Part:BBa_K5218018

p1300-GRF3-GUS

Source：plasmid derived from pCAMBIA1300 backbone with Gibson Cloning system.

Lengths：12727 bp

Usage：Functional characterization of GRF3 promoter in transgenic tobacco.

Figure 1. Map of p1300-GRF3-GFP construct.

Characterisation

The GRF3 promoter is described in basic part BBa_K5218017. 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.

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 2. 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[1], 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.

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

Reference

1.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.

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]