CN116676315A - Cephalosporium camellia gene CcUOX and application thereof - Google Patents

Abstract

The invention provides a c & u & ox gene of c & lt/EN & gt and application thereof, which relate to pathogenicity and purine metabolism, belong to the technical field of microbial genetic engineering, and are derived from c & lt/EN & gt, wherein a cDNA sequence consisting of 906 nucleotides is shown as SEQ ID NO. 1; the protein coded by the CcUOX gene provided has an amino acid sequence consisting of 302 amino acids and shown in SEQ ID NO. 2; the CcUOX gene can be applied to the field of anthracnose resistance gene engineering of tea trees, and can be applied to the fields of food science and engineering; the engineering strain expressing the CcUOX gene can be used for degrading, utilizing and repairing the environment of caffeine waste, and can be applied to uric acid degradation; the CcUOX can be used as a gene target in the design and screening of the sterilizing bacteria of the colletotrichum camellia.

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 spore fungus gene CcUOX in the fields of prevention and treatment of anthracnose of camellia plants, improvement of plant disease resistance and food safety.

Background

Colletotrichum fungi are asexual fungi belonging to the class of Coelomyces, melanomyces, melanomycetaceae, and their sexuality is ascomyceta, amanomycetaceae, amanomyces GlomerellaSpauld et Schrenck, and belongs to a class of plant pathogenic fungi distributed globally. 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 named CcUOX, has a sequence shown as SEQ ID NO.1 and is a CcUOX gene open reading frame, and consists of 906 nucleotides, wherein the length of a coding region formed by the gene is 302 nucleotides; the amino acid sequence of the CcUOX protein coded by the CcUOX gene of the camellia sinensis is shown as SEQ ID NO: 2.

The deletion of the CcUOX gene causes the reduction of the pathogenicity of the camellia sinensis on host plants, the CcUOX gene and the protein coded by the CcUOX gene can be used as targets for the development of anti-camellia sinensis medicaments, and the compound capable of inhibiting the expression of the gene and the expression, modification and positioning of the protein of the CcUOX gene can be screened, so that the occurrence of the camellia sinensis can be effectively controlled and used as a novel bactericide.

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 camellia thorn fungus CcUOX gene shown in SEQ ID No.1 or a protein coded by the camellia thorn fungus CcUOX gene and shown in SEQ ID No.2 as a control target in preparation of the camellia thorn fungus bactericide.

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

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 c muox gene of the c muox camellia.

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, so that the biocontrol strain with reduced pathogenicity is obtained.

The invention proves that the deletion of the CcUOX gene leads to the obvious reduction of the pathogenicity of the thorn 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 the CcUOX gene provided by the invention is that the expression of the gene and a 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 expression of the CcUOX gene 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 c muox gene of c muox of camellia through 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, and the expression of the CcUOX gene is promoted by the over-expression vector to obtain an over-expression strain with enhanced uric acid metabolic capability, which is used for repairing environmental treatments such as caffeine, uric acid and the like.

The cDNA sequence of the functional gene CcUOX which is derived from the fungus pathogenicity and the tolerance to caffeine of the colletotrichum camellia and can metabolize uric acid and consists of 906 nucleotides is shown as SEQ ID NO. 1; the protein coded by the CcUOX gene provided has an amino acid sequence consisting of 302 amino acids and shown in SEQ ID NO. 2; the CcUOX gene can be applied to the field of anthracnose resistance genetic engineering of tea trees; the CcUOX gene can be applied to the fields of food science and engineering; the engineering strain expressing the CcUOX gene can be used for degradation, utilization and environmental repair of caffeine waste; the engineering strain expressing the CcUOX gene can be applied to uric acid degradation; the CcUOX can be used as a gene target in the design and screening of the sterilizing bacteria of the colletotrichum camellia.

Drawings

FIG. 1 is a graph showing the prediction of amino acid function of a Cephalosporium camellia CcUOX; wherein the CcUOX gene is numbered CcaCcLH18_02012;

FIG. 2 is a schematic representation of the knockout strategy (gene replacement by homologous recombination) of the C.camellia sinensis CcUOX gene: wherein CCA is a wild type strain, pXEH is a knockout vector, and DeltaCcUOX is a CcUOX gene deletion mutant;

FIG. 3 shows PCR-validated electrophoretogram and qPCR expression profile of CcUOX gene deletion mutant:

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. Panel B shows that wild-type cDNA has an expression level and knockouts ΔCcUOX-2, ΔCcUOX-44, ΔCcUOX-50 have no expression level using the CcUOX gene qPCR primer;

FIG. 4 is a pathogenicity comparison analysis of knockout mutant ΔCcUOX versus wild type strain C.camelliae and complement ΔCcUOX-C: wherein DeltaCcUOX is a knockout mutant, C.camelliae is wild type, deltaCcUOX-C-CcUOX is complement of a complement-supplementing tea tree anthracnose CcUOX gene, and a host is tea tree leaves;

FIG. 5 is a comparative analysis of the resistance of knockout mutant ΔCcUOX to 1mg/ml caffeine with wild type strain C.camelliae: wherein Δccoox is a knockout mutant and c.camelliae is wild type;

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

FIG. 7 is a comparative analysis of the degradability 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 CcUOX genes in example 1.

The CcUOX gene of tea tree anthracnose consists of 906 nucleotides and the encoded protein product consists of 302 amino acids. Blast alignment was found to encode urate oxidase as shown in figure 1.

The knockdown of the ccox gene of example 2 was genetically complementary.

1) Knock-out vector construction

Designing a primer by utilizing DNAMAN, synthesizing the primer by Shanghai biological technology limited company, and amplifying a fragment about 750bp upstream of a CcUOX gene by adopting the primers CcUOX-UP-F (5'-CGGAATTCTGAGATGAACCAACAGACC-3') and CcUOX-UP-R (5'-CGGGGTACCGTTGGCGGTTGTGGTTG-3') and taking genomic DNA of a tea tree anthracnose bacterial strain C.camelliae as a template; the primers CcUOX-DN-F (5'-CGGGATCCGTTGGGGCGCTGTTGAAA-3') and CcUOX-DN-R (5'-ACGCGTCGACAGAGGAAGAGAGCCGTGA-3') were used to amplify a fragment of about 900bp downstream of the CcUOX gene using genomic DNA of the tea plant anthracnose strain C.camelliae as template. Reaction system (25 μl): takara PrimerSTAR Max DNA Polymerase (2×) 12.5 μl; 1. Mu.L of each of the upstream and downstream primers (10. Mu.M); 1. Mu.L of template DNA; ddH ₂ O9.5. Mu.L. Amplification procedure: (1) 98 ℃ for 10s; (2) 53℃for 15s; (3) 10s at 72℃the 1-3 steps were cycled 35 times. The PCR products were recovered and purified using a TAKARA gel recovery kit, and the upstream and downstream fragments were ligated with the 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 the Agrobacterium tumefaciens strain AGL-1 containing the binary vector CcUOX is 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:90mL ddH) ₂ O;1mL of 50% glycerol; 1mL of 20% glucose; 4ml1M MES;2mL M-N buffer;1 mLTracell; 80 mu L K-buffer;250 μL of 20% NH N0;100 μL of 1% CaCl;1ml of 0.01% FeS0; 200. Mu.L 19.2% As), first at 90mL ddH ₂ Adding medicines except FeS0 and As into O, mixing, taking 5ml of the shaken bacterial solution into a centrifuge tube with volume of 15ml, centrifuging for 5 minutes at 4000 turns, discarding the supernatant, adding 5ml of IM culture medium (without FeS0 and As), blowing once, centrifuging for 5 minutes at 4000 turns, discarding the supernatant, adding FeS0 and As into the IM culture medium, taking 5ml into a centrifuge tube with volume of 15ml, blowing once, transferring to an empty bottle, and shaking for 4 hours at 28 ℃. Culturing the rest of IM liquidThe base is placed in a refrigerator (newspaper package) at 4 ℃.

b. Spore-producing culture of tea tree anthracnose

Activating C.camelliae strain, inoculating the bacterial cake to 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 5×10 with hemocytometer ⁵ /mL。

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 temperature of 28 ℃ in a dark place. 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. After 4-7 days, the expanded colonies were picked up and subjected to secondary screening on screening media containing the same antibiotics.

d. Verification of transformants

Transformants were screened by PCR amplification using hygromycin primers and the CcUOX gene primer. The amplification results were confirmed to be CcUOX gene deletion mutants in that hygromycin resistance gene inner primers HpTa (5'-GTCGTTTGACAAGATGGTTCA-3') and HpTb (5'-CGTCTGCTGCTCCATACAA-3') could be amplified to a 993bp fragment (as shown in FIG. 3A); two primers (5'-CGGTCCTTATCTCAATCCATC-3') were designed using the left arm downstream and right arm upstream nucleotide sequences (5'-GATGCGGGAAGAAAATGC-3'), and the normal knockout homozygote was a hygromycin gene replaced by a gene of interest by crossover 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). At the same time, transcription was not smoothly performed at the RNA level due to deletion of the CcUOX gene, and expression was not detected using the QPCR primers (5'-TTCAGCAACCGAAACACC-3') and (5'-TGTAGACGAGGCAGACAACG-3') for the CcUOX gene. 3 independent CcUOX gene deletion mutants were selected from transformants: deltaCcUOX-2, deltaCcUOX-44, deltaCcUOX-50 were used for subsequent functional analysis.

Example 3, effect of the CcUOX gene on the pathogenicity of colletotrichum tea tree anthracnose.

Inoculating spore liquid: culturing each strain under the same culture condition for producing spore, collecting and concentrating spore liquid, diluting the spore liquid obtained by centrifugation to 1×10 with sterile water ⁶ Mixing the two parts per mL fully, sucking 20 mu L of spore liquid by using a liquid-transferring gun, dripping the spore liquid to two sides of the leaves of the host plant with the same batch size and good state, carrying out moisturizing culture on the inoculated host, observing the disease condition, and photographing and recording; inoculating a fungus cake: activating the strain on PDA, when the strain grows for 4d, using a puncher to punch out uniform bacterial cakes, reversely buckling the bacterial cakes on leaves of host plants, carrying out moisturizing culture on the inoculated hosts, observing the disease condition, and taking photos 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 result shows that after the CcUOX is knocked out, the pathogenicity of the anthrax on a host plant is greatly reduced, and the pathogenicity of complement is basically the same as that of a wild type, which indicates that the CcUOX participates in regulating and controlling the pathogenicity process of the anthrax on the host plant and is an essential key gene for maintaining the virulence of the anthrax on the host plant.

Example 4, effect of the CcUOX gene on caffeine inhibition of colletotrichum tea.

The center of a PDA culture medium plate (diameter 5.5 cm) with pure PDA culture medium and caffeine concentration of 1mg/ml is respectively connected with a bacterial cake with the diameter of 0.6cm, and the bacterial cake is cultivated in darkness in a constant temperature incubator at 28 ℃ for 24 hours at intervals, and the colony diameter (unit: cm) is measured until hypha on a certain plate is 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 addition of 1mg/ml caffeine, the inhibition rate of the wild-type strain was 39.11% for 96 hours; the knockout mutant has obvious inhibition effect, the inhibition rate of 96h can reach 56.93%, and a significant difference exists between the knockout mutant and the wild type, as shown in figure 5.

After the original gene CcUOX was complemented/overexpressed with the CcUOX knockout mutant Δccuox, an enhancement of resistance to caffeine was shown in fig. 6.

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

After sterilization, spores of the C.Camelliae wild-type strain and the ΔCcUOX mutant strain were inoculated with 0.1% uric acid added to CM liquid medium, and shaking culture was performed for 48 hours, and the change in uric acid content was detected by hydrogen peroxide chromogenic reaction as shown in FIG. 7, ΔCcUOX-2 was pink, ΔCcUOX-44 was pink, and C.Camelliae wild-type was colorless. The strain of CcUOX over-expressed protein can be used for reducing uric acid in purine-rich foods such as seafood, beer, tea garden waste, coffee garden waste, tea grounds, coffee grounds, etc.

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

181 AACACAATCT 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 description of the above embodiments, it will be apparent to those skilled in the art that the above example methods may be implemented by means of a superposition of some variants plus the necessary general techniques; of course, the method can also be realized by simplifying some important technical features. Based on such understanding, the technical solution of the present invention essentially or partly contributes to the prior art is: and the whole construction method is matched with the structures described in the various embodiments of the invention.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and not for limiting the same; although the invention has been described in detail with reference to the foregoing embodiments, it will be understood by those of ordinary skill in the art that: the technical scheme described in the foregoing embodiments can be modified or some or all of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit of the invention.

Claims (10)

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

2. A protein encoded by the c muox gene of c muox as claimed in claim 1, wherein: the amino acid sequence of the protein is shown in SEQ ID NO: 2.

3. The CcUOX gene of claim 1 or the protein of claim 2, wherein: the regulation and control of the pathogenicity of the camellia sinensis is realized by improving the tolerance to caffeine.

4. A ccouox gene or/and protein as claimed in claim 3, wherein: the activity or/and the expression quantity of the gene or/and the coded protein of the gene of the campylobacter sphaeroides CcUOX in the campylobacter sphaeroides is increased to promote pathogenicity of pathogenic bacteria.

5. Use of the ccuoox gene according to claim 1 for the preparation of a medicament for controlling cercospora camellia, characterized in that: the medicament comprises an inhibitor of the CcUOX gene or an inhibition vector containing the CcUOX gene.

6. Use of the ccuoox gene of claim 1 to increase disease resistance in camellia plants.

7. The use according to claim 6, wherein: the use of the CcUOX gene mutant induces immunity in plants of the genus Camellia.

8. Use of a ccuoox gene according to claim 6 or a protein according to claim 2 for modulating resistance of a c.

9. The use of the ccuoox gene over-expressed protein of claim 1 in uric acid metabolism of a strain.

10. Use of the over-expressed protein according to claim 9 for reducing uric acid in a purine-rich food.