Biotransformation of Benzyl Alcohol by Pseudomonas cepacia

To cDNAs encoding class I chitinases of rice were expressed in Escherichia coli. The cDNAs were fused to the MS2-polymerase gene in an expression vector, pEx31. The fusion proteins, expressed under the control of the λP_L-promoter, showed the chitinase activity independent of the existence of the hevein domain. The enzymatic hydrolysis of colloidal chitin by the fusion proteins showed that the proteins were endo-type enzymes.     

Diffusion ordered spectroscopy as a complement to size exclusion chromatography in oligosaccharide analysis

A series of N-acetyl-chitooligosaccharides (GlcNAc)(1-6) have been studied by a nuclear magnetic resonance (NMR) method, diffusion ordered spectroscopy (DOSY). DOSY has also been applied to two additional synthetic related oligosaccharides [GlcNH(2)-(GlcNAc)(4) and GlcNH(2)-(GlcNAc)(2)-GlcNAcSO(3)Na]. A plot of the log of the determined diffusion coefficients (logD) of (GlcNAc)(n) versus the log of molecular weight was linear (6 points, R(2) = 0.995). The molecular weights of the two synthetic chitin derivatives could be estimated to within 10% error. The processed NMR data of all the chitooligosaccharides was also plotted in a polyacrylamide gel-like format to aid visual interpretation. Moreover, the logD value of the NMR signal resonances of a chitin-binding protein (hevein) changed as a function of a given titrated ligand, (GlcNAc)(6). Evidence for a 2:1 hevein:(GlcNAc)(6) complex is detected by DOSY at high hevein:(GlcNAc)(6) ratios. This data is consistent with published analytical ultracentrifugation and isothermal titration calorimetry data. A 1:1 complex is preferred at higher ligand concentrations. DOSY can complement size exclusion chromatography in carbohydrate research with the advantage that oligosaccharides are more readily detected by NMR.     

橡胶树凝集因子hevein基因及其启动子序列的分离与分析

橡胶树 (HeveabrasiliensisMuell._Arg .)凝集因子hevein是引起橡胶粒子凝集的主要因素 ,它是胶乳中黄色体内主要的蛋白质 ,具有几丁结合的功能。通过PCR技术扩增并克隆了橡胶树hevein基因共 6 80bp的序列。继而通过步移法分离启动子区域 130 6bp的序列 ,序列含典型的TATA盒和CAAT盒以及ABA效应元件的同源序列。为证实该基因在乳管中特异表达 ,利用Northernblot分析hevein基因在胶乳和叶片中的表达 ,同时 ,分析乙烯和ABA处理后hevein基因的表达。结果表明 ,hevein基因主要在胶乳中表达 ,乙烯和ABA对基因的表达有诱导作用。     

巴西橡胶树HbHEV3基因的克隆和表达分析

根据EST序列信息,通过RT-PCR获得了一个编码橡胶素前体的基因,命名为HbHEV3。HbHEV3编码区cDNA长度为630bp,编码209个氨基酸。预测的HbHEV3蛋白包含1个信号肽,1个具有几丁质结合特性的Hevein结构域和1个Barwinn结构域,HbHEV3与橡胶树及其他植物中类似蛋白具有很高的同源性。分离获得了HbHEV3起始密码子上游1 050bp的启动子序列,该序列含有众多应答激素和胁迫信号元件。实时荧光定量PCR分析结果表明,HbHEV3在所检测的组织中均有表达,其中在胶乳中的表达量最高,乙烯诱导能显著上调胶乳中HbHEV3的表达。研究表明,HbHEV3可能参与了橡胶树乙烯介导的防御反应,并在橡胶凝集过程中具有重要功能。     

橡胶树凝集因子hevein基因及其启动子序列的分离与分析

橡胶树( Hevea brasiliensis Muell.-Arg.)凝集因子hevein是引起橡胶粒子凝集的主要因素,它是胶乳中黄色体内主要的蛋白质,具有几丁结合的功能.通过PCR技术扩增并克隆了橡胶树hevein基因共680 bp的序列.继而通过步移法分离启动子区域1 306 bp的序列,序列含典型的TATA盒和CAAT盒以及ABA效应元件的同源序列.为证实该基因在乳管中特异表达,利用Northern blot分析hevein基因在胶乳和叶片中的表达,同时,分析乙烯和ABA处理后hevein基因的表达.结果表明,hevein基因主要在胶乳中表达,乙烯和ABA对基因的表达有诱导作用.     

In vitro transient expression system of latex C-serum was used for analysis of hevein promoter in response to abscisic acid in Hevea brasiliensis

Hevein has been found to be an essential element in coagulation of rubber particles in latex of rubber trees. In a previous study, we cloned a 1 241-bp fragment of a 5＇ upstream region of the hevein gene by genome walking. This fragment was analyzed by a 5＇ end nested deletion method in the present study, fused with a uidA （gus） gene to produce a series of tested constructs, which were transferred into C-serum of latex and the Gus activities were detected. Results showed that the fragment from -749 to -292 was sufficient for expression of gus gene in latex, and the fragment from -292 to -168 was crucial in response to abscisic acid inducement. In a transient transgenic test of rubber leaf with particle bombardment, construct Hev749 conferred gus-specific expression in veins, in which the latex tubes mainly distributed. This implies that the fragment from -749 to -292 was laticiferous-specific.     

Methyl jasmonate induces expression of a novel Brassica juncea chitinase with two chitin-binding domains

We have cloned a 1.3 kb Brassica juncea cDNA encoding BjCHI1, a novel acidic chitinase with two chitin-binding domains that shows 62% identity to Nicotiana tabacum Chia1 chitinase. BjCHI1 is structurally unlike Chia1 that has one chitin-binding domain, but resembles Chia5 chitinase UDA1, the precursor of Urtica dioica agglutinin; however there is only 36.9% identity between them. We propose that BjCHI1 should be classified under a new class, Chia7. The spacer and the hinge region of BjCHI1 are proline-rich, like that of Beta vulgaris Ch1, a Chia6 chitinase with half a chitin-binding domain. Northern blot analysis showed that the 1.3 kb BjCHI1 mRNA is induced by wounding and methyl jasmonate (MeJA) treatment but is unaffected by ethylene, salicylic acid (SA) or abscisic acid (ABA). This is the first report on MeJA induction of chitinase gene expression and further suggests that wound-related JA-mediated signal transduction is independent of that involving SA. Western blot analysis using polyclonal antibodies against BjCHI1 showed a cross-reacting band with an apparent molecular mass of 37 kDa in wounded tissues of B. juncea, revealing that, unlike UDA1, BjCHI1 is not cleaved post-translationally at the hinge. Expression of recombinant BjCHI1 in Escherichia coli BL21(DE3) inhibited its growth while crude extracts from E. coli JM109 expressing recombinant BjCHI1 showed chitinase activity. Results from polymerase chain reaction (PCR) suggest that genes encoding chitinases with single or double chitin-binding domains exist in B. juncea.     

Hevein,an allergenic lectin from rubber latex,activates human neutrophils' oxidative burst

Hevein is an N-acetyl-D-glucosamine (GlcNAc) specific lectin that has been hypothesized to participate in the IgE-mediated allergic reactions in patients with latex allergy. In this work we assessed the specificity and biological effect of hevein purified from rubber latex on human leukocytes, using epifluorescence microscopy and flow cytometry. Purified human granulocytes were stimulated in vitro with hevein, and production of oxidative radicals was measured by reduction of nitroblue tetrazolium formazan. Histochemical staining and flow cytometry showed that hevein recognizes specifically monocytes (CD14+) and neutrophils (CD16+), but not lymphoid cells. Hevein induced oxidative response in purified granulocytes; this effect was 1.3–1.5-fold higher than the effect observed with the lectin WGA (wheat germ agglutinin), or other lectins with different sugar specificity. The induced reactions and cellular recognition by hevein were inhibited with GlcNAc and its oligomers; as well as by glycoproteins containing tri-and tetra-antennary N-glycosydically linked glycans. Our findings suggest that neutrophils are the main target for latex hevein; this lectin induces production of oxidative radicals, which seem to play an important role in tissue damage during latex allergy.     

Antimicrobial Peptides from Plants

Peptides with antimicrobial properties are present in most if not all plant species. All plant antimicrobial peptides isolated so far contain even numbers of cysteines (4, 6, or 8), which are all pairwise connected by disulfide bridges, thus providing high stability to the peptides. Based on homologies at the primary structure level, plant antimicrobial peptides can be classified into distinct families including thionins, plant defensins, lipid transfer proteins, and he vein- and knottin-type antimicrobial peptides. Detailed three-dimensional structure information has been obtained for one or more members of these peptide families. All antimicrobial peptides studied thus far appear to exert their antimicrobial effect at the level of the plasma membrane of the target microorganism, but the different peptide types are likely to act via different mechanisms. Antimicrobial peptides can occur in all plant organs. In unstressed organs, antimicrobial peptides are usually most abundant in the outer cell layer lining the organ, which is consistent with a role for the antimicrobial peptides in constitutive host defense against microbial invaders attacking from the outside. Thionins are predominantly located intracellularly but are also found in the extracellular space, whereas most plant defensins and lipid transfer proteins are deposited exclusively in the extracellular space. In a number of plant species, a strong induction of genes expressing either thionins, plant defensins, or lipid transfer proteins has been observed on infection of the leaves by microbial pathogens. Hence, antimicrobial peptides can also take part in the inducible defense response of plants. Constitutive expression in transgenic plants of heterologous antimicrobial peptide genes has been achieved, which in some cases has led to enhanced resistance to particular microbial plant pathogens.