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  • Research Background

    Heavy metal pollution has become a global problem, attracting broad attention. The presence of cadmium, mercury, copper and other heavy metals in the soil causes damage to the growth and development of plants and can easily enter the human-practices body through the food chain, posing a health hazard. These heavy metals mainly come from natural and human-practices activities, such as geochemical background values, industrial waste, pesticides, fertilizers, and urban garbage. Therefore, it is particularly important to test for total metals in soil.

    In nature, some fungi can spontaneously bioluminescence, such as species belonging to Armillaria, Mycena, Omphalotus and other genera. This is due to the metabolic pathway provided by the Fungal Bioluminescence Pathway (FBP) within the fungal biomass. The pathway converts caffeic acid into a molecule of luciferin. The luciferin enzyme Luz then directly oxidizes the luciferin to produce a bioluminescent signal. Through the FBP system, the dynamic process of plants can be detected, developmental and pathogenic mechanisms can be elucidated. It is particularly suitable for experiments on plants that need to grow in soil without the need for additional luciferin or other substrates.

    Scientists have discovered that plants can slow down the absorption and utilization of heavy metals in their bodies by changing cell wall composition, metal precipitation and rhizosphere microorganisms. They can also respond to heavy metal stress by changing gene expression, such as the NRAMP gene family and SRO gene family. This suggests that there are cis-acting elements in the promoter sequences of such gene families that recognize heavy metal ions.

  • Research purpose and significance

    Based on the above, our goal is to combine the FBP system with gene promoters that respond to heavy metal stress, and modify plants through genetic engineering techniques to make them capable of responding to soil heavy metals and continuously and stably emitting spontaneous fluorescence. Compared with traditional time-consuming and laborious detection methods, this plant can more quickly and effectively reflect changes in the concentration of heavy metal elements in soil through biological luminescence, providing a reliable indication for ecological environment protection and food safety.

  • Methods

  • (1) Promoter screening and primer design

    Collect genes related to heavy metal stress by reviewing existing literature. Process the samples for transcriptome sequencing to detect the expression of stress-responsive genes, and screen for significantly up-regulated and significantly expressed stress-responsive genes. Predict promoter-cis-acting elements using PlantCare, design primers using NCBI Primer Blast, and check primer specificity.

  • (2) GUS+GFP validation experiment

    Construct a plasmid containing a specific promoter using double enzyme digestion and homologous recombination methods, transform it into E. coli, and verify it by bacterial liquid PCR and sequencing. Transform the successfully constructed plasmid into Agrobacterium tumefaciens, verify it by bacterial liquid PCR and sequencing, then infect plant leaves, flowers, fruits and other parts. Finally, conduct GUS and GFP experiments to verify the temporal and spatial expression of the gene and the expression intensity of the promoter.

  • (3) FBP luminescence pathway verification experiment

    The verified promoter from (2) was connected to the front of the LUZ gene using a double enzyme digestion and homologous recombination method. The constructed vector was transformed into Agrobacterium tumefaciens and then infected the plants. The samples were treated with different concentrations of heavy metal elements to detect whether the plants could emit light and record the spontaneous luminescence intensity.

  • Reference

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