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1.
The properties of morrenain b II, a proteinase isolated from the latex of Morrenia brachystephana, were compared with those of morrenain o II, a proteinase obtained from the latex of Morrenia odorata. Both peptidases were purified to homogeneity by acetone precipitation followed by cation exchange chromatography. The enzymes have pI values higher than 9.3 and similar molecular masses (close to 26 kDa) as determined by SDS-PAGE. They display maximum proteolytic activity within an alkaline pH range, and also exhibit esterolytic activity. The N-terminal sequences of morrenain o II and morrenain b II show a high degree of homology between each other and to other cysteine plant proteinases.  相似文献   

2.
A new papain-like cysteine peptidase isolated from latex of Philibertia gilliesii Hook. et Arn., Apocynaceae (formerly Asclepiadaceae) has been purified and characterized. The enzyme, named philibertain g I, is the most basic component present in latex extracts and was purified by acetone fractionation followed by cation exchange chromatography (SP-Sepharose HR) using FPLC system. Homogeneity was confirmed by SDS-PAGE and mass spectroscopy (MS). Molecular mass of the enzyme was 23,530 Da (MALDI-TOF MS), its isoelectric point was >10.25, and maximum proteolytic activity (casein) was achieved at pH 7–8. The new protease was inhibited by E-64 a cysteine peptidases inhibitor. Km was 0.15 mM, using PFLNA as substrate. The N-terminal sequence of philibertain g I (LPASVDWRKEGAVLPIRHQGQCG) was compared with those of twenty plant proteases. Philibertain g I showed the higher degree of identity (73%) with caricain, one of the Carica papaya endopepetidases.  相似文献   

3.
Alpha 1 chain (Colα1(I)) and alpha 2 chain (Colα2(I)) are universal components of type I collagen in tetrapods, but rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio) have a third: alpha 3 chain (Colα3(I)). This study tests whether Colα3(I) is a duplicate of Colα1(I) by whole-genome duplication (WGD) that occurred early in the ray-fin fish lineage. We also examine how their promoter sequence was modified after WGD. We cloned Colα1(I), Colα2(I) and Colα3(I) cDNAs and their promoters from flounder (Paralichthys olivaceus) and obtained corresponding sequences from the genome databanks of two pufferfishes Takifugu rubripes and Tetraodon nigroviridis, by BLAST-Search using flounder sequences. Phylogenetic analysis of N-terminal sequences of ca. 100 amino acids, including signal peptide and N-propeptide sequences before short triple helical domain, indicates that Colα3(I), found only in teleosts, is a duplicate of Colα1(1) by WGD. Colα1(I) and Colα3(I) genes begin to be transcribed at different stages of Takifugu embryogenesis, suggesting that their structure of promoter is modified differently after WGD. In flounder, Takifugu and Tetraodon, the structure of proximal region of promoter is highly conserved within Colα1(I) and within Colα3(I); no homology is apparent except for the TATA element motif between Colα1(I) and Colα3(I) of each species. Unexpectedly, zebrafish Colα1(I) promoter is more homologous to Colα3(I) of flounder and fugu than Colα1(I) is. These results suggest that each duplicated Colα1(I) gene promoter inherited a unique structure after WGD, but the manner of modification differed between the phylogenetically separated zebrafish and flounder/pufferfish lineages.  相似文献   

4.
The lutoid-body (bottom) fraction of latex from the rubber tree (Hevea brasiliensis) contains a limited number of major proteins. These are the chitin-binding protein hevein, its precursor and C-terminal fragment of the precursor, a basic chitinase/lysozyme, and a β-1,3-glucanase. The content and properties of the latter enzyme differ between lutoid-body fractions from four different rubber clones (cultivars). While the enzyme from clone GT.1 is a glycoprotein with carbohydrate attached to two glycosylation sites, the enzymes from other clones contain little or no carbohydrate. Latex from clone GT.1 has a higher β-1,3-glucanase content than those from the other three clones, but with a significantly lower specific activity. The enzyme exhibits a pH optimum at 4.5, but there is a second one at 6.7. Peptides isolated from β-1,3-glucanase of clone GT.1 showed that the enzyme is heterogeneous at the C-terminus, probably as a result of removal of a vacuolar targeting sequence by an endopeptidase, followed by further removal of C-terminal residues by a carboxypeptidase-like activity. This incomplete digestion can be related to glycosylation at the extreme C-terminus of the mature enzyme. Non-glycosylated Hevea β-1,3-glucanases exhibit less C-terminal heterogeneity. A homologue of the antifungal protein osmotin was isolated from rubber clones which are less susceptible to fungal diseases. Another identified protein is identical to a citrate binding protein (CBP), already sequenced as cDNA, but with cleaved-off N-terminal signal and C-terminal vacuolar targeting peptides. Four C-terminal propeptides of vacuolar proteins in Hevea are positively identified, which is a valuable contribution to previously known examples of this type of processing.  相似文献   

5.
滨海沙地条件恶劣,季节气候环境差异较大,植物生存困难。厚藤是南方滨海沙地广泛分布的重要固沙植物。为探究厚藤对不同季节环境变化的适应机制,研究其叶片生理性状的季节变化,该文以广西滨海沙地自然生长的厚藤为实验材料,分别测定了不同季节厚藤叶片的叶绿素含量、渗透物质含量、抗氧化酶活性、叶绿素荧光参数等生理指标,并进行相关性分析和主成分分析。结果表明:(1)叶绿素含量随季节变化的趋势一致,春季均显著大于其他三个季节,但叶绿素a/b在各季节间无显著变化。(2)叶绿素荧光参数的Fv/FmFv/Fo具有相同的变化趋势,整体表现为夏冬季显著高于春秋季。(3)脯氨酸含量随季节逐渐增大,冬季时含量最高; 可溶性糖含量冬季显著高于其他三个季节; 丙二醛(MDA)含量各季节间差异不显著。(4)春季的超氧化物歧化酶(SOD)活性和过氧化氢酶(CAT)活性显著高于其他季节,夏秋冬季节间无显著差异; 过氧化物酶(POD)活性在各季节间差异不显著。(5)相关性分析和主成分分析显示,各生理指标与气候因子间存在一定的关联。温度和日照数显著影响可溶性糖含量; 叶绿素含量和抗氧化酶活性能够较好地反映厚藤对季节气候变化的响应。综上可知,厚藤可通过调节叶绿素a与叶绿素b含量使叶绿素a/b保持稳定,同时提高渗透调节物质含量和抗氧化酶活性以适应季节变化,其中光合作用和抗氧化酶系统是影响其季节性适应能力的关键。  相似文献   

6.
Spontaneous latex exudation is the main symptom of papaya sticky (meleira) disease caused by the Papaya meleira virus (PMeV), a double-stranded RNA (dsRNA) virus. This paper describes different effects of PMeV on papaya latex. Latex samples were subjected to different histochemical tests to evaluate their chemical composition. Additionally, the integrity of the latex particles was assessed by transmission and scanning electron microscopy analysis. Biochemical and micro- and macro-element measurements were performed. PMeV dsRNA extraction was performed to evaluate the interaction of the virus with the latex particles. Sticky diseased latex was positive for alkaloid biosynthesis and showed an accumulation of calcium oxalate crystals. PMeV also increased H2O2 synthesis within sticky diseased laticifers. The protein, sugar and water levels were altered, probably due to chemical changes. The morphology of the latex particles was further altered; PMeV particles seemed to be bound to the latex particles. The alkaloid and H2O2 biosynthesis in the papaya laticifers indicate a papaya defense response against PMeV. However, such efforts failed, as the virus affected the plant latex. The effects described here suggest some advantages of the infection process, including facilitating the movement of the virus within the papaya plant.  相似文献   

7.
Myrosinases (EC 3.2.1.147) are β-thioglucoside glucosidases present in Brassicaceae plants. These enzymes serve to protect plants against pathogens and insect pests by initiating breakdown of the secondary metabolites glucosinolates into toxic products. Several forms of myrosinases are present in plants but the properties and role of different isoenzymes are not well understood. The dicot plant model organism Arabidopsis thaliana seems to contain six myrosinase genes (TGG1TGG6). In order to compare the different myrosinases, cDNAs corresponding to TGG1 from leaves and TGG4 and TGG5 from roots were cloned and overexpressed in Pichia pastoris. The His-tagged recombinant proteins were purified using affinity chromatography and the preparations were homogenous according to SDS–PAGE analysis. Myrosinase activity was confirmed for all forms and compared with respect to catalytic activity towards the allyl-glucosinolate sinigrin. There was a 22-fold difference in basal activity among the myrosinases. The enzymes were active in a broad pH range, are rather thermostable and active in a wide range of salt concentrations but sensitive to high salt concentrations. The myrosinases showed different activation–inhibition responses towards ascorbic acid with maximal activity around 0.7–1 mM. No activity was registered towards desulphosinigrin and this compound did not inhibit myrosinase activity towards sinigrin. All myrosinases also displayed O-β-glucosidase activity, although with lower efficiency compared to the myrosinase activity. The differences in catalytic properties among myrosinase isozymes for function in planta are discussed.  相似文献   

8.
The cleavage patterns of mitochondrial DNA (mtDNA) by 17 restriction endonucleases were compared between eight lines of musk shrews derived from different wild-caught stocks. Enzymatic digestion byBamHI,PvuII,XbaI, andXhoI showed a cleavage pattern common to all lines that were from five Japanese islands (Nag, Ize, OKI, TKU, and Tr), Bangladesh (BAN), Sri Lanka (SRI), and Java (Bog), and every line lacked cleavage sites forSalI andSmaI. Different cleavage patterns were detected by the remaining 11 enzymes. Within the BAN line, the presence of at least two types of mtDNAs was proved by six enzymes and was not contradictory to the maternal pedigrees going up to the wild ancestors of the stock. More than 30 cleavage sites of the shrew mtDNA were mapped by double-digestion methods. Nucleotide diversities of mtDNA were calculated from these maps and were estimated to be less than 0.5% among Japanese and Bog lines but to be 3.8% between BAN and the other seven lines and 2.3% within the BAN line. These results indicate that BAN shrews differentiate from the other lines to the intersubspecific extent reported in mice previously.  相似文献   

9.
10.
Plants are continually exposed to external and internal DNA-damaging agents. Although lesions can be removed by different repair processes, damages often remain in the DNA during replication. Synthesis of template damages requires the replacement of replicative enzymes by translesion synthesis polymerases, which are able to perform DNA synthesis opposite specific lesions. These proteins, in contrast to replicative polymerases, operate at low processivity and fidelity. DNA polymerase η and Rev 1 are two proteins found in eukaryotes that are involved in translesion DNA synthesis. In Arabidopsis, DNA polymerase η and Rev 1 are encoded by AtPOLH and AtREV1 genes, respectively. Transgenic plants over-expressing AtPOLH showed increased resistance to ultraviolet light. Only plants with moderate AtREV1 over-expression were obtained, indicating that this enzyme could be toxic at high levels. Transgenic plants that over-expressed or disrupted AtREV1 showed reduced germination percentage, but the former exhibited a higher stem growth rate than the wild type during development.  相似文献   

11.
Polyphenol oxidase from the latex of opium poppy was purified to the electrophoretic homogeneity by affinity chromatography using p-aminobenzoic acid as a ligand coupled to Sepharose CL-4B by divinyl sulphone activation method. The purified enzyme was used to prepare the polyclonal antibodies. The purified latex PPO exhibited high diphenolase activity in comparison with almost unmeassurable monophenolase activity. Both of these activities were sensitive to the activation with sodium dodecyl sulphate. Two isoforms (65 and 40 kDa) of latex PPO were separated by the gel filtration. There were no differences in substrate specifity (weak monophenolase and high diphenolase activity) and sensitivity to inhibitors between these isoforms, but they showed differences in electrophoretic mobility. This revised version was published online in July 2006 with corrections to the Cover Date.  相似文献   

12.
Summary The locus, I2, which in tomato confers resistance against Fusarium oxysporum f. sp. lycopersici race 2, was introgressed into Lycopersicon esculentum from the wild species L. pimpinellifolium (P.I. 126915). We searched for restriction fragment length polymorphisms (RFLPs) between nearly isogenic lines (NILs) in clones that map to the region introgressed from the wild species. Since I2 maps to chromosome 11, we used DNA clones from this chromosome as hybridization probes to Southern blots containing bound DNA of the NILs digested with 23 restriction enzymes. Of the 14 chromosome 11 clones, 9 exhibited polymorphism. These clones were further hybridized to verification filters that contained DNA from resistant and susceptible L. esculentum varieties digested with the enzymes that gave the polymorphism. One clone, TG105, was found to be associated with I2; 19 susceptible lines showed a different RFLP with this probe than 16 resistant lines, including the original L. pimpinellifolium accession used as a source for the resistance gene. These results together with our mapping analysis indicate that TG105 is closely linked to the resistance gene.  相似文献   

13.
Allergen protein detection was performed by a surface imprinted layer combined with an interdigitated capacitance (IDC) transducer that allowed label-free measurements. The immobilized imprinted polymers are the probes that bind to rubber allergen proteins extracted from products such as rubber gloves. Copolymers made from methacrylic acid–vinylpyrrolidone–dihydroxyethylene-bisacrylamide (MAA–NVP–DHEBA) are soluble in aqueous solution and eliminate the denaturation of protein. When deposited as a coating onto an IDC microelectrode transduction system, such materials lead to sensors that produce capacitance responses that are clearly dependent on the concentration of the latex protein (10–900 ng ml−1) in pH 7.4 buffer. The biosensor can detect Hev b1 within minutes and with a detection limit of 10 ng ml−1. Different but related hevein allergenic proteins isolated from natural rubber latex from the rubber tree (Hev b1, Hev b2, and Hev b3) were distinguished by the imprinted material, depending on the dimension and conformation of these proteins with a selectivity factor of 4. They recognized Hevea latex proteins better than non-Hev b proteins, such as lysozyme, ovalbumin, and bovine serum albumin, by a factor of 2. Moreover, the sensor exhibited good operational stability of up to 180 days when used continuously at room temperature.  相似文献   

14.
Cultured fibroblasts from patients with the lysosomal storage disease, mucolipidosis II, produce complex glycosylated lysosomal enzymes which are preferentially excreted presumably due to the absence of specific phosphomannosyl recognition residues needed for intracellular retention. Complex glycosylated hydrolases are also produced by fibroblasts from patients with mucolipidosis I but an abnormal excretion is not apparent in this disorder. Intra- and extracellular distribution, lectin binding, and specific endocytosis were criteria used to compare the properties of intra- and extracellular β-hexosaminidase derived from mucolipidosis I and normal fibroblast cultures. Mucolipidosis I fibroblasts did not hyperexcrete β-hexosaminidase when maintained in serum-free medium. Using the specifity of ricin binding to terminal galactosyl residues, the most galactosylated forms of the enzyme derived from mucolipidosis I cell extracts and culture fluids were found in the mucolipidosis I cell extracts (50% of total enzyme). Mucolipidosis I-excreted β-hexosaminidase which was eluted from ricin-120-Sepharose, was a high-uptake form in endocytosis experiments while unbound enzyme was a low-uptake form. These data suggest that β-hexosaminidase molecules contained phosphomanosyl residues necessary for receptor-mediated endocytosis as well as galactosyl residues on the same molecule. The co-existence of complex chains with high-mannose chains did not interfere with the phosphomannose-mediated endocytosis of β-hexosaminidase nor with the retention of endogenous enzyme. We can speculate that since complex oligosaccharide chains in the mucolipidosis I cellular enzyme persist due to a sialidase deficiency, more extensive sialylation of cellular enzyme in normal fibroblasts probably occurs at some point during post-translational processing. However, the presence of sialidase in normal cells initiates complex chain trimming in the lysosomes resulting in a less glycosylated end product.  相似文献   

15.
Mitochondrial DNA (mtDNA) from the cryptomonad Pyrenomonas salina was isolated by CsCl-buoyant density centrifugation of whole-cell DNA in the presence of Hoechst dye 33258. mtDNA consists of circular molecules about 47 kb in size as estimated from restriction enzyme analysis. A physical map for six restriction enzymes (Bam HI, Bge I, Eco RI, Pst I, Sac I and Sac I) has been constructed. Genes coding for the small subunit of rRNA, cytochrome oxidase subunits I and II, and apocytochrome b were localized on this map using Southern blot hybridization with heterologous gene probes from Oenothera. Genes for 5S rRNA and NADH dehydrogenase subunit 5 are absent from P. salina mtDNA. The mitochondrial genome, being the first analysed to this extent in chromophytic algae, should be valuable for taxonomic and phylogenetic studies.  相似文献   

16.
In the present report, a total of 75 Fusarium spp isolates (35 of the Gibberella fujikuroi species complex, 26 of F. oxysporum, 7 of F. graminearum, 5 of F. culmorum, 1 of F. cerealis, and 1 of F. poae) from different hosts were characterized morphologically, physiologically and genetically. Morphological characterization was performed according to macroscopic and microscopic aspects. Physiological characterization was based on their ability to produce fumonisin B1 (FB1), fumonisin B2 (FB2), zearalenone (ZEA) and type B trichothecenes (deoxynivalenol, nivalenol and 3-acetyldeoxynivalenol). FB1, FB2, and ZEA were determined by liquid chromatography and trichothecenes by gas chromatography. Molecular characterization of isolates was carried out using an optimized and simple method for isolation of DNA from filamentous fungi and polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLP) of the intergenic spacer region (IGS) of the rDNA. The results indicated that G. fujikuroi complex isolates can be␣divided into low and high fumonisin producers. The haplotypes obtained with HhaI, EcoRI, AluI, PstI and XhoI enzymes provided very characteristic groupings of G. fujikuroi isolates as a function of host type and fumonisin producing capacity. F. graminearum, F. culmorum and F. cerealis isolates were high ZEA␣and type B trichothecene producers, while F. oxysporum and the G. fujikuroi complex isolates did not show this ability. The haplotypes obtained with CfoI, AluI, HapII, XhoI, EcoRI and PstI enzymes permitted to discern these five Fusarium species and G. fujikuroi complex isolates but the restriction patterns of the IGS region did not show any relationship with the geographic origin of isolates.  相似文献   

17.
Asclepain f is a papain-like protease previously isolated and characterized from latex of Asclepias fruticosa. This enzyme is a member of the C1 family of cysteine proteases that are synthesized as preproenzymes. The enzyme belongs to the alpha + beta class of proteins, with two disulfide bridges (Cys22-Cys63 and Cys56-Cys95) in the alpha domain, and another one (Cys150-Cys201) in the beta domain, as was determined by molecular modeling. A full-length 1,152 bp cDNA was cloned by RT-RACE-PCR from latex mRNA. The sequence was predicted as an open reading frame of 340 amino acid residues, of which 16 residues belong to the signal peptide, 113 to the propeptide and 211 to the mature enzyme. The full-length cDNA was ligated to pPICZα vector and expressed in Pichia pastoris. Recombinant asclepain f showed endopeptidase activity on pGlu-Phe-Leu-p-nitroanilide and was identified by PMF-MALDI-TOF MS. Asclepain f is the first peptidase cloned and expressed from mRNA isolated from plant latex, confirming the presence of the preprocysteine peptidase in the latex.  相似文献   

18.
Hemocytes of the solitary ascidian Halocynthia roretzi released phenoloxidase in response to sheep red blood cells and yeast cells but not to latex beads. Phenoloxidase was also released from the hemocytes by treatments with zymosan and lipopolysaccharides but not with β1–3 glucan. EDTA scarcely inhibited the activity of phenoloxidase but inhibited the release of the enzyme. Phenoloxidase was purified from H. roretzi hemocytes by SP-Sephadex chromatography and Sephadex G-100 gel filtration. The molecular weight of the purified enzyme was estimated to be 62 000. Phenoloxidase activity was strongly inhibited by diethyldithiocarbamate, phenylthiourea and reducing agents. H. roretzi phenoloxidase was characterized as a metalloenzyme that required copper ions for the expression of full activity. The phenoloxidase showed antibacterial activity in the presence of -(3,4-dihydroxy)-phenylalanine and H. roretzi plasma. Thus, it can be concluded that phenoloxidase released from H. roretzi hemocytes functions as a humoral factor in the defense system of H. roretzi.  相似文献   

19.
Summary Screening the tryptophan (Trp)-dependent indole-3-acetic acid (IAA) production of different Azospirillum species revealed that A. irakense KA3 released 10 times less IAA into the medium than A. brasilense Sp7. A cosmid library of strain Sp7 was transferred into A. irakense KA3 with the aim of characterizing genes involved in IAA biosynthesis. Trp-dependent IAA production was increased in two transconjugants which both contained an identical 18.5 kb HindIII fragment from Sp7. After Tn5 mutagenesis, cosmids carrying Tn5 insertions at 36 different positions of the 18.5 kb fragment were isolated and transferred into strain KA3. IAA production by the recipient strains was screened by HPLC. The Tn5 insertions of 4 clones with decreased IAA production were mapped on a 2 kb Sall — SphI fragment. Recombination of Tn5 insertions at this locus into the genome of strain Sp7 led to Trp auxotrophic mutants. A 5.2 kb EcoRI — SalI fragment including the kb SalI — SphI fragment was sequenced and six open reading frames were identified. Three of them were clustered and their deduced amino acid sequences showed significant similarity to TrpG, TrpD and TrpC, which are enzymes involved in tryptophan biosynthesis. One of the remaining open reading frames probably encodes an acetyltransferase. The region responsible for the enhanced Trp-dependent IAA production in strain KA3 corresponded to trpD, coding for the phosphoribosyl anthranilate transferase.  相似文献   

20.
披针叶茴香对变化光环境的表型可塑性   总被引:1,自引:0,他引:1  
植物对变化光环境的表型可塑性大小影响其在林下生境中分布、生长和更新。为探讨披针叶茴香在不同光环境下的整体表型可塑性及其适应机制,采用遮荫试验模拟5种光照条件(100%、52%、33%、15%和6%相对光照强度),研究了不同光环境下披针叶茴香叶片形态、生理、解剖结构、根系形态以及生物量分配等的变化。结果表明:叶生物量在5种光照处理之间差异不显著,但叶面积和比叶面积均随光照强度减弱显著增加。遮荫处理增加了叶绿素a、叶绿素b和类胡萝卜素的含量,但叶绿素a/b比值随光照强度减弱而降低。遮荫降低了非结构性碳水化合物(淀粉和可溶性糖)和可溶性蛋白的含量,增加了叶片氮和磷含量,对叶片氮/磷比影响较小。在52%和33%相对光照处理下,叶片中硝酸盐含量最低,而在100%和6%相对光照处理下硝酸盐积累较多。根生物量、细根和粗根的长度、表面积以及比根长和比根表面积在5种光照处理之间均没有显著差异,根系氮含量在低光环境(15%和6%相对光照处理)中显著降低。随光照强度减弱,披针叶茴香采取保守生存策略,并没有增加叶生物量的分配,而是分配较多的生物量给枝条和树干,储存能量。综合来看,披针叶茴香具有较宽的光生态幅,在6%—100%光照强度下均能正常生长,遮荫有利于披针叶茴香地上和总生物量的积累,52%的相对光照条件下生长最佳。变化光环境下根系性状和整体结构的可塑性相对较低,叶片生理性状的可塑性在披针叶茴香适应光环境变化过程中发挥了主要作用。  相似文献   

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