CN115960932B - Anthrax CsOxdC gene and its application - Google Patents

Abstract

The invention provides an anthrax CsOxdC gene and its application. The gene contains three introns and two Cupin structural domains. Experiments have confirmed that the deletion of this gene can reduce the resistance of fungi to azole drugs such as fludioxonil, indicating that the CsOxdC gene can be used to regulate the resistance of fungi to azole drugs, and the CsOxdC gene can be used as a target of azole drugs.

Description

Anthrax CsOxdC gene and its application

Technical field

The invention relates to the field of biotechnology, and in particular to an anthrax CsOxdC gene and its application.

Background technique

Oxalate decarboxylase (Oxdc) is a homogeneous polymerase containing Mn ²⁺ and belongs to the Cupin protein superfamily; it can catalyze the conversion of oxalic acid into formic acid and CO ₂ without cofactors, and oxalic acid is a filamentous fungus. The most common organic acids secreted can inhibit the growth of fungi that are more sensitive to acidity or oxalic acid, thereby affecting competition between fungal species. On the other hand, many fungal species tolerate high concentrations of toxic metals in their environment by secreting oxalic acid. Oxalate decarboxylase is one of the main catalytic enzymes for the metabolic degradation of oxalic acid in plants and microorganisms. This enzyme was first discovered in white rot fungi. It is mainly derived from fungi such as Aspergillus niger, Sclerotinia sclerotiorum, Enoki mushrooms and brown rot fungi. Currently, the most thoroughly studied is the bacterial OxdC from Bacillus subtilis. OxdC is one of the most abundant bacterial cell wall proteins expressed in B. subtilis under acidic stress conditions. This enzyme protects bacterial cells from low pH by consuming protons through decarboxylation of oxalate. Effects of coercion. Like Bacillus subtilis, low pH induced the Oxdc activity of the basidiomycete Flammulinavelutipes, the ascomycete Sclerotinia sclerotiorum and Aspergillusniger. The oxdc gene was cloned from F. velutipes and transferred into Brassica napus. The transgenic plants had obvious delay and ability to resist sclerotinia infection. After being transferred into tomatoes, the oxalic acid in the transgenic tomatoes was reduced and the nutrient content was increased; After being introduced into tobacco, the oxalic acid content in transgenic tobacco decreased and showed obvious resistance to plant programmed death induced by oxalic acid and NLP. However, there are currently no reports on the involvement of oxalate decarboxylase OxdC in the regulation of sensitivity to azole drugs in fungi.

Contents of the invention

In view of the shortcomings of the existing technology, the present invention provides a CsOxdC gene of Colletotrichum anthracis and its application.

The solution of the present invention includes the following aspects:

A Colletotrichum CsOxdC gene, which contains the nucleotide sequence shown in SEQ ID NO: 1.

A CsOxdC gene knockout mutant includes the following preparation steps: the first round of PCR, designing primer pairs CsOxdC-UF/CsOxdC-UR and CsOxdC-DF/CsOxdC-DR before and after the CsOxdC gene coding reading frame, and using PCR amplification Obtain the upper arm sequence and the lower arm sequence after the C terminus of the CsOxdC gene. CsOxdC-UR and CsOxdC-DF respectively contain the chlorsulfuron resistance gene linker sequence. Design the primer pair S1F/S2R to amplify the chlorsulfuron resistance gene ILV1. ;

In the second round of PCR, the products amplified by the first round of PCR are fused using the primers CsOxdC-UF/CsOxdC-UR;

In the third round of PCR, the fusion product of the second round of PCR was enriched using the primers CsOxdC-UF/CsOxdC-DR; the enriched fragments were introduced into the protoplasts of Colletotrichum anthracis of the rubber tree and screened through the medium containing chlorsulfuron , and then PCR verification is obtained;

The nucleotide sequence of the CsOxdC gene is shown in SEQ ID NO: 1;

The sequence of primer CsOxdC-UF is: 5’-CCAACATGCCGTCGAAACC-3’

The sequence of primer CsOxdC-UR is:

5’-AGATTGGGGCACTGTGGCGTTGGCACGATGGGCGTGAAGTAGGAAGG-3’

The sequence of primer CsOxdC-DF is:

5’-TATTGCACGGGAATTGCATGCTCTCACGGGAGCACTGCCACAATGGAA-3’

The sequence of primer CsOxdC-DR is: 5’-CTGCTTCTAGTTCGAGAAGCG-3’

The sequence of primer S1F is: 5’-GTGCCAACGCCACAGTGCCCCACA-3’

The sequence of primer S2R is: 5’-GTGAGAGCATGCAATTCCCGTGCAATA-3’.

The present invention also relates to the application of the Colletotrichum CsOxdC and the CsOxdC gene knockout mutant in regulating the sensitivity of fungi to azole drugs.

Further, the present invention relates to the application of the CsOxdC of Colletotrichum anthracis as the target of azole drugs.

Further, the present invention relates to the application of the CsOxdC gene knockout mutant in reducing the sensitivity of fungi to azole drugs.

Further, the azole agent is fludioxonil.

Compared with the prior art, the beneficial effects of the present invention are:

The present invention clones the CsOxdC gene and constructs a CsOxdC gene knockout mutant. Through functional experiments, it is confirmed that the gene is involved in regulating the sensitivity of fungi to azole agents such as fludioxonil. Therefore, the CsOxdC gene can be used as a drug target, and drugs that can inhibit the expression of the CsOxdC gene can be used as fungicides to inhibit the growth of fungi.

Description of the drawings

Figure 1: Schematic diagram of CsOxdC gene knockout.

Figure 2: PCR verification results of the gene deletion mutant ΔCsOxdC.

Figure 3: Colony growth morphology of wild-type HN08 strain and mutant ΔCsOxdC strain in CM medium with different concentrations of fludioxonil (5d).

Detailed ways

In order to facilitate skilled persons to understand the technical content of the present invention, the present invention will be further described in detail below with reference to specific embodiments and drawings.

Example 1 Cloning of Colletotrichum rubber tree CsOxdC gene

Based on the sequence fragments obtained by yeast two-hybrid technology in the early stage, we used BLAST technology to search the full-length gene sequence of C. siamense oxalate decarboxylase CsOxdC in NCBI and designed primer pairs.

CsOxdC-F (5’-ATGCATCTCCCGCTCCTCT-3’)/CsOxdC-R (5’-AAGTTCGTCGGTCTGAGTGC-3’) used the cDNA and DNA of the rubber tree Colletotrichum C. siamenseHN08 as templates to amplify the target bands respectively. Sequence analysis showed that the obtained sequence contained the complete coding open reading frame. The DNA sequence size is 1576bp and the cDNA sequence size is 1395bp. The gene contains 3 introns and two Cupin domains, indicating that CsOxdC is a gene in the Cupin protein superfamily. This gene was named CsOxdC.

Example 2 Obtaining CsOxdC gene knockout mutant

Based on homologous recombination technology and the biological characteristics of Colletotrichum, the upstream and downstream fragment sequences were obtained from the Colletotrichum genome database.

In the first round of PCR, before and after the coding reading frame of the CsOxdC gene, the primer pair CsOxdC-UF/CsOxdC-UR and CsOxdC-DF/CsOxdC-DR was designed, and PCR amplification was used to obtain the upper arm sequence of the CsOxdC gene and the lower arm sequence after the C terminus. CsOxdC-UR and CsOxdC-DF respectively contain the chlorsulfuron resistance gene linker sequence, and the primer pair S1F/S2R was designed to amplify the chlorsulfuron resistance gene (ILV1);

In the second round of PCR, the CsOxdC upper arm and lower arm fragments and the ILV1 fragment amplified by the first round of PCR were fused using the primers CsOxdC-UF/CsOxdC-DR;

In the third round of PCR, the fusion product of the second round of PCR was enriched using the primers CsOxdC-UF/CsOxdC-DR (see Figure 1 for a schematic diagram); the enriched fragments were introduced into Colletotrichum hevea HN08 using the PEG-mediated protoplast transformation method. Protoplasts were screened by DCM medium containing chlorsulfuron methyl (100 μg/ml). A total of 1 batch was transformed, and 6 transformants were obtained.

The genomic DNA sequences of 6 transformants were extracted in batches and verified by PCR (see Figure 2). Transformant △CsOxdC-3 was in line with expectations. The CsOxdC gene was amplified from HN08 (lane 4) using the primer pair CsOxdC-OF/OR. A 1576 bp fragment of the coding sequence, but not ΔCsOxdC (lane 8). Primer CsOxdC-UouF/S1R amplified a 2164 bp fragment from ΔCsOxdC (lane 6), but HN08 could not (lane 2). Primer S2F/CsOxdC-DouR amplified the downstream sequence of CsOxdC gene and the 2537bp fragment of ILV1 gene part from ΔCsOxdC (lane 7), but HN08 could not (lane 3). The primers CsOxdC-UouF/CsOxdC-DouR were used to amplify a 4085bp fragment from ΔCsOxdC (lane 5) and a 2844bp fragment from HN08 (lane 1), confirming that the 1576bp fragment of the CsOxdC gene was replaced by the 2817bp fragment of the ILV1 gene. The PCR results preliminarily showed that the CsOxdC gene in transformant △CsOxdC-3 has been replaced by the ILV1 gene.

Using the CsOxdC-UouF/CsOxdC-DouR primer pair (CsOxdC-UouF: GCGTCATCGCCATTCC; CsOxdC-DouR: CCGAGGTAGTGATCACGAACATG), a 4085bp long band was amplified and sent to BGI for sequencing. Sequence analysis showed that the target gene CsOxdC has been blocked by ILV1 Gene replacement. It was confirmed that the transformant △CsOxdC-3 was a CsOxdC gene deletion mutant.

Example 3 CsOxdC gene regulates fungal resistance to the fungicide fludioxonil

In CM medium containing fludioxonil at different concentrations, as the concentration of fludioxonil increased, the growth rate of the CsOxdC gene deletion mutant gradually decreased, as shown in Figure 3. This result shows that CsOxdC can regulate the sensitivity of fungi to azole agents such as fludioxonil, and the deletion of this gene can reduce the resistance of Colletotrichum to azole agents such as fludioxonil. Therefore, the CsOxdC gene can be used as a drug target, and drugs that can inhibit the expression of the CsOxdC gene can be used as fungicides to inhibit the growth of fungi.

The above are only some preferred embodiments of the present invention and are not intended to limit the present invention. The protection scope of the present invention is not limited to the above contents. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention shall be included in the protection scope of the present invention.

Claims (4)

1. A CsOxdC gene knockout mutant comprising the following preparation steps:

in the first PCR, before and after a CsOxdC gene codes a reading frame, designing primer pairs CsOxdC-UF/CsOxdC-UR and CsOxdC-DF/CsOxdC-DR, obtaining an upper arm sequence of the CsOxdC gene and a lower arm sequence after a C end by utilizing PCR amplification, respectively containing chlorimuron-ethyl resistance gene joint sequences, and designing primer pairs S1F/S2R to amplify to obtain chlorimuron-ethyl resistance genes ILV1;

a second PCR, wherein the product obtained by the first PCR amplification is fused by using a primer CsOxdC-UF/CsOxdC-DR;

a third round of PCR, the products fused by the second round of PCR are enriched by using a primer CsOxdC-UF/CsOxdC-DR; introducing the enriched fragments into rubber tree anthrax protoplast, screening by chlorimuron-ethyl-containing culture medium, and then performing PCR verification to obtain the bacillus subtilis;

the nucleotide sequence of the CsOxdC gene is shown in SEQ ID NO:1 is shown in the specification;

the primer CsOxdC-UF has the sequence: 5'-CCAACATGCCGTCGAAACC-3'

The primer CsOxdC-UR has the sequence:

5’-AGATGTGGGGCACTGTGGCGTTGGCACGATGGGCGTGAAGTAGGAAGG-3’

the primer CsOxdC-DF has the sequence as follows:

5’-TATTGCACGGGAATTGCATGCTCTCACGGGAGCACTGCCACAATGGAA-3’

the sequence of the primer CsOxdC-DR is: 5'-CTGCTTCTAGTTCGAGAAGCG-3'

The sequence of primer S1F is: 5'-GTGCCAACGCCACAGTGCCCCACA-3'

The sequence of primer S2R is: 5'-GTGAGAGCATGCAATTCCCGTGCAATA-3'.

2. The use of the CsOxdC gene knockout mutant of claim 1 for modulating the sensitivity of anthrax to fludioxonil.

3. The application of the CsOxdC gene in the aspect of serving as an action target point of pyrrole medicaments is characterized in that the nucleotide sequence of the CsOxdC gene is shown as SEQ ID NO: 1.

4. The use of the CsOxdC gene knockout mutant of claim 1 to reduce the sensitivity of anthrax to fludioxonil.