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Molecular cloning and characterization of a trehalose-6-phosphate synthase/phosphatase from Dunaliella viridis

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Abstract

Dunaliella is a group of green algae with exceptional stress tolerance capability, and is considered as an important model organism for stress tolerance study. Here we cloned a TPS (trehalose-6-phosphate synthase) gene from Dunaliella viridis and designated it as DvTPS (D. viridis trehalose-6-phosphate synthase/phosphatase).The DvTPS cDNA contained an ORF of 2793 bp encoding 930 aa. DvTPS had both TPS and TPP domain and belonged to the Group II TPS/TPP fusion gene family. Southern blots showed it has a single copy in the genome. Genome sequence analysis revealed that it has 18 exons and 17 introns. DvTPS had a constitutive high expression level under various NaCl culture conditions, however, could be induced by salt shock. Promoter analysis indicated there were ten STREs (stress response element) in its promoter region, giving a possible explanation of its inducible expression pattern upon salt shock. Yeast functional complementation analysis showed that DvTPS had neither TPS nor TPP activity. However, DvTPS could improve the salt tolerance of yeast salt sensitive mutant G19. Our results indicated that despite DvTPS showed significant similarity with TPS/TPP, its real biological function is still remained to be revealed.

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Abbreviations

aa:

Amino acids(s)

bp:

Base pair(s)

kb:

1000 bp

ORF:

Open reading frame

UTR:

Untranslated region(s)

′ (prime):

Denotes a truncated gene at the indicated side

TPS:

Trehalose-6-phosphate synthase

TPP:

Trehalose-6-phosphate phosphatase

STRE:

Stress response element

T6P:

Trehalose-6-phosphate

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Acknowledgments

We thank Prof. Johan M. Thevelein (Katholieke Universiteit Leuven, Belgium) for providing yeast tps1Δ mutant, Prof. Hohmann Stefan (Göteborg University, Sweden) for providing yeast tps2Δ mutant, Prof. Alonso Rodriguez Navarro (Polytechnic University, Madrid) for providing yeast G19 mutant. This work was supported by the National Natural Sciences Foundation of China (30871278, 30970242 and 3047119), the Ministry of Agriculture of China (2008ZX08003-005, 2008ZX08003-001), Shanghai Municipal Commission of Science and Technology (09DZ2271800), and Key Discipline “Molecular Physiology” of Shanghai Municipal Education Commission.

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Authors and Affiliations

  1. Shanghai Key Laboratory of Bio-Energy Crops, School of Life Science, Shanghai University, 99 Shangda Road, Shanghai, 200444, People’s Republic of China

    Nan Zhang, Fei Wang, Xiangzong Meng, Saifan Luo, Zhengkai Xu & Rentao Song

  2. Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Science, Chinese Academy of Science, Shanghai, 200032, People’s Republic of China

    Xiangzong Meng, Hongyun Dong & Zhengkai Xu

  3. Institute of Plant Protection, Jilin Academy of Agricultural Sciences, Gongzhuling, 136100, People’s Republic of China

    Qiyun Li

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Correspondence to Rentao Song.

Electronic supplementary material

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11033_2010_354_MOESM1_ESM.doc

Supplement Fig. 1 List of the trehalose biosynthetic proteins involved in this paper. The taxonomic groups are shown on the left side; the accession numbers or protein ids for each protein are indicated. VcTPS is derived from JGI Volvox carteri f. nagariensis version 1.0 while the rest are from GenBank. Colors show each different protein type. (DOC 1387 kb)

Supplement Fig. 2 (A, B) Alignment of TPS or TPP domain from various Group II TPS genes including DvTPS. (DOC 16948 kb)

11033_2010_354_MOESM3_ESM.doc

Supplement Fig. 3 Cis-elements analysis of the 1.6 kb promoter region. Nucleotides are numbered on the left. Transcription factor-binding sites are underlined. Arrows indicate the predicted transcription start site (or Inr). (DOC 5628 kb)

11033_2010_354_MOESM4_ESM.doc

Supplement Fig. 4 Southern blot analysis of the DvTPS gene. Genomic DNA (5ug) of D. viridis was digested with HindIII or BamHI. The filter was probed with DIG-labeled DvTPS cDNA fragments without these restriction enzyme sites. The sizes of the bands are also indicated on the figure. (DOC 83 kb)

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Zhang, N., Wang, F., Meng, X. et al. Molecular cloning and characterization of a trehalose-6-phosphate synthase/phosphatase from Dunaliella viridis . Mol Biol Rep 38, 2241–2248 (2011). https://doi.org/10.1007/s11033-010-0354-1

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