Transgenic moss, ozone monitoring biosensor and preparation method and application thereof

Abstract

The invention belongs to the technical field of biological gene modification, and particularly relates to transgenic moss, an ozone monitoring biosensor and a preparation method and application of the transgenic moss and the ozone monitoring biosensor. The transgenic moss is obtained by transfecting a target gene into moss cells, the target gene comprises a ZAT12 promoter, and the sequence of the gene ZAT12 is as shown in SEQ ID NO: 1. The transgenic moss is transferred into an oxygen response element ZAT12 promoter, and an ozone specific signal of the transgenic moss can be actively enhanced, so that high-sensitivity (5ppb) detection of ozone can be realized.

Description

Technical Field

[0001] This invention relates to the field of bio-genetic modification technology, specifically to transgenic moss, ozone monitoring biosensors, their preparation methods, and applications. Background Technology

[0002] As one of the major air pollutants, ozone biomonitoring technology has long relied on the sensitive responses of plants to ozone. Currently, higher plants such as beans, white clover, poplars, and pines, as well as mosses, are used as ozone bioindicators. Among them, mosses, due to their unique biological characteristics (lacking true roots and vascular tissue, relying on direct atmospheric absorption of water and nutrients, and having leaves that are mostly single-celled without a waxy cuticle), are extremely sensitive to air pollutants (including ozone)—ozone can directly penetrate their cell membranes, and pollutants can efficiently enter their bodies with precipitation / aerosols. This characteristic theoretically makes mosses a potential high-sensitivity ozone biosensor, but their current applications are limited to large-scale, long-term comprehensive air quality assessments (where ozone is only one of the influencing factors), and specific ozone monitoring technologies have not yet been developed.

[0003] The ozone response mechanisms of mosses and higher plants differ significantly, limiting their monitoring performance: ① Absorption efficiency: Mosses lack true stomata, and ozone is mainly absorbed through epidermal permeation, resulting in lower absorption efficiency compared to the direct stomata of higher plants. Although their monolayer cell structure is thin, extracellular polysaccharides (EPS) can slow ozone diffusion. ② Damage buffering: Mosses are naturally resilient and contain various antioxidant enzymes (such as APX and SOD), which can buffer ozone damage in the short term and delay the appearance of visible symptoms. ③ Gene response: The expression of homologous defense genes in mosses (such as PpZAT12) is delayed, and the lack of a vascular bundle system leads to low signal transduction efficiency. ④ Detection threshold: Existing transgenic moss sensors (such as those using GFP / Luciferase reporter systems) require the accumulation of high doses of ozone (e.g., 50 ppb × 24 h) to generate a detectable signal, which cannot meet the needs of rapid monitoring of low-concentration ozone. In addition, monitoring technologies for wild-type mosses mostly rely on passive responses (such as chlorophyll decay), lacking specificity and sensitivity.

[0004] There are significant gaps in the application of existing ozone biomonitoring technologies in the field of moss: On the one hand, although moss is highly sensitive to air pollutants, ozone-specific monitoring technologies are not yet mature—existing transgenic mosses (such as the CRISPR-edited antioxidant gene of *Sphaerocera spp.* by the Peking University team in 2023) only achieve an ozone sensitivity threshold of 50 ppb, which cannot meet the high-sensitivity detection requirements for low concentrations (such as 5 ppb) of ozone; on the other hand, existing patents for moss (such as EP2899283A) only focus on its heavy metal adsorption performance and do not involve ozone monitoring; other plant sensor patents (such as the Arabidopsis GUS reporting system in US20150020290A1) have also failed to solve the core problem of moss-specific ozone detection. In addition, the application of plant-based ozone monitoring technologies is limited (for example, the "intelligent green wall" in patent CN110412193A does not involve ozone-specific monitoring).

[0005] Therefore, developing a highly sensitive (e.g., 5 ppb) and highly stable ozone biosensor based on moss is of great significance for improving air pollution monitoring technology. Summary of the Invention

[0006] Based on this, the present invention achieves highly sensitive (5ppb) ozone detection with high stability by transplanting ozone-responsive elements (e.g., ZAT12 promoter) from higher plants (e.g., Bel-W3 tobacco) and antioxidant enhancers (APX1 / SOD) from Arabidopsis thaliana into mosses.

[0007] In order to achieve the above object, the present invention can adopt the following technical solutions:

[0008] In one aspect, the present invention provides a transgenic moss, which is obtained by transfecting a target gene into moss cells. The target gene includes the ZAT12 promoter, and the sequence of the ZAT12 gene is shown in SEQ ID NO: 1.

[0009] Preferably, the target gene further includes one or more of the antioxidant enhancer APX1, the antioxidant enhancer SOD, or a reporter gene; wherein the sequence of the antioxidant enhancer APX1 is shown in SEQ ID NO: 2, and the sequence of the antioxidant enhancer SOD is shown in SEQ ID NO: 3.

[0010] Preferably, the moss is *Moss styracifolium*.

[0011] Another aspect of the present invention provides an ozone monitoring biosensor comprising the aforementioned transgenic moss.

[0012] Preferably, the ozone monitoring biosensor further includes a carrier and a water-absorbing component, with the transgenic moss attached to the carrier and the water-absorbing component disposed on the side where the transgenic moss is not attached.

[0013] More preferably, the aforementioned water-absorbing component is a water-retaining cotton.

[0014] Preferably, the method for preparing the above-mentioned carrier is selected from any of the following:

[0015] (a) Using 3D printing to assemble cordierite (Mg2Al4Si5O) 18 The substrate is printed into a porous ceramic substrate and then sintered to obtain a carrier.

[0016] (b) Using 3D printing to fabricate a carrier from a porous ceramic plate raw material, wherein the porous ceramic plate raw material is obtained by mixing 10%-20% by volume of activated carbon, 3%-7% by mass of methylcellulose and 1%-3% by mass of α-tricalcium phosphate.

[0017] More preferably,

[0018] In the aforementioned carrier (a), the porous ceramic substrate has a pore size of 10 μm-50 μm and a porosity of 40%-60%; and / or

[0019] In the aforementioned carrier (a), a coating is formed by coating the surface of the carrier with a paint, which is obtained by mixing activated carbon with 1%-3% by mass of binder, 60%-80% by mass of deionized water, and 0.3%-0.7% by mass of dispersant; and / or

[0020] In the above-mentioned carrier (b), the activated carbon is loaded with 0.05%-0.15% nano-silver by mass.

[0021] In another aspect, the present invention provides a method for preparing the above-mentioned ozone monitoring biosensor, the method comprising:

[0022] (1) Genetically modified moss was prepared into a suspension containing an adhesive agent;

[0023] (2) Ball mill the suspension to break it down to fragments ≤100μm;

[0024] (3) Spray the suspension after ball milling onto a carrier equipped with a water-absorbing component and let it stand to solidify to obtain an ozone monitoring biosensor.

[0025] In another aspect, the present invention provides the application of the above-mentioned transgenic moss or the above-mentioned ozone monitoring biosensor in the detection of ozone.

[0026] The beneficial effects of this invention include:

[0027] (1) The transgenic moss provided by the present invention can actively enhance its ozone-specific signal by introducing the oxygen-responsive element ZAT12 promoter, thereby achieving high-sensitivity (5ppb) detection of ozone.

[0028] (2) The transgenic moss provided by the present invention can improve the ROS scavenging ability of moss cells by introducing the oxygen-responsive element ZAT12 promoter and Arabidopsis antioxidant enhancer APX1 and / or SOD, thereby avoiding excessive cell death, improving the survival rate of transgenic moss, and thus improving stability.

[0029] (3) The transgenic moss provided by the present invention can quantify the ozone exposure level by introducing a reporter gene, and realize visual ozone detection.

[0030] (4) The ozone monitoring biosensor provided by the present invention can utilize the natural attachment characteristics of transgenic moss rhizoids, combined with a porous ceramic carrier, to propose a soil-free, low-maintenance building integration scheme. At the same time, it can utilize the physiological state of moss (such as chlorophyll fluorescence) to reflect the heat island effect and realize multi-parameter monitoring. Attached Figure Description

[0031] Figure 1 This is a schematic diagram of the structure of a biosensor for ozone monitoring; in the diagram, 1: transgenic *Sphagnum moss*; 2: porous ceramic plate carrier; 3: water-storing cotton; 4: water level line. Detailed Implementation

[0032] The examples are provided to better illustrate the present invention, but are not intended to limit the present invention to the examples. Therefore, non-essential improvements and adjustments to the embodiments made by those skilled in the art based on the above-mentioned invention still fall within the scope of protection of the present invention.

[0033] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "comprise" and the like are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0034] In a first aspect, embodiments of the present invention provide a transgenic moss, which is obtained by transfecting a target gene into moss cells. The target gene includes the ZAT12 promoter, and the sequence of the ZAT12 gene is shown in SEQ ID NO: 1.

[0035] It should be noted that existing moss monitoring technologies mostly rely on the passive response of wild-type mosses (such as chlorophyll decay), while this invention actively enhances its ozone-specific signal through synthetic biology, that is, by implanting the ozone-responsive element (ZAT12 promoter) of higher plants (such as Bel-W3 tobacco) into mosses, which can achieve high-sensitivity (5ppb) detection of ozone.

[0036] It should also be noted that Bel-W3, as an ozone-sensitive indicator tobacco, is a cultivar specifically developed for ozone biomonitoring, originating in Maryland, USA (Heggestad Laboratory, 1960s). Heggestad elucidated the high ozone sensitivity of the Bel-W3 tobacco cultivar (10 times more sensitive than ordinary varieties), laying the foundation for its use as an ozone biomonitor. Scofield discovered that ozone induces visible damage to tobacco leaves by inducing ethylene synthesis, with typical leaf symptoms—chlorotic spots (white / brown necrotic spots)—appearing after 8 hours of exposure to 5-10 ppb. Unlike high-yield, high-aroma commercial flue-cured tobacco varieties used as raw materials for cigarettes (Yunnan Tobacco Research Institute, 2000s), Yunyan 78, which has a strong ROS scavenging ability, only shows slight yellowing leaf symptoms at concentrations above 50-80 ppb, and is easily confused with other stresses. Bel-W3 leaves are thin and brittle, with a high stomatal density (≥300 / mm²). 2 It has a large stomatal aperture and does not close in response to ozone, exhibits low levels of antioxidant enzyme (SOD, APX) activity, and the leaf injury area is linearly correlated with ozone concentration (R). 2 Tobacco (>0.9) has been listed as a core indicator species for ozone pollution monitoring in Europe. The European ICPVegetation Programme (EU-ICPVegetation Programme) lists tobacco as a baseline bioindicator for ozone and regularly publishes the "Ozone Injury Manual" to guide standardized assessments. The Bel-W3 tobacco network deployed in cities such as São Paulo uses standardized leaf damage area to inversely determine ozone exposure (AOT40 index). Tobacco monitoring in the suburbs of Beijing shows that when the annual average ozone concentration is 60 ppb, the Bel-W3 leaf damage rate reaches 85%.

[0037] In some specific examples, the target gene mentioned above also includes one or more of the antioxidant enhancer APX1, the antioxidant enhancer SOD, or a reporter gene; wherein the sequence of the antioxidant enhancer APX1 is shown in SEQ ID NO: 2, and the sequence of the antioxidant enhancer SOD is shown in SEQ ID NO: 3.

[0038] It should be noted that, in addition to transferring the ozone-responsive element ZAT12 promoter into moss to achieve high-sensitivity (5ppb) ozone detection, the Arabidopsis antioxidant enhancer (APX1 / SOD) can also be transplanted into moss simultaneously to achieve a balance in ozone detection resistance. Specifically, transplanting the ozone-responsive element ZAT12 promoter into moss can promote activation under ozone stress (response threshold 5ppb), driving the expression of downstream reporter genes. At the same time, transplanting the Arabidopsis antioxidant enhancer into moss can improve the intracellular ROS scavenging capacity of moss cells, avoid excessive cell death, improve the survival rate of transgenic moss, and thus improve stability.

[0039] It should also be noted that the ZAT12 pathway can serve as a target for biosensor design. Bel-W3 addresses ozone stress by regulating antioxidant enzymes (SOD, APX) through the ZAT12 transcription factor. Editing the RAD4 gene increased tobacco ozone sensitivity by 3 times (reducing the response threshold to 3 ppb). The luminescence intensity of luciferase expressed in transgenic tobacco after ozone exposure can be quantified using portable photometric methods. Ozone detection (LOD = 2 ppb) can be completed within 15 minutes by immobilizing tobacco suspension cells on a microfluidic chip.

[0040] It should also be noted that reporter genes such as GFP / Luciferase can be introduced into moss to enable the visual detection of ozone.

[0041] In some specific examples, the moss mentioned above is Physcomitrellapatens.

[0042] It should be noted that *Sphaerophyte simonii* has a clear genome, efficient homologous recombination, and tolerance to extreme environments, but requires optimization of ozone permeability (due to insufficient stomatal development); *Mossia lesions* is naturally drought / ozone tolerant and has long-term survival (>5 years), but has low genetic transformation efficiency (requiring viral vector assistance); lichen-type fungi and symbiotic algae enhance photosynthesis and are widely distributed in the wild, but their symbiotic systems interfere with transgenic expression. Furthermore, *Sphaerophyte simonii* has a simple and malleable structure, making it an ideal basis for low-maintenance biosensors. *Sphaerophyte simonii* is a non-vascular plant; its roots, unlike those of higher plants (such as tobacco), have a filamentous structure, lacking an endodermis and vascular bundle differentiation. This gives it unique advantages in low-maintenance ozone monitoring systems: ① Although it cannot anchor the plant, it can mechanically fix it, for example, by embedding it in 3D-printed porous ceramics, or by forming a rhizoid network through spray culture, suitable for deployment on non-horizontal surfaces such as walls; ② Although it cannot actively transport water and nutrients, it can absorb water through capillary action at a rate of up to 1 μL / h / cm. 2This meets the needs of drought recovery. At the same time, ozone-induced reactive oxygen species (ROS) can be rapidly diffused to the whole plant through rhizoids (30% slower than leaves but more persistent); ③ Rhizoid-specific promoters (such as PpRSL1) can drive transgenic expression and enhance environmental signal capture; ④ Compared with the vascular plant tobacco, which requires regular irrigation and fertilization, sphagnum moss does not require soil and can survive by spraying water once a week during drought, and the rhizoid base can survive continuously.

[0043] It should also be noted that the method of transferring the target gene into moss is known in the art. For example, Agrobacterium can be used as a vector for transfer, or the gene gun method can be used to embed the ZAT12::APX1-2A-GFP-hptII vector in gold particles and deliver the target gene into the moss through physical penetration.

[0044] Secondly, embodiments of the present invention provide an ozone monitoring biosensor, comprising the aforementioned transgenic moss.

[0045] It should be noted that traditional biosensors require regular maintenance (such as irrigation and substrate replacement), while this invention utilizes the natural attachment characteristics of moss rhizoids to combine transgenic moss with a porous ceramic carrier, proposing a soil-free, low-maintenance building integration solution. Ozone exposure levels are quantified using a fluorescent reporter gene (GFP), while the physiological state of the moss (such as chlorophyll fluorescence) reflects the heat island effect, enabling multi-parameter monitoring. It can be integrated with intelligent building management systems to trigger ventilation equipment to adjust ozone concentration.

[0046] In some specific examples, the ozone monitoring biosensor also includes a carrier and a water-absorbing component, with the transgenic moss attached to the carrier and the water-absorbing component positioned on the side where the transgenic moss is not attached.

[0047] It should be noted that the method of placing the water-absorbing component on the side not covered by the genetically modified moss is well known in the art, such as by watering and bonding, or by other methods.

[0048] In some specific examples, the aforementioned water-absorbing component is a water-retaining cotton.

[0049] It should be noted that the water level line is set at the lower 1 / 3 height of the water-storing cotton, and the water level line contains 60% of the water-saturated absorption capacity of the water-storing cotton.

[0050] In some specific examples, the preparation method of the above-mentioned carrier is selected from any of the following:

[0051] (a) Using 3D printing to assemble cordierite (Mg2Al4Si5O) 18 The substrate is printed into a porous ceramic substrate and then sintered to obtain a carrier.

[0052] (b) Using 3D printing to fabricate a carrier from a porous ceramic plate raw material, wherein the porous ceramic plate raw material is obtained by mixing 10%-20% by volume of activated carbon, 3%-7% by mass of methylcellulose and 1%-3% by mass of α-tricalcium phosphate.

[0053] In some specific examples,

[0054] In the aforementioned carrier (a), the porous ceramic substrate has a pore size of 10 μm-50 μm and a porosity of 40%-60%; and / or

[0055] In the aforementioned carrier (a), a coating is formed on the surface of the carrier by applying a coating material. The coating material is obtained by mixing activated carbon with 1%-3% by mass of binder, 60%-80% by mass of deionized water, and 0.3%-0.7% by mass of dispersant; and / or

[0056] In the above-mentioned carrier (b), the activated carbon is loaded with 0.05%-0.15% nano-silver by mass.

[0057] It should be noted that the above method of applying paint to form a coating can be achieved by applying the paint twice with a soft brush to form a coating.

[0058] Thirdly, embodiments of the present invention provide a method for preparing the above-mentioned ozone monitoring biosensor, the method comprising:

[0059] (1) Genetically modified moss was prepared into a suspension containing an adhesive agent;

[0060] (2) Ball mill the suspension to break it down to fragments ≤100μm;

[0061] (3) Spray the suspension after ball milling onto a carrier equipped with a water-absorbing component and let it stand to solidify to obtain an ozone monitoring biosensor.

[0062] Fourthly, embodiments of the present invention provide an application of the above-mentioned transgenic moss or the above-mentioned ozone monitoring biosensor in ozone detection.

[0063] It should be noted that the aforementioned genetically modified moss or ozone monitoring biosensor can be used for ozone concentration detection, suitable for real-time ozone monitoring in urban, polar, or desert environments; it is also suitable for applications such as kindergartens or nursing homes, triggering LED alarms for early warning when ozone levels exceed standards; furthermore, it is applicable to traffic pollution hotspots, such as tunnel entrances and viaduct walls (monitoring NOx-derived ozone); school / hospital building envelopes (children's health protection); and industrial zone boundaries: replacing traditional walls (simultaneously adsorbing SO2 / ozone). Specific settings can be configured according to specific needs.

[0064] In order to better understand the present invention, the content of the present invention is further explained below with reference to specific examples, but the content of the present invention is not limited to the following examples.

[0065] In the following example, the 3D printing parameters are as follows: the planar dimensions of the above-mentioned single plate can be 50×50mm or 150×150mm, with a thickness of 2-5mm (<1mm is prone to brittleness, >8mm significantly increases gas diffusion resistance) (≤3mm is a flat plate, >3mm contains internal spiral microchannels). Alternating layers of 20μm and 50μm pore sizes are designed along the thickness direction (mimicking the vascular bundle structure of plants) (interlayer connectivity ≥80%), and the surface layer is distributed with Φ20-30μm anchor point micropores (anchor point density of 625 points / cm²). 2 The bottom features a water-retaining cotton connecting layer (containing Φ50μm connecting holes) for moss rhizoids to penetrate and fix (moss rhizoid penetration diameter is 15-40μm, ozone adsorption flow resistance (pressure drop <50Pa)). Moss and agarose composite gel (1:1 mixture) can be filled into the micropores (20-30μm) at the anchoring point to form local water-retaining units. An extrusion-type ceramic 3D printer (0.4mm hard alloy nozzle) was used, and the distance between the nozzle and the substrate was calibrated to 0.12mm in an environment of 25±2℃ and 40-50% humidity. The slurry was prepared by mixing cordierite powder (D50 = 5μm, 65%) with deionized water (30%), ammonium citrate (2%), and PVA (3%). The mixture was then subjected to planetary ball milling (300rpm, 2 hours) and vacuum degassing (-0.1MPa, 15 minutes) to control the viscosity at 2000-3000mPa·s. During printing, the extrusion pressure was 0.2-0.3MPa, the speed was 5mm / s in straight sections and 3mm / s in corner areas, the layer height was 0.1mm, the substrate temperature was 40℃, and the interlayer dwell time was 10 seconds. The printing sequence was: bottom layer → alternating aperture layer (20μm layer line spacing 0.3mm, 50μm layer line spacing 0.6mm) → surface anchor points and microchannels (>3mm). After completion, the mixture was allowed to stand for 2 hours (25℃).

[0066] Example 1

[0067] This invention provides a transgenic *Moss simonii* species, obtained through the following specific method:

[0068] (1) Wild-type P. patens Gransden strain was selected and cultured on BCDAT solid medium (Coolaber's customized complete medium for P. patens, 1.5% agar, pH 5.8). The medium was then exposed to light (light intensity 50 μmol / m²) at 25℃. 2 / s)16h, then 8h in darkness for growth;

[0069] (2) Take 7-day-old green protonemata, grind them into fragments (about 10 cell clusters), pre-treat with 0.4M mannitol for 1 hour for permeation treatment to enhance DNA absorption capacity.

[0070] (3) Wash 60 mg of 1.0 μm gold particles (Bio-Rad) with 1 mL of anhydrous ethanol, centrifuge and discard the supernatant; then add 50 μL of 2.5 M CaCl2, 20 μL of 0.1 M spermidine and 10 μg of [unclear - possibly a specific ingredient or solution] in sequence. ZAT12::APX1-2A-GFP-hptII custom plasmid (containing ZAT12 promoter, APX1 gene, P2A self-cleaving peptide sequence, GFP reporter gene, hptII selection marker gene, NOS terminator; GFP-hptII integrates gene expression monitoring and transformant screening) (concentration ≥1 μg / μL, dissolved in TE buffer), after mixing, DNA adsorption was performed, vortexed for 3 minutes (2500 rpm), allowed to stand for 1 minute, centrifuged and the supernatant was discarded, and resuspended in 100 μL of anhydrous ethanol; each dish of moss (9 cm in diameter) was bombarded once with a vacuum of 28 inches of mercury, a bombardment distance of 6 cm, and a helium pressure of 1100 psi, and immediately transferred to a stress-free medium (Coolaber custom-made small-bowl moss medium, without selective antibiotics, maintaining stable intracellular and extracellular osmotic pressure, preventing cytoplasmic leakage due to mechanical damage after gene gun bombardment), and allowed to recover for 24 hours;

[0071] (4) Initial screening (7-10 days) (set based on the efficacy of hygromycin and the proliferation rate of moss cells to ensure that the initial screening is completed within the window period when positive clones are identifiable and not under excessive stress). The culture medium is BCDAT + 50 mg / L hygromycin B. Untransformed cells gradually turn brown and die, while positive clones remain green. Single clone isolation is performed. Green clumps with a diameter >1 mm are selected with sterile forceps and transferred to fresh BCDAT solid culture medium containing 30 mg / L hygromycin. The cells are passaged once every 2 weeks.

[0072] (5) PCR verification was performed on clones that survived after the second passage (the primer amplification verification was performed by Microspection Detection Technology Group, using specific primers for exogenous genes (such as APX1-2A-GFP). The amplification products were detected by 1.5% agarose gel electrophoresis (containing EB). A single bright band at 387bp was a positive clone, and the negative control (untransformed wild-type moss) did not have this band, while the positive control (plasmid containing exogenous gene) had the same band). False positive samples that were not transformed were excluded to ensure that the correct Pleurotus ostreatus cells with integrated target plasmids were used in subsequent experiments.

[0073] (6) After genomic DNA detection, GFP expression was verified. The moss was exposed to 80 ppb ozone for 4 hours for induction, and fluorescence was detected by confocal microscopy (excitation 488 nm / emission 510 nm). Compared with the uninduced sample, obvious green fluorescence was observed. After culturing for 3 generations in a stress-free medium, the moss was placed in BCDAT medium containing hygromycin (30 mg / L). The line could grow normally (without browning or death), which was consistent with the growth status of the line cultured in hygromycin medium for a long time, confirming that the resistance was not lost. The transgenic moss line stably expressing ZAT12::APX1-2A-GFP-hptII (hereinafter referred to as transgenic moss) was obtained.

[0074] Example 2

[0075] This invention provides an ozone monitoring biosensor, comprising: a porous ceramic plate carrier and a transgenic *Sphaerocarpus spp.* constructed in Example 1. The transgenic *Sphaerocarpus spp.* is attached to the porous ceramic plate carrier, and a porous water-retaining cotton (2 mm thick, 90% porosity) is connected to the bottom of the porous ceramic plate carrier, automatically replenishing water through capillary action (see reference). Figure 1 (As shown). The specific preparation method of the ozone biomonitoring component is as follows:

[0076] (1) Preparation of porous ceramic plate carrier

[0077] 1) Using 3D printing to produce cordierite (Mg2Al4Si5O) 18 The filaments are printed into porous ceramic substrates (pore size 10μm-50μm, porosity 40%-60%), and then sintered at 1200℃ for 3 hours (to ensure sufficient diffusion and sintering of cordierite particles to form Mg2Al4Si5O). 18 Crystal phase (enhancing mechanical strength);

[0078] 2) A porous ceramic substrate carrier is formed by applying two coatings to the surface of a porous ceramic substrate using a scraping method;

[0079] The coating thickness is 18 μm (the thickness is controlled by the number of immersions), and the pore size retention rate of the porous ceramic substrate after coating is 75% (SEM verification).

[0080] The preparation method of the coating includes: applying activated carbon powder (particle size 1-10μm, specific surface area >1000m²) to the activated carbon powder. 2 The mixture is prepared by adding 2% sodium alginate (binder), 70% deionized water, and 0.5% ammonium polyacrylate (dispersant) to the mixture and mixing them evenly.

[0081] (2) Preparation of ozone monitoring biosensor

[0082] 1) The transgenic *Moss simonii* prepared in Example 1 was rinsed three times with sterile water and then placed on sterile filter paper to absorb surface moisture. It was then cut into 1-2 mm segments with sterile scissors. 5.0 g (corresponding to 100 mL final volume) of the pretreated moss tissue was weighed according to the weight-to-volume ratio (w / v). 0.01 mL of Tween-20 was dissolved in 10 mL of sterile water to prepare a pre-diluted solution. At the same time, stress-free culture medium was prepared as a solvent. The moss tissue was placed in a sterile beaker, 80 mL of stress-free culture medium was added and gently stirred to disperse it. Then, the Tween-20 pre-diluted solution was added and stirring was continued for 1 minute. The volume was adjusted to 100 mL with stress-free culture medium and transferred to a sterile conical flask. The mixture was magnetically stirred at 100 rpm for 5 minutes to ensure uniform mixing and prepare a 5% biomass suspension (containing 0.01% Tween-20 as a cohesive agent). The suspension was ball-milled until the fragments were ≤100 μm.

[0083] 2) The porous ceramic plate carrier prepared above was sterilized by ultraviolet light for 30 minutes (wavelength 254nm);

[0084] 3) Spraying the ball-milled suspension (spraying air pressure: 0.2 MPa, nozzle diameter: 0.3 mm, spraying volume: 0.5 mL / cm²) 2 Apply the curing agent to the porous ceramic substrate and then allow it to stand at 25°C and 90% RH (relative humidity) for 48 hours to cure.

[0085] 4) For the first 3 days after the transgenic moss was implanted (including the curing time), the RH was maintained at 100%, and then reduced by 5% daily to the target humidity of 50% to obtain an ozone monitoring biosensor.

[0086] Example 3

[0087] Example 3 is largely the same as Example 2, except that the porous ceramic plate carrier is different. Otherwise, it is the same as Example 2, and an ozone monitoring biosensor is prepared. The preparation method of the porous ceramic plate carrier in Example 3 includes:

[0088] 3D printing was used to fabricate porous ceramic plate carriers from porous ceramic plate raw materials. The porous ceramic plate raw materials were obtained by mixing 15% activated carbon (by volume), 5% methylcellulose (by mass), and 2% citric acid (by mass) with α-tricalcium phosphate (ceramic substrate). Additionally, the activated carbon was loaded with 0.1% nano-silver (AgNP, 10nm particle size) (preparation: commercial activated carbon (20-40 mesh particle size) was soaked in 1M hydrochloric acid for 24 hours to remove impurities, washed with distilled water until pH 6.8-7.2, vacuum dried at 80℃ for 12 hours, and then ground through a 100-mesh sieve; based on the calculation of "nano-silver accounting for 0.1% of activated carbon mass", 0.01g of silver nitrate was dissolved in 100mL of deionized water to prepare a 0.1g / L Ag... +Add 10g of pretreated activated carbon to the solution, and magnetically stir at 200rpm for 30 minutes at 25℃ to allow Ag to react. + Adsorbed onto the surface of activated carbon, 0.1M sodium borohydride solution was added dropwise at a molar ratio of NaBH4 to AgNO3 of 2:1. The mixture was stirred in the dark for 1 hour until the solution turned light gray (AgNO3). + Restored to Ag 0 After the reaction, the activated carbon was centrifuged and washed three times at 8000 rpm (until no chloride ion residue was found), and then vacuum dried at 60°C for 6 hours to obtain a supported activated carbon with a uniform dispersion of about 10 nm.

[0089] Comparative Example 1

[0090] Comparative Example 1 is largely the same as Example 2. The difference between Comparative Example 1 and Example 2 is that the porous ceramic plate carrier is different. Otherwise, they are the same as in Example 2. An ozone monitoring biosensor was prepared. In Comparative Example 1, the porous ceramic plate carrier is an ordinary ceramic plate (a 1060 type alumina ceramic plate (containing 95% Al2O3, with the remainder being SiO2 and MgO additives), with a size of 50×50×2mm, a smooth surface (no porous structure, porosity <5%), and a thickness error of ±0.1mm.

[0091] Performance testing

[0092] (I) Visual Detection of Transgenic Small Bowl Moss

[0093] This invention quantifies ozone exposure levels using GFP fluorescence signals monitored optically. Specifically, it involves: preparing 5% biomass suspensions (containing 0.01% Tween-20) of both the transgenic *Sphaerocarpus spp.* (constructed in Example 1) stably expressing ZAT12::APX1-2A-GFP-hptII and wild-type *Sphaerocarpus spp.*, uniformly coating them onto porous ceramic plate carriers (transgenic samples) from Example 2 and ordinary ceramic plates (wild-type samples) from Comparative Example 1. After humidity acclimatization (100% RH for the first 3 days, then reduced to 50% RH), carriers with consistent growth were selected for later use. Six ozone concentration gradients were set up (control group 0 ppb, low concentrations 20 ppb and 40 ppb, medium concentrations 60 ppb and 80 ppb, high concentration 100 ppb), with three replicates per group. The samples were placed in a 10L ozone exposure chamber at 25°C and a light intensity of 100 μmol·m⁻¹. -2 ·s -1Under the established conditions, the samples were continuously exposed for 2 hours, while the control group was circulated with the same amount of clean air. Qualitative observation was performed using a Zeiss LSM880 confocal microscope with a 488nm excitation filter and a 510nm emission filter, a fixed shooting distance of 30cm, and an exposure time of 500ms. Quantitative analysis was conducted using a mobile app (such as "Moss Fluorescence Monitor") equipped with the OpenCV algorithm. Results showed that the transgenic *Moss simonii* exhibited weak green fluorescence at 0 ppb (intensity 500-800), significantly enhanced fluorescence at 20 ppb (1200-1500, pale green visible to the naked eye), a significant increase at 40 ppb (2000-2500, uniformly distributed green fluorescence), strong green fluorescence at 60 ppb (3000-3500, covering the entire vector), and extremely strong fluorescence at 80 ppb (4000-4500, clear saturation edges). The fluorescence intensity reached its peak at ppb (5000-5500, without quenching), while all concentration groups of wild-type *Sphagnum moss* showed no green fluorescence (APP detection intensity value ≤300), only turning brown at 80-100 ppb due to chlorophyll degradation; the mobile APP converted fluorescence intensity to concentration using the formula "ozone concentration (ppb) = 0.02 × fluorescence intensity value - 10", with an error ≤±5 ppb within the range of 20-100 ppb. The fluorescence intensity of transgenic samples showed a linear positive correlation with ozone concentration (R0). 2 =0.97), the wild type can only qualitatively reflect the damage caused by high concentrations of ozone and has no quantitative value. This process realizes the visualization and quantification of ozone concentration through the change of fluorescence gradient, which can accurately distinguish the 5ppb concentration difference.

[0094] (II) Performance Testing of Transgenic Small Bowl Moss

[0095] To further verify the performance of the transgenic *Sphaerocera minor*, transgenic *Sphaerocera minor* expressing ZAT12::APX1-2A-GFP-hptII constructed in Example 1 and wild-type *Sphaerocera minor* were used to prepare 5% biomass suspensions (containing 0.01% Tween-20). These suspensions were evenly coated onto the porous ceramic plate carrier (transgenic sample) of Example 2 and the ordinary ceramic plate (wild-type sample) of Comparative Example 1. After humidity acclimatization (100% RH for the first 3 days, then reduced to 50% RH), carriers with consistent growth were selected for later use. The samples were placed in a 10L ozone exposure chamber and exposed to ozone environments of 5 ppb (transgenic group) and 80 ppb (wild-type group), respectively, at 25°C and a light intensity of 100 μmol·m⁻¹. -2 ·s -1 After continuous exposure for 2 hours under certain conditions, fluorescence response tests were performed on transgenic moss under 5 ppb ozone stress and wild-type moss under 80 ppb ozone stress (using a chlorophyll fluorometer (e.g., Walz PAM-2500), with a light intensity of 1 μmol·m⁻¹). -2 ·s -13000 μmol·m saturated pulse light -2 ·s -1 The initial fluorescence (Fo) and maximum fluorescence (Fm) were recorded, and the ratio was calculated using the formula Fv / Fm=(Fm-Fo) / Fm. Measurements were taken every 2 hours for 24 hours. SOD activity was measured (using the xanthine oxidase method: 0.1g of moss tissue was added to 1mL of pre-cooled 50mM phosphate buffer (pH 7.8, containing 0.1mM EDTA), homogenized in an ice bath, and centrifuged at 12000rpm for 10 minutes at 4℃. The supernatant was used as the crude enzyme extract. The reaction system consisted of 2.8mL 50mM phosphate buffer, 0.1mL 130mM methionine, 0.1mL 750μM nitroblue tetrazolium (NBT), and 0.1mL... The mixture consisted of 20 μM riboflavin and 0.1 mL of crude enzyme extract, and reacted at 25 °C in the dark for 20 minutes. The control group used buffer instead of enzyme solution. Absorbance (OD value) was measured at 560 nm. SOD activity was defined as the amount of enzyme required to inhibit 50% of NBT photoreduction, and results were expressed as U / mg protein. H2O2 content was determined using the ammonium molybdate colorimetric method: 0.1 g of moss tissue was added to 1 mL of pre-cooled acetone, ground in an ice bath, centrifuged at 8000 rpm for 10 minutes at 4 °C, and 0.5 mL of the supernatant was collected and then added to 0.1 mL of the solution. 5% ammonium molybdate and 0.2 mL concentrated ammonia were mixed and allowed to stand for 10 minutes. For colorimetric analysis, the OD value was measured at 400 nm, and the content was calculated using a 0-100 μM H2O2 standard curve. The results were expressed as nmol / gFW. The 48-hour survival rate and cell death rate were determined (survival rate was calculated by transferring the moss vector to a stress-free medium after exposure, counting the number of surviving clones after 48 hours (judgment criteria: green color and new leaf growth), and calculating as (surviving clones / total clones) × 100%; cell death rate was calculated by taking moss gametophytes, staining with 0.4% trypan blue solution for 10 minutes, rinsing with distilled water, and counting the proportion of blue dead cells in 100 cells under a 10×40x optical microscope, repeating 3 times and taking the average). New shoot elongation was also determined (the initial length of 30 new shoots was measured with calipers before exposure (accurate to 0.01 mm), and measured daily after exposure, calculating the elongation over 48 hours (final length - initial length), and taking the average as the final result).

[0096] Laboratory preliminary performance test data showed that under 80 ppb ozone stress, the fluorescence response (Fv / Fm ratio) of wild-type moss was only 0.35±0.05, indicating severe damage to the photosynthetic system. The transgenic *Sphaerocarpus spp.* constructed in Example 1 achieved a ratio of 0.68±0.03 under 5 ppb ozone, demonstrating stronger photosynthetic system stability. Regarding antioxidant capacity, the transgenic moss exhibited significantly higher SOD activity than the wild type, and its H2O2 content was only 41% of the wild type, indicating less oxidative damage. In terms of survival and growth, the 48-hour survival rate of the transgenic moss was far higher than that of the wild type, its cell death rate was significantly lower, and its new shoot elongation was five times that of the wild type. The transgenic *Sphaerocarpus spp.* exhibits higher sensitivity and stronger tolerance to low-concentration ozone stress. Its stress response can be reflected through microscopic indicators such as fluorescence and enzyme activity, while the degree of damage can be reflected through macroscopic indicators such as survival rate and growth. It is an ideal material for biomonitoring in low-concentration ozone environments and is suitable for smart cities and extreme environment remediation. The response data of transgenic moss under 5 ppb ozone stress are compared with those of wild-type moss under 80 ppb ozone stress, as shown in Table 1 below.

[0097] Table 1. Response data of transgenic *Sphaerocarpus spp.* under 5 ppb ozone stress and data of wild-type *Sphaerocarpus spp.* under 80 ppb ozone stress.

[0098]

[0099]

[0100] In addition, this invention also tested the resistance of the transgenic moss constructed in Example 1 to interference factors such as SO2 and NOx. A 5% biomass suspension (containing 0.01% Tween-20) of the transgenic *Sphaerocarpus spp.* stably expressing ZAT12::APX1-2A-GFP-hptII constructed in Example 1 was prepared and uniformly coated onto the porous ceramic plate carrier (transgenic sample) of Example 2. After humidity acclimatization, the sample was placed in gaseous environments polluted by 5 ppb O3, 50 ppb SO2, 100 ppb NO2, and a mixture of O3 and SO2, respectively, at 25°C and a light intensity of 100 μmol·m⁻¹. -2 ·s -1 After 4 hours of continuous exposure under the specified conditions, fluorescence response tests and 48-hour survival rate determinations were performed (using the same methods as above). Statistical analysis was performed using SPSS 26.0 with one-way ANOVA (Dunnett-t test compared with the pure O3 group; p < 0.05 was marked as *, p < 0.01 as **). Specificity tests were performed by GC-MS analysis to exclude cross-reactions from other factors (such as SO2 and NOx). Finally, stable lines were propagated, and field fixation substrates were developed. The test results are shown in Table 2 below.

[0101] Table 2. Response data and specific defect analysis under various stresses.

[0102]

[0103] (*p<0.05 compared to the pure O3 group, **p<0.01; test temperature 25℃, RH 60%)

[0104] (III) Water retention rate test of porous ceramic plate carrier

[0105] The water retention rate test was only conducted on porous ceramic plate carriers without transgenic moss (excluding the interference of biomass on water content). The specific procedure was as follows: 50mm × 50mm × 5mm carriers from Examples 2, 3, and Comparative Example 1 were taken, ultrasonically cleaned with deionized water, and dried at 105℃ to constant weight (recorded as m0). Then, 500 "wet-dry cycles" were performed (soaking at 25℃ for 24 hours, draining, and weighing the absorbed water m1; drying at 40℃ for 12 hours, and weighing the dry water m2). The initial water retention rate refers to the water retention capacity of the first cycle (according to...). (Calculations) show that Example 2 had an initial water retention rate of 90%, and Example 3 had an initial water retention rate of 88%. After 500 cycles, the water retention rates of Examples 2 and 3 were 73.8% and 72.16%, respectively, still greater than the initial value of 82%. In contrast, Comparative Example 1 had an initial water retention rate of 55%, and after cycling, the water retention rate was 24.75%, retaining only 45%. The difference is due to the porous structure of Examples 2 and 3 maintaining stable water absorption channels, while the dense structure of Comparative Example 1 is prone to cracking after cycling.

[0106] (iv) Performance testing of ozone monitoring biosensors

[0107] The attachment efficiency of the transgenic *Moss simonii* and the respiration rate of the ozone monitoring biosensor constructed in Examples 2 and 3 were tested, respectively. Details are as follows:

[0108] (1) Adhesion efficiency test

[0109] Five 10mm × 10mm samples were taken from each of the sensors used in Example 2 and Example 3 to ensure consistent moss growth on the sample surfaces. The samples were stained in the dark using 0.1% Calcofluor White staining solution (dissolved in pH 7.2 PBS buffer). After 15 minutes, the samples were gently rinsed three times with distilled water to avoid dispersing the rhizoid structure and to retain only the specifically bound staining signal. The samples were observed using a Zeiss LSM880 confocal microscope with excitation light set to 350nm and emission light to 450nm, focusing on the 10-50μm micropore region to clearly capture the distribution morphology of the rhizoids within the pores. Ten fields of view were randomly selected from each group, and the proportion of micropore area covered by rhizoids to the total pore area was calculated using ImageJ software. The data showed that the rhizoid coverage rate in Example 2 was 68% ± 5%, and in Example 3 it reached 72% ± 4%, both exceeding the 60% threshold.

[0110] (2) Respiratory rate detection

[0111] The sensors from Examples 2 and 3 were placed in 50 mL sealed chambers, each fitted with a Hansatech Oxygraph+ oxygen electrode. Uninoculated moss-free plates were used as a control group. Tests were conducted in a dark environment at 25°C to avoid interference from photosynthesis on oxygen consumption and to ensure that only respiration was monitored. During monitoring, the O2 consumption rate in the chamber was recorded in real time (unit: μmol O2·h⁻¹·cm⁻¹). -2 The ratio of O2 consumption rate after moss coverage to bare plate ventilation rate (cavity oxygen diffusion rate) was calculated to reflect the effect of carrier ventilation efficiency on moss respiration. The O2 consumption rate maintenance rate in Example 2 was 85% ± 3%, and in Example 3 it was 82% ± 4%, both meeting the standard of ≥80% bare plate ventilation rate.

[0112] (VI) Urban Applications

[0113] A LoRa sensor can be powered by moss secretions (such as glucose) to cover an urban grid: The ozone monitoring biosensor of Example 2 / Example 3 integrates a microbial fuel cell (MFC) at the bottom, utilizing glucose secreted by moss (0.1-0.3 mg / cm³ per day) through a carbon cloth anode (loaded with Shewanella oneidensis) and a Pt / C cathode. 2It generates electricity (0.6-0.8V) to power the LoRa module (standby current ≤5μA), and is equipped with a glucose sensor and a 1F supercapacitor to ensure continuous operation. It is deployed in green belts, rooftops and other areas at a density of 20 5cm×5cm units per square meter. It is built in a star topology and uploads data to the cloud platform every 30 minutes through the LoRa gateway (covering 1-3km). The unit spacing is 22cm and a 5mm gap is reserved to prevent water accumulation. During operation and maintenance, water is replenished weekly (5mL / unit). It can be self-sustaining during the rainy season. The MFC anode is replaced every 6 months. The platform generates an ozone heat map through AI processing to support grid-based management and control.

[0114] The physiological state of moss (such as chlorophyll fluorescence) can also be used to reflect the heat island effect, enabling multi-parameter monitoring: using the ozone monitoring biosensor of Example 3, a chlorophyll fluorescence probe, an NTC temperature sensor (±0.5℃), and an SHT30 temperature and humidity sensor are integrated at the edge of the carrier. Relying on the LoRa star network of Scheme 1, the Fv / Fm ratio, carrier temperature, and environmental humidity data are uploaded synchronously every hour; urban deployment selects typical areas such as commercial areas and parks, with 3 monitoring points in each area, and 4 units arranged in a 2m×2m grid at each point, covering hard paved areas, green areas, and transition areas. Fluorescence detection is performed once daily at 9:00, 14:00, and 20:00. Temperature and humidity are averaged every 10 minutes. The heat island effect is quantified using the ternary equation "heat island intensity (°C) = 0.8 × (carrier temperature - suburban control temperature) - 0.3 × (Fv / Fm) + 0.05 × (humidity deviation)", and divided into three levels: mild (1-2°C, Fv / Fm 0.6-0.7), moderate (2-4°C, 0.5-0.6), and severe (>4°C, <0.5). A "heat island-ozone overlay map" is generated using the city cloud platform. An early warning is issued when the heat island intensity is >3°C and ozone is >80ppb. Water replenishment is performed in the same manner. The probe is calibrated every 3 months using a standard fluorescent plate (Fv / Fm = 0.83) (error ≤ ±0.02). This achieves coordinated governance of "pollution and thermal environment".

[0115] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not limiting. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the purpose and scope of the technical solutions of the present invention, which should all be covered by the scope of the claims of the present invention.

Claims

1. A genetically modified moss, characterized in that, It is obtained by transfecting the target gene into moss cells. The target gene includes the ZAT12 promoter, and the sequence of the ZAT12 gene is shown in SEQ ID NO:

1.

2. The transgenic moss according to claim 1, characterized in that, The target gene also includes one or more of the antioxidant enhancer APX1, the antioxidant enhancer SOD, or a reporter gene; wherein the sequence of the antioxidant enhancer APX1 is shown in SEQ ID NO: 2, and the sequence of the antioxidant enhancer SOD is shown in SEQ ID NO:

3.

3. The transgenic moss according to claim 1 or 2, characterized in that, The moss is *Moss styracifolium*.

4. An ozone monitoring biosensor, characterized in that, Including the genetically modified moss as described in any one of claims 1 to 3.

5. The ozone monitoring biosensor according to claim 4, characterized in that, The ozone monitoring biosensor also includes a carrier and a water-absorbing component. The transgenic moss is attached to the carrier, and the water-absorbing component is located on the side where the transgenic moss is not attached.

6. The ozone monitoring biosensor according to claim 5, characterized in that, The absorbent component is a water-retaining cotton.

7. The ozone monitoring biosensor according to any one of claims 4 to 6, characterized in that, The method for preparing the carrier is selected from any of the following: (a) Using 3D printing to assemble cordierite (Mg2Al4Si5O) 18 The substrate is printed into a porous ceramic substrate and then sintered to obtain a carrier. (b) Using 3D printing to fabricate a carrier from a porous ceramic plate raw material, wherein the porous ceramic plate raw material is obtained by mixing 10%-20% by volume of activated carbon, 3%-7% by mass of methylcellulose and 1%-3% by mass of α-tricalcium phosphate.

8. The ozone monitoring biosensor according to claim 7, characterized in that, In carrier (a), the porous ceramic substrate has a pore size of 10 μm-50 μm and a porosity of 40%-60%; and / or In carrier (a), a coating is formed by coating the surface of the carrier with a paint, which is obtained by mixing activated carbon with 1%-3% by mass of binder, 60%-80% by mass of deionized water and 0.3%-0.7% by mass of dispersant; and / or In carrier (b), activated carbon is loaded with 0.05%-0.15% nano-silver by mass.

9. The method for preparing the ozone monitoring biosensor according to any one of claims 4 to 8, characterized in that, Preparation methods include: (1) Genetically modified moss was prepared into a suspension containing an adhesive agent; (2) Ball mill the suspension to break it down to fragments ≤100μm; (3) Spray the suspension after ball milling onto a carrier equipped with a water-absorbing component and let it stand to solidify to obtain an ozone monitoring biosensor.

10. The application of the genetically modified moss according to any one of claims 1 to 3 or the ozone monitoring biosensor according to any one of claims 4 to 8 in the detection of ozone.

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