Application of Physcomitrella patens aux mutants as an indicator of cadmium hypersensitivity

Abstract

The application discloses Physcomitrella patens aux The application discloses a mutant of Physcomitrella patens aux The mutant is knocked out of at least one gene relative to the wild type PpAUX The application discloses a mutant of Physcomitrella patens aux The mutant is significantly hypersensitive to cadmium stress, especially in the aspects of the regeneration of rhizoids and the proliferation of gametophytes, and the mutant has stronger response difference than the wild type, and can be applied to the fields of rapid detection of cadmium pollution in soil / water, environmental risk early warning and biological monitoring, and has the advantages of high sensitivity, stable response, strong visualization, simple operation and extremely low cost.

Description

Technical Field

[0001] This invention relates to the field of cadmium ultrasensitive detection technology, specifically to *Moss sclerotium*. aux Application of mutants as cadmium-sensitive indicator materials. Background Technology

[0002] Cadmium (Cd) is a highly toxic, easily migrating, and bioaccumulative heavy metal pollutant. Long-term exposure to low concentrations can cause serious harm to ecosystems and human health. Traditional cadmium detection relies on instrumental methods such as atomic absorption spectrometry and ICP-MS, which are accurate but have limitations such as complex pretreatment, long detection cycles, expensive instruments, and difficulty in rapid on-site screening.

[0003] Bioindicator methods can directly reflect the bioavailability and toxic effects of pollutants. Among them, bryophyte detection is a highly regarded environmental monitoring method. Its basic principle is that, compared to higher plants, bryophytes, due to their lower evolutionary level, have a lower resistance to environmental toxicity. Under environmental pollution, they can rapidly exhibit a series of significant symptoms and changes in growth indicators. These changes can be used to comprehensively evaluate the degree of environmental pollution. This characteristic of bryophytes can be utilized for the detection of heavy metal pollution in the atmosphere, soil, and water. However, this method faces the following practical problems in cadmium pollution detection: bryophytes in nature have developed certain adaptations to polluted environments during their long-term growth. Relevant case studies show that directly using them for cadmium detection is ineffective, especially for low concentrations of cadmium.

[0004] Therefore, the development of cadmium-sensitive bioindicator materials is of great significance for the rapid detection of low-concentration cadmium pollution in soil and water. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention provides a small-sized *Moss simonii*. aux The application of mutants as cadmium-sensitive indicator materials aims to address the shortcomings of existing bryophyte detection methods in the ultrasensitive detection of cadmium pollution.

[0006] The present invention specifically adopts the following technical solution: In a first aspect, the present invention provides a type of *Sphaerocarpus spp.* that can be used as a cadmium-sensitive indicator material. aux The mutant, *Moss simonii* aux The mutant had at least one knockout compared to the wild type. PpAUX Genes, among which PpAUX Genes are selected PpAUX1 , PpAUX2 , PpAUX3 , PpAUX4, PpAUX5 and PpAUX6 .

[0007] Preferred, small bowl moss aux The mutant strain had its gene knocked out compared to the wild-type *Sclerotium spp.* PpAUX1, PpAUX3 or PpAUX4 Specifically, the aux Mutants are selected from PpAUX1 Single gene knockout mutant (i.e.) aux1 ), PpAUX3 Single gene knockout mutant (i.e.) aux3 )and PpAUX4 Single gene knockout mutant (i.e.) aux4 ).

[0008] As a model moss, *Sclerotium simonii* is easy to cultivate and its phenotype is easy to observe, making it an ideal environmental indicator organism. AUX1 / LAX The family gene encodes an auxin influx carrier that regulates plant root development and stress response; however, there is currently no indication of its application in *Moss sphaerocephala*. aux Patents and literature reports related to the use of mutants for ultrasensitive detection of cadmium pollution. This invention is the first experimental discovery of *Moss styrax*. aux Mutants, especially aux1, aux3 or aux4 Single-gene deletion mutants are significantly hypersensitive to cadmium stress, especially in rhizoid regeneration and gametophyte proliferation, showing a stronger difference in response than wild-type mutants, and can be used as hypersensitive indicator materials for cadmium pollution.

[0009] In the above applications, *Moss styrax* aux The mutant, as a cadmium-sensitive indicator material, can rapidly detect low concentrations of cadmium in soil and water bodies (such as irrigation water), and can also be used in scenarios such as environmental risk early warning, biological monitoring, and pollution ecological risk assessment.

[0010] Secondly, the present invention provides a method utilizing the aforementioned *Sclerotium simonii*. aux A rapid detection method for cadmium pollution in mutants.

[0011] The above-mentioned rapid detection method for cadmium pollution may specifically include the following steps: separating wild-type and... aux The mutants were cultured in the soil or water body to be tested, or in a culture medium prepared with leachate from the water or soil body to be tested, and the wild type and mutants were observed and compared. aux The number of rhizoids, spreading area, and number of newly formed gametophytes in the mutant, and under cadmium pollution aux The mutant had fewer rhizoids, a smaller spreading area, and fewer newly formed gametophytes than the wild type. The term "spreading area" in this invention specifically refers to the spreading and colonization area of ​​the protonemata-rhizoid complex.

[0012] Preferably, in the above-mentioned rapid detection method for cadmium pollution, the *Sphaerocarpus spp.* is used. aux The mutant had the gene knocked out compared to the wild type. PpAUX1, PpAUX3 orPpAUX4 .

[0013] Preferably, in the above-mentioned rapid detection method for cadmium pollution, the concentration of cadmium ions in the soil / water body or the culture medium prepared from its leachate is 50 nmol / L - 5000 nmol / L.

[0014] Thirdly, the present invention provides a cadmium detection kit, which includes *Moss spp.* aux Mutant, and this small bowl-shaped moss aux The mutant had at least one knockout compared to the wild type. PpAUX Genes, among which aux Genes include PpAUX1 , PpAUX2 , PpAUX3 , PpAUX4, PpAUX5 and PpAUX6 .

[0015] Preferably, in the above-mentioned cadmium detection kit, *Moss styrax* is included. aux The mutant had the gene knocked out compared to the wild type. PpAUX1, PpAUX3 or PpAUX4 .

[0016] In this invention, *Sclerotium spp.* aux The construction of mutants can refer to existing technologies, as long as the target gene can be specifically knocked out; moreover, the term "knockout" in this invention refers to complete gene knockout or only knocking down / reducing gene expression levels. For example, in some embodiments of this invention, a targeted knockout vector was constructed using homologous recombination, and combined with *Symplocos spicata* protoplast transformation technology, *Symplocos spicata* was successfully obtained. aux Mutant materials aux1 , aux3 and aux4 .

[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: This invention discloses for the first time the small-bowl moss PpAUX The application of gene knockout mutants as cadmium-sensitive bioindicators. Furthermore, experiments in this invention demonstrate that under cadmium chloride stress of 50 nmol / L - 5000 nmol / L, aux1 , aux3 and aux4 The number of rhizoids regenerated, the spreading area, and the number of newly formed gametophytes in the gene deletion mutant were significantly lower than those in the wild type, and it exhibited a specific, dose-dependent hypersensitive phenotype to cadmium stress.

[0018] Small bowl moss auxThe mutant is used for cadmium biomonitoring and has advantages such as high sensitivity, stable response, strong visualization, simple operation and extremely low cost. It can achieve screening and semi-quantitative determination of ultra-low concentration cadmium pollution and is suitable for detection of environmental samples such as farmland soil, surface water and farmland irrigation water. Attached Figure Description

[0019] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0020] Figure 1 The small bowl moss in Example 1 PpAUX Visualization and analysis of gene and protein domain structures; Figure 2 Sequence alignment of the protein domains of PpAUX from *Sclerotium simonii* and AtAUX1 from *Arabidopsis thaliana* in Example 1; Figure 3 The pTN182-PpAUX vector spectrum in Example 2; Figure 4 The mutant of *Scleroderma purpurea* in Example 2 aux1 , aux3 and aux4 Design of identification primers; Figure 5 The mutant of *Scleroderma purpurea* in Example 2 aux1 , aux4 DNA and RNA level identification results; Figure 6 The mutant of *Scleroderma purpurea* in Example 2 aux3 DNA level identification results; Figure 7 For example, the small bowl moss WT in Example 3, aux1 , aux4 Phenotypic comparison of materials; Figure 8 The small lichen WT that grew for 3 weeks under normal conditions in Example 3. aux1 , aux4 Comparison of rhizoid phenotypes in materials; Figure 9 The small lichen WT that grew for 2 weeks under normal conditions in Example 3. aux1 , aux4 Phenotypic comparison of the number of newly formed gametophytes in materials; Figure 10 The different concentrations of CdCl2 used to treat *Moss sphaerocephala* WT in Example 4 aux1 , aux3 , aux4Comparison of the number of false roots in the materials; Figure 11 The different concentrations of CdCl2 used to treat *Moss sphaerocephala* WT in Example 4 aux1 , aux3 , aux4 Comparison of the root structure and spreading area of ​​the materials; Figure 12 The different concentrations of CdCl2 used to treat *Moss sphaerocephala* WT in Example 4 aux1 , aux3 , aux4 Comparison of the number of newly formed gametes in the materials. Detailed Implementation

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention.

[0022] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0023] Example 1 This study identified and analyzed members of the PpAUX moss species *Phyllostachys pubescens*. The specific results are as follows: The amino acid sequences of four AUX1 / LAX proteins from Arabidopsis thaliana were used as input sequences and analyzed in the Phytozome database (https: / / phytozome-next.jgi.doe.gov / ). The results showed that eight proteins from *Phyllostachys spp.* shared high homology with *Arabidopsis thaliana* AtAUX1, but after screening, only six proteins, like the *Arabidopsis thaliana* AUX1 / LAX members, possessed the PLN03074 transmembrane amino acid transporter domain. These six *Phyllostachys spp.* AUX family members were subsequently named PpAUX1 (Pp3c15_640), PpAUX2 (Pp3c22_23050), PpAUX3 (Pp3c3_9870), PpAUX4 (Pp3c6_27190), PpAUX5 (Pp3c9_3590), and PpAUX6 (Pp3c9_620).

[0024] Further domain analysis of these six PpAUX members of *Sclerotium spp.* revealed that all six proteins contain only the cl00456 domain, belonging to the SLC5-6-like_sbd superfamily. Figure 1Protein sequence alignment of Arabidopsis thaliana AtAUX1 and PpAUX1-6 in *Phyllostachys pubescens* was performed using the CLUSTALW online software. The results showed high homology among members of the PpAUX family in *Phyllostachys pubescens*, suggesting potential functional similarities among PpAUX family members. Figure 2 ).

[0025] Example 2 This example utilizes the principle of homologous recombination to provide a method for constructing *Moss spp.* aux The method for mutants includes the following steps: (1) Construction of the knockout vector.

[0026] Querying the Phytozome database PpAUX1 (Pp3c15_640) PpAUX3 (Pp3c3_9870) PpAUX4 The gene sequence (Pp3c6_27190) was used to construct knockout vectors pTN182-PpAUX1, pTN182-PpAUX3, and pTN182-PpAUX4, respectively. The vector maps are shown below. Figure 3 As shown. The above knockout vectors were obtained through the following operations: ① Vector linearization: The vector was digested with restriction endonuclease at 37℃ for 3 h. The enzyme digestion system used is shown in Table 1. After the enzyme digestion, the vector was inactivated at 65℃ for 15-20 min and the linearized vector was recovered.

[0027] Table 1 Enzyme digestion reaction system

[0028] The restriction endonucleases used to construct the pTN182-PpAUXs vector were EcoRI and NdeRI.

[0029] ② Amplification of the target fragment: Prepare the PCR reaction system as shown in Table 2, and perform amplification according to the procedure shown in Table 3 to obtain the target fragment; in Table 3, T1 is the annealing temperature of the short primer, T2 is the annealing temperature of the long primer, and T0 is the temperature increase in each cycle (T0 = (T2-T1) / 10). After the reaction, spot the sample, perform electrophoresis at 140 V for 13~18 min, cut off the gel containing the target size fragment, recover the fragment, and purify the product.

[0030] Table 2 PCR reaction system

[0031] Table 3 PCR amplification program

[0032] Table 4 Primers used for two PpAUX amplifications

[0033] ③ Seamless cloning: The linearized vector obtained in step ① is ligated with the target fragment obtained in step ②. The reaction is carried out in a water bath (50℃, 1 hour). After the reaction, the resulting ligation product is stored at -20℃.

[0034] ④ Heat shock transformation of Escherichia coli strain DH5α: Place DH5α competent cells on ice and wait for them to thaw completely; take out the ligation product obtained in step ③ and thaw it on ice; transfer 10~50 µL of competent cells to a 1.5 mL centrifuge tube, add an appropriate amount of ligation product, and gently mix with a pipette tip; let stand in an ice bath for 20~30 minutes, then heat shock in a 42°C water bath for 1 minute (without shaking), and finally let stand in an ice bath for 2 minutes; add 500 µL of antibiotic-free liquid LB medium and incubate at 37°C at 200 rpm for 1 hour; spread the bacterial solution on solid LB medium containing antibiotics and incubate upside down at 37°C for 12~15 hours.

[0035] ⑤ Identification of positive clones by bacterial culture PCR and sequencing: Single clones were picked from 300-500 µL of liquid LB medium containing antibiotics and cultured at 37℃ and 200 rpm until the culture became turbid; 10 µL of PCR reaction system was prepared according to Table 5, and the reaction procedure was executed according to Table 7. After the reaction, an electrophoresis gel was prepared and the sample was loaded; bacterial cultures with the correct band size were sent to a sequencing company for sequencing; finally, plasmids were extracted from the bacterial culture samples with correct sequencing.

[0036] Table 5 Colony PCR Reaction System

[0037] Table 6 Primers used for colony PCR

[0038] Table 7 Colony PCR Reaction Procedure

[0039] (2) Small bowl moss aux Construction of mutants.

[0040] Protoplast transformation of *Symplocos macrantha*: All *Symplocos macrantha* genetic material was obtained by PEG-mediated protoplast transformation (aseptic technique). This is an existing technique and can be performed according to the reference (doi: 10.1111 / pce.15572).

[0041] like Figure 4 As shown, based on the above operations, this example yielded a total of 3 species of *Moss sphaerocephala*. auxmutant materials, i.e. aux1, aux4 and aux3 .

[0042] (3) aux Identification of mutant materials.

[0043] To verify whether the targeted knockout of the target gene has been successfully integrated into the genome, corresponding identification primers were designed based on the knockout principles of each mutant (see...). Figure 4 (as shown), thus utilizing PCR to investigate wild-type WT and mutant WT. aux The genomic DNA was used for verification; simultaneously, its cDNA was used as a template to detect the RNA expression level of the gene by RT-PCR. The PCR and RT-PCR amplification systems are shown in Table 8, and the primers used are shown in Table 9.

[0044] Table 8 Reaction System

[0045] Table 9 Primer sequences used for positive identification

[0046] right aux1 , aux4 DNA and RNA levels were identified, and the test results were as follows: Figure 5 As shown: both the P1+P2 primer pair and the P3+P4 primer pair yielded positive bands, indicating the presence of the 35S promoter. Neo The R resistance gene and the Term termination sequence were completely inserted into the target position; the expression of the transcript was detected using the P9+P10 primer pair, and it was found that... aux1 in materials PpAUX1 The transcripts completely disappeared; aux4 The material was also found to be completely lacking in transcripts upon testing.

[0047] right aux3 DNA level identification was performed, and the test results were as follows: Figure 6 As shown, positive bands were obtained in all cases. This result indicates that... aux3 The materials were tested and the construction was successful.

[0048] Example 3 Using the wild type as a control, this case tested *Moss sclerotium*. aux The growth performance of the mutant, along with specific experiments and results, is as follows: Gametophyte gametophyte WT and mutant aux1 , aux4 Planted in substrate blocks and cultured at 22℃ with 16 hours of light for approximately 45 days, the plants were observed under a microscope and found to show improvements compared to the wild type. aux1 , aux4There was no significant difference in the growth and development status of the mutant materials. Figure 7 ).

[0049] Polishing the wild type and mutant of *Scleroderma purpurea* separately aux The gametophytes were cultured in a 25°C incubator for 14 days, then polished a second time and cultured again at 25°C until new gametophytes emerged. To observe the growth of the rhizoids, after the culture medium solidified, half of each plant was surgically removed. Single new gametophytes from mutant and wild-type materials with similar growth were selected and transferred to culture medium separately. They were then cultured upright in a 25°C incubator, and the growth of their rhizoids was observed and recorded after 3 weeks. We found that under normal conditions, compared with the wild type, aux1 , aux4 There was no significant difference in rhizoid growth between the mutant materials. Figure 8 ).

[0050] Further analysis using a flat placement method quantified the number of newly formed gametophytes after 2 weeks, revealing that under normal circumstances... aux1 , aux4 The mutant material showed no significant difference from the wild type. Figure 9 ).

[0051] Example 4 This example verifies the application of the *Sclerotium spp.* mutant as a cadmium hypersensitive indicator material. Specific experiments and results are as follows: Wild-type and mutant of *Sphagnum moss* were taken respectively. aux1 , aux3 , aux4 Uniformly growing single-plant gametophyte materials were obtained, roots were removed, and they were placed on BCD medium with a concentration of 50 nmol / L or 5000 nmol / L CdCl2 for stress treatment. After the rootless gametophytes were cultured vertically for 14 days, the number of rhizoids, spreading area and number of new gametophytes of each material were observed.

[0052] The number of rhizoids was calculated as follows: The rhizoids newly emerging from each rootless gametophyte were directly counted under a stereomicroscope. Rhizoids longer than 0.5 mm that newly grew from the base of the gametophyte were used as the standard for countable rhizoids. The total number of rhizoids for each gametophyte was recorded.

[0053] The method for calculating the spreading area is as follows: A complete image of the protonema and newly formed rhizoids at the base of the gametophyte is taken using a stereomicroscope. The distribution range of the entire rhizoid system on the culture medium surface is manually outlined using ImageJ software, and the spreading area (mm²) is calculated after calibration with a scale.

[0054] The number of newly formed gametophytes was calculated by directly counting the total number of newly formed gametophytes in each sector region under a stereomicroscope or through images. The criteria for identifying newly formed gametophytes were: small plantlets that sprouted from the base of the original inoculated gametophytes or the surrounding culture medium, had independent pseudo-stem-leaf structures, and had a diameter greater than 0.5 mm.

[0055] Test results as follows Figures 10-12 As shown. Figure 10 To compare the number of rhizoids in various materials under different concentrations of CdCl2 stress treatment, from Figure 10 It can be seen that at different concentrations, aux1 , aux3 , aux4 The number of false roots is significantly reduced compared to WT, indicating that the present invention... aux The mutant is sensitive to low concentrations (5000 nmol / L) or even very low concentrations (50 nmol / L) of cadmium stress, which can be indicated and observed by the number of rhizoids. Figure 11 To compare the rhizoids and spreading area of ​​various materials under different concentrations of CdCl2 stress treatment, from Figure 11 It can be seen that under low concentration cadmium stress (5000 nmol / L), aux1 , aux3 , aux4 The rhizoids and spreading area are significantly reduced compared to WT, indicating that the present invention... aux The mutant is sensitive to low concentrations (5000 nmol / L) of cadmium stress, which can be indicated and observed by rhizoids and spreading area; however, this indicator is not effective for extremely low concentrations of cadmium stress (50 nmol / L). Figure 12 To compare the number of newly formed gametes in various materials under CdCl2 stress treatment, from... Figure 12 It can be seen that when the cadmium stress treatment concentration of 500 nmol / L is selected within the indicated range (50 nmol / L-5000 nmol / L), it has a positive effect on wild-type and... aux When mutants are treated, aux1 , aux3 , aux4 The number of newly generated gametes was significantly reduced compared to WT, indicating that the present invention... aux The mutant is sensitive to cadmium stress, and this can be indicated and observed by increasing the number of gametophytes.

[0056] In conclusion, *Moss styrax* PpAUX1 , PpAUX3 , PpAUX4 Single-gene deletion mutants are significantly hypersensitive to cadmium stress, especially in rhizoid regeneration and gametophyte proliferation, showing a stronger difference in response than wild-type mutants, and can be used as hypersensitive indicator materials for cadmium pollution.

[0057] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. Small-bowl moss aux Application of mutants as cadmium-sensitive indicator materials, including *Sphagnum moss*. aux The mutant had at least one knockout compared to the wild type. PpAUX Gene.

2. The application according to claim 1, characterized in that, The small bowl moss aux The mutant had the gene knocked out compared to the wild type. PpAUX1, PpAUX3 or PpAUX4 .

3. The application according to claim 1, characterized in that, Small bowl moss aux The mutant can be used as a highly sensitive indicator for the rapid detection of cadmium in soil or water.

4. The application according to claim 3, characterized in that, By comparing small bowl moss aux Cadmium detection was achieved by differentiating mutants from wild-types in terms of the number of rhizoids, spreading area, and / or the number of newly formed gametophytes.

5. A rapid detection method for cadmium pollution, characterized in that, Using small bowl moss aux The mutant was tested, and the *Moss simonii* was also tested. aux The mutant had the gene knocked out compared to the wild type. PpAUX1, PpAUX3 or PpAUX4 .

6. The method according to claim 5, characterized in that, Includes the following steps: Wild-type and small-bowl moss were respectively divided into aux The mutants were planted in the soil or water body to be tested, or wild-type and wild-type *Sphaerocarpus spp.* were respectively used. aux The mutants were cultured in a medium prepared with leachate from the water and soil samples to be tested, and the wild type and mutants were observed and compared. aux The number of rhizoids, spreading area, and number of newly formed gametophytes in the mutant, and under cadmium pollution aux The mutant had fewer rhizoids, a smaller spreading area, and fewer newly formed gametophytes than the wild type.

7. The method according to claim 6, characterized in that, In the soil, water, or culture medium, the concentration of cadmium ions is 50 nmol / L - 5000 nmol / L.

8. The method according to claim 5, characterized in that, Using homologous recombination to respectively PpAUX1, PpAUX3 or PpAUX4 Single-gene knockout was performed to obtain *Pseudomonas aeruginosa*. aux Mutant.

9. A cadmium detection kit, characterized in that, Including small bowl moss aux Mutant, and the small bowl-shaped moss aux The mutant had at least one knockout compared to the wild type. PpAUX Gene.

10. The cadmium detection kit according to claim 9, characterized in that, The small bowl moss aux The mutant had the gene knocked out compared to the wild type. PpAUX1, PpAUX3 or PpAUX4 .

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