Promoter of mentha citronellol synthase gene McLS and application thereof

Abstract

The application discloses a promoter of a mentha citral synthase gene McLS and application thereof, and belongs to the technical field of plant genetic engineering. The nucleotide sequence of the promoter of the mentha citral synthase gene McLS is shown in SEQ ID NO. 1. The application takes a mint leaf as a material, clones the promoter of the mentha citral synthase gene McLS, constructs a plant expression vector pMcLS::GUS on the basis, and transplants the plant expression vector into tobacco seeds to cultivate and screen a transgenic tobacco plant with increased GUS enzyme activity in young leaves. GUS staining experiments show that the McLS promoter has a starting activity in short-stalk glandular hairs of young leaves and stems of the transgenic tobacco, does not start expression in long-stalk glandular hairs, and also has a starting activity in guard cells of stomata of the young leaves. After ABA stress, the GUS enzyme activity in the transgenic tobacco leaves is significantly increased.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, and more specifically, relates to the promoter of the peppermint limonene synthase gene McLS and its application. Background Technology

[0002] Limonene is a monocyclic monoterpene found abundantly in citrus, mint, and pine trees. It has three isomers: D-limonene, L-limonene, and DL-limonene. Limonene is a major fragrance component in plant essential oils and also possesses significant biological activities, such as anti-inflammatory, anticancer, antidiabetic, antigenotoxic, and immunomodulatory effects. In mint, limonene is a precursor in the synthesis of volatile substances. It is formed by the cyclization of GPP catalyzed by limonene synthase. This reaction determines the overall reaction rate of the mint monoterpene synthesis pathway; therefore, limonene synthase is a key rate-limiting enzyme in the synthesis of limonene and other mint monoterpenes. Limonene synthase is a typical monocyclizing enzyme in angiosperms, with a molecular weight of approximately 70 kDa, a pI value of approximately 5.3, and an optimal reaction pH of around 7.0. Limonene synthase genes have been isolated and identified from various plants, but the limonene promoter sequence in mint has not yet been reported.

[0003] A promoter, located upstream of the coding region of a gene, is a specific DNA sequence that participates in transcription initiation and regulation by recognizing and binding to RNA polymerase (RNApol). Based on the type of RNA polymerase it recognizes, eukaryotic promoters are mainly classified into RNApol I, RNApol II, and RNApol III promoters. RNApol I promoters primarily participate in the transcription of rRNA precursor genes; RNApol III promoters typically participate in the transcription of 5S rRNA, tRNA, and a small portion of miRNAs; while RNApol II promoters participate in the transcriptional regulation of many protein-coding genes, as well as mRNA and most miRNAs. Based on their expression type, RNApol II promoters can be classified as constitutive, tissue-specific, and inducible promoters. Although CaMV35S and Ubiquitin promoters have been widely used in the genetic transformation of dicotyledons and monocotyledons, as constitutive promoters, they drive strong expression of exogenous genes in all developmental stages and various tissues and organs of plants, leading to excessive energy consumption and stunted plant growth and development. To eliminate the strong expression of exogenous genes in all tissues and ensure their expression only in specific tissues of transgenic plants, a suitable promoter is needed. Organ-specific promoters, also known as tissue-specific promoters, are specific promoters that drive the transcriptional expression of target genes in specific tissues, organs, or developmental stages of plants, avoiding unnecessary expression of exogenous genes and thus conserving the overall energy consumption of the plant. Summary of the Invention

[0004] In view of the above-mentioned problems existing in the prior art, the technical problem to be solved by the present invention is to provide a promoter for the peppermint limonene synthase gene McLS for genetic improvement breeding; another technical problem to be solved by the present invention is to provide the application of the promoter for the peppermint limonene synthase gene McLS for genetic improvement breeding.

[0005] To solve the above-mentioned technical problems, the technical solution adopted by the present invention is as follows:

[0006] The promoter of the peppermint limonene synthase gene McLS has the nucleotide sequence shown in SEQ ID NO.1.

[0007] The application of the promoter of the peppermint limonene synthase gene McLS in promoting increased GUS enzyme activity in young tobacco leaves, wherein the nucleotide sequence of the promoter of the peppermint limonene synthase gene McLS is shown in SEQ ID NO.1, includes the following steps:

[0008] (1) Construct a vector for the promoter of the peppermint limonene synthase gene McLS;

[0009] (2) The vector of the promoter of the constructed peppermint limonene synthase gene McLS was transformed into tobacco seeds.

[0010] (3) Transgenic tobacco plants with increased GUS enzyme activity in young leaves were obtained through cultivation and screening.

[0011] The vector is a plant expression vector.

[0012] The plant expression vector is pMcLS::GUS.

[0013] The application of the promoter of the peppermint limonene synthase gene McLS in the expression of the GUS gene in the short-stalked glandular hairs of young tobacco leaves, the nucleotide sequence of the promoter of the peppermint limonene synthase gene McLS is shown in SEQ ID NO.1.

[0014] The application of the promoter of the peppermint limonene synthase gene McLS in the expression of the GUS gene in the short stalk glandular hairs of tobacco stem, the nucleotide sequence of the promoter of the peppermint limonene synthase gene McLS is shown in SEQ ID NO.1.

[0015] The application of the promoter of the peppermint limonene synthase gene McLS in the expression of the GUS gene in the stomatal guard cells of young leaves, the nucleotide sequence of the promoter of the peppermint limonene synthase gene McLS is shown in SEQ ID NO.1.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0017] This invention uses peppermint leaves as material. The McLS promoter of the peppermint limonene synthase gene was cloned, and a plant expression vector pMcLS::GUS was constructed based on this. This vector was then transferred into tobacco seeds, and transgenic tobacco plants with increased GUS enzyme activity in young leaves were obtained through cultivation and screening. GUS staining experiments showed that wild-type tobacco did not stain, while the 35S promoter transgenic positive control was expressed in all tissues. The McLS promoter showed initiation activity in the short-stalked glandular trichomes of young leaves and stems of transgenic tobacco, but not in the long-stalked glandular trichomes. Additionally, the McLS promoter also showed initiation activity in the guard cells of young leaf stomata. Under ABA stress, the GUS enzyme activity in the young leaves of transgenic tobacco was the highest. Attached Figure Description

[0018] Figure 1 This is an electrophoresis image of the PCR amplification of the McLS promoter of the peppermint limonene synthase gene, where lane 1 is the McLS promoter;

[0019] Figure 2 A schematic diagram of the transcription factor binding site of the McLS promoter of the peppermint limonene synthase gene.

[0020] Figure 3This is a diagram of enzyme digestion verification of the pMcLS-pGC-GUS(G5) recombinant plasmid, where the approximately 1000bp band in lane 1 is the McLS promoter;

[0021] Figure 4 The PCR identification diagram of transgenic tobacco (lanes 1-6 show bands indicating positive plants, lane 7 shows wild-type plants without bands);

[0022] Figure 5 GUS staining effect of pMcLS::GUS transgenic tobacco leaves (A is transgenic positive seedling, B is wild type, C is tobacco positive control transformed with 35S promoter; a is young leaf, b is short-stalked glandular hairs of young leaf, c is short-stalked glandular hairs of stem, d is long-stalked glandular hairs).

[0023] Figure 6 Colorimetric images of short-stalked glandular hairs in young leaves of pMcLS::GUS transgenic tobacco (a), short-stalked glandular hairs in stems (b), and stomatal guard cells on young leaves (c).

[0024] Figure 7 Determination of GUS enzyme activity in different tissues of transgenic tobacco;

[0025] Figure 8 Determination of GUS enzyme activity in transgenic tobacco leaves treated with 5 μMABA Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, the technical means used in the following embodiments are all conventional means well known to those skilled in the art.

[0027] The plant material used in this invention is *Mentha canadensis* Linn., a plant belonging to the genus *Mentha* of the Lamiaceae family, cultivated in the nursery of the Jiangsu Provincial Institute of Botany, Chinese Academy of Sciences, Xuanwu District, Nanjing City, Jiangsu Province. The tobacco variety used in this invention is "K326," cultivated in the greenhouse of the Jiangsu Provincial Institute of Botany, Chinese Academy of Sciences, Xuanwu District, Nanjing City, Jiangsu Province.

[0028] Example 1

[0029] 1. Extraction of peppermint genomic DNA

[0030] Place approximately 300 mg of peppermint leaves into a 1.5 mL centrifuge tube, along with 3 steel balls. Place the centrifuge tube in a tissue homogenizer and vibrate it up and down to grind the leaves using the steel balls. Add 600 μL of 2×CTAB extraction buffer and shake the centrifuge tube up and down to mix the homogenized solution. Place the centrifuge tube in a 65°C water bath and gently shake it every 30 minutes. After 1-2 hours, remove the tube and cool it to room temperature. Add 600 μL of chloroform-isoamyl alcohol (24:1). Shake well, incubate at 4°C for 30 min, centrifuge at 4°C and 12000 rpm for 10-12 min to obtain supernatant; pipette 400-450 μL of supernatant into another new 1.5 mL centrifuge tube, add 1 / 10 volume of 3M NaAc (pH 4.8) and an equal volume of isopropanol (pre-cooled at 4°C), immediately invert to mix to facilitate DNA precipitation; centrifuge at 4°C and 12000 rpm for 10 min, discard supernatant; rinse with 600 μL of 70% ethanol (pre-cooled at 4°C), centrifuge at 4°C and 12000 rpm for 10 min, discard supernatant, invert the centrifuge tube on a spread paper towel, and after a few minutes, upright the centrifuge tube to air dry at room temperature; dissolve in 40-100 μL of 1×TE buffer or ddH2O and store at -20°C for later use.

[0031] 2. Amplification of the McLS promoter of the peppermint limonene synthase gene

[0032] Based on the previously obtained mint limonene synthase gene McLS sequence (Genebank IDJX555976) in our laboratory, a search was conducted in the Peppermint Genome Database (http: / / langelabtools.wsu.edu / mgr / ) to find homologous genes. Primers for amplifying the mint limonene synthase gene McLS promoter were designed based on the upstream promoter sequence of this gene. The primer sequences are shown below:

[0033] pMcLS-F: 5'-GAATTATTATTCATGTCA-3',

[0034] pMcLS-R: 5'-AAGGAAAGATTAATCATG-3'.

[0035] Using peppermint genomic DNA as a template, the promoter of the peppermint limonene synthase gene McLS was amplified by PCR using pMcLS-F and pMcLS-R. The PCR reaction system followed the instructions of PhantaMax Super-Fidelity DNA Polymerase (Vazyme, Nanjing). The PCR program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 30 s, 55℃ annealing for 30 s, 72℃ extension for 2 min, for 35 cycles; 72℃ extension for 5 min.

[0036] The PCR products were subjected to agarose gel electrophoresis. Figure 1 The sample was sent to the company for sequencing, and the nucleotide sequence of the promoter of the peppermint limonene synthase gene McLS is shown in SEQ ID NO.1.

[0037] Core functional elements were analyzed from the promoter sequence of the peppermint limonene synthase gene McLS obtained by sequencing, such as... Figure 2 As shown, this promoter contains the basic conserved elements of eukaryotic promoters, namely the CAAT-box and TATA-box, as well as transcription factor binding sites and plant hormone response elements such as ABA.

[0038] The PCR product was ligated into the pCE2-TAvector vector from Vazyme for sequencing analysis, resulting in the proMcLS-pCE2-TA recombinant plasmid containing the McLS promoter sequence of the peppermint limonene synthase gene.

[0039] Example 2

[0040] 1. Vector double enzyme digestion

[0041] The pGC-GUS(G5) vector was double-digested using KpnI enzyme, and the resulting double-digested vector was subjected to agarose gel electrophoresis. Figure 3 Then, the gel was cut and recovered using a kit.

[0042] 2. Construction of expression vectors containing the GUS gene

[0043] Design primers to introduce the restriction enzyme site KpnⅠ. The primer sequences are shown below:

[0044] pMcLS-G5-F:

[0045] 5'-CGCGTTGGGAGTCCCTCGAGGGTACCGAATTATTATTCATGTCA-3', pMcLS-G5-R:

[0046] 5'-ACTCATTCTAGAGAATTCAAGCTTGGGTACCAAGGAAAGATTAATCATG-3'.

[0047] PCR amplification was performed using the recombinant plasmid pMcLS-pCE2-TA as a cloning template, and the system is as follows:

[0048] pMcLS-G5-F 2μL pMcLS-G5-R 2μL pMcLS-pCE2-TA 0.5μL 2×PhantaMaxBuffer 25μL dNTPMax(10mM) 1μL Phanta 1μL <![CDATA[ddH2O]]> 19.5μL

[0049] The PCR product was transformed into E. coli, and the specific steps are as follows:

[0050] 100 μL of competent Escherichia coli DH5α cells (TSINGKE, Beijing) were thawed on ice, and the PCR product was added. The cells were then incubated on ice for 30 min. After heat shock in a 42°C water bath for 90 s, the cells were quickly transferred to ice and placed for 2 min. 1 mL of antibiotic-free LB medium was added to the competent cells containing the plasmid, and the cells were incubated at 200 rpm in a shaker at 37°C for 1 h. After centrifugation at 5000 g for 2 min, most of the supernatant was discarded in a clean bench, leaving approximately 200 μL. The bacterial pellet was resuspended by pipetting and transferred to a solid culture medium containing 50 mg / mL Kanabolic acid. The plate was spread evenly and inverted in a 37°C incubator for 12-16 h.

[0051] Single clones were selected for bacterial culture PCR detection using primers (5'-GAATTATTATTCATGTCA-3') and (5'-AAGGAAAGATTAATCATG-3'). Positive clones were identified as expression vectors containing the GUS gene, pMcLS::GUS.

[0052] 3. Transformation of Agrobacterium

[0053] The obtained peppermint McLS promoter plant expression vector pMcLS::GUS was transformed into Agrobacterium. The specific steps are as follows: 100 μL of competent Agrobacterium cells EHA105 (TSINGKE, Beijing) were thawed on ice, 5 μL of recombinant plasmid was added, and the cells were incubated on ice for 5 min, in liquid nitrogen for 5 min, in a 37℃ water bath for 5 min, and on ice for 5 min. 1 mL of antibiotic-free LB medium was added to the competent cells containing the plasmid, and the cells were cultured at 200 rpm in a shaker at 28℃ for 2 h. After centrifugation at 5000g for 2 min, most of the supernatant was discarded in a clean bench, leaving about 200 μL. The bacterial pellet was resuspended by pipetting and transferred to a solid medium containing the specified antibiotic. The plate was spread evenly and incubated upside down in a 37℃ incubator for 2-3 days. Single colonies were picked for PCR identification.

[0054] 4. Tobacco Conversion Method

[0055] Take an appropriate amount of mature tobacco K326 seeds and pour them into a 1.5 mL centrifuge tube. Add 1 mL of 75% ethanol and soak for 1 min. Use a pipette to remove the 75% ethanol, avoiding the seeds, and wash the seeds once with sterile water. Add 1 mL of 2% sodium hypochlorite and soak for 10 min, then wash three times with sterile water. Transfer the seeds to MS solid medium, about 30 seeds per plate. Seal the plates and culture them in a culture room at 25℃, 16 h light / 8 h dark, and 1500 Lux light intensity for about 1 week until the tobacco seeds germinate. Transfer the germinated tobacco seedlings to tissue culture bottles containing MS solid medium and culture for about 3 weeks. They can be used for transformation. Pick Agrobacterium containing recombinant plasmids in a clean bench and transfer them to LB liquid medium containing 50 mg / L LRFP and 100 mg / L Spec. Culture overnight in a shaker at 200 rpm and 28℃. Transfer 1 mL of the overnight cultured Agrobacterium to 25 mL of medium containing the same antibiotics. In LB liquid medium, culture at 200 rpm and 28℃ on a shaker for 3-4 hours until the OD600 reaches 0.4-0.6. Centrifuge at 5000g for 5 minutes to collect Agrobacterium cells, resuspend the cells in MS salt infection solution containing 100 μM s, and finally adjust the OD600 to 0.4-0.6. Incubate at room temperature in the dark for 2-4 hours. Take tobacco leaves in good growth condition, cut off the leaf margins and veins in a clean bench, cut them into small pieces, and place them in the infection solution. Shake at 200 rpm and 28℃ for 15-20 minutes. After the tobacco leaves are infected with the bacterial solution, blot off the excess bacterial solution with absorbent paper and place them on tobacco co-culture medium with filter paper on the surface. Co-culture at 25℃ in the dark for 4-6 days. After co-culturing for 4-6 days, transfer the tobacco leaves to a medium containing 500 mg / L Cef and 100 mg / L Cef. On the tobacco selection differentiation medium of Spec, the plants were cultured in a culture room at 25°C, with 16h light / 8h darkness and a light intensity of 1500 Lux until shoots differentiated. The shoots were then cut off and placed on the same selection rooting medium containing 500mg / L Cef and 100mg / L Spec, and allowed to root.

[0056] The results are as follows Figure 4 As shown, PCR was used to identify transgenic positive plants, and six pMcLS::GUS transgenic tobacco lines were successfully obtained.

[0057] 5. GUS expression activity in transgenic positive plants

[0058] Wild-type tobacco was used as the negative control, and tobacco with the 35S promoter was used as the positive control. GUS staining was performed on various tissue parts of the peppermint McLS promoter transgenic tobacco using a GUS staining kit (Coolaber, Beijing). The steps were as follows: Tissue parts of the McLS promoter transgenic tobacco and the negative control wild-type tobacco were immersed in GUS staining solution and placed overnight in a 37°C incubator in the dark. Except for the parts without chlorophyll, the remaining samples were destained in 75% ethanol 2-3 times until the negative control material turned white. The presence of blue areas was observed under a stereomicroscope, and the blue areas indicated GUS gene expression.

[0059] The results are as follows Figure 5 , 6 As shown, wild-type tobacco did not show any color staining, and the 35S promoter transgenic positive control was expressed in all tissues; the McLS promoter was active in the short-stalked glandular hairs of young leaves and stems of transgenic tobacco, but not in the long-stalked glandular hairs. In addition, the McLS promoter was also active in the guard cells of stomata in young leaves.

[0060] 6. ProMcLS-initiated GUS expression activity detection

[0061] 1) T1 generation seedlings of transgenic tobacco with McLS promoter and wild-type tobacco control group were transferred to MS medium containing 5 μMABA, with three biological replicates per group. They were subjected to stress for 7 days in a culture room at 25°C, 16h light / 8h dark, and light intensity of 1500 Lux.

[0062] 2) Total protein extraction

[0063] Total protein was extracted from young leaves of McLS promoter transgenic tobacco and wild-type tobacco after ABA stress treatment using PBS buffer (Jiancheng, Nanjing). 900 μL of PBS buffer was added to a glass mortar, and approximately 100 mg of fresh young tobacco leaves was ground into a homogenate. The homogenate was transferred to a 1.5 mL centrifuge tube and centrifuged at 8000 rpm for 5 min at 4°C. The supernatant was transferred to another clean 1.5 mL centrifuge tube, which was the protein extract, and placed on ice for later use.

[0064] 3) Quantitative determination of GUS expression level

[0065] Using wild-type tobacco as a negative control, the GUS expression level in young leaves of transgenic tobacco with the McLS promoter after ABA stress treatment was determined using a GUS gene quantitative detection kit (Coolaber, Beijing). The specific steps are as follows: Dilute 4-MU with stop solution to a concentration of 1-10 μM to obtain the standard solution. Store in the dark and use a fluorescence spectrophotometer to measure the fluorescence value of 4-MU for each sample at an excitation wavelength of 365 nm and an emission wavelength of 456 nm. Plot a standard curve M = kn, where M is the fluorescence value and n is the concentration of 4-MU. Add 900 μL of stop solution to three 1 mL centrifuge tubes and incubate at 37℃. Take 100 μL of protein extract and add 100 μL of... The 4-MUG substrate solution was incubated at 37°C to prepare the reaction solution. At 0 min, 10 min, and 20 min, 60 μL of the reaction solution was added to 180 μL of the stop solution in the incubator. The mixture was then stored in the dark. The fluorescence value of 4-MU in each sample was measured using a fluorescence spectrophotometer at an excitation wavelength of 365 nm and an emission wavelength of 456 nm. The fluorescence value of the 4-MU generated in the reaction solution was calibrated using the fluorescence value of the standard solution. The content of the fluorescent substance produced by the reaction in the sample was calculated from the standard curve. The fluorescence intensity change per unit mass of protein per unit time was calculated by dividing the change in fluorescence intensity per unit time by the amount of protein participating in the reaction, which is the GUS enzyme activity.

[0066] The results are as follows Figure 7 As shown, ProMcLS promoter expression in different tissues of transgenic tobacco exhibits significant tissue specificity, with the highest GUS enzyme activity observed in young leaves.

[0067] The results are as follows Figure 8 As shown, the GUS enzyme activity in transgenic tobacco leaves treated with ABA was significantly increased compared to the untreated control group.

[0068] The above embodiments are for illustrating the implementation schemes disclosed in this invention and should not be construed as limiting the invention. Furthermore, various modifications listed herein, as well as variations in the methods and compositions of the invention, will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been specifically described in conjunction with various specific preferred embodiments, it should be understood that the invention should not be limited to these specific embodiments. In fact, various modifications as described above that are obvious to those skilled in the art to obtain the invention should be included within the scope of this invention.

Claims

1. The promoter of the peppermint limonene synthase gene McLS, the nucleotide sequence of which is shown in SEQ ID NO.

1.

2. The application of the promoter of the peppermint limonene synthase gene McLS in promoting increased GUS enzyme activity in young tobacco leaves, wherein the nucleotide sequence of the promoter of the peppermint limonene synthase gene McLS is shown in SEQ ID NO.1; the application includes the following steps: (1) Construct the proMcLS-pCE2-TA recombinant plasmid containing the McLS promoter sequence of the peppermint limonene synthase gene; use the recombinant plasmid pMcLS-pCE2-TA as a template to construct the expression vector pMcLS::GUS containing the GUS gene. (2) The constructed expression vector pMcLS::GUS containing the GUS gene was transformed into tobacco seeds; (3) Transgenic tobacco plants with increased GUS enzyme activity in young leaves were obtained through cultivation and screening.

3. The application of the promoter of the peppermint limonene synthase gene McLS in the expression of the GUS gene in the short-stalked glandular trichomes of young tobacco leaves, wherein the nucleotide sequence of the promoter of the peppermint limonene synthase gene McLS is shown in SEQ ID NO.1; the application includes the following steps: (1) Construct the proMcLS-pCE2-TA recombinant plasmid containing the McLS promoter sequence of the peppermint limonene synthase gene; use the recombinant plasmid pMcLS-pCE2-TA as a template to construct the expression vector pMcLS::GUS containing the GUS gene. (2) The constructed expression vector pMcLS::GUS containing the GUS gene was transformed into tobacco seeds; (3) Transgenic tobacco plants expressing the GUS gene in the short stalk glandular hairs of young tobacco leaves were cultivated and screened.

4. The application of the promoter of the peppermint limonene synthase gene McLS in the expression of the GUS gene in the short stalk glandular hairs of tobacco stems, wherein the nucleotide sequence of the promoter of the peppermint limonene synthase gene McLS is shown in SEQ ID NO.1; the application includes the following steps: (1) Construct the proMcLS-pCE2-TA recombinant plasmid containing the McLS promoter sequence of the peppermint limonene synthase gene; use the recombinant plasmid pMcLS-pCE2-TA as a template to construct the expression vector pMcLS::GUS containing the GUS gene. (2) The constructed expression vector pMcLS::GUS containing the GUS gene was transformed into tobacco seeds; (3) Transgenic tobacco plants expressing the GUS gene in the short stalk glandular hairs of tobacco stems were cultivated and screened.

5. The application of the promoter of the peppermint limonene synthase gene McLS in the expression of the GUS gene in the stomatal guard cells of young leaves, wherein the nucleotide sequence of the promoter of the peppermint limonene synthase gene McLS is shown in SEQ ID NO.1; the application includes the following steps: (1) Construct the proMcLS-pCE2-TA recombinant plasmid containing the McLS promoter sequence of the peppermint limonene synthase gene; use the recombinant plasmid pMcLS-pCE2-TA as a template to construct the expression vector pMcLS::GUS containing the GUS gene. (2) The constructed expression vector pMcLS::GUS containing the GUS gene was transformed into tobacco seeds; (3) Transgenic tobacco plants with GUS gene expression initiated in the guard cells of young leaves were obtained by culturing and screening.

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