Design

1 Project overview

Our project primarily utilized genetic engineering and hybridization methods to construct three Drosophila lines, Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 (genotype), Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4, and Drosophila UAS-MTF-1;MRE-Hid/ptc-GAL4, each of which can be used as a visual monitoring system for heavy metals. In response to different heavy metal concentrations in the growing environment, Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 shows observably abnormally small eye sizes, while Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4 and Drosophila UAS-MTF-1;MRE-Hid/ptc-GAL4 show abnormally small sizes of wings. The phenotype can be shown in the first generation within 5-10 days. Our system is sensitive, cost-effective, and environmentally friendly, requiring minimum equipment and professional personnel, and is especially of great value to remote and underdeveloped regions.

2 Working principle of engineered Drosophila

The Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 or Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4 or Drosophila UAS-MTF-1;MRE-Hid/ptc-GAL4 line was engineered to have the following genes: MTF-1 driven by UAS, Hid driven by MRE, and a GMR-GAL4 or Vg-GAL4 or ptc-GAL4 (notated as GMR-GAL4/Vg-GAL4/ptc-GAL4) driver for eye or wing development.

MTF-1 (metal-responsive transcription factor-1) is a transcription factor that can be activated by heavy metals. In the cell nucleus of Drosophila cells, MTF-1 binds to MRE to recruit RNA polymerase, in turn increasing the expression of Mto/Mtn to detoxicate metals. We replaced the Mto/Mtn with head involution defective (Hid) to visualize the transcription process. Hid gene induces cellular apoptosis, decreasing the development of tissues in which its transcription is promoted. Cellular apoptosis in organs during development can be shown by organ size. UAS only triggers transcription of the downstream gene when exposed to transcription factor GAL4 (Busson & Pret, 2007). In the GMR-GAL4/Vg-GAL4/ptc-GAL4 driver, GAL4 is expressed during eye or wing development.

Therefore, during the eye/wing development of the Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 or Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4 or Drosophila UAS-MTF-1;MRE-Hid/ptc-GAL4 line, GAL4 is expressed due to the GMR-GAL4/Vg-GAL4/ptc-GAL4 driver, binds to UAS and activates the expression of MTF-1. When the Drosophila grows under heavy metal pollution, MTF-1 is activated and binds to MRE, which activates the expression of Hid. Hid induces apoptosis in the eyes/wings, resulting in the abnormally small sizes of the eyes/wings. The degree of the shrinkage of eyes/wings is related to the concentration of heavy metal ions.

Figure 1 Working principle of engineered flies for heavy metal contamination

3 General design

To construct the Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 or Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4 or Drosophila UAS-MTF-1;MRE-Hid/ptc-GAL4 line, we have three missions.

Mission 1: Construction of pUAST-MTF-1 and pMRE-Hid plasmids for MTF-1 gene driven by UAS and Hid gene driven by MRE.

Mission 2: Construction of Drosophila S2 cell lines with genotype UAS-MTF-1 and genotype MRE-Hid.

Mission 3: Hybridization of Drosophila UAS-MTF-1 and Drosophila MRE-Hid to obtain Drosophila UAS-MTF-1/MRE-Hid. Hybridization of Drosophila UAS-MTF-1/MRE-Hid and Drosophila GMR-GAL4/Vg-GAL4/ptc-GAL4 to obtain Drosophila UAS-MTF-1;MRE-Hid/GMR-GAL4 or Drosophila UAS-MTF-1;MRE-Hid/Vg-GAL4 or Drosophila UAS-MTF-1;MRE-Hid/ptc-GAL4 line.

Figure 2 Flow chart of the construction of the engineered Drosophila

4 Detailed design

Mission 1： Construction of pUAST-MTF-1 and pMRE-Hid plasmids

pUAST vector system is highly effective for generating transgenic flies. It possesses consecutive sites for a wide range of restriction enzymes, increasing its ability to recombine with other DNA fragments. It contains UAS and Hsp70. We inserted MTF-1 gene in the pUAST vector downstream of UAS and Hsp70 by enzyme cutting and linking, and constructed pUAST-MTF-1 plasmid. MTF-1 gene was derived from Drosophila melanogaster (D. melanogaster).

To construct pMRE-Hid plasmid, we replaced UAS with MRE in the pUAST vector and inserted Hid gene in the modified pUAST vector downstream of MRE and Hsp70 by enzyme cutting and linking. Hid gene was derived from D. melanogaster.

PCR and gene sequencing were used for verification.

Figure 3 pUAST vector

Figure 4 pUAST-MTF-1 and pMRE-Hid plasmids

Mission 2： Construction of Drosophila S2 cell lines with genotype of UAS-MTF-1 and MRE-Hid. Step 1: Cellular confirmation: Construction of a Drosophila S2 cell line with genotype UAS-MTF-1/MRE-Hid/Ac-Gal4

To confirm if the combination of UAS-MTF-1, MRE-Hid and GMR-GAL4/Vg-GAL4/ptc-GAL4 driver in Drosophila can respond to heavy metal ions, we first constructed a Drosophila S2 cell line with genotype UAS-MTF-1/MRE-Hid/Ac-Gal4 via co-transfection of pUAST-MTF-1, pMRE-Hid and pAc-Gal4 plasmids. pAc-Gal4 plasmid was provided by Genetic Lab, School of Life Science and Technology, Tongji University.

Real-time PCR and Western Blot were used to determine the levels of MTF-1 and Hid mRNA and proteins in the Drosophila UAS-MTF-1/MRE-Hid/Ac-Gal4 cells treated with different concentrations of ZnCl2 and CdCl2.

Step 2: Construction of Drosophila S2 cell lines with genotype of UAS-MTF-1 and MRE-Hid

pUAST-MTF-1 and pMRE-Hid were micro-injected into embryos of Drosophila W1118 respectively to obtain Drosophila UAS-MTF-1 and Drosophila MRE-Hid.

Figure 5 Flow chart of cell experiments

Mission 3：Generation of Drosophila lines with genotype of UAS-MTF-1;MRE-Hid/GMR-GAL4 or UAS-MTF-1;MRE-Hid/Vg-GAL4 or UAS-MTF-1;MRE-Hid/ptc-GAL4

Drosophila UAS-MTF-1 was crossed with Drosophila MRE-Hid to obtain the offspring with genotype of UAS-MTF-1;MRE-Hid. This progeny was crossed with Drosophila GMR-GAL4/Vg-GAL4/ptc-GAL4 to obtain the progeny with genotype of UAS-MTF-1;MRE-Hid/GMR-GAL4 or UAS-MTF-1;MRE-Hid/Vg-GAL4 or UAS-MTF-1;MRE-Hid/ptc-GAL4. Drosophila GMR-GAL4/Vg-GAL4/ptc-GAL4 line was provided by Genetic Lab, School of Life Science and Technology, Tongji University

The above three Drosophila lines were cultured with different concentrations of ZnCl2 and CdCl2. The larvae and F1 generation adults were collected. Imaginal discs were dissected from the larvae.

The stable transformation of sweet potato was performed using the protocol of Yang et al (2011) .

Acridine Orange (AO) staining and Death Caspase-1 (Dcp-1) staining were used to detect cell death and apoptosis, respectively, in the eye and wing imaginal discs. The sizes of eyes/wings of the adults were measured and analyzed.

Figure 6 Flow chart of experiments with Drosophila lines with genotype of UAS-MTF-1;MRE-Hid/GMR-GAL4 or UAS-MTF-1;MRE-Hid/Vg-GAL4 or UAS-MTF-1;MRE-Hid/ptc-GAL4

Supplementary task: Generation of a Drosophila line with genotype of UAS-MTF-1;MRE-GFP/GMR-GAL4

In addition, we also constructed a Drosophila line that show different degrees of fluorescence in the eyes in response to different heavy metal concentrations in the growing environment.

We used the same methods in the Mission 1-3, and replaced Hid with GFP to construct a Drosophila line with genotype of UAS-MTF-1;MRE-GFP/GMR-GAL4. GFP (green fluorescent protein) is a specialized protein that emits green fluorescence when exposed to excitation light. The fluorescence in the eye imaginal discs from larvae after culturing with different concentrations of heavy metal ions was photographed under fluorescent microscope, and the fluorescence intensity of GFP was calculated by Photoshop.

Figure 7 Flow chart of experiments with a Drosophila line with genotype of UAS-MTF-1;MRE-GFP/GMR-GAL4

Reference

Andrews, G. K. (2001). Cellular zinc sensors: MTF-1 regulation of gene expression. Biometals, 14(3-4), 223-237. doi: 10.1023/a:1012975626361

Busson, D., & Pret, A. M. (2007). GAL4/UAS targeted gene expression for studying Drosophila Hedgehog signaling. Methods in molecular biology (Clifton, N.J.), 397, 161–201.