CN116445532A - Rhizopus stolonifer plasmid vector - Google Patents
Rhizopus stolonifer plasmid vector Download PDFInfo
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- CN116445532A CN116445532A CN202310339255.XA CN202310339255A CN116445532A CN 116445532 A CN116445532 A CN 116445532A CN 202310339255 A CN202310339255 A CN 202310339255A CN 116445532 A CN116445532 A CN 116445532A
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Abstract
The invention belongs to the field of plasmid vector construction, and provides a plasmid vector, a construction method and application thereof. The plasmid vector is formed by modifying pAN7-1, and the plasmid vector is formed by inserting a rhizopus stolonifer promoter gene and a resistance gene into pAN7-1 multiple cloning sites, wherein the resistance gene is positioned at the downstream of the rhizopus stolonifer promoter gene. The sections of the rhizopus stolonifer promoter gene selected by the invention can efficiently start genes connected at the back, so that the transformation efficiency of the plasmid vector is greatly improved. The invention establishes a method for efficiently transforming the rhizopus stolonifer by using the agrobacterium-mediated exogenous gene, which can provide assistance in the aspects of genetic modification of the rhizopus stolonifer, development of useful genes, separation of drug target genes and the like.
Description
Technical Field
The invention belongs to the technical field of plasmid vector construction, and in particular relates to a rhizopus stolonifer plasmid vector pAN-RS-GH constructed based on pAN7-1 and application thereof.
Background
Rhizopus stolonifer is a common mould, and is of the genus rhizopus, order zygomycetes, family mucorales. Is characterized by having slender hyphae, not being separated and being in a multinuclear state. Asexual reproduction forms spores for reproduction, and sexual reproduction forms thick-walled zygospores.
Rhizopus stolonifer as a plant pathogen also has development value and is an important biological resource to be developed. Plant pathogens successfully infect host plants, and must be able to overcome the plant's defensive response and also be able to cross the physical protective barriers of plants such as the plant epidermis. Therefore, plant pathogens tend to produce more extracellular degrading enzymes and other effectors than saprophytes, degrading plant defense responses and subjecting plant epidermal cell wall components to the necessary degradation.
With the development of modern biotechnology, molecular biology technology has been combined with traditional biology technology, so as to greatly promote the development of biological research and biological industry, and both the molecular modification of biological cells and cloning of useful genes, a transformation system for transforming exogenous genes into cells needs to be established, which is a precondition for the molecular modification and research of the cells. Aiming at the fungus rhizopus stolonifer, the gene transformation efficiency of the fungus is lower at present.
Plasmid pAN7-1 is a common expression vector for gene transformation of filamentous fungi, and can be used for gene transformation by Agrobacterium-mediated methods. The recombinant DNA is characterized in that the recombinant DNA contains a plurality of multiple cloning sites and restriction enzyme cutting sites, the promoter is GPD, the terminator is TRPC, and the size is 6756 bp. Which contains an ampicillin gene as a selectable marker. However, the present group of subjects found that transformation efficiency was low when the plasmid was used to transform rhizopus stolonifer in the previous study.
Therefore, the subject group constructs a new screening marker, integrates the screening marker into a vector for agrobacterium transformation, and establishes a method for efficiently transforming the rhizopus stolonifer by using an agrobacterium-mediated exogenous gene, thereby providing assistance in the aspects of genetic modification of the rhizopus stolonifer, development of useful genes, separation of drug target genes and the like.
Disclosure of Invention
The invention aims to provide a brand-new plasmid vector, which solves the problem of low conversion efficiency when the existing plasmid is converted into rhizopus stolonifer.
Specifically, the invention provides a plasmid vector pAN-RS-GH, and the nucleotide sequence is shown as SEQ ID NO. 1. The plasmid vector is formed by modifying pAN7-1 (the nucleotide sequence of which is shown as SEQ ID NO: 2), inserting a rhizopus stolonifer promoter gene (the nucleotide sequence of which is shown as SEQ ID NO: 3) and a resistance gene into pAN7-1 multiple cloning sites, wherein the resistance gene is a hygromycin resistance gene (the nucleotide sequence of which is shown as SEQ ID NO: 4), and the resistance gene is positioned at the downstream of the rhizopus stolonifer gene promoter gene.
Plasmid pAN7-1 is a common expression vector for gene transformation of filamentous fungi, and can be used for gene transformation by Agrobacterium-mediated methods. The invention also adopts pAN7-1 as a primary vector.
The invention selects the rhizopus stolonifer promoter gene as a strong promoter for regulating and controlling the high expression of exogenous genes (such as GFP), and the transformation efficiency of the constructed plasmid vector transformed into rhizopus stolonifer is far higher than that of the transformation vector commonly used at present. Taking the currently commonly used vector pAN7-1 as an example, under the same transformation conditions, when there are only 6 transformants obtained by taking pAN7-1 as the vector on average, 14-20 transformants can be obtained by using the plasmid vector of the present invention.
Further, the resistance gene is a hygromycin resistance gene. The original vector pAN7-1 contains an ampicillin resistance gene, but the promoter is a 35S promoter, so that the expression efficiency in fungi is low, and the expression in rhizopus stolonifer is hardly carried out. The research shows that compared with the control vector pAN7-1, the expression level of hygromycin resistance gene in the plasmid vector constructed by the invention is higher than 10 times, which proves that the hygromycin resistance gene promoter gene can be obviously regulated and controlled to be highly expressed in the rhizopus stolonifer, thereby being beneficial to the screening of transformants.
Furthermore, a reporter gene is inserted downstream of the rhizopus stolonifer promoter gene, and the expression of the target gene can be calibrated by the reporter gene, wherein the reporter gene is GFP gene (the nucleotide sequence is shown as SEQ ID NO: 5).
The invention also provides a construction method for constructing the plasmid vector, which comprises the following steps.
Amplifying DNA fragments respectively comprising the rhizopus stolonifer promoter gene, the hygromycin resistance gene and the GFP reporter gene.
The DNA fragment was inserted into the multicloning site of pAN7-1 vector by recombination reaction to obtain the plasmid vector.
Specifically, the 5 'and 3' extreme ends of the amplified PCR products are respectively provided with a completely consistent sequence corresponding to the extreme ends of the adjacent fragments by designing specific primers, and the directional cloning is completed under the action of recombinase.
Further, the rhizopus stolonifer promoter gene, hygromycin resistance gene and GFP reporter gene were designed as follows.
The primers for amplifying the rhizopus stolonifer promoter gene are as follows:
an upstream primer: AAATAATTGAGGATGCGCATCGATGGAGGCGATACGAA
A downstream primer: CCTTGCCAGGTCTCACTTTGGGATGCCAGTTCCGAGGGCT
The primers for amplifying GFP gene are:
an upstream primer: ATGGTGAGCAGCGAGGAGGAG
A downstream primer: TCTAGAGTACAGTTACTTCTCGTCCATGCCG
Primers for amplifying hygromycin resistance gene are:
an upstream primer: ATGAAAAGAAAGCCTCTCACCG
A downstream primer: ACGACGGCCAGTTCTACGCCACATCTATTCCTTTGCCC.
Furthermore, the invention also provides an application of the plasmid vector, and an agrobacterium-mediated rhizopus stolonifer gene transformation method, which is characterized by comprising the following steps.
Inserting the target gene into the constructed plasmid vector, and transforming into agrobacterium to obtain agrobacterium containing recombinant plasmid.
And co-culturing agrobacterium containing the recombinant plasmid with rhizopus stolonifer to obtain a transformant.
The invention has the beneficial effects that.
The plasmid vector constructed by the invention can stably and effectively integrate a target gene into a rhizopus stolonifer genome, enables the target gene to be efficiently expressed in the rhizopus stolonifer, and can provide assistance in genetic modification of the rhizopus stolonifer, development of useful genes, separation of drug target genes and the like.
Additional features and advantages of the invention will be set forth in the detailed description which follows.
Drawings
The above and other objects, features and advantages of the present invention will become more apparent by describing in more detail exemplary embodiments thereof with reference to the attached drawings.
FIG. 1 is a map of plasmid vector pAN-RS-GH.
FIG. 2 is a map of plasmid vector pAN7-1.
FIG. 3 shows the conversion of two vectors into Rhizopus stolonifer.
Description of the embodiments
Preferred embodiments of the present invention will be described in more detail below. While the preferred embodiments of the present invention are described below, it should be understood that the present invention may be embodied in various forms and should not be limited to the embodiments set forth herein.
The specific conditions not specified in the examples were either conventional or manufacturer-recommended. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
EXAMPLE 1 construction of the rhizopus stolonifer plasmid vector pAN-RS-GH.
1. Purchase of plasmid pAN7-1.
Plasmid pAN7-1 of the invention was purchased from Shanghai Ke Lei Biotechnology Co. The recombinant DNA is characterized in that the recombinant DNA contains a plurality of multiple cloning sites and restriction enzyme cutting sites, the promoter is GPD, the terminator is TRPC, and the size is 6756 bp. Which contains an ampicillin gene as a selectable marker.
2. And (5) designing a primer.
Searching in a rhizopus stolonifer genome database to find a rhizopus stolonifer promoter gene, and designing a primer capable of obtaining the rhizopus stolonifer promoter gene by a PCR method. Based on the hygromycin resistance gene sequence on plasmid pCB1003, primers were designed which can amplify the hygromycin resistance gene by PCR method. Based on the GFP gene sequence on plasmid pEGFP, primers were designed which can obtain the GFP gene by PCR. The sequence of each primer is as follows:
the primers for amplifying the rhizopus stolonifer promoter gene are as follows:
an upstream primer: AAATAATTGAGGATGCGCATCGATGGAGGCGATACGAA
A downstream primer: CCTTGCCAGGTCTCACTTTGGGATGCCAGTTCCGAGGGCT
The primers for amplifying GFP gene are:
an upstream primer: ATGGTGAGCAGCGAGGAGGAG
A downstream primer: TCTAGAGTACAGTTACTTCTCGTCCATGCCG
Primers for amplifying hygromycin resistance gene are:
an upstream primer: ATGAAAAGAAAGCCTCTCACCG;
a downstream primer: ACGACGGCCAGTTCTACGCCACATCTATTCCTTTGCCC.
And (5) carrying out PCR amplification to obtain the target gene.
(1) And amplifying the rhizopus stolonifer promoter gene.
| Taq DNA polymerase buffer | 5μL |
| dNTPs | 5μL |
| Upstream primer | 2.5μL |
| Downstream primer | 2.5μL |
| Rhizopus stolonifer genome | 2μL |
| Taq DNA polymerase | 0.5μL |
| ddH2O | 32.5μL |
| All of which | 50μL |
The reaction procedure is: 94 ℃ for 5min; cycling at 94℃for 30s,56℃for 30s,72℃for 4.5min, 35; extending at 72 ℃ for 10min; preserving at 4 ℃.
(2) GFP gene amplification.
| Taq DNA polymerase buffer | 5μL |
| dNTPs | 5μL |
| Upstream primer | 2.5μL |
| Downstream primer | 2.5μL |
| pCB1003 plasmid | 2μL |
| Taq DNA polymerase | 0.5μL |
| ddH2O | 32.5μL |
| All of which | 50μL |
The reaction procedure is: 94 ℃ for 5min;94℃for 30s,57℃for 30s,72℃for 4.5min,35 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
(3) Hygromycin resistance gene amplification.
| Taq DNA polymerase buffer | 5μL |
| dNTPs | 5μL |
| Upstream primer | 2.5μL |
| Downstream primer | 2.5μL |
| pEGFP plasmid | 2μL |
| Taq DNA polymerase | 0.5μL |
| ddH2O | 32.5μL |
| All of which | 50μL |
The reaction procedure is: 94 ℃ for 5min;94℃for 30s,55℃for 30s,72℃for 4.5min,35 cycles; extending at 72 ℃ for 10min; preserving at 4 ℃.
Construction of the vector.
Plasmid pAN7-1 was digested with restriction enzymes XhoI and HindIII, and the large fragment was recovered in a gel. Then, the PCR fragment obtained above was purified by using a PCR fragment purification kit, and a novel vector was constructed by recombination using a Vazyme company CloneExpress Multis kit by a one-step cloning method. The recombination system is as follows:
| 5×CE Multis Buffe | 4μL |
| pAN7-1 cleavage product | 2μL(50ng) |
| Rhizopus stolonifer promoter gene amplification product | 2μL(25ng) |
| GFP gene amplification products | 2μL(25ng) |
| Hygromycin resistance gene amplification products | 2μL(25ng) |
| Exnase Multis enzyme | 2μL |
| All of which | 20μL |
Example 2 preparation of rhizopus stolonifer protoplasts.
Preparation of reagents and culture media.
0.7M sodium chloride solution: 40.95g of sodium chloride is dissolved in 1000mL of redistilled water, and the constant volume is carried out for conventional high-temperature sterilization.
1M Tris-Cl: tris (hydroxymethyl) aminomethane 121.14 was dissolved in 600mL of redistilled water, ph=7.5 was adjusted with concentrated hydrochloric acid, and then volume was set to 1L with redistilled water, and conventional high temperature sterilization was performed for use.
Cell wall degrading enzyme solution: 100mg of crashase and 100mg of muramidase were weighed on a balance, dissolved in 10mL of 0.7M sodium chloride solution, and sterilized by filtration through a filter membrane having a diameter of 0.22. Mu.m, and prepared for use.
STC solution: 21.8604g of sorbitol and 0.735g of calcium chloride were weighed and dissolved in about 60mL of water, 1mL of 1M Tris-Cl was added, and the volume was fixed to 100mL.
Potato dextrose agar medium (PDA): cutting 200g of potato into small pieces, adding water, boiling for 15min, filtering with 2 layers of gauze, discarding potato pieces, adding 20g of glucose and 20g of agar into filtrate, adding distilled water to volume to 1000mL, and sterilizing at conventional temperature.
Potato Dextrose Broth (PDB): cutting 200g of potato into small pieces, adding water, boiling for 15min, filtering with 2 layers of gauze, discarding potato pieces, adding 20g of glucose into filtrate, adding distilled water to volume to 1000mL, and sterilizing at conventional temperature.
Preparation of protoplasts.
The rhizopus stolonifer strain stored in the laboratory is inoculated on a PDA plate, 5-8 rhizopus stolonifer strains are picked by a picking needle after being cultured for 5 days at 25 ℃, and the rhizopus stolonifer strains are inoculated in 100mL of PDB liquid culture medium and are cultured for 2 days at 25 ℃.
In an ultra clean bench, mycelium was filtered with four layers of gauze, then the mycelium was rinsed 3 times with 0.7M NaCl, the water was drained with sterilized filter paper and absorbent paper, 1g mycelium was weighed and placed in a 50mL sterilized centrifuge tube, 10mL of the prepared cell wall degrading enzyme solution was added, and enzymatic hydrolysis was performed on a shaker at 28℃at 80 rpm for 3.5 hours.
The enzymatic solution was filtered through two sterilized layers of paper, and the protoplasts remaining on the paper were rinsed twice with 10mL of NaCl at a time. The residue was removed, and the filtrate was transferred to a 50ml centrifuge tube, placed in a centrifuge, and centrifuged at 5000rpm for 10min at 4 ℃.
The supernatant was carefully discarded, 15mL of STC was added, and the centrifuge tube was gently shaken to suspend the pellet.
The 50ml centrifuge tube was placed in a centrifuge and centrifuged at 3000rpm for 10min at 4 ℃.
The supernatant was carefully discarded, 1mL of STC was added, and the centrifuge tube was gently shaken to suspend the pellet.
Counting protoplast with a blood cell counting plate, adding STC solution according to the counting result, and adjusting the concentration of protoplast to 10 6 And each mL.
The obtained protoplast is packed into sterilized 1.5mL centrifuge tubes, 100 mu L each tube is preserved at-80 ℃ for standby
Example 3 Agrobacterium-mediated transformation of rhizopus stolonifer DNA.
1. Preparation of rhizopus stolonifer strain.
The rhizopus stolonifer strain stored in the laboratory is inoculated on a PDA plate, and is cultured for 5 days at 25 ℃ for later use.
2. Preparation of plasmids.
The pAN-RS-GH plasmid of example 1 was extracted and a control plasmid pAN7-1 was prepared. The concentration of the plasmid was adjusted to 0.25. Mu.g/. Mu.L.
3. The plasmid vector was transformed into agrobacterium.
(1) Preparation of LB medium: LB solid medium: 10g of tryptone, 5g of yeast extract, 10g of sodium chloride, 15g of agar, 1000mL of water, pH=7.0 and conventional high-temperature sterilization. LB liquid medium: agar was not added to LB solid medium, and the same was the same as above.
(2) Agrobacterium strain AGL1 was streaked on LB plates and cultured at 28℃for 2 days. Then, 1 single colony was transferred to 5mL of LB liquid medium and cultured overnight at 28℃with shaking.
(3) Transferring 2mL of the culture solution into a triangular flask containing 50mL of LB liquid medium, culturing at 28 ℃ for 6 hours with shaking, measuring the absorbance, and obtaining the OD 600 Culture was stopped when =0.5 to 1.0.
(4) The cultured bacterial liquid was placed on ice and then transferred to a 50mL centrifuge tube and centrifuged at 5000rpm for 5min at 4 ℃.
(5) The supernatant was discarded and the pellet was pelleted with 1mL of pre-chilled 20mM CaCl 2 The solution was suspended and dispensed into pre-chilled 1.5mL centrifuge tubes, 0.1mL per tube.
(6) Two plasmids (4. Mu.L) with the concentration adjusted to 0.25. Mu.g/. Mu.L were added to a 1.5mL centrifuge tube containing 0.1mL of Agrobacterium, covered with a cap, gently mixed, and rapidly frozen in liquid nitrogen.
(7) The 1.5mL centrifuge tube was removed, and placed in a 37℃water bath for 5min to thaw.
(8) 1mL of LB culture solution is added into a 1.5mL centrifuge tube, and the mixture is subjected to light shaking culture at 100rpm for 2 to 4 hours at 28 ℃.
(9) Putting the centrifuge tube into a centrifuge, centrifuging at 10000rpm for 5min, discarding the supernatant, suspending the precipitate, coating on an LB plate, standing for 30min on the front surface, inverting a culture dish after bacterial liquid is completely absorbed by a culture medium, and culturing for 2-4 days at 28 ℃.
4. Agrobacterium is used to transform rhizopus stolonifer.
(1) Dissolving solutionPreparing liquid: agrobacterium medium IM:0.8mL K-Phosphate-buffer,20mL MN-buffer,1mL 1% CaCl 2 ·2H 2 O，10mL 0.01％FeSO4，5mL spore elements，2.5mL 20％NH 4 NO 3 10mL 50%glycerol,40mL 1M MES,10mL 20%glucose water was added to 1L and 1.5% agar powder was added to the solid medium.
100mM Acetosyringone Stock (AS): 1.962g acetosyringone was dissolved in dimethyl sulfoxide (DMSO), fixed to a volume of 100mL and sterilized by filtration through a microporous filter membrane with a pore size of 0.22. Mu.m.
Agrobacteria induction medium AIM: 200. Mu.L of acetosyringone stock solution was added to 100mL of IM medium to give a final acetosyringone concentration of 200. Mu.M.
Recovery medium YPS: peptone 5g, yeast extract 3.5g, glucose 10g, sucrose 342.3g, water 1000ml, ph=7.0. And (3) conventional high-temperature high-pressure sterilization.
(2) An Agrobacterium single colony was selected from freshly cultured LB plates and inoculated into 5mL of LB liquid medium for overnight culture at 28℃at 200 rpm.
(3) The next day, 400. Mu.L of the culture solution was transferred to 5mL of an induction liquid medium (AIM) and incubated at 28℃for 5 to 6 hours at an OD of about 0.15 to give an OD of the bacterial solution 600 Reaching 0.5 to 0.6.
(4) Activation of rhizopus stolonifer conversion material: mu.L of recovery medium was added to a 1.5mL centrifuge tube containing 100. Mu.L of protoplasts, and the culture was resumed at 28℃for 5 to 6 hours.
(5) After 100mL of the IM solid medium was melted, the mixture was cooled to 50℃at room temperature, and after gentle shaking and mixing, the mixture was poured into a 6cm disposable plastic petri dish to prepare an induction plate. After the medium in the dish had solidified, a sterilized cellulose film of 5cm diameter was covered on the surface of the medium.
(6) Mixing 100 μl of the cultured rhizopus stolonifer (containing carrier) bacterial solution with 200 μl of conversion material, uniformly coating the mixed solution on the surface of nitrocellulose membrane on an induction plate, and co-culturing at 22deg.C for 48 hr.
(7) After 100mL of PDA solid medium was melted, the mixture was cooled to 50℃at room temperature, and after gentle shaking and mixing, the mixture was poured into a 6cm disposable plastic petri dish to prepare a screening plate. The nitrocellulose membrane on the induction plate was cut into strips of about 0.5cm, transferred to the screening plate with about 0.5cm between the strips, and placed in culture at 28℃for 7 days, and hyphae were seen to grow onto the medium between the nitrocellulose membrane strips.
(8) The grown mycelia were picked up and transferred to PDA plates containing 10. Mu.g/mL hygromycin and incubated at 28℃for 5 days to confirm successful transformation. If growth is possible, successful transformation can be confirmed.
Example 4 detection of transformants.
1. And (5) detecting stability of the transformant.
From the obtained transformants, 10 transformants were randomly selected, and cultured at 28℃for 5 times on PDA medium plates, and then further cultured at 28℃for 5 days on PDA plates containing 10. Mu.g/mL hygromycin. As a result, all of the 10 transformants were allowed to grow, indicating that the stability of the transformants was good and that the transferred resistance gene was stable.
2. And (5) detecting green fluorescence of the transformant.
Transformants obtained by random selection were inoculated on PDA plates and incubated at 28℃for 5 days. A small amount of mycelium is picked up by a picking needle, placed on a glass slide, made into an observation glass slide, and placed on a stage of a microscope. Ultraviolet light (450-490 nm) is turned on, and green fluorescence is observed. The result shows that the mycelium of the transformant of the vector pAN-RS-GH constructed by the invention can emit stronger fluorescence, while the transformant of pAN7-1 serving as a control emits weaker fluorescence.
3. And (5) detecting the transformant by PCR.
The method comprises the steps of extracting a small amount of rhizopus stolonifer genome DNA, carrying out PCR amplification on hygromycin resistant genes and GFP genes in transformants by adopting the PCR method, and detecting PCR products by 1% agarose gel electrophoresis after the PCR reaction is finished, wherein electrophoresis strips of the hygromycin resistant genes of about 1100bp and electrophoresis strips of the GFP genes of about 650bp appear in all 10 transformants, which indicates that the hygromycin resistant genes and the GFP genes are stably integrated into the rhizopus stolonifer genome.
The foregoing description of embodiments of the invention has been presented for purposes of illustration and description, and is not intended to be exhaustive or limited to the embodiments disclosed. Many modifications and variations will be apparent to those of ordinary skill in the art without departing from the scope and spirit of the various embodiments described.
Claims (4)
1. The rhizopus stolonifer plasmid vector is characterized in that the nucleotide sequence of the plasmid vector pAN-RS-GH is shown as SEQ ID NO. 1.
2. The method for constructing a plasmid vector according to claim 1, wherein the method for constructing comprises the steps of:
(1) Amplifying DNA fragments respectively comprising the rhizopus stolonifer promoter gene, the hygromycin resistance gene and the GFP reporter gene;
(2) The DNA fragment was inserted into the multicloning site of pAN7-1 vector by recombination reaction to obtain the plasmid vector.
3. The construction method of claim 2, wherein the rhizopus stolonifer promoter gene, hygromycin resistance gene and GFP reporter gene are designed as follows:
the primers for amplifying the rhizopus stolonifer promoter gene are as follows:
an upstream primer: AAATAATTGAGGATGCGCATCGATGGAGGCGATACGAA
A downstream primer: CCTTGCCAGGTCTCACTTTGGGATGCCAGTTCCGAGGGCT
The primers for amplifying GFP gene are:
an upstream primer: ATGGTGAGCAGCGAGGAGGAG
A downstream primer: TCTAGAGTACAGTTACTTCTCGTCCATGCCG
Primers for amplifying hygromycin resistance gene are:
an upstream primer: ATGAAAAGAAAGCCTCTCACCG
A downstream primer: ACGACGGCCAGTTCTACGCCACATCTATTCCTTTGCCC.
4. Use of a plasmid vector according to claim 1, wherein the agrobacterium-mediated transformation of the rhizogenes gene comprises the steps of:
(1) Inserting a target gene into the plasmid vector of claim 1, and transforming into agrobacterium to obtain agrobacterium containing recombinant plasmid;
(2) And co-culturing agrobacterium containing the recombinant plasmid with rhizopus stolonifer to obtain a transformant.
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