Identification of Alternaria brassicicola genes expressed in planta during pathogenesis of Arabidopsis thaliana

Alternaria brassicicola is a necrotrophic fungal pathogen that causes black spot disease on cruciferous plants including economically important Brassica species. The purpose of this study was to identify fungal genes expressed during infection of Arabidopsis. In order to identify candidate genes involved in pathogenicity, we employed suppression subtractive hybridization (SSH) between RNA isolated from A. brassicicola spores incubated in water and on the leaf surface of the Arabidopsis ecotype Landsberg. Two populations of cDNA were created from total RNA extracted after 24h when approximately 80% of the spores had germinated either on the leaf surface or in water. Following SSH, expression of clones was examined using dot-blot macro-arrays and virtual Northern blots. 47 cDNA clones differentially expressed between Alternaria infected Arabidopsis leaves and spore germination in water were selected for sequencing. Seventy-seven percent (36) of the cDNAs had significant homology to fungal sequences from databases examined, including available fungal genomes, while 13% (11) had no homology to sequences in the databases. All 36 genes had significant matches with genes of fungal origin, while 11 genes did not have significant hits in the databases examined. Five sequences were expressed on the plant leaf surface but not during spore germination in water according to virtual Northern blots. These five cDNAs were predicted to encode a cyanide hydratase, arsenic ATPase, formate dehydrogenase, major Alternaria allergen, and one unknown. RT-PCR was used to examine the expression of these five genes during infection of Brassica oleraceae var. capitata (cabbage), in vitro growth in nutrient rich media, and infection of Arabidopsis thaliana. Four of these genes are expressed in the nutrient rich medium, while the unknown gene P3F2 was only expressed during plant infection. The results of this study provide the first insight into genes expressed during A. brassicicola infection of Brassica species that may be involved in fungal pathogenesis.     

Differential gene expression in fully-grown oocytes between gynogenetic and gonochoristic crucian carps.

Silver crucian carp (Carassius auratus gibelio) is a unique triploid bisexual species that can reproduce by gynogenesis. As all other gynogenetic animals, it keeps its chromosome integrity by inhibiting the first meiosis division (no extrusion of the first pole body). To understand the molecular events governing this reproduction mode, suppression subtractive hybridization was used to identify the genes differentially expressed in fully-grown oocytes of the gynogenetic and gonochoristic crucian carp (gyno-carp and gono-carp). From two specific subtractive cDNA libraries, the clones screened out by dot blots and virtual Northern blots were chosen to clone full-length cDNA by RACE. Four differentially expressed genes were obtained. Two are novel genes and are expressed specifically in the oocytes. The gyno-carp stores much more mRNA of cyclin A2, a new member of the fish A-type cyclin gene, in its fully-grown oocyte than in the gono-carp. The last gene is histone H2A. The histone H2As of these two closely related crucian carps are quite different in the C-terminus. Preliminary characterization of the four genes has been analyzed by nucleotide and deduced amino acid sequence and Northern analysis.     

Cloning and characterization of full-length human ribosomal protein L15 cDNA which was overexpressed in esophageal cancer

The aim of this investigation was trying to identify the genes differentially expressed in esophageal cancer. By combining suppression subtractive hybridization (SSH) with reverse Northern high density blots, a gene named EC45 was obtained, which dramatically overexpressed in 70% esophageal cancer (18/26). EC45 was mapped to 3p12-3p11.2 by radiation hybrid mapping (RH mapping). The putative full length EC45 cDNA (1987 bp) was identified by cDNA libraries screening of esophageal cancer. EC45 encoded 204 amino acids, and it shared a 100% similarity with ribosomal protein L15 (635 bp, mRNA) in ORF, but no similarity in 5' UTR or 3' UTR. Northern blot panel of multiple adult human normal tissues showed EC45 distributed in almost normal tissues tested. All these data suggested that EC45, encoding ribosomal protein L15 and overexpressing in esophageal cancer might play a possible role in carcinogenesis of esophagus.     

新型小鼠淋巴组织特异性肌动蛋白结合蛋白相关序列cDNA克隆

 应用抑制性差减杂交技术 ( SSH)克隆两种不同小鼠胸腺基质细胞的差异表达基因 ,获得新基因片段 C55.通过 Gen Bank检索及 RT- PCR扩增出一个全长 1 .4kb的 c DNA.杂交分析认为它是一个完整的 c DNA序列 .c DNA序列分析表明 ,它拥有一个 636bp的开放读码框架 ,编码 2 1 2个氨基酸 .同源序列比较发现 ,它编码一个肌动蛋白相关蛋白的新成员 ,该序列与多种已知的肌动蛋白相关蛋白 SM2 2 α及其同源蛋白在氨基酸水平上有 62 %～ 95%的同源性 .Northern杂交分析显示 ,该基因 m RNA转录本在两种不同胸腺基质细胞中的表达存在显著差异 . RT- PCR分析显示 ,该基因特异表达于小鼠淋巴相关组织中 ,而在非淋巴组织中无表达 .     

Size-selection of cDNA libraries for the cloning of cDNAs after suppression subtractive hybridization

Here we describe the establishment of size-selected cDNA libraries for the cloning of full-length cDNAs that were initially identified by suppression subtractive hybridization (SSH) technology as being differentially expressed. First, the SSH-cDNA fragments were used as 32P-probes to verify their level and differential pattern of expression by virtual Northern and to establish their corresponding full-length cDNA size. Second, cDNAs were separated by size on agarose gels and used to construct size-selected cDNA plasmid libraries, which were then screened by colony hybridization with the SSH-cDNA fragments. We conclude that the described approach complements SSH technology by allowing efficient cloning and characterization of the corresponding full-length cDNA from any desired cell type or species. This approach will give researchers the ability to specifically target and study differentially expressed genes in an efficient manner for functional genomic studies.     

Suppression subtractive hybridization cloning of cDNAs of differentially expressed genes in dovetree (<Emphasis Type="Italic">Davidia involucrata</Emphasis>) bracts

A subtracted cDNA library forDavidia involucrata was constructed using suppression subtractive hybridization (SSH). mRNA isolated from young leaves was used as a “driver,” and mRNAs isolated from young bracts were used as “testers.” The differentially expressed cDNA fragments in bracts were identified by differential screening. Of the 16 clones selected randomly from the screened library, 8 were known genes found in GenBank, and 2 had no similar sequences. Northern blot analysis revealed that the expression level of P1A5 cDNAs selected randomly was dominantly expressed in bracts. This indicates that SSH can be used to clone differentially expressed cDNAs inD. involucrata bracts.     

利用抑制消减杂交分离受褐飞虱取食下调的水稻基因

为了分离受褐飞虱取食抑制的水稻基因,采用抑制消减杂交的方法,以正常生长的水稻幼苗为目标群体,以褐飞虱胁迫32 h的水稻幼苗作为对照群体,构建了含200个重组质粒的SSH cDNA文库.随机挑选50个重组质粒进行反向Northern差异筛选后,再经Northern杂交验证,得到2个受褐飞虱取食抑制的基因:一个是Lhca,编码水稻光系统Ⅰ天线蛋白;另一个基因(bpHd002)与肌苷-5'-单磷酸脱氢酶基因有同源性.以BpHd002为探针筛选水稻幼苗cDNA文库分离出该基因的全长cDNA(BpHd002A).其长度为1 285bp,含有一由519 bp组成的完整的阅读框,编码的蛋白质具有两个CBS结构域.     

Characterization of an ethylene-inducible, calcium-dependent nuclease that is differentially expressed in cucumber flower development

? Production of unisexual flowers is an important mechanism that promotes cross-pollination in angiosperms. We previously identified primordial anther-specific DNA damage and organ-specific ethylene perception responsible for the arrest of stamen development in female flowers, but little is known about how the two processes are linked. ? To identify potential links between the two processes, we performed suppression subtractive hybridization (SSH) on cucumber (Cucumis sativus L.) stamens of male and female flowers at stage 6, with stamens at stage 5 of bisexual flowers as a control. ? Among the differentially expressed genes, we identified an expressed sequence tag (EST) encoding a cucumber homolog to an Arabidopsis calcium-dependent nuclease (CAN), designated CsCaN. Full-length CsCaN cDNA and the respective genomic DNA sequence were cloned and characterized. The CsCaN protein exhibited calcium-dependent nuclease activity. CsCaN showed ubiquitous expression; however, increased gene expression was detected in the stamens of stage 6 female flowers compared with male flowers. As expected, CsCaN expression was ethylene inducible. It was of great interest that CsCaN was post-translationally modified. ? This study demonstrated that CsCaN is a novel cucumber nuclease gene, whose DNase activity is regulated at multiple levels, and which could be involved in the primordial anther-specific DNA damage of developing female cucumber flowers.     

利用抑制差减杂交技术分离马铃薯晚疫病抗性相关基因

以晚疫病病原菌混合小种接种处理48h的马铃薯水平抗性材料(R-gene-free)叶片为目的材料,以未处理材料作为对照,用抑制差减杂交技术构建了一个富集晚疫病抗性相关基因的差减文库。应用反向Northern技术对840个克隆进行斑点杂交筛选,筛选出150个病原诱导后信号明显增强的克隆。26个片段测序结果表明：部分片段基因功能与抗病性明显相关。7个差异表达片段与GenBank EST数据库中已有晚疫病原诱导马铃薯叶片得到的EST有很高同源性(达95％～100％);部分片段核苷酸或氨基酸序列分别与番茄、烟草、拟南芥等的EST序列或氨基酸序列有较高同源性;另有4个基因片段在GenBank EST数据库中未找到明显的同源序列,可能为新发现的基因片段。     

Identification of a differentially expressed gene PPP1CB between porcine Longissimus dorsi of Meishan and Large WhitexMeishan hybrids

To study the molecular basis of heterosis, suppression subtractive hybridization was used to investigate the differences in gene expression between porcine Longissimus dorsi of F1 hybrids Large WhitexMeishan and their female parents Meishan. From two specific subtractive cDNA libraries, the clones selected by reverse Northern high-density blot screening were chosen to clone full-length cDNA by rapid amplification of cDNA ends. An expression-upregulated gene for Meishan skeletal muscle, designated protein phosphatase 1, catalytic subunit, beta isoform (PPP1CB), was identified. Porcine PPP1CB contains an open reading frame encoding 327 amino acid residues with 13 and 1763 nucleotides in the 5' and 3' untranslated regions, respectively. A DNA fragment of 721 nucleotides was amplified and a mutation that creates/disrupts a restriction site for endonuclease RsaI was found. The derived amino acid sequence of PPP1CB has high homology with the PPP1CB of three species, Mus musculus (99%), human (99%) and mouse (100%). The tissue expression analysis indicated that the swine PPP1CB gene is generally expressed in most tissues. The possible role of PPP1CB and its relation to porcine heterosis are discussed.     

用改良消减杂交筛选类风湿性关节炎滑膜细胞中高表达基因

 为了研究类风湿性关节炎 (rheumatoid arthritis,RA)滑膜细胞 (fibroblast- like synovialcells,FLS)过度增殖和破坏软骨的分子机理 ,利用改良消减杂交法以骨性关节炎 (osteoarthritis,OA)病人滑膜细胞为对照 ,筛选 RA滑膜细胞中的高表达基因 .将得到的基因片段克隆入质粒载体 ,通过反向点杂交排除假阳性克隆后 ,将阳性克隆进行核酸序列分析 ,最后用 Northern杂交方法检测一些高表达基因在 RA和 OA病人滑膜细胞中的表达水平 .结果显示 ,共分离到 1 50个 RA高表达基因片段 ,其中长于 1 0 0 0 bp的片段占 8% (1 2 /1 50 ) ,长于 40 0 bp的片段占 36.7% (55/1 50 ) ,在大于 40 0 bp的片段中 ,假阳性率为 2 3.7% (1 3/55) .在测序的 1 8个片段中 ,已知基因有 1 2个 ,其中包括 IGF- 1结合蛋白 (IGFBP)特异性丝氨酸蛋白酶、层粘连蛋白受体和组织蛋白酶 B等 .新序列有 6个 ,其中两个序列分别与 Ring- box蛋白 1和 SON DNA结合蛋白同源 .对 IGFBP特异性丝氨酸蛋白酶、层粘连蛋白受体和组织蛋白酶 B基因的 Northern杂交分析显示 ,在 RA病人滑膜细胞中 ,这些基因的表达水平高于 OA病人滑膜细胞 .这些结果提示 ,这种改良消减杂交法是一种简便有效的分离差异表达基因的方法 ;IGF- 1结合蛋白特异性丝氨酸蛋白酶、层粘     

应用抑制性消减杂交技术筛选流感病毒感染宿主应答基因

从宿主系统寻找病毒感染特异性相关的生物大分子是研究病毒药物靶标和诊断标志物的新方向 .为了筛选宿主细胞中流感病毒感染特异性基因 ,采用抑制性消减杂交技术 (SSH) ,以流感病毒A 鲁防 93 9(H3N2 )感染MDCK细胞及正常MDCK细胞为材料 ,构建病毒感染特异性差减cDNA文库 ,PCR法扩增鉴定其中插入片段大小 .从差减文库中随机挑取 10 0个克隆进行测序 ,用生物信息学方法对其同源性和基因功能进行分析和预测 .结果显示 ,成功构建了流感病毒感染特异性差减cDNA文库 ,文库中cDNA片段长度在 2 5 0～ 10 0 0bp之间 .从文库中随机选取 10 0个克隆测序 ,获得了 95个有效序列 ,经blast同源性分析发现 ,大部分基因为参与宿主细胞能量代谢和蛋白质生物合成过程中的基因 ;其中 19个为无任何功能线索的新基因片段 .流感病毒感染特异性差减cDNA文库的建立和筛选出病毒感染应答候选新基因cDNA片段 ,为发现新型流感病毒药靶和诊断标志物以及病毒感染机制研究打下基础     

用改良消减杂交结合选择性PCR法构建老年性白内障消减cDNA文库

为构建含较多大片段的高质量的老年性白内障消减cDNA文库 ,利用生物素标记、磁珠分离的改良消减杂交法获得差异cDNA .利用选择性PCR法扩增其中大片段差异cDNA ,将其与T 载体进行T A连接并转化入大肠杆菌 ,成功构建老年性白内障消减cDNA文库 .共获得 4 0 0 0余个克隆 ,随机挑取的 2 2个克隆中 ,≥ 10 0 0bp的片段有 7个 ,占 31 8% ,≥ 75 0bp有 15个 ,占 6 8 2 % .将≥ 75 0bp的 15个克隆进行反向点杂交 ,排除其中假阳性克隆 ,阳性克隆经测序并与GenBank比较 ,得到 6个已知基因、1个新基因 ,6个已知基因中 4个为全长基因 ,说明所得cDNA片段较大 ,文库质量较高 .改良消减杂交法结合选择性PCR法可以快速有效地获得大片段高质量的消减cDNA文库 ,为进一步筛选、鉴定老年性白内障致病相关基因奠定了基础     

雌核发育银鲫原肠期胚胎和尾芽期胚胎差异表达基因的呈现

构建了雌核发育银鲫原肠期胚胎和尾芽期胚胎间的抑制性差减杂交cDNA质粒文库。对原肠期’739个和尾芽期816个PCR阳性克隆进行斑点杂交,得到72个原肠期和98个尾芽期斑点杂交阳性克隆。测序和基因数据库比对结果表明：72个原肠期斑点杂交阳性克隆中,包括19个已知基因的cDNA片段和31个没有同源性的cDNA片段;98个尾芽期斑点杂交阳性克隆中,包括52个已知基因的cDNA片段和37个没有同源性的cDNA片段。采用虚拟Northern杂交和RT-PCR证实了部分基因在银鲫胚胎发育过程中的差异表达。这些差异表达基因的呈现为进一步研究银鲫胚胎发育的分子机制奠定了基础。     

Isolation and functional characterization of PgTIP1, a hormone-autotrophic cells-specific tonoplast aquaporin in ginseng

The suppression subtractive hybridization technique was used to identify differentially expressed genes between hormone-autotrophic and hormone-dependent Panax ginseng callus lines. A tonoplast intrinsic protein cDNA (PgTIP1) was found to be highly and specifically expressed in hormone-autotrophic ginseng cells, which was slightly up-regulated by cytokinin while significantly down-regulated when treated with auxin. PgTIP1 encodes a polypeptide of 250 amino acids which shows sequence and structure similarity with tonoplast aquaporins in plants. The water channel activity of PgTIP1 was demonstrated by its expression in Xenopus laevis oocytes. When over-expressed in Arabidopsis thaliana, PgTIP1 substantially altered the plant's vegetative and reproductive growth and development. Arabidopsis plants over-expressing PgTIP1 showed significantly enhanced seed size and seed mass plus greatly increased growth rate compared with those of the wild type. Moreover, the seeds from PgTIP1 over-expressing Arabidopsis had 1.85-fold higher fatty acid content than the wild-type control. These results demonstrate a significant function of PgTIP1 in the growth and development of plant cells.