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
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] Taqman Quantitative PCR technique was adopted to detect the copies of exogenous nos terminator in transgenic hybrid soybean.[Method] With soybean Lectin as the endogenous reference gene,and gene complex DN...[Objective] Taqman Quantitative PCR technique was adopted to detect the copies of exogenous nos terminator in transgenic hybrid soybean.[Method] With soybean Lectin as the endogenous reference gene,and gene complex DNA in non-GMO soybeans as the endogenous reference standard,the method of gradient dilution was used for separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies,and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation.[Result] The standard curve equation of endogenous reference gene is y=-3.422x＋35.201,R2=0.998;and the standard curve equation of exogenous gene is y=-3.348x＋34.890,R2=0.999.Nos terminator and its lower boundary sequences in transgenic soybean is of single copy.[Conclusion] The study has provided a theoretical basis for determining exogenous gene copies in transgenic soybean.展开更多
To elucidate the differential gene expression patterns in soybeans during infection by Phytophthora sojae,a cDNA library for suppression subtractive hybridization （SSH） was constructed with cDNAs from soybean cultiv...To elucidate the differential gene expression patterns in soybeans during infection by Phytophthora sojae,a cDNA library for suppression subtractive hybridization （SSH） was constructed with cDNAs from soybean cultivar Suinong 10 treated with sterile distilled water as the driver and cDNAs from Suinong 10 inoculated with P.sojae as the tester.A total of 2 067 recombinant colonies from the SSH library were randomly picked,amplified,and sequenced.After discarding 312 poor quality expressed sequence tags （EST）,1 755 high quality ESTs were assembled and edited to 1 384 tentatively unique genes （TUG）,in which,586 showed significant homology to known sequences,and 798 had low homology or no match with the known sequences.A cDNA microarray containing 307 singletons from the 586 TUGs and 222 singletons from the 798 TUGs was developed to characterize differentially expressed cDNAs in the SSH library,and eight cDNAs were identified to be up-regulated after microarray analysis and then confirmed by real-time PCR.They were homologous to the protein 10,and were also related to some proteins in disease resistance response,such as pathogen-related protein,phenylalanine ammonia-lyase,isoflavone reductase,WRKY transcription factor 31,major allergen Pru ar 1,and pleiotropic drug resistance protein 12.Most of the up-regulated cDNAs encode enzymes of phytoalexin biosynthesis and pathogenesis-related proteins involved in plant disease resistance.Here,we fist reported the Pru ar 1 in soybeans.The findings of this research have contributed to better understanding of soybean resistance to P.sojae at the molecular level.展开更多
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.
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Funded by Program of Technology Bureau of Harbin(2010RFQXN101)Sub-project of Transgenic Significant Specific Project(2008ZX08012-001)~~
文摘[Objective] Taqman Quantitative PCR technique was adopted to detect the copies of exogenous nos terminator in transgenic hybrid soybean.[Method] With soybean Lectin as the endogenous reference gene,and gene complex DNA in non-GMO soybeans as the endogenous reference standard,the method of gradient dilution was used for separately calculate Ct value of endogenous reference gene and plasmid DNA and correlation standard curve equation of logarithm of copies,and then to calculate the copies of samples through substituting thus-obtained Ct into the standard curve equation.[Result] The standard curve equation of endogenous reference gene is y=-3.422x＋35.201,R2=0.998;and the standard curve equation of exogenous gene is y=-3.348x＋34.890,R2=0.999.Nos terminator and its lower boundary sequences in transgenic soybean is of single copy.[Conclusion] The study has provided a theoretical basis for determining exogenous gene copies in transgenic soybean.
基金supported by the Program for New Century Excellent Talents in Universities,Ministry of Education,China(NCET-09-164)the National Natural Science Foundation of China(30671317,30971811,31071439,and 31110103001)+1 种基金the Program for New Century Excellent Talents in Universities in Heilongjiang Province,China(NCET-06-007)the Natural Science Foundation of Heilongjiang Province,China(C200814)
文摘To elucidate the differential gene expression patterns in soybeans during infection by Phytophthora sojae,a cDNA library for suppression subtractive hybridization （SSH） was constructed with cDNAs from soybean cultivar Suinong 10 treated with sterile distilled water as the driver and cDNAs from Suinong 10 inoculated with P.sojae as the tester.A total of 2 067 recombinant colonies from the SSH library were randomly picked,amplified,and sequenced.After discarding 312 poor quality expressed sequence tags （EST）,1 755 high quality ESTs were assembled and edited to 1 384 tentatively unique genes （TUG）,in which,586 showed significant homology to known sequences,and 798 had low homology or no match with the known sequences.A cDNA microarray containing 307 singletons from the 586 TUGs and 222 singletons from the 798 TUGs was developed to characterize differentially expressed cDNAs in the SSH library,and eight cDNAs were identified to be up-regulated after microarray analysis and then confirmed by real-time PCR.They were homologous to the protein 10,and were also related to some proteins in disease resistance response,such as pathogen-related protein,phenylalanine ammonia-lyase,isoflavone reductase,WRKY transcription factor 31,major allergen Pru ar 1,and pleiotropic drug resistance protein 12.Most of the up-regulated cDNAs encode enzymes of phytoalexin biosynthesis and pathogenesis-related proteins involved in plant disease resistance.Here,we fist reported the Pru ar 1 in soybeans.The findings of this research have contributed to better understanding of soybean resistance to P.sojae at the molecular level.
