【作者】 叶露鹏； 钱秋杰； 张玉玉； 尤征英； 车佳倩； 宋佳； 钟伯雄；

【Author】 Lupeng Ye;Qiujie Qian;Yuyu Zhang;Zhengying You;Jiaqian Che;Jia Song;Boxiong Zhong;Institute of Sericulture & Apiculture, College of Animal Sciences, Zhejiang University;

【机构】 浙江大学动物科学学院蚕桑蜜蜂研究所；

【摘要】 Promoter is one of the most important regulatory elements in regulating target genes spatiotemporal expression. However,the majority of studies are focus on core or proximal promoter regions, so that the information far away from 5’-fianking region of proximal promoter is often neglected. In this study, a long length sequence(about 4-kb) of sericin1(Ser1) promoter was cloned from silkworm genome, which was further validated as a kind of conserved and tissue-specific promoter by bioinformatic analysis. The4-kb length of Ser1 promoter was divided into three sections and each section was predicted including a lot of potential transcriptional factor binding sites(TFBSs). The transgenic experiment suggested that the TFBSs facilitated enhancement of promoter driving activity. To our surprise, the combination of multi-copy proximal Ser1 promoters could not improve the transcriptional level of exogenous gene but reduction. We speculated that multi-copy promoters would disperse transcription pre-initiation complexes, therefore the driving force of promoters were weakened. The correlation analysis between two contiguous genes, target foreign gene(firefly luciferase, FLuc) and marker gene(enhanced green fluorescent protein, EGFP), was carried out.The results indicated that the correlation was highly significant between FLuc and EGFP regardless of different insertion sites.Moreover, the ELISA assay revealed the correlation between transcriptional and translational level of EGFP were also very significant. Therefore, the exogenous genes expression could be simply and conveniently predicted by using an adjacent marker gene.This is a low-cost, time- and labour-saving approach for large-scale screening high foreign protein expression transgenic strains. In sum, all those results provide important clues for researchers to further investigate underlying molecular mechanism of promoters,and a new way is discovered to quickly and accurately predict exogenous genes expression in transgenic studies.更多还原

【Abstract】 Promoter is one of the most important regulatory elements in regulating target genes spatiotemporal expression. However,the majority of studies are focus on core or proximal promoter regions, so that the information far away from 5’-fianking region of proximal promoter is often neglected. In this study, a long length sequence(about 4-kb) of sericin1(Ser1) promoter was cloned from silkworm genome, which was further validated as a kind of conserved and tissue-specific promoter by bioinformatic analysis. The4-kb length of Ser1 promoter was divided into three sections and each section was predicted including a lot of potential transcriptional factor binding sites(TFBSs). The transgenic experiment suggested that the TFBSs facilitated enhancement of promoter driving activity. To our surprise, the combination of multi-copy proximal Ser1 promoters could not improve the transcriptional level of exogenous gene but reduction. We speculated that multi-copy promoters would disperse transcription pre-initiation complexes, therefore the driving force of promoters were weakened. The correlation analysis between two contiguous genes, target foreign gene(firefly luciferase, FLuc) and marker gene(enhanced green fluorescent protein, EGFP), was carried out.The results indicated that the correlation was highly significant between FLuc and EGFP regardless of different insertion sites.Moreover, the ELISA assay revealed the correlation between transcriptional and translational level of EGFP were also very significant. Therefore, the exogenous genes expression could be simply and conveniently predicted by using an adjacent marker gene.This is a low-cost, time- and labour-saving approach for large-scale screening high foreign protein expression transgenic strains. In sum, all those results provide important clues for researchers to further investigate underlying molecular mechanism of promoters,and a new way is discovered to quickly and accurately predict exogenous genes expression in transgenic studies.更多还原