Soybean transformation by ovary-drip was improved by optimizing the length of the transformation pathway by cutting the styles. These modifications facilitated soybean transformation manipulation and improved transfor...Soybean transformation by ovary-drip was improved by optimizing the length of the transformation pathway by cutting the styles. These modifications facilitated soybean transformation manipulation and improved transformation reproducibility and efficiency. Using a linear minimal gus gene cassette as the foreign DNA, a maximum transformation frequency of 11% was obtained in flowers of the soybean cultivar ‘Liaodou 14’ with their styles mostly removed, whereas removal of only the stigma, partial style cutting and partial ovary cutting gave transformation frequencies of 0%, 1%, and 2%, respectively. An average transformation frequency of 8.2% was obtained when 619 flowers from three soybean cultivars (‘Liaodou 14’, ‘Liaodou 13’, and ‘Tiefeng 29’) were transformed by this optimized method. Southern blotting analysis showed that the gus reporter gene (encoding β-glucuronidase) was stably inherited with a simple pattern. Reverse transcription-polymerase chain reaction (RT-PCR) and GUS staining confirmed the expression of the gus gene in transgenic plants.展开更多
The exogenous gene was integrated into Dunaliella salina successfully by using LiAc/PEG mediating method for the first time. According to the results of histochemical staining, transgenic D. salina was blue, showing t...The exogenous gene was integrated into Dunaliella salina successfully by using LiAc/PEG mediating method for the first time. According to the results of histochemical staining, transgenic D. salina was blue, showing that the exogenous GUS gene was successfully expressed in the cells of D. salina. Meanwhile, the effects of growth state of D. salina, plasmid concentration and temperature on its transformation efficiency were studied, and the transformation conditions were optimized. The results show that the optimum conditions for the genetic transformation of D. salina are shown as follows： D. salina was in the early logarithmic phase; plasmid DNA concentration was 600 μg/ml; temperature was 29 ℃, and transformation efficiency was up to 74.8‰ under the best conditions. According to the results of PCR amplification and PCR-Southern hybridization, the target gene had been integrated into genome and was hereditary.展开更多
[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants...[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants,after 48 hours＇ pre-cultivation on MS medium in the absence of phytohormone,GUS gene was transmitted into embryos of Ginkgo biloba mediated by three kinds of Agrobacterium.Transient expression of GUS gene activity was observed through histochemical staining,and the influencing factors of the expression of GUS gene were analyzed.And the expression vector of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the biosynthesis approach of biobalide precursor of Ginkgo biloba was constructed.[Result] A more suitable genetic transformation scheme was obtained as follows：taking embryos of Ginkgo biloba as explants,using EHA105 Agrobacterium with pCAMBIA1304＋ for infection,co-culture for 3 days and GUS staining.The results showed that transient expression rate of GUS after transformation was higher.[Conclusion] The research provide a more effective method for further study on the transgene of Ginkgo biloba.展开更多
Different types of explants of China Rose （Rosa chinensis Jacq.） were placed on a Schenk and Hildebrandt （SH） medium containing L-proline and 2,4-dichlorophenoxyacetic acid （2,4-D）. Organogenesis was observed on...Different types of explants of China Rose （Rosa chinensis Jacq.） were placed on a Schenk and Hildebrandt （SH） medium containing L-proline and 2,4-dichlorophenoxyacetic acid （2,4-D）. Organogenesis was observed on callus induced from both whole leaf and petiole and the high frequency of organogenesis was observed on the whole leaf. Shoot regeneration was obtained via or- ganogenesis. The effects of pH and concentrations of antibiotics on maintenance of organogenesis capacity were investigated in sub- sequent subcultures. The pH value was found to play a critical role in retaining organogenesis capacity. The binary vector pBI121, carrying the gus gene coding for fl-glucuronidase （GUS） and the npt II gene mediated by Agrobacterium tumefaciens, was used for transformation of organogenic callus using 50 mg·L^-1 geneticin for selection. Six regenerated lines showed GUS activity, of which five were verified for the presence ofnpt Ⅱ gene by PCR.展开更多
Hevein has been found to be an essential element in coagulation of rubber particles in latex of rubber trees. In a previous study, we cloned a 1 241-bp fragment of a 5＇ upstream region of the hevein gene by genome wa...Hevein has been found to be an essential element in coagulation of rubber particles in latex of rubber trees. In a previous study, we cloned a 1 241-bp fragment of a 5＇ upstream region of the hevein gene by genome walking. This fragment was analyzed by a 5＇ end nested deletion method in the present study, fused with a uidA （gus） gene to produce a series of tested constructs, which were transferred into C-serum of latex and the Gus activities were detected. Results showed that the fragment from -749 to -292 was sufficient for expression of gus gene in latex, and the fragment from -292 to -168 was crucial in response to abscisic acid inducement. In a transient transgenic test of rubber leaf with particle bombardment, construct Hev749 conferred gus-specific expression in veins, in which the latex tubes mainly distributed. This implies that the fragment from -749 to -292 was laticiferous-specific.展开更多
基金Project (No. JY03-B-18-02) supported by the National R & D Project of Transgenic Crops of Ministry of Science and Technology of China
文摘Soybean transformation by ovary-drip was improved by optimizing the length of the transformation pathway by cutting the styles. These modifications facilitated soybean transformation manipulation and improved transformation reproducibility and efficiency. Using a linear minimal gus gene cassette as the foreign DNA, a maximum transformation frequency of 11% was obtained in flowers of the soybean cultivar ‘Liaodou 14’ with their styles mostly removed, whereas removal of only the stigma, partial style cutting and partial ovary cutting gave transformation frequencies of 0%, 1%, and 2%, respectively. An average transformation frequency of 8.2% was obtained when 619 flowers from three soybean cultivars (‘Liaodou 14’, ‘Liaodou 13’, and ‘Tiefeng 29’) were transformed by this optimized method. Southern blotting analysis showed that the gus reporter gene (encoding β-glucuronidase) was stably inherited with a simple pattern. Reverse transcription-polymerase chain reaction (RT-PCR) and GUS staining confirmed the expression of the gus gene in transgenic plants.
基金Supported by National Natural Science Foundation of China(31472260)~~
文摘The exogenous gene was integrated into Dunaliella salina successfully by using LiAc/PEG mediating method for the first time. According to the results of histochemical staining, transgenic D. salina was blue, showing that the exogenous GUS gene was successfully expressed in the cells of D. salina. Meanwhile, the effects of growth state of D. salina, plasmid concentration and temperature on its transformation efficiency were studied, and the transformation conditions were optimized. The results show that the optimum conditions for the genetic transformation of D. salina are shown as follows： D. salina was in the early logarithmic phase; plasmid DNA concentration was 600 μg/ml; temperature was 29 ℃, and transformation efficiency was up to 74.8‰ under the best conditions. According to the results of PCR amplification and PCR-Southern hybridization, the target gene had been integrated into genome and was hereditary.
文摘[Objective] The research aimed to provide reference for increasing the genetic transformation efficiency of Ginkgo biloba mediated by Agrobacterium.[Method] Taking the mature embryos of Ginkgo biloba seeds as explants,after 48 hours＇ pre-cultivation on MS medium in the absence of phytohormone,GUS gene was transmitted into embryos of Ginkgo biloba mediated by three kinds of Agrobacterium.Transient expression of GUS gene activity was observed through histochemical staining,and the influencing factors of the expression of GUS gene were analyzed.And the expression vector of 1-deoxy-D-xylulose-5-phosphate reductoisomerase in the biosynthesis approach of biobalide precursor of Ginkgo biloba was constructed.[Result] A more suitable genetic transformation scheme was obtained as follows：taking embryos of Ginkgo biloba as explants,using EHA105 Agrobacterium with pCAMBIA1304＋ for infection,co-culture for 3 days and GUS staining.The results showed that transient expression rate of GUS after transformation was higher.[Conclusion] The research provide a more effective method for further study on the transgene of Ginkgo biloba.
文摘Different types of explants of China Rose （Rosa chinensis Jacq.） were placed on a Schenk and Hildebrandt （SH） medium containing L-proline and 2,4-dichlorophenoxyacetic acid （2,4-D）. Organogenesis was observed on callus induced from both whole leaf and petiole and the high frequency of organogenesis was observed on the whole leaf. Shoot regeneration was obtained via or- ganogenesis. The effects of pH and concentrations of antibiotics on maintenance of organogenesis capacity were investigated in sub- sequent subcultures. The pH value was found to play a critical role in retaining organogenesis capacity. The binary vector pBI121, carrying the gus gene coding for fl-glucuronidase （GUS） and the npt II gene mediated by Agrobacterium tumefaciens, was used for transformation of organogenic callus using 50 mg·L^-1 geneticin for selection. Six regenerated lines showed GUS activity, of which five were verified for the presence ofnpt Ⅱ gene by PCR.
基金the National Natural Science Foundation of China (39960065)
文摘Hevein has been found to be an essential element in coagulation of rubber particles in latex of rubber trees. In a previous study, we cloned a 1 241-bp fragment of a 5＇ upstream region of the hevein gene by genome walking. This fragment was analyzed by a 5＇ end nested deletion method in the present study, fused with a uidA （gus） gene to produce a series of tested constructs, which were transferred into C-serum of latex and the Gus activities were detected. Results showed that the fragment from -749 to -292 was sufficient for expression of gus gene in latex, and the fragment from -292 to -168 was crucial in response to abscisic acid inducement. In a transient transgenic test of rubber leaf with particle bombardment, construct Hev749 conferred gus-specific expression in veins, in which the latex tubes mainly distributed. This implies that the fragment from -749 to -292 was laticiferous-specific.
