Gene CcUOX of Colletotrichum camelliae and its application

Abstract

The invention provides a CcUOX gene of Colletotrichum camelliae and an application thereof. The CcUOX gene is related to pathogenicity and purine metabolism, belongs to the technical field of microbial genetic engineering, comes from Colletotrichum camelliae, and its cDNA sequence consisting of 906 nucleotides is shown in SEQ ID NO:1; a protein encoded by the provided CcUOX gene has an amino acid sequence consisting of 301 amino acids as shown in SEQ ID NO:2; the CcUOX gene can be applied in the field of tea tree anthracnose resistance genetic engineering, and can be applied in the fields of food science and engineering; an engineering strain expressing the CcUOX gene can be used for the degradation, utilization and environmental restoration of caffeine waste, and can be applied to uric acid degradation; and CcUOX can be used as a gene target in the design and screening of fungicidal bacteria for Colletotrichum camelliae.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial genetic engineering, and specifically relates to a gene of Colletotrichum camelliae CcUOX Applications in the prevention and control of anthracnose in Camellia plants, improvement of plant disease resistance, and food safety. Background Art

[0002] Anthrax Colletotrichum The fungus is an asexual fungus belonging to the class Coelomycetes, the order Melanconiales, the family Melanconiaceae, and its sexual form belongs to the phylum Ascomycota, the family Glomerellaceae, and the genus Glomerellaceae. Glomerella Spauld et Schrenck., belongs to a class of plant pathogenic fungi with global distribution. The host range of this genus is wide, from gymnosperms to angiosperms, from monocots to dicots. This genus of pathogenic fungi often harms the roots, stems, leaves, flowers, fruits and seedlings of forest trees, fruit trees, vegetables, flowers, medicinal plants and field crops, which can cause plant wilt, fruit rot, leaf spots and other symptoms, causing serious economic losses. Based on its scientific and economic importance, this genus of fungi is listed as the eighth largest plant pathogenic fungi in the world.

[0003] Tea contains abundant secondary metabolites such as tea polyphenols, theanine, alkaloids and their glycosides, proanthocyanidins and anthocyanidin compounds, which give tea a special quality. Caffeine is one of the important purine bases in tea, and its structural feature is that the N at the 1, 3, and 7 positions of xanthine is connected to 3 methyl groups. The content and distribution of caffeine vary in different varieties and different parts of different tea trees. Since the caffeine of tea trees is mainly synthesized in chloroplasts, it is mainly distributed in the new shoots of tea trees, and the content gradually decreases with the maturity and aging of the leaves. Studies have shown that caffeine may be related to plant disease resistance. The higher the caffeine content, the stronger the disease resistance.

[0004] Therefore, the study of the interaction between caffeine biosynthesis in tea plants and pathogens is of great significance for revealing the disease resistance mechanism of tea plants. Summary of the invention

[0005] The present invention provides a method for regulating Colletotrichum camelliae ( Colletotrichum camelliae ) The pathogenicity gene is derived from Colletotrichum camelliae, named CcUOX , as shown in SEQ ID NO: 1, CcUOX The gene open reading frame consists of 906 nucleotides, and the total length of the coding region is 301 nucleotides; the camellia thorn colletotrichum CcUOX The amino acid sequence of the CcUOX protein encoded by the gene is shown in SEQ ID NO:2.

[0006] Said CcUOX The deletion of the gene reduces the pathogenicity of Colletotrichum japonicum to host plants. CcUOX The gene and the protein it encodes can be used as targets in the development of anti-Camellia thorny Colletotrichum agents. Screening for compounds that can inhibit the expression, modification and localization of the gene and its protein can effectively control the occurrence of Camellia thorny Colletotrichum and serve as a new fungicide.

[0007] Furthermore, the host plant is one of the plants of the genus Camellia, such as tea tree, oil tea, and camellia.

[0008] Another object of the present invention is to provide a fungus of Colletotrichum camelliae as shown in SEQ ID No.1. CcUOX The gene or the protein encoded by the gene as shown in SEQ ID No. 2 is used as a control target in the preparation of a fungicide for Colletotrichum spp.

[0009] Another object of the present invention is to provide a biocontrol agent for preventing and controlling Colletotrichum camelliae, wherein the biocontrol agent contains a biocontrol agent for preventing or inhibiting Colletotrichum camelliae. CcUOX Genetically engineered vectors for gene expression.

[0010] Furthermore, the biocontrol agent is any one or more of A to C:

[0011] A. Reduce the pathogenicity of Colletotrichum japonicum;

[0012] B. Reduce the tolerance of Colletotrichum camelliae to caffeine;

[0013] C. Reduce the degradation of uric acid.

[0014] Furthermore, the plant is a host plant of Colletotrichum camelliae.

[0015] Furthermore, the biocontrol agent contains Colletotrichum camelliae CcUOX Gene knockout vector.

[0016] Another object of the present invention is to provide a method for preventing and controlling Colletotrichum camelliae, wherein the knockout expression vector blocks or inhibits Colletotrichum camelliae. CcUOX By regulating the expression of genes, biocontrol strains with reduced pathogenicity are obtained.

[0017] The present invention proves CcUOX The deletion of the gene significantly reduces the pathogenicity of Colletotrichum camelliae, reduces tolerance to caffeine, and reduces uric acid metabolism. Therefore, screening compounds that can prevent the expression of the gene and the expression, modification, and localization of its protein can effectively control the occurrence of Colletotrichum camelliae, thereby contributing to the development of new fungicides. That is, the present invention provides CcUOXAn important use of the gene is that the expression of the gene and the protein product it encodes can serve as important candidate target sites for the design and screening of anti-Camellia spinulosa drugs.

[0018] Another object of the present invention is to provide a recombinant protein, which is over-induced by the over-expression vector CcUOX The expression of the gene was used to obtain a complementary strain to reduce uric acid in high-purine foods.

[0019] Furthermore, the high-purine foods are seafood, beer, animal offal, beef and mutton, etc.

[0020] Another object of the present invention is to provide a method for reducing purine alkaloids in food, by inducing Colletotrichum spp. CcUOX The gene is expressed to obtain the expressed protein, which is used to metabolize uric acid in food.

[0021] Another object of the present invention is to provide a method for reducing purine base pollution in soil or water environment, by promoting the overexpression vector CcUOX By regulating the expression of the gene, an overexpression strain with enhanced uric acid metabolism ability is obtained, which is used for environmental management such as repairing caffeine and uric acid.

[0022] The present invention provides a functional gene from Colletotrichum camelliae that has fungal pathogenicity, tolerance to caffeine and can metabolize uric acid CcUOX , whose cDNA sequence consisting of 906 nucleotides is shown in SEQ ID NO: 1; provided CcUOX The protein encoded by the gene has an amino acid sequence consisting of 301 amino acids as shown in SEQ ID NO: 2; CcUOX The gene can be used in the field of tea tree anthracnose resistance genetic engineering; CcUOX Genes can be used in food science and engineering; expression CcUOX Genetically engineered strains can be used for the degradation, utilization and environmental remediation of caffeine waste; CcUOX Genetically engineered strains can be used for uric acid degradation; CcUOX It can be used as a gene target in the design and screening of fungicides against Colletotrichum camelliae. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 The prediction of amino acid functions of CcUOX in Colletotrichum camelliae; CcUOX The gene number is CcaCcLH18_02012;

[0024] Figure 2 Colletotrichum camelliae CcUOXSchematic diagram of gene knockout strategy (gene replacement by homologous recombination): CCA is the wild-type strain, pXEH is the knockout vector, ΔCcUOX is CcUOX gene deletion mutants;

[0025] Figure 3 for CcUOX PCR verification electrophoresis and qPCR expression diagram of gene deletion mutants:

[0026] In Figure A, two primers are designed using the nucleotide sequences of the left arm downstream and the right arm upstream. The normal knockout homozygote replaces the target gene with the hygromycin gene through crossover and homologous recombination. Therefore, through PCR, a band that matches the size of the hygromycin band can be amplified, which is a knockout homozygote. Figure B uses CcUOX Gene qPCR primers, wild-type cDNA has expression level while knockout body Δ CcUOX- 2. Δ CcUOX- 44. Δ CcUOX- 50 no expression;

[0027] Figure 4 The knockout mutant Δ CcUOX Wild-type strain C.camelliae and complement Δ CcUOX -C pathogenicity comparison analysis: CcUOX For knockout mutants, C. camelliae is the wild type, Δ CcUOX -C-CcUOX is to replenish tea tree anthracnose CcUOX The complement of the gene, the host is the tea tree leaf;

[0028] Figure 5 The knockout mutant Δ CcUOX Wild-type strain C.camelliae Comparative analysis of tolerance to 1mg / ml caffeine: CcUOX For knockout mutants, C.camelliae is wild type;

[0029] Figure 6 Complement Δ CcUOX -C-CcUOX and wild-type strain C.camelliae Comparative analysis of tolerance to 1mg / ml caffeine;

[0030] Figure 7 Wild-type strain C. camelliae And comparative analysis of the mutants' ability to degrade 0.1% uric acid. DETAILED DESCRIPTION

[0031] In order to better describe the present invention, the following is further illustrated by specific examples. The methods in the following examples are conventional methods unless otherwise specified.

[0032] Embodiment 1, CcUOX Gene correlation analysis.

[0033] Tea tree anthracnose CcUOX The gene consists of 906 nucleotides and the encoded protein product consists of 301 amino acids. Blast comparison revealed that it encodes urate oxidase, such as Figure 1 shown.

[0034] Embodiment 2, CcUOX Gene knockout and genetic complementation.

[0035] 1) Knockout vector construction

[0036] Primers were designed using DNAMAN and synthesized by Shanghai Shenggong Biotechnology Co., Ltd. CcUOX -UP-F(5'-CGGAATTCTGAGATGAACCAACAGACC -3') and CcUOX -UP-R (5'-CGGGGTACCGTTGGCGGTTGTGGTTG -3'), using tea tree anthracnose fungus strain C. camelliae Amplification of genomic DNA as template CcUOX The approximately 750 bp fragment upstream of the gene; using primers CcUOX -DN-F(5'-CGGGATCCGTTGGGGCGCTGTTGAAA -3') and CcUOX -DN-R (5'-ACGCGTCGACAGAGGAAGAGAGCCGTGA -3'), using tea tree anthracnose fungus strain C. camelliae Amplification of genomic DNA as template CcUOX A fragment of about 900 bp downstream of the gene. Reaction system (25 μL): Takara PrimerSTAR Max DNA Polymerase (2×) 12.5 μL; upstream and downstream primers (10 μM) 1 μL each; template DNA 1 μL; ddH2O 9.5 μL. Amplification procedure: (1) 98°C 10 s; (2) 53°C 15 s; (3) 72°C 10 s, step 1-3 was cycled 35 times. The PCR product was recovered and purified using the TAKARA gel recovery kit, and the samples were purified using EcoRI + KpnI as well as BamHI + SalI Connect the upstream and downstream fragments to the pXEH vector to construct a knockout vector (strategy as Figure 2 as shown).

[0037] 2) Transformation of tea tree anthracnose pathogen

[0038] a. Cultivation of Agrobacterium tumefaciens AGL-1

[0039] Pick the binary vector CcUOX A single colony of the Agrobacterium tumefaciens strain AGL-1 was inoculated into LB liquid medium containing 50 μg / mL kanamycin and 25 μg / mL rifampicin, and cultured for 36 h until OD 600 =about 0.8. Prepare IM liquid culture medium (100mL: 90 mL ddH2O; 1 mL 50% glycerol; 1 mL 20% glucose; 4 mL 1M MES; 2 mL MN buffer; 1 mL Tracelement; 80μL K-buffer; 250μL 20% NH4N03; 100μL 1% CaCl2; 1 mL 0.01% FeS04; ​​200μL 19.2% As). First, add the drugs except FeS04 and As to 90 mL ddH2O and mix well. Take 5 ml of the shaken bacterial liquid and put it in a 15 ml centrifuge tube. Centrifuge at 4000 rpm for 5 minutes, discard the supernatant, add 5 ml of IM culture medium (without FeS04, As), blow it, centrifuge it at 4000 rpm for 5 minutes, discard the supernatant, add FeS04, As to the IM culture medium, take 5 ml and add it to a 15 ml centrifuge tube, blow it and transfer it to an empty bottle, and shake it at 28℃ for 4 hours. The remaining IM liquid medium was placed in a 4°C refrigerator (wrapped in newspaper).

[0040] b. Spore culture of tea anthracnose pathogen

[0041] activation C. camelliae The strain was inoculated with PDB medium (20% boiled and filtered potato, 2% glucose) and cultured at 28°C for 3-8 days. The supernatant was filtered with gauze and observed under a microscope. The spore concentration was adjusted to 5×10 using a hemocytometer. 5 / mL.

[0042] c. Co-culture of Agrobacterium tumefaciens and conidia of tea anthracnose pathogen

[0043] Mix the Agrobacterium culture liquid induced in IM liquid medium with anthrax spores of adjusted concentration in equal volumes, take 100 μL and spread it on IM solid medium with cellophane on it, spread it on 6 plates, seal the plates, and culture at 28°C in the dark for 2 days. After co-culture, transfer the cellophane to PDA medium containing 100 μg / mL hygromycin and continue to culture under the same conditions. After 4 to 7 days, pick the expanded colonies and place them on the screening medium containing the same antibiotics for secondary screening.

[0044] d. Verification of transformants

[0045] Using hygromycin primers and CcUOXGene primers were used to screen transformants by PCR amplification. The amplification results were determined to be consistent with the following results. CcUOX Gene deletion mutant: The internal primers HpTa (5'-GTCGTTTGACAAGATGGTTCA-3') and HpTb (5'-CGTCTGCTGCTCCATACAA-3') of the hygromycin resistance gene can amplify a 993 bp fragment (e.g. Figure 3 A); two primers (5'-CGGTCCTTATCTCAATCCATC-3') (5'-GATGCGGGAAGAAAATGC-3') were designed using the nucleotide sequences of the left arm downstream and the right arm upstream. The normal knockout homozygote replaces the target gene with the hygromycin gene through crossover and homologous recombination. Therefore, through PCR, a band that can be amplified to match the size of the hygromycin band is a knockout homozygote (such as Figure 3 B). At the same time, further at the RNA level CcUOX If the gene is missing, transcription cannot proceed smoothly. CcUOX The gene QPCR primers (5'-TTCAGCAACCGAAACACC-3') and (5'-TGTAGACGAGGCAGACAACG-3') could not detect its expression. Three independent CcUOX gene deletion mutants were screened from the transformants: Δ CcUOX- 2. Δ CcUOX- 44. Δ CcUOX- 50 for subsequent functional analysis.

[0046] Embodiment 3, CcUOX The role of genes in the pathogenicity of tea anthracnose pathogen.

[0047] Spore inoculation: Each strain was cultured under the same culture conditions to produce spores, the spores were collected and concentrated, and the spores obtained by centrifugation were diluted to 1×10 6 / mL and mix thoroughly. Use a pipette to draw 20μL of spore liquid and drip it on both sides of the leaves of the same batch of host plants of the same size and in good condition. Keep the inoculated host moisturized and cultured. Pay attention to observe the disease situation and take photos to record it. Inoculation of bacterial cake: activate the strain on PDA. When it grows for 4 days, use a puncher to punch out a uniform bacterial cake, turn it upside down on the leaves of the host plant, keep the inoculated host moisturized and cultured. Pay attention to observe the disease situation and take photos to record it. By combining spore liquid and bacterial cake, explore the differences in pathogenicity of wild type, knockout body and complement body to host plants, such as Figure 4 The results showed that when the CcUOX After that, the pathogenicity of anthrax to host plants was greatly reduced, while the pathogenicity of complements was basically the same as that of the wild type, indicating that CcUOXIt is involved in regulating the pathogenicity of anthrax to host plants and is an indispensable key gene for anthrax to maintain its virulence to host plants.

[0048] Embodiment 4, CcUOX The role of genes in caffeine's inhibition of tea plant anthracnose.

[0049] Place a 0.6-cm-diameter bacterial cake in the center of a pure PDA medium plate and a PDA medium plate (diameter 5.5 cm) containing 1 mg / ml caffeine. Culture the plate in the dark at 28°C in a constant temperature incubator. Measure the colony diameter (unit: cm) every 24 h until the hyphae on a certain plate cover the entire plate.

[0050] Calculate the inhibition rate

[0051]

[0052] On PDA medium, the growth of the knockout mutant and the wild-type strain was basically the same, and the knockout mutant even grew better on PDA. However, after adding 1 mg / ml of caffeine, the 96h inhibition rate of the wild-type strain was 39.11%, while the knockout mutant had a more obvious inhibition effect, with a 96h inhibition rate of 56.93%, which was significantly different from the wild-type. Figure 5 shown.

[0053] will be knocked out CcUOX The mutant ΔCcUOX complements / overexpresses the original gene CcUOX Afterwards, Figure 6 Shown is an increased resistance to caffeine.

[0054] Example 5. CcUOX Analysis of the role of genes in uric acid degradation.

[0055] Add 0.1% uric acid to CM liquid culture medium and inoculate after sterilization C. Camelliae Wild-type strain and Δ CcUOX The spores of the mutant strain were shaken for 48 hours, and the changes in uric acid content were detected by hydrogen peroxide colorimetric reaction. Figure 7 As shown, Δ CcUOX-2 Pink, Δ CcUOX-44 Pink, C. Camelliae The wild type is colorless. CcUOX The strain overexpressing the protein can be used to reduce uric acid in purine-rich foods, such as seafood, beer, tea garden waste, coffee garden waste, tea dregs, coffee dregs, etc.

[0056] SEQ ID No: 1: CcUOX Nucleotide sequence: 906 bp

[0057] 1ATGCCCGTTC TCGCCTCCGC CCGCTACGGC AAGGACAATG TCCGCGTCTA CAAGGTCGAG

[0058] 61CGCCACGGCG ACTCCCAGAC CGTCACCGAG ATGACCGTCT GCTGCCTCCT CGAGGGCGAG

[0059] 121ATCGAGACCT CCTACACCGT CGCCGACAAC AGCGTCGTCG TCGCCACCGA CTCCATCAAG

[0060] 181AACACAATCT ACATCAAGGC CAAGGAGAAC CCCATCAACC CGCCAGAGCT CTACGCCTCC

[0061] 241ATCCTCGGCA CCCACTTCCT CGACACCTAC AAGCACATCC ACGCCGCCAA CATCAAGATC

[0062] 301GTCCAGCACC GCTGGACCCG CATGACCGTC GACGGCAAGC CGCACCCGCA CTCCTTCTTC

[0063] 361CGCGACGGCG AGGAGACCCG CAACGTCGAG GCCCGCATCT CCCGCAAGGA CGGCATCGCC

[0064] 421CTCACCTCCA CCATCCAGAA GCTCACCGTC CTCAAGAGCA CCGGCTCCGC CTTCCACGGC

[0065] 481TTCGTCCGCG ACGAGTACAC CACCCTGCCC GAGACCTGGG ACCGCATCCT CTCCACCGAC

[0066] 541GTCGACGCCC AGTGGTCCTG GAAGTTCCCC GACGTCGCCG CCGTCAGGGC CGCCGTCCCC

[0067] 601AAGTTCGACC CGGCCTGGCA GGCCGCCCGC GACATCACCA TGAAGACTTT CGCCGAGGAC

[0068] 661GAGAGCGCCA GCGTGCAGAA CACAATGTAC AAGATGTGCG AGCAGATCCT CGCCGCCGTG

[0069] 721CCAGAGGTCC TGACCGTCAC GTACACGCTC CCCAACAAGC ACTACTTTGA GATCGACCTC

[0070] 781AGCTGGCACA ACGGCCTGAA GAACACGGGC AAAGACGCCG AGGTGTACGC GCCGCAGTCC

[0071] 841GGCCCGAACG GTCTCATCAG GTGCGAGGTC GCGCGCTCTG CCAAGAACGA GACGGCCAAG

[0072] 901TTGTAA。

[0073] SEQ ID No:2: Amino acid sequence:

[0074] 1MPVLASARYG KDNVRVYKVE RHGDSQTVTE MTVCCLLEGE IETSYTVADN SVVVATDSIK

[0075] 61NTIYIKAKEN PINPPELYAS ILGTHFLDTY KHIHAANIKI VQHRWTRMTV DGKPHPHSFF

[0076] 121RDGEETRNVE ARISRKDGIA LTSTIQKLTV LKSTGSAFHG FVRDEYTTLP ETWDRILSTD

[0077] 181VDAQWSWKFP DVAAVRAAVP KFDPAWQAAR DITMKTFAED ESASVQNTMY KMCEQILAAV

[0078] 241PEVLTVTYTL PNKHYFEIDL SWHNGLKNTG KDAEVYAPQS GPNGLIRCEV ARSAKNETAK

[0079] 301 L*.

[0080] The serial numbers of the above embodiments of the present invention are for description only and do not represent the advantages and disadvantages of the embodiments. Through the description of the above implementation methods, those skilled in the art can clearly understand that the above embodiment methods can be implemented by means of some deformations plus the necessary general technology superposition; of course, it can also be implemented by simplifying some important technical features. Based on this understanding, the technical solution of the present invention is essentially or the part that contributes to the prior art is: the overall construction method, and the structure described in each embodiment of the present invention.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Colletotrichum spp. QUR The gene, whose nucleotide sequence is shown in SEQ ID NO: 1, said QUR The gene knockout resulted in reduced pathogenicity of Colletotrichum japonicum to host plants.

2. A Colletotrichum camelliae as claimed in claim 1 QUR The protein encoded by the gene is characterized by: The amino acid sequence of the protein is shown in SEQ ID NO:

2.

3. Knockout as claimed in claim 1 QUR Use of the gene or the protein as claimed in claim 2 in reducing the resistance of Colletotrichum spp. to caffeine stress.

4. Overexpression of the QUR Application of strains expressing genes in uric acid metabolism.

5. Use of overexpressing the protein as claimed in claim 2 to reduce uric acid in foods rich in purine bases.

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