Construction and characterization of normalized cDNA library of maize inbred Mo17 from multiple tissues and developmental stages

Comprehensive complementary DNA (cDNA) library is a valuable resource for functional genomics. In this study, we set up a normalized cDNA library of Mo17 (MONL) by saturation hybridization with genomic DNA, which contained expressed genes of eight tissues and organs from inbred Mo17 of maize (Zea mays L.). In this library, the insert sizes range from 0.4 kb to 4 kb and the average size is 1.18 kb. 10.830 clones were spotted on nylon membrane to make a cDNA microarray. Randomly picked 300 clones from the cDNA library were sequenced. The cDNA microarry was hybridized with pooled tissue mRNA probes or housekeeping gene cDNA probes. The results showed the normalized cDNA library comprehensively includes tissue-specific genes in which 71% are unique ESTs (expressed sequence tags) based on the 300 sequences analyzed. Using BLAST program to compare the sequences against online nucleotide databases, 88% sequences were found in ZmDB or NCBI, and 12% sequences were not found in existing nucleotide databases. More than 73% sequences are of unknown function. The library could be extensively used in developing DNA markers, sequencing ESTs, mining new genes, identifying positional cloning and candidate gene, and developing microarrays in maize genomics research.     

Analysis of expressed sequence tags of the water flea Daphnia magna.

To study gene expression in the water flea Daphnia magna we constructed a cDNA library and characterized the expressed sequence tags (ESTs) of 7210 clones. The EST sequences clustered into 2958 nonredundant groups. BLAST analyses of both protein and DNA databases showed that 1218 (41%) of the unique sequences shared significant similarities to known nucleotide or amino acid sequences, whereas the remaining 1740 (59%) showed no significant similarities to other genes. Clustering analysis revealed particularly high expression of genes related to ATP synthesis, structural proteins, and proteases. The cDNA clones and EST sequence information should be useful for future functional analysis of daphnid biology and investigation of the links between ecology and genomics.     

47个早期人胚胎低丰度表达基因ESTs筛选及结果分析

构建高质量ｃＤＮＡ文库在基因克隆、ｍＲＮＡ差异展示、表达序列标签测序和基因定位等研究中具有十分重要的作用。为了从早期胚胎中分离人类新基因,构建了受精后３周龄的人ｃＤＮＡ文库,用标记的一链ｃＤＮＡ探针对该文库的６５０８个克隆子进行菌落原位杂交,得到１６７７个无任何杂交信号的低丰度表达克隆子,从中随机挑选了４７个进行５′端部分测序,将测序结果与三大基因库进行序列同源性比较,发现１８个克隆（３８．３％）     

Analysis of expressed genes in ethylene-treated potato leaves using expressed sequence tags

To isolate useful and interesting plant genes in large quantities, random sequencing of cDNA clones from potato leaf library treated with ethylene was performed. Partial sequences of randomly selected 210 clones with the insert of longer than 500 base pair (bp) as well as poly (A) tail have been compared with sequences in GeneBank, EMBL and DDBJ nucleic acid databases and fostered 193 expressed sequence tags (ESTs). The 210 cDNA clones identified are related to various aspect of metabolic pathways such as glycolysis, amino acid synthesis, translation mechanism, ribosome synthesis, hormone response, stress response, regulation of gene expression, and signal transduction. Among the 193 ESTs, 12 ESTs (29 cDNA clones) appeared more than once and 181 ESTs appeared once regarded as a solitary group. Out of 210 clones, 29 clones (13.8%) have no similarity to the known nucleotide sequences and could serve as a potentially useful resource for plant molecular biology referring to particular genes. Nucleotide sequencing to generate more ESTs from ethylene-induced as well as non-induced potato leaf is in progress as well.     

Expressed sequence tags of Chinese cabbage flower bud cDNA.

We randomly selected and partially sequenced cDNA clones from a library of Chinese cabbage (Brassica campestris L. ssp. pekinensis) flower bud cDNAs. Out of 1216 expressed sequence tags (ESTs), 904 cDNA clones were unique or nonredundant. Five hundred eighty-eight clones (48.4%) had sequence homology to functionally defined genes at the peptide level. Only 5 clones encoded known flower-specific proteins. Among the cDNAs with no similarity to known protein sequences (628), 184 clones had significant similarity to nucleotide sequences registered in the databases. Among these 184 clones, 142 exhibited similarities at the nucleotide level only with plant ESTs. Also, sequence similarities were evident between these 142 ESTs and their matching ESTs when compared using the deduced amino acid sequences. Therefore, it is possible that the anonymous ESTs encode plant-specific ubiquitous proteins. Our extensive EST analysis of genes expressed in floral organs not only contributes to the understanding of the dynamics of genome expression patterns in floral organs but also adds data to the repertoire of all genomic genes.     

玉米耐铝毒基因的分离

以抑制消减杂交（SSH）为手段,以玉米对铝敏感的自交系Mo17和耐铝的自交系TL94B为材料,分别构建它们的正向和反向消减文库,分别筛选获得了124、47、103和64个阳性克隆。对文库的鉴定表明,插入片段分布在0.25-1．0kb之间,阳性克隆率在18％左右。对338个阳性克隆进行测序,得到232种表达序列标签（EST）,其中70．2％的EST可推测其功能。结果表明,玉米的铝离子胁迫反应涉及胁迫因子的信号传导、响应基因的转录表达与调控、物质的合成与运输、细胞结构和功能的改变等。     

Generation of EST and Microarray Resources for Functional Genomic Studies on Chicken Intestinal Health

Abstract

Expressed sequenced tags (ESTs) and microarray resources have a great impact on the ability to study host response in mice and humans. Unfortunately, these resources are not yet available for domestic farm animals. The aim of this study was to provide genomic resources to study chicken intestinal health, in particular malabsorption syndrome (MAS), which affects mainly the intestine. Therefore a normalized and subtracted cDNA library containing more than 7000 clones was prepared. Randomly chosen clones were sequenced for control purposes. New ESTs were found and multiple ESTs not identified in the chicken intestine before were observed. The number of non‐specific ESTs in this cDNA library was low. Based on this normalized and subtracted library a cDNA microarray was made. In a preliminary hybridization experiment with the microarray, genes were identified to be up‐ or downregulated in MAS infected chickens. This indicates that the generated resources are valuable tools to investigate chicken intestinal health by whole genome expression analysis approaches.     

Generation of EST and microarray resources for functional genomic studies on chicken intestinal health

Expressed sequenced tags (ESTs) and microarray resources have a great impact on the ability to study host response in mice and humans. Unfortunately, these resources are not yet available for domestic farm animals. The aim of this study was to provide genomic resources to study chicken intestinal health, in particular malabsorption syndrome (MAS), which affects mainly the intestine. Therefore a normalized and subtracted cDNA library containing more than 7000 clones was prepared. Randomly chosen clones were sequenced for control purposes. New ESTs were found and multiple ESTs not identified in the chicken intestine before were observed. The number of non-specific ESTs in this cDNA library was low. Based on this normalized and subtracted library a cDNA microarray was made. In a preliminary hybridization experiment with the microarray, genes were identified to be up- or downregulated in MAS infected chickens. This indicates that the generated resources are valuable tools to investigate chicken intestinal health by whole genome expression analysis approaches.     

青杄均一化cDNA文库构建及EST序列分析

以青杄花粉和针叶为材料,将青杄全长cDNA与Gateway供体载体pDONR222重组,构建了其非剪切型全长cDNA原始文库,利用基因组DNA饱和杂交技术对原始cDNA文库进行均一化处理,构建青杄的均一化全长cDNA文库。文库的总库容量为1.1×106CFU/mL,平均插入片段长度大于1.0 kb,重组率大于95%。定量RT-PCR检测表明,青杄高丰度表达基因EF1-α在均一化cDNA文库中的表达量下降了约41倍。接着对文库中随机的5 144个克隆进行了测序,获得高质量的有效EST(expressedsequence tag)序列为5 144条,经拼接共获得单一基因(unigene)为2 717个,其中包括片段重叠群(contig)628个和单一EST序列(singlet)2 089个。NCBI同源比对分析表明,其中1 887个序列unigenes获得分子功能注释,这些EST涉及细胞生长、信号转导、转录、抗逆、能量代谢等功能。这些数据有助于对青杄的相关功能蛋白及分子机制开展进一步的研究。     

Structural analysis of a Lotus japonicus genome. I. Sequence features and mapping of fifty-six TAC clones which cover the 5.4 mb regions of the genome.

A total of 56 TAC clones with an average insert size of 100 kb were isolated from a TAC library of the Lotus japonicus genome based on the expressed sequences tags (ESTs), cDNA and gene information, and their nucleotide sequences were determined according to the shot-gun based strategy. The total length of the sequenced regions is 5,473,195 bp. By comparison with the sequences in protein and EST databases and analysis with computer programs for gene modeling, a total of 605 potential protein-encoding genes with known or predicted functions, 69 gene segments, and 172 pseudogenes were identified. The average density of the genes assigned so far is 1 gene/8120 bp. Introns were identified in approximately 78% of the potential genes. There was an average of 3.8 introns per gene and the average length of the introns was 375 bp. DNA markers were generated based on the nucleotide sequences obtained, and each clone was mapped onto the linkage map using the F2 mapping population derived from a cross of L. japonicus Gifu B-129 and Miyakojima MG-20. The sequence data, gene information and mapping information are available through the World Wide Web at http://www.kazusa.or.jp/lotus/.     

Cloning and characterization of chitin synthase gene fragments from Penicillium chrysogenum

Abstract We constructed a 3'-directed cDNA library of cleistothecia and Hülle cells of Aspergillus nidulans to examine gene expression patterns of the sexual structures and to have probes necessary to isolate sexual structure-specific genes. Sequencing of 360 randomly selected cDNA clones yielded 272 expressed sequence tags (ESTs), most of which probably represent frequently or less expressed genes in sexual structures of A. nidulans . Among the 272 ESTs, 33 ESTs (87 cDNA clones) appeared more than once and 2 ESTs appeared 6 times; 9 ESTs matched GenBank entries. When compared with sequences obtained from a mycelial 3'-directed cDNA library of A. nidulans , 28 out of 33 ESTs seem to be sexual structure-specific. Northern blot analyses of 20 ESTs showed that 17 are sexual structure-specific. The remaining three ESTs also hybridized with RNA isolated from vegetative mycelia. These results suggest that analyses of ESTs from different cell types or tissues can readily demonstrate gene expression patterns of specific cell types and identify cell type-specific cDNA probes.     

Identification and characterization of differentially expressed ESTs of <Emphasis Type="Italic">Gossypium barbadense</Emphasis> infected by <Emphasis Type="Italic">Verticillium dahliae</Emphasis> with suppression subtractive hybridization

The wilt defense reaction of cotton is a complicated continuous process and involves a battery of genes. In this study, we adopted the suppression subtractive hybridization (SSH) technique to isolate differentially expressed ESTs from Gossypium barbadense variety 7124 during the Verticillium wilt defense process. An array of 1165 clones from the subtractive library has been screened with reverse northern blotting, of which 131 ESTs were considered as overexpressed and 16 ESTs were downregulated. Sequence analysis and blast search showed that 83 ESTs were homologous to 45 unique sequences in the databases. Among all these differentially expressed ESTs, at least three kinds of genes were characterized. The majority of ESTs with a deduced identity as aerobic metabolism enzymes were strongly expressed in the infection process. Likewise, ESTs similar to those reported for pathogen-related protein genes were also picked out in this study. These ESTs, in combination with other kinase-like genes and a defensin-like EST, constituted an assembly of genes which responded during pathogenic infection. These results imply that sea-island cotton undergoes strong oxidative stress and results in a series of defense responses when attacked by V. dahliae. To our knowledge, this is the first report on the isolation of global ESTs during the sea-island cotton defense reaction.__________From Molekulyarnaya Biologiya, Vol. 39, No. 2, 2005, pp. 214–223.Original English Text Copyright © 2005 by Zuo, Wang, Wu, Chai, Sun, Tang.This article was submitted by the authors in English.