monitored by in situ FTIR spectroscopy. The results will be helpful for synthesis of cBN at moderate conditions.     

Optimization of butanol production from corn straw hydrolysate by <Emphasis Type="Italic">Clostridium acetobutylicum</Emphasis> using response surface method

Butanol is a new kind of very potential biofuels.Enzymatic hydrolysis of corn stalk was utilized in this study to produce butanol by Clostridium acetobutylicum CICC 8008.Plackett-Burman (P-B) design and Central Composite Design (CCD) were adopted to screen crucial factors during fermentation as well as the optimization of experimental conditions.The result demonstrated that among the seven factors,namely,Yeast extract,(NH 4) 2 SO 4,KH 2 PO 4,MgSO 4,FeSO 4,CuSO 4 and CaCO 3,only CaCO 3 was selected as the most critical factor.The optimization experiment results for CaCO 3 usage,temperature and reaction time by CCD were determined to be 5.04 g/L,35°C and 70 h,respectively.A corresponding mathematical model was established to predict the fermentation experiment and maximum butanol yield of 6.57 g/L was acquired.The result of verification experiment under the optimum conditions showed that 6.20 g/L was the maximum butanol yield.This demonstrated that statistical method was a powerful tool for the optimization of butanol production from enzymatic hydrolysis of corn stalk.     

Asian origin for Polystichum （Dryopteridaceae） based on rbcL sequences

Chloroplast rbcL sequences of 60 species of Polystichum sensu lato (s.l.), including 23 new sequences from southwest China, were used to assess the phylogenetic relationships within the genus. On the basis of estimated evolution rate of rbcL gene and the genetic distance data that passed relative-rate tests, we further estimated the divergence times between some clades of the genus. The phylogenetic relationships were inferred using the neighbor-joining and maximum-parsimony methods, both methods producing trees with completely congruent topology. These trees reveal that all species of Polystichum s.l. in this study (including Cyrtomium and Cyrtomidictyum) form a monophyletic group. The basal split in Polystichum s.l. separates a clade with all Asian members from a clade containing other species from all over the world. The phylogenetic and divergence time estimation results lead us to suggest that Polystichum s.l. originated in Asia in the late Late Cretacous (≈76 Ma) and migrated into other places in the world in early Eocene(≈46 Ma).     

Deletion analysis of oligomycin PKS genes （olmA） in Streptomyces arermitilis

Gene deletion vector pXL05(pKC1139::△olmA1 △olmA4) was used to disrupt oligomycin PKS encoding genes (olmA ) in Streptomyces avermitilis CZ8-73, the producer of anthelmintic avermectins B and the cell growth inhibitor oligomycin, olmA gene cluster in the chromosome was displaced by deletion allele on the plasmid via double crossover. Four of disruptants were confirmed by Southern blotting. Shaking flask experiments and HPLC analyses showed that the four mutants no longer produced the toxic oligomycin, but only made four components of avermectins B, which were avermectin Bla, Blb, B2a, B2b. The yields of avermectins B in these mutants were separately equal to those in CZ8-73. This revealed that olmA genes deletion did not affect the biosynthesis of avermectins. The deletion mutants were proved to be genetically stable, and thus might be promising strains in industrial production of avermectins B.     

Transmembrane structure and function of dctPQM encoding C4－dicarboxylate transport proteins from nitrogen－fixing P. stutzeri A1501

C4-dicarboxylate transport proteins of diazotroph Pseudomonas stutzeri were encoded by dctPQM genes. Nucleotide sequence analysis indicated that dctP, dctQ,and dctM grouped together. Its nucleotide and amino acid sequence shared high homology with that of dctP gene encoding periplasmic C4-dicarboxylate-binding protein and dctQM genes encoding C4-dicarboxylate transport proteins from the free-living nitrogen-fixing Aotobacter vinelandii.Structural analysis showed that DctP of P. stutzeri did not include membrane-spanning regions, and DctQ and DctM contained 5 and 12 transmembrane segments, respectively.The fragment containing the complete dctPQM genes was cloned into the Tn5 transposon region of suicide mobilization plasmid pSZ2L The resultant plasmid was named pSZY6.By triparental mating, Tn5 transposon carrying the dctPQM genes inserted into the genome of the wild type strain A1501,randomly. The recombinant strain A-142 which harboured an extra copy of dctPQM genes was constructed and identified by PCR amplification of npt II gene. When A-142 was grown in minimal medium with different concentrations (20,10 and 5mmol/L) of C4-dicarboxylates succinate, malate, or fumarate as the sole carbon source, the rate of nitrogen fixation assayed by acetylene reduction was significantly higher than that of the wild-type strain A150L This result was established that an extra copy of dctPQM genes could increase the activity of nitrogen fixation of P. stutzeri strain A1501.     

Ubi1 intron-mediated enhancement of the expression of Bt cry1Ah gene in transgenic maize （Zea mays L.）

The cry1Ah gene was one of novel insecticidal genes cloned from Bacillus thuringiensis isolate BT8. Two plant expression vectors containing cry1Ah gene were constructed. The first intron of maize ubiqutinl gene was inserted between the maize Ubiquitin promoter and cry1Ah gene in one of the plant expressing vectors （pUUOAH）. The two vectors were introduced into maize immature embryonic calli by microprojectile bombardment, and the reproductively plants were acquired. PCR and Southern blot analysis showed that foreign genes had been integrated into maize genome and inherited to the next generation stably. The ELISA assay to T1 and T2 generation plants showed that the expression of CrylAh protein in the construct containing the ubil intron （pUUOAH） was 20% higher than that of the intronless construct （pUOAH）. Bioassay results showed that the transgenic maize harboring cry1Ah gene had high resistance to the Asian corn borers and the insecticidal activity of the transgenic maize containing the ubil intron was higher than that of the intronless construct. These results indicated that the maize ubil intron can enhance the expression of the Bt cry1Ah gene in transgenic maize efficiently     

Rate constants for the reaction of ozone with <Emphasis Type="Italic">n</Emphasis>-butyl, <Emphasis Type="Italic">s</Emphasis>-butyl and <Emphasis Type="Italic">t</Emphasis>-butyl methyl sulfides

The rate constants for the ozone reactions with n-butyl methyl sulfide （n-BMS, CHaCH2CH2CH2SCH3）, sec-butyl methyl sulfide （s-BMS, CH3CH2（CH3）CHSCHa） and tert-butyl methyl sulfide （t-BMS, （CH3）3CSCH3） were measured using our smog chamber under supposedly pseudo-first-order conditions at 30002 K and 760 Torr. The experimental determined rate constants for n-butyl, s-butyl and t-butyl methyl sulfide are （1.23 ± 0.06）×10-19, （5.08 ± 0.19）×10-20 and （2.26 ± 0.14）×10-20 cm3 molecule-1· s-1, respectively. The reactivity-structure relationship of the reactions was discussed and used to illustrate the mechanism of the ozone reaction with thioethers. The results enrich the kinetics data of atmospheric chemistry.     

Agrobacterium tumefaciens-mediated Transformation of CryⅠA(b) gene to Trichoderma harzianum

In this study, Cry ⅠA(b) gene was successfully transferred into the biocontrol fungus Trichoderma harzianum with an efficiency of 60-180 transformants per 10^6 spores by using Agrobacterium tumefaciens-mediated transformation. Putative transformants were analyzed to test the presence of Cry ⅠA(b) gene by Southern blot. Most transformants contained a single T-DNA copy. RT-PCR analysis showed that the Cry ⅠA(b) gene was transcribed. Antifungal activities and insecticidal activities of the transformants were examined. There was no obvious difference in antifungal activities between the transformants and their wild strains. The modified mortalities of the transformants T1 and T2 were 69.57% and 91.30%, respectively. The tranformation system mediated by A. tumefaciens proved to be a powerful tool for the filamentous fungi transformation and functional genomic study with its high transformation frequency, simplicity of T-DNA integration, and genetic stability of transformants.     

Analysis of type II toxin-antitoxin genes <Emphasis Type="Italic">all</Emphasis> 3211-<Emphasis Type="Italic">asl</Emphasis> 3212 in <Emphasis Type="Italic">Anabaena</Emphasis> PCC 7120

The type II toxin-antitoxin genes are responsible for the phenotypic switch to a quasi-dormant state that enables cell survival under stresses, a similar function to heterocyst of cyanobacteria. In this paper, we particularly study the role of gene pair all3211-asl3212 under Spectinomycin stress to reveal how the type II toxin-antitoxin involved in environmental stress responses. Bioinformatics prediction shows that toxin protein gene All3211 is homologous to MazF, a member of mazEF family that encoding nucleases. We clone gene all3211-asl3212 into expression vectors to identify its molecular characteristics. Deletion mutant strains of all3211-asl3212 are selected in a tri-parental mating screen. Phenotype comparisons of mutant and wild type reveals no difference of single-deletion-mutants in pigment integrity, the sensitivity to antibiotics, and heterocyst formation. The results show that deletion mutation of single TAS gene pair all3211-asl3212 results in limited effects on the cellular growth of PCC 7120. Thus, we suggest that dosage compensating might be provided from redundant genes or bypass pathways to offset obvious phenotypic differences.     

Engineering <Emphasis Type="Italic">Deinococcus radiodurans</Emphasis> into biosensor to monitor radioactivity and genotoxicity in environment

Based on a genetically modified radioresistant bacteria Deinococcus radiodurans, we constructed a real time whole cell biosensor to monitor radioactivity and genotoxicity in highly radioactive environment. The enhanced green fluorescence protein （eGFP） was fused to the promoter of the crucial DNA damage-inducible recA gene from D. radiodurans, and the consequent DNA fragment （PrecA-egfp） carried by plasmid was introduced into D. radiodurans R1 strain to obtain the biosensor strain DRG300. This engineered strain can express eGFP protein and generate fluorescence in induction of the recA gene promoter. Based on the correlation between fluorescence intensity and protein expression level in live D. radiodurans cells, we discovered that the fluorescence induction of strain DRG300 responds in a remarkable dose-dependent manner when treated with DNA damage sources such as gamma radiation and mitomycin C. It is encouraging to find the widely detective range and high sensitivity of this reconstructed strain comparing with other whole cell biosensors in former reports. These results suggest that the strain DRG300 is a potential whole cell biosensor to construct a detective system to monitor the biological hazards of radioactive and toxic pollutants in environment in real time.     

Adsorption and reduction of palladium (Pd<Superscript>2+</Superscript>) by <Emphasis Type="Italic">Bacillus licheniformis</Emphasis> R08

Preliminary study on the mechanism of Pd²⁺ biosorption by resting cells of Bacillus licheniformis R08 biomass has been carried out by means of chemical kinetics and AAS, TEM, XRD and FTIR methods. The results showed that at 30℃ and pH 3.5, when dry R08 biomass powder (800 mg/L) was mixed with Pd²⁺ (100 mg/L) for 45 min, the rate constant k of biosorption of Pd²⁺ attained a maximum of 5.97×10^-2 min^-1 and the half life period of the reaction reached 12 min. The part of Pd²⁺ adsorbed by R08 biomass was reduced to elemental, cell-bound Pd⁰ at the same condition. The cell wall of R08 biomass was the primary location for accumulating Pd²⁺ , and aldoses, i. e. hydrolysate of a part of polysaccharides on the peptidoglycan layer in the acidic medium, serving as the electron donor, in situ reduced the Pd²⁺ to Pd⁰.     

The composition and distribution of metal clusters in the MoFe protein from a nifZ deletion strain （DJ 194） of Azotobacter vinelandii

Through the anaerobic chromatography on the columns of DEAE 52, Q-Sepharose and Sephacryl S-200, a nitrogenase MoFe protein (△nifZ Av1) was obtained from a nifZ deleted mutant of Azotobacter vinelandii (stain DJ194).The results of Western blotting after anoxic native electrophoresis and SDS-PAGE showed that △nifZ Av1 was similar to wild type MoFe protein (OP Av1) at the electrophoretic mobility, molecular weight and subunit composition. Furthermore, △nifZ Avl was also similar to OP Av1 at the molybdenum content, EPR signal (g≈4.3, 3.65 and 2.01), and the molar extinction coefficient (△ε) of circular dichroism (CD)at 660 nm region. All of these indicated that, besides having the same α2β2 composition as OP Av1, the △nifZ Av1 also contained equal amount of reductive FeMoco in the spin state of S=3/2 to OP Av1. However, the iron content and substrate (C2H2, H^ and N2)-reduction activity of △nifZ Av1 were 74% and 46%-50% of those of OP Av1, respectively. Furthermore, the △ε at around 450 nm, which reflects P-cluster in Av1, was obviously lower than that of OP Av1. It suggested that the difference between △nifZ Avl and OP Av1 resulted from P-cluster rather than FeMoco, and from the half number of P-cluster in △nifZ Av1, but the composition or redoxstate of P-cluster in △nifZ Av1 were not changed. Thus it could propose that △nifZ Av1 is composed of two different αβsubunit pairs. One is a FeMoco-and P-cluster-containing pair, and the other is a P-cluster-deficient but FeMoco-containing pair. Since the deletion of nifZ gene leads to the deficiency of only one of two P-clusters in a α2β2 tetramer, the assembly of P-cluster may not simply depend on one gene product, and so a possible mechanism of NifZ is supposed here.