The PDM1 gene encodes a pentatricopeptide repeat protein of the PLS subfamily.It is essential for rpoA polycistronic processing in Arabidopsis.In this study,we performed functional analysis of PDM1 in rpoA monocistron...The PDM1 gene encodes a pentatricopeptide repeat protein of the PLS subfamily.It is essential for rpoA polycistronic processing in Arabidopsis.In this study,we performed functional analysis of PDM1 in rpoA monocistronic cleavage and chloroplast development.The pdm1 mutants display an albino lethal phenotype with severe chloroplast development defects.When the construct of PDM1 fused with the GFP gene was introduced into Arabidopsis protoplasts,the GFP signal was exclusively observed in chloroplasts.This shows that PDM1 is localized to chloroplast.In the wild type,the rpoA transcript of about 990 nt is processed.In pdm1,this transcript is absent.However,Western blot showed that the RpoA protein in pdm1 is accumulated at a level approximately 1/3 of the wild type.This suggests that certain other transcripts processed from L23-L2-S19-L22-S3-L16-L14S8-L36-S11-rpoA polycistronic precursor may be used as templates for protein translation.To determine whether PDM1 can bind to the rpoA pre-mRNA,we generated a transgenic Arabidopsis line with PDM1 fused to FLAG tag that was capable of complementing the pdm1-1 mutant phenotype.RNA immunoprecipitation analysis showed that PDM1 is associated with S11-rpoA intergenic sequence.This indicates that PDM1 may bind to the S11-rpoA intergenic region to perform rpoA processing.展开更多
[Objective] This study aimed to construct Brassica napus chloroplast multi- cistron double cross-over expression vector, to lay the foundation for the genetic engi- neering research of Brassica napus chloroplast. [Met...[Objective] This study aimed to construct Brassica napus chloroplast multi- cistron double cross-over expression vector, to lay the foundation for the genetic engi- neering research of Brassica napus chloroplast. [Method] Two primers were designed based on the known Brassica napus chloroplast DNA sequences AF267640 and Z50868 in GenBank. By using PCR method, two Brassica napus L. chloroplast DNA fragments were obtained, which were named RbcL and ACCD. The two Brassica na- pus chloroplast DNA homologous fragments were then cloned into plasmid pMD18-T to obtain recombinant plasmid pHBM715. Tandem expression cassette harboring spectinomycin-resistant gene aadA, mannanase gene man and green fluorescent pro- tein gene gfp was cloned into the plasmid pHBM715, thereby constructing Brassica napus chloroplast multicistron double cross-over expression vector pHBM716, which was transformed into Escherichia coil for expression and identification. [Result] Plate qualitative analysis was conducted for the functional identification of expression cas- sette in the constructed Brassica napus chloroplast multicistron double cross-over ex- pression vector, results showed that the three genes of the same multicistron were all expressed in E. coil [Conclusion] This study successfully constructed Brassica napus chloroplast multicistron double cross-over expression vector, which laid the foundation for the genetic engineering of Brassica napus chloroplast.展开更多
基金supported by the National Natural Science Foundation of China (31100180)
文摘The PDM1 gene encodes a pentatricopeptide repeat protein of the PLS subfamily.It is essential for rpoA polycistronic processing in Arabidopsis.In this study,we performed functional analysis of PDM1 in rpoA monocistronic cleavage and chloroplast development.The pdm1 mutants display an albino lethal phenotype with severe chloroplast development defects.When the construct of PDM1 fused with the GFP gene was introduced into Arabidopsis protoplasts,the GFP signal was exclusively observed in chloroplasts.This shows that PDM1 is localized to chloroplast.In the wild type,the rpoA transcript of about 990 nt is processed.In pdm1,this transcript is absent.However,Western blot showed that the RpoA protein in pdm1 is accumulated at a level approximately 1/3 of the wild type.This suggests that certain other transcripts processed from L23-L2-S19-L22-S3-L16-L14S8-L36-S11-rpoA polycistronic precursor may be used as templates for protein translation.To determine whether PDM1 can bind to the rpoA pre-mRNA,we generated a transgenic Arabidopsis line with PDM1 fused to FLAG tag that was capable of complementing the pdm1-1 mutant phenotype.RNA immunoprecipitation analysis showed that PDM1 is associated with S11-rpoA intergenic sequence.This indicates that PDM1 may bind to the S11-rpoA intergenic region to perform rpoA processing.
基金Supported by National 863 Project of China (2002AA227011)Natural Science Foundation of Hubei Province (2003ABAI18)Natural Science Foundation of Shandong Province (ZR2010HQ054)~~
文摘[Objective] This study aimed to construct Brassica napus chloroplast multi- cistron double cross-over expression vector, to lay the foundation for the genetic engi- neering research of Brassica napus chloroplast. [Method] Two primers were designed based on the known Brassica napus chloroplast DNA sequences AF267640 and Z50868 in GenBank. By using PCR method, two Brassica napus L. chloroplast DNA fragments were obtained, which were named RbcL and ACCD. The two Brassica na- pus chloroplast DNA homologous fragments were then cloned into plasmid pMD18-T to obtain recombinant plasmid pHBM715. Tandem expression cassette harboring spectinomycin-resistant gene aadA, mannanase gene man and green fluorescent pro- tein gene gfp was cloned into the plasmid pHBM715, thereby constructing Brassica napus chloroplast multicistron double cross-over expression vector pHBM716, which was transformed into Escherichia coil for expression and identification. [Result] Plate qualitative analysis was conducted for the functional identification of expression cas- sette in the constructed Brassica napus chloroplast multicistron double cross-over ex- pression vector, results showed that the three genes of the same multicistron were all expressed in E. coil [Conclusion] This study successfully constructed Brassica napus chloroplast multicistron double cross-over expression vector, which laid the foundation for the genetic engineering of Brassica napus chloroplast.
