Identification by subtractive suppression hybridization of bacteria-induced genes expressed in Manduca sexta fat body

Insect immune processes are mediated by programs of differential gene expression. To understand the molecular regulation of the immune response in the tobacco hornworm, Manduca sexta, the relevant subset of differentially expressed genes of interest must be identified, cloned and studied in detail. In this study, suppression subtractive hybridization, a PCR-based method for cDNA subtraction was performed to identify mRNAs from fat body of immunized larvae that are not present (or present at a low level) in control larvae. A subtracted cDNA library enriched in immune-inducible genes was constructed. Northern blot analysis of a sample of clones from our subtracted library indicated that >90% of the clones randomly selected from the subtracted library are immune inducible. Sequence analysis of 238 expressed sequence tags (ESTs) revealed that 120 ESTs, representing 54 distinct genes or gene families, had sequences identical or similar to previously characterized genes, some of which have been confirmed to be involved in innate immunity. These ESTs were categorized into seven groups, including pattern recognition proteins, serine proteinases and their inhibitors, and antimicrobial proteins. 112 ESTs, about 47.5% of the library, showed no significant similarity to any known genes. The sequences identified in this M. sexta library reflect our knowledge of insect immune strategies and may facilitate better understanding of insect immune responses.     

家蝇幼虫消减文库的构建及差异表达基因的鉴定

为了鉴定家蝇Musca domestica免疫相关基因,应用抑制性消减杂交技术,构建刺激家蝇幼虫差异表达cDNA消减文库。以大肠杆菌Escherichia coli和金黄色葡萄球菌Staphylococcus aureus诱导12 h的家蝇幼虫与未诱导的家蝇幼虫为消减杂交对象,获得了差异表达基因的cDNA片段,将其与T/A载体连接并转化大肠杆菌DH5α,构建了刺激家蝇幼虫cDNA消减文库。PCR检测发现,文库的阳性克隆中插入的cDNA片段大小在200~1 000 bp之间,随机挑选了161个含大小不等差异片段的克隆进行测序和同源性分析,鉴定了36种蛋白的基因片段,包括抗菌肽、酶、核糖体蛋白、其他功能蛋白以及功能不明的蛋白。用半定量RT-PCR分析了其中6种蛋白基因的表达,结果显示：防御素和攻击素基因在细菌刺激后24 h内明显上调表达,而溶菌酶、酚氧化酶原活化因子、糜蛋白酶和蛋白质合成起始因子基因在细菌刺激后0-4 h内表达受抑制,12 h后上调表达。该研究结果为家蝇免疫相关基因的克隆和家蝇免疫防御机制的探讨奠定了良好的基础。     

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。     

人参植物与皂苷生物合成相关的差减cDNA文库构建及基因差异表达分析

应用抑制差减杂交技术,分别以源于4年和1年生人参根组织cDNA群体作为检测子(tester)与驱赶子(driver),成功构建了与人参植物皂苷生物合成相关的差减cDNA文库,并时从中筛选的阳性cDNA克隆进行DNA测序及其序列分析、PCR及Northern印迹杂交鉴定．结果显示,获得的13个克隆为新基因序列．其中6个差减克隆系人参植物根生长发育阶段差异表达基因．目前,6个差异表达新基因的结构与功能仍在进一步研究中．     

Construction and characterization of a cDNA library from floral organs and fruitlets of <Emphasis Type="Italic">Citrus reticulata</Emphasis>

To explore and isolate genes related to flowering and fruit development, we constructed a cDNA library from floral organs and fruitlets of Ponkan mandarin (Citrus reticulata Blanco). A total of 661 high-quality expressed sequence tags (ESTs) were generated and submitted to GenBank with the accession numbers from GO343532 to GO344192. All these ESTs were assembled into 43 contigs and 296 singletons (totally 339 unigenes). The BLAST2GO software was employed to annotate the unigenes, among which 77 ones had no significant homology with the sequences in NCBI non-redundant proteins database by BLASTX analysis. Additionally, gene ontology (GO) analysis revealed an overview of sequences distribution, which implied some specially expressed genes related to flower and fruit development. Furthermore, some abundantly expressed unigenes involved in several crucial metabolic pathways related to fruit quality were highlighted and three types of homologues of miraculin-like protein2 were analyzed by both semiquantitative RT-PCR and real-time PCR. The results showed different expression profiles of these genes, which meant that they contribute distinctly to fruit development.     

Identification of stress-induced genes from the drought tolerant semi-arid legume crop horsegram (Macrotyloma uniflorum (Lam.) Verdc.) through analysis of subtracted expressed sequence tags

Abiotic stresses adversely affect the agricultural productivity worldwide. Horsegram (Macrotyloma uniflorum (Lam.) Verdc.) is a legume crop that can tolerate severe adverse environmental conditions such as drought, salinity and heavy metal contamination. As a first step towards characterization of genes that contribute to combating abiotic stresses, construction and analysis of subtracted cDNA library is reported here. Using this strategy a total of 1050 ESTs were isolated, sequenced, 959 high quality ESTs were obtained and clustered. Further, our analysis revealed that of these 531 sequences are unique and 30% of these have no homology to known proteins in the database. This observation has great relevance since horsegram is a stress-adapted legume crop. Further, to validate the identified differentially expressed genes, expression profiles of selected clones were analyzed using reverse-northern, northern blot analysis and we show that indeed these clones are differentially expressed under various abiotic stress conditions. The implications of the analyzed genes in abiotic stress tolerance also discussed.     

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.     

日本血吸虫期别差异表达基因文库的构建及分析

为从期别差异表达基因分析入手研究血吸虫的生长发育机制,应用抑制性消减杂交 (suppressed subtractive hybridization , SSH) 技术首次构建了日本血吸虫尾蚴、虫卵和成虫的期别差异表达基因文库 . 经消减效率分析和三种文库克隆的 EST 的期别差异性鉴定,表明所建文库质量较高,为在整个基因组水平分离血吸虫的差异表达基因提供了重要材料 . 由三个文库选择 257 个插入片段大于 500 bp 的克隆测定了 EST 序列 . 同源性分析结果表明 257 个 EST 代表 182 种血吸虫基因,其中有 22 种为血吸虫已知基因,有 128 种为血吸虫已知 EST ,有 32 种为新发现的血吸虫基因 . 对 EST 编码蛋白的功能预测结果显示：尾蚴消减文库的基因多与运动、能量代谢、转录调节及致病性相关;虫卵消减文库的基因可能参与信号转导、细胞粘附、蛋白质和碳水化合物的代谢以及抗氧化反应;成虫消减文库的基因多参与蛋白质的合成、转运及分解代谢,参与虫体的运动等 . 大规模分离、分析血吸虫期别差异表达基因将对从分子水平去解读血吸虫的生长发育机制,筛选高效疫苗候选抗原、药物靶标及诊断制剂有重要意义 .     

RhoA signaling modulates cyclin D1 expression in human lung fibroblasts; implications for idiopathic pulmonary fibrosis

Background

To elucidate further the pathogenesis of sporadic, idiopathic pulmonary arterial hypertension (IPAH) and identify potential therapeutic avenues, differential gene expression in IPAH was examined by suppression subtractive hybridisation (SSH).

Methods

Peripheral lung samples were obtained immediately after removal from patients undergoing lung transplant for IPAH without familial disease, and control tissues consisted of similarly sampled pieces of donor lungs not utilised during transplantation. Pools of lung mRNA from IPAH cases containing plexiform lesions and normal donor lungs were used to generate the tester and driver cDNA libraries, respectively. A subtracted IPAH cDNA library was made by SSH. Clones isolated from this subtracted library were examined for up regulated expression in IPAH using dot blot arrays of positive colony PCR products using both pooled cDNA libraries as probes. Clones verified as being upregulated were sequenced. For two genes the increase in expression was verified by northern blotting and data analysed using Student's unpaired two-tailed t-test.

Results

We present preliminary findings concerning candidate genes upregulated in IPAH. Twenty-seven upregulated genes were identified out of 192 clones examined. Upregulation in individual cases of IPAH was shown by northern blot for tissue inhibitor of metalloproteinase-3 and decorin (P < 0.01) compared with the housekeeping gene glyceraldehydes-3-phosphate dehydrogenase.

Conclusion

Four of the up regulated genes, magic roundabout, hevin, thrombomodulin and sucrose non-fermenting protein-related kinase-1 are expressed specifically by endothelial cells and one, muscleblind-1, by muscle cells, suggesting that they may be associated with plexiform lesions and hypertrophic arterial wall remodelling, respectively.     

Variability of gene expression profiles in human blood and lymphoblastoid cell lines

Background

Infection of plants by pathogens and the subsequent disease development involves substantial changes in the biochemistry and physiology of both partners. Analysis of genes that are expressed during these interactions represents a powerful strategy to obtain insights into the molecular events underlying these changes. We have employed expressed sequence tag (EST) analysis to identify rice genes involved in defense responses against infection by the blast fungus Magnaporthe oryzae and fungal genes involved in infectious growth within the host during a compatible interaction.

Results

A cDNA library was constructed with RNA from rice leaves (Oryza sativa cv. Hwacheong) infected with M. oryzae strain KJ201. To enrich for fungal genes, subtraction library using PCR-based suppression subtractive hybridization was constructed with RNA from infected rice leaves as a tester and that from uninfected rice leaves as the driver. A total of 4,148 clones from two libraries were sequenced to generate 2,302 non-redundant ESTs. Of these, 712 and 1,562 ESTs could be identified to encode fungal and rice genes, respectively. To predict gene function, Gene Ontology (GO) analysis was applied, with 31% and 32% of rice and fungal ESTs being assigned to GO terms, respectively. One hundred uniESTs were found to be specific to fungal infection EST. More than 80 full-length fungal cDNA sequences were used to validate ab initio annotated gene model of M. oryzae genome sequence.

Conclusion

This study shows the power of ESTs to refine genome annotation and functional characterization. Results of this work have advanced our understanding of the molecular mechanisms underpinning fungal-plant interactions and formed the basis for new hypothesis.     

Genes expressed in sugarcane maturing internodal tissue

To explore gene expression during sugarcane culm maturation, we performed a partial sequence analysis of random clones from maturing culm total and subtracted cDNA libraries. Database comparisons revealed that of the 337 cDNA sequences analysed, 167 showed sequence homology to gene products in the protein databases, while 111 matched uncharacterised plant expressed sequence tags (ESTs) only. The remaining cDNAs showed no database match and could represent novel genes. The majority of ESTs corresponded to a variety of genes associated with general cellular metabolism. ESTs homologous to various stress response genes were also well represented. Analysis of ESTs from the subtracted library identified genes that may be preferentially expressed during culm maturation. This research has provided a framework for functional gene analysis in sugarcane sucrose-accumulating tissues.     

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.     

利用抑制差减杂交技术分离受水稻抗性调控的褐飞虱基因

为分离受水稻抗性调控的褐飞虱Nilaparvata lugens基因, 以取食感虫水稻台中1号和高抗水稻B5的2叶1芯秧苗24 h的褐飞虱4龄若虫为起始材料, 采用抑制差减杂交技术构建了两个群体间的正反向差减cDNA文库。通过斑点杂交从差减文库中筛选代表受水稻抗性调控的基因的cDNA克隆, 进行测序和功能分析, 挑选具功能的基因进行Northern杂交验证。结果表明, 通过斑点杂交筛选到的98个阳性克隆代表92个互不重复的单基因, 其中25个与动物的已知蛋白基因存在较高的同源性。Northern杂交表明, 这25个基因有11个表达上调, 8个表达下调, 提示它们可能在褐飞虱适应抗性水稻过程中发挥了重要作用。本研究结果为克隆上述新基因的全长cDNA序列及进一步研究其在褐飞虱与水稻互作中的功能奠定了基础。