Recently, a novel green fluorescent protein eYGFPuv has been identified in the marine organism Chiridius poppei which displays high fluorescence intensity and can be visible by eyes in dark. Although strong green fluo...Recently, a novel green fluorescent protein eYGFPuv has been identified in the marine organism Chiridius poppei which displays high fluorescence intensity and can be visible by eyes in dark. Although strong green fluorescence was achieved in transgenic petunia, 3 expression cassettes (about 8 kb) complicate its application. In this study, to confirm whether 1 expression cassette could be used as a transgenic marker in prokaryotes and eukaryotes, eYGFPuv was cloned into prokaryotic expression vector pET28α-eYGFPuv- His and plant binary expression vector 35S::eYGFPuv. Compared to EGFP, eYGFPuv protein exhibited stronger dazzling green fluorescence in E. coli under excited light at 365 nm and maintains steadily over a long period of time without degradation. When transiently expressed in tobacco leaves, eYGFPuv protein displayed strong green fluorescence. Moreover, the fluorescence of eYGFPuv protein also could be directly observed in living plant, and thus can be used easily as a marker to screen transformed lines in transgenic research. Overall, compared to previous studies on eYGFPuv tandem repeats, our data confirmed that single eYGFPuv sequence still possesses high fluorescence intensity and quenching resistance. Furthermore, because of small size of expression cassette,it is suitable for efficient transformation in both prokaryotic and eukaryotic organisms.展开更多
The important agronomic traits of seed size and seed oil content in oilseed rape were af-fected by maternal effect. The maternal tissue endosperm, generated by fertilization of the diploid central cell in the female ...The important agronomic traits of seed size and seed oil content in oilseed rape were af-fected by maternal effect. The maternal tissue endosperm, generated by fertilization of the diploid central cell in the female gametophyte, is essential for embryo and seed development. Imprinting is primarily re-stricted to the endosperm of flowering plants and is associated with differential DNA methylation of paren-tal alleles. Therefore, it is necessary to find out whether the endosperm influences these traits. If so, whether DNA methylation participated in these processes？ To answer these questions, isolation of pure liquid endosperm qualified for extraction genomic DNA and RNA was the prerequisite. However, it was not easy to conduct this isolation due to endosperm is encapsulated by tiny seed coat and it adjoins to de-veloping embryo. This research adopted a procedure for isolation pure lipid endosperm from 25DAF （days after flowering） ovules of Brassica napus and improved procedure for subsequent DNA extraction. With the optimized procedures, we successfully extracted genomic DNA of liquid endosperm from 500 mg o-vules with high concentration （ ≥2. 5μg/μl, 200μl） and high purity, which were proved by the subse-quent analysis.展开更多
基金supported by the National Natural Science Foundation of China (31500237)
文摘Recently, a novel green fluorescent protein eYGFPuv has been identified in the marine organism Chiridius poppei which displays high fluorescence intensity and can be visible by eyes in dark. Although strong green fluorescence was achieved in transgenic petunia, 3 expression cassettes (about 8 kb) complicate its application. In this study, to confirm whether 1 expression cassette could be used as a transgenic marker in prokaryotes and eukaryotes, eYGFPuv was cloned into prokaryotic expression vector pET28α-eYGFPuv- His and plant binary expression vector 35S::eYGFPuv. Compared to EGFP, eYGFPuv protein exhibited stronger dazzling green fluorescence in E. coli under excited light at 365 nm and maintains steadily over a long period of time without degradation. When transiently expressed in tobacco leaves, eYGFPuv protein displayed strong green fluorescence. Moreover, the fluorescence of eYGFPuv protein also could be directly observed in living plant, and thus can be used easily as a marker to screen transformed lines in transgenic research. Overall, compared to previous studies on eYGFPuv tandem repeats, our data confirmed that single eYGFPuv sequence still possesses high fluorescence intensity and quenching resistance. Furthermore, because of small size of expression cassette,it is suitable for efficient transformation in both prokaryotic and eukaryotic organisms.
文摘The important agronomic traits of seed size and seed oil content in oilseed rape were af-fected by maternal effect. The maternal tissue endosperm, generated by fertilization of the diploid central cell in the female gametophyte, is essential for embryo and seed development. Imprinting is primarily re-stricted to the endosperm of flowering plants and is associated with differential DNA methylation of paren-tal alleles. Therefore, it is necessary to find out whether the endosperm influences these traits. If so, whether DNA methylation participated in these processes？ To answer these questions, isolation of pure liquid endosperm qualified for extraction genomic DNA and RNA was the prerequisite. However, it was not easy to conduct this isolation due to endosperm is encapsulated by tiny seed coat and it adjoins to de-veloping embryo. This research adopted a procedure for isolation pure lipid endosperm from 25DAF （days after flowering） ovules of Brassica napus and improved procedure for subsequent DNA extraction. With the optimized procedures, we successfully extracted genomic DNA of liquid endosperm from 500 mg o-vules with high concentration （ ≥2. 5μg/μl, 200μl） and high purity, which were proved by the subse-quent analysis.
