Part:BBa_K5218021

MDH1 promoter

Promoter of MALATE DEHYDROGENASE 1 gene (MDH1) from Arabidopsis thaliana.

Base Pairs：340 bp

Function：MDH1 plays a criticle role in the redox homeostasis and central metabolism between organelle compartments. Cytoplasmic MDH1 is considered to be the key role of chloroplasts or mitochondria in plant cells to transfer reduction equivalent to other destinations [1]. Recent report showed MDH1 was responsive to HCHO stress [2].

Figure 1. HCHO stress response pathway in Arabidopsis.

Figure adopted from X. Zhao et al, 2023 [2]

Introduction

Formaldehyde (HCHO) is a common environmental and occupational pollutant, widely used in both industrial and consumer products. Exposure to formaldehyde can result in serious health issues, including upper respiratory illnesses and cancer. Therefore, developing effective monitoring and purification technologies for HCHO is essential.

The engineering objective of this project is to generate brighter autoluminescent plants using synthetic biology approaches, and explore its potential applications. Team BGI-MammothEdu-South 2024 selected a set of promoter elements candidate and tested their regulatory functions in Fungal Bioluminescence Pathway (FBP) via eGFP and GUS reporter system (pS1300-GFP, pS1300-GUS plasmid). The AtMDH1 gene ID is AT1G04410, transcript ID is NM_100321.3 (NCBI). Team BGI-MammothEdu-South extracted the Arabidopsis thaliana leaf genomic DNA and cloned the AtMDH1 promoter with specific primer pairs.

Characterisation

The expression pattern of AtMDH1 was firstly investigated on the AtGenExpress eFP database. The result showed that AtMDH1 was highly expressed in roesette, leaf and seedling; moderately expressed in silique and shoot apex.

Figure 2. AtMDH1 expression pattern.

The cloning strategy for p1300-AtMDH1-GUS construct is as follows: The pS1300-GUS vector was first digested with BamH I to remove the super promoter and get linear p1300-GUS, which was ligated with AtMDH1 promoter fragment via Gibson Cloning system. In the construct, GUS reporter gene was driven by AtMDH1 promoter, followed by NOS terminator. The recombinant plasmid p1300-AtMDH1-GUS was transformed into E. coli TOP10 competent cells and verified through colony PCR and sequencing.

Figure 3. Generation of p1300-AtMDH1-GUS construct.

A. PCR product on agarose gel electrophoresis. Lane M, 5000bp DNA marker; lane 1&2, AtMDH1 promoter PCR prodoct.

B. Single colonies of p1300-AtMDH1-GUS transformants on LB kanamycin+ plate.

C. Colony PCR product on agarose gel electrophoresis. Lane 1-16, 16 single colonies tested.

D. AtMDH1 promoter sequence validated through sequencing.

E. Single colonies of p1300-AtMDH1-GUS transformants on LB kanamycin+ rifampicin+ plate.

F. Colony PCR of GV3101 transformants product on agarose gel electrophoresis. Lane 1-8, 8 single colonies tested.

Nicotiana benthamiana tobacco line FBP-22[3], in which the Fungal Bioluminescence Pathway (FBP, includes LUZ, H3H, CPH and HispS gene) was introduced was used as control and genetical engineering material to verify the function of AtMDH1 promoter. The p1300-AtMDH1-GUS construct was transformed into Agrobacterium GV3101 strain, followed by transient transformation of tobacco leaf through injection. The transgenic tobacco plants were stressed with 2mM HCHO (treatment) or H2O (control) for 36 hours, and leaf samples were collected 12 hours after treatment for GUS staining procedure. After destaining, the leaf tissues were photographed.</p>

The result displayed that for the negative control 0.5x PBS, no GUS signal was found in either HCHO or H2O group. When transgene was introduced, GUS signal was detected in H2O group, in which MDH1-GUS signal being the strongest, follwed by vector control pS1300-GUS, GRF3-GUS and GS1-GUS. However, after HCHO stress, 3 promoter-GUS showed different levels of signal reduction, compared to the enhanced signal in vector control pS1300-GUS. The result indicated that the three promoter candidates in this project were negatively responsive to HCHO stress, and GRF3 had the strongest phenotype.

Figure 4. Promoter candidates negatively responded to HCHO stress in dissected leaf GUS staining.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  COMPATIBLE WITH RFC[25]
• 1000
  COMPATIBLE WITH RFC[1000]

Reference

1.Wang, Q.J., Sun, H., Dong, Q.L., Sun, T.Y., Jin, Z.X., Hao, Y.J., et al., (2016) The enhancement of tolerance to salt and cold stresses by modifying the redox state and salicylic acid content via the cytosolic malate dehydrogenase gene in transgenic apple plants. Plant Biotechnol J 14, 1986–1997.

2.Xing Zhao, Xueting Yang, Yunfang Li, Hongjuan Nian, Kunzhi Li. (2023) 14-3-3 proteins regulate the HCHO stress response by interacting with AtMDH1 and AtGS1 in tobacco and Arabidopsis. Journal of Hazardous Materials, 458, 132036

3.Zheng, P., Ge, J., Ji, J., Zhong, J., Chen, H., Luo, D., Li, W., Bi, B., Ma, Y., Tong, W., Han, L., Ma, S., Zhang, Y., Wu, J., Zhao, Y., Pan, R., Fan, P., Lu, M. and Du, H. (2023), Metabolic engineering and mechanical investigation of enhanced plant autoluminescence. Plant Biotechnol J, 21: 1671-1681.