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In vivo characterization of plant promoter element interaction using synthetic promoters

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Abstract

Short directly-repeated (DR) DNA enhancer elements of plant viral origin were analyzed for their ability, both individually and in combination, to influence in vivo transcription when inserted upstream from a minimal CaMV35S promoter. Synthetic promoters containing multiple copies and/or combinations of DR cassettes were tested for their effect upon reporter gene (luciferase) expression using an Agrobacteria-based leaf-infiltration transient assay and within stably transformed plants (Nicotiana tabacum). Transgenic plants harboring constructs containing different numbers or combinations of DR cassettes were further tested to look for tissue-specific expression patterns and potential promoter response to the infiltration process employed during transient expression. Multimerization of DR elements produced enhancer activity that was in general additive, increasing reporter activity in direct proportion to the number of DR cassettes within the test promoter. In contrast, combinations of different DR cassettes often functioned synergistically, producing reporter enhancement markedly greater then the sum of the combined DR activities. Several of the DR constructs responded to Agrobacteria (lacking T-DNA) infiltration of transgenic leaves by an induction (2 elements) or reduction (1 element) in reporter activity. Combinations of DR cassettes producing the strongest enhancement of reporter activity were used to create two synthetic promoters (SynPro3 and SynPro5) that drive leaf reporter activities at levels comparable to the CaMV35S promoter. Characterization of these synthetic promoters in transformed tobacco showed strong reporter expression at all stages of development and in most tissues. The arrangement of DR elements within SynPro3 and SynPro5 appears to play a role in defining tissue-specificity of expression and/or Agrobacteria-infusion responsiveness.

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Abbreviations

Agst :

Agropine synthase terminator

CaMV35S:

Cauliflower Mosaic Virus 35S promoter

HcPro:

Viral Helper component Protein (suppressor of silencing)

GUSi:

Intron-modified β-Glucuronidase

IM:

Infiltration media

FLUC:

Firefly (Photinus pyralis) luciferase

FiLUC:

Intron modified Firefly luciferase gene

Nost :

Nopaline synthase terminator

NptII :

Neomycin phosphotransferase II

pAg7:

Gene 7 terminator

PClSV:

Peanut chlorotic streak virus (promoter)

PHV:

Pepper Huasteco Virus

Pnos:

Nopaline synthase promoter

PTGS:

Post transcriptional gene silencing

PVY:

Potato Virus Y

R0 :

Primary transgenic line

R1 :

Progeny of self fertilized R0 plants

RiLUC:

Renilla reniformis (sea pansy) intron-modified luciferase gene

RLUC:

Intron modified Renilla reniformis luciferase

RLU:

Relative Light Units

TrAP:

Transcription Activating Protein

35St :

CaMV35S terminator

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Acknowledgements

Our appreciation goes out to Drs. John Burke, Junping Chen and Zhanguo Xin for critical reading of the manuscript, Erick Armijo and Derek Pugh for helping with luciferase and GUS assays, Kay McCrary and DeeDee Laumbach for tobacco transformation and David Wheeler for excellent technical assistance. Mention of a commercial or proprietary product does not constitute an endorsement by the USDA. USDA offers its programs to all eligible persons regardless of race, color, age, sex, or national origin.

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  1. Plant Stress and Water Conservation Laboratory, United States Department of Agriculture—Agricultural Research Service, 3810 4th Street, Lubbock, Texas, 79415, USA

    Christopher Ian Cazzonelli & Jeff Velten

  2. Australian National University, School of Biochemistry and Molecular Biology, Building 41, Linnaus Way, Canberra, ACT, 0200, Australia

    Christopher Ian Cazzonelli

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Cazzonelli, C.I., Velten, J. In vivo characterization of plant promoter element interaction using synthetic promoters. Transgenic Res 17, 437–457 (2008). https://doi.org/10.1007/s11248-007-9117-8

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