甜橙辣椒红／辣椒玉红素合成酶同源基因的克隆（简报）

The complete sequence of orange homologous capsanthin/capsorubin synthase gene is 3788 bp long with a coding sequence of 1512 bp, which encodes a polypeptide of 503 amino acids. The 5' upstream sequence is 1721 bp long and the 3' downstream sequence is 555 bp long. The amino acid sequence of this gene is 78% and 69% identical to the genes from carrot and pepper, respectively. It is also partially homologous to plant neoxanthin synthase, lycopene beta-cyclase and lycopene epsilon cyclase genes. Isolation of the gene provides a framework for elucidation of the mechanisms involved in inability of citrus to produce capsanthin and capsorubin.     

小鼠2—细胞胚胎单个卵裂球体外培养及其发育形成囊胚的玻璃化冷冻保存技术的研究（简报）

The present experiments were designed to study the effects of glucose, EDTA, glutamine on the in vitro development of single blastomeres from 2-cell embryos in mouse, and the efficiency of cryopreservation of blastocysts from single blastomers with different vitrification. Single blastomeres derived from female ICR x male BDF1 2-cell embryos were cultured in mKRB with or without glucose, EDTA and glutamine, respectively. The expanded blastocyst rates were significantly different between in mKRB with glucose and without glucose (34% vs 65%); The blastomeres were cultured in mKRB with EDTA and glutamine but glucose, the expanded blastocyst rate (90%) was significantly higher than other groups. The blastocysts derived from single blastomeres were vitrified in liquid nitrogen after equilibration in GFS40 for 0.5-2 min, the survival rate 24%-51%. The blastocysts were pretreated in mPBS with 10% glycerol for 5 min, followed by exposure to GFS40 at 25 degrees C for 0.5 min, then vitrified in liquid nitrogen(two-step method), the survival rate was 61%. However, the survival rates increased to 64% and 70% when the blastocysts were vitrified(one-step method) ater equilibration in EFS40 at 25 degrees C for 0.5-1 min.     

Effects of kanamycin on tissue culture and somatic embryogenesis in cotton

The aminoglycoside antibiotic kanamycin was evaluated for its effects on callus initiation from hypocotyl and cotyledon explants, proliferation of non-embryogenic and embryogenic calli, initiation and development of somatic embryos in cotton (Gossypium hirsutum L.). On this basis, the potential use of kanamycin as a selective agent in genetic transformation with the neomycin phosphotransferase II gene as the selective marker gene was evaluated. Cotton cotyledon and hypocotyl explants, and embryogenic calluses were highly sensitive to kanamycin. Kanamycin at 10 mg/L or higher concentrations reduced callus formation, with complete inhibition at 60 mg/L. Kanamycin inhibited embryogenic callus growth and proliferation, as well as the initiation and development of cotton somatic embryos. The sensitivity of embryogenic callus and somatic embryos to kanamycin was different during the initiation and development stages. Kanamycin was considered as a suitable selective agent for transformed callus formation and growth of non-embryogenic callus. Forty to sixty mg/L was the optimal kanamycin concentration for the induction and proliferation of transformed callus. The concentration of kanamycin must be increased (from 50 to 200 mg/L) for the selection of transformation embryogenic callus and somatic embryos. A scheme for selection of transgenic cotton plants when kanamycin is used as the selection agent is discussed.     

弱氧化醋杆菌阿拉伯糖醇脱氢酶基因克隆及其在大肠杆菌中的表达

The partial genomic library of Acetobacter suboxydans was constructed using Yeast\| E.coli shuttle plasmid YEp352 as vector.Two positive transformants,designated as DH5α(pAD91) and DH5α(pAD98),were obtained by screening the growth of transformants on the agar plate in which D\|arabitol was used as the sole carbon source.The results of Southern blot and restriction endonuclease analysis showed that the two recombinants are identical.The insert is about 2.3kb.Arabitol dehydrogenase activity assay indic…     

Transmembrane-4 superfamily proteins associate with activated protein kinase C (PKC) and link PKC to specific beta(1) integrins

Translocation of conventional protein kinases C (PKCs) to the plasma membrane leads to their specific association with transmembrane-4 superfamily (TM4SF; tetraspanin) proteins (CD9, CD53, CD81, CD82, and CD151), as demonstrated by reciprocal co-immunoprecipitation and covalent cross-linking experiments. Although formation and maintenance of TM4SF-PKC complexes are not dependent on integrins, TM4SF proteins can act as linker molecules, recruiting PKC into proximity with specific integrins. Previous studies showed that the extracellular large loop of TM4SF proteins determines integrin associations. In contrast, specificity for PKC association probably resides within cytoplasmic tails or the first two transmembrane domains of TM4SF proteins, as seen from studies with chimeric CD9 molecules. Consistent with a TM4SF linker function, only those integrins (alpha(3)beta(1), alpha(6)beta(1), and a chimeric "X3TC5" alpha(3) mutant) that associated strongly with tetraspanins were found in association with PKC. We propose that PKC-TM4SF-integrin structures represent a novel type of signaling complex. The simultaneous binding of TM4SF proteins to the extracellular domains of the integrin alpha(3) subunit and to intracellular PKC helps to explain why the integrin alpha3 extracellular domain is needed for both intracellular PKC recruitment and PKC-dependent phosphorylation of the alpha(3) integrin cytoplasmic tail.     

Directed evolution of operon of trehalose-6-phosphate synthase/phosphatase from Escherichia coli

Trehalose is a nonspecific protective agent for biomacromolecules. Trehalose-6-phosphate synthase (OtsA)/phosphatase (OtsB), which is encoded by the gene operon otsBA located at -42 of the Escherichia coli genome, is the main enzyme system that catalyzes the synthesis of trehalose in E. coli. We cloned the operon and modified it by directed evolution. Unlike in the previously reported work, we modified the whole operon and screened the positive mutant simultaneously. Thus we believe that the gene complex solves the negative effects between two enzymes if one of them diversifies its structure or functions and finds the form most suitable for trehalose synthesis. It thus mimics the natural process, in which the functional improvement of organisms is related to alterations in coordinated enzymes. The evolution procedure was carried out in a sequence of error-prone PCR, shuffling PCR, and then strict screening of the mutants. After screening of a library of more than 4000 colonies, about 15 positive colonies were analyzed, resulting in a higher concentration of trehalose than control. One of them, E. coli TS7, shows 12.3-fold higher trehalose synthesis ability than E. coli DH5alpha. In contrast, we introduced the cDNA sequence of the tps1 gene from Saccharomyces cerevisiae, which has 54% identity with the gene otsA, as one of the templates in shuffling PCR. By hybrid evolution and screening, we obtained 10 positive colonies with higher concentrations of trehalose than control. E. coli TS22 appears to have 5.3-fold higher trehalose synthesis ability than E. coli DH5alpha and 1.6-fold more than E. coli DEF3(pOTS11). This result demonstrated that coevolution and hybrid evolution, as powerful protocols in protein engineering, are effective in modifying enzyme. It indicates that repeating the process of genomic evolution in nature is feasible.     

Replication of DNA templates containing 5-formyluracil, a major oxidative lesion of thymine in DNA.

5-Formyluracil (5-foU) is a major lesion of thymine produced in DNA by ionizing radiation and various chemical oxidants. To assess its biochemical effects on DNA replication, 22mer oligonucleotide templates containing an internal 5-foU at defined sites were synthesized by the phosphoramidite method and examined for ability to serve as a template for various DNA polymerases in vitro . Klenow fragments with and without 3'-->5'exonuclease of DNA polymerase I, Thermus thermophilus DNA polymerase (exonuclease-deficient) and Pyrococcus furiosus DNA polymerase (exonuclease-proficient) read through the site of 5-foU in the template. Primer extension assays revealed that the 5-foU directed not only incorporation of dAMP but also dCMP opposite the lesion during DNA synthesis. Misincorporation opposite 5-foU was unaffected by 3'-->5' exonuclease activity. DNA polymerases had different dissociation rates from a dCMP/T mispair and from a dCMP/5-foU mispair. The incorporation of an 'incorrect' nucleotide was dependent on the sequence context and DNA polymerase used. These results suggest that 5-foU produced in DNA has mutagenic potential leading to T-->G transversions during DNA synthesis.     

Salicylic acid activates a 48-kD MAP kinase in tobacco.

The involvement of phosphorylation/dephosphorylation in the salicylic acid (SA) signal transduction pathway leading to pathogenesis-related gene induction has previously been demonstrated using kinase and phosphatase inhibitors. Here, we show that in tobacco suspension cells, SA induced a rapid and transient activation of a 48-kD kinase that uses myelin basic protein as a substrate. This kinase is called the p48 SIP kinase (for SA-Induced Protein kinase). Biologically active analogs of SA, which induce pathogenesis-related genes and enhanced resistance, also activated this kinase, whereas inactive analogs did not. Phosphorylation of a tyrosine residue(s) in the SIP kinase was associated with its activation. The SIP kinase was purified to homogeneity from SA-treated tobacco suspension culture cells. The purified SIP kinase is strongly phosphorylated on a tyrosine residue(s), and treatment with either protein tyrosine or serine/threonine phosphatases abolished its activity. Using primers corresponding to the sequences of internal tryptic peptides, we cloned the SIP kinase gene. Analysis of the SIP kinase sequence indicates that it belongs to the MAP kinase family and that it is distinct from the other plant MAP kinases previously implicated in stress responses, suggesting that different members of the MAP kinase family are activated by different stresses.