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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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Related in: MedlinePlus

Histochemical localization of GUS activity in transgenic tobacco and Arabidopsis seedlings generated for the respective promoter-GUS constructs.(a) Histochemical staining of transgenic tobacco seedlings expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× magnification) attached to a CCD camera. (b) Histochemical staining of transgenic tobacco stem cross sections expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× magnification) attached to a CCD camera. (c) Histochemical staining of transgenic tobacco leaf petiole cross sections expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× maginification) attached to a CCD camera. (d) Histochemical staining of transgenic Arabidopsis seedling expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× maginification) attached to a CCD camera.
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pone-0031931-g010: Histochemical localization of GUS activity in transgenic tobacco and Arabidopsis seedlings generated for the respective promoter-GUS constructs.(a) Histochemical staining of transgenic tobacco seedlings expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× magnification) attached to a CCD camera. (b) Histochemical staining of transgenic tobacco stem cross sections expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× magnification) attached to a CCD camera. (c) Histochemical staining of transgenic tobacco leaf petiole cross sections expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× maginification) attached to a CCD camera. (d) Histochemical staining of transgenic Arabidopsis seedling expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× maginification) attached to a CCD camera.

Mentions: Histochemical staining using X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) of transgenic tobacco seedlings (T1 generation, 21 days old) generated for the GUS construct with CaMV35S; FuasFScp and FSuasFcp promoter were presented in Figure 10a, while those of stem cross sections of transgenic tobacco plants and of leaf petioles were presented in (Figures 10b and 10c, respectively). Histochemical staining of transgenic Arabidopsis seedlings expressing GUS directed by CaMV35S, FuasFScp and FSuasFcp promoters were shown in Figure 10d.


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)

Histochemical localization of GUS activity in transgenic tobacco and Arabidopsis seedlings generated for the respective promoter-GUS constructs.(a) Histochemical staining of transgenic tobacco seedlings expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× magnification) attached to a CCD camera. (b) Histochemical staining of transgenic tobacco stem cross sections expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× magnification) attached to a CCD camera. (c) Histochemical staining of transgenic tobacco leaf petiole cross sections expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× maginification) attached to a CCD camera. (d) Histochemical staining of transgenic Arabidopsis seedling expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× maginification) attached to a CCD camera.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3303778&req=5

pone-0031931-g010: Histochemical localization of GUS activity in transgenic tobacco and Arabidopsis seedlings generated for the respective promoter-GUS constructs.(a) Histochemical staining of transgenic tobacco seedlings expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× magnification) attached to a CCD camera. (b) Histochemical staining of transgenic tobacco stem cross sections expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× magnification) attached to a CCD camera. (c) Histochemical staining of transgenic tobacco leaf petiole cross sections expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× maginification) attached to a CCD camera. (d) Histochemical staining of transgenic Arabidopsis seedling expressing GUS under the control of respective promoter constructs. Photographs were taken using Leica DM LS2 microscope (at 10× maginification) attached to a CCD camera.
Mentions: Histochemical staining using X-gluc (5-bromo-4-chloro-3-indolyl-β-D-glucuronide) of transgenic tobacco seedlings (T1 generation, 21 days old) generated for the GUS construct with CaMV35S; FuasFScp and FSuasFcp promoter were presented in Figure 10a, while those of stem cross sections of transgenic tobacco plants and of leaf petioles were presented in (Figures 10b and 10c, respectively). Histochemical staining of transgenic Arabidopsis seedlings expressing GUS directed by CaMV35S, FuasFScp and FSuasFcp promoters were shown in Figure 10d.

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