Profiling of differentially expressed genes in ectomycorrhizal fungus Pisolithus tinctorius responding to mycorrhiza helper Brevibacillus reuszeri MPt17

A cDNA library of the ectomycorrhizal (ECM) fungus Pisolithus tinctorius Pt2 after interaction with the mycorrhiza helper bacterium (MHB) Brevibacillus reuszeri MPt17 was constructed by suppression subtraction hybridization. Total RNA from B. reuszeri MPt17 exudates treated Pi. tinctorius Pt2 was used as a “tester” and total RNA from nonbacterial treated Pi. tinctorius Pt2 was used as a “driver.” Among the differentially expressed sequences, a BLASTX in the NCBI non-redundant protein sequence database revealed that 75% of the non-redundant sequences (147 out of 196) were highly similar to known proteins (E-value < e^?5). Twelve sequences were annotated as mycelium development function combining with a potential functional categories using gene ontology. Quantitative real-time PCR analysis showed that all of the 3 symbiosis regulated acidic polypeptide genes were all up-regulated in the MPt17-treated Pt2. These results provide evidence that the MHB B. reuszeri MPt17 could significantly change the expression of symbiosis-related genes and genes in mycelium development in ECM fungus, and also support the hypothesis that the MHB functions as helper though promotion on fungal mycelium.     

糙皮侧耳原基期差异表达基因分析

为解析糙皮侧耳原基期与菌丝期差异表达的基因,本研究以原基期cDNA为检测子（tester）、双核菌丝期cDNA为驱赶子（driver）,采用抑制性消减杂交法（suppression subtractive hybridization,SSH）构建了糙皮侧耳SSH cDNA文库。菌液PCR验证SSH cDNA文库插入cDNA片段后,挑取了2 055个差异转化子,差异转化子经3次反向Northern杂交筛选,得423个信号差异显著的克隆;阳性克隆测序后,经NCBI数据库Blastn和Blastx比对,共得206条差异表达序列（expressed sequence tag,EST）,重复序列去除后,有46个基因参与了细胞急救和防御、能量代谢、转录和蛋白调控、膜蛋白和信号转导,18个基因编码未知功能的推定蛋白,5个无任何同源性的新基因。挑取10个差异表达基因进行半定量RT-PCR,发现这些序列在原基期的表达水平显著高于菌丝期。结果表明,本研究成功构建了糙皮侧耳原基期与菌丝期SSH cDNA文库,为进一步分离糙皮侧耳生长发育相关基因并研究糙皮侧耳的发育机制奠定了基础。     

Identification of genes differentially expressed in hemocytes of Scylla paramamosain in response to lipopolysaccharide

Although the crab Scylla paramamosain has been cultured in China for a long time, little knowledge is available on how crabs respond to infection by bacteria. A forward suppression subtractive hybridization (SSH) cDNA library was constructed from their hemocytes and the up-regulated genes were identified in order to isolate differentially expressed genes in S. paramamosain in response to bacterial lipopolysaccharide (LPS). A total of 721 clones on the middle scale in the SSH library were sequenced. Among these genes, 271 potentially functional genes were recognized based on the BLAST searches in NCBI and were categorized into seven groups in association with different biological processes using AmiGO against the Gene Ontology database. Of the 271 genes, 269 translatable DNA sequences were predicted to be proteins, and the putative amino acid sequences were searched for conserved domains and proteins using the CD-Search service and BLASTp. Among 271 genes, 179 (66.1%) were annotated to be involved in different biological processes, while 92 genes (33.9%) were classified as an unknown-function gene group. It was noted that only 18 of the 271 genes (6.6%) had previously been reported in other crustaceans and most of the screened genes showed less similarity to known sequences based on BLASTn results, suggesting that 253 genes were found for the first time in S. paramamosain. Furthermore, two up-regulated genes screened from the SSH library were selected for full-length cDNA sequence cloning and in vivo expression study, including Sp-superoxide dismutase (Sp-Cu-ZnSOD) gene and Sp-serpin gene. The differential expression pattern of the two genes during the time course of LPS challenge was analyzed using real-time PCR. We found that both genes were significantly expressed in LPS-challenged crabs in comparison with control. Taken together, the study primarily provides the data of the up-regulated genes associated with different biological processes in S. paramamosain in response to LPS, by which the interesting genes or proteins potentially involved in the innate immune defense of S. paramamosain will be investigated in future.     

Identification of symbiosis-regulated genes in Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza by differential hybridization of arrayed cDNAs

Ectomycorrhiza development alters gene expression in the fungal and plant symbionts. The identification of a large number of genes expressed exclusively or predominantly in the symbiosis will contribute greatly to the understanding of the development of the ectomycorrhizal symbiosis. We have constructed a cDNA library of 4-day-old Eucalyptus globulus-Pisolithus tinctorius ectomycorrhiza and sequenced 850 cDNAs cloned randomly or obtained through suppression subtractive hybridization (SSH). Based on the absence of a database match, 43% of the ectomycorrhiza ESTs are coding for novel genes. At the developmental stage analysed (fungal sheath formation), the majority of the identified sequences represented 'housekeeping' proteins, i.e. proteins involved in gene/protein expression, cell-wall proteins, metabolic enzymes, and components of signalling systems. We screened arrayed cDNAs to identify symbiosis-regulated genes by using differential hybridization. Comparisons of signals from free-living partners and symbiotic tissues revealed significant differences in expression levels (differential expression ratio >2.5) for 17% of the genes analysed. No ectomycorrhiza-specific gene was detected. The results successfully demonstrate the use of the cDNA array and SSH systems as general approaches for dissecting symbiosis development, and provide the first global picture of the cellular functions operating in ectomycorrhiza.     

Suppression subtractive hybridization identifies differentially expressed genes in Brassica napus chlorophyll-reduced mutant

Suppressive subtraction hybridization (SSH) was used to identify differentially expressed genes caused by a chlorophyll-reduced mutation in B. napus. The cDNA fragments, derived from SSH positive subtractive library (tester: normal wild type, driver: mutant) were cloned into pMD18-T vector. Two hundred SSH cDNA clones were screened by dot blot array, and 151 clones were identified as differentially expressed cDNA fragments in Cr3529 line. Thirty-six positive clones which showed marked expression differences were selected and sequenced. After redundant cDNAs were removed, 33 differentially expressed unique cDNA section clones were obtained. Among the 33 clones, two clones possess different parts of the cDNA sequence of the same gene coding geranylgeranyl reductase, four clones belong to unknown proteins, and the rest share homology to genes of diverse class. Sequence analysis showed that at least 12 genes were discovered to be related to the photosynthesis, seven of them coded the proteins which belong to the subunit of photosystem 2. RNA gel blot analysis showed that compared with 3529, the gene expression of the chlorophyll a/b-binding protein Lhcb2 in photosystem 2 declined markedly in the cotyledons and seedling leaves of Cr3529, indicating that the reduced light-harvesting complex 2 accumulation in thylakoid membrane of Cr3529 was due to the decrease of the related gene mRNA level for translation.     

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 .     

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．     

Differential gene expression profile in Pseudomonas putida NBRIC19-treated wheat (Triticum aestivum) plants subjected to biotic stress of Parthenium hysterophorus

The inoculation of Pseudomonas putida NBRIC19 protected wheat plant from phytotoxic effect of Parthenium hysterophorus (Parthenium) and enhanced root length, shoot length, dry weight, spike length and chlorophyll content. With the aim to screen for genes differentially expressed in P. putida NBRIC19-inoculated wheat grown along with Parthenium (WPT), the suppression subtractive hybridization (SSH) methodology was employed. The SSH analysis was performed with WPC (uninoculated wheat grown along with Parthenium) as driver and WPT as tester. The cDNA library, enriched with differentially expressed ESTs (expressed sequence tags), were constructed from WPT. Following an initial screen of 165 ESTs in our library, 32 ESTs were identified, annotated and further validated by semiquantitative RT-PCR. The differentially expressed ESTs were associated with general stress response, defense response, growth and development, metabolic process, photosynthesis, signal transduction, and some other with unknown function. Five ESTs showing downregulation in expression level in response to Parthenium got upregulated due to P. putida NBRIC19 inoculation and further validated by quantitative real time PCR analysis at different time intervals viz. 15, 30, 45 and 90 days. SSH has been implemented for the first time to gain insights into molecular events underlying successful role of P. putida NBRIC19 in providing protection to wheat against Parthenium. The information generated in this study provides new clues to aid the understanding of genes corresponding to differentially expressed ESTs putatively involved in allelopathic interactions. Further characterization and functional analysis of these genes may provide valuable information for future studies of the molecular mechanism by which plants adapt to allelopathic effect of Parthenium.     

Differential expression of fungal genes at preinfection and mycorrhiza establishment between Terfezia boudieri isolates and Cistus incanus hairy root clones

Changes in gene expression by isolates of Terfezia boudieri during mycorrhization with Cistus incanus hairy roots were followed. Four fungus-hairy root clone combinations were cultivated under two sets of conditions, in which the root and the fungus were separated by a cellophane sheet or were allowed physical contact. One of the combinations produced endomycorrhizas, the other three solely ectomycorrhizas. Fragments isolated by cDNA-AFLP analysis from cellophane-separated cultures (preinfection) were used to identify differentially expressed genes by reverse Northern analysis. Genes showing no homology to known sequences constituted the largest group under both growth conditions. Some fungal genes were expressed transiently, while others exhibited altered expression patterns as conditions changed from individually growing through the preinfection stage to mycorrhizas. Genes expressed exclusively under combinations allowing either ectomycorrhiza or endomycorrhiza under a particular condition were detected. Our results point, for the first time, to some of the genes that might be involved in determining the type of association that will be formed: ecto- or endomycorrhiza.     

Identification of genes associated with fruit ripening in Ziziphus jujuba using suppression subtractive hybridization approach

Owing to the high nutritional value and extensive medicinal use of its products, Chinese jujube (Ziziphus jujuba Mill) is one of the most important fruit crops in China. However, jujube fruits are highly perishable and thus have a short shelf life, which is a serious hindrance to the industry. Better understanding of the molecular mechanisms underlying jujube fruit softening is fundamental to overcome the problem. Thus, both forward and reverse suppression subtractive hybridization (SSH) cDNA libraries were constructed to identify differentially expressed genes for fruit at half-red ripening stage and complete red stage. As a result of dot blot confirmation, a total of 154 differentially expressed genes were identified. After removed low-quality regions and screened for vector contamination, blasted with the non-redundant NCBI databases, 78.6 % of sequences exhibited high homology to previously identified or putative proteins. All the ESTs were annotated and classified according to the terms of the three main Gene Ontology vocabularies using the Blast2GO software. Furthermore, the quantitative real-time PCR was carried out for 17 genes to validate the genes differentially expressed from the SSH libraries. And the full-length sequences of galactose oxidase and aldehyde dehydrogenase genes were obtained. It is the first step to explore the functional genomics and regulatory networks during the storage period of jujube fruit. The identification of the genes differentially expressed is helpful to understand the ripening and softening of the jujube fruit at the molecular level.     

Identification of Differentially Expressed Genes of Trichinella spiralis Larvae after Exposure to Host Intestine Milieu

Although it has been known for many years that T. spiralis muscle larvae (ML) can not invade intestinal epithelial cells unless they are exposed to the intestinal milieu and activated into intestinal infective larvae (IIL), which genes in IIL are involved in the process of invasion is still unknown. In this study, suppression subtractive hybridization (SSH) was performed to identify differentially expressed genes between IIL and ML. SSH library was constructed using cDNA generated from IIL as the ‘tester’. About 110 positive clones were randomly selected from the library and sequenced, of which 33 T. spiralis genes were identified. Thirty encoded proteins were annotated according to Gene Ontology Annotation in terms of molecular function, biological process, and cellular localization. Out of 30 annotated proteins, 16 proteins (53.3%) had binding activity and 12 proteins (40.0%) had catalytic activity. The results of real-time PCR showed that the expression of nine genes (Ts7, Ndr family protein; Ts8, serine/threonine-protein kinase polo; Ts11, proteasome subunit beta type-7; Ts17, nudix hydrolase; Ts19, ovochymase-1; Ts22, fibronectin type III domain protein; Ts23, muscle cell intermediate filament protein OV71; Ts26, neutral and basic amino acid transport protein rBAT and Ts33, FACT complex subunit SPT16) from 33 T. spiralis genes in IIL were up-regulated compared with that of ML. The present study provide a group of the potential invasion-related candidate genes and will be helpful for further studies of mechanisms by which T. spiralis infective larvae recognize and invade the intestinal epithelial cells.     

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.     

Identification of ESTs differentially expressed in green and albino mutant bamboo (Bambusa edulis) by suppressive subtractive hybridization (SSH) and microarray analysis

To gain a better understanding of gene expression in bamboo (Bambusa edulis Murno), we have used a combination of suppressive subtractive hybridization (SSH), microarray hybridization analysis, sequencing, and bioinformatics to identify bamboo genes differentially expressed in a bamboo albino mutant. Ten expressed sequence tags (ESTs) were found to be differentially expressed; these were isolated and sequenced. RT-PCR analysis of these ESTs supported the results of the microarray analysis. Six ESTs that were nucleus-encoded exhibited differential expression patterns in the green wild-type bamboo relative to the albino mutant. These genes (exception being the Rubisco small subunit) were non-photosynthesis-related genes. The development of a specific SSH cDNA library in which most of the chloroplast-encoded or photosynthesis-related genes had been subtracted proved to be useful for studying the function of non-photosynthesis-related genes in the albino bamboo mutants with aberrant chloroplast genome. The combined use of this SSH library with microarray analysis will provide a powerful analytical tool for future studies of the bamboo genome.