To investigate the expression profile of maize genes induced by submergence, a subtracted cDNA library of maize seedling roots was constructed using suppression subtractive hybridization (SSH). The cDNA of maize seedl...To investigate the expression profile of maize genes induced by submergence, a subtracted cDNA library of maize seedling roots was constructed using suppression subtractive hybridization (SSH). The cDNA of maize seedling roots treated with submergence (ST) was used as tester and what from untreated roots (UT) as driver. Products of the secondary PCR from the forward subtraction were cloned into T/A vector and transferred into Escherichia coli strain JM10B by electroporation. Four hundred and eight randomly chosen transformants carrying cDNA fragments were screened with PCR-Select Deferential Screening Kit. One hundred and eighty-four cDNA clones were identified as, submergence specifically induced or highly expressed. After sequencing and removing redundant cDNAs, we got 95 submergence-induced cDNA clones. Of the 95 cDNA clones, 68 contain the regions with 60%-90% identity to their homolog in GenBank, 21 are expected to be novel genes, only 6 correspond to the published maize sequences.展开更多
Phosphorus is one of the three essential macroelements for plant growth. Plants respond to phosphorus starvation through adaptive mechanisms involved in morphological, biochemical and molecular changes. To investigate...Phosphorus is one of the three essential macroelements for plant growth. Plants respond to phosphorus starvation through adaptive mechanisms involved in morphological, biochemical and molecular changes. To investigate the molecular background of the adaptive mechanisms, the suppression subtractive hybridization (SSH) method was used to construct a rice phosphorus-starvation ( Pi-starvation) induced cDNA library. Through screening of the cDNA library and sequencing of the enriched cDNAs, 18 known genes and 47 novel genes were identified. The known genes are involved in different metabolic processes, including phosphate uptake and transport, signal transduction, protein synthesis and degradation, carbon metabolism and stress response. Northern analysis was performed to detect the expression patterns of some known genes and novel genes under different phosphorus levels. Different expression patterns of the selected genes were identified, which suggests that genes involved in different pathways may have different responses to Pi-starvation.展开更多
In this study, a forward cDNA library was constructed by suppression subtractive hybridization using seedling leaves of CN165, a drought-tolerant maize inbred line. In the suppression subtractive hybridization （SSH）...In this study, a forward cDNA library was constructed by suppression subtractive hybridization using seedling leaves of CN165, a drought-tolerant maize inbred line. In the suppression subtractive hybridization （SSH） library, 672 positive clones were picked up randomly. After polymerase chain reaction （PCR） of each clone, all the single clones were sequenced. Totally 598 available sequences were obtained. After cluster analysis of the EST sequences, 80 uniESTs were obtained, among which 57 uniESTs were contigs and 23 uniESTs were singlets. The results of BLASTN showed that all the uniESTs had homologous sequences in the nr database. The BLASTX results indicated that 68 uniESTs had significant protein homology, 8 uniESTs with homology of unknown proteins and putative proteins, and 4 uniESTs without protein homology. Those drought stress-induced genes were involved in many metabolism pathways to regulate plant growth and development under drought stress.展开更多
AIM： To screen for metronidazole （MTZ）-resistance associated gene fragments of H pylori by suppression subtractive hybridization （SSH）. METHODS： Five MTZ-resistant （tester, T） and 1 MTZ- susceptible （driver, ...AIM： To screen for metronidazole （MTZ）-resistance associated gene fragments of H pylori by suppression subtractive hybridization （SSH）. METHODS： Five MTZ-resistant （tester, T） and 1 MTZ- susceptible （driver, D） clinical H pylori isolates were selected. Genomic DNAs were prepared and submitted to Rsa I digestion. Then two different adaptors were ligated respectively to the 5＇-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNAs. The specific inserts of tester strains were screened and MTZ-resistance related gene fragments were identified by dot blotting. RESULTS： Among the randomly selected 120 subtractive colonies, 37 DNA fragments had a different number of DNA copies （≥ 2 times） in resistant and susceptible strains and 17 of them had a significantly different number of DNA copies （≥ 3 times）. Among the sequences obtained from the 17 DNA fragments, new sequences were found in 10 DNA fragments and duplicated sequences in 7 DNA fragments, representing respectively the sequences of depeptide ABC transporter periplasmic dipeptide-binding protein （dppA）, permease protein （dppB）, ribosomal protein S4 （rps4）, ribonuclease Ⅲ （rnc）, protease （pqqE）, diaminopimelate epimerase （dapF）, acetatekinase （ackA）, H pylori plasmid pHP51 and Hpylori gene 1334. CONCLUSION： Gene fragments specific to MTZ-resistant H pylori strains can be screened by SSH and may be associated with MTZ-resistant Hpylori.展开更多
AIM: To identify potential diagnostic target genes in early reperfusion periods following warm liver ischemia before irreversible liver damage occurs. METHODS: We used two strategies (SSH suppression subtractive hybri...AIM: To identify potential diagnostic target genes in early reperfusion periods following warm liver ischemia before irreversible liver damage occurs. METHODS: We used two strategies (SSH suppression subtractive hybridization and hybridization of cDNA arrays) to determine early changes in gene expression profiles in a rat model of partial WI/R, comparing postischemic and adjacent nonischemic liver lobes. Differential gene expression was verified (WI/R; 1 h/2 h) and analyzed in more detail after warm ischemia (1 h) in a reperfusion time kinetics (0, 1, 2 and 6 h) and compared to untreated livers by Northern blot hybridizations. Protein expression was examined on Western blots and by immunohistochemistry for four differentially expressed target genes (Hsp70, Hsp27, Gadd45a and IL-1rI). RESULTS: Thirty-two individual WI/R target genes showing altered RNA levels after confirmation by Northern blot analyzes were identified. Among them, six functionally uncharacteristic expressed sequences and 26 known genes (12 induced in postischemic liver lobes, 14 with higher transcriptional expression in adjacent nonischemic liver lobes). Functional categories of the verified marker genes indicate on the one hand cellular stress and tissue damage but otherwise activation of protective cellular reactions (AP-1 transcription factors, apoptosis related genes, heat shock genes). In order to assign the transcriptional status to the biological relevant protein level we demonstrated that Hsp70, Hsp27, Gadd45a and IL-1rI were clearly up-regulated comparing postischemic and untreated rat livers, suggesting their involvement in the WI/R context. CONCLUSION: This study unveils a WI/R response gene set that will help to explore molecular pathways involved in the tissue damage after WI/R. In addition, these genes especially Hsp70 and Gadd45a might represent promising new candidates indicating WI/R liver damage.展开更多
Soybean is planted worldwide and its productivity is significantly hampered by salinity. Development of salt tolerant cultivars is necessary for promoting soybean production. Despite wealth of information generated on...Soybean is planted worldwide and its productivity is significantly hampered by salinity. Development of salt tolerant cultivars is necessary for promoting soybean production. Despite wealth of information generated on salt tolerance mechanism, its basics still remain elusive. A continued effort is needed to understand the salt tolerance mechanism in soybean using suitable molecular tools. To better understand the molecular basis of the responses of soybean to salt stress and to get an enrichment of critical salt stress responsive genes in soybean, suppression subtractive hybridization libraries （SSH） are constructed for the root tissue of two cultivated soybean genotypes, one was tolerant and the other was sensitive to salt stress. To compare the responses of plants in salt treatment and non-treatment, SSH1 was constructed for the salt-tolerant cultivar Wenfeng 7 and SSH2 was constructed for the salt-sensitive cultivar Union. From the two SSH cDNA libraries, a total of 379 high quality ESTs were obtained. These ESTs were then annotated by performing sequence similarity searches against the NCBI nr （National Center for Biotechnology Information protein non-redundant） database using the BLASTX program. Sixty-three genes from SSH1 and 49 genes from SSH2 could be assigned putative function. On the other hand, 25 ESTs of SSH1 which may be not the salt tolerance-related genes were removed by comparing and analyzing the ESTs from the two S SH libraries, which increased the proportion of the genes related to salt tolerance in S SH 1. These results suggested that the novel way could realize low background of SSH and high level enrichment of target cDNAs to some extent.展开更多
For the purpose of screening and analyzing the differentially expressed genes from the salivary gland of Rhipicephalus haemaphysaloides, two salivary gland-subtracted cDNA libraries of partially fed female ticks and f...For the purpose of screening and analyzing the differentially expressed genes from the salivary gland of Rhipicephalus haemaphysaloides, two salivary gland-subtracted cDNA libraries of partially fed female ticks and fed male ticks were constructed using suppression subtractive hybridization （SSH）. A total of 247 female expression sequence tags （ESTs） and 168 male ESTs were obtained from the two SSH cDNA libraries. It is predicted that 25 female ESTs and 44 female ESTs contain the 5＂ and 3＂ ends, respectively, and that 53 male ESTs and 74 male ESTs contain the 5＂ and 3＂ ends, respectively. To identify the subtraction rate of the two SSH cDNA libraries, the RT-PCR method was used to test 24 female ESTs and 21 male ESTs selected randomly but not repeatedly. The results showed that there were 13 upregulated or differentially expressed genes in the partially fed salivary gland of the female R. haemaphysaloides and that the differentially expressed rate was 54%. In addition, they indicated that there were 9 upregulated or differently expressed genes in the fed salivary gland of the male R. haemaphysaloides and that the differentially expressed rate was 43%. Putative translations of 141 （57%） female ESTs and 125 （74%） male ESTs had similarity to GenBank sequences, and 32 （23%） female ESTs and 29 （23%） male ESTs exhibited similarity to tick proteins, which showed that most of the proteins in the libraries were mainly related to the feeding blood physiology of the ticks.展开更多
[Objectives]This study was conducted to construct forward and reserve libraries of suppression subtractive hybridization (SSH) in the testis of male tilapia under the stress of methomyl by using SSH technology.[Method...[Objectives]This study was conducted to construct forward and reserve libraries of suppression subtractive hybridization (SSH) in the testis of male tilapia under the stress of methomyl by using SSH technology.[Methods]Using male tilapia as the test animal,the forward and reserve libraries of SSH in the testis of tilapia under the stress of methomyl were constructed by using the SSH technology.[Results]45 expressed sequence tags (ESTs) were obtained,and 25 expressed sequence tags were successfully noted,including 13 forward libraries and 12 reverse libraries.The genes with confirmed functions were classified into five types.Genes related to catalytic activity and cell characteristics were up-regulated,while genes related to structural molecule's activity and biological process were down-regulated.The expression amount of integrin β1 was up-regulated,while serine/threonine protein kinase pim-3,Ca^2+-ATPase,Na^+-K^+-ATPase and ribosomal protein L22 were down-regulated.[Conclusions]The research results could lay a foundation for revealing the molecular mechanism of methomyl's reproductive toxicity to tilapia.展开更多
Plants reshape their transcriptomes, proteomes and metabolomes in response to insect damage. In this study, we used suppression subtractive hybridization to investigate the transcriptomes of two cotton varieties （CCR...Plants reshape their transcriptomes, proteomes and metabolomes in response to insect damage. In this study, we used suppression subtractive hybridization to investigate the transcriptomes of two cotton varieties （CCRI41 and CCRI23） under Apolygus lucorum damage. From the CCRI23 libraries we obtained 92 transcripts and from the CCRI41 libraries we obtained 96 transcripts. 26 and 63 of the transcripts from CCRI23 and CCRI41, respectively, had known functions. Using reverse transcription PCR, we detected expression proifle of genes with known functions. Ultimately, we identiifed eight signiifcantly regulated genes, including one downregulated and four upregulated genes from the CCRI41 libraries, and one downregulated and two upregulated genes from the CCRI23 libraries. Only the gene encoding the polyphenol oxidase （PPO） is involved in plant defense against insect herbivores, and the others are related to improving tolerance to insect damage. Quantitative real-time PCR was used to study changes in expression levels during A. lucorum damage in CCRI23 and CCRI41. Signiifcantly regulated genes from CCRI23 showed a response in CCRI23 but not response in CCRI41. Similarly, signiifcantly regulated genes from CCRI41 showed a response in CCRI41 but not response in CCRI23. The results showed that, among transcriptomes of cotton varieties, there are different responses to A. lucorum damage.展开更多
AIM： To clone and identify human genes transactivated by PSITP5 by constructing a cDNA subtractive library with suppression subtractive hybridization （SSH） technique. METHODS： SSH and bioinformatics techniques wer...AIM： To clone and identify human genes transactivated by PSITP5 by constructing a cDNA subtractive library with suppression subtractive hybridization （SSH） technique. METHODS： SSH and bioinformatics techniques were used for screening and cloning of the target genes transactivated by PS1TP5 protein. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1（-）- myc-his（A）-PS1TP5 and pcDNA3.1（-）-myc-his（A） empty vector, respectively, and SSH technique was employed to analyze the differentially expressed DNA sequence between the two groups. After digestion with restriction enzyme Rsa Ⅰ, small size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. The tester cDNA was hybridized with driver cDNA twice and subjected to nested PCR for two times, and then subcloned into T/A plasmid vectors to set up the subb-active library. Amplification of the library was carried out with E.. coil strain DH5α. The cDNA was sequenced and analyzed in GenBank with Vector NTI 9.1 and NCBI BLAST software after PCR amplification. RESULTS： The subtractive library of genes transactivated by PS1TP5 was constructed successfully. The amplified library contained 90 positive clones. Colony PCR showed that 70 clones contained 200-1000-bp inserts. Sequence analysis was performed in 30 clones randomly, and the full-length sequences were obtained by bioinformatics technique. Altogether 24 coding sequences were obtained, which consisted of 23 known and 1 unknown.One novel gene with unknown functions was found and named as PSITP5TP1 after being electronically spliced, and deposited in GenBank （accession number： DQ487761）. CONCLUSION： PSITP5 is closely correlated with immunoregulation, carbohydrate metabolism, signal transduction, formation mechanism of hepatic fibrosis, and occurrence and development of tumor. Understanding PSlTP5 transactive proteins may help to bring some new clues for further studying the biological functions of pre-S1 protein.展开更多
Suppression subtractive hybridization (SSH) was employed to investigate bioluminescence in Panellus stipticus (Bull.) P. Karst. by detecting proteins differentially expressed in bioluminescent and luminescent strains....Suppression subtractive hybridization (SSH) was employed to investigate bioluminescence in Panellus stipticus (Bull.) P. Karst. by detecting proteins differentially expressed in bioluminescent and luminescent strains. Comparisons of luminescent and non-luminescent monokaryon cultures of North American strains revealed differences in transcript levels of proteins responsible for post-translational modification (PTM) of enzymes. A similar comparison of a luminescent strain of P. stipticus from North America with a non-luminescent European strain revealed the presence of extracellular manganese superoxide dismutase (MnSOD) in the luminescent form, in addition to proteins involved in PTM. The application of MnSOD-specific inhibitors to luminescent mycelium resulted in the rapid loss of luminescence. The relevance to luminescence of proteins involved in PTM is discussed, together with a possible role for MnSOD that considers the potential for SODs to form stable complexes with catechols revealed in previously published research. In light of the recent discovery that hispidine may be the precursor of fungal luciferin, we consider a hypothetical mechanism for fungal luminescence in which the ο-hydroquinone moiety of a hispidine derivative ligates with the extracellular form of MnSOD producing a semiquinone-radical complex, with the resultant semiquinonato complex potentially reacting with molecular oxygen or other reactive oxygen species to produce sufficiently excited intermediates to emit light on relaxation.展开更多
A cDNA subtractive library enriched for dark-induced up-regulated ESTs was constructed by suppression subtractive hybridization(SSH) from leaf tissues of soybean cultivar DongNong L13 treated with short-day(8-h light/...A cDNA subtractive library enriched for dark-induced up-regulated ESTs was constructed by suppression subtractive hybridization(SSH) from leaf tissues of soybean cultivar DongNong L13 treated with short-day(8-h light/16-h dark) and long-day(16-h light/8-h dark) conditions.A total of 148 clones were sequenced,representing 76 unique ESTs which corresponded to about 20% of 738 clones from the cDNA library and showed a significant up-regulation of at least three fold verified by dot blot hybridization.The putative functions of ESTs were predicted by Blastn and Blastx.The 43 differentially expressed genes identified by subtractions were classified according to their putative functions generated by Blast analysis.Genetic functional analysis indicated that putative proteins encoded by these genes were related to diverse functions during organism development,which include biological regulation pathways such as transcription,signal transduction and programmed cell death,protein,nucleic acid and carbohydrate macromolecule degradation,the cell wall modification,primary and secondary metabolism and stress response.Two soybean transcription factors enhanced in SD conditions,GAMYB-binding protein and DNA binding protein RAV cDNAs that may be involved in SD soybean photoperiod response,had been isolated using 5'-and 3'-rapid amplification of cDNA ends(RACE)(Genbank Accession numbers DQ112540 and DQ147914).展开更多
Background and Objective The invasion and metastasis are not only the malignant markers and characteristics of lung cancer, but also the leading cause of failure to treatment and patient’
Objective To construct and screen the suppression subtractive hybridization (SSH) library of human renal cell carcinoma (RCC). Methods Poly A+ RNA was isolated from RCC lines 786-O(tester) and renal cell(RC) lines HK-...Objective To construct and screen the suppression subtractive hybridization (SSH) library of human renal cell carcinoma (RCC). Methods Poly A+ RNA was isolated from RCC lines 786-O(tester) and renal cell(RC) lines HK-2 ( driver), respectiely. SSH procedure was performed according to the protocol of the PCR-Select cDNA Subtraction Kit ( Clontech), and PCR products were cloned into pT-Adv vector and transformed E. coli TOP10F’. All positive clones picked out were digested and some of which were sequenced. Results The SSH library contained 362 clones with SSH cDNA fragments distributed mainly from 0.3 to 0.9 kb. Among 50 clones sequenced randomly,2 represented unknown genes and the other 48 derived from 36 known genes. Conclusion The quality of the SSH library of human RCC is reliable and is construction is the basis for further screening differentially expressed genes of RCC. 6 refs,4 figs, 1 tab.展开更多
The modular multilevel converter(MMC)has become a promising topology for widespread power converter applications.However,an evident circulating current flowing between the phases will increase system losses and compli...The modular multilevel converter(MMC)has become a promising topology for widespread power converter applications.However,an evident circulating current flowing between the phases will increase system losses and complicate the heatsink design.This paper proposes a novel hybrid model predictive control method for MMCs.This method utilizes an indirect structure MPC and a sorting algorithm to implement current tracking and capacitor voltages balancing,considerably resulting in reduced calculation burden.In addition,different from the conventional MPC solutions,we add a simple proportional-integral(PI)controller to suppress circulating current through modifying the submodule(SM)inserted number,which is parallel to the MPC loop.This hybrid control solution combines both advantages of MPC and linear control,evidently resulting in improved performance of circulating current.Finally,the MATLAB/Simulink results of an 11-level MMC system verify the effectiveness of the proposed solution.展开更多
BACKGROUND: Interferon-alpha (IFN-alpha) is an important cytokine with multiple functions, but the target genes transactivated by IFN-alpha remain largely unknown. A study of such genes will help to understand the mec...BACKGROUND: Interferon-alpha (IFN-alpha) is an important cytokine with multiple functions, but the target genes transactivated by IFN-alpha remain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-alpha. To isolate the gene transcripts specifically upregulated by IFN-alpha in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis. METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-alpha protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-alpha (rIFN-alpha, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-alpha as driver. The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed significant homology to other known proteins. RESULTS: The subtractive cDNA library of genes upregulated by IFN-alpha was constructed successfully, rIFN-alpha upregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc finger RNA binding protein, Dickkopf homolog 1 (DKK1) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). CONCLUSIONS: These results suggest that rIFN-alpha can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-alpha.展开更多
A set of basic deformation modes for hybrid stress finite elements are directly derived from the element displacement field. Subsequently, by employing the so-called united orthogonal conditions, a new orthogonalizati...A set of basic deformation modes for hybrid stress finite elements are directly derived from the element displacement field. Subsequently, by employing the so-called united orthogonal conditions, a new orthogonalization method is proposed. The result- ing orthogonal basic deformation modes exhibit simple and clear physical meanings. In addition, they do not involve any material parameters, and thus can be efficiently used to examine the element performance and serve as a unified tool to assess different hybrid elements. Thereafter, a convenient approach for the identification of spurious zero-energy modes is presented using the positive definiteness property of a flexibility matrix. More- over, based on the orthogonality relationship between the given initial stress modes and the orthogonal basic deformation modes, an alternative method of assumed stress modes to formulate a hybrid element free of spurious modes is discussed. It is found that the orthogonality of the basic deformation modes is the sufficient and necessary condition for the suppression of spurious zero-energy modes. Numerical examples of 2D 4-node quadrilateral elements and 3D 8-node hexahedral elements are illustrated in detail to demonstrate the efficiency of the proposed orthogonal basic deformation mode method.展开更多
In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression sub-tractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88genes. After ...In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression sub-tractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88genes. After two rounds of screening by reverse Northern analysis, twenty genes were proved to be up-regulated, including seventeen known genes and three genes with unknown function. All these genes werefirstly associated with UL. Three genes with notable difference were selected for Northern confirmationOur results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showedup-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obviousexpression in prostate, testis, liver, heart and skeletal muscle.展开更多
文摘To investigate the expression profile of maize genes induced by submergence, a subtracted cDNA library of maize seedling roots was constructed using suppression subtractive hybridization (SSH). The cDNA of maize seedling roots treated with submergence (ST) was used as tester and what from untreated roots (UT) as driver. Products of the secondary PCR from the forward subtraction were cloned into T/A vector and transferred into Escherichia coli strain JM10B by electroporation. Four hundred and eight randomly chosen transformants carrying cDNA fragments were screened with PCR-Select Deferential Screening Kit. One hundred and eighty-four cDNA clones were identified as, submergence specifically induced or highly expressed. After sequencing and removing redundant cDNAs, we got 95 submergence-induced cDNA clones. Of the 95 cDNA clones, 68 contain the regions with 60%-90% identity to their homolog in GenBank, 21 are expected to be novel genes, only 6 correspond to the published maize sequences.
文摘Phosphorus is one of the three essential macroelements for plant growth. Plants respond to phosphorus starvation through adaptive mechanisms involved in morphological, biochemical and molecular changes. To investigate the molecular background of the adaptive mechanisms, the suppression subtractive hybridization (SSH) method was used to construct a rice phosphorus-starvation ( Pi-starvation) induced cDNA library. Through screening of the cDNA library and sequencing of the enriched cDNAs, 18 known genes and 47 novel genes were identified. The known genes are involved in different metabolic processes, including phosphate uptake and transport, signal transduction, protein synthesis and degradation, carbon metabolism and stress response. Northern analysis was performed to detect the expression patterns of some known genes and novel genes under different phosphorus levels. Different expression patterns of the selected genes were identified, which suggests that genes involved in different pathways may have different responses to Pi-starvation.
文摘In this study, a forward cDNA library was constructed by suppression subtractive hybridization using seedling leaves of CN165, a drought-tolerant maize inbred line. In the suppression subtractive hybridization （SSH） library, 672 positive clones were picked up randomly. After polymerase chain reaction （PCR） of each clone, all the single clones were sequenced. Totally 598 available sequences were obtained. After cluster analysis of the EST sequences, 80 uniESTs were obtained, among which 57 uniESTs were contigs and 23 uniESTs were singlets. The results of BLASTN showed that all the uniESTs had homologous sequences in the nr database. The BLASTX results indicated that 68 uniESTs had significant protein homology, 8 uniESTs with homology of unknown proteins and putative proteins, and 4 uniESTs without protein homology. Those drought stress-induced genes were involved in many metabolism pathways to regulate plant growth and development under drought stress.
基金Supported by the Natural Science Foundation of ZhejiangProvince, No. 29801
文摘AIM： To screen for metronidazole （MTZ）-resistance associated gene fragments of H pylori by suppression subtractive hybridization （SSH）. METHODS： Five MTZ-resistant （tester, T） and 1 MTZ- susceptible （driver, D） clinical H pylori isolates were selected. Genomic DNAs were prepared and submitted to Rsa I digestion. Then two different adaptors were ligated respectively to the 5＇-end of two aliquots of the tester DNA fragments and SSH was made between the tester and driver DNAs. The specific inserts of tester strains were screened and MTZ-resistance related gene fragments were identified by dot blotting. RESULTS： Among the randomly selected 120 subtractive colonies, 37 DNA fragments had a different number of DNA copies （≥ 2 times） in resistant and susceptible strains and 17 of them had a significantly different number of DNA copies （≥ 3 times）. Among the sequences obtained from the 17 DNA fragments, new sequences were found in 10 DNA fragments and duplicated sequences in 7 DNA fragments, representing respectively the sequences of depeptide ABC transporter periplasmic dipeptide-binding protein （dppA）, permease protein （dppB）, ribosomal protein S4 （rps4）, ribonuclease Ⅲ （rnc）, protease （pqqE）, diaminopimelate epimerase （dapF）, acetatekinase （ackA）, H pylori plasmid pHP51 and Hpylori gene 1334. CONCLUSION： Gene fragments specific to MTZ-resistant H pylori strains can be screened by SSH and may be associated with MTZ-resistant Hpylori.
文摘AIM: To identify potential diagnostic target genes in early reperfusion periods following warm liver ischemia before irreversible liver damage occurs. METHODS: We used two strategies (SSH suppression subtractive hybridization and hybridization of cDNA arrays) to determine early changes in gene expression profiles in a rat model of partial WI/R, comparing postischemic and adjacent nonischemic liver lobes. Differential gene expression was verified (WI/R; 1 h/2 h) and analyzed in more detail after warm ischemia (1 h) in a reperfusion time kinetics (0, 1, 2 and 6 h) and compared to untreated livers by Northern blot hybridizations. Protein expression was examined on Western blots and by immunohistochemistry for four differentially expressed target genes (Hsp70, Hsp27, Gadd45a and IL-1rI). RESULTS: Thirty-two individual WI/R target genes showing altered RNA levels after confirmation by Northern blot analyzes were identified. Among them, six functionally uncharacteristic expressed sequences and 26 known genes (12 induced in postischemic liver lobes, 14 with higher transcriptional expression in adjacent nonischemic liver lobes). Functional categories of the verified marker genes indicate on the one hand cellular stress and tissue damage but otherwise activation of protective cellular reactions (AP-1 transcription factors, apoptosis related genes, heat shock genes). In order to assign the transcriptional status to the biological relevant protein level we demonstrated that Hsp70, Hsp27, Gadd45a and IL-1rI were clearly up-regulated comparing postischemic and untreated rat livers, suggesting their involvement in the WI/R context. CONCLUSION: This study unveils a WI/R response gene set that will help to explore molecular pathways involved in the tissue damage after WI/R. In addition, these genes especially Hsp70 and Gadd45a might represent promising new candidates indicating WI/R liver damage.
基金the National Natural Science Foundation of China (30771358)
文摘Soybean is planted worldwide and its productivity is significantly hampered by salinity. Development of salt tolerant cultivars is necessary for promoting soybean production. Despite wealth of information generated on salt tolerance mechanism, its basics still remain elusive. A continued effort is needed to understand the salt tolerance mechanism in soybean using suitable molecular tools. To better understand the molecular basis of the responses of soybean to salt stress and to get an enrichment of critical salt stress responsive genes in soybean, suppression subtractive hybridization libraries （SSH） are constructed for the root tissue of two cultivated soybean genotypes, one was tolerant and the other was sensitive to salt stress. To compare the responses of plants in salt treatment and non-treatment, SSH1 was constructed for the salt-tolerant cultivar Wenfeng 7 and SSH2 was constructed for the salt-sensitive cultivar Union. From the two SSH cDNA libraries, a total of 379 high quality ESTs were obtained. These ESTs were then annotated by performing sequence similarity searches against the NCBI nr （National Center for Biotechnology Information protein non-redundant） database using the BLASTX program. Sixty-three genes from SSH1 and 49 genes from SSH2 could be assigned putative function. On the other hand, 25 ESTs of SSH1 which may be not the salt tolerance-related genes were removed by comparing and analyzing the ESTs from the two S SH libraries, which increased the proportion of the genes related to salt tolerance in S SH 1. These results suggested that the novel way could realize low background of SSH and high level enrichment of target cDNAs to some extent.
基金the National Natural Science Foundation of China (31172095)
文摘For the purpose of screening and analyzing the differentially expressed genes from the salivary gland of Rhipicephalus haemaphysaloides, two salivary gland-subtracted cDNA libraries of partially fed female ticks and fed male ticks were constructed using suppression subtractive hybridization （SSH）. A total of 247 female expression sequence tags （ESTs） and 168 male ESTs were obtained from the two SSH cDNA libraries. It is predicted that 25 female ESTs and 44 female ESTs contain the 5＂ and 3＂ ends, respectively, and that 53 male ESTs and 74 male ESTs contain the 5＂ and 3＂ ends, respectively. To identify the subtraction rate of the two SSH cDNA libraries, the RT-PCR method was used to test 24 female ESTs and 21 male ESTs selected randomly but not repeatedly. The results showed that there were 13 upregulated or differentially expressed genes in the partially fed salivary gland of the female R. haemaphysaloides and that the differentially expressed rate was 54%. In addition, they indicated that there were 9 upregulated or differently expressed genes in the fed salivary gland of the male R. haemaphysaloides and that the differentially expressed rate was 43%. Putative translations of 141 （57%） female ESTs and 125 （74%） male ESTs had similarity to GenBank sequences, and 32 （23%） female ESTs and 29 （23%） male ESTs exhibited similarity to tick proteins, which showed that most of the proteins in the libraries were mainly related to the feeding blood physiology of the ticks.
基金Supported by Basic Scientific Research Fund for Chinese Academy of Fishery Sciences(2015C02XK01)Youth Natural Scientific Foundation of Jiansu Province(BK20150117)Earmarked Fund for China Agriculture Research System(CARS-46)
文摘[Objectives]This study was conducted to construct forward and reserve libraries of suppression subtractive hybridization (SSH) in the testis of male tilapia under the stress of methomyl by using SSH technology.[Methods]Using male tilapia as the test animal,the forward and reserve libraries of SSH in the testis of tilapia under the stress of methomyl were constructed by using the SSH technology.[Results]45 expressed sequence tags (ESTs) were obtained,and 25 expressed sequence tags were successfully noted,including 13 forward libraries and 12 reverse libraries.The genes with confirmed functions were classified into five types.Genes related to catalytic activity and cell characteristics were up-regulated,while genes related to structural molecule's activity and biological process were down-regulated.The expression amount of integrin β1 was up-regulated,while serine/threonine protein kinase pim-3,Ca^2+-ATPase,Na^+-K^+-ATPase and ribosomal protein L22 were down-regulated.[Conclusions]The research results could lay a foundation for revealing the molecular mechanism of methomyl's reproductive toxicity to tilapia.
基金supported by the National Natural Science Foundation of China (31201518)
文摘Plants reshape their transcriptomes, proteomes and metabolomes in response to insect damage. In this study, we used suppression subtractive hybridization to investigate the transcriptomes of two cotton varieties （CCRI41 and CCRI23） under Apolygus lucorum damage. From the CCRI23 libraries we obtained 92 transcripts and from the CCRI41 libraries we obtained 96 transcripts. 26 and 63 of the transcripts from CCRI23 and CCRI41, respectively, had known functions. Using reverse transcription PCR, we detected expression proifle of genes with known functions. Ultimately, we identiifed eight signiifcantly regulated genes, including one downregulated and four upregulated genes from the CCRI41 libraries, and one downregulated and two upregulated genes from the CCRI23 libraries. Only the gene encoding the polyphenol oxidase （PPO） is involved in plant defense against insect herbivores, and the others are related to improving tolerance to insect damage. Quantitative real-time PCR was used to study changes in expression levels during A. lucorum damage in CCRI23 and CCRI41. Signiifcantly regulated genes from CCRI23 showed a response in CCRI23 but not response in CCRI41. Similarly, signiifcantly regulated genes from CCRI41 showed a response in CCRI41 but not response in CCRI23. The results showed that, among transcriptomes of cotton varieties, there are different responses to A. lucorum damage.
基金Supported by the National Natural Science Foundation,No. 30371288Beijing Natural Science Foundation,No. 5042024
文摘AIM： To clone and identify human genes transactivated by PSITP5 by constructing a cDNA subtractive library with suppression subtractive hybridization （SSH） technique. METHODS： SSH and bioinformatics techniques were used for screening and cloning of the target genes transactivated by PS1TP5 protein. The mRNA was isolated from HepG2 cells transfected with pcDNA3.1（-）- myc-his（A）-PS1TP5 and pcDNA3.1（-）-myc-his（A） empty vector, respectively, and SSH technique was employed to analyze the differentially expressed DNA sequence between the two groups. After digestion with restriction enzyme Rsa Ⅰ, small size cDNAs were obtained. Then tester cDNA was divided into two groups and ligated to the specific adaptor 1 and adaptor 2, respectively. The tester cDNA was hybridized with driver cDNA twice and subjected to nested PCR for two times, and then subcloned into T/A plasmid vectors to set up the subb-active library. Amplification of the library was carried out with E.. coil strain DH5α. The cDNA was sequenced and analyzed in GenBank with Vector NTI 9.1 and NCBI BLAST software after PCR amplification. RESULTS： The subtractive library of genes transactivated by PS1TP5 was constructed successfully. The amplified library contained 90 positive clones. Colony PCR showed that 70 clones contained 200-1000-bp inserts. Sequence analysis was performed in 30 clones randomly, and the full-length sequences were obtained by bioinformatics technique. Altogether 24 coding sequences were obtained, which consisted of 23 known and 1 unknown.One novel gene with unknown functions was found and named as PSITP5TP1 after being electronically spliced, and deposited in GenBank （accession number： DQ487761）. CONCLUSION： PSITP5 is closely correlated with immunoregulation, carbohydrate metabolism, signal transduction, formation mechanism of hepatic fibrosis, and occurrence and development of tumor. Understanding PSlTP5 transactive proteins may help to bring some new clues for further studying the biological functions of pre-S1 protein.
文摘Suppression subtractive hybridization (SSH) was employed to investigate bioluminescence in Panellus stipticus (Bull.) P. Karst. by detecting proteins differentially expressed in bioluminescent and luminescent strains. Comparisons of luminescent and non-luminescent monokaryon cultures of North American strains revealed differences in transcript levels of proteins responsible for post-translational modification (PTM) of enzymes. A similar comparison of a luminescent strain of P. stipticus from North America with a non-luminescent European strain revealed the presence of extracellular manganese superoxide dismutase (MnSOD) in the luminescent form, in addition to proteins involved in PTM. The application of MnSOD-specific inhibitors to luminescent mycelium resulted in the rapid loss of luminescence. The relevance to luminescence of proteins involved in PTM is discussed, together with a possible role for MnSOD that considers the potential for SODs to form stable complexes with catechols revealed in previously published research. In light of the recent discovery that hispidine may be the precursor of fungal luciferin, we consider a hypothetical mechanism for fungal luminescence in which the ο-hydroquinone moiety of a hispidine derivative ligates with the extracellular form of MnSOD producing a semiquinone-radical complex, with the resultant semiquinonato complex potentially reacting with molecular oxygen or other reactive oxygen species to produce sufficiently excited intermediates to emit light on relaxation.
文摘A cDNA subtractive library enriched for dark-induced up-regulated ESTs was constructed by suppression subtractive hybridization(SSH) from leaf tissues of soybean cultivar DongNong L13 treated with short-day(8-h light/16-h dark) and long-day(16-h light/8-h dark) conditions.A total of 148 clones were sequenced,representing 76 unique ESTs which corresponded to about 20% of 738 clones from the cDNA library and showed a significant up-regulation of at least three fold verified by dot blot hybridization.The putative functions of ESTs were predicted by Blastn and Blastx.The 43 differentially expressed genes identified by subtractions were classified according to their putative functions generated by Blast analysis.Genetic functional analysis indicated that putative proteins encoded by these genes were related to diverse functions during organism development,which include biological regulation pathways such as transcription,signal transduction and programmed cell death,protein,nucleic acid and carbohydrate macromolecule degradation,the cell wall modification,primary and secondary metabolism and stress response.Two soybean transcription factors enhanced in SD conditions,GAMYB-binding protein and DNA binding protein RAV cDNAs that may be involved in SD soybean photoperiod response,had been isolated using 5'-and 3'-rapid amplification of cDNA ends(RACE)(Genbank Accession numbers DQ112540 and DQ147914).
基金supported by a grant from the key project of the National Natural Science Foundation of China (to Qinghua ZHOU)(No. 30430300)National Natural Science Foundation of China (to Qinghua ZHOU)(No. 30670922)INTERNATION Scienc and Techniquie COOPRATION PROGRAM OFCHINA (ISCP) (to Qinghua ZHOU)(No.2006DFB32330)
文摘Background and Objective The invasion and metastasis are not only the malignant markers and characteristics of lung cancer, but also the leading cause of failure to treatment and patient’
文摘Objective To construct and screen the suppression subtractive hybridization (SSH) library of human renal cell carcinoma (RCC). Methods Poly A+ RNA was isolated from RCC lines 786-O(tester) and renal cell(RC) lines HK-2 ( driver), respectiely. SSH procedure was performed according to the protocol of the PCR-Select cDNA Subtraction Kit ( Clontech), and PCR products were cloned into pT-Adv vector and transformed E. coli TOP10F’. All positive clones picked out were digested and some of which were sequenced. Results The SSH library contained 362 clones with SSH cDNA fragments distributed mainly from 0.3 to 0.9 kb. Among 50 clones sequenced randomly,2 represented unknown genes and the other 48 derived from 36 known genes. Conclusion The quality of the SSH library of human RCC is reliable and is construction is the basis for further screening differentially expressed genes of RCC. 6 refs,4 figs, 1 tab.
基金This work was partially supported by the National Natural Science Foundation of China(11847104)General Program of National Natural Science Foundation of China(51977124)+2 种基金Shandong Natural Science Foundation(ZR2019QEE001)Natural Science Foundation of Jiangsu Province(BK20190204)National Distinguished Expert(Youth Talent)Program of China(31390089963058)。
文摘The modular multilevel converter(MMC)has become a promising topology for widespread power converter applications.However,an evident circulating current flowing between the phases will increase system losses and complicate the heatsink design.This paper proposes a novel hybrid model predictive control method for MMCs.This method utilizes an indirect structure MPC and a sorting algorithm to implement current tracking and capacitor voltages balancing,considerably resulting in reduced calculation burden.In addition,different from the conventional MPC solutions,we add a simple proportional-integral(PI)controller to suppress circulating current through modifying the submodule(SM)inserted number,which is parallel to the MPC loop.This hybrid control solution combines both advantages of MPC and linear control,evidently resulting in improved performance of circulating current.Finally,the MATLAB/Simulink results of an 11-level MMC system verify the effectiveness of the proposed solution.
文摘BACKGROUND: Interferon-alpha (IFN-alpha) is an important cytokine with multiple functions, but the target genes transactivated by IFN-alpha remain largely unknown. A study of such genes will help to understand the mechanism of function of IFN-alpha. To isolate the gene transcripts specifically upregulated by IFN-alpha in HepG2 cells, we conducted suppressive subtractive hybridization (SSH) analysis. METHODS: SSH was used to analyze the target genes transactivated by recombinant IFN-alpha protein, and a subtractive cDNA library was constructed from HepG2 cells treated with recombinant IFN-alpha (rIFN-alpha, 2000 IU/ml) for 16 hours as tester, and cells not treated with rIFN-alpha as driver. The SSH PCR products from the library were cloned into pGEM-T easy vector and with BLASTX, the positive clones were randomly selected, sequenced and compared to the database in GenBank of the 35 differentially expressed gene fragments from the library, 6 clones showed significant homology to other known proteins. RESULTS: The subtractive cDNA library of genes upregulated by IFN-alpha was constructed successfully, rIFN-alpha upregulated the expression of the RAN binding protein 5 (RANBP5), NADH dehydrogenase, exosome component 3 (EXOSC3), zinc finger RNA binding protein, Dickkopf homolog 1 (DKK1) and acetyl-coenzyme A acetyltransferase 2 (ACAT2). CONCLUSIONS: These results suggest that rIFN-alpha can upregulate the expression of important genes to exert its functions, and provide new clues for discovering the molecular mechanisms of action of IFN-alpha.
基金Project supported by the National Natural Science Foundation of China(No.10972188)the Fundamental Research Funds for the Central Universities of China(No.2010121073)the Scientific Program of Fujian Province of China(No.2007F3096)
文摘A set of basic deformation modes for hybrid stress finite elements are directly derived from the element displacement field. Subsequently, by employing the so-called united orthogonal conditions, a new orthogonalization method is proposed. The result- ing orthogonal basic deformation modes exhibit simple and clear physical meanings. In addition, they do not involve any material parameters, and thus can be efficiently used to examine the element performance and serve as a unified tool to assess different hybrid elements. Thereafter, a convenient approach for the identification of spurious zero-energy modes is presented using the positive definiteness property of a flexibility matrix. More- over, based on the orthogonality relationship between the given initial stress modes and the orthogonal basic deformation modes, an alternative method of assumed stress modes to formulate a hybrid element free of spurious modes is discussed. It is found that the orthogonality of the basic deformation modes is the sufficient and necessary condition for the suppression of spurious zero-energy modes. Numerical examples of 2D 4-node quadrilateral elements and 3D 8-node hexahedral elements are illustrated in detail to demonstrate the efficiency of the proposed orthogonal basic deformation mode method.
基金This work was supported by Nationa1 NaturalScience Fundation of China No.39700148 and LifeScience Special fund of CAS supported by ChineseMinisery of Finance.
文摘In searching for differentially expressed genes in human uterine leiomyomas (ULs), suppression sub-tractive hybridization was used to construct an UL up-regulated library, which turned out to represent 88genes. After two rounds of screening by reverse Northern analysis, twenty genes were proved to be up-regulated, including seventeen known genes and three genes with unknown function. All these genes werefirstly associated with UL. Three genes with notable difference were selected for Northern confirmationOur results proved the authenticity of the twenty genes. One gene named Phospholipase A2 (PLA2) showedup-regulation in 4/6 of the patients and investigation of tissue distribution indicated that it had obviousexpression in prostate, testis, liver, heart and skeletal muscle.
