CN116676315B - Gene CcUOX of Colletotrichum camelliae and its application - Google Patents

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

Cephalosporium camellia gene CcUOX and application thereof

Technical Field

The invention belongs to the technical field of microbial genetic engineering, and in particular relates to application of a camellia thorn fungus gene CcUOX in the fields of prevention and control of anthracnose of camellia plants, improvement of plant disease resistance and food safety.

Background

The Colletotrichum fungus is a asexual fungus belonging to the class of the plant pathogenic fungi distributed globally, belonging to the class of the ascomycetes Coelomycetes, the order of the Colletotrichum Melanconiales and the family of the Colletotrichum Melanconiaceae, the sexuality of which is ascomycota Ascomycota, the small Cong Keke Glomerellaceae and the small plexus GlomerellaSpauld et Schrenck. The fungus has wide host range, and is parasitic from bare plant to angiosperm and from monocotyledonous plant to dicotyledonous plant. The pathogenic fungi of the genus, which often harm the roots, stems, leaves, flowers, fruits and seedlings of woods, fruit trees, vegetables, flowers, medicinal plants and field crops, can cause symptoms such as plant wilt, fruit rot, leaf spot and the like, and cause serious economic loss, are classified as eighth plant pathogenic fungi worldwide based on the importance of science and economy.

The tea contains abundant secondary metabolites such as tea polyphenol, theanine, alkaloids, glycosides, proanthocyanidins, anthocyanin compounds and the like, and the special quality of the tea is provided. Caffeine is one of the important purine bases of tea, and is structurally characterized in that N at the 1, 3 and 7 positions of xanthine is connected with 3 methyl groups. The content and distribution of caffeine in different varieties and different parts of different tea trees are different, and because the caffeine of the tea trees is mainly synthesized in chloroplasts, the caffeine is mainly distributed at the young shoot part of the tea trees, and the content is gradually reduced along with the maturity and aging of leaves. It has been demonstrated that caffeine may be related to plant disease resistance, with higher caffeine content being more resistant to disease.

Therefore, the research of the interaction of the biosynthesis of the caffeine and pathogenic bacteria of the tea tree is of great significance for revealing the disease-resistant mechanism of the tea tree.

Disclosure of Invention

The pathogenic gene for regulating and controlling the colletotrichum camellia (Colletotrichum camelliae) is derived from colletotrichum camellia, is CcUOX, has a CcUOX gene open reading frame shown in SEQ ID NO. 1, consists of 906 nucleotides, and has a coding region length of 301 nucleotides, wherein the amino acid sequence of CcUOX protein coded by the colletotrichum camellia CcUOX gene is shown in SEQ ID NO. 2.

The deletion of CcUOX genes causes the reduction of the pathogenicity of the camellia thorn fungus on host plants, ccUOX genes and proteins coded by the genes can be used as targets for developing anti-camellia thorn fungus medicaments, and compounds capable of inhibiting the expression of the genes and the expression, modification and positioning of the proteins can be screened, so that the occurrence of the camellia thorn fungus can be effectively controlled and used as novel bactericides.

Further, the host plant is one of plants of camellia, camellia oleifera, camellia japonica and the like.

The invention also aims to provide an application of the thorn fungus CcUOX gene of the thorn fungus camellia shown as SEQ ID No.1 or the coded protein shown as SEQ ID No.2 thereof as a control target in the preparation of the thorn fungus camellia bactericide.

The invention also aims to provide a biocontrol microbial inoculum for preventing and controlling the camellia thorn fungus, which contains a genetic engineering vector for blocking or inhibiting the gene expression of the camellia thorn fungus CcUOX.

Further, the biocontrol microbial inoculum is one or more of A to C:

A. Reduce the pathogenicity of the echinococcus camellia;

B. reducing the tolerance of the disc sporotrichum camellia to caffeine;

C. Reducing degradation of uric acid.

Further, the plant is a host plant of the colletotrichum camellia.

Further, the biocontrol microbial inoculum contains a knockout vector of the gene CcUOX of the colletotrichum camellia sinensis.

The invention also aims to provide a method for preventing and controlling the camellia thorn fungus, and the expression of the camellia thorn fungus CcUOX gene is blocked or inhibited by the knockout expression vector to obtain the biocontrol strain with reduced pathogenicity.

The invention proves that the deletion of CcUOX genes leads to the obvious reduction of the pathogenicity of the thorn-dish camellia fungus, the reduction of the tolerance to caffeine and the reduction of the uric acid metabolic capability. Therefore, the compound which can prevent the gene expression and the expression, modification and localization of the protein can be screened, and the occurrence of the colletotrichum camellia can be effectively controlled, thereby being beneficial to developing a novel bactericide. Namely, one important application of CcUOX gene provided by the invention is that the expression of the gene and the protein product encoded by the gene can be used as important candidate target sites for designing and screening anti-camellia thorn disc spore medicaments.

It is still another object of the present invention to provide a recombinant protein, which is obtained by excessively inducing CcUOX gene expression by the over-expression vector, and is useful for reducing uric acid in high purine foods.

Further, the high-purine food is seafood, beer, animal viscera, beef and mutton, etc.

It is still another object of the present invention to provide a method for reducing purine bases in foods, which induces the expression of the gene CcUOX of the colletotrichum camellia sinensis via the over-expression vector to obtain an expression protein for metabolizing uric acid in foods.

The invention also aims to provide a method for reducing purine base pollution in soil or water environment, wherein the expression of CcUOX genes is promoted by the over-expression vector, and over-expression strain with enhanced uric acid metabolic capability is obtained and is used for repairing environmental treatments such as caffeine, uric acid and the like.

The functional gene CcUOX which is derived from the colletotrichum camellia and has fungal pathogenicity, caffeine tolerance and uric acid metabolism, a cDNA sequence composed of 906 nucleotides is shown as SEQ ID NO. 1, a protein coded by CcUOX gene and composed of 301 amino acids is shown as SEQ ID NO. 2, a CcUOX gene can be applied in the field of tea tree anthracnose resistance genetic engineering, a CcUOX gene can be applied in the fields of food science and engineering, an engineering strain expressing CcUOX gene can be used for degrading, utilizing and environmental repairing caffeine waste, an engineering strain expressing CcUOX gene can be applied for uric acid degradation, and CcUOX can be used as a gene target for designing and screening colletotrichum camellia sterilization.

Drawings

FIG. 1 is a diagram of amino acid functional prediction of a. Camellia sinensis CcUOX, wherein the CcUOX gene number is CcaCcLH18_02012;

FIG. 2 is a schematic diagram of a knockout strategy (gene replacement by homologous recombination) for the gene CcUOX of Cephalosporium camellia, wherein CCA is a wild-type strain, pXEH is a knockout vector, and Delta CcUOX is a CcUOX gene deletion mutant;

FIG. 3 shows PCR-validated electrophoresis patterns and qPCR expression patterns of CcUOX gene deletion mutants:

Wherein, the two primers are designed by using the nucleotide sequences at the downstream of the left arm and the upstream of the right arm in the diagram A, and the normal knockout homozygote replaces the target gene with the hygromycin gene through cross exchange and homologous recombination. Therefore, by PCR, one band conforming to the hygromycin band size can be amplified, namely the knockout homozygote. FIG. B shows that the wild type cDNA has an expression level and the knockouts Δ CcUOX-2, Δ CcUOX-44, and Δ CcUOX-50 have no expression level using CcUOX gene qPCR primers;

FIG. 4 shows a comparative analysis of pathogenicity of knockout mutant Δ CcUOX with wild type strain C.camelliae and complement Δ CcUOX-C, wherein Δ CcUOX is knockout mutant, C. camelliae is wild type, Δ CcUOX-C-CcUOX is complement of the gene of B.anthracis CcUOX, host is tea leaves;

FIG. 5 is a comparative analysis of the tolerance of knockout mutant Δ CcUOX to 1mg/ml caffeine with wild-type strain C.camelliae, wherein Δ CcUOX is knockout mutant and C.camelliae is wild-type;

FIG. 6 is a comparative analysis of the tolerance of complement Delta CcUOX-C-CcUOX to 1mg/ml caffeine with wild-type strain C.camelliae;

FIG. 7 is a comparative analysis of the degradation ability of wild type strain C. camelliae and mutants to 0.1% uric acid.

Detailed Description

In order to better describe the present invention, the following description will be given by way of specific examples, which are conventional methods unless otherwise indicated.

Correlation analysis of the genes of examples 1 and CcUOX.

The gene CcUOX of tea tree anthracnose consists of 906 nucleotides, and the coded protein product consists of 301 amino acids. Blast alignment was found to encode urate oxidase as shown in figure 1.

The knockdown of the genes of examples 2, ccUOX was genetically complementary.

1) Knock-out vector construction

The primer is designed by DNAMAN, and is synthesized by Shanghai biological technology limited company, wherein the primer CcUOX-UP-F (5'-CGGAATTCTGAGATGAACCAACAGACC-3') and CcUOX-UP-R (5'-CGGGGTACCGTTGGCGGTTGTGGTTG-3') are adopted, the genome DNA of the tea tree anthracnose bacterial strain C. camelliae is taken as a template to amplify a fragment of about 750bp at the upstream of CcUOX genes, the primer CcUOX-DN-F (5'-CGGGATCCGTTGGGGCGCTGTTGAAA-3') and CcUOX-DN-R (5'-ACGCGTCGACAGAGGAAGAGAGCCGTGA-3') are adopted, and the genome DNA of the tea tree anthracnose bacterial strain C. camelliae is taken as a template to amplify a fragment of about 900bp at the downstream of CcUOX genes. The reaction system (25. Mu.L) was TAKARA PRIMERSTAR Max DNA Polymerase (2X) 12.5. Mu.L, 1. Mu.L of each of the upstream and downstream primers (10. Mu.M), 1. Mu.L of the template DNA, and 9.5. Mu.L of ddH ₂ O. Amplification procedure (1) 98℃for 10s, (2) 53℃for 15s, and (3) 72℃for 10s were cycled between 1 and 3 steps for 35 times. The PCR products were recovered and purified using a TAKARA gel recovery kit, and the upstream and downstream fragments were ligated with pXEH vector using EcoRI+KpnI and BamHI+SalI, respectively, to construct a knockout vector (strategy shown in FIG. 2).

2) Transformation of tea tree anthracnose

A. Cultivation of Agrobacterium tumefaciens AGL-1

A single colony of Agrobacterium tumefaciens strain AGL-1 containing binary vector CcUOX was picked and inoculated into LB liquid medium containing 50. Mu.g/mL kanamycin and 25. Mu.g/mL rifampicin, and shake cultured for 36h to OD ₆₀₀ = 0.8 or so. Preparation of IM liquid Medium (100 mL: 90 mL ddH ₂ O; 1mL of 50% glycerol; 1mL of 20% glucose ;4 mL1M MES;2 mL M-N buffer;1 mLTracelement;80μL K-buffer;250μL20%NH₄N0₃;100μL1%CaCl₂;1 mL0.01%FeS0₄;200μL19.2% As）,) first, in 90 mL ddH ₂ O, with the addition of drugs except for FeS0 ₄ and As, mixing well, taking 5mL of the shaken bacterial liquid into a 15-mL centrifuge tube, rotating 4000, centrifuging for 5 minutes, discarding the supernatant, adding 5mL of IM medium (without FeS0 ₄, as), blowing once, rotating 4000, centrifuging for 5 minutes, discarding the supernatant, adding FeS0 ₄, as into the IM medium, taking 5mL of the IM medium, adding into a 15-mL centrifuge tube, blowing once into an empty bottle, shaking at 28 ℃ for 4h, and placing the rest of IM liquid medium into a4 ℃ refrigerator (newspaper package).

B. Spore-producing culture of tea tree anthracnose

Activating strain C. camelliae, inoculating the bacterial cake on PDB culture medium (20% of potato is boiled and filtered, 2% of glucose), shaking culturing at 28deg.C for 3-8 days, filtering supernatant with gauze, observing with microscope, and regulating spore concentration to 5× ⁵/mL with hemocytometer.

C. co-culture of agrobacterium tumefaciens and tea tree anthracnose conidium

Mixing the agrobacteria liquid induced in the IM liquid culture medium with the equal volume of anthrax spores with the adjusted concentration, taking 100 mu L of the agrobacteria liquid, coating the agrobacteria liquid on the IM solid culture medium with the glass paper, coating 6 plates, sealing the plates, and culturing for 2 days at the dark place at 28 ℃. After the co-cultivation, the cellophane was transferred to PDA medium containing 100. Mu.g/mL hygromycin and cultivation was continued under the same conditions. And after 4-7 days, picking the expanded bacterial colony on a screening culture medium containing the same antibiotics for secondary screening.

D. verification of transformants

Transformants were screened by PCR amplification using hygromycin primers and CcUOX gene primers. The amplification result is determined to be CcUOX gene deletion mutant, namely, the hygromycin resistance gene inner primers HpTa (5'-GTCGTTTGACAAGATGGTTCA-3') and HpTb (5'-CGTCTGCTGCTCCATACAA-3') can be amplified to 993bp fragments (shown in figure 3A), two primers (5'-CGGTCCTTATCTCAATCCATC-3') are designed by utilizing the nucleotide sequences of the downstream left arm and the upstream right arm (5'-GATGCGGGAAGAAAATGC-3'), and the normal knockout homozygote replaces the target gene with the hygromycin gene through cross exchange and homologous recombination. Therefore, by PCR, one band conforming to the hygromycin band size can be amplified, i.e., a knockout homozygote (as shown in FIG. 3B). Meanwhile, the transcription activity cannot be smoothly carried out on the RNA level due to the CcUOX gene deletion, and the CcUOX gene QPCR primers (5'-TTCAGCAACCGAAACACC-3') and (5'-TGTAGACGAGGCAGACAACG-3') cannot be used for detecting the expression. From the transformants, 3 independent CcUOX gene deletion mutants, Δ CcUOX-2, Δ CcUOX-44, and Δ CcUOX-50 were selected for subsequent functional analysis.

Example 3, ccUOX Gene effect on tea tree anthracnose pathogen pathogenicity.

Spore liquid inoculation, namely, culturing each strain under the same culture condition to produce spores, collecting and concentrating the spore liquid, diluting the spore liquid obtained by centrifugation to 1X 10 ⁶ pieces/mL by using sterile water, fully and uniformly mixing, sucking 20 mu L of spore liquid by using a pipetting gun, dripping the spore liquid to two sides of a host plant leaf with the same batch size and good state, carrying out moisturizing culture on the inoculated host, observing the disease condition, photographing and recording, inoculating a bacterial cake, namely, activating the strain on a PDA, beating a uniform bacterial cake by using a puncher when the bacterial cake grows for 4d, reversely buckling on the host plant leaf, carrying out moisturizing culture on the inoculated host, observing the disease condition, and taking a photograph and recording. The difference in pathogenicity of wild type, knockdown and complement to host plants was investigated by means of spore fluid and bacterial cake combination, as shown in figure 4. The results show that the pathogenicity of the anthrax on the host plant is greatly reduced after CcUOX is knocked out, and the pathogenicity of the complement is basically the same as that of the wild type, which indicates that CcUOX participates in regulating and controlling the pathogenicity process of the anthrax on the host plant and is an essential key gene for the anthrax to maintain the virulence of the host plant.

Example 4, ccUOX Gene effect on caffeine inhibits tea tree anthracnose.

Bacterial cakes with the diameter of 0.6 cm are respectively connected to the centers of a pure PDA culture medium plate (with the diameter of 5.5 cm) and a PDA culture medium plate with the concentration of caffeine of 1mg/ml, and are cultivated in darkness in a 28 ℃ constant temperature incubator, and the colony diameter (unit: cm) is measured at intervals of 24 hours until hyphae on a certain plate are fully paved on the whole plate.

Calculating inhibition rate

On PDA medium, the growth condition of the knockout mutant is basically consistent with that of the wild strain, and even the growth condition of the knockout mutant on PDA is better. However, after 1mg/ml of caffeine is added, the inhibition rate of the wild strain for 96 hours is 39.11%, the inhibition effect of the knockout mutant is obvious, the inhibition rate for 96 hours can reach 56.93%, and a significant difference exists between the wild strain and the knockout mutant, as shown in fig. 5.

After the CcUOX knocked-out mutant Δ CcUOX was complemented/overexpressed with the original gene CcUOX, it was shown that resistance to caffeine was enhanced as shown in fig. 6.

Example 5 analysis of the effect of the ccuox gene on uric acid degradation.

Adding 0.1% uric acid into CM liquid culture medium, sterilizing, inoculating spores of C. CAMELLIAE wild type strain and delta CcUOX mutant strain, shaking culturing for 48 hr, and detecting uric acid content change by hydrogen peroxide chromogenic reaction as shown in figure 7, delta CcUOX-2 is pink, delta CcUOX-44 is pink, and C. CAMELLIAE wild type is colorless. CcUOX the use of strains overexpressing proteins to reduce uric acid in purine-rich foods such as seafood, beer, tea garden waste, coffee garden waste, tea grounds, coffee grounds, and the like.

SEQ ID No. 1: ccUOX nucleotide sequence: 906bp

1ATGCCCGTTC TCGCCTCCGC CCGCTACGGC AAGGACAATG TCCGCGTCTA CAAGGTCGAG

61CGCCACGGCG ACTCCCAGAC CGTCACCGAG ATGACCGTCT GCTGCCTCCT CGAGGGCGAG

121ATCGAGACCT CCTACACCGT CGCCGACAAC AGCGTCGTCG TCGCCACCGA CTCCATCAAG

181AACACAATCT ACATCAAGGC CAAGGAGAAC CCCATCAACC CGCCAGAGCT CTACGCCTCC

241ATCCTCGGCA CCCACTTCCT CGACACCTAC AAGCACATCC ACGCCGCCAA CATCAAGATC

301GTCCAGCACC GCTGGACCCG CATGACCGTC GACGGCAAGC CGCACCCGCA CTCCTTCTTC

361CGCGACGGCG AGGAGACCCG CAACGTCGAG GCCCGCATCT CCCGCAAGGA CGGCATCGCC

421CTCACCTCCA CCATCCAGAA GCTCACCGTC CTCAAGAGCA CCGGCTCCGC CTTCCACGGC

481TTCGTCCGCG ACGAGTACAC CACCCTGCCC GAGACCTGGG ACCGCATCCT CTCCACCGAC

541GTCGACGCCC AGTGGTCCTG GAAGTTCCCC GACGTCGCCG CCGTCAGGGC CGCCGTCCCC

601AAGTTCGACC CGGCCTGGCA GGCCGCCCGC GACATCACCA TGAAGACTTT CGCCGAGGAC

661GAGAGCGCCA GCGTGCAGAA CACAATGTAC AAGATGTGCG AGCAGATCCT CGCCGCCGTG

721CCAGAGGTCC TGACCGTCAC GTACACGCTC CCCAACAAGC ACTACTTTGA GATCGACCTC

781AGCTGGCACA ACGGCCTGAA GAACACGGGC AAAGACGCCG AGGTGTACGC GCCGCAGTCC

841GGCCCGAACG GTCTCATCAG GTGCGAGGTC GCGCGCTCTG CCAAGAACGA GACGGCCAAG

901TTGTAA。

SEQ ID No. 2 amino acid sequence:

1MPVLASARYG KDNVRVYKVE RHGDSQTVTE MTVCCLLEGE IETSYTVADN SVVVATDSIK

61NTIYIKAKEN PINPPELYAS ILGTHFLDTY KHIHAANIKI VQHRWTRMTV DGKPHPHSFF

121RDGEETRNVE ARISRKDGIA LTSTIQKLTV LKSTGSAFHG FVRDEYTTLP ETWDRILSTD

181VDAQWSWKFP DVAAVRAAVP KFDPAWQAAR DITMKTFAED ESASVQNTMY KMCEQILAAV

241PEVLTVTYTL PNKHYFEIDL SWHNGLKNTG KDAEVYAPQS GPNGLIRCEV ARSAKNETAK

301 L*。

The foregoing embodiment numbers of the present invention are merely for the purpose of description, and do not represent the advantages or disadvantages of the embodiments. From the above description of the embodiments, it will be clear to those skilled in the art that the above embodiment method may be implemented by means of superposition of some variants and necessary general techniques, or may of course be implemented by simplifying some important technical features. Based on the understanding, the technical scheme of the invention is essentially or the part contributing to the prior art is an integral construction method and is matched with the structures described in the various embodiments of the invention.

It should be noted that the above embodiments are merely for illustrating the technical solution of the present invention and not for limiting the same, and although the present invention has been described in detail with reference to the above embodiments, it should be understood by those skilled in the art that the technical solution described in the above embodiments may be modified or some or all of the technical features may be equivalently replaced, and these modifications or substitutions do not deviate from the essence of the corresponding technical solution from the scope of the technical solution of the embodiments of the present invention.

Claims (5)

1. A nucleotide sequence of the gene CcUOX of the colletotrichum camellia is shown as SEQ ID NO.1, and the knockout of the CcUOX gene leads to the reduction of the pathogenicity of the colletotrichum camellia on host plants.

2. A protein encoded by the gene of the Paecilomyces camellia sinensis CcUOX as set forth in claim 1, wherein the amino acid sequence of the protein is shown in SEQ ID NO. 2.

3. Use of a knock-out CcUOX gene according to claim 1 or a protein according to claim 2 to reduce resistance of a c.

4. Use of a strain overexpressing the CcUOX gene according to claim 1 in uric acid metabolism.

5. Use of overexpression of a protein according to claim 2 for reducing uric acid in a purine-rich food.