Purification and Some Enzymatic Properties of Acid Protease A and B of Scytalidium lignicolum ATCC 24568

Three kinds of acid proteases were purified from the culture filtrate of Scytalidium lignicolum ATCC 24568. About 3 mg of A–1, 6 mg of A–2 and 60 mg of B were obtained from one liter of culture broth. These purified enzymes were monodisperse by physicochemical criteria such as ultracentrifugal analysis and disc electrophoresis.

A–1 and A–2 were very similar to each other on their enzymatic properties except the small difference of isoelectric point. A–1 and A–2 were active between pH 3.0~3.5 toward casein, and stable between pH 2.5 and 5.5 for 20 hr at 37°C. Both enzymes were strongly inhibited by NBS, but not by EDTA, DFP and sulfhydryl reagents.

B was most active at pH 2.0, and stable at pH values between 1.5 and 5.0. This enzyme was also inhibited by NBS and KMnO₄, but not by EDTA, DFP and sulfhydryl reagents.

The molecular weights and isoelectric points of A–1, A–2 and B were 43,000, pH 3.6; 43,000, pH 3.8 and 22,000, pH 3.2, respectively.

A–1 and A–2 were not inhibited by S–PI and synthetic pepsin inhibitor such as diazoacetyl-dl-norleucine methylester (DAN) and 1,2-epoxy-3-(p-nitrophenoxy)-propane (EPNP). B was inhibited by EPNP, but not by S–PI and DAN.     

Expression and Secretion of Scytalidopepsin B,an Acid Protease from Scytalidium lignicolum,in Yeast

An expression and secretion system for scytalidopepsin B, an acid protease from Scytalidium lignicolum, was constructed in yeast. Saccharomyces cerevisiae AH22 was transformed with an yeast-E. coli shuttle vector, pAM82, in which an yeast invertase signal segment and the cDNA encoding the pro- and mature enzyme regions were inserted. The transformant was found to secret a pepstatin-insensitive acid protease, when cultured aerobically in a low phosphate (Pi) medium. Amino terminal amino acid sequencing analysis indicated that the recombinant acid protease was accurately processed and secreted as a mature form.     

香菇菌丝后熟转色形成的活性氧作用与自噬特征

为了解活性氧(reactive oxygen species,ROS)在香菇菌丝后熟转色形成中的作用及其自噬细胞学特征,以香菇工厂化菌株KS11为研究材料,分析其在菌丝后熟转色过程中4个时间点(30、45、60、75 d)的活性氧含量(ROS)、丙二醛(MDA)含量、NADPH氧化酶浓度、抗氧化酶活性以及外源活性氧和DPI对其影响的表型试验,利用透射电镜观察该过程菌丝细胞自噬特征变化,并运用实时荧光定量PCR对自噬基因Atg8的表达水平进行比较分析。结果表明:(1) H₂O₂作为主要的活性氧因子在菌丝后熟转色形成中呈现显著动态变化,后熟转色过程中不断升高,并在转色中第60天呈高峰值。(2) NADPH氧化酶浓度与H₂O₂含量变化呈紧密正相关。(3)外源施加一定浓度H₂O₂显著促进香菇菌丝后熟转色,且DPI作为NADPH氧化酶抑制剂显著抑制了香菇菌丝后熟转色的发生。(4)香菇菌丝后熟转色过程中,细胞自噬特征逐渐增强,并在转色中后期最显著。上述结果表明以H     

香菇C_(91-3)转录本Unigene14872基因Pkinase结构域的克隆表达及其生物信息学分析

旨在克隆香菇C91-3转录本Unigene 14872基因中的Pkinase结构域,并对其进行生物信息学分析。提取香菇C91-3菌丝体中总RNA,根据转录组测序结果,用3'-Full RACE、5'-Full RACE方法获得Unigene 14872基因全长。用NCBI对其进行生物信息学分析,预测其具有丝氨酸/苏氨酸蛋白激酶(Pkinase)结构域。设计引物PCR扩增Pkinase结构域,然后克隆至pET32a(+)载体,再热转化至JM109中保存并进行质粒测序。重组质粒pET32a(+)-Pkinase构建成功,并成功诱导表达重组蛋白,初步鉴定该蛋白具有抗肿瘤活性,为进一步研究重组蛋白的活性及作用机制奠定基础。     

香菇锰过氧化物酶基因（LeMnP1）生物信息学分析及高温胁迫下的表达

本研究对香菇锰过氧化物酶基因（manganese peroxidase 1,MnP1）进行了生物信息学分析,选用香菇出发菌株18和诱变菌株18N44作为实验材料,经测定高温胁迫后菌丝恢复生长速度,分析了高温胁迫过程中香菇锰过氧化物酶基因（LeMnP1）的表达水平及酶活性。结果表明：LeMnP1定位于细胞外,该蛋白属于过氧化物酶超家族,与其他担子菌具有较高的同源性。18N44在高温胁迫后其恢复能力明显强于18;在高温胁迫过程中,18N44中LeMnP1的基因表达水平均高于18,呈现先上升后下降的趋势;在未处理时,18N44的MnP活性显著高于18,随着高温胁迫时间延长,其酶活性显著下降,18的LeMnP1基因表达量及酶活性在整个高温胁迫过程中基本维持稳定。据此推测锰过氧化物酶基因的相对高表达,可能是18N44高温胁迫后恢复生长快的原因之一。     

A streamlined, bi-organelle, multiplex PCR approach to species identification: Application to global conservation and trade monitoring of the great white shark, Carcharodon carcharias

The great white shark, Carcharodoncarcharias, is the most widely protectedelasmobranch in the world, and is classified asVulnerable by the IUCN and listed on AppendixIII of CITES. Monitoring of trade in whiteshark products and enforcement of harvest andtrade prohibitions is problematic, however, inlarge part due to difficulties in identifyingmarketed shark parts (e.g., dried fins, meatand processed carcasses) to species level. Toaddress these conservation and managementproblems, we have developed a rapid, moleculardiagnostic assay based on species-specific PCRprimer design for accurate identification ofwhite shark body parts, including dried fins. The assay is novel in several respects: Itemploys a multiplex PCR assay utilizing bothnuclear (ribosomal internal transcribed spacer2) and mitochondrial (cytochrome b) locisimultaneously to achieve a highly robustmeasure of diagnostic accuracy; it is verysensitive, detecting the presence of whiteshark DNA in a mixture of genomic DNAs from upto ten different commercially fished sharkspecies pooled together in a single PCR tube;and it successfully identifies white shark DNAfrom globally distributed animals. Inaddition to its utility for white shark trademonitoring and conservation applications, thishighly streamlined, bi-organelle, multiplex PCRassay may prove useful as a general model forthe design of genetic assays aimed at detectingbody parts from other protected and threatenedspecies.     

Multiplex PCR and RFLP approaches for identification of rabbitfish (Siganus) species using mitochondrial gene regions

Molecular assays are described for the identification of six rabbitfish (Siganus) species. A multiplex PCR assay using primers targeting the mitochondrial cytochrome b region simultaneously identifies four species: Siganus canaliculatus, S. fuscescens, S. javus, and S. spinus. Subsequent RFLP assays of multiplex amplicons differentiate between S. virgatus and S. corallinus based on diagnostic fragments from the mitochondrial cytochrome oxidase I region. Assays were validated with known specimens demonstrating accuracy of the molecular identification. Applied to morphologically indistinguishable early developmental stages, these assays can facilitate studies on species-specific spatio-temporal patterns of larval dispersal and population connectivity to aid fishery management.     

伞菌DNA简易序列分析引出的思考

目的:伞菌物种以子实体形态特征为分类依据,研究以菌丝体代替子实体进行种质资源鉴定的遗传证据。方法:以常见的食用伞菌香菇、平菇子实体的不同部位组织及其分离菌丝体为供试材料,制备了12个随机引物介导的RAPD-PCR指纹,把DNA指纹图谱转化为简易的序列数据,经生物信息处理软件比较分析。结果:香菇不同菌株子实体DNA相似性系数在0.886～0.986之间,平菇不同菌株子实体DNA相似系数在0.779～0.976之间。对于供试的单个子实体而言,香菇和平菇子实体的菌盖、菌褶、菌柄及其组织分离菌丝体,与以此为菌种栽培得到的子实体相比较,所获得的有限DNA指纹图谱全部相同。结论:揭示了香菇和平菇不同菌株的遗传多样性,初步反映了伞菌不同发育阶段在分子水平上的遗传变异和亲缘关系,争论了以菌丝体代替子实体使用RAPD手段进行种质资源鉴定与系统发育分析引出的真菌遗传问题。     

从脱毒棉籽粕和食用菌中提取食用核酸的工艺探讨

目的：利用核酸含量高的植物原料,探索食用核酸提取新工艺。方法：以脱毒棉籽粕粉和平菇子实体为原料,采用去离子水一次抽提-等电点与乙醇分次沉析法和NaCl溶液分次抽提-等电点与乙醇一次沉析法从中提取核酸。结果：两种原料采用两种不同工艺,提取核酸制品的A260／A280值均大于1．6,其中水提法大于1．6,盐提法大于1．8。核酸提取率在1．0％以上,其中水提法为1．07％和2．05％,盐提法为1．74％和2．48％。结论：脱毒棉籽粕粉和平菇子实体均是提取核酸的良好原料,后者优于前者。水提法和盐提法均适合于对脱毒棉籽粕粉和平菇子实体中食用核酸的提取,盐提法更为合理。     

Studies on the Structure of Gluco-oligosaccharides Obtained from the Partial Acid Hydrolyzate of Sclerotia of Sclerotinia libertiana

The defatted sclerotia powder was partially hydrolyzed with dilute acid, and the material obtained was fractionated by carbon column chromatography, separated into two disaccharides, three trisaccharides and three tetrasaccharides, respectively. In these hydrolyzates α, α-trehalose, Iaminaribiose, and gentiobiose were identified.     

Molecular Detection of <Emphasis Type="Italic">Xenostrobus securis</Emphasis> and <Emphasis Type="Italic">Mytillus Galloprovincialis</Emphasis> Larvae in Galician Coast (Spain)

The mussel species Xenostrobus securis from New Zealand was detected in the Spanish coast recently, in the mouth of the Verdugo River into the Vigo Ria. In view of the great importance of the farm mussel sector in this region, the presence of this alien species greatly concerned producers and administration authorities, because of its potential medium- or long-term effects on the autochthonous species, Mytilus galloprovincialis, an important marine resource widely exploited in this location. The goal of this study was to develop a DNA-based technique to identify X. securis and M. galloprovincialis larvae in plankton samples, which would allow monitoring for the presence of X. securis in different points of the Vigo Ria. The techniques used were simplex and multiplex polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP), and fragment analysis. The application of this system to planktonic samples could be an effective means to assess the presence of the alien species, allowing monitoring if its dispersion is increasing, or on the contrary, if its distribution is restricted to the mouth of the Verdugo River, where X. securis was first detected. In addition, the application of this system at different times could be useful to assess the presence of larvae of these two species in the plankton.     

Rapid identification of Bactrocera latifrons (Dipt., Tephritidae) by real-time PCR using SYBR Green chemistry

Abstract:  The solanum fruit fly, Bactrocera latifrons (Hendel), is a major agricultural pest in Asia and Hawaii, and it is important to prevent its widespread invasion in plant quarantine. In this study we introduced a real-time polymerase chain reaction (PCR) essay, using SYBR Green I dye, to rapidly identify B. latifrons on an ABI PRISM 7700 sequence detection system. A latifron-specific PCR primer set was obtained based on mtDNA COI gene of B. latifrons . Nine Bactrocera fruit flies, B. latifrons , Bactrocera dorsalis , Bactrocera papayae , Bactrocera carambolae , Bactrocera philippinensis , Bactrocera occipitalis , Bactrocera correcta , Bactrocera cucurbitae and Bactrocera tau , were used to determine the specificity of primers lati1 and lati2. A series of genomic DNA dilutions of B. latifrons (0.01, 0.1, 1, 10, 20, 40 and 100 ng) were used to assess the sensitivity of the SYBR Green PCR. Template DNA concentration was one of the sources of variability in cycle threshold values (CT) and the optimum DNA concentration was between 1 and 20 ng. Genomic DNA isolated from larvae, pupae and adult specimens of B. latifrons were used to assess the specificity of the SYBR Green PCR. Melting curve analysis and agarose gel electrophoresis was employed to check the specificity of PCR products. Similar amplification plots were obtained using DNA from the three different stages of B. latifrons with primer set lati1/lati2. The melting temperature ( T _m) of PCR products was 77.5 ± 0.1°C, and the length of the amplified fragment 366 bp. Given the specificity and sensitivity of the assay, combined with high speed, low cost and the possibility of automating, SYBR Green PCR can be used as a rapid and specific technique for pest species identification in plant quarantine.     

基于Taqman-MGB探针的亚稀褶红菇实时荧光定量PCR检测方法

本研究建立了一种基于Taqman-MGB探针的亚稀褶红菇Russula subnigricans实时荧光定量PCR检测方法。根据亚稀褶红菇与其近似种的内转录间隔区（internal transcribed spacers,ITS）序列差异,设计合成1对引物和1条特异性Taqman-MGB探针,并用常见有毒红菇种类进行验证。结果显示,引物特异性良好,仅亚稀褶红菇出现荧光信号,完成整个检测过程只需2h。该法能够为毒蘑菇中毒的快速检测提供技术支持。     

A novel multiplex PCR assay for the identification of Indian crocodiles

Illegal hunting has been a major threat for the survival of wildlife fauna, including the three crocodile species that India harbours: Crocodylus palustris, Crocodylus porosus and Gavialis gangeticus. Although law prevents trade on these species, illicit hunting for trade continues to threaten the survival of these endangered species; conservation strategies therefore require a rapid molecular identification technique for Indian crocodiles. A multiplex polymerase chain reaction (PCR) assay with species-specific primers, considered as one of the most effective molecular techniques, is described herein. The primers were designed to yield species-specific sized amplicons. The assay discriminates the three Indian crocodile species unambiguously within a short time period using only simple agarose gel electrophoresis. We recommend this multiplex PCR assay to be used in the identification of Indian crocodile species.     

Identification of New World Leishmania species from Peru by biochemical techniques and multiplex PCR assay

We have characterized diverse strains or species of Leishmania isolated in humans that are currently circulating throughout Peru, by means of isoenzymatic characterization, kDNA analysis by restriction enzymes, and multiplex PCR assay. The cluster analysis gave five groups. Cluster 1 includes L. (L.) donovani together with the isolates LP4 and LP7, forming the donovani complex. Thus, this complex corresponds to the New World visceral form, L. (L.) chagasi. Cluster 2 is formed by the isolates LP1-LP3, LP6, LP10, LP9, and LP11, phylogenetically intermediate between Cluster 1 and Cluster 3, or they can be treated as hybrids. Cluster 3 is divided into two subgroups: one formed by L. (V.) peruviana, together with the isolates LP14 and LP5, and the second one formed by L. (V.) brazilensis and the isolate LP8. These two subgroups form part of the brazilensis complex. The three strains of L. (L.) infantum [L. (L.) infantum I and II and la LSI] make up Cluster 4. In Cluster 5, we include the three Mexican strains (LM1-LM3) forming one subgroup while we would place L. (L.) amazonensis in another subgroup. These two subgroups would comprise the complex mexicana.     

Rapid and Simultaneous Identification of Body Parts from the Morphologically Similar Sharks Carcharhinus obscurus and Carcharhinus plumbeus (Carcharhinidae) Using Multiplex PCR

Many commercially exploited carcharhinid sharks are difficult to identify to species owing to extensive morphological similarities. This problem is severely exacerbated when it comes to identifying detached shark fins, and the finless and headless shark carasses typically sold in markets. To assist in the acquisition of urgently needed conservation and management data on shark catch and trade, we have developed a highly streamlined approach based on multiplex polymerase chain reaction (PCR) that uses species-specific primers derived from nuclear ribosomal ITS2 sequences to achieve rapid species identification of shark body parts. Here we demonstrate the utility of this approach for identifying fins and flesh from two globally distributed, morphologically very similar carcharhinid sharks (Carcharhinus obscurus and Carcharhinus plumbeus) intensively targeted in fisheries worldwide, and often confused for each other even as whole animals. The assay is conducted in a 4-primer multiplex format that is structured to simultaneously achieve the following efficiency and cost-reduction objectives: it requires only a single-tube amplification reaction for species diagnosis, it incorporates an internal positive control to allow detection of false-negative results, and it is novel in that it allows species identification even when DNAs from two species are combined in the same tube during the PCR reaction. The latter innovation reduces the required effort for screening a set of unknown samples by 50%. The streamlined approach illustrated here should be amenable for use in a shark conservation and management context where large numbers of samples typically need to be screened; the approach shown may also provide a model for a rapid diagnostic method applicable to species identification in general. Received September 15, 2000; accepted December 15, 2000     

RFLP and semi-multiplex PCR-based identification of four eel species from the south-western Indian Ocean region

To make the assignment of elvers to species more easily attainable and reliable, unpublished and recently published mitochondrial DNA sequences were used to design molecular determination approaches not based on sequencing. Two methods were proposed based on 16SrRNA single nucleotide polymorphism analysis, which improve identification of the four eel species living in the south-western Indian Ocean region.     

一株番茄表面着生醋酸菌的分离鉴定及产酸条件优化

醋酸菌是食醋酿造过程中的关键菌种,性能优良的菌种对于产品品质的提升意义重大。以分离自番茄表面的产醋酸菌为研究对象,通过生理生化指标鉴定、16S rRNA编码基因比对及系统发育树构建等方法对其种类进行鉴定,并通过单因素实验、正交实验对鉴定为醋酸菌的菌株进行培养条件优化。结果表明,所分离的3株醋酸生产菌中,BQ-1被鉴定为醋酸杆菌属(Acetobacteraceae),在以酵母粉为主要氮源,蔗糖为主要碳源的培养基中,其最高产酸量为1823 g·L-1。由于该菌株在番茄表面具有很强的生长能力,因此有望应用于番茄果醋的酿造。     

A multiplex PCR for detection of genes encoding exfoliative toxins from Staphylococcus hyicus

AIMS: To develop a multiplex PCR for detection of genes encoding the exfoliative toxins ExhA, ExhB, ExhC and ExhD from Staphylococcus hyicus and to estimate the prevalence of exfoliative toxins among Staph. hyicus isolates from Danish pig herds with exudative epidermitis (EE). METHODS AND RESULTS: A multiplex PCR employing specific primers for each of the genes encoding four different exfoliative toxins was developed and evaluated using a collection of Staph. hyicus with known toxin type and a number of other staphylococcal species. A total of 314 Staph. hyicus isolates from pigs with EE were screened by multiplex PCR and the combined results of the present and previous investigations showed that ExhA, ExhB, ExhC and ExhD was found in 20, 33, 18 and 22%, respectively, of 60 cases of EE investigated. CONCLUSIONS: This study has provided a new tool for detection of toxigenic Staph. hyicus and a more comprehensive picture of the prevalence of the Staph. hyicus exfoliative toxins in Danish pig herds. SIGNIFICANCE AND IMPACT OF THE STUDY: The multiplex PCR can be used in studies on the prevalence of toxigenic Staph. hyicus elucidating the epidemiology of EE in pigs. The multiplex PCR is currently being used for selection of Staph. hyicus isolates for production of autogenous vaccine.