The cis-acting regulatory elements, e.g., promoters and ribosome binding sites （RBSs） with various desired properties, are building blocks widely used in synthetic biology for fine tuning gene expression. In the las...The cis-acting regulatory elements, e.g., promoters and ribosome binding sites （RBSs） with various desired properties, are building blocks widely used in synthetic biology for fine tuning gene expression. In the last decade, acquisition of a controllable regulatory element from a random library has been established and applied to control the protein expression and metabolic flux in different chassis cells. However, more rational strategies are still urgently needed to improve the efficiency and reduce the laborious screening and multifaceted characterizations. Building precise computational models that can predict the activity of regulatory elements and quantitatively design elements with desired strength have been demonstrated tremendous potentiality. Here, recent progress on construction of cis- acting regulatory element library and the quantitative predicting models for design of such elements are reviewed and discussed in detail.展开更多
The cis-regulatory elements of promoters regulate temporal and spatial expression of genes. DNA inethylation, histone methylation and histone acetylation are the main types of epigenetic modifications, which play impo...The cis-regulatory elements of promoters regulate temporal and spatial expression of genes. DNA inethylation, histone methylation and histone acetylation are the main types of epigenetic modifications, which play important roles in plant growth and development. DNA methylation could seilenco transposons, affect gene imprinting and gene expression. In this study, we found that granule bound starch synthase 1 （GBSSI） gene is expressed specifically in wheat endosperm rath- er than in the embryo. We also analyzed the cis-elements within this promoter region and found some seed-specific elements. In order to confirm the tissue specifici- ty, we cloned 4k bp sequences upstream of GBSS1 gene to link to vector with GUS and this construct was transferred to tobacco by Agrobacterium mediated transfor- marion. The results showed that wheat GBSS1 promoter mediated the seed-specific expression of GUS gene, hut not mediated expression in embryo. In addition, we found that GBSSI promoter is methylated in wheat embryo and de-methylated in wheat endosperm. Our study might provide the molecular basis for specific expres- sion of GBSSI gene.展开更多
The human genome contains millions of DNA regulatory elements and a large number of gene clusters,most of which have not been tested experimentally.The clustered regularly interspaced short palindromic repeats(CRISPR)...The human genome contains millions of DNA regulatory elements and a large number of gene clusters,most of which have not been tested experimentally.The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)programed with a synthetic single-guide RNA(sgRNA)emerges as a method for genome editing in virtually any organisms.Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs.Specifically,we found that,in cultured human cells and mice,efficient precise inversions of DNA fragments ranging in size froma few tens of bp to hundreds of kb could be generated.In addition,DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks(DSBs)on two homologous chromosomes(chromatids).Moreover,junctions of combinatorial inversions and duplications of the protocadherin(Pcdh)gene clusters induced by Cas9 with four sgRNAs could be detected.In mice,we obtained founders with alleles of precise inversions,duplications,and deletions of DNA fragments of variable sizes by CRISPR.Interestingly,we found that very efficient inversions were mediated by microhomology-mediated end joining(MMEJ)through short inverted repeats.We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice.Finally,we applied this CRISPR method to a regulatory element of the Pcdha cluster and found a new role in the regulation of members of the Pcdhg cluster.This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters.展开更多
The promoter region of a drought and abscisic acid （ABA） inducible gene, osr40c1, was isolated from a salt-tolerant indica rice variety Pokkali, which is 670 bp upstream of the putative translation start codon. In ...The promoter region of a drought and abscisic acid （ABA） inducible gene, osr40c1, was isolated from a salt-tolerant indica rice variety Pokkali, which is 670 bp upstream of the putative translation start codon. In silico promoter analysis of resulted sequence showed that at least 15 types of putative motifs were distributed within the sequence, including two types of common promoter elements, TATA and CAAT boxes. Additionally, several putative cis-acing regulatory elements which may be involved in regulation of osr40c1 expression under different conditions were found in the 5′-upstream region of osr40c1. These are ABA-responsive element, light-responsive elements （ATCT-motif, Box I, G-box, GT1-motif, Gap-box and Sp1）, myeloblastosis oncogene response element （CCAAT-box）, auxin responsive element （TGA-element）, gibberellin-responsive element （GARE-motif） and fungal-elicitor responsive elements （Box E and Box-W1）. A putative regulatory element, required for endosperm-specific pattern of gene expression designated as Skn-1 motif, was also detected in the Pokkali osr40c1 promoter region. In conclusion, the bioinformatic analysis of osr40c1 promoter region isolated from indica rice variety Pokkali led to the identification of several important stress-responsive cis-acting regulatory elements, and therefore, the isolated promoter sequence could be employed in rice genetic transformation to mediate expression of abiotic stress induced genes.展开更多
基金This work was supported by the National Basic Research Program of China （973 Program, grant No. 2012CB721104）, the National High Technology Research and Development Program （863 Program, grant No. 2012AA02A701）, the National Natural Science Foundation of China （grant Nos. 31170101 and 31301017）, and the Natural Science Foundation of Guangdong Province, China （grant No. 2015A030310317）.
文摘The cis-acting regulatory elements, e.g., promoters and ribosome binding sites （RBSs） with various desired properties, are building blocks widely used in synthetic biology for fine tuning gene expression. In the last decade, acquisition of a controllable regulatory element from a random library has been established and applied to control the protein expression and metabolic flux in different chassis cells. However, more rational strategies are still urgently needed to improve the efficiency and reduce the laborious screening and multifaceted characterizations. Building precise computational models that can predict the activity of regulatory elements and quantitatively design elements with desired strength have been demonstrated tremendous potentiality. Here, recent progress on construction of cis- acting regulatory element library and the quantitative predicting models for design of such elements are reviewed and discussed in detail.
基金Supported by Agricultural Scientific and Technological Innovation Project of Shandong Academy of Agricultural Sciences(CXGC2016D01)
文摘The cis-regulatory elements of promoters regulate temporal and spatial expression of genes. DNA inethylation, histone methylation and histone acetylation are the main types of epigenetic modifications, which play important roles in plant growth and development. DNA methylation could seilenco transposons, affect gene imprinting and gene expression. In this study, we found that granule bound starch synthase 1 （GBSSI） gene is expressed specifically in wheat endosperm rath- er than in the embryo. We also analyzed the cis-elements within this promoter region and found some seed-specific elements. In order to confirm the tissue specifici- ty, we cloned 4k bp sequences upstream of GBSS1 gene to link to vector with GUS and this construct was transferred to tobacco by Agrobacterium mediated transfor- marion. The results showed that wheat GBSS1 promoter mediated the seed-specific expression of GUS gene, hut not mediated expression in embryo. In addition, we found that GBSSI promoter is methylated in wheat embryo and de-methylated in wheat endosperm. Our study might provide the molecular basis for specific expres- sion of GBSSI gene.
基金supported by grants to Q.W.from the National Natural Science Foundation of China(31171015 and 31470820)the Science and Technology Commission of Shanghai Municipality(13XD1402000 and 14JC1403600).
文摘The human genome contains millions of DNA regulatory elements and a large number of gene clusters,most of which have not been tested experimentally.The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated nuclease 9(Cas9)programed with a synthetic single-guide RNA(sgRNA)emerges as a method for genome editing in virtually any organisms.Here we report that targeted DNA fragment inversions and duplications could easily be achieved in human and mouse genomes by CRISPR with two sgRNAs.Specifically,we found that,in cultured human cells and mice,efficient precise inversions of DNA fragments ranging in size froma few tens of bp to hundreds of kb could be generated.In addition,DNA fragment duplications and deletions could also be generated by CRISPR through trans-allelic recombination between the Cas9-induced double-strand breaks(DSBs)on two homologous chromosomes(chromatids).Moreover,junctions of combinatorial inversions and duplications of the protocadherin(Pcdh)gene clusters induced by Cas9 with four sgRNAs could be detected.In mice,we obtained founders with alleles of precise inversions,duplications,and deletions of DNA fragments of variable sizes by CRISPR.Interestingly,we found that very efficient inversions were mediated by microhomology-mediated end joining(MMEJ)through short inverted repeats.We showed for the first time that DNA fragment inversions could be transmitted through germlines in mice.Finally,we applied this CRISPR method to a regulatory element of the Pcdha cluster and found a new role in the regulation of members of the Pcdhg cluster.This simple and efficient method should be useful in manipulating mammalian genomes to study millions of regulatory DNA elements as well as vast numbers of gene clusters.
文摘The promoter region of a drought and abscisic acid （ABA） inducible gene, osr40c1, was isolated from a salt-tolerant indica rice variety Pokkali, which is 670 bp upstream of the putative translation start codon. In silico promoter analysis of resulted sequence showed that at least 15 types of putative motifs were distributed within the sequence, including two types of common promoter elements, TATA and CAAT boxes. Additionally, several putative cis-acing regulatory elements which may be involved in regulation of osr40c1 expression under different conditions were found in the 5′-upstream region of osr40c1. These are ABA-responsive element, light-responsive elements （ATCT-motif, Box I, G-box, GT1-motif, Gap-box and Sp1）, myeloblastosis oncogene response element （CCAAT-box）, auxin responsive element （TGA-element）, gibberellin-responsive element （GARE-motif） and fungal-elicitor responsive elements （Box E and Box-W1）. A putative regulatory element, required for endosperm-specific pattern of gene expression designated as Skn-1 motif, was also detected in the Pokkali osr40c1 promoter region. In conclusion, the bioinformatic analysis of osr40c1 promoter region isolated from indica rice variety Pokkali led to the identification of several important stress-responsive cis-acting regulatory elements, and therefore, the isolated promoter sequence could be employed in rice genetic transformation to mediate expression of abiotic stress induced genes.
