[Objective]The study was to analyze the expression of the deletion fragments from the promoter of a glycosyltransferase gene induced both by MeJA and SA cloned from tobacco W38（sm-Ngt） in transgenic tobacco plants.[...[Objective]The study was to analyze the expression of the deletion fragments from the promoter of a glycosyltransferase gene induced both by MeJA and SA cloned from tobacco W38（sm-Ngt） in transgenic tobacco plants.[Method]Using T1 seedlings of sm-Ngt transgenic tobacco lines containing Gus gene controlled by five 5＇ flank deletion promoter fragments different in length as experimental materials,GUS histochemical staining and fluorometric analysis of T1 seedlings treated with MeJA and SA for 16 h were conducted to analyze the effect of MeJA and SA treatment on the expression of 5＇ flank deletion promoter fragments.[Result]Of five 5＇ flank deletion promoter fragments transgenic plant lines,30 d old T1 seedlings containing 220-0 bp promoter fragment performed worst in GUS staining（showing least staining spots）,those containing-524-0 bp and-468-0 bp promoter fragment both performed best.In the plants not treated with MeJA and SA,activities of GUS driven by-524-0 bp and-468-0 bp deletion promoter fragments were enormously higher than that driven by-1 150-0,-800-0 or-220 0 bp,and which were proved to be not resulted from insert copy number by Southern blot.For GUS expression,promoter fragment-800-0 bp expression was doubly induced by both MeJA and SA,while fragment-1 150-0 was induced by MeJA.[Conclusion]There are activity enhancement elements within-524--220 bp of the sm-Ngt in promoter and activity down regulation elements within-1 150--524 bp region,as well as MeJA and SA doubly inducing activity regulation elements in this promoter.展开更多
Both bolting and flowering times influence taproot and seed production in radish. FLOWERING LOCUS C （FLC） plays a key role in plant flowering by functioning as a repressor. Two genomic DNA sequences, a 3 046-bp from...Both bolting and flowering times influence taproot and seed production in radish. FLOWERING LOCUS C （FLC） plays a key role in plant flowering by functioning as a repressor. Two genomic DNA sequences, a 3 046-bp from an early- and a 2 959-bp from a late-bolting radish line were isolated and named as RsFLC1 and RsFLC2, respectively, for they share approximately 87.03% sequence identity to the FLC cDNA sequences. The genomic DNA sequences, 1 466-bp and 1 744-bp, flanking the 5＇-regions of RsFLC1 and RsFLC2, respectively, were characterized. Since both of them harbor the basic promoter elements, the TATA box and CAAT box, they were designated as PRsFLC1 and PRsFLC2. The transcription start site （TSS） was identified at 424 and 336 bp upstream of the start codon in PRsFLC1 and PRsFLC2, respectively, cis-regulatory elements including CGTCA （MeJA-responsive） and ABRE （abscisic acid-responsive） motifs were found in both promoters, while some cis-regulatory elements including TCAelement and GARE-motif were present only in PRsFLCI. These sequence differences lead to the diversity of promoter core elements, which could partially result in the difference of bolting and flowering time in radish line NauDY13 （early-bolting） and Naulu 127 （late-bolting）. Furthermore, to investigate the activity of these promoters, a series of 5＇-deletion fragment-GUS fusions were constructed and transformed into tobacco. GUS activity was detected in PRsFLCI-（1 to 4）-GUS-PSlaG-3 and PRsFLC2-（1 to 4）-GUS-PS1aG-3 transgenic tobacco leaf discs, and this activity progressively decreased from PRsFLC-1-GUS-PSlaG-3 to PRsFLC-5-GUS-PS1aG-3. Deletion analysis indicated that the cis-regulatory elements located at -395 bp to ＋1 bp may be critical for specifying RsFLC gene transcription.展开更多
Three MARs(matrix attachment regions)fragments were cloned from tobacco(Nicotiana tabacum)(MAR1), yeast(Saccharomyces cerevisiae)(MAR3)and kidney bean(Phaseolus vulgaris)(MAR5)which ranged 984, 822 and 782bp, respecti...Three MARs(matrix attachment regions)fragments were cloned from tobacco(Nicotiana tabacum)(MAR1), yeast(Saccharomyces cerevisiae)(MAR3)and kidney bean(Phaseolus vulgaris)(MAR5)which ranged 984, 822 and 782bp, respectively. Sequence analysis showed that all thefragments had fairly high A/T content (73, 62 and 75%, respectively),harbored differentnumber and different type of some characteristic motifs of MARs, such as A-box and T-box,etc. The results of in vitro binding assay showed that the three MARs fragments derivedfrom different organisms could bind specifically to the matrix extracted from the tobacconuclei with different strength, which also demonstrated that these MARs fragments arefunctionally conserved during evolution. By using these MARs fragments to flank the β-glucuronidase (GUS) reporter gene and bialaphos resistance(bar) selectable marker gene,and then introducing the resulting plant expression vectors containing MARs-uidA-bar-MARs into tobacco through Agrobacterium mediated procedures, the effects of MARs sequenceson the expression of transgenes in tobacco were investigated and compared. The GUSactivity in individual transformants showed that, comparing to the controls withoutadditional MARs, the overall transgene expression level in transformants with MARs hadbeen greatly increased while the variations in transgene expression among transformantswere decreased in different degrees. In accordance with the results of sequence analysisand in vitro binding assay in which MAR1 fragment showed the strongest binding strength,this MARs fragment also showed the greatest effect in increasing transgene overallexpression level.展开更多
Xylem-specific promoter could regulate efficient expression of foreign genes in xylem. Deletion analysis method was applied to obtain 5＇ deletion promoter fragments MU2 andMU4 with different distances from transcript...Xylem-specific promoter could regulate efficient expression of foreign genes in xylem. Deletion analysis method was applied to obtain 5＇ deletion promoter fragments MU2 andMU4 with different distances from transcription start site of xylem-specific promoter MDCcsAP through PCR procedure. Then CaMV35S promoter in plant expression vector pBI121 was replaced by amplified fragments. The recombinant plasmids fused with corresponding gene segments and GUS reporter gene were obtained and transformed into Agrobacterium. The results of the tobacco transient transformation test showed that MU2 andMU4 had the promoter function, and both of their activity were significantly higher than that of CaMV35S promoter, and they had the characteristics of xylem tissue specificity as well. Research on xy- lem-specific promoter structure and function could lay a foundation for the determination of necessary components to the xylem tissue specificity and cis-acting ele- ments with induction activity, so as to enable the regulation of efficient expression of exogenous genes in specific time and specific tissues in plants through these cis-acting elements.展开更多
In order to analyze the Oslbglu4 phenotype, the inducible promoter of the transgenic rice which knock-down the Oslbglu4 expression was assessed. The result showed that 30 μM dexamethasone（DEX） had the stronger indu...In order to analyze the Oslbglu4 phenotype, the inducible promoter of the transgenic rice which knock-down the Oslbglu4 expression was assessed. The result showed that 30 μM dexamethasone（DEX） had the stronger induction effect than 10 μM DEX by β-Glucuronidase （GUS） staining, qRT-PCR further verified the Oslbglu4 gene deletion. The effect of DEX and its solvent absolute ethanol on seed development was measured, and no significant effect was observed. The conclusion is that final concentration of DEX at 30 μM is suitable for pOp6 promoter induction.展开更多
基金Supported by Natural Science Foundation of Hubei Province(2004ABA123)~~
文摘[Objective]The study was to analyze the expression of the deletion fragments from the promoter of a glycosyltransferase gene induced both by MeJA and SA cloned from tobacco W38（sm-Ngt） in transgenic tobacco plants.[Method]Using T1 seedlings of sm-Ngt transgenic tobacco lines containing Gus gene controlled by five 5＇ flank deletion promoter fragments different in length as experimental materials,GUS histochemical staining and fluorometric analysis of T1 seedlings treated with MeJA and SA for 16 h were conducted to analyze the effect of MeJA and SA treatment on the expression of 5＇ flank deletion promoter fragments.[Result]Of five 5＇ flank deletion promoter fragments transgenic plant lines,30 d old T1 seedlings containing 220-0 bp promoter fragment performed worst in GUS staining（showing least staining spots）,those containing-524-0 bp and-468-0 bp promoter fragment both performed best.In the plants not treated with MeJA and SA,activities of GUS driven by-524-0 bp and-468-0 bp deletion promoter fragments were enormously higher than that driven by-1 150-0,-800-0 or-220 0 bp,and which were proved to be not resulted from insert copy number by Southern blot.For GUS expression,promoter fragment-800-0 bp expression was doubly induced by both MeJA and SA,while fragment-1 150-0 was induced by MeJA.[Conclusion]There are activity enhancement elements within-524--220 bp of the sm-Ngt in promoter and activity down regulation elements within-1 150--524 bp region,as well as MeJA and SA doubly inducing activity regulation elements in this promoter.
基金supported by the grants from the National Natural Science Foundation of China (31171956)the National Key Technologies R&D Program of China (2012BAD02B01)+2 种基金the Key Technologies R&D Program of Jiangsu Province, China (BE2013429)the Priority Academic Program Development of Jiangsu Higher Education Institutions of China (PAPD)Jiangsu Agricultural Science and Technology Innovation Fund, China (JASTIF, CX(12)2006, CX(13)2007)
文摘Both bolting and flowering times influence taproot and seed production in radish. FLOWERING LOCUS C （FLC） plays a key role in plant flowering by functioning as a repressor. Two genomic DNA sequences, a 3 046-bp from an early- and a 2 959-bp from a late-bolting radish line were isolated and named as RsFLC1 and RsFLC2, respectively, for they share approximately 87.03% sequence identity to the FLC cDNA sequences. The genomic DNA sequences, 1 466-bp and 1 744-bp, flanking the 5＇-regions of RsFLC1 and RsFLC2, respectively, were characterized. Since both of them harbor the basic promoter elements, the TATA box and CAAT box, they were designated as PRsFLC1 and PRsFLC2. The transcription start site （TSS） was identified at 424 and 336 bp upstream of the start codon in PRsFLC1 and PRsFLC2, respectively, cis-regulatory elements including CGTCA （MeJA-responsive） and ABRE （abscisic acid-responsive） motifs were found in both promoters, while some cis-regulatory elements including TCAelement and GARE-motif were present only in PRsFLCI. These sequence differences lead to the diversity of promoter core elements, which could partially result in the difference of bolting and flowering time in radish line NauDY13 （early-bolting） and Naulu 127 （late-bolting）. Furthermore, to investigate the activity of these promoters, a series of 5＇-deletion fragment-GUS fusions were constructed and transformed into tobacco. GUS activity was detected in PRsFLCI-（1 to 4）-GUS-PSlaG-3 and PRsFLC2-（1 to 4）-GUS-PS1aG-3 transgenic tobacco leaf discs, and this activity progressively decreased from PRsFLC-1-GUS-PSlaG-3 to PRsFLC-5-GUS-PS1aG-3. Deletion analysis indicated that the cis-regulatory elements located at -395 bp to ＋1 bp may be critical for specifying RsFLC gene transcription.
基金supported by the National High Tech R&D Program(863 Program)of China(2001AA212161)the National Natural Science Foundation of China(30170747).
文摘Three MARs(matrix attachment regions)fragments were cloned from tobacco(Nicotiana tabacum)(MAR1), yeast(Saccharomyces cerevisiae)(MAR3)and kidney bean(Phaseolus vulgaris)(MAR5)which ranged 984, 822 and 782bp, respectively. Sequence analysis showed that all thefragments had fairly high A/T content (73, 62 and 75%, respectively),harbored differentnumber and different type of some characteristic motifs of MARs, such as A-box and T-box,etc. The results of in vitro binding assay showed that the three MARs fragments derivedfrom different organisms could bind specifically to the matrix extracted from the tobacconuclei with different strength, which also demonstrated that these MARs fragments arefunctionally conserved during evolution. By using these MARs fragments to flank the β-glucuronidase (GUS) reporter gene and bialaphos resistance(bar) selectable marker gene,and then introducing the resulting plant expression vectors containing MARs-uidA-bar-MARs into tobacco through Agrobacterium mediated procedures, the effects of MARs sequenceson the expression of transgenes in tobacco were investigated and compared. The GUSactivity in individual transformants showed that, comparing to the controls withoutadditional MARs, the overall transgene expression level in transformants with MARs hadbeen greatly increased while the variations in transgene expression among transformantswere decreased in different degrees. In accordance with the results of sequence analysisand in vitro binding assay in which MAR1 fragment showed the strongest binding strength,this MARs fragment also showed the greatest effect in increasing transgene overallexpression level.
基金Supported by National Science Foundation of China(31000305)Fund from Education Department of Hebei Province(QN2015182)+1 种基金Innovation Fund for Forestry Discipline of Hebei Agricultural University(LXXK2014-1)Fund for Overseas Research and Study by Young and Middle-aged Backbone Teachers in Hebei Agriculture University(JWYX2015)
文摘Xylem-specific promoter could regulate efficient expression of foreign genes in xylem. Deletion analysis method was applied to obtain 5＇ deletion promoter fragments MU2 andMU4 with different distances from transcription start site of xylem-specific promoter MDCcsAP through PCR procedure. Then CaMV35S promoter in plant expression vector pBI121 was replaced by amplified fragments. The recombinant plasmids fused with corresponding gene segments and GUS reporter gene were obtained and transformed into Agrobacterium. The results of the tobacco transient transformation test showed that MU2 andMU4 had the promoter function, and both of their activity were significantly higher than that of CaMV35S promoter, and they had the characteristics of xylem tissue specificity as well. Research on xy- lem-specific promoter structure and function could lay a foundation for the determination of necessary components to the xylem tissue specificity and cis-acting ele- ments with induction activity, so as to enable the regulation of efficient expression of exogenous genes in specific time and specific tissues in plants through these cis-acting elements.
基金Supported by Guizhou International Cooperation Project on Science and Technology[No.QiankehewaiG(2013)7040]The 20th Project of The Joint Committee on Scientific and Technical Cooperation between The Government of the Kingdom of Thailand and The Government of the People’s Republic of China(No.20-606J)China.Suranaree University of Technology grant number SUT3-304-54-12-29,Thailand
文摘In order to analyze the Oslbglu4 phenotype, the inducible promoter of the transgenic rice which knock-down the Oslbglu4 expression was assessed. The result showed that 30 μM dexamethasone（DEX） had the stronger induction effect than 10 μM DEX by β-Glucuronidase （GUS） staining, qRT-PCR further verified the Oslbglu4 gene deletion. The effect of DEX and its solvent absolute ethanol on seed development was measured, and no significant effect was observed. The conclusion is that final concentration of DEX at 30 μM is suitable for pOp6 promoter induction.
