Cloning and Functional Analysis of Phosphoethanolamine Methyltransferase Promoter from Maize (Zea mays L.)

Abstract

Betaine, a non-toxic osmoprotectant, is believed to accumulate considerably in plants under stress conditions to maintain the osmotic pressure and promote a variety of processes involved in growth and development. Phosphoethanolamine N-methyltransferase (PEAMT), a key enzyme for betaine synthesis, is reported to be regulated by its upstream promoter. In the present investigation, by using the transgenic approach, a 1048 bp long promoter region of ZmPEAMT gene from Zea mays was cloned and functionally characterized in tobacco. Computational analysis affirmed the existence of abiotic stress responsive cis-elements like ABRE, MYC, HST, LST etc., as well as pathogen, wound and phytohormone responsive motifs. For transformation in tobacco, four 5'-deletion constructs of 826 bp (P2), 642 bp (P3), 428 bp (P4) and 245 bp (P5) were constructed from the 1048 bp (P1) promoter fragment. The transgenic plants generated through a single event exhibited a promising expression of GUS reporter protein in the leaf tissues of treated with salt, drought, oxidative and cold stress as well as control plants. The GUS expression level progressively reduced from P1 to P5 in the leaf tissues, whereas a maximal expression was observed with the P3 construct in the leaves of control plants. The expression of GUS was noted to be higher in the leaves of osmotically- or salt-treated transgenic plants than that in the untreated (control) plants. An effective expression of GUS in the transgenic plants manifests that this promoter can be employed for both stress-inducible and constitutive expression of gene(s). Due to this characteristic, this potential promoter can be effectively used for genetic engineering of several crops.

Keywords: functional analysis; maize; phosphoethanolamine N-methyltransferase gene (peamt); promoter.

Figure 1

Nucleotide sequence and distribution map of cis-acting element of ZmPEAMT gene promoter in maize. Different colors and shapes represented different cis-acting elements of ZmPEAMT gene promoter.

Figure 2

The different length of 5′-deletion of the PEAMT gene promoter in maize. The M, represented the DL2000 and 1, 2, 3, 4, 5 represented the PEAMT gene promoter P1, P2, P3, P4, P5. The size of the different promoter is 1048, 826, 642, 428 and 245 bp, respectively.

Figure 3

Histochemical analysis of GUS in transformed tobacco leaves with different length fragment of the PEAMT gene promoter. The positive control was transformed tobacco with pCAMBIA1301 vector. The negative control was wild type tobacco. P1 was transformed tobacco with 1048 bp promoter. P2 was transformed tobacco with 826 bp promoter. P3 was transformed tobacco with 642 bp promoter. P4 was transformed tobacco with 428 bp promoter. P5 was transformed tobacco with 245 bp promoter, respectively.

Figure 4

Analysis of GUS activity for PEAMT deletions in transformed tobacco leaves under different stress conditions. The drought stress condition was simulated by 15% PEG-6000 (m/v) solution. The salt stress condition was simulated by 100 mmol L⁻¹ NaCl solution. The cold stress condition was simulated by 4 °C. The oxidative stress was simulated by 100 mmol L⁻¹ H₂O₂ solution. Different letters represented significant difference among treatments at p < 0.05. The t-test was performed to compare differences between the inoculation treatments under drought stress conditions. Data presented as treatment means ± S.E. (n = 3).