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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (04): 620-626.doi: 10.3724/SP.J.1006.2018.00620

• RESEARCH NOTES • Previous Articles    

Cloning and Regulation Function Analysis of TaSC Promoter from Salt Tolerant Wheat

Bo JIAO(), Feng BAI, Yan-Yan LI, Jia LU, Xiao ZHANG, Yi-Ru CAO, Rong-Chao GE, Bao-Cun ZHAO*()   

  1. College of Life Science, Hebei Normal University, Shijiazhuang 050024, Hebei, China
  • Received:2017-08-22 Accepted:2018-01-08 Online:2018-01-26 Published:2018-01-26
  • Contact: Bao-Cun ZHAO E-mail:408850814@qq.com;baocunzh@126.com
  • Supported by:
    This study was supported by the National Natural Science Foundation of China (30871471) and the Natural Science Foundation of Hebei Province (C2011205085).

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Abstract:

High salinity is one of the major abiotic stress factors in wheat. Exploring stress related genes from salt-tolerant wheat varieties and analyzing their regulatory mechanism are helpful for elucidating the salt tolerance mechanism in wheat. In this study, the promoter sequence of a salt-tolerant related gene TaSC, designated ProTaSC, was cloned from salt-tolerant wheat mutant RH8706-49 by TAIL-PCR and silicon cloning method. A series of cis-acting elements including abscisic acid response element (ABRE), MYB protein binding site (MBS), TATA-box and CAAT-box were predicted in the promoter region. Among them ABRE and MBS are involved in abiotic stress responses. Beta-glucuronidase gene was used as reporter to study the expression characteristic of ProTaSC, showing that the full-length fragment and two 5'-progressive deletion fragments (681 bp and 1096 bp) were able to trigger GUS expression. However, GUS expression was undetectable when the fragment was less than 343 bp. These results suggest that the full-length promoter has promoting activity and the sequence between -681 to -343 nucleotides is the basic core region of ProTaSC. ProTaSC is a tissue-specific promoter because GUS gene driven by full-length ProTaSC was expressed in root, leaf, anther, sepals, and mature pods, but not in stem, petal, young fruit, and seed of Arabidopsis harboring ProTaSC:GUS. Quantification of GUS activity assay showed that ProTaSC was induced significantly by NaCl (200 mmol L-1) and ABA (10 μmol L-1) in the transgenic Arabidopsis seedlings, indicating ProTaSC is a functional sequence induced by NaCl or ABA treatment.

Key words: wheat, salt-tolerant wheat mutant RH8706-49, TaSC promoter, TAIL-PCR, expression activity

Fig. 1

Schematic diagrams of 5' deletion fragments of TaSC promoter with different lengths for constructing GUS reporter vectors"

Fig. 2

Cloning of TaSC promoter A: electrophoretic pattern of genomic DNA of RH8706-49; B: TAIL-PCR amplification of TaSC promoter; C: amplification of TaSC promoter from RH8706-49; M: DL-2000 DNA ladder in 2000, 1000, 750, 500, 200, and 100 bp (from the top to the bottom); 1: profile of the tertiary TAIL-PCR amplification with SP3 and AD4 ( target band shown by the arrow); 2: profile of the secondary TAIL-PCR amplification with SP2 and AD4; 3: the amplification profile with SP3 primer only; 4: Amplification of TaSC promoter ProTaSC (target band shown by the arrow)."

Fig. 3

DNA Sequences of TaSC promoter in salt-tolerant wheat mutant RH8706-49 The amplification fragment of TAIL-PCR is shown by blue italic letters. TATA box and CAAT box are underlined with dotted and solid lines respectively. Shadow shows other functional cis-acting elements whose name remarks under the sequence. Among them, ABRE element (CACGTG) and MBS element (CAACTG) are related to stress response."

Fig. 4

GUS assay results of transgenic Arabidopsis harboring different lengths of ProTaSC promotor fragmentsA: transferred with the 1419 bp promotor; B: transferred with the 1096 bp promotor fragment; C: transferred with the 681 bp promotor fragment; D: transferred with the 343 bp promotor fragment; E: transferred with the 152 bp promotor fragment."

Fig. 5

GUS assay results of different tissues of Arabidopsis harboring full-length promoter ProTaSCA: root; B: stem leaf; C: rosette leaf; D: floral, young pot and stem; E: blooming flower; F: mature pod."

Fig. 6

Quantification of GUS activity in transgenic Arabidopsis under different stresses** and *** indicate significant between the stress treatment and the control (0 h) at the 0.01 and 0.001 probability level, respectively (t-test)."

Supplementary Fig. 6

Alignment of ProTaSC+TaSC sequence from salt-tolerant wheat RH8706-49 and genomic DNA sequence from Chinese spring (Triticum aestivum L.)ProTaSC+TaSC: the cDNA sequence (557 bp) and its promoter sequence (1419 bp) of TaSC gene in RH8706-49. Red bars show the start (ATG) and stop codon (TAA) of TaSC. Traes_5DL_50BA3A: the 4266 bp contiguous genomic DNA sequence of Chinese spring, containing 2290 bp of introns."

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