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Development and functional analysis of novel genetic promoters using DNA shuffling, hybridization and a combination thereof.

Ranjan R, Patro S, Pradhan B, Kumar A, Maiti IB, Dey N - PLoS ONE (2012)

Bottom Line: Xanthi) protoplasts.It was observed that most of the shuffled promoters showed reduced activity compared to the two parent promoters (F and FS) and the CaMV35S promoter.In silico studies (computer simulated analyses) revealed that the reduced promoter activities of the shuffled promoters could be due to their higher helical stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Gene Function and Regulation, Institute of Life Sciences, Department of Biotechnology, Government of India, Chandrasekherpur, Bhubaneswar, Odisha, India.

ABSTRACT

Background: Development of novel synthetic promoters with enhanced regulatory activity is of great value for a diverse range of plant biotechnology applications.

Methodology: Using the Figwort mosaic virus full-length transcript promoter (F) and the sub-genomic transcript promoter (FS) sequences, we generated two single shuffled promoter libraries (LssF and LssFS), two multiple shuffled promoter libraries (LmsFS-F and LmsF-FS), two hybrid promoters (FuasFScp and FSuasFcp) and two hybrid-shuffled promoter libraries (LhsFuasFScp and LhsFSuasFcp). Transient expression activities of approximately 50 shuffled promoter clones from each of these libraries were assayed in tobacco (Nicotiana tabacum cv. Xanthi) protoplasts. It was observed that most of the shuffled promoters showed reduced activity compared to the two parent promoters (F and FS) and the CaMV35S promoter. In silico studies (computer simulated analyses) revealed that the reduced promoter activities of the shuffled promoters could be due to their higher helical stability. On the contrary, the hybrid promoters FuasFScp and FSuasFcp showed enhanced activities compared to F, FS and CaMV 35S in both transient and transgenic Nicotiana tabacum and Arabidopsis plants. Northern-blot and qRT-PCR data revealed a positive correlation between transcription and enzymatic activity in transgenic tobacco plants expressing hybrid promoters. Histochemical/X-gluc staining of whole transgenic seedlings/tissue-sections and fluorescence images of ImaGene Green™ treated roots and stems expressing the GUS reporter gene under the control of the FuasFScp and FSuasFcp promoters also support the above findings. Furthermore, protein extracts made from protoplasts expressing the human defensin (HNP-1) gene driven by hybrid promoters showed enhanced antibacterial activity compared to the CaMV35S promoter.

Significance/conclusion: Both shuffled and hybrid promoters developed in the present study can be used as molecular tools to study the regulation of ectopic gene expression in plants.

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A schematic map of the parent promoters (F and FS), hybrid promoters (FuasFScp and FSuasFcp) and DNA shuffling strategy.(a) At the top, the coordinates of the respective promoters Figwort mosaic virus (FMV) full-length transcript promoter (F, −249 to +64), FMV sub-genomic transcript promoter (FS, −270 to +31), and two hybrid promoters (FuasFScp, −343 to +31; and FSuasFcp, −449 to +64), the relative position of the TATA box, transcription start site (TSS, +1), upsteam activation sequence (uas) and core-promoter (cp) regions marked with arrow were shown. (b) A schematic presentation of creating promoter libraries by DNA shuffling of single (F or FS), multiple (F and FS) and hybrid promoters (FuasFScp, FSuasFcp) was presented. The construction strategies of generating hybrid promoters (FuasFScp and FSuasFcp), the single shuffled libraries (LssF and LssFS), multiple shuffled libraries (LmsFFS and LmsFSF), and hybrid promoter shuffled libraries (LhsFuasFScp and LhsFSuasFcp) were described in “Materials and Methods” section.
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pone-0031931-g001: A schematic map of the parent promoters (F and FS), hybrid promoters (FuasFScp and FSuasFcp) and DNA shuffling strategy.(a) At the top, the coordinates of the respective promoters Figwort mosaic virus (FMV) full-length transcript promoter (F, −249 to +64), FMV sub-genomic transcript promoter (FS, −270 to +31), and two hybrid promoters (FuasFScp, −343 to +31; and FSuasFcp, −449 to +64), the relative position of the TATA box, transcription start site (TSS, +1), upsteam activation sequence (uas) and core-promoter (cp) regions marked with arrow were shown. (b) A schematic presentation of creating promoter libraries by DNA shuffling of single (F or FS), multiple (F and FS) and hybrid promoters (FuasFScp, FSuasFcp) was presented. The construction strategies of generating hybrid promoters (FuasFScp and FSuasFcp), the single shuffled libraries (LssF and LssFS), multiple shuffled libraries (LmsFFS and LmsFSF), and hybrid promoter shuffled libraries (LhsFuasFScp and LhsFSuasFcp) were described in “Materials and Methods” section.

Mentions: A schematic map of parent promoters (F and FS) and hybrid promoters (FuasFScp and FSuasFcp was shown in Figure 1(a). The 195 bp long Fuas (−249 to −54, upstream activation sequence of F promoter), 314 bp long Fcp (−238 to +64, TATA box containing core-promoter sequence of F promoter) [22]; 210 bp long FSuas (−270 to −60, upstream activation sequence of FS promoter) and 182 bp long FScp (−151 to +31, TATA box containing core-promoter sequence of FS promoter) [23] were PCR amplified using promoter specific primer pairs (Table 1) having appropriate sequence to generate EcoRI and HincII sites at the 5′ end and SmaI and HindIII sites at the 3′ end. PCR amplifications of these promoter fragments were carried out as per protocol described earlier [24]. PCR-amplified fragments were restricted with EcoRI and HindIII, gel-purified and cloned into the corresponding sites of pBS (K+). The resulting plasmids were designated as pBSFuas, pBSFcp, pBSFSuas and pBSFScp respectively. The integrity of DNA sequences of these clones was verified by DNA sequencing as described earlier [5].


Development and functional analysis of novel genetic promoters using DNA shuffling, hybridization and a combination thereof.

Ranjan R, Patro S, Pradhan B, Kumar A, Maiti IB, Dey N - PLoS ONE (2012)

A schematic map of the parent promoters (F and FS), hybrid promoters (FuasFScp and FSuasFcp) and DNA shuffling strategy.(a) At the top, the coordinates of the respective promoters Figwort mosaic virus (FMV) full-length transcript promoter (F, −249 to +64), FMV sub-genomic transcript promoter (FS, −270 to +31), and two hybrid promoters (FuasFScp, −343 to +31; and FSuasFcp, −449 to +64), the relative position of the TATA box, transcription start site (TSS, +1), upsteam activation sequence (uas) and core-promoter (cp) regions marked with arrow were shown. (b) A schematic presentation of creating promoter libraries by DNA shuffling of single (F or FS), multiple (F and FS) and hybrid promoters (FuasFScp, FSuasFcp) was presented. The construction strategies of generating hybrid promoters (FuasFScp and FSuasFcp), the single shuffled libraries (LssF and LssFS), multiple shuffled libraries (LmsFFS and LmsFSF), and hybrid promoter shuffled libraries (LhsFuasFScp and LhsFSuasFcp) were described in “Materials and Methods” section.
© Copyright Policy
Related In: Results  -  Collection

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pone-0031931-g001: A schematic map of the parent promoters (F and FS), hybrid promoters (FuasFScp and FSuasFcp) and DNA shuffling strategy.(a) At the top, the coordinates of the respective promoters Figwort mosaic virus (FMV) full-length transcript promoter (F, −249 to +64), FMV sub-genomic transcript promoter (FS, −270 to +31), and two hybrid promoters (FuasFScp, −343 to +31; and FSuasFcp, −449 to +64), the relative position of the TATA box, transcription start site (TSS, +1), upsteam activation sequence (uas) and core-promoter (cp) regions marked with arrow were shown. (b) A schematic presentation of creating promoter libraries by DNA shuffling of single (F or FS), multiple (F and FS) and hybrid promoters (FuasFScp, FSuasFcp) was presented. The construction strategies of generating hybrid promoters (FuasFScp and FSuasFcp), the single shuffled libraries (LssF and LssFS), multiple shuffled libraries (LmsFFS and LmsFSF), and hybrid promoter shuffled libraries (LhsFuasFScp and LhsFSuasFcp) were described in “Materials and Methods” section.
Mentions: A schematic map of parent promoters (F and FS) and hybrid promoters (FuasFScp and FSuasFcp was shown in Figure 1(a). The 195 bp long Fuas (−249 to −54, upstream activation sequence of F promoter), 314 bp long Fcp (−238 to +64, TATA box containing core-promoter sequence of F promoter) [22]; 210 bp long FSuas (−270 to −60, upstream activation sequence of FS promoter) and 182 bp long FScp (−151 to +31, TATA box containing core-promoter sequence of FS promoter) [23] were PCR amplified using promoter specific primer pairs (Table 1) having appropriate sequence to generate EcoRI and HincII sites at the 5′ end and SmaI and HindIII sites at the 3′ end. PCR amplifications of these promoter fragments were carried out as per protocol described earlier [24]. PCR-amplified fragments were restricted with EcoRI and HindIII, gel-purified and cloned into the corresponding sites of pBS (K+). The resulting plasmids were designated as pBSFuas, pBSFcp, pBSFSuas and pBSFScp respectively. The integrity of DNA sequences of these clones was verified by DNA sequencing as described earlier [5].

Bottom Line: Xanthi) protoplasts.It was observed that most of the shuffled promoters showed reduced activity compared to the two parent promoters (F and FS) and the CaMV35S promoter.In silico studies (computer simulated analyses) revealed that the reduced promoter activities of the shuffled promoters could be due to their higher helical stability.

View Article: PubMed Central - PubMed

Affiliation: Department of Gene Function and Regulation, Institute of Life Sciences, Department of Biotechnology, Government of India, Chandrasekherpur, Bhubaneswar, Odisha, India.

ABSTRACT

Background: Development of novel synthetic promoters with enhanced regulatory activity is of great value for a diverse range of plant biotechnology applications.

Methodology: Using the Figwort mosaic virus full-length transcript promoter (F) and the sub-genomic transcript promoter (FS) sequences, we generated two single shuffled promoter libraries (LssF and LssFS), two multiple shuffled promoter libraries (LmsFS-F and LmsF-FS), two hybrid promoters (FuasFScp and FSuasFcp) and two hybrid-shuffled promoter libraries (LhsFuasFScp and LhsFSuasFcp). Transient expression activities of approximately 50 shuffled promoter clones from each of these libraries were assayed in tobacco (Nicotiana tabacum cv. Xanthi) protoplasts. It was observed that most of the shuffled promoters showed reduced activity compared to the two parent promoters (F and FS) and the CaMV35S promoter. In silico studies (computer simulated analyses) revealed that the reduced promoter activities of the shuffled promoters could be due to their higher helical stability. On the contrary, the hybrid promoters FuasFScp and FSuasFcp showed enhanced activities compared to F, FS and CaMV 35S in both transient and transgenic Nicotiana tabacum and Arabidopsis plants. Northern-blot and qRT-PCR data revealed a positive correlation between transcription and enzymatic activity in transgenic tobacco plants expressing hybrid promoters. Histochemical/X-gluc staining of whole transgenic seedlings/tissue-sections and fluorescence images of ImaGene Green™ treated roots and stems expressing the GUS reporter gene under the control of the FuasFScp and FSuasFcp promoters also support the above findings. Furthermore, protein extracts made from protoplasts expressing the human defensin (HNP-1) gene driven by hybrid promoters showed enhanced antibacterial activity compared to the CaMV35S promoter.

Significance/conclusion: Both shuffled and hybrid promoters developed in the present study can be used as molecular tools to study the regulation of ectopic gene expression in plants.

Show MeSH
Related in: MedlinePlus