全部 标题 作者
关键词 摘要

OALib Journal期刊
ISSN: 2333-9721
费用:99美元

查看量下载量

相关文章

更多...
PLOS ONE  2012 

Development and Functional Analysis of Novel Genetic Promoters Using DNA Shuffling, Hybridization and a Combination Thereof

DOI: 10.1371/journal.pone.0031931

Full-Text   Cite this paper   Add to My Lib

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.

References

[1]  Goodrich JA, Tjian R (2010) Modes of transcriptional regulation: Unexpected roles for core promoter recognition factors in cell-type-specific transcription and gene regulation. Nat Rev Genetics 11: 549–558.
[2]  Venter M, Botha FC (2010) Synthetic promoter engineering. In: Pua E-C, Davey MR, editors. Plant Developmental Biology- Biotechnological Perspective. 2. : 393–414. Springer-Verlag Berlin Heidelberg.
[3]  Bhullar S, Chakravarthy S, Advani S, Datta S, Pental D, et al. (2003) Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping. Plant Physiol 132: 988–998.
[4]  Bestwick RK, Kellogg JA (2000) Synthetic Hybrid Tomato E4/E8 Plant Promoter - Patent 6118049.
[5]  Kumar D, Patro S, Ranjan R, Sahoo DK, Maiti IB, et al. (2011) Development of useful recombinant promoter and its expression analysis in different plant cells using Confocal Laser Scanning Microscopy. PLoS ONE 6(9): e24627. doi:10.1371/journal.pone.0024627.
[6]  Comai L, Moran P, Maslyar D (1990) Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS elements. Plant Mol Biol 15: 373–381.
[7]  Ni M, Cui D, Einstein J, Narasimhulu S, Vergara CE (1995) Strength and tissue specificity of chimeric promoters derived from the octopine and mannopine synthase genes. The Plant J 7: 661–676.
[8]  Ranjan R, Patro S, Kumari S, Kumar D, Dey N, et al. (2011) Efficient chimeric promoters derived from full-length and sub-genomic transcript promoters of Figwort mosaic virus (FMV). Journal of Biotechnology 152: 58–62.
[9]  Lee LY, Kononov ME, Bassuner B, Frame BR, Wang K, et al. (2007) Novel Plant Transformation Vectors Containing the Superpromoter. Plant Physiol 145: 1294–1300.
[10]  Rushton PJ, Reinstadler A, Lipka V, Lippok B, Somssich IE (2002) Synthetic plant promoters containing defined regulatory elements provide novel insights into pathogen- and wound-induced signaling. Plant Cell 14: 749–762.
[11]  Venter M (2007) Synthetic Promoters: genetic control through cis engineering. Trends in Plant Sci 12: 118–124.
[12]  Edelman GM, Meech R, Owens GC, Jones FS (2000) Synthetic promoter elements obtained by nucleotide sequence variation and selection for activity. Proc Nat Acad Sci (USA) 97: 3038–3043.
[13]  Bhullar S, Datta S, Advani S, Chakravarthy S, Gautam T, et al. (2007) Functional Analysis of Cauliflower mosaic virus 35S promoter: re-evalution of the role of subdomains B5, B4 and B2 in Promoter activity. Plant biotechnology J 5: 696–708.
[14]  Alper H, Fischer C, Nevoigt E, Stephanopoulos G (2005) Tuning genetic control through promoter engineering. PNAS 102: 12678–12683.
[15]  Stemmer WP (1994a) DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci USA 91: 10747–10751.
[16]  Stemmer WP (1994b) Rapid evolution of a protein in vitro by DNA shuffling. Nature 370: 389–391.
[17]  Xiong A, Peng R, Liu J, Zhuang J, Shan Y, et al. (2007) High efficiency and throughput system in directed evolution in vitro of reporter gene. Appl Microbiol Biotechnol 74: 160–168.
[18]  Castle LA, Siehl DL, Gorton R, Patten PA, Chen YH, et al. (2004) Discovery and directed evolution of a glyphosate tolerance gene. Science 304: 1151–1154.
[19]  Keenan RJ, Siehl DL, Gorton R, Castle LA (2005) DNA Shuffling as a tool for protein crystallization. Proc Natl Acad Sci USA 102: 8887–8892.
[20]  Hibbert EG, Baganz F, Hailes HC, Ward JM, Lye GJ, et al. (2005) Directed evolution of biocatalytic processes. Biomol Eng 22: 11–19.
[21]  Otten LG, Quax WJ (2005) Directed evolution: selecting today's biocatalysts. Biomol Eng 22: 1–9.
[22]  Maiti IB, Gowda S, Kiernan J, Ghosh SK, Shepherd RJ (1997) Promoter/leader deletion analysis and plant expression vectors with Figwort Mosaic Virus (FMV) full-length transcript (Flt) promoter containing single and double enhancer domains. Transgenic Res 6: 143–156.
[23]  Bhattacharyya S, Dey N, Maiti IB (2002) Analysis of cis-sequence of subgenomic transcript promoter from the Figwort mosaic virus and comparison of promoter activity with the Cauliflower mosaic virus promoters in monocot and dicot cells. Virus Res 90: 47–62.
[24]  Dey N, Maiti IB (1999) Structure and promoter/leader deletion analysis of mirabilis mosaic virus (MMV) full-length transcript promoter in transgenic plants. Plant Mol Biol 40: 771–782.
[25]  Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6: 3901–3907.
[26]  Bradford MM (1976) A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254.
[27]  Thompson JD, Higgins DG, Gibson TJ (1994) CLUSTAL W: Improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position specific gap penalties and weight matrix choice. Nucl Acids Res 22: 4673–4680.
[28]  Huang Y, Kowalski D (2003) WEB-THERMODYN: Sequence analysis software for profiling DNA helical stability. Nucleic Acids Research 31: 3819–3821.
[29]  Sahoo DK, Ranjan R, Kumar D, Kumar A, Sahoo BS, et al. (2009) An alternative method of promoter assessment by confocal laser scanning microscopy. Journal Virological Methods 161: 114–121.
[30]  Chen H, Nelson RS, Sherwood JL (1994) Enhanced recovery of transformants of Agrobacterium tumefaciens after freeze-thaw transformation and drug selection. Biotechniques 16: 664–670.
[31]  Cazzonelli CI, Velten J (2008) In vivo characterization of plant promoter element interaction using synthetic promoters. Transgenic Research 17: 437–457.
[32]  Zhang X, Henriques R, Lin SS, Niu QW, Chua NH (2006) Agrobacterium-mediated transformation of Arabidopsis thaliana using the floral dip method. Nature Protocol 1: 641–6.
[33]  Maiti IB, Dey N, Pattanaik S, Dahlman DL, Rana RL, et al. (2003) Antibiosis-type insect resistance in transgenic plants expressing a teratocyte secretory protein (TSP14) gene from a hymenopteran endoparasite (Microplitis croceipes). Plant Biotechnology Journal 1: 209–219.
[34]  Fleming JA, Manzara T, Gruissem W, Kuhlemeier C (1996) Fluorescent imaging of GUS activity and RT-PCR analysis of gene expression in the shoot apical meristem. The Plant journal 4: 745–754.
[35]  Vazquez F, Gonzalez EA, Garabal JI, Valderrama S, Blanco J, et al. (1996) Development and evaluation of an ELISA to detect Escherichia coli K88 (F44) fimbrial antibody levels. J Med Microbial 44: 453–463.
[36]  Nitschke M, Wiehl S, Baer PC, Kreft B (2000) Bactericidal Activity of Renal Tubular Cells: The Putative Role of Human ?-Defensins. Exp Nephrol 10: 332–337.
[37]  Morey C, Mookherjee S, Rajasekaran G, Bansal M (2011) DNA Free Energy-Based Promoter Prediction and Comparative Analysis of Arabidopsis and Rice Genomes. Plant Physiology 156: 1300–15.
[38]  Remans T, Grof CPL, Ebert PR, Schenk PM (2005) Functional Promoter Analysis Using an Approach Based on an In Vitro Evolution Strategy. Biotechniques 38: 209–216.
[39]  Kuluev BR, Knyazev AV, Lebedev YP, Iljassowa AA, Chemeris AV (2010) Construction of Hybrid Promoters of Caulimoviruses and Analysis of Their Activity in Transgenic Plants. Russian Journal of Plant Physiology 57: 582–589.
[40]  Kanhere A, Bansal M (2005) Structural properties of promoters: similarities and differences between prokaryotes and eukaryotes. Nucleic Acids Research 33: 3165–3175.
[41]  Varkey J, Nagraj R (2005) Antibacterial activity of Human Neutrophil defensin HNP-1 analogs without cysteines. Antimicrobial agents and chemotherapy 49: 4561–4566.
[42]  Xiong YQ, Yeaman MR, Bayer AS (1999) In vitro antibacterial activities of platelet microbicidal protein and neutrophil defensin against Staphylococcus aureus are influenced. by antibiotics differing in mechanism of action. Antimicrobial Agents and Chemotherapy 43: 1111–1117.

Full-Text

Contact Us

service@oalib.com

QQ:3279437679

WhatsApp +8615387084133