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Plant Cell Reports

, Volume 38, Issue 8, pp 899–914 | Cite as

Identification of sugar response complex in the metallothionein OsMT2b gene promoter for enhancement of foreign protein production in transgenic rice

  • Chia-Yu Chang
  • Kuo-Wei Lee
  • Chung-Shen Wu
  • Yu-Hsing Huang
  • Ho-Chun Chang
  • Chien-Lung Chen
  • Chen-Tung Li
  • Min-Jeng Li
  • Chung-Fu Chang
  • Peng-Wen ChenEmail author
Original Article

Abstract

Key message

A 146-bp sugar response complex MTSRC is identified in the promoter of rice metallothionein OsMT2b gene conferring high-level expression of luciferase reporter gene and bioactive recombinant haFGF in transgenic rice.

Abstract

A rice subfamily type 2 plant metallothionein (pMT) gene, OsMT2b, encoding a reactive oxygen species (ROS) scavenger protein, has been previously shown to exhibit the most abundant gene expression in young rice seedling. Expression of OsMT2b was found to be regulated negatively by ethylene and hydrogen peroxide in rice stem node under flooding stress, but little is known about its response to sugar depletion. In this study, transient expression assay and transgenic approach were employed to characterize the regulation of the OsMT2b gene expression in rice. We found that the expression of OsMT2b gene is induced by sugar starvation in both rice suspension cells and germinated embryos. Deletion analysis and functional assay of the OsMT2b promoter revealed that the 5′-flanking region of the OsMT2b between nucleotides − 351 and − 121, which contains the sugar response complex (− 266 to − 121, designated MTSRC) is responsible for high-level promoter activity under sugar starvation. It was also found that MTSRC significantly enhances the Act1 promoter activity in transgenic rice cells and seedlings. The modified Act1 promoter, Act1-MTSRC, was used to produce the recombinant human acidic fibroblast growth factor (haFGF) in rice cells. Our result shows that the bioactive recombinant haFGF is stably produced in transformed rice cell culture and yields are up to 2% of total medium proteins. Our studies reveal that MTSRC serves as a strong transcriptional activator and the Act1-MTSRC promoter can be applicable in establishing an efficient expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings.

Keywords

Rice metallothionein OsMT2b promoter Act1 promoter Transcriptional activator Sugar response complex (SRC) Human acidic fibroblast growth factor (haFGF) 

Notes

Acknowledgements

We thank Dr. Carol PeiYin Wu for critical review of the manuscript. This work was supported by grants from the Ministry of Science and Technology (NSC96-2311-B-415-003-MY3 and 105A11) of Taiwan.

Compliance with ethical standards

Conflict of interest

The authors declare that there are no conflicts of interest.

Supplementary material

299_2019_2411_MOESM1_ESM.pptx (59 kb)
OsMT2b promoter sequence in Japonica rice variety Lomello (GenBank accession no. AF048750). The nucleotides are numbered relative to the transcription start site (+1) of OsMT2b gene. The consensus sequences of G box (5’-TACGTG-3’) and the TA box (5’-TATCCA-3’), and putative TATA box are highlighted and underlined (PPTX 59 kb)
299_2019_2411_MOESM2_ESM.pptx (105 kb)
The nucleotide sequence of the T-DNA region of pAAct1-MTSRC::ShaFGF. The T-DNA containsCaMV35S::hpt and Act1-MTSRC::ShaFGF chimeric genes. Nucleotide and deduced amino acid sequence of theαAct1-MTSRC chimeric promoter and the codon optimized αAmy3 SP-haFGF gene are shown. The three copies of MTSRC and the signal peptide (SP) of αAmy3 gene are indicated (PPTX 105 kb)
299_2019_2411_MOESM3_ESM.pptx (1.7 mb)
The level of OsMT2b mRNA significantly decreased in hypoxic embryos during seed germination and seedling growth in rice. Seeds were germinated and grown in air (Air) or under hypoxic conditions (Hypoxia, 1% O2) with an oxygen-controlled system (Model 101, Prosperous Instrument Co., Ltd.) at 28 ℃ in the dark for up to 3 days. Total RNA was purified from aerobic and hypoxic embryos at indicated time points and RNA gel blot was performed using the OsMT2b specific cDNA probe (PPTX 1754 kb)
299_2019_2411_MOESM4_ESM.pptx (53 kb)
The MTSRC confers higher promoter activity than αAmy3 SRC in rice embryo. a Schematic representation of constructs for αAmy3 SRC and MTSRC promoter analysis. b Transient expression assay of luciferase activity in rice embryos. Rice embryos were transfected with plasmids pαAmy3SRC::Luc, pMTSRC::Luc, or p35mA::Luc by particle bombardment. Transfected embryos were incubated at 28 ℃ for 24 hours in glucose-containing (+ Glu, open bar) or glucose-free (-Glu, filled bar) cultured medium. X indicates fold induction by sugar starvation. Error bars indicate the standard deviations of data collected from three replicate experiments for each construct. The plasmid p35mA::Luc was used as a negative control in these assays for sugar-starvation responses (PPTX 53 kb)
299_2019_2411_MOESM5_ESM.pptx (52 kb)
The MTSRC significantly enhances the Act1 promoter activity and confers higher promoter activity than αAmy3 SRC in rice embryo. a Schematic representation of constructs for Act1, Act1-αAmy3SRC, and Act1-MTSRC promoter activity analysis. b Transient luciferase activity assay in rice embryos. Rice embryos were transfected with plasmids pAct1::Luc, pAct1-αAmy3SRC::Luc, or pAct1-MTSRC::Luc by particle bombardment. Transfected embryos were incubated at 28 ℃ for 24 hours in glucose-containing (+ Glu, open bar) or glucose-free (-Glu, filled bar) cultured medium. X indicates fold induction by sugar starvation. Error bars indicate the standard deviations of luciferase activity readings from three replicate experiments for each construct. Significant differences between the wild type and modified Act1 promoters are at P ≤ 0.01 (t-test) (PPTX 52 kb)

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Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of BioAgricultural SciencesNational Chiayi UniversityChiayiTaiwan
  2. 2.PRIT Biotech Co., Ltd.ChunanTaiwan

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