Regulatory

Part:BBa_K5218017

Designed by: Amy Fu, Robert, Lichuan Chen, Xiaojuan Wang   Group: iGEM24_BGI-MammothEdu-South   (2024-09-03)


GRF3 promoter

Promoter of GENERAL REGULATORY FACTOR 3 gene (GRF3, also known as 14-3-3) from Arabidopsis thaliana.

Base Pairs：1783 bp

Function：GRF3 is widely involved in the regulation of material transport, growth and development and nutrient metabolism[1]. GRF3 proteins are also known to regulate the response of plants to HCHO stress.

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 XbaI site found at 1203
  • 12
    COMPATIBLE WITH RFC[12]
  • 21
    INCOMPATIBLE WITH RFC[21]
    Illegal BglII site found at 222
    Illegal BglII site found at 1746
  • 23
    INCOMPATIBLE WITH RFC[23]
    Illegal XbaI site found at 1203
  • 25
    INCOMPATIBLE WITH RFC[25]
    Illegal XbaI site found at 1203
  • 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.

Plant 14-3-3 proteins are key regulators that play significant roles in plant growth, development, and stress response processes [3]. GRF3 proteins are reported to regulate the response of plants to HCHO stress by interacting with AtMDH1 and AtGS1 [2].

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 AtGRF3 gene ID is AT5G38480, transcript ID is NM_123209.4 (NCBI). Team BGI-MammothEdu-South extracted the Arabidopsis thaliana leaf genomic DNA and cloned the AtGRF3 promoter with specific primer pairs.

Characterisation

The expression pattern of AtGRF3 was firstly investigated on the AtGenExpress eFP database. The result showed that AtGRF3 was highly expressed in imbibed seed and shoot apex; moderately expressed in leaf and flowers.

Figure 2. AtGRF3 expression pattern.

The cloning strategy for p1300-GRF3-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 HCHO-responsive GRF3 promoter fragment via Gibson Cloning system. In the construct, GUS reporter gene was driven by GRF3 promoter, followed by NOS terminator. The recombinant plasmid p1300-GRF3-GUS was transformed into E. coli TOP10 competent cells and verified through colony PCR and sequencing.

Figure 3. Generation of p1300-GRF3-GUS construct.

A. PCR and digestion product on agarose gel electrophoresis. Lane M, 5000bp DNA marker; lane 1, cicular pS1300-GUS plasmid; lane 2, BamH I digestion of pS1300-GUS; lane 3, GRF3 promoter PCR prodoct.
B. Single colonies of p1300-GRF3-GUS transformants on LB kanamycin+ plate.
C. Colony PCR product on agarose gel electrophoresis. Lane 1-16, 16 single colonies tested.
D. GRF3 promoter sequence validated through sequencing.
E. Single colonies of p1300-GRF3-GUS transformants on LB kanamycin+ rifampicin+ plate.
F&G. Colony PCR of GV3101 transformants product on agarose gel electrophoresis. Lane 1-8, 8 single colonies tested.

Nicotiana benthamiana tobacco line FBP-22[4], 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 GRF3 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.</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.

Next, the GRF3 promoter, which showed the most significant phenotypic differences, was selected to replace the super promoter in the pS1300-RtTAL-GFP and pS1300-AtC3H-GFP constructs, resulting in p1300-pGRF3-RtTAL-GFP (BBa_K5218023) and p1300-pGRF3-AtC3H-GFP (BBa_K5218024). This combination integrates a formaldehyde-responsive regulatory module with a luminescence-enhancing gene to create luminescent plants that respond to formaldehyde.</p>


Figure 5. Generation of p1300-pGRF3-RtTAL-GFP and p1300-pGRF3-AtC3H-GFP construct.

A. PCR product on agarose gel electrophoresis. Lane M, 1kb plus DNA marker; lane 1&2, MDH1 promoter; lane 3&4, GS1 promoter; lane 5&6, GRF3 promoter. Lane 1/3/5 products contain RtTAL recombination overhang; lane 2/4/6 products contain AtC3H recombination overhang;
B. Single colonies of p1300-pGRF3-RtTAL-GFP transformants on LB kanamycin+ plate.
C. Single colonies of p1300-pGRF3-AtC3H-GFP transformants on LB kanamycin+ plate.
D. Single colonies of p1300-pGRF3-RtTAL-GFP transformants on LB kanamycin+ rifampicin+ plate.
E. Single colonies of p1300-pGRF3-AtC3H-GFP transformants on LB kanamycin+ rifampicin+ plate.
F. Colony PCR of p1300-pGRF3-RtTAL-GFP in E.coli. Lane M, 5000bp DNA marker. Lane 1-24, 24 single colonies tested.
G. MDH1 promoter sequence validated through sequencing.
H. Colony PCR of p1300-pGRF3-AtC3H-GFP in E.coli. Lane M, 5000bp DNA marker. Lane 1-24, 24 single colonies tested.
I. MDH1 promoter sequence validated through sequencing.
J. Colony PCR of p1300-pGRF3-RtTAL-GFP in GV3101. Lane M, 5000bp DNA marker. Lane 1-8, 8 single colonies tested.
K. Colony PCR of p1300-pGRF3-AtC3H-GFP in GV3101. Lane M, 5000bp DNA marker. Lane 1-8, 8 single colonies tested.


Figure 6. p1300-pGRF3-RtTAL-GFP plasmid map.

Figure 7. p1300-pGRF3-AtC3H-GFP plasmid map.
The p1300-pGRF3-RtTAL-GFP and p1300-pGRF3-AtC3H-GFP constructs were transformed into Agrobacterium GV3101 strain, followed by infiltration to generate transient expression lines.

<p>Upon 2mM HCHO treatment for 36 hours, the bioluminescence intensity was repressed in pS1300-GFP control lines compared to water treatment, whereas the pS1300-RtTAL-GFP and p1300-pGRF3-RtTAL-GFP lines could compensate the repression. The results showed that RtTAL could stably enhance the bioluminescence of plants, and GRF3 has some level of responsiveness to HCHO. The enhancement of light intensity by GRF3 promoter is limited though. It is worth memtioning that the batch of tobacco materials for this experiment went through significant injury stress in the process of injection, which may affected the bioluminescence analysis and interpretation. Unfortunately, due to limited time, we did not conduct a second test on new batch of plants.

Figure 8. pGRF3-RtTAL in response to HCHO.

Upon 2mM HCHO treatment for 36 hours, the bioluminescence intensity was clearly repressed in pS1300-GFP control lines, whereas the pS1300-AtC3H-GFP and p1300-pGRF3-AtC3H-GFP lines could compensate the repression. The results showed that AtC3H could stably enhance the bioluminescence of plants, and GRF3 is indeed responsive to HCHO. Yet the enhancement of light intensity by promoter GRF3 is limited.

Figure 9. pGRF3-AtC3H in response to HCHO.


Reference

1.Liu, Q., Zhang, S., Liu, B. (2016) 14-3-3 proteins: Macro-regulators with great potential for improving abiotic stress tolerance in plants. Biochem Biophys Res Commun 477, 9–13.

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.Zhao, X., Li, F., Li, K. (2021) The 14-3-3 proteins: regulators of plant metabolism and stress responses. Plant Biol 23, 531–539.

4.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.

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