Betalain,an economically valuable water-soluble natural plant pigment,is prized for its strong antioxidant activity,making it popular as a dietary supplement and a visual marker for plant transformation.However,market...Betalain,an economically valuable water-soluble natural plant pigment,is prized for its strong antioxidant activity,making it popular as a dietary supplement and a visual marker for plant transformation.However,market demand significantly outstrips current production capacity.This study reports the development of an efficient push-and-pull multigene strategy based on polycistronic expression and metabolic flux regulation to enhance betalain biosynthesis in transgenic maize(Zea mays L.)endosperm.We engineered a novel enhanced RUBY(eRUBY)system derived from the original polycistronic RUBY construct(CYP76AD1P2ADODA1P2ADOPA5GT unit,abbreviated CDG)by introducing arogenate dehydrogenase(ADHα)to increase the L-tyrosine substrate supply.All the genes were driven by the endosperm-specific promoter.Fusion of ADHαinto a single polycistronic eRUBY construct(CDGA)produced significantly higher betanin(6.88 mg g−1 dry weight)and isobetanin(1.81 mg g−1 dry weight)levels than in CDG+A,which stacked the ADHαcassette independently with CDG.The high betalain accumulation in CDGA lines(which also exhibited higher transgene copy number)resulted in a 2.85–7.58-fold improvement in endosperm antioxidant capacity compared to WT(versus 2.48–2.80-fold in CDG+A).Importantly,transgenic plants maintained a normal phenotype.Transcriptome and metabolome analyses further indicated that metabolism of phenylalanine,alanine,aspartate,and glutamate contributes to betalain production.Hybridization with sweet corn successfully created a high-sugar eRUBY maize variety.Collectively,these results demonstrate the successful development of a novel maize germplasm with significantly enhanced nutritional value through high betalain accumulation.展开更多
The need for co-ordinate,high-level,and stable expression of multiple genes is essential for the engineering of biosynthetic circuits and metabolic pathways.This work outlines the functionality and design of IRES-and ...The need for co-ordinate,high-level,and stable expression of multiple genes is essential for the engineering of biosynthetic circuits and metabolic pathways.This work outlines the functionality and design of IRES-and 2 A-peptide-based constructs by comparing different strategies for co-expression in polycistronic vectors.In particular,2 A sequences are small peptides,mostly derived from viral polyproteins,that mediate a ribosome-skipping event such that several,different,separate proteins can be generated from a single open reading frame.When applied to metabolic engineering and synthetic gene circuits,2 A peptides permit to achieve co-regulated and reliable expression of various genes in eukaryotic cells.展开更多
Generation of induced pluripotent stem （iPS） cells from somatic cells has been achieved successfully by simultaneous viral transduction of defined reprogramming transcription factors （TFs）. However, the process re...Generation of induced pluripotent stem （iPS） cells from somatic cells has been achieved successfully by simultaneous viral transduction of defined reprogramming transcription factors （TFs）. However, the process requires multiple viral vectors for gene delivery. As a result, generated iPS cells harbor numerous viral integration sites in their genomes. This can increase the probability of gene mutagenesis and genomic instability, and present significant barriers to both research and clinical application studies of iPS cells. In this paper, we present a simple lentivirus reprogramming system in which defined factors are fused in-frame into a single open reading frame （ORF） via self-cleaving 2A sequences. A GFP marker is placed downstream of the transgene to enable tracking of transgene expression. We demonstrate that this polycistronic expression system efficiently generates iPS cells. The generated iPS cells have normal karyotypes and are similar to mouse embryonic stem cells in morphology and gene expression. Moreover, they can differentiate into cell types of the three embryonic germ layers in both in vitro and in vivo assays. Remarkably, most of these iPS cells only harbor a single copy of viral vector. This system provides a valuable tool for generation of iPS cells, and our data suggest that the balance of expression of transduced reprogramming TFs in each cell is essential for the reprogramming process. More importantly, when delivered by non-integrating gene-delivery systems, this re-engineered single ORF will facilitate efficient generation of human iPS cells free of genetic modifications.展开更多
基金supported by grants from the Biological Breeding-National Science and Technology Major Project(2024ZD04077)the Invigorate the Seed Industry of Guangdong Province(2024-NPY-00-044)+3 种基金the National Natural Science Foundation of China(32272120)the Guangxi Science and Technology Major Project(GKAA24206023)the National Key Research and Development Program of China(2024YFF1000800)the Guangdong Basic Research Center of Excellence for Precise Breeding of Future Crops Major Project(FCBRCE-202502,FCBRCE-202504).
文摘Betalain,an economically valuable water-soluble natural plant pigment,is prized for its strong antioxidant activity,making it popular as a dietary supplement and a visual marker for plant transformation.However,market demand significantly outstrips current production capacity.This study reports the development of an efficient push-and-pull multigene strategy based on polycistronic expression and metabolic flux regulation to enhance betalain biosynthesis in transgenic maize(Zea mays L.)endosperm.We engineered a novel enhanced RUBY(eRUBY)system derived from the original polycistronic RUBY construct(CYP76AD1P2ADODA1P2ADOPA5GT unit,abbreviated CDG)by introducing arogenate dehydrogenase(ADHα)to increase the L-tyrosine substrate supply.All the genes were driven by the endosperm-specific promoter.Fusion of ADHαinto a single polycistronic eRUBY construct(CDGA)produced significantly higher betanin(6.88 mg g−1 dry weight)and isobetanin(1.81 mg g−1 dry weight)levels than in CDG+A,which stacked the ADHαcassette independently with CDG.The high betalain accumulation in CDGA lines(which also exhibited higher transgene copy number)resulted in a 2.85–7.58-fold improvement in endosperm antioxidant capacity compared to WT(versus 2.48–2.80-fold in CDG+A).Importantly,transgenic plants maintained a normal phenotype.Transcriptome and metabolome analyses further indicated that metabolism of phenylalanine,alanine,aspartate,and glutamate contributes to betalain production.Hybridization with sweet corn successfully created a high-sugar eRUBY maize variety.Collectively,these results demonstrate the successful development of a novel maize germplasm with significantly enhanced nutritional value through high betalain accumulation.
文摘The need for co-ordinate,high-level,and stable expression of multiple genes is essential for the engineering of biosynthetic circuits and metabolic pathways.This work outlines the functionality and design of IRES-and 2 A-peptide-based constructs by comparing different strategies for co-expression in polycistronic vectors.In particular,2 A sequences are small peptides,mostly derived from viral polyproteins,that mediate a ribosome-skipping event such that several,different,separate proteins can be generated from a single open reading frame.When applied to metabolic engineering and synthetic gene circuits,2 A peptides permit to achieve co-regulated and reliable expression of various genes in eukaryotic cells.
文摘Generation of induced pluripotent stem （iPS） cells from somatic cells has been achieved successfully by simultaneous viral transduction of defined reprogramming transcription factors （TFs）. However, the process requires multiple viral vectors for gene delivery. As a result, generated iPS cells harbor numerous viral integration sites in their genomes. This can increase the probability of gene mutagenesis and genomic instability, and present significant barriers to both research and clinical application studies of iPS cells. In this paper, we present a simple lentivirus reprogramming system in which defined factors are fused in-frame into a single open reading frame （ORF） via self-cleaving 2A sequences. A GFP marker is placed downstream of the transgene to enable tracking of transgene expression. We demonstrate that this polycistronic expression system efficiently generates iPS cells. The generated iPS cells have normal karyotypes and are similar to mouse embryonic stem cells in morphology and gene expression. Moreover, they can differentiate into cell types of the three embryonic germ layers in both in vitro and in vivo assays. Remarkably, most of these iPS cells only harbor a single copy of viral vector. This system provides a valuable tool for generation of iPS cells, and our data suggest that the balance of expression of transduced reprogramming TFs in each cell is essential for the reprogramming process. More importantly, when delivered by non-integrating gene-delivery systems, this re-engineered single ORF will facilitate efficient generation of human iPS cells free of genetic modifications.
