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Acta Agronomica Sinica ›› 2018, Vol. 44 ›› Issue (7): 1000-1009.doi: 10.3724/SP.J.1006.2018.01000

• CROP GENETICS & BREEDING · GERMPLASM RESOURCES · MOLECULAR GENETICS • Previous Articles     Next Articles

Cloning and Expression Analysis of Strong Inducible Promoter P1502-ZmPHR1 Responding to Low Phosphorus Stress in Maize

Rui-Juan YANG1,2,Jian-Rong BAI2,*(),Lei YAN2,Liang SU2,Xiu-Hong WANG3,Rui LI2,Cong-Zhuo ZHANG2   

  1. 1 College of Biology Engineering, Shanxi University, Taiyuan 030006, Shanxi, China
    2 Institute of Crop Sciences, Shanxi Academy of Agricultural Sciences, Taiyuan 030031, Shanxi, China
    3 Research Center of Modern Agriculture, Shanxi Academy of Agricultural Sciences, Taiyuan 030031, Shanxi, China
  • Received:2018-01-02 Accepted:2018-03-26 Online:2018-07-10 Published:2018-04-10
  • Contact: Jian-Rong BAI E-mail:zwsjrbai@126.com
  • Supported by:
    This study was supported by the Agricultural Science and Technology Innovation Program of Shanxi Academy of Agricultural Sciences (ZDSY1507).

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Abstract:

ZmPHR1, a member of transcription factor MYB-CC family, enhanced phosphate absorption and improved plant growth when it was overexpressed under low-phosphate conditions. In order to elucidate the transcriptional regulatory mechanism of the promoter of ZmPHR1, its 5′ upstream sequence P2205-ZmPHR1 was cloned from P-efficient maize inbred line 478. The full length of P2205-ZmPHR1 and four sequence deletion fragments in different lengths fused with the reporter gene GUS were transformed into Arabidopsis, respectively. The results of histochemical and quantitative fluorometric GUS assay showed that in the interval from 0 to -1502 bp, the response time and activity of sequence deletion fragments were increased with increasing fragment length. However, the activities were decreased or unchanged when the length of fragments were longer than 1502 bp. It was considered that the interval from 0 to -972 bp was function region and that from -972 bp to -1502 bp was function enhancing region. The results of qPCR analysis in transgenic plants at different time points (0, 1, 2, 3, and 4 days) after P-stress treatment showed that the activities of P1502-ZmPHR1 were influenced by both external signals and internal gene products, having feedback regulation. This study indicates that P1502-ZmPHR1 is a strong inducible promoter responding to low phosphorus stress and its functional activity is regulated by both external and internal phosphate concentration. These fundings provide an ideal promoter for crop improvement under low phosphate conditions and a basis for its application.

Key words: maize, low phosphorus stress, strong inducible promoter, P1502-ZmPHR1

Table 1

Primers of PCR"

引物名称
Primer name		 序列
Sequence (5'-3')
SF1 GTCGACTTGTGATCGCAGTACGAAAA
SF2 GTCGACCATCACACCTGTACTTGGC
SF3 GTCGACTGCTGGGCTTTTGATTCC
SF4 GTCGACCAGGTGGACCCACGTATC
SF5 GTCGACCCTGTCGTGTTGGACTTG
SR AGATCTacCATTCAGAGCAGTGAACAAGCTC
qGUS-F CCGTCCCAAGCAGTTACAAT
qGUS-R CGATACGCTGATCCTTCAGATAG
AtACT2-qRT-F GGAATCGTTCACAGAAAATG
AtACT2-qRT-R CAAACAAATGGAGAAGCAAA

Supplementary Fig. 1

5' upstream sequence alignments of maize 478 and B73 ZmPHR1 gene"

Fig. 1

Amplified fragment of P2205-ZmPHR1"

Fig. 2

Nucleotide sequence and putative cis-acting elements of P2205-ZmPHR1"

Fig. 3

Schematic show of cis-acting elements in 5° terminal deletion fragments of P2205-ZmPHR1"

Fig. 4

Amplified fragment in 5° terminal deletion fragments of P2205-ZmPHR1 M: marker; 1: P1763-ZmPHR1::GUS; 2: P1502-ZmPHR1::GUS; 3: P972-ZmPHR1::GUS; 4: P482-ZmPHR1::GUS."

Fig. 5

PCR analysis of T1 transgenic Arabidopsis plants M: marker; 1-4: TP2205-ZmPHR1; 5-8: TP1763-ZmPHR1; 9-12: TP1502-ZmPHR1; 13-16: TP972-ZmPHR1; 17-20: TP482-ZmPHR1."

Fig. 6

GUS activity in transgenic Arabidopsis under low phosphorus stress"

Fig. 7

Quantification of GUS activity in transgenic Arabidopsis under low phosphorus stress * and ** indicate significant difference between stress treatment and the control (0 h) at the 0.05 and 0.01 probability levels, respectively (t-test)."

Fig. 8

Relative expression of GUS in TP1502-ZmPHR1 transgenic Arabidopsis under low phosphorus stress"

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