Molecular Functional Characterisation of MechlPPDK Promoter in Transgenic Tobacco

Abstract

Chloroplastic pyruvate phosphate dikinase (PPDK) (chlPPDK) is a key enzyme in the photosynthesis of C4 plants. PPDK is expressed in high abundance in C4 plants but only in trace amounts in C3 plants. The existing research reveals a higher expression of MechlPPDK in cultivated cassava varieties than that in wild cassava W14. However, knowledge about the transcriptional regulation of the MechlPPDK gene in cassava (Manihot esculenta Crantz) is insufficient. Therefore, we aim to identify the transcription profile of MechlPPDK and the core promoter region of MechlPPDK. We cloned the MechlPPDK coding sequence and its 5′-flanking sequence from the cassava variety Ku50. A series of deletion constructions fused to a uidA reporter gene were performed in the 5′-flanking sequence and stably transformed into tobacco. We found that P1 (from −590 bp to +114 bp) had the highest promoter activity among P1 to P3. The 5′-flanking sequence of MechlPPDK responded differently to varied abiotic stresses. Our results will further the understanding of the regulation of MechlPPDK expression.

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Abbreviations

CDS:

Coding sequence

EDTA:

Ethylene diamine tetraacetic acid

PEPC:

Phosphoenolpyruvate carboxylase

PPDK:

Pyruvate phosphate dikinase

RACE:

Rapid-amplification of cDNA ends

TSS:

Transcriptional start site

GUS:

β-glucuronidase

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Acknowledgements

This study was funded by the National Natural Science Foundation of China (Grant No. 31701509) and the China Agriculture Research System (CARS-12).

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Contributions

Kaimian Li and Wenquan Wang directed and designed the experiments. Haiyan Wang performed most of the research and drafted the manuscript. Xu Shen planted the transformed tobacco plants, and Cheng Lu performed some field experiments. All authors have read and approved the final manuscript.

Corresponding authors

Correspondence to Haiyan Wang or Kaimian Li or Wenquan Wang.

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Communicated by: Elliosha Hajari

Electronic supplementary material

ESM 2
figure6

Fig. A1. Structure of MechlPPDK gene. Location of exons and introns are indicated by box and line between boxes. The numbers above the box indicate length of exons. Red box indicates 5’untranslated region and white box indicates 3’untranslated region. ATG and TGA indicate translation initiation codon and termination codon (PNG 897 kb)

ESM 3
figure7

Fig.A2. The map of pCAMBIA1381Z:pMechPPDK (PNG 870 kb)

ESM 1

(DOCX 58 kb)

12042_2020_9257_MOESM2_ESM.tif

High Resolution Image (TIF 33 kb)

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High Resolution Image (TIF 148 kb)

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Wang, H., Shen, X., Lu, C. et al. Molecular Functional Characterisation of MechlPPDK Promoter in Transgenic Tobacco. Tropical Plant Biol. 13, 287–295 (2020). https://doi.org/10.1007/s12042-020-09257-0

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Keywords

  • Abiotic stress
  • Manihot esculenta
  • Pyruvate phosphate dikinase
  • Promoter
  • Regulatory elements