Molecular cloning, expression and biochemical property analysis of AtKP1, a kinesin gene from Arabidopsis thaliana

Kinesins are common in a variety of eukaryotic cells with diverse functions. A cDNA encoding a member of the Kinesin-14B subfamily is obtained using 3＇-RACE technology and named AtKP1 （for Arabidopsis kinesin protein 1）. This cDNA has a maximum open reading frame of 3.3 kb encoding a polypeptide of 1087 aa. Protein domain analysis shows that AtKP1 contains the motor domain and the calponin homology domain in the central and amino-terminal regions, respectively. The carboxyl-terminal region with 202 aa residues is diverse from other known kinesins. Northern blot analysis shows that AtKP1 is widely expressed at a higher level in seedlings than in mature plants. 2808 bp of the AtKP1 promoter region is cloned and fused to GUS. GUS expression driven by the AtKP1 promoter region shows that AtKP1 is mainly expressed in vasculature of young organs and young leaf trichomes, indicating that AtKP1 may participate in the differentiation or development of Arabidopsis thaliana vascular bundles and trichomes. A truncated AtKP1 protein containing the putative motor domain is expressed in E. coil and affinity-purified. In vitro characterizations indicate that the polypeptide has nucleotide-dependent microtubule-binding ability and microtubule-stimulated ATPase activity.     

Effects of cadmium, 17β-estradiol and their interaction in the male Chinese loach （Misgurnus anguillicaudatus）

To assess the interaction effect between cadmium (Cd) and 17-estradiol (E2),male Chinese loaches (Misgurnus anguillicaudatus) were exposed to E2 (1 g/L) and Cd (50 or 500 g/L) alone and in combination using a semi-static waterborne exposure system.The effects of E2 on the accumulation and distribution of Cd,as well as the effects of Cd on vitellogenin (Vtg) synthesis induced by E2,were investigated.Cd mainly accumulated in the kidneys,liver,intestines,and gills,with little amounts in muscles,bones,and gonads.Co-exposure with E2 did not change the main targets for Cd.E2 could induce Vtg synthesis in male Chinese loaches,and co-exposure with 50 or 500 g/L Cd could inhibit the Vtg induced by 1 g/L E2.Compared with the results reported in the literature,it can be concluded that factors such as fish species,Cd dosage,and manner of exposure might make contributions to the interaction between Cd and E2.Our results also suggested that male Chinese loaches are susceptible to Cd,and can be recommended as a potential sentinel species to study the ecotoxicology of heavy metals.     

The permeability effect of microcystin-RR on <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Microcystins are a kind of cyclic hepatoxins produced by many species of cyanobacteria. Most previous work have been done on the toxic effects of microcystins on animals and plants. However, the reports about the effect of microcystins on microbial cells are very limited. In this work, the permeability of MC-RR on the cell outer membrane of Escherichia coli (E. coli) and Bacillus subtilis (B. subtilis) was discussed. The permeability effect of MC-RR on the cell outer membrane of E. coli and B. subtilis under different concentrations was demonstrated by a rapid and sustained reduction in the A₆₇₅ values of lysozyme-treated cells. The decrease of the absorbance values showed a time- and dose-effect. The extravasations of protein and carbonhydrate increased with the increment of the treated-concentration of MC-RR. The results showed that MC-RR could increase the permeability of cell outer membranes of E. coli and B. subtilis. The synergistic effects of MC-RR and lysozyme on bacteria indicated that MC-RR might play an ecological role in bacteria in combination with other substances in some aquatic environments.     

Transpositional behaviour of the Ds element in the Ac/Ds system in rice

Insertional mutagenesis based on maize Activator/Dissociator (Ac/Ds) transposons is becoming a ma- jor approach used to produce a saturated mutant collection in rice. In this research, Ds-T-DNA trans- formed homozygotes were crossed with Ac-T-DNA transformed homozygotes in order to establish an Ac/Ds transposon system in rice. The successive investigation of Ds transposition from F1 to F5 gen- erations indicated that the frequencies of germinal transposition increased over successive genera- tions and reached 54.2% in F3 generation. The Ds transposition pattern revealed that a Ds transposition induced an approximately 170-bp deletion of T-DNA sequence and another Ds transposition carried a 272-bp T-DNA sequence. Using thermal asymmetric interlaced PCR (TAIL-PCR), some flanking se- quences of the Ds element were amplified. Analyses of 17 Ds-flanking sequences showed that five Ds were inserted into gene regions. The Ds could transpose not only to the linked sites but also to the unlinked sites. The frequency of inter-chromosomal transposition of Ds was 33.3%.     

The R- and AR-indices： Complementing the h-index

Based on the foundation laid by the h-index we introduce and study the R- and AR-indices. These new indices eliminate some of the disadvantages of the h-index, especially when they are used in combina-tion with the h-index. The R-index measures the h-core’s citation intensity, while AR goes one step further and takes the age of publications into account. This allows for an index that can actually in-crease and decrease over time. We propose the pair (h, AR) as a meaningful indicator for research evaluation. We further prove a relation characterizing the h-index in the power law model.     

Asymmetric epoxidation of <Emphasis Type="Italic">α</Emphasis>,<Emphasis Type="Italic">β</Emphasis>-unsaturated ketones using <Emphasis Type="Italic">α</Emphasis>,<Emphasis Type="Italic">α</Emphasis>-diarylprolinols as catalysts

Asymmetric epoxidation of α,β-unsaturated ketones has been extensively studied and several important procedures have been developed in the last decade. This review addresses the most significant advances in asymmetric epoxidation of α,β-unsaturated ketones using proline-derived α,α-diarylprolinols as catalysts. Special attention has been paid to the enantioselective epoxidation of chalcones, α,β-unsaturated trifluoromethyl, trichloromethyl ketones and β,γ-unsaturated α-keto esters based on the reseach of our group.     

Osmotically stress-regulated the expression of glutathione peroxidase 3 in Arabidopsis

Gene expression of glutathione peroxidase 3 (ATGPX3) in response to osmotic stress was analyzed in Arabidopsis using ATGPX3 promoter-glucuronidase (GUS) transgenic plants. High levels of GUS ex- pression were detected under osmotic stress in ATGPX3 promoter-GUS transgenic plants. Compared with the wild type, the growth and development of ATGPX3 mutants (atgpx3-1) were more sensitive to mannitol. In addition, the expression of RD29A, ABI1, ABI2 and RbohD in atgpx3-1 was induced by ABA stress. These results suggest that ATGPX3 might be involved in the signal transduction of osmotic stress.     

A novel gene <Emphasis Type="Italic">R049</Emphasis> identified in uropathogenic <Emphasis Type="Italic">Escherichia coli</Emphasis> provides partial protection in mice from colonization

Uropathogenic Escherichia coli (UPEC) is the most common causative organism of human urinary tract infection (UTI). Several UPEC virulence factors have been identified, but more are yet to be found. We previously identified a novel 789-bp-long DNA fragment (named R049) in UPEC strain 132 using a suppressive subtractive hybridization technique. In the present study, we used genome walking to elongate the sequence of this fragment to obtain the whole gene sequence and examined the role of this gene product in generating protective immunity. Through bioinformatic analysis, we predicted that this gene is a 1311-bp open reading frame (ORF), which we designated ORF_R049 (GenBank accession No.: EF488001). We further constructed a prokaryotic expression system to express full recombinant R049 protein and isolated and purified the protein through IPTG induction and nickel affinity chromatography. Using mouse immunosera generated by the purified protein, we confirmed the natural expression and outer membrane localization of the protein in wild-type strain UPEC132 by Western blotting. To test the potential of this protein as a vaccine candidate, we immunized mice with the recombinant protein before challenging them with UPEC132 through the urinary tract. The results showed significantly reduced bacterial colonization in the urine and kidneys of the immunization group compared with the control group. However, the degree of renal pathological damage was not significantly improved in the immunized mice. Our study has identified a novel gene of UPEC which can generate protective immunity against UTI. This novel gene provides a promising new vaccine candidate.     

Cloning and expression of <Emphasis Type="Italic">AtPLC6</Emphasis>, a gene encoding a phosphatidylinositol-specific phospholipase C in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

A full-length cDNA clone corresponding to a putative phosphatidylinositol-specific phospholipase C(PIPLC) was isolated from Arabidopsis thaliana by screening a cDNA library and using RT-PCR strategy.The cDNA,designated AtPLC6,encodes a putative polypeptide of 578 amino acid residues with a calculated molecular mass of 66251.84 D and a pI of 7.24. The sequence analysis indicates that the polypeptide contains X, Y, EF-hand and C2 domains.The overall structure of putative AtPLC6 protein, like other plant PI-PLCs,is most similar to that of mammalian PLCδ The recombinant AtPLC6 protein expressed in E. coil was able to hydrolyze phosphatidylinositol 4,5-biophosphate (PIP2) to generate inositol 1,4,5-trisphate (IP3) and 1,2-diacylglycerol (DAG).The protein hydrolyzes PIP2 in a Ca^2 -dependent manner and the optimum concentration of Ca^2 is 10μmol/L.These results suggested that AtPLC6 gene encodes a genuine PIPLC.Northern blot analysis showed that the AtPLC6 gene is expressed at low level in all examined tissues, such as roots,stems,leaves,flowers,siliques and seedlings under normal growth conditions.The gene is strongly induced under low temperature and weakly induced under various stresses,such as ABA, high-salt stress and heat. These results suggested that AtPLC6 might be involved in the signal-transduction pathways of cold responses of the plants.     

Transfer of a eubacteria-type cell division site-determining factor CrldfnD 8ene to the nucleus from the chloroplast genome in Chlamydomonas reinhardtii

MinD is a ubiquitous ATPase that plays a crucial role in selection of the division site in eubacteria, chloroplasts, and probably Archaea. In four green algae, MesosUgma viride, Nephroselmis olivacea, Chlorella vulgaris and Prototheca wickerhamii, MinD homologues are encoded in the plastid genome. However, in Arabidopsis, MinD is a nucleus-encoded, chloroplast-targeted protein involved in chloroplast division, which suggests that MinD has been transferred to the nucleus in higher land plants. Yet the lateral gene transfer （LGT） of MinD from plastid to nucleus during plastid evolution remains poorly understood. Here, we identified a nucleus-encoded MinD homologue from unicellular green alga Chlamydomonas reinhardtii, a basal species in the green plant lineage. Overexpression of CrMinD in wild type E. coil inhibited cell division and resulted in the filamentous cell formation, clearly demonstrated the conservation of the MinD protein during the evolution of photosynthetic eukaryotes. The transient expression of CrMinD-egfp confirmed the role of CrMinD protein in the regulation of plastid division. Searching all the published plastid genomic sequences of land plants, no MinD homologues were found, which suggests that the transfer of MinD from plastid to nucleus might have occurred before the evolution of land plants.     

Roles of a sustained activation of NCED3 and the synergistic regulation of ABA biosynthesis and catabolism in ABA signal production in Arabidopsis

ABA, acting as a stress signal, plays crucial roles in plant resistance to water stress. Because ABA signal production is based on ABA biosynthesis, the regulation of NCED, a key enzyme in the ABA biosynthesis pathway, is normally thought of as the sole factor controlling ABA signal production. Here we demonstrate that ABA catabolism in combination with a synergistic regulation of ABA biosynthesis plays a crucial role in governing ABA signal production. Water stress induced a significant accumulation of ABA, which exhibited different patterns in detached and attached leaves. ABA catabolism followed a temporal trend of exponential decay for both basic and stress ABA, and there was little difference in the catabolic half-lives of basic ABA and stress ABA. Thus, the absolute rate of ABA catabolism, i.e. the amount of ABA catabolized per unit time, increases with increased ABA accumulation. From the dynamic processes of ABA biosynthesis and catabolism, it can be inferred that stress ABA accumulation may be governed by a synergistic regulation of all the steps in the ABA biosynthesis pathway. Moreover, to maintain an elevated level of stress ABA sustained activation of NCED3 should be required. This inference was supported by further findings that the genes encoding major enzymes in the ABA biosynthesis pathway, e.g. NCED3, AAO3 and ABA3 were all activated by water stress, and with ABA accumulation progressing, the expressions of NCED3, AAO3 and ABA3 remained activated. Data on ABA catabolism and gene expression jointly indicate that ABA signal production is controlled by a sustained activation of NCED3 and the synergistic regulation of ABA biosynthesis and catabolism.     

Studies of new EST-SSRs derived from Gossypium barbadense

Existing cotton EST-SSR markers are mostly derived from Gossypium arboreum and Gossypium hir-sutum, but EST-SSR markers from Gossypium barbadense are scarce. One hundred and nineteen EST-SSRs were developed based on 98 unique ESTs from a cDNA library constructed in our laboratory using developing fibers from G. barbadense cv. Pima3-79. Among the SSRs, trinucleotide AAG appeared at a high frequency of 11.76%. 36 accessions (consisting of 13 diploids of the A genome, 11 diploids of the D genome and 12 allotetraploids of the AD genome) were employed to test new EST-SSRs. 76 EST-SSRs were successfully amplified, and 313 polymorphic fragments were yielded, with an average of 4.11 fragments per primer pair. The PIC ranged from 0.17 to 0.95 with an average of 0.53. Based on Jaccard’s genetic similarity coefficient, these 36 accessions were clustered into three groups. 21 EST-SSRs exhibited polymorphisms in BC1 population ((Emian22 × Pima3-79) × Emian22), 24 polymor- phic loci were generated, while 22 of the 24 polymorphic loci were integrated with our interspecific BC1 backbone genetic linkage map, and anchored in 12 chromosomes. This study effectively proved that EST-SSRs from G. barbadense are valuable for genetic diversity analysis and genetic mapping.     

Synthesis,characterization and photoluminescence of aluminum <Emphasis Type="Italic">N</Emphasis>-aryloxo functionalized <Emphasis Type="Italic">β</Emphasis>-ketoiminate complexes

The synthesis, characterization and luminescent properties of aluminum complexes containing a dianionic N-aryloxo functionalized β-ketoiminate ligand are presented. 4-(2-Hydroxy-5-R-phenyl)imino-2-pentanone (R = Me, L¹H₂; R = tert-butyl, L²H₂) ligands reacted with AlEt₃ in tetrahydrofuran to give the aluminum complexes (L¹AlEt)₂ (1) and (L²AlEt)₂ (2) in reasonable isolated yields. X-ray diffraction revealed that complexes 1 and 2 have solvent-free centrosymmetric dimeric structures, and each aluminum center has distorted trigonal bipyramidal geometry. At room temperature, complexes 1 and 2 exhibit blue photoluminescence in acetonitrile with maximum emission wavelengths of 419 and 413 nm, respectively.     

Developmental sequence of Cambrian embryo Markuelia

Based on more exquisitely preserved specimens of Markuelia hunanensis recently recovered from Middle and Upper Cambrian in western Hunan and in the light of Synchrotron radiation X-ray tomo-graphic microscopy, the developmental sequence from cleavage through organogenesis to the pre-hatching of Cambrian embryo Markuelia, especially the developmental sequence during the pre-hatching stage, i.e. from the earliest period when the scalids and tail spines only took shape to the latest period (just about hatching), is established. This developmental sequence provides a pattern of embryonic development during the pre-hatching stage, which has not been established in the living scalidophorans (priapulids, loriciferans and kinorhynchs). Thus, it not only enriches our knowledge on the embryonic development of the extant descendants of Markuelia, but also opens a new window to the evolution and development of the animal.