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
Cytoplasmic effects (CEs) have been discovered to influence a diverse array of agronomic traits in crops, and understanding the underlying mechanisms can help accelerate breeding programs. Seed oil content (SOC) is of...Cytoplasmic effects (CEs) have been discovered to influence a diverse array of agronomic traits in crops, and understanding the underlying mechanisms can help accelerate breeding programs. Seed oil content (SOC) is of great agricultural, nutritional, and economic importance. However, the genetic basis of CEs on SOC (CE-SOC) remains enigmatic. In this study, we use an optimized approach to sequence the cytoplasmic (plastid and mitochondrial) genomes of allotetraploid oilseed rape (Brassica napus) cultivars, 51218 and 56366, that bear contrasting CE-SOC. By combining comparative genomics and genome-wide transcriptome analysis, we identify mitochondria-encoded orf188 as a potential CE-SOC determinant gene. Functional analyses in the model system Arabidopsis thaliana and rapeseed demonstrated that orf188 governs CE-SOC and could significantly increase SOC, strikingly, through promoting the yield of ATP. Consistent with this finding, transcriptional profiling with microarray and RNA sequencing revealed that orf188 affects transcriptional reprogramming of mitochondrial energy metabolism to facilitate ATP production. Intriguingly, orf188 is a previously uncharacterized chimeric gene, and the presence of this genetic novelty endows rapeseed with positive CE-SOC. Our results shed light on the molecular basis of CEs on a key quantitative trait in polyploid crops and enrich the theory of maternal control of oil content, providing new scientific guidance for breeding high-oil rapeseed germplasms.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.
基金supported by grants from the National Key Basic Research Program of China (2015CB150200)the National Natural Science Foundation of China (31871664)+1 种基金the Agricultural Science and Technology Innovation Project of the Chin ese Academy of Agricultural Sciences (CAAS-ASTIP-OCRI)Fundamental Research Funds for Central Non-profit Scientific Institution.
文摘Cytoplasmic effects (CEs) have been discovered to influence a diverse array of agronomic traits in crops, and understanding the underlying mechanisms can help accelerate breeding programs. Seed oil content (SOC) is of great agricultural, nutritional, and economic importance. However, the genetic basis of CEs on SOC (CE-SOC) remains enigmatic. In this study, we use an optimized approach to sequence the cytoplasmic (plastid and mitochondrial) genomes of allotetraploid oilseed rape (Brassica napus) cultivars, 51218 and 56366, that bear contrasting CE-SOC. By combining comparative genomics and genome-wide transcriptome analysis, we identify mitochondria-encoded orf188 as a potential CE-SOC determinant gene. Functional analyses in the model system Arabidopsis thaliana and rapeseed demonstrated that orf188 governs CE-SOC and could significantly increase SOC, strikingly, through promoting the yield of ATP. Consistent with this finding, transcriptional profiling with microarray and RNA sequencing revealed that orf188 affects transcriptional reprogramming of mitochondrial energy metabolism to facilitate ATP production. Intriguingly, orf188 is a previously uncharacterized chimeric gene, and the presence of this genetic novelty endows rapeseed with positive CE-SOC. Our results shed light on the molecular basis of CEs on a key quantitative trait in polyploid crops and enrich the theory of maternal control of oil content, providing new scientific guidance for breeding high-oil rapeseed germplasms.
