运用差异展示分离特异性表达的基因

在高等生物中含有约100000个不同的基因,其中仅有15％的基因在任何个体细胞中均表达．因此分离特异的目的基因便显得十分重要.差异展示是通过部分扩增mRNA的逆转录产物、经测序胶电泳,分离到差异性表达的基因.它与消减杂交相比是分离特异表达基因的更有效的手段．虽然这种方法在实际运用中存在着这样或那样的困难,但随着对这种技术的不断改进,它将会有越来越广泛的用途．     

mRNA差异展示研究胃癌差异表达基因

以胃癌细胞株GC7901与胃粘膜细胞株GES-1为对比材料,利用mRNA差异展示技术,研究胃粘膜至胃癌过程的差异表达基因,为建立胃癌预警系统打下基础.获得8个未知的与胃癌相关的cDNA,命名为GCYS-1,2,3,4,5,6,7,20,完成其克隆及序列分析,RNA印迹证实GCYS-1至7片段与GCYS-20基因在GC7901细胞株中呈高表达,在GES-1细胞株中呈低表达.此8个序列在GenBank数据库中登录,接受号为AF054162～AF056168及AF219140.     

Homeobox Genes Expressed During Echinoderm Arm Regeneration

Regeneration in echinoderms has proved to be more amenable to study in the laboratory than the more classical vertebrate models, since the smaller genome size and the absence of multiple orthologs for different genes in echinoderms simplify the analysis of gene function during regeneration. In order to understand the role of homeobox-containing genes during arm regeneration in echinoderms, we isolated the complement of genes belonging to the Hox class that are expressed during this process in two major echinoderm groups: asteroids (Echinaster sepositus and Asterias rubens) and ophiuroids (Amphiura filiformis), both of which show an extraordinary capacity for regeneration. By exploiting the sequence conservation of the homeobox, putative orthologs of several Hox genes belonging to the anterior, medial, and posterior groups were isolated. We also report the isolation of a few Hox-like genes expressed in the same systems.     

Colonization of Sclerotinia sclerotiorum sclerotia by a biocontrol isolate of Trichoderma harzianum,and effects on myceliogenic germination

Sclerotia of Sclerotinia sclerotiorum were incubated on cultures of Trichoderma harzianum. Myceliogenic germination decreased by 50% within 1 day and continued to decrease over time. Quantitative PCR showed a decrease in Sclerotinia DNA for older sclerotia, but not fresh sclerotia. Trichoderma DNA increased and persisted inside older sclerotia but not fresh sclerotia.     

Cytostatic effect of L-lysine-alpha-oxidase from Trichoderma harzianum Rifai and Trichoderma viride

L-lysine-alpha-oxidase, a new fungal enzyme catalyzing oxidative L-lysine deamination, was shown to have an inhibitory effect on the in vitro synthesis of DNA, RNA and proteins in human carcinoma ovarian (CaOv) cells.     

Chromosomal Mapping of HSPCB and MYL1 Expressed Abundantly in the Bovine Fetus

Abstract

Chromosomal mapping of expressed sequence tags for HSPCB and MYL1 expressed abundantly in the bovine fetus was performed by analyzing bovine/murine somatic cell hybrid DNAs with polymerase chain reaction (PCR) using primers specific for those 3^′‐untranslated regions. HSPCB and MYL1 were assigned to bovine chromosomes 23 and 2, respectively.     

Rapid and Reliable Differential Display from Minute Amounts of Tissue: Mass Cloning and Characterization of Differentially Expressed Genes from Loblolly Pine Embryos

Performing RNA differential display analysis on small tissue samples is difficult since much RNA, the initial template for the reaction, is lost during conventional isolation procedures. We have developed a rapid method which employs oligo-dT beads to capture mRNA from cell lysates. Subsequent reactions are primed directly from the beads, thus RT and PCR reactions can be completed within a few hours of tissue harvest. This approach allows us to perform differential display on a single pine embryo. We describe strategies for distinguishing classes of co-migrating bands excised from differential display gels and outline a PCR-based method for confirming differential expression of large numbers of cloned bands in cases where RNA quantities are limiting.     

利用mRNA荧光差异显示技术规模化筛选棉纤维特异表达基因

以陆地棉（Gossypium hirsutum L.）品种TM-1开花后9d、21d、27d三个不同发育时期的棉花纤维为材料,利用mRNA荧光差异显示 (FDD) 技术,筛选到109个差异显示的cDNA片段。在此基础上,结合两轮反Northern杂交筛选和Northern杂交分析,获得了多个仅在棉花纤维细胞中特异表达或在纤维中优先表达基因的cDNA片段,序列测定和数据库搜索分析表明,这些cDNA片段中的多数还未有报道。本工作为克隆上述基因的全长cDNA,并进一步研究它们在棉纤维发育中的功能奠定了基础。     

Production and preliminary characterization of RNA-depolymerase from Trichoderma harzianum

Trichoderma harzianum produced RNA-depolymerase with maximum activity after 72 to 120 h of growth. Addition of K₂HPO₄ repressed enzyme production by the fungus. The optimal activity was at pH 7.8 and 40 to 50°C. The enzyme was stable at pH 3.2 to 9.0 and 80% of activity remained after 60 min at 40°C. EDTA and p-chloromercuribenzoate had no effect on the enzyme activity.E.S. Vasileva-Tonkova is with the Institute of Microbiology, Bulgarian Academy of Sciences, Acad, G. Bonchev str., B1. 26, 1113 Sofia, Bulgaria     

Purification and characterization of two d-xylanases from Trichoderma harzianum

Two endo-1,4-β-d-xylanases (1,4-β-d-xylan xylanohydrolase, EC 3.2.1.8) from Trichoderma harzianum E58 have been purified by ultrafiltration and chromatography on carboxymethyl-Sepharose, phenyl-Sepharose and Sephadex G-75. The d-xylanases were shown to be homogeneous by the criteria of dodecyl sulphate polyacrylamide gel electrophoresis and isoelectric focusing. The molecular weights were estimated to be 20 000 and 29 000, with pl values of 9.4 and 9.5, respectively. Typically, 456 mg of the 20 000 dalton and 1.9 mg of the 29 000 dalton d-xylanases were purified from 4.2 litre of culture filtrate with specific activities of 370 and 75 U mg^?1, respectively. Optimum d-xylanase activities were obtained when the enzymes were incubated at pH 5, 50°C, for the 20 000 dalton protein and pH 5, 60°C for the 29 000 dalton protein.     

宫颈癌差异表达基因筛选及功能分析

目的:筛选参与宫颈癌发生、发展的关键基因,为临床诊疗提供新的靶点。方法:在NCBI-GEO数据库中筛选多组宫颈癌基因表达检测数据集,利用GEO2R分析工具筛选各组数据集的差异表达基因;应用R分析筛选不同数据集之间共有的差异表达基因;利用DAVID在线分析对差异表达基因进行功能聚类和通路分析;利用STRING分析差异表达基因编码蛋白之间的相互作用关系。结果:共选择6组表达数据集,筛选得到59个差异表达基因(宫颈癌组织vs正常组织),表达差异至少达2倍,其中包含50个表达上调基因及9个表达下调基因。这些差异表达基因参与细胞周期、DNA复制、细胞分裂等生物进程。蛋白互作分析表明,这些差异表达基因多数存在相互作用。结论:利用生物信息学方法对不同来源的基因检测数据进行整合分析,有助于更准确的筛选对宫颈癌发生、发展过程具有重要作用的关键基因,本文筛选的宫颈癌差异基因为进一步研究宫颈癌发生、发展的分子机制及临床诊疗提供思路。     

Viability of dry Trichoderma harzianum spores under storage

Spores of the potential biocontrol agent Trichoderma harzianum P1 were prepared without (M1) and with heat shock (40?°C for 90?min) after fermentation (M2), filtered into a paste and dried over silica gel. M1 and M2 exhibited high viability (55%) and similar initial trehalose contents (4.0 and 5.4%, respectively) after slow drying. No significant differences in viability were found between treatments during storage for 110 days under different temperatures, T (8, 33 and 42?°C) and water activities, a _w (0.03, 0.33 and 0.75). Viability of spores, after storage at a _w =0.03 were 100 and 70% for 8 and 33?°C, respectively. During storage, decrease in trehalose content and viability was faster at a _w =0.75 and 42?°C. Loss of viability was modeled by a first order kinetic model depending on 1/T and a _w . M2 (with heat shock) showed slightly higher trehalose contents than M1 which resulted in 100% viability after 52 days at 8?°C.     

Regulation of chitinase synthesis in Trichoderma harzianum.

The production of chitinase by Trichoderma species is of interest in relation to their use in biocontrol and as a source of mycolytic enzymes. Fourteen isolates of the genus were screened to identify the most effective producer of chitinase. The best strain for chitinase was Trichoderma harzianum 39.1, and this was selected for study of the regulation of enzyme synthesis. Washed mycelium of T. harzianum 39.1 was incubated with a range of carbon sources. Chitinase synthesis was induced on chitin-containing medium, but repressed by glucose and N-acetylglucosamine. Production of the enzyme was optimal at a chitin concentration of 0.5%, at 28 degrees C, pH 6.0 and was independent of the age of the mycelium. The synthesis of chitinase was blocked by both 8-hydroxyquinoline and cycloheximide, inhibitors of RNA and protein synthesis, respectively. The mode of chitinase synthesis in this fungus is discussed.     

Hypocrea lixii, the teleomorph of Trichoderma harzianum

Cultures derived from ascospores of Hypocrea lixii (= H. nigricans, H. lentiformis) produced the morphological species Trichoderma harzianum in pure culture. Trichoderma harzianum, the most commonly found species of the genus, is also one of the most species frequently used in biocontrol of plant pathogens. It has not been connected previously to a teleomorph. The connection was confirmed by DNA sequence analysis. Similar to the anamorph, the teleomorph collections have a wide geographic distribution. Described in the 19^th century, Hypocrea lixii is epitypified by a collection from Thailand.     

Genetic diversity and vegetative compatibility among Trichoderma harzianum isolates

Trichoderma harzianum is the collective name of a set of asexual fungal strains which exhibit heterogeneity in genome structure, DNA sequence and behavior. Contour-clamped homogeneous field (CHEF) electrophoresis of the chromosomes of ten isolates of T. harzianum revealed six clearly distinct electrophoretic karyotypes. Of the ten isolates analyzed, four (GH12, G109, Y and YF) could be classified in a single group with identical karyotypes, while the strains T35 and 315 formed a second group. The genome size characteristic of the different isolates fell into a broad range varying from 29.6 to 56.1?Mb. Gene assignments to the resolved chromosomes showed that all genes analyzed were localized on equivalent chromosomes in the isolates belonging to the same group. Analysis of randomly amplified polymorphic DNAs from the ten isolates confirmed the classification into groups and allowed us to distinguish between isolates T35 and 315, as well as between isolates GH12, G109, Y and YF. Direct confrontation assays using isolates of the same group showed compatible interactions, whereas the same experiment carried out with isolates of different groups showed an incompatible interaction characterized by an area of cell damage. Microscopic observation of the compatible interactions showed hyphal fusions between the isolates, similar to those described for vegetative compatible groups in other fungi. The molecular karyotypes correlated well with the compatibility of the isolates. In addition, we have evaluated both electrophoretic karyotype and randomly amplified polymorphic DNAs analysis as criteria for grouping isolates within the genus according to their capacity for biocontrol of plant pathogens.     

黄瓜抗黑星病相关基因的差异表达分析

以抗黑星病黄瓜材料HX1为试材,接种黑星病菌（Cladosporium cucumerinum）2h、8h、20h、32h和72h的叶片作为试验方（Tester）,相应的未接种叶片作为对照方（Driver）,利用SSH技术,构建了黑星病菌侵染初期的正向和反向cDNA-SSH文库。用巢式引物PCR检测插入片段,获得了200个阳性克隆,通过测序,除去重复序列,共得到105个Unique ESTs。与非冗余蛋白数据库进行BLASTx比对,结果显示,17条ESTs未找到同源序列,88条非重复序列和已知基因的同源性较高,占全部ESTs序列的83.8%,其中86条ESTs与非冗余蛋白数据库已知功能的蛋白具有高度的相似性。结合高密度点阵膜杂交差异筛选,阳性率为75.0%。经初步分析这些序列的功能,差异表达的ESTs功能涉及能量和基础代谢、信号转导、蛋白和核酸代谢、光合作用及逆境中特异表达的基因等方面。为研究黄瓜抗黑星病基因提供了依据。