To understand the expansion ofmulticopy microRNA （miRNA） families in plants, we localized the reported miRNA genes from Arabidopsis and rice to their chromosomes, respectively, and observed that 37% of 117 miRNA gen...To understand the expansion ofmulticopy microRNA （miRNA） families in plants, we localized the reported miRNA genes from Arabidopsis and rice to their chromosomes, respectively, and observed that 37% of 117 miRNA genes from Arabidopsis and 35% of 173 miRNA genes from rice were segmental duplications in the genome. In order to characterize whether the expression diversification has occurred among plant multicopy miRNA family members, we designed PCR primers targeting 48 predicted miRNA precursors from 10 families in Arabidopsis and rice. Results from RT-PCR data suggest that the transcribed precursors of members within the same miRNA family were present at different expression levels. In addition, although miRl60 and miR162 sequences were conserved in Arabidopsis and rice, we found that the expression patterns of these genes differed between the two species. These data suggested that expression diversification has occurred in multicopy miRNA families, increasing our understanding of the expression regulation of miRNAs in plants.展开更多
Using luc gene as a reporter to study the activation of Rhizobium meliloti nif/fix genes in thedevelopment of symbiosis,the authors observed that nodule development and nitrogen fixation were inhibitedby both multicop...Using luc gene as a reporter to study the activation of Rhizobium meliloti nif/fix genes in thedevelopment of symbiosis,the authors observed that nodule development and nitrogen fixation were inhibitedby both multicopy promoters of nifHDK and fixABCX.The phenotype of R.meliloti containing multicopynif/fix promoters appeared exactly like that of nifA mutant.Using lacZ as a reporter,the authors got the same re-sults.By contrast,the rhizobia containing low-copy promoters of nif/fix genes were normal fornodule development and nitrogen fixation.These results substantiate the evidence that the product of nifAgene not only acts as a transcriptional activator of nif/fix genes,but also plays an important role in thedevelopment of root nodules.展开更多
In the biotechnological industry,multicopy gene integration represents an effective strategy to maintain a high-level production of recombinant proteins and to assemble multigene biochemical pathways.In this study,we ...In the biotechnological industry,multicopy gene integration represents an effective strategy to maintain a high-level production of recombinant proteins and to assemble multigene biochemical pathways.In this study,we developed copper-induced in vivo gene amplification in budding yeast for multicopy gene expressions.To make copper as an effective selection pressure,we first constructed a copper-sensitive yeast strain by deleting the CUP1 gene encoding a small metallothionein-like protein for copper resistance.Subsequently,the reporter gene fused with a proline-glutamate-serine-threonine-destabilized CUP1 was integrated at the δ sites of retrotransposon(Ty)elements to counter the copper toxicity at 100 μM Cu^(2+).We further demonstrated the feasibility of modulating chromosomal rearrangements for increased protein expression under higher copper concentrations.In addition,we also demonstrated a simplified design of integrating the expression cassette at the CUP1 locus to achieve tandem duplication under high concentrations of copper.Taken together,we envision that this method of copper-induced in vivo gene amplification would serve as a robust and useful method for protein overproduction and metabolic engineering applications in budding yeast.展开更多
基金We acknowledge Prof HerváVaucheret for kindly supplying the agol-27 mutant and Dr Xuemei Chen for helpful suggestions and reviewing this manuscript.This work was supported by the Fund of National Key Basic Research Developments Program of the Ministry of Science and Technology China(2001CB109002)National Natural Science Foundation of China(30370893)+2 种基金Shanghai Municipal Committee of Science and Technology(03JC14061)the Program for New Century Excellent Talents in University(NCET-04-0403)the ShuGuang Scholarship(04SG15).
文摘To understand the expansion ofmulticopy microRNA （miRNA） families in plants, we localized the reported miRNA genes from Arabidopsis and rice to their chromosomes, respectively, and observed that 37% of 117 miRNA genes from Arabidopsis and 35% of 173 miRNA genes from rice were segmental duplications in the genome. In order to characterize whether the expression diversification has occurred among plant multicopy miRNA family members, we designed PCR primers targeting 48 predicted miRNA precursors from 10 families in Arabidopsis and rice. Results from RT-PCR data suggest that the transcribed precursors of members within the same miRNA family were present at different expression levels. In addition, although miRl60 and miR162 sequences were conserved in Arabidopsis and rice, we found that the expression patterns of these genes differed between the two species. These data suggested that expression diversification has occurred in multicopy miRNA families, increasing our understanding of the expression regulation of miRNAs in plants.
基金the National Natural Science Foundation of China.
文摘Using luc gene as a reporter to study the activation of Rhizobium meliloti nif/fix genes in thedevelopment of symbiosis,the authors observed that nodule development and nitrogen fixation were inhibitedby both multicopy promoters of nifHDK and fixABCX.The phenotype of R.meliloti containing multicopynif/fix promoters appeared exactly like that of nifA mutant.Using lacZ as a reporter,the authors got the same re-sults.By contrast,the rhizobia containing low-copy promoters of nif/fix genes were normal fornodule development and nitrogen fixation.These results substantiate the evidence that the product of nifAgene not only acts as a transcriptional activator of nif/fix genes,but also plays an important role in thedevelopment of root nodules.
基金supported by the National Natural Science Foundation of China(grant no.32270087)Daan Gene(20223160A0063)ZhenSheng Biotech.
文摘In the biotechnological industry,multicopy gene integration represents an effective strategy to maintain a high-level production of recombinant proteins and to assemble multigene biochemical pathways.In this study,we developed copper-induced in vivo gene amplification in budding yeast for multicopy gene expressions.To make copper as an effective selection pressure,we first constructed a copper-sensitive yeast strain by deleting the CUP1 gene encoding a small metallothionein-like protein for copper resistance.Subsequently,the reporter gene fused with a proline-glutamate-serine-threonine-destabilized CUP1 was integrated at the δ sites of retrotransposon(Ty)elements to counter the copper toxicity at 100 μM Cu^(2+).We further demonstrated the feasibility of modulating chromosomal rearrangements for increased protein expression under higher copper concentrations.In addition,we also demonstrated a simplified design of integrating the expression cassette at the CUP1 locus to achieve tandem duplication under high concentrations of copper.Taken together,we envision that this method of copper-induced in vivo gene amplification would serve as a robust and useful method for protein overproduction and metabolic engineering applications in budding yeast.
