Part:BBa_K5218019

GS1 promoter

Promoter of Glutamine synthetase 1 gene (GS1) from Arabidopsis thaliana.

Base Pairs：997 bp

Function：Plant GS catalyzes the synthesis of glutamine from glutamic acid and ammonium ions and acts as a key enzyme in the nitrogen metabolism pathway. GS is crucial in mitigating stress damage such as low or high temperature, salinity, drought and oxidation [1]. Recent report showed GS1 was responsive to HCHO stress [2].

Figure 1. HCHO stress response pathway in Arabidopsis.

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

Sequence and Features

Assembly Compatibility:

• 10
  INCOMPATIBLE WITH RFC[10]

  Illegal PstI site found at 591
  
• 12
  INCOMPATIBLE WITH RFC[12]

  Illegal PstI site found at 591
  
• 21
  INCOMPATIBLE WITH RFC[21]

  Illegal BglII site found at 529
  
• 23
  INCOMPATIBLE WITH RFC[23]

  Illegal PstI site found at 591
  
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal PstI site found at 591
  
• 1000
  COMPATIBLE WITH RFC[1000]

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 AtGS1 gene ID is AT5G57440, transcript ID is NM_125126.3 (NCBI). Team BGI-MammothEdu-South extracted the Arabidopsis thaliana leaf genomic DNA and cloned the AtGS1 promoter with specific primer pairs.

Characterisation

The expression pattern of AtGS1 was firstly investigated on the AtGenExpress eFP database. The result showed that AtGS1 was highly expressed in imbibed seed, roesette and shoot apex; moderately expressed in leaf and flowers.

Figure 2. AtGS1 expression pattern.

The cloning strategy for p1300-GS1-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 GS1 promoter fragment via Gibson Cloning system. In the construct, GUS reporter gene was driven by GS1 promoter, followed by NOS terminator. The recombinant plasmid p1300-GS1-GUS was transformed into E. coli TOP10 competent cells and verified through colony PCR and sequencing.

Figure 3. Generation of p1300-GS1-GUS construct.

A. PCR product on agarose gel electrophoresis. Lane M, 5000bp DNA marker; lane 1&2, GS1 promoter PCR prodoct.

B. Single colonies of p1300-GS1-GUS transformants on LB kanamycin+ plate.

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

D. GS1 promoter sequence validated through sequencing.

E. Single colonies of p1300-GS1-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 GS1 promoter. The p1300-GRF3-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.

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.

Reference

1.Li, J., Li, W., Xu, L., Wang, M., Zhou, W., Li, S., et al., (2022) Acclimation of sugar beet in morphological, physiological and BvAMT1.2 expression under low and high nitrogen supply. PLoS One 17, e0278327.

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.