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
Objective: To screen and clone differentially expressed genes of dermal papillae cells (DPC) with aggregative behavior, and to explore the molecular mechanism of their aggregation. Methods: Total RNAs were extracted f...Objective: To screen and clone differentially expressed genes of dermal papillae cells (DPC) with aggregative behavior, and to explore the molecular mechanism of their aggregation. Methods: Total RNAs were extracted from DPC with and without aggregative behavior and double strand cDNAs were synthesized by using SMART cDNA synthesis, respectively. The cDNA fragments of differentially expressed genes in DPCs with aggregative behavior were isolated by suppression subtractive hybridization. Positive clones were screened by PCR method and verified by cDNA dot blot, Northern blot and then analyzed through homologous retrieving. Results: A subtractive cDNA library of DPC with aggregative behavior has been successfully constructed. The result of screening and cloning of the library showed that, DPC with aggregative behavior could expresse genes related to homologous aggregation, proliferation and cycle control, including known genes (capping protein, paladin, vascular endothelial growth factor), hematopoietic stem/progenitor cells (HSPC) related clone (HSPC011 and HSPC016) and a new gene. Conclusion: The construction of subtracted library of DPC lays solid foundation for screening and cloning new and specific genes related to aggregative behavior of DPC. Several genes might be cooperatively involved in the homologous aggregation, proliferation and cycle control of DPC. Among these genes, capping protein and palladin might be closely related to the aggregative behavior of dermal papilla cells, and VEGF and HSPC related clone would be responsible for the status of higher proliferation of dermal papilla cells.展开更多
Objective To construct a morphine tolerance model in primarily cultured striatal neurons, and screen the differentially expressed genes in this model using suppression subtractive hybridization （SSH）. Methods Sbtra...Objective To construct a morphine tolerance model in primarily cultured striatal neurons, and screen the differentially expressed genes in this model using suppression subtractive hybridization （SSH）. Methods Sbtracted cDNA libraries were constructed using SSH from normal primarily cultured striatal neurons and long-term morphine treated striatal neurons （10^-5 mol/L for 72 hours）. To check reliability of the cell culture model, RT-PCR was performed to detect the cAMP-responsive element-binding protein （CREB） mRNA expression. The subtracted clones were prescreened by PCR. The clones containing inserted fragments from forward libraries were sequenced and submitted to GenBank for homology analysis. And the expression levels of genes of interest were confirmed by RT-PCR. Results CREB mRNA expression showed a significant increase in morphine treated striatal neurons （62.85± 1.98） compared with normal striatal neurons （28.43 ± 1.46, P〈0.01）. Thirty-six clones containing inserted fragments were randomly chosen for sequence analysis. And the 36 clones showed homology with 19 known genes and 2 novel genes. The expression of 2 novel genes, mitochondrial carrier homolog 1 （Mtchl ; 96.81±2.04 vs. 44.20±1.31, P〈0.01） and thyrnoma viral proto-oncogene 1 （Akt1 ; 122.10±2.17 vs 50.11±2.01, P〈0.01）, showed a significant increase in morphine-treated striatal neurons compared with normal striatal neurons. Conclusions A reliable differential cDNA library of striatal neurons treated with long-term morphine is constructed. Mtchl and Aktl might be the candidate genes for the development of morphine tolerance.展开更多
SSH was used to analyze gene transactivation during root formation of Larix cuttings. Two subtractive cDNA libraries were constructed from clone 31-6 as tester or driver and clone 15-4 as driver or tester. The SSH PCR...SSH was used to analyze gene transactivation during root formation of Larix cuttings. Two subtractive cDNA libraries were constructed from clone 31-6 as tester or driver and clone 15-4 as driver or tester. The SSH PCR products from the libraries were cloned into a pGEM-T easy vector and after PCR and dot blot analysis, positive clones were selected, sequenced and compared to the database in GenBank with BLASTX. The results of a sequence assembly in two libraries show that 521 UniEST （expressed sequence tag） were obtained. These 521 UniEST belong to metabolism, signal pathways, transport, resistance, developmental processes, local- ization, unknown proteins and ＂no hits found＂. All of these suggest that subtractive cDNA libraries during root formation of Larix cuttings were constructed successfully.展开更多
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).
文摘Objective: To screen and clone differentially expressed genes of dermal papillae cells (DPC) with aggregative behavior, and to explore the molecular mechanism of their aggregation. Methods: Total RNAs were extracted from DPC with and without aggregative behavior and double strand cDNAs were synthesized by using SMART cDNA synthesis, respectively. The cDNA fragments of differentially expressed genes in DPCs with aggregative behavior were isolated by suppression subtractive hybridization. Positive clones were screened by PCR method and verified by cDNA dot blot, Northern blot and then analyzed through homologous retrieving. Results: A subtractive cDNA library of DPC with aggregative behavior has been successfully constructed. The result of screening and cloning of the library showed that, DPC with aggregative behavior could expresse genes related to homologous aggregation, proliferation and cycle control, including known genes (capping protein, paladin, vascular endothelial growth factor), hematopoietic stem/progenitor cells (HSPC) related clone (HSPC011 and HSPC016) and a new gene. Conclusion: The construction of subtracted library of DPC lays solid foundation for screening and cloning new and specific genes related to aggregative behavior of DPC. Several genes might be cooperatively involved in the homologous aggregation, proliferation and cycle control of DPC. Among these genes, capping protein and palladin might be closely related to the aggregative behavior of dermal papilla cells, and VEGF and HSPC related clone would be responsible for the status of higher proliferation of dermal papilla cells.
基金Supported by the National Natural Science Foundation of China (81070961,30770676,and 30870932)the Natural Science Foundation of Shandong Province (ZR2009DZ004)the Science and Technology Bureau Foundation of Shandong Province (2006GG2202037)
文摘Objective To construct a morphine tolerance model in primarily cultured striatal neurons, and screen the differentially expressed genes in this model using suppression subtractive hybridization （SSH）. Methods Sbtracted cDNA libraries were constructed using SSH from normal primarily cultured striatal neurons and long-term morphine treated striatal neurons （10^-5 mol/L for 72 hours）. To check reliability of the cell culture model, RT-PCR was performed to detect the cAMP-responsive element-binding protein （CREB） mRNA expression. The subtracted clones were prescreened by PCR. The clones containing inserted fragments from forward libraries were sequenced and submitted to GenBank for homology analysis. And the expression levels of genes of interest were confirmed by RT-PCR. Results CREB mRNA expression showed a significant increase in morphine treated striatal neurons （62.85± 1.98） compared with normal striatal neurons （28.43 ± 1.46, P〈0.01）. Thirty-six clones containing inserted fragments were randomly chosen for sequence analysis. And the 36 clones showed homology with 19 known genes and 2 novel genes. The expression of 2 novel genes, mitochondrial carrier homolog 1 （Mtchl ; 96.81±2.04 vs. 44.20±1.31, P〈0.01） and thyrnoma viral proto-oncogene 1 （Akt1 ; 122.10±2.17 vs 50.11±2.01, P〈0.01）, showed a significant increase in morphine-treated striatal neurons compared with normal striatal neurons. Conclusions A reliable differential cDNA library of striatal neurons treated with long-term morphine is constructed. Mtchl and Aktl might be the candidate genes for the development of morphine tolerance.
基金supported by the National S&T Support Projects for the 11th Five-Year Plan (2006BA-D01A14)the National High-Tech "863" Program of China (2006AA100109)the National "973" Program (2009CB119107)
文摘SSH was used to analyze gene transactivation during root formation of Larix cuttings. Two subtractive cDNA libraries were constructed from clone 31-6 as tester or driver and clone 15-4 as driver or tester. The SSH PCR products from the libraries were cloned into a pGEM-T easy vector and after PCR and dot blot analysis, positive clones were selected, sequenced and compared to the database in GenBank with BLASTX. The results of a sequence assembly in two libraries show that 521 UniEST （expressed sequence tag） were obtained. These 521 UniEST belong to metabolism, signal pathways, transport, resistance, developmental processes, local- ization, unknown proteins and ＂no hits found＂. All of these suggest that subtractive cDNA libraries during root formation of Larix cuttings were constructed successfully.
