Reverse Libraries Research Articles

Application of SSH and quantitative real time PCR to construction of gene expression profiles from scallop Chlamys farreri in response to exposure to tetrabromobisphenol A

TBBPA-induced genes were identified using suppression subtractive hybridization (SSH) from Chlamys farreri. A total of 203 and 44 clones from SSH forward and reverse library were respectively obtained including cellular process, immune system process, response to stimulus, metabolic process and signaling etc. Differential gene expressions were compared between scallops from control and TBBPA treatment groups (400μg/L, 15 days) using quantitative real time RT-PCR. For further research, eight significant genes expression from scallops exposed to TBBPA (0; 100; 200; 400μg/L) sampling at 0, 1, 3, 6 and 15 days, were utilized for Q-RT-PCR. The results revealed that the expression level of most selected cDNAs was dominantly up-regulated or down-regulated in the TBBPA-induced scallops. These findings provide basic genomic information of the bivalve and the selected genes may be the potential molecular biomarkers for TBBPA pollution in aquatic environment.

Abstract 342: RNA-seq Profiling of Individually Microdissected Renal Tubule Segments

Control of renal tubule NaCl transport plays an important role in blood pressure regulation. In the past, transport studies and biochemical assays have been conducted in microdissected renal tubule segments to discover mechanisms of transport regulation. Here, we extend the use of microdissected renal tubules to transcriptomic analysis using the RNA-seq method with small sample amplification. The feasibility of the approach was tested in microdissected cortical thick ascending limbs (cTAL) isolated by hand dissection from collagenase-incubated rat renal cortex. After lysis of isolated cTAL cells, poly(A)-targeted reverse transcription and library preparation were performed ( Nature Protocols 5:516-535). Paired-end sequencing of the cDNA library was done using an Illumina HiSeq 2000 sequencer. The generated 50-bp reads were aligned to the UCSC rat reference genome ( Bioinformatics 25, 1754-60). Reads per kilobase exon model normalized by million mapped reads (RPKM) were calculated. In an initial sample of 5 mm cTALs, 451,694,588 total reads were obtained of which 235,735,784 reads (52%) were mapped and properly paired. In this experiment, 5410 RefSeq genes were identified. To assess accuracy, we surveyed G-protein coupled receptors (GPCRs) and transport proteins that were identified. Among the GPCRs known to be expressed in cTAL ( Avpr2, Pth1r, Gcgr, Calcr, Casr , and Ptger3 ) only the calcitonin receptor was not found. Among the transporter proteins generally accepted to be expressed in cTAL ( Slc12a1, Kcnj1, Clcnkb, Cldn16, Slc9a3, Slc2a4 and Atp1a1 ) only Slc9a3 was not found. In contrast, transporter proteins characteristic of neighboring tubule segments were not found. In addition, gene-set analysis showed overrepresentation of mitochondrial genes among transcripts with high RPKM . When longer lengths of cTAL were used, the number of mapped transcripts increased, but not linearly (15 mm, 8455 genes; 25 mm, 12,110 genes). Experiments with a second segment, medullary TALs, gave a profile very similar to that of the cTAL. These studies establish the feasibility of RNA-seq-based transcriptomic profiling in microdissected renal tubule segments and ‘open the door’ for profiling of gene expression along the entire renal tubule.

Profiling of differentially expressed genes in hepatopancreas of white shrimp (Litopenaeus vannamei) exposed to long-term low salinity stress

The Pacific white shrimp, Litopenaeus vannamei, is a euryhaline crustacean capable of tolerating a wide range of ambient salinity (0.5–40psu). To investigate the effect of long-term low salinity stress on gene expression in the hepatopancreas in shrimp, we performed suppression subtractive hybridization (SSH) in juvenile L. vannamei exposed to long-term low salinity. The shrimp (initial body weight, 0.27±0.01g) were cultured at salinity 2 psu and salinity 30 psu for 56days. We then constructed forward and reverse subtractive cDNA libraries. We used bioinformatics tools and vector screening to select a total of 200 (80 from forward, 120 from reverse) randomly selected clones over 100 nucleotides in length for further analysis. Nineteen contigs and 54 singletons were generated from a total of 73 consensuses. The consensuses, upon a sequence homology search using BLASTX (NCBI), revealed that 24.66% (18/73) of them had no significant match to reported sequences in the database, suggesting that they had not previously been found and that they were probably associated with stress-regulated functions. The remaining 75.34% (55/73) of the consensuses encoded proteins were matched to a wide range of functions including immune-related functions, metabolism, ribosomal activity, transfer activity, and apoptosis. The most common group in these SSH libraries was immune-related proteins and enzymes (11/17). Quantitative RT-PCR results confirmed that the relative expression of 5 differentially expressed genes encoding hemocyanin, chitinase, ecdysteroid-regulated protein, trypsin and chymotrypsin 1 was decreased 2-, 1.45-, 11.11-, 1.33- and 1.54-fold, respectively, in the reverse library. This subtractive cDNA library provides a basis for the study of the genetic response of shrimp to environmental stress.

Airborne Signals from a Wounded Leaf Facilitate Viral Spreading and Induce Antibacterial Resistance in Neighboring Plants

Many plants release airborne volatile compounds in response to wounding due to pathogenic assault. These compounds serve as plant defenses and are involved in plant signaling. Here, we study the effects of pectin methylesterase (PME)-generated methanol release from wounded plants (“emitters”) on the defensive reactions of neighboring “receiver” plants. Plant leaf wounding resulted in the synthesis of PME and a spike in methanol released into the air. Gaseous methanol or vapors from wounded PME-transgenic plants induced resistance to the bacterial pathogen Ralstonia solanacearum in the leaves of non-wounded neighboring “receiver” plants. In experiments with different volatile organic compounds, gaseous methanol was the only airborne factor that could induce antibacterial resistance in neighboring plants. In an effort to understand the mechanisms by which methanol stimulates the antibacterial resistance of “receiver” plants, we constructed forward and reverse suppression subtractive hybridization cDNA libraries from Nicotiana benthamiana plants exposed to methanol. We identified multiple methanol-inducible genes (MIGs), most of which are involved in defense or cell-to-cell trafficking. We then isolated the most affected genes for further analysis: β-1,3-glucanase (BG), a previously unidentified gene (MIG-21), and non-cell-autonomous pathway protein (NCAPP). Experiments with Tobacco mosaic virus (TMV) and a vector encoding two tandem copies of green fluorescent protein as a tracer of cell-to-cell movement showed the increased gating capacity of plasmodesmata in the presence of BG, MIG-21, and NCAPP. The increased gating capacity is accompanied by enhanced TMV reproduction in the “receivers”. Overall, our data indicate that methanol emitted by a wounded plant acts as a signal that enhances antibacterial resistance and facilitates viral spread in neighboring plants.

Gene Expression Profiling in the Thiamethoxam Resistant and Susceptible B-biotype Sweetpotato Whitefly,Bemisia tabaci

Thiamethoxam has been used as a major insecticide to control the B-biotype sweetpotato whitefly, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae). Due to its excessive use, a high level of resistance to thiamethoxam has developed worldwide over the past several years. To better understand the molecular mechanisms underlying this resistance in B. tabaci, gene profiles between the thiamethoxam-resistant and thiamethoxam-susceptible strains were investigated using the suppression subtractive hybridization (SSH) library approach. A total of 72 and 52 upand down-regulated genes were obtained from the forward and reverse SSH libraries, respectively. These expressed sequence tags (ESTs) belong to several functional categories based on their gene ontology annotation. Some categories such as cell communication, response to abiotic stimulus, lipid particle, and nuclear envelope were identified only in the forward library of thiamethoxam-resistant strains. In contrast, categories such as behavior, cell proliferation, nutrient reservoir activity, sequence-specific DNA binding transcription factor activity, and signal transducer activity were identified solely in the reverse library.To study the validity of the SSH method, 16 differentially expressed genes from both forward and reverse SSH libraries were selected randomly for further analyses using quantitative realtime PCR (qRT-PCR). The qRT-PCR results were fairly consistent with the SSH results; however, only 50% of the genes showed significantly different expression profiles between the thiamethoxam-resistant and thiamethoxam-susceptible whiteflies. Among these genes, a putative NAD-dependent methanol dehydrogenase was substantially over-expressed in the thiamethoxamresistant adults compared to their susceptible counterparts. The distributed profiles show that it was highly expressed during the egg stage, and was most abundant in the abdomen of adult females.

Identification of differentially expressed genes preferably related to drought response in pigeon pea (Cajanus cajan) inoculated by arbuscular mycorrhizae fungi (AMF)

Pigeon pea is an ideal crop for sustainable agriculture systems in Karst areas of southwest China, which frequently suffers from the formidable water deficit. Physiologically, arbuscular mycorrhizae (AM)-colonized pigeon pea (Cajanus cajan) demonstrated a further enhanced tolerance to drought stress. To elucidate the molecular mechanism underlying the elevated tolerance, suppression subtractive hybridization (SSH) were employed to dig up the differentially expressed genes using mixed cDNAs prepared from drought-stressed and unstressed pigeon pea seedlings inoculated by AM fungi (AMF) in the present work. Both forward and reverse SSH cDNA library were constructed and a total of 768 clones were obtained. Dot-blotting expression analysis identified that 142 clones were upregulated, and 49 were downregulated during water stress. After sequencing, 182 unique expressed sequence tags (ESTs) were obtained via blast analysis, among which 142 (78%) exhibited high homology to previously identified or putative proteins, however, 40 (22%) showed no homology in the database. The upregulated (102) and downregulated (40) ESTs with significant protein homology might be sorted into 16 and 12 functional categories respectively, which involved in a broad spectrum of biological pathways. Furthermore, semi-quantitative reverse transcription (RT)-PCR was carried out for the 35 differentially expressed genes whose putative functions implicated in abiotic stress tolerances in other species, and it was verified these differentially expressed genes highly involved in drought stress tolerance of AM-colonized pigeon pea.

Identification of differentially expressed genes in Oncomelania hupensis chronically infected with Schistosoma japonicum

Oncomelania hupensis is the unique intermediate host of Schistosoma japonicum. The schistosome–snail interaction is biomedically important. To identify differentially expressed transcripts in O. hupensis chronically infected with S. japonicum, suppression subtractive hybridization (SSH) was used to construct a cDNA library in each direction for transcripts that are more abundantly enriched in head–foot part of the infected O. hupensis and for those that are more abundantly enriched in the uninfected, as head–foot part contains hemocytes and hemolymph which are associated with the snail internal defense system. After differential screening, 39 transcripts were identified, including nine and 30 transcripts enriched in infected and uninfected snails, respectively. Some of the transcripts have similar homology to available sequences in current databases, including transposase, caveolin-like protein, pancreatic trypsin inhibitor-like protein, prosaposin, glutathione s-transferase (GST), and several hypothetical proteins, while most of the transcripts do not match with any sequences in available databases. The identified transcripts were involved functionally in cell growth, metabolism, signal transduction, and immune responses. Two forward library transcripts and 11 reverse library transcripts were selected for real-time PCR, and 10 of them were confirmed to be consistent with the SSH results. It is intriguing to continue functional studies for some genes such as pancreatic trypsin inhibitor; a hypothetical protein (HS576367) related to calcium ion binding; GST; and several unknown proteins (HS576353 and HS576355). These identified differentially expressed genes may be key targets for understanding the molecular mechanism of co-existence during which the snail is unable to rid itself of the schistosome in chronic infection stage.

Identification of genes differentially expressed in maize (Zea mays L.) during Rhizoctonia Solani Kühn infection by suppression subtractive hybridization

Banded leaf and sheath blight (BLSB) caused by Rhizoctonia solani Kuhn is an emerging problem in maize worldwide. To study gene expression in maize plants infected by R. solani Kuhn, we constructed two suppression subtractive hybridization (SSH) library using complementary DNA (cDNA) from uninoculated plants as a driver and those from inoculated plants as a tester. After two cycles of hybridization, 84 cDNA clones from the forward and reverse SSH library were obtained and sequenced. Gene analysis indicated that these clones represented 51 single genes, and the functions of 35 genes could be assigned using existing databases. Thirty-five expression sequences tags (ESTs) were classified in fuctional categories, such as: transcription, regulation, protein processing, metabolism, defense, disease response and other functions. The expression of 15 genes was analyzed by semi-quantitative reverse-transcription-polymerase chain reaction (RT-PCR) and found to be up-regulated or down-regulated in response to R. solani Kuhn re-infection. In conclusion, this study provides a basis for a molecular understanding of host reaction in response to pathogen. Key words: Banded leaf and sheath blight, expressed sequence tag, gene expression, maize, suppression subtractive hybridization.

Construction of Leave Library by SSH and Preliminary Analysis of Genes Responsible for Heat Sress in Incarvillea zhongdiannensis

High temperature might be one of the main limiting factors to be introduced and domesticated into lower altitude for Incarvillea zhongdiannensis. To further reveal the underlying molecular mechanism in I.zhongdiannensis responding to high temperature, the suppression subtractive hybridization approach (SSH) was used in this study. The results showed the quality of forward or reverse subtraction cDNA library is good enough for further SSH experiments. Total sixty expressed tags (ESTs) with high quality were randomly obtained by SSH and sequencing. According to BLAST screening and functional annotation, 50 ESTs with known function were classified into 9 broad categories such as defense/stress, photosynthesis, metabolism, energy, signal transduction and transcription, protein fate, transporter, biosynthesis, cell structure and cell growth. There were six ESTs significantly matched with hypothetical proteins. Four ESTs with no hits possibly encode novel genes and play important role in I.zhongdiannensis responding to high temperature stress.

Identification and characterization of low temperature stress responsive genes in Poncirus trifoliata by suppression subtractive hybridization

Trifoliate orange ( Poncirus trifoliata (L.) Raf.) is extremely cold hardy when fully acclimated, but knowledge relevant to the molecular events underlying the acclimation is still limited so far. In this study, forward (4 °C over 25 °C) and reverse (25 °C over 4 °C) suppression subtractive hybridization (SSH) libraries were constructed in order to identify the genes involved in cold acclimation in trifoliate orange. After reverse northern blotting analysis and sequencing, a total of 105 and 117 non-redundant differentially expressed sequence tags (ESTs) were obtained from the forward and reverse libraries, respectively. Blast2go analysis revealed that 91 ESTs, 31 from the forward library and 60 from the reverse library, displayed significant sequence homology to the genes with known or putative functions. They were categorized into various functional groups, including catalytic activity, binding protein, structural molecule, enzyme regulator, molecular transducer, electron carrier, and transport activity/transcription regulation. Expression analysis of the selected ESTs by reverse transcriptase polymerase chain reaction was consistent with the results of differential screening. In addition, time-course expression patterns of the genes further confirmed that they were responsive to low temperature treatment. Among the genes of known functions, many are related to maintenance of cell wall integrity, adjustment of osmotic potential and maintenance of reactive oxygen species homeostasis, implying that these physiological processes might be of paramount significance in rendering protective mechanisms against the low temperature stress. The data presented here gain an insight into the molecular changes underlying the cold acclimation of trifoliate orange, and the results can be of reference for unraveling candidate genes that hold great potential for genetic engineering in an effort to create novel germplasms with enhanced cold stress tolerance.

Down-regulated HS6ST2 in osteoarthritis and Kashin-Beck disease inhibits cell viability and influences expression of the genes relevant to aggrecan metabolism of human chondrocytes

Primary OA and Kashin-Beck disease (KBD) show similar pathological changes in articular cartilage. The objective was to screen differentially expressed genes between OA and normal cartilage, confirm the candidate gene expression among OA, KBD and normal cartilage, and then clarify its role in vitro. Differentially expressed genes in OA cartilage were screened by suppression subtractive hybridization (SSH) and verified by real-time quantitative PCR (Q-PCR) analysis. Heparan sulphate 6-O-sulphotransferase 2 (HS6ST2) expression was identified by Q-PCR and immunohistochemistry. After suppressing HS6ST2 by RNA interference in C28/I2 human chondrocyte line, the effects were analysed through determining the cell viability by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), the aggrecan contents by toluidine blue staining and the mRNA expression levels of SRY-type high mobility group box 9 (SOX9), AGC1, MMP3, a disintegrin and metalloproteinase domain with thrombospondin motifs 4 (ADAMTS4) and ADAMTS5 by Q-PCR. HS6ST2 in the reverse subtraction library was identified as a down-regulated gene in OA and KBD at both mRNA and protein levels. The percentage of safranion O staining area was correlated positively with the percentage of HS6ST2-positive chondrocytes in OA and KBD cartilage. After HS6ST2-specific short interfering RNA (siRNA) transfection to C28/I2 cells, the cell viability was inhibited significantly, and the mRNA expression levels of SOX9 and AGC1 were reduced markedly, while MMP3 expression was increased significantly. CONCLUSION; HS6ST2 down-regulation was identified in both OA and KBD cartilage. The findings first suggest that HS6ST2 may participate in the pathogenesis of OA and KBD by influencing aggrecan metabolism.

Construction of differentially expressed genes library of bighead carp ( Aristichthys nobilis) exposed to microcystin-lr using ssh and expression profile of related genes

Microcystins (MCs) are hepatotoxic cyclic heptapeptides produced by cyanobacteria (blue-green algae). There are more than 70 MCs variants of which the most common and widely studied is MC-LR. We screened the hepatocellular differentially expressed genes against MC-LR in the bighead carp ( Aristichthys nobilis). Suppression subtractive hybridization was used to construct the forward subtracted and reverse subtracted cDNA libraries, and one hundred and thirty two positive clones (seventy one in forward library and sixty one in reverse library) were randomly selected and sequenced. Finally, fifty five reliable sequences from the forward subtracted library were used in a homology search by BLASTn and BLASTx, as were 57 reliable sequences from the reverse subtracted library. Furthermore, eight analyzed sequences from the forward subtracted cDNA library and seven from the reverse subtracted library were found to be non-homologous sequences. The screening identified genes induced by MC-LR in both libraries that are involved in various processes, such as energy metabolism, immunity, and apoptosis. Some are cytoskeleton- and transportation-related genes, while signal transduction-related genes were also found. Significant genes, such as the apoptosis-related gene p53 and the proto-oncogene c-myc, are involved in inhibition of the MC-LR response in the reverse subtracted library. In addition, several immune-related genes, which play an important role in antioxidation and detoxification of MC-LR, were characterized and identified in both of the subtracted libraries. The study provides the basic data to further identify the genes and molecular mechanism of detoxification of microcystins.

Use of suppressive subtractive hybridization to identify differentially expressed genes in ayu ( Plecoglossus altivelis) associated with Listonella anguillarum infection

Suppressive subtractive hybridization (SSH) was employed to identify differentially expressed genes in ayu ( Plecoglossus altivelis) associated with Listonella anguillarum infection. 800 random clones were selected from forward and reverse subtractive libraries and 787 were successfully sequenced. After assembling, 105 contigs and 414 singletons were finally obtained, some of which were immune-related genes. A real-time quantitative PCR (RT-qPCR) analysis of the expression patterns of 28 transcripts showed that the false-positive rate was approximately 7.1%. Furthermore, Wap65-2 was overexpressed in Escherichia coli, purified and used for antiserum preparation. Western blot analysis revealed that serum Wap65-2 of ayu significantly increased after bacterial infection, suggesting that it was a positive acute-phase protein (APP).

Construction and analysis of liver suppression subtractive hybridization library of silver carp ( Hypophthalmichthys molitrix) intraperitoneally injected with microcystin-LR

Microcystin-LR (MC-LR) is the most frequently studied cyclic heptapeptide hepatotoxin produced by cyanobacteria. The toxin accumulates rapidly in the liver where it exerts most of its damage, but the molecular mechanisms behind its toxicity remain unclear. Here, suppression subtractive hybridization (SSH) was used to identify alterations in gene transcription of the silver carp ( Hypophthalmichthys molitrix) after exposure to MC-LR. After hybridization and cloning, the forward and reverse subtractive cDNA libraries were obtained. At random, 150 positive clones (70 forward and 80 reverse) were selected and sequenced from the subtractive libraries, which gave a total of 88 gene fragment sequences (48 forward and 40 reverse). Sequencing analysis and homology searches showed that these ESTs represented 75 unique genes and 13 duplicates. Of the 75 unique genes, 38 shared high homology with fish genes of known functions, including immune-related genes, transporters and some involved in cell metabolism. Four sequenced genes (Fs59, Fs70, Rs2 and Rs15) were analyzed further using semi-quantitative RT-PCR. The genes from the forward library (Fs59 and Fs70) were found to be transcriptionally upregulated, while the genes from the reverse library (Rs2 and Rs15) were found to be transcriptionally downregulated. These results confirmed the successful construction of the subtractive cDNA library that was enriched for genes that were differentially transcribed in the silver carp liver challenged with MC-LR.

Construction of cDNA subtractive library from pearl oyster (Pinctada fucata Gould) with red color shell by SSH

The molecular basis of color polymorphism in the shells of the pearl oyster Pinctada fucata is largely unknown. We developed a red-shelled family line and used suppression subtractive hybridization (SSH) to screen for differentially expressed genes in red- and non-red-shelled pearl oysters. We constructed forward and reverse cDNA subtractive libraries consisting of 2 506 and 797 clones, respectively. Among 343 randomly selected clones in the forward library, 304 sequences were identified in GenBank using BLASTx and BLASTn. Of the 304 sequences, 13 showed no similarity to known sequences and 291 were matched with known genes of the pearl oyster, including shematrin-1, shematrin-2, shematrin-6, shematrin-7, nacrein, nacrein-like protein, aspein for shell matrix protein, glycine-rich protein, mantle gene 5, 28S, EST00031, EST00036, 16S, and COI. In the reverse library, 7 clones were sequenced and analyzed by BLAST. Two sequences shared similarity with EST00036 from the P. fucata subtraction cDNA library, four with the P. fucata mitochondrial gene for 16S rRNA and 1 with P. fucata shematrin-2. We evaluated the expression of 12 genes from the forward library using RT PCR. Two sequences matched with 16S and COI so were considered to be false positives. The remaining 10 sequences were differentially expression in the red-shelled pearl oysters. Our results suggest that differential expression of these genes may be related to color variation in the red-shelled family line of the pearl oyster.

Identification and characterization of differential gene expression in the mesocarp and kernel of oil palm nuts using suppression subtractive hybridization

Suppression subtracted hybridization (SSH) and dot blotting were used to identify differential gene expression in the mesocarp and kernel of oil palm nuts. The different types of nut tissue show differences in fatty acid anabolism and the synthesis of other important compounds. In total, 302 clones from forward SSH libraries and 238 clones from reverse SSH libraries were identified following differential screening, respectively. Among these, 120 clones from the forward SSH library and 81 clones from the reverse SSH library, showed tenfold or more differential expression levels, and were sequenced. Sequence analysis revealed that 76 clones (28 from the forward SSH library and 48 from the reverse SSH library) represent non-redundant cDNA inserts. The differential expression of 39 subset genes in the two different tissues was further confirmed by RT-PCR analysis. Functionally annotated blasting against the GenBank non-redundant protein database classified all 76 candidate genes into six categories, according to their putative functions. Interestingly, our results show that a group of significantly differentially expressed genes are involved in processes associated with oil palm nut maturation, such as the synthesis of medium-chain saturated fatty acids and phytic acid, nut development, and stress/defense responses. This study describes some relationships between gene expression and metabolic pathways in mature oil palm nuts, and contributes to our understanding of oil palm nut ESTs.

Identification of differentially expressed genes in dormant (banjhi) bud of tea (Camellia sinensis (L.) O. Kuntze) using subtractive hybridization approach

Growth regulation associated with dormancy is an essential element in plant's life cycle that leads to changes in expression of large number of genes. Forward and reverse suppression subtractive hybridization (SSH) libraries were developed to identify and characterize the genes associated with bud (banjhi) dormancy in tea (Camellia sinensis (L.) O. Kuntze). Efficiency of subtraction was confirmed by comparing the abundance of β-actin gene. A total of 17 and 45 unique sequences were obtained from forward and reverse SSH library respectively. Many of the differentially regulated genes have unknown (41.1% and 26.7%) or hypothetical functions (11.7% and 2.2%) in forward and reverse SSH library respectively, while others have a role in cell growth and metabolism. Further, semi-quantitative RT-PCR was carried out for selected genes to validate the quality of ESTs from SSH library. Gene Ontology analysis identified a greater association of these ESTs in cellular metabolic pathways and their relevance to bud dormancy. Based on the EST data, the putative role of identified genes from tea is discussed in relation to dormancy, which includes various metabolic and signalling pathways. We demonstrated that SSH is an efficient tool for enriching up- and down-regulated genes related to bud dormancy in tea. This study represents an attempt to investigate banjhi dormancy in tea under field conditions, and the findings indicate that there is a potential to develop new approaches to modulate dormancy in this species.

Construction and analysis of suppression subtractive cDNA libraries of continuous monoculture Rehmannia glutinosa

To explore the molecular mechanism of continuous monoculture problem by constructing the cDNA libraries of continuous monoculture Rehmannia glutinosa. To use the suppression subtractive hybridization (SSH) technique to construct the forward and reverse subtractive cDNA libraries of continuous monoculture R. glutinosa to adopt blue-white colony screening and PCR to detect the positive clones which would be sequenced and analyzed by bioinformatics. The subtracted cDNA libraries of continuous monoculture R. glutinosa. were successfully constructed, and the result showed that the forward and reverse subtracted libraries obtained 300 positive clones, respectively. The forward and reverse libraries got different ESTs, and produced 232 (forward library) and 214 (reverse library) unique ESTs by sequencing. Based on homology search of BLASTX and BLASTN in NCBI, 200 and 195 of unique ESTs were homologous to known genes in the forward and reverse libraries, respectively. Categories of orthologous group (COG) showed that the forward and reverse libraries got 60 and 61 ESTs with the corresponding gene annotation, involving 21 metabolic pathways. The information of differential expression genes in continuous monoculture R. glutinosa, and their functional annotation of differentially expressed genes indicate that continuous monoculture has a profound effect on expression of the genes in R. glutinosa. Furthermore, the research analyzed several key genes in response to replant problem, which provided a foundation for revealing the molecular mechanism of continuous monoculture R. glutinosa.

Expression Profile of Early Responsive Genes Under Salt Stress in Upland Cotton (Gossypium hirsutum L.)

Cotton (Gossypium spp.) is an important fiber and oil crop. High soil salinity affects cotton growth and production severely. To identify genes in response to salt stress and clarify salt tolerance mechanism in cotton, suppression subtractive hybridization (SSH) libraries were constructed from cotton roots under salt stress. A total of 1,131 expressed sequence tags (ESTs) from both forward and reverse libraries were assembled into 468 uniESTs and grouped into 11 functional categories according to Gene Ontology prediction. The results showed that many physiological processes of cotton were influenced by salt stress. Some signaling elements and transcription factors, which might play important roles in salt stress response, were carefully discussed. The expression patterns of 21 selected genes under salt stress were validated by parallel method semi-quantitative reverse transcriptase-polymerase chain reaction (semi-quantitative RT-PCR), which were consistent with SSH results. An interaction network of salt-responsive genes was constructed and three molecular regulatory pathways of cotton were deduced. Our findings might result in further understanding of salt stress response in cotton and contribute to genetically modified cotton with enhanced salt tolerance.

Differential expression of genes in the earthworm Eisenia fetida following exposure to Escherichia coli O157:H7

In this study, suppression subtractive hybridization was used to construct forward and reverse cDNA libraries to identify genes involved in the response of Eisenia fetida after exposure to Escherichia coli O157:H7. We cloned 1428 cDNAs or expressed sequence tags (ESTs), of which 738 were confirmed to be differentially expressed on dot blotting analysis. A total of 394 good-quality ESTs (GenBank dbEST accession numbers HO001170–HO001563) were obtained from the raw clone sequences after cleaning. The genes were associated with metabolism (10%), transport (10%), translation (5%), immunity (2%), and the cytoskeleton (1%). Thirteen candidates were selected to assess expression levels in earthworms exposed to artificially contaminated soil by real-time PCR. The translated amino acid sequences of clones were similar to fibrinolytic protease 1, extracellular globin-3, myosin essential light chain, lumbrokinase, lysozyme, ferritin, ATP synthase F0 subunit 6, and hsp 70. Characterization of differential gene expression in the earthworm E. fetida on exposure to E. coli O157:H7 expands our understanding of the molecular mechanisms of interactions at the earthworm–pathogen interface.