[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
supported by grants from the National Natural Science Foundation of China (30671178);the Shanxi Province Science Foundation for Youths, China (2014021029-2)
Using the hypocotyl and cotyledon explants of Brassica napus L. cuhivar Qingza No. 5 as receptors, hormone combinations in bud differentiation medi- um, bud growth medium and rooting medium were optimized to establish...Using the hypocotyl and cotyledon explants of Brassica napus L. cuhivar Qingza No. 5 as receptors, hormone combinations in bud differentiation medi- um, bud growth medium and rooting medium were optimized to establish an efficient plantlet regeneration system of B. napus cuhivar Qingza No. 5. The results showed that the highest differentiation efficiency of hypocotyls of B. napus cuhivar Qingza No. 5 reached about 90%, which was three times that of cotyledons. The appropriate differentiation medium was MSB + 5 mg/L thidiazuron (TDZ) +7.5 mg/L AgNO3 + 0.1 mg/L NAA + 2 mg/L proline (L-pro) + 250 mg/L casein acid hydrolysate (CH) + 3% sucrose; the appropriate growth medium was 1/2 MSB + 1 mg/L IBA + 2 mg/L L-pro + 250 mg/L CH + 1.5% sucrose; the ap- propriate rooting medium was 1/2 MSB + 0.2 mg/L IAA + 1.5% sucrose. On this basis, a binary expression vector harboring insect resistance gene B12 was constructed and introduced into B. napus hypocotyls by Agrobacterium-mediated transformation. Positive plants were screened using hygromycin and carbenicillin. Transgenic plants were verified by PCR and GUS histochemical staining. The results showed that insect resistance gene B12 was successfully integrated into the nu- clear genome of B. napus plants and could be expressed normally. Leaves of transgenic plants with high expression levels were collected for indoor inoculation test with Plutella xylotella larvae to evaluate insect resistance of transgenic plants.展开更多
The characterization of flower specific promoter is critical during flower development by cucumber transformation technology.AGAMOUS(AG)is an organ identity gene that is required for carpel and stamen development in A...The characterization of flower specific promoter is critical during flower development by cucumber transformation technology.AGAMOUS(AG)is an organ identity gene that is required for carpel and stamen development in Arabidopsis.The promoter and second intron of AG contain multiple regulatory elements that confer proper spatial and temporal expression.Cucumber is an important vegetable with unisexual flowers.Cucumber MADS-box 1(CUM1)is the AG homolog in cucumber,belonging to the eu AG lineage along with AG.In situ hybridization showed that CUM1 was specifically expressed in the stamens and carpels of cucumber.GUS staining indicated that the second intron of CUM1 confers stamen-specific expression,while the promoter of CUM1 drives both stamen-and carpel-specific expression during the early stages of flower development,but is restricted to carpel-and connectivum-specific expression during the late stages of flower development.Furthermore,a yeast one-hybrid assay demonstrated that two auxin response factors(Cs ARF13 and Cs ARF17)had bound directly to the second intron of CUM1.Our data suggest that different regulatory circuits operate in AG homologs in plant species with distinct sex types.展开更多
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.
基金supported by grants from the National Natural Science Foundation of China (30671178)the Shanxi Province Science Foundation for Youths, China (2014021029-2)
文摘supported by grants from the National Natural Science Foundation of China (30671178);the Shanxi Province Science Foundation for Youths, China (2014021029-2)
基金Supported by National Natural Science Foundation of China(31301703)Agricultural Science and Technology Independent Innovation Fund of Jiangsu Province[CX(14)5068]
文摘Using the hypocotyl and cotyledon explants of Brassica napus L. cuhivar Qingza No. 5 as receptors, hormone combinations in bud differentiation medi- um, bud growth medium and rooting medium were optimized to establish an efficient plantlet regeneration system of B. napus cuhivar Qingza No. 5. The results showed that the highest differentiation efficiency of hypocotyls of B. napus cuhivar Qingza No. 5 reached about 90%, which was three times that of cotyledons. The appropriate differentiation medium was MSB + 5 mg/L thidiazuron (TDZ) +7.5 mg/L AgNO3 + 0.1 mg/L NAA + 2 mg/L proline (L-pro) + 250 mg/L casein acid hydrolysate (CH) + 3% sucrose; the appropriate growth medium was 1/2 MSB + 1 mg/L IBA + 2 mg/L L-pro + 250 mg/L CH + 1.5% sucrose; the ap- propriate rooting medium was 1/2 MSB + 0.2 mg/L IAA + 1.5% sucrose. On this basis, a binary expression vector harboring insect resistance gene B12 was constructed and introduced into B. napus hypocotyls by Agrobacterium-mediated transformation. Positive plants were screened using hygromycin and carbenicillin. Transgenic plants were verified by PCR and GUS histochemical staining. The results showed that insect resistance gene B12 was successfully integrated into the nu- clear genome of B. napus plants and could be expressed normally. Leaves of transgenic plants with high expression levels were collected for indoor inoculation test with Plutella xylotella larvae to evaluate insect resistance of transgenic plants.
基金supported by The National Key Research and Development Program of China[2016YFD0101007]the National Natural Science Foundation of China[31572132]
文摘The characterization of flower specific promoter is critical during flower development by cucumber transformation technology.AGAMOUS(AG)is an organ identity gene that is required for carpel and stamen development in Arabidopsis.The promoter and second intron of AG contain multiple regulatory elements that confer proper spatial and temporal expression.Cucumber is an important vegetable with unisexual flowers.Cucumber MADS-box 1(CUM1)is the AG homolog in cucumber,belonging to the eu AG lineage along with AG.In situ hybridization showed that CUM1 was specifically expressed in the stamens and carpels of cucumber.GUS staining indicated that the second intron of CUM1 confers stamen-specific expression,while the promoter of CUM1 drives both stamen-and carpel-specific expression during the early stages of flower development,but is restricted to carpel-and connectivum-specific expression during the late stages of flower development.Furthermore,a yeast one-hybrid assay demonstrated that two auxin response factors(Cs ARF13 and Cs ARF17)had bound directly to the second intron of CUM1.Our data suggest that different regulatory circuits operate in AG homologs in plant species with distinct sex types.