A polyketide synthase from asparagus stem blight pathogen and its encoding gene and application

Abstract

The present invention relates to a polyketide synthase of asparagus stem blight pathogen and its encoding gene and application, belonging to the field of biotechnology. The present invention provides a polyketide synthase, the amino acid sequence of the polyketide synthase is shown in SEQ ID NO.1; studies have shown that compared with the wild-type strain XT3, the pigment production of the polyketide synthase encoding gene pks-pa knockout strain XT3-KN is significantly reduced, and compared with the polyketide synthase encoding gene pks-pa knockout strain XT3-KN, the polyketide synthase encoding gene pks-pa introduced strain XT3-KN-RV has significantly improved pigment production ability, indicating that the polyketide synthase with an amino acid sequence as shown in SEQ ID NO.1 and the gene encoding the polyketide synthase with a nucleotide sequence as shown in SEQ ID NO.2 play an important role in the pigment production process of Phomopsis asparagus.

Description

Technical Field

[0001] The invention relates to a polyketide synthase of asparagus stem blight pathogen, a coding gene and application thereof, and belongs to the field of biotechnology. Background Art

[0002] Synthetic pigments are artificially synthesized pigments with bright colors, strong tinting power, and a variety of hues, but they are toxic. These toxicities come from the arsenic, lead, copper, phenol, aniline, ether, chlorides, and sulfates in synthetic pigments, all of which can cause varying degrees of harm to the human body. In 1982, China promulgated the "Hygienic Standards for the Use of Food Additives," which stipulates that only five synthetic pigments can be used and sets maximum usage limits. For example, the pure pigment content of synthetic pigments must not be less than 85-99%, the arsenic content in 1 kilogram of synthetic pigments should be less than 1 mg, the lead content less than 10 mg, and the copper content less than 20 mg. Per 100 grams of pigment, the phenol content should not exceed 5 mg, the aniline content should not exceed 4 mg, and the various chlorides should not exceed 0.5%. These regulations are intended to limit impurities in pigments to reduce toxicity to the human body.

[0003] Natural pigments are food pigments obtained from natural resources, primarily extracted from animal and plant tissues and microbial cultures. Natural pigments not only color foods, but a significant portion also possesses physiological activity. The vast majority of natural pigments are safe and have no side effects. Therefore, obtaining large quantities of natural pigments from various natural resources and exploring their physiological activity are crucial to alleviating and resolving the toxicity issues associated with synthetic pigments.

[0004] Phomopsis asparagi is the causative agent of asparagus stem blight, a fungal disease with a relatively narrow host range and a high degree of damage. In fungal taxonomy, it belongs to the genus Phomopsis in the family Phomopsisaceae, order Ascomycetes, order Ascomycetes. The pathogenic colonies of Phomopsis asparagi on PDA plates are initially white, then develop a distinct yellow-green color, indicating that Phomopsis asparagi is very likely to contain genes encoding pigment synthesis. If the genes encoding pigment synthesis in Phomopsis asparagi can be identified and used to construct genetically engineered bacteria capable of synthesizing pigments, and then ferment to obtain natural pigments, it would be very important to promote the large-scale acquisition of natural pigments. Currently, there is no research on the use of genes encoding pigment synthesis from Phomopsis asparagi to construct genetically engineered bacteria and then ferment to obtain natural pigments. Summary of the Invention

[0005] To address the above problems, the present invention provides a polyketide synthase (PKS), the amino acid sequence of which is shown in SEQ ID NO.1.

[0006] In one embodiment of the present invention, the polyketide synthase is derived from the pathogen Phomopsis asparagi NCXT3, which causes asparagus stem blight.

[0007] In one embodiment of the present invention, the polyketide synthase is an iterative type I WA subclass polyketide synthase derived from Asparagus NCXT3.

[0008] The present invention also provides a gene encoding the polyketide synthase.

[0009] In one embodiment of the present invention, the nucleotide sequence of the gene is shown as SEQ ID NO.2.

[0010] The present invention also provides a recombinant plasmid carrying the above gene.

[0011] In one embodiment of the present invention, the expression vector of the recombinant plasmid is myp821 plasmid or pMD18 plasmid.

[0012] The present invention also provides a host cell, which carries the above gene or the above recombinant plasmid.

[0013] In one embodiment of the present invention, the host cell is Phomopsis asparagus, Escherichia coli or Agrobacterium tumefaciens.

[0014] The present invention also provides the use of the polyketide synthase, the gene, the recombinant plasmid, the host cell or the method in producing pigments or polyketide compounds.

[0015] The present invention also provides the use of Phomopsis asparagus NCXT3 in producing polyketide synthase, pigment or polyketide compound. The amino acid sequence of the polyketide synthase is shown in SEQ ID NO.1, and the deposit number of Phomopsis asparagus NCXT3 is CGMCC No.40315.

[0016] The technical solution of the present invention has the following advantages:

[0017] 1. This invention provides a polyketide synthase, the amino acid sequence of which is shown in SEQ ID NO. 1. Studies have shown that, compared with the wild-type Asparagus strain XT3, the pigment production of the Pks-pa gene-encoding polyketide synthase knockout strain XT3-KN from Asparagus strain XT3 is significantly reduced. Furthermore, compared with the Pks-pa gene-encoding polyketide synthase knockout strain XT3-KN from Asparagus strain XT3, the pigment production capacity of the Pks-pa gene-encoding polyketide synthase introduced from Asparagus strain XT3 is significantly improved. This indicates that the polyketide synthase with the amino acid sequence shown in SEQ ID NO. 1 and the gene encoding the polyketide synthase with the nucleotide sequence shown in SEQ ID NO. 2 play an important role in the pigment production process of Asparagus strain. Therefore, the polyketide synthase with the amino acid sequence shown in SEQ ID NO. 1 and the gene encoding the polyketide synthase with the nucleotide sequence shown in SEQ ID NO. 2 have great application prospects in pigment production. Moreover, polyketide synthase can catalyze the synthesis of polyketide compounds. Therefore, the polyketide synthase with an amino acid sequence as shown in SEQ ID NO.1 and the gene encoding the polyketide synthase with a nucleotide sequence as shown in SEQ ID NO.2 also have great application prospects in the production of polyketide compounds.

[0018] Biomaterial Deposit

[0019] A strain of Phomopsis asparagi NCXT3, taxonomically named Phomopsis asparagi, was deposited in the General Microbiology Center of the China Culture Collection Administration on September 16, 2022, with the deposit number CGMCC No. 40315, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 : PCR amplification results of genomic DNA of Phomopsis asparagus strains XT1~XT4. Figure 1 In the figure, A: PCR was performed using degenerate primers LC1 / LC2 to identify the WA type; B: PCR was performed using degenerate primers LC3 / LC4 to identify the PMSAS type.

[0021] Figure 2 : Cloning of the full-length cDNA sequence of the polyketide synthase encoding gene. Figure 2In the figure, A: total RNA of Phomopsis asparagus strain XT3; B: RT-PCR amplification results, M is DNA marker DL2000, 1 is Phomopsis asparagus strain XT3, and 2 is ddH2O; C is the amplification result of the 3' end sequence, M is DNA marker DL2000, 1 is Phomopsis asparagus strain XT3, and 2 is ddH2O; D is the amplification result of the 5' end sequence, M is DNA marker DL2000, 1 is Phomopsis asparagus strain XT3, and 2 is ddH2O.

[0022] Figure 3 : Cloning of the full-length gDNA sequence of the polyketide synthase encoding gene. Figure 3 In the figure, A: PCR amplification results of Phomopsis asparagus strain XT3, M is DNA marker DL2000, 1 to 7 are the PCR amplification results of Phomopsis asparagus strain XT3 using primer pairs NGP1F / R, NGP2F / R, NGP3F / R, NGP4F / R, NGP5F / R, NGP6F / R and NGP7F / R, respectively, 8 is ddH2O; B: TAIL-PCR amplification results of polyketide synthase encoding genes using primers SP1, SP2 and SP3, respectively, M1 is λ-HindIII digest; 1-3 are the amplification results of the 1st, 2nd, and 3rd rounds of TAIL-PCR using AP1 primers; 4-6 are the amplification results of the 1st, 2nd, and 3rd rounds of TAIL-PCR using AP2 primers; 7-9 are the amplification results of the 1st, 2nd, and 3rd rounds of TAIL-PCR using AP3 primers; 10-12 are the amplification results of the 1st, 2nd, and 3rd rounds of TAIL-PCR using AP4 primers; 13-15 are positive controls for SP1, SP2, and SP3 primers; M2 is DNA Marker DL2000; C: PCR amplification results using primers designed using existing gDNA sequences and sequences obtained by TAIL-PCR; M is DNA marker DL2000; 1 is from Phomopsis asparagus strain XT3; and 2 is ddH2O; D: Amplification results verifying the sequences obtained by TAIL-PCR; M1 is λ-HindIII digest; 1 is the amplification result from GP2 / GYR1 primers; and M2 is DNA Marker DL2000.

[0023] Figure 4 : Intron analysis of polyketide synthase encoding genes (gDNA 1-600).

[0024] Figure 5 : Intron analysis of polyketide synthase encoding genes (gDNA: 601-1200).

[0025] Figure 6Intron analysis of the polyketide synthase encoding gene (gDNA 1201–1800).

[0026] Figure 7 : Intron analysis of polyketide synthase encoding genes (gDNA 1801-2400).

[0027] Figure 8 : Intron analysis of polyketide synthase encoding genes (gDNA 2401-3000).

[0028] Figure 9 Intron analysis of the polyketide synthase encoding gene (gDNA 3001-3600).

[0029] Figure 10 Intron analysis of the polyketide synthase encoding gene (gDNA 3601–4200).

[0030] Figure 11 Intron analysis of the polyketide synthase encoding gene (gDNA 4201–4800).

[0031] Figure 12 : Intron analysis of polyketide synthase encoding genes (gDNA 4801-5400).

[0032] Figure 13 Intron analysis of the polyketide synthase encoding gene (gDNA 5401-6000).

[0033] Figure 14 Intron analysis of the polyketide synthase encoding gene (gDNA 6001-6600).

[0034] Figure 15 : Intron analysis of polyketide synthase encoding genes (gDNA 6601-7200).

[0035] Figure 16 : Intron analysis of polyketide synthase encoding genes (gDNA 7201-7800).

[0036] Figure 17 Intron analysis of the polyketide synthase encoding gene (gDNA 7801-8400).

[0037] Figure 18 Intron analysis of the polyketide synthase encoding gene (gDNA 8401-9000).

[0038] Figure 19 : Intron analysis of polyketide synthase encoding genes (gDNA 9001-9273).

[0039] Figure 20 : Sequence alignment of polyketide synthase genes cDNAs from P. asparagi and Phomopsis sp. (GenBank accession number HQ197938). Figure 20 There are 28 different bases in total.

[0040] Figure 21 : Phylogenetic analysis of polyketide synthase-encoding genes. Figure 21In the expression vectors, ‘Aspergillus nigerXM001393410.2’ represents the Aspergillus niger polyketide synthase-encoding gene (GenBank accession number XM001393410.2), ‘Aspergillus oryzae XM 001825112.2’ represents the Aspergillus oryzae polyketide synthase-encoding gene (GenBank accession number XM 001825112.2), ‘Aspergillus flavus XM002376376.1’ represents the Aspergillus flavus polyketide synthase-encoding gene (GenBank accession number XM002376376.1), and ‘Aspergillus clavatus XM 001275050.1’ represents the Aspergillus clavatus polyketide synthase-encoding gene (GenBank accession number XM 001275050.1, ‘Phomopsis asparagi XM001275050’ represents the gene encoding polyketide synthase of Phomopsis asparagi (GenBank accession number XM001275050), ‘Phomopsis sp.EU862539.1’ represents the gene encoding polyketide synthase of Phomopsis sp. (GenBank accession number EU862539.1), ‘Penicillium griseofulvum U89769.1’ represents the gene encoding polyketide synthase of Penicillium griseofulvum (GenBank accession number U89769.1), ‘Colletotrichum higginsianumAB508803.1’ represents the gene encoding polyketide synthase of Colletotrichum higginsianum (GenBank accession number AB508803.1), ‘Colletotrichum lagenarium D83643.1' represents the gene encoding the polyketide synthase of Colletotrichum lagenarium (GenBank accession number D83643.1), 'Gibberella fujikuroi AJ278141.1' represents the gene encoding the polyketide synthase of Gibberella fujikuroi (GenBank accession number AJ278141.1), and 'Gibberella zeae AY706311.1' represents the gene encoding the polyketide synthase of Gibberella zeae (GenBank accession number AY706311.1).

[0041] Figure 22 Prediction of polyketide synthase protein. Figure 22 It includes hydrophilic / hydrophobic properties, alpha region, beta region, turn region, coil region, alpha amphiphilic region, beta amphiphilic region and flexible region.

[0042] Figure 23 : PCR amplification results of PKSF and PKSR of wild-type Phomopsis asparagus strain XT3. Figure 23 Middle, M: DNA marker DL1000, 1-3: PCR amplification of PKSF, 4: ddHO2 (primer pair SF1 / SR1), 5-7: PCR amplification of PKSR, 8: ddHO2 (primer pair XF1 / XR1).

[0043] Figure 24 : Plasmid map of the gene knockout vector myp821-pks-pa.

[0044] Figure 25 :The color production results of wild-type Phomopsis asparagus strain XT3 and the polyketide synthase encoding gene pks-pa knockout strain XT3-KN derived from Phomopsis asparagus strain XT3. Figure 25 Middle, A: wild-type Phomopsis asparagus strain XT3; B: polyketide synthase encoding gene pks-pa knockout strain XT3-KN derived from Phomopsis asparagus strain XT3.

[0045] Figure 26 :The color production results of the polyketide synthase encoding gene pks-pa knockout strain XT3-KN from Phomopsis asparagus strain XT3 and the polyketide synthase encoding gene pks-pa introduced strain XT3-KN-RV from Phomopsis asparagus strain XT3. Figure 26 In the middle, A: the polyketide synthase encoding gene pks-pa from the Phomopsis asparagus strain XT3 was knocked out in the strain XT3-KN; B: the polyketide synthase encoding gene pks-pa from the Phomopsis asparagus strain XT3 was introduced into the strain XT3-KN-RV. DETAILED DESCRIPTION

[0046] The following examples are provided for a better understanding of the present invention and are not intended to limit the best mode of implementation. They do not limit the content and scope of protection of the present invention. Any product identical or similar to the present invention obtained by anyone under the guidance of the present invention or by combining the features of the present invention with other prior arts shall fall within the scope of protection of the present invention.

[0047] If no specific experimental steps or conditions are specified in the following examples, the experiments were carried out according to the conventional experimental steps or conditions described in the literature in the field. If no manufacturer is specified for the reagents or instruments used, they are all commercially available conventional reagents.

[0048] The culture medium and its preparation process involved in the following embodiments are as follows:

[0049] Potato dextrose agar: Wash and peel 200 g of fresh potatoes, cut into small pieces, add water and boil for 20 minutes (unless the potatoes can be pierced by a glass rod). Filter the extract through four layers of gauze to obtain an extract. Add 20 g of glucose and 15 g of agar to the extract, and add water to 1000 mL to obtain potato dextrose agar.

[0050] Example 1: Acquisition and identification of polyketide synthase and its encoding gene

[0051] 1. Experimental Materials

[0052] 1.1. Extraction of nucleic acids used in the experiment

[0053] The bacterial suspensions of Phomopsis asparagi strains XT1, XT2, XT3 and XT4 (Phomopsis asparagi strains XT1, XT2, XT3 and XT4 are recorded in the literature “YangYQ, Sun Q, Li CM, Chen HF, Zhao F, Huang JH, Zhou JS, Li XM, LanB. Biological Characteristics and Genetic Diversity of Phomopsis asparagi, Causal Agent of Asparagus Stem Blight. Plant Disease, 2020, 104(11): 2898-2904”, wherein Phomopsis asparagi strain XT3 is Phomopsis asparagi NCXT3, with a deposit number of CGMCC No. 40315) were streaked on potato dextrose agar and cultured at 26°C under black light for 6 days to obtain mycelia; genomic DNA (gDNA) was extracted from the mycelia using a universal DNA extraction kit (TaKaRa, Japan); and the genomic DNA was extracted using a Takara DNA extraction kit (TaKaRa, Japan). Total RNA was extracted from mycelia using the Minibest Plant RNA Extraction Kit (TaKaRa, Japan).

[0054] 1.2. Design of primers used in the experiment

[0055] Based on the sequences of conserved polyketide synthase-encoding genes on the NCBI website (GenBank accession numbers: EU862537.1, EU862538.1, and EU862539.1), four pairs of primers for amplifying putative polyketide synthase-encoding genes were designed using GenBank 5.0 software (primers are shown in Table 1).

[0056] Table 1 Primers used for amplifying the presumed polyketide biosynthesis encoding gene.

[0057] coding Primers Primer sequence (5'→3') Annealing temperature Fragment size PKS-1 PKS-1F / PKS-1R SEQ ID NO.3 / SEQ ID NO.4 54℃ 710bp PKS-2 PKS-2F / PKS-2R SEQ ID NO.5 / SEQ ID NO.6 56℃ 680bp PKS-3 PKS-3F / PKS-3R SEQ ID NO.7 / SEQ ID NO.8 55℃ 735bp PKS-4 PKS-4F / PKS-4R SEQ ID NO.9 / SEQ ID NO.10 54℃ 480bp

[0058] 2. Experimental process

[0059] 2.1 Type Identification

[0060] Genomic DNA of Phomopsis asparagus strains XT1, XT2, XT3, and XT4 were amplified by PCR using degenerate primers LC1 (SEQ ID NO.11, N is a degenerate base, N=A or G or C or T) / LC2 (SEQ ID NO.12) and LC3 (SEQ ID NO.13) / LC5 (SEQ ID NO.14), respectively. The amplified products were analyzed by agarose gel electrophoresis. Based on the analysis results, the polyketide synthase encoding genes of Phomopsis asparagus strains XT1, XT2, XT3, and XT4 were typed (type identification refers to the literature "Bingle LEH, Simpson TJ, Lazarus CM Ketosynthase domain probes identify two subclasses of offungal polyketide synthase genes [J]. Fungal Genet. Biol., 1999, 26:209-223").

[0061] 2.2. Acquisition of full-length cDNA sequences

[0062] RT-PCR was performed on total RNA from Phomopsis asparagus strains XT1, XT2, XT3, and XT4 using four pairs of primers for amplifying putative polyketide synthase-encoding genes (PKS-1F / PKS-1R, PKS-2F / PKS-2R, PKS-3F / PKS-3R, and PKS-4F / PKS-4R). cDNAs encoding putative polyketide synthase genes from these strains were obtained. RT-PCR was performed in a 20 μL volume, including 5 μL of 2×1Step Buffer, 1 μL of each upstream and downstream primer, 0.5 μL of PrimeScript 1Step Enzyme Mix, 1 μL of total RNA, and 11.5 μL of RNase-free ddH2O.

[0063] The amplified product of total RNA (i.e., the cDNA of the putative polyketide synthase encoding gene) was separated by agarose gel electrophoresis (1%, g / 100 mL). The expected band was extracted from the gel using a micro agarose gel DNA extraction kit (TaKaRa, Japan), and the expected band was purified using a PCR purification kit (Qiagen, Germany) to obtain a purified total RNA product. The PMD20-T vector (TaKaRa, Japan) and the purified total RNA product were digested with the restriction endonuclease EcoR V, and the digested PMD20-T vector and the purified total RNA product were ligated to obtain recombinant plasmid PMD20-T-purified product-1. The recombinant plasmid PMD20-T-purified product-1 was transformed into Escherichia coli DH5α competent cells (purchased from TaKaRa, Japan) to obtain recombinant Escherichia coli DH5α / PMD20-T-purified product-1.

[0064] by Using the RACE 5' / 3' Kit (TaKaRa, Japan), PrimeScript 1st Strand cDNASynthesis Kit (TaKaRa, Japan), Tks Gflex DNA Polymerase (TaKaRa, Japan), and MiniBESTAgarose Gel DNA Extraction Kit (TaKaRa, Japan) as auxiliary reagents, the 3'-RACE and 5'-RACE genes of recombinant Escherichia coli DH5α / PMD20-T-purified product-1 were sequenced.

[0065] Oligo dT-anchor primer was used as a primer and total RNA was used as a template to synthesize the first-strand cDNA using PrimeScript RTase (TaKaRa, Japan); outer PCR amplification of the first-strand cDNA was performed using 3'-RACE outer primer CF4 (SEQ ID NO.17), and inner PCR amplification of the first-strand cDNA was performed using 3'-RACE inner primer CF5 (SEQ ID NO.18) to obtain the 3' end sequence; wherein, outer PCR and inner PCR were both performed in a volume of 50 μL, including 10 mM Tris-HCl (pH 8.4), 1.8 mM MgCl2, 50 mM KCl, 0.20 mM each dNTP, 0.25 μM each primer, 20 ng of cDNA product and 5 U of Tks Gflex DNA Polymerase (TaKaRa, Japan); outer PCR was performed using a cycle of 94°C for 1 min, 98°C for 10 s, 55°C for 15 sec, and 68°C for 1 min; Inner PCR was performed using a cycle of 94°C for 1 min, 98°C for 10 s, 55°C for 15 sec, and 68°C for 3 min.

[0066] First-strand cDNA was synthesized from total RNA using SMARTScribe Reverse Transcriptase (TaKaRa, Japan) and cDNA synthesis primer CR2 (SEQ ID NO.19); outer PCR amplification of the first-strand cDNA was performed using 5'-RACE outer primer CR6 (SEQ ID NO.20), and inner PCR amplification of the first-strand cDNA was performed using LATaq enzyme (TaKaRa, Japan) and 5'-RACE inner primer CR7 (SEQ ID NO.21) to obtain 5' end sequences; both outer PCR and inner PCR were performed in a volume of 50 μL, including 10 mM Tris-HCl (pH 8.4), 1.8 mM MgCl2, 50 mM KCl, 0.20 mM of each dNTP, 0.25 μM of each primer, 20 ng of cDNA product, and 5 U of Tks Gflex DNA Polymerase (TaKaRa, Japan); outer PCR amplification was performed using LATaq enzyme (TaKaRa, Japan) and 5'-RACE inner primer CR7 (SEQ ID NO.21). PCR was performed at 94°C for 1 min, 98°C for 10 s, 55°C for 15 sec, and 68°C for 1 min; InnerPCR was performed at 94°C for 1 min, 98°C for 10 s, 55°C for 15 sec, and 68°C for 3 min.

[0067] The 3' and 5' end sequences were amplified by PCR using the primer pair YZF (SEQ ID NO. 22) / YZR (SEQ ID NO. 23) to obtain the full-length cDNA sequence of the polyketide synthase-encoding gene pks-pa. PCR was performed in a 50 μL volume containing 10 mM Tris-HCl (pH 8.4), 1.8 mM MgCl2, 50 mM KCl, 0.20 mM of each dNTP, 0.25 μM of each primer, 2 μL of reverse transcription reaction solution, and 5 U of Tks Gflex DNA Polymerase (TaKaRa, Japan). PCR was performed using cycles of 94°C for 1 min, 98°C for 10 s, 55°C for 15 sec, and 68°C for 1 min.

[0068] 2.3 Obtaining the full-length gDNA sequence

[0069] With reference to the literature "Sambrook, J., Fritsch, EF, & Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2nd ed [M]. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY", genomic DNA (gDNA) of Phomopsis asparagus strain XT3 was PCR amplified using primers GPS-1, GPS-2, GPS-3, GPS-4, GPS-5, GPS-6 and GPS-7 (primers are shown in Table 2); wherein, PCR amplification was performed in a volume of 20 μL, including 10 mM Tris-HCl (pH 8.4), 1.8 mM MgCl2, 50 mM KCl, 0.20 mM of each dNTP, 0.25 μM of each primer, 2 μL reverse transcription reaction solution and 5 U of Tks Gflex. DNA Polymerase (TaKaRa, Japan); PCR amplification used annealing at 95°C for 2 min, followed by 35 cycles of 94°C for 30 s, temperature change from 54°C to 56°C for 40 s, and finally extension at 72°C for 10 min.

[0070] Genomic DNA amplification products were separated by agarose gel electrophoresis (1%, g / 100 mL). The expected band was extracted from the gel using a micro-agarose gel DNA extraction kit (TaKaRa, Japan), and the expected band was purified using a PCR purification kit (Qiagen, Germany) to obtain purified genomic DNA. The PMD20-T vector (TaKaRa, Japan) and the purified genomic DNA were digested with restriction endonuclease EcoR V. The digested PMD20-T vector and the purified genomic DNA were then ligated to obtain recombinant plasmid PMD20-T-purified product-2. The recombinant plasmid PMD20-T-purified product-2 was transformed into Escherichia coli DH5α competent cells (purchased from TaKaRa, Japan) to obtain recombinant Escherichia coli DH5α / PMD20-T-purified product-2.

[0071] Using Tks Gflex DNA Polymerase (TaKaRa, Japan) and MiniBEST Agarose Gel DNA Extraction Kit (TaKaRa, Japan) as auxiliary reagents, and employing the Genome Walking Kit (TaKaRa, Japan), first, second, and third rounds of TAIL-PCR were performed on recombinant E. coli DH5α / PMD20-T-purified product-2 using primers SP1 (SEQ ID NO.24), SP2 (SEQ ID NO.25), and SP3 (SEQ ID NO.26), respectively. The TAIL-PCR products were obtained by performing the TAIL-PCR in 50 μL volumes, including 20 ng genomic DNA, 8 μL dNTP Mixture (2.5 mM each), 5 μL 10x LA Taq Buffer II, and LA Taq. 0.5 μL, 1 μL of upstream and downstream primers, add ddH2O to 50 μL; TAIL-PCR adopted 94℃1 min, 98℃1 min, then 5 cycles (94℃30 s, temperature changed from 60℃ to 68℃1 min, 72℃3 min), 94℃30 s, 25℃3 min, 72℃3 min, then 15 cycles (94℃30 s, temperature changed from 60℃ to 68℃1 min, 72℃3 min), and finally extension at 72℃ for 10 min.

[0072] The amplified product of recombinant Escherichia coli DH5α / PMD20-T-purified product-2 was separated by agarose gel electrophoresis (1%, g / 100 mL), and the expected band was extracted from the gel using a micro agarose gel DNA extraction kit (TaKaRa, Japan), and the expected band was purified using a PCR purification kit (Qiagen, Germany) to obtain the purified product of recombinant Escherichia coli DH5α / PMD20-T-purified product-2; the restriction endonuclease EcoR V enzymatically digested the PMD20-T vector (TaKaRa, Japan) and the purified product of recombinant Escherichia coli DH5α / PMD20-T-purified product-2, and then ligated the enzymatically digested PMD20-T vector and the purified product to obtain recombinant plasmid PMD20-T-purified product-3; the recombinant plasmid PMD20-T-purified product-3 was transformed into Escherichia coli DH5α competent cells (purchased from TaKaRa, Japan) to obtain recombinant Escherichia coli DH5α / PMD20-T-purified product-3.

[0073] by The RACE 5′ / 3′ Kit (TaKaRa, Japan), PrimeScript 1st Strand cDNA Synthesis Kit (TaKaRa, Japan), Tks Gflex DNA Polymerase (TaKaRa, Japan), and MiniBESTAgarose Gel DNA Extraction Kit (TaKaRa, Japan) were used as auxiliary reagents to perform gene sequencing on the recombinant Escherichia coli DH5α / PMD20-T-purified product-3.

[0074] Referring to the literature "Sambrook, J., Fritsch, EF, & Maniatis, T. (1989). Molecular cloning: a laboratory manual, 2nd ed [M]. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY", the purified product was amplified by PCR using GP1 (SEQ ID NO. 27) / GP2 (SEQ ID NO. 28) as primers to obtain the full-length gDNA sequence of the polyketide synthase encoding gene pks-pa. The PCR amplification was performed in a 50 μL volume, including 10 mM Tris-HCl (pH 8.4), 1.8 mM MgCl2, 50 mM KCl, 0.20 mM each dNTP, 0.25 μM each primer, 20 ng of cDNA product, and 5 U of Tks Gflex DNA Polymerase (TaKaRa, Japan). The PCR amplification was performed by cycling at 94 °C for 1 min, 98 °C for 10 s, 55 °C for 15 sec, and 68 °C for 1 min.

[0075] To verify the full-length gDNA sequence of the polyketide synthase-encoding gene pks-pa, genomic DNA (gDNA) of Phomopsis asparagus strain XT3 was amplified by PCR using primers GP2 (SEQ ID NO. 29) / GYR1 (SEQ ID NO. 30). The PCR amplification products were obtained and analyzed by agarose gel electrophoresis.

[0076] Table 2 Primers used to amplify genes encoding polyketide biosynthesis

[0077]

[0078]

[0079] 2.4 Analysis and prediction of polyketide synthase-encoding genes

[0080] The coding region of the full-length cDNA sequence encoding the polyketide synthase gene was analyzed using DNAstar software. The full-length cDNA sequence of the polyketide synthase gene was aligned with the full-length gDNA sequence using GeneDoc software to identify introns in the gDNA sequence. Sequences of polyketide synthase genes from closely related fungi of Phomopsis asparagus strain XT3 were analyzed using GeneDoc software. A phylogenetic tree was constructed using MEGA4 software to analyze the phylogenetic relationships of polyketide synthase genes from Phomopsis asparagus strain XT3 and other closely related fungi. Protein prediction of the polyketide synthase from Phomopsis asparagus strain XT3 was performed using BioEditor software.

[0081] 2.5. Vector construction and genetic transformation

[0082] Primers were designed using Primer 5.0 software based on the full-length cDNA and gDNA sequences of the polyketide synthase encoding gene pks-pa from Phomopsis asparagi strain XT3. SF1 (SEQ ID NO. 45) containing a Hind III restriction site and its protective gene (CCCAAGCTT) was used as an upstream primer, and SR1 (SEQ ID NO. 46) containing a Pst I restriction site and its protective gene (AACTGCAG) was used as a downstream primer. The PKSF fragment (upstream fragment of the polyketide synthase encoding gene pks-pa in the gene knockout vector) was amplified using total RNA as a template. The PKSR fragment (downstream fragment of the polyketide synthase encoding gene pks-pa in the gene knockout vector) was amplified using upstream primer XF2 (SEQ ID NO. 47) and downstream primer XR2 (SEQ ID NO. 48). PCR amplification was performed in a 50 μL volume containing 10 mM Tris-HCl (pH 8.4), 1.8 mM MgCl2, 50 mM KCl, 0.25 mM each dNTP, 0.2 μM each primer, 20 ng template DNA and 1 U LATaq (TaKaRa, Japan); PCR amplification was performed for 30 cycles (98°C for 10 s, 55°C for 15 s, and 72°C for 1 min).

[0083] PCR products of the PKSF fragment were separated by agarose gel electrophoresis (1%, g / 100 mL). The expected band was extracted from the gel using a Gel Extraction Kit (OMEGA, USA), and the expected band was purified using a PCR purification kit (Qiagen, Germany) to obtain the purified PKSF fragment. DNA of plasmid myp821 (obtained from South China Agricultural University, with kanamycin and hygromycin antibiotics) was extracted from overnight cultured *E. coli* DH5α using a MiniBEST Plasmid Purification Kit Ver. 2.0 (TaKaRa, Japan). Restriction endonucleases Hind III and Pst... I After enzymatic digestion of the plasmid DNA and the purified product of the PKSF fragment, the enzymatically digested plasmid DNA and the purified product were ligated using T4-DNA ligase (TaKaRa, Japan) to insert the PKSF fragment upstream of the myp821 fragment of the ligation product; the above operation was repeated to insert the PKSR fragment downstream of the myp821 fragment of the ligation product to obtain the gene knockout vector myp821-pks-pa for the polyketide synthase encoding gene pks-pa.

[0084] PCR amplification of the pks-pa gene knockout vector myp821-pks-pa was performed using hph gene-specific primer pair ph-F (SEQ ID NO.49) / ph-R (SEQ ID NO.50), specific primer pair SF1 (SEQ ID NO.51) / SR1 (SEQ ID NO.52) for amplifying the pks-pa fragment PKSF of the polyketide synthase gene, and specific primer pair XF2 (SEQ ID NO.53) / XR2 (SEQ ID NO.54) for amplifying the pks-pa fragment PKSR of the polyketide synthase gene. PCR amplification was performed in a 20 μL volume containing 10 mM Tris-HCl (pH 8.4), 1.5 mM MgCl2, 50 mM KCl, 0.25 mM per dNTP, 0.2 μM per primer, and 20 ng of template. DNA and 1U of Taq DNA polymerase (TaKaRa, Japan); PCR amplification using hph gene-specific primer pairs was performed at 95℃ for 2 min, 30 cycles (94℃ for 40 s, 56℃ for 40 s, 72℃ for 1 min), and a final amplification at 72℃ for 10 min; PCR amplification using specific primer pairs for the polyketide synthase gene pks-pa PKSF and PKSR fragments was performed at 95℃ for 2 min, 35 cycles (94℃ for 35 s, 55℃ for 40 s, 72℃ for 1 min), and a final amplification at 72℃ for 10 min.

[0085] With reference to the literature “Yang YQ, Yang M., Li MH, Li Y., He XX, Zhou EX (2011). Establishment of Agrobacterium tumefaciens-mediated transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1IA [J]. Rice Sci., 18, 297-303”, the amplified product of the gene knockout vector myp821-pks-pa (the nucleotide sequence of the gene knockout vector myp821-pks-pa is shown in SEQ ID NO.55) was transformed into the protoplasts of Phomopsis asparagus strain XT3 to obtain transformants carrying the hph gene primer; with reference to the literature “Yang YQ, Yang M., Li M.H., Li Y., He XX, Zhou EX (2011). Establishment of Agrobacterium tumefaciens-mediated transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1IA [J]. Rice Sci., 18, 297-303”, the amplified product of the gene knockout vector myp821-pks-pa (the nucleotide sequence of the gene knockout vector myp821-pks-pa is shown in SEQ ID NO.55) was transformed into the protoplasts of Phomopsis asparagus strain XT3 to obtain transformants carrying the hph gene primer; solani AG-1IA[J].Rice Sci.,18,297-303” were used to verify the transformants carrying the hph gene primers. To exclude the contamination of plasmid DNA, the references were “Yang YQ, Yang M., Li MH, Li Y., He X.X., Zhou EX (2011). Establishment of Agrobacterium tumefaciens-mediated transformation system for rice sheath blight pathogen Rhizoctonia solani AG-1IA[J].Rice Sci.,18,297-303” and “H., Ieki H., Matsuda I. PCR detection of Ti and Ri plasmids from phytopathogenic Agrobacterium strains[J]."Appl EnvironMicrobiol, 1995, 61(2):828-831" Transformants carrying hph gene primers were cultured for 5 generations on PDA plates, and then verified by PCR using Vir gene primers to obtain the XT3-KN strain, a polyketide synthase gene-encoding gene knockout strain derived from the Asparagus spp. strain XT3.

[0086] 2.6 Verification of pathogenicity and pigment production

[0087] References to the literature "Yang YQ, Li JH,Zhou JS,LiX.M.,Lan B.(2020).Biological Characteristics and Genetic DiversityofPhomopsis asparagi,CausalAgent ofAsparagus Stem Blight[J].Plant Disease, 104(11): 2898-2904”, the conidia spray method was used to compare the virulence difference between the wild-type Asparagus cochinchinensis strain XT3 and the Asparagus cochinchinensis strain XT3-KN with the polyketide synthase encoding gene pks-pa knocked out; if the virulence of the Asparagus cochinchinensis strain XT3-KN with the polyketide synthase encoding gene pks-pa knocked out was significantly weaker than that of the wild-type Asparagus cochinchinensis strain XT3, it can be verified that the polyketide synthase encoding gene pks-pa from the Asparagus cochinchinensis strain XT3 is role in pathogenicity; the wild-type Phomopsis asparagus strain XT3 and the polyketide synthase encoding gene pks-pa knockout strain XT3-KN derived from Phomopsis asparagus strain XT3 were cultured on PDA plates at 26°C for 10 days, and their color production abilities were compared; if the pigment production of the polyketide synthase encoding gene pks-pa knockout strain XT3-KN derived from Phomopsis asparagus strain XT3 was significantly lower than that of the wild-type Phomopsis asparagus strain XT3, the role of the polyketide synthase encoding gene pks-pa derived from Phomopsis asparagus strain XT3 in pigment production can be verified.

[0088] 2.7. Construction of gene transfer vectors and genetic transformation

[0089] The pMD18-T vector (TaKaRa, Japan) and the full-length cDNA sequence of the polyketide synthase encoding gene pks-pa from Phomopsis asparagi strain XT3 were digested with the restriction endonuclease EcoRV, and the digested pMD18-T vector and the full-length cDNA sequence were ligated to obtain the gene introduction vector pMD18-pks-pa. I. After enzyme digestion of the gene introduction vector pMD18-pks-pa and the pUCATPH co-transformation vector with the hygromycin B (HygB) resistance gene (recorded in the document "Beijing Tian Enze Gene Technology Co., Ltd."), the enzyme-digested gene introduction vector pMD18-pks-pa and the pUCATPH co-transformation vector were co-transformed into the protoplasts of the pks-pa knockout transformant of the polyketide synthase encoding gene of the Asparagus asparagus strain XT3, thereby obtaining transformants in which the polyketide synthase encoding gene pks-pa from the Asparagus asparagus strain XT3 was restored; transformants were screened using a PDA plate containing 100 μg / mL hygromycin B, single spores of the transformants were isolated, and genomic DNA was extracted for PCR verification to obtain the strain XT3-KN-RV in which the polyketide synthase encoding gene pks-pa from the Asparagus asparagus strain XT3 was introduced. The polyketide synthase encoding gene pks-pa knockout strain XT3-KN from Phomopsis asparagus strain XT3 and the polyketide synthase encoding gene pks-pa introduced strain XT3-KN-RV from Phomopsis asparagus strain XT3 were cultured on PDA plates at 26℃ for 6 days to compare their color production abilities.

[0090] 3. Experimental results

[0091] 3.1 Type Identification

[0092] like Figure 1 As shown, the genomic DNA of Phomopsis asparagus strains XT1, XT2, XT3 and XT4 can all amplify an 800 bp band using the degenerate primers LC1 / LC2, but cannot amplify a band using the degenerate primers LC3 / LC5, which proves that the polyketide biosynthesis type of the polyketide synthase encoding gene in Phomopsis asparagus strains XT1, XT2, XT3 and XT4 is the iterative type I WA subtype.

[0093] 3.2. Acquisition of full-length cDNA sequences

[0094] like Figure 2As shown, all four primer pairs used to amplify the putative polyketide synthase encoding gene were able to amplify the total RNA of *Asparagus cochinchinensis* strains XT1, XT2, XT3, and XT4. The total RNA was cloned into *E. coli* DH5α, and the corresponding cDNA sequences were obtained after sequencing. The shock assay results showed that all obtained cDNA sequences were indeed encoding the polyketide synthase gene. However, these four sequences could not be spliced ​​together; the longest gDNA sequence, 733 bp, was obtained from the primer pair PKS-3F / PKS-3R. To obtain the full-length cDNA sequence of the polyketide synthase encoding gene, 3'-RACE and 5'-RACE experiments were performed, yielding cDNA fragments of approximately 6.18 kb and 2.21 kb, respectively. Subsequently, the two cDNA fragments were cloned into *E. coli* DH5α, and the corresponding cDNA sequences were obtained by sequencing the positive transformants. The above three sequences were derived from RT-PCR, 3'-RACE and 5'-RACE, respectively. They were assembled together to obtain a 9.082 kb full-length cDNA sequence of the polyketide synthase encoding gene (SEQ ID NO. 15).

[0095] 3.3. Acquisition of full-length gDNA sequences

[0096] like Figure 3 As shown, bands of approximately 1.65 kb, 0.68 kb, 2.00 kb, 0.85 kb, 1.05 kb, 1.20 kb, and 2.15 kb were amplified from the genomic DNA (gDNA) of *Pseudomonas asparagus* strain XT3. The relevant gDNA fragments were cloned into *E. coli* DH5α competent cells, and the corresponding gDNA sequences were obtained by sequencing positive transformants. TAIL-PCR results showed that the third round of AP1 amplified a band of approximately 0.75 kb, and the third round of AP4 amplified a band of approximately 0.70 kb, which were confirmed as the target bands by sequencing and alignment. Primers were designed based on the obtained sequences for PCR, yielding a bandwidth of approximately 0.75 kb, and the relevant sequence was obtained after sequencing. PCR verification using primers for GP2 / GYR1 yielded a band of approximately 2.20 kb, consistent with the expected result. The fragment was cloned and sequenced to obtain the sequence, which was then compared to the existing sequence to confirm that the fragment was continuous. The obtained sequence fragments were assembled together to obtain a 9.336 kb full-length gDNA sequence (SEQ ID NO. 16).

[0097] 3.4 Analysis and prediction of polyketide synthase-encoding genes

[0098] The full-length cDNA sequence encoding the pks-pa gene (9.082 kb) was obtained by RT-PCR, 3'-RACE, 5'-RACE and sequence splicing. The ORF search results showed that the cDNA sequence encoding the pks-pa gene contained a complete ORF (7.752 kb). The full-length gDNA sequence of the pks-pa gene (9.336 kb) was obtained by PCR, TAIL-PCR and sequence assembly. Intron analysis results showed that there were 5 introns in the gDNA sequence of the polyketide synthase encoding gene pks-pa ( Figures 4 to 19 ).

[0099] The full-length cDNA sequence of the polyketide synthase encoding gene pks-pa from Phomopsis asparagus strain XT3 was compared with the full-length cDNA sequence of the polyketide synthase encoding gene from Phomopsis sp. (NCBI accession number: HQ197938). The results showed that there was a 28-base difference in the polyketide synthase encoding genes between the two ( Figure 20 ).

[0100] Phylogenetic analysis of polyketide synthase encoding genes of fungi such as Asparagus asparagus revealed that polyketide synthase encoding genes of different fungi of the same genus clustered on the same branch, i.e. Aspergillus, Gibberella, Penicillium, Colletotrichum and Phomopsis were clustered on the same branch ( Figure 21 ), indicating that there is polymorphic differentiation in polyketide synthase encoding genes among different fungi.

[0101] Protein prediction results show that the biosynthesis of polyketides is hydrophilic, and there are α region, β region, turn region, coil region, α amphiphilic region, β amphiphilic region and active region ( Figure 22 ).

[0102] 3.5 Vector construction and genetic transformation

[0103] The PKSF fragment was PCR amplified using the SF1 / SR1 primer pair, yielding a fragment of approximately 1.00 kb (the target band was extracted from the PCR gel). The PKSF fragment was inserted into the plasmid myp821 between the Hind III and Pst I sites on one side, yielding the intermediate construct myp821-pksf. The PKSR fragment was PCR amplified using the same method, yielding another fragment of approximately 1.00 kb (the target band was extracted from the PCR gel). Figure 23). The PKSR fragment was inserted into the vector myp821-PKSF to obtain the gene knockout vector myp821-pks-pa ( Figure 24 ).

[0104] Specific PCR amplification of the hph gene showed that the expected band of approximately 500 bp was amplified from all genomic DNA extracted from the strain XT3-KN (pks-pa knockout strain for polyketide synthase), while no band was amplified from the wild-type strain XT3. A single expected band of approximately 730 bp was amplified from genomic DNA extracted from strains AGL-1, EHA105, MP90, and LBA4404, while no corresponding sequence was detected in the DNA bands amplified from the wild-type strain XT3 and the strain XT3-KN (pks-pa knockout strain for polyketide synthase), indicating that the genomic DNA from the wild-type strain XT3 and the strain XT3-KN (pks-pa knockout strain for polyketide synthase) were not contaminated with Agrobacterium tumefaciens.

[0105] 3.6 Verification of pathogenicity and pigment production

[0106] The results of pathogenicity (virulence) assays of wild-type Phomopsis asparagus strain XT3 and the strain XT3-KN, in which the polyketide synthase gene pks-pa was knocked out, showed that the disease index of the strain XT3-KN, in which the polyketide synthase gene pks-pa was knocked out, did not improve significantly. Compared with wild-type Phomopsis asparagus strain XT3, the strain XT3-KN, in which the polyketide synthase gene pks-pa was knocked out, showed a significant decrease in pigment production ( Figure 25 ), indicating that the polyketide synthase encoding gene pks-pa from Phomopsis asparagus strain XT3 plays an important role in the pigment production process of Phomopsis asparagus.

[0107] 3.7. Construction of gene transfer vectors and genetic transformation

[0108] Compared with the XT3-KN strain, which had its polyketide synthase gene pks-pa knocked out from XT3 of *Pseudomonas asparagus* strain XT3, the pigment production capacity of the XT3-KN-RV strain, which had its polyketide synthase gene pks-pa introduced from XT3 of *Pseudomonas asparagus* strain XT3, was significantly improved. Figure 26 ), further indicating that the polyketide synthase encoding gene pks-pa from Phomopsis asparagus strain XT3 plays an important role in the pigment production process of Phomopsis asparagus.

[0109] Obviously, the above embodiments are merely examples for clarity of explanation and are not intended to limit the implementation methods. Those skilled in the art will readily appreciate that other variations or modifications based on the above descriptions are possible. It is not necessary and impossible to enumerate all implementation methods here. Obvious variations or modifications arising therefrom remain within the scope of protection of the present invention.

Claims

1. A polyketide synthase, characterized in that The amino acid sequence of the polyketide synthase is shown in SEQ ID NO.

1.

2. A gene, characterized in that The gene encodes the polyketide synthase according to claim 1.

3. A gene according to claim 2, characterized in that The nucleotide sequence of the gene is shown in SEQ ID NO.

2.

4. A recombinant plasmid, characterized in that: The recombinant plasmid carries the gene according to claim 2 or 3.

5. The recombinant plasmid according to claim 4, characterized in that The expression vector of the recombinant plasmid is myp821 plasmid or pMD18 plasmid.

6. A host cell, characterized in that The host cell carries the gene according to claim 2 or 3 or the recombinant plasmid according to claim 4 or 5.

7. A host cell according to claim 6, characterized in that The host cell is Phomopsis asparagus, Escherichia coli or Agrobacterium tumefaciens.

Images