Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodoph...Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were con-structed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5’- and 3’-flanking regions (Tub5’ and Tub3’) up- and down-stream of β-glucuronidase (GUS) gene (gusA), respectively, into pA, a derivative of pCAT?3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3’. These con-structs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5’-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when com-pared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35 S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.展开更多
Agrobacterium-mediated genetic transformation of Sophora japonica was standardized using the Agrobacterium tumefa- ciens strain LBA4404 that harbored the binary vector pBI121 containing genes for fl-glucuronidase （GU...Agrobacterium-mediated genetic transformation of Sophora japonica was standardized using the Agrobacterium tumefa- ciens strain LBA4404 that harbored the binary vector pBI121 containing genes for fl-glucuronidase （GUS） and neomycin phos- photransterase （npt II）. S. japonica transformants were selected by the ability of the leaf explants to produce kanamycin-resistant calli that regenerated into kanamycin-resistant plantlets. Successful transformation was confirmed by histochemical assay for GUS activity, PCR analysis and Southern blot. The period of nearly two months was required for the regeneration of transgenic plantlets fi＇om the explants. The transformed plants resembled their parents in morphology.展开更多
[Objective] This study aimed to reveal the expression pattern of foreign genes regulated by tomato rbcS3A promoter in transgenic tomato. [Method] Rubisco small subunit promoter rbcS3A was cloned by PCR, fused to the u...[Objective] This study aimed to reveal the expression pattern of foreign genes regulated by tomato rbcS3A promoter in transgenic tomato. [Method] Rubisco small subunit promoter rbcS3A was cloned by PCR, fused to the upstream of Gus coding region in a binary vector, and transformed into tomato plants mediated by Agrobacterium. Histochemical staining on PCR positive plants was performed to ana- lyze the expression pattern of the foreign gene regulated by the tomato rbcS3A pro- moter in transgenic tomato. [Result] A total of 15 positive plants were obtained, ac- counting for 33.3%. Histochemical staining showed that the expression level of Gus fusion gene was highest in mature leaf, lower in reproductive organs such as fruit, and not detected in seed. [Conclusion] More positive seedlings were obtained using the modified tissue culture method. Under the control of tomato rbcS3A promoter, exogenous gene highly expressed in transgenic plant leaves, but did not express in seeds and tomato pulp.展开更多
【目的】从棉花无短绒突变体GZnn中分离棉纤维发育相关的转录因子,并对其转录激活功能和表达模式进行初步分析。【方法】通过RACE(rapid amplification of the cDNA ends)和染色体步行(genome walking)技术,获得GhMS3的cDNA序列及基因组...【目的】从棉花无短绒突变体GZnn中分离棉纤维发育相关的转录因子,并对其转录激活功能和表达模式进行初步分析。【方法】通过RACE(rapid amplification of the cDNA ends)和染色体步行(genome walking)技术,获得GhMS3的cDNA序列及基因组DNA序列。利用生物信息学方法对获得的DNA序列及推定的氨基酸序列进行分析,采用酵母单杂交系统验证GhMS3蛋白的转录激活功能,运用GUS组织化学染色法在转基因烟草中分析该基因的表达模式。【结果】获得GhMS3的基因组DNA以及上游1174bp的启动子序列。氨基酸序列比对发现GhMS3是R2R3 MYB转录因子。酵母试验表明,GhMS3蛋白具体外转录激活功能,C端体外转录激活功能较强,在PGhMS3:GUS转基因烟草中,GUS主要在表皮毛、根毛以及细胞分裂旺盛区域表达。【结论】从棉花无短绒突变体GZnn中分离到的R2R3 MYB转录因子GhMS3,具有组织特异性表达模式并且其编码蛋白具有体外转录激活功能,是否参与植物表皮细胞分化有待于进一步研究。展开更多
文摘Endogenous tubulin promoter has been widely used for expressing foreign genes in green algae, but the efficiency and feasibility of endogenous tubulin promoter in the economically important Porphyra yezoensis (Rhodophyta) are unknown. In this study, the flanking sequences of beta-tubulin gene from P. yezoensis were amplified and two transient expression vectors were con-structed to determine their transcription promoting feasibility for foreign gene gusA. The testing vector pATubGUS was constructed by inserting 5’- and 3’-flanking regions (Tub5’ and Tub3’) up- and down-stream of β-glucuronidase (GUS) gene (gusA), respectively, into pA, a derivative of pCAT?3-enhancer vector. The control construct, pAGUSTub3, contains only gusA and Tub3’. These con-structs were electroporated into P. yezoensis protoplasts and the GUS activities were quantitatively analyzed by spectrometry. The results demonstrated that gusA gene was efficiently expressed in P. yezoensis protoplasts under the regulation of 5’-flanking sequence of the beta-tubulin gene. More interestingly, the pATubGUS produced stronger GUS activity in P. yezoensis protoplasts when com-pared to the result from pBI221, in which the gusA gene was directed by a constitutive CaMV 35 S promoter. The data suggest that the integration of P. yezoensis protoplast and its endogenous beta-tubulin flanking sequences is a potential novel system for foreign gene expression.
文摘Agrobacterium-mediated genetic transformation of Sophora japonica was standardized using the Agrobacterium tumefa- ciens strain LBA4404 that harbored the binary vector pBI121 containing genes for fl-glucuronidase （GUS） and neomycin phos- photransterase （npt II）. S. japonica transformants were selected by the ability of the leaf explants to produce kanamycin-resistant calli that regenerated into kanamycin-resistant plantlets. Successful transformation was confirmed by histochemical assay for GUS activity, PCR analysis and Southern blot. The period of nearly two months was required for the regeneration of transgenic plantlets fi＇om the explants. The transformed plants resembled their parents in morphology.
文摘[Objective] This study aimed to reveal the expression pattern of foreign genes regulated by tomato rbcS3A promoter in transgenic tomato. [Method] Rubisco small subunit promoter rbcS3A was cloned by PCR, fused to the upstream of Gus coding region in a binary vector, and transformed into tomato plants mediated by Agrobacterium. Histochemical staining on PCR positive plants was performed to ana- lyze the expression pattern of the foreign gene regulated by the tomato rbcS3A pro- moter in transgenic tomato. [Result] A total of 15 positive plants were obtained, ac- counting for 33.3%. Histochemical staining showed that the expression level of Gus fusion gene was highest in mature leaf, lower in reproductive organs such as fruit, and not detected in seed. [Conclusion] More positive seedlings were obtained using the modified tissue culture method. Under the control of tomato rbcS3A promoter, exogenous gene highly expressed in transgenic plant leaves, but did not express in seeds and tomato pulp.
文摘【目的】从棉花无短绒突变体GZnn中分离棉纤维发育相关的转录因子,并对其转录激活功能和表达模式进行初步分析。【方法】通过RACE(rapid amplification of the cDNA ends)和染色体步行(genome walking)技术,获得GhMS3的cDNA序列及基因组DNA序列。利用生物信息学方法对获得的DNA序列及推定的氨基酸序列进行分析,采用酵母单杂交系统验证GhMS3蛋白的转录激活功能,运用GUS组织化学染色法在转基因烟草中分析该基因的表达模式。【结果】获得GhMS3的基因组DNA以及上游1174bp的启动子序列。氨基酸序列比对发现GhMS3是R2R3 MYB转录因子。酵母试验表明,GhMS3蛋白具体外转录激活功能,C端体外转录激活功能较强,在PGhMS3:GUS转基因烟草中,GUS主要在表皮毛、根毛以及细胞分裂旺盛区域表达。【结论】从棉花无短绒突变体GZnn中分离到的R2R3 MYB转录因子GhMS3,具有组织特异性表达模式并且其编码蛋白具有体外转录激活功能,是否参与植物表皮细胞分化有待于进一步研究。
