Application of UvMCM1 gene in inhibition of biosynthesis of melanostin and prevention and treatment of false smut

Abstract

The invention belongs to the technical field of molecular biology, and particularly relates to application of a UvMCM1 gene in inhibition of biosynthesis of melanostin and / or prevention and treatment of false smut, and the base sequence of the Uvmcm1 gene is as shown in SEQ ID NO.1. The invention also relates to application of the UvMCM1 gene in inhibition of biosynthesis of melanostin and / or prevention and treatment of false smut. The Uvmcm1 gene is knocked out through a CRISPR / Cas9 (clustered regularly interspaced short palindromic repeats / CRISPR associated 9) technology, and a mutant knockout Uvmcm1 strain is obtained. The detection on the biosynthesis and pathogenicity of the ustilaginoidea virens shows that compared with a wild strain, the biosynthesis and pathogenicity of the ustilaginoidea virens in the Uvmcm1 mutant are obviously reduced.

Description

Technical Field

[0001] This invention belongs to the field of molecular biology technology, specifically relating to a... UvMCM1 Application of genes in inhibiting smuttoxin biosynthesis and / or controlling rice false smut. Background Technology

[0002] Rice is an important food crop, and its high yield is a pillar of the agricultural economy. It is also a model organism for life science research, playing a vital role in ensuring economic development and promoting scientific research. Rice false smut is mainly caused by the rice false smut fungus. (Ustilaginoidea virens) Rice false smut infects the rice panicle, forming dark green or yellowish-green spherical sclerotia on the grains. Furthermore, the sclerotin and smuttoxin produced by the rice false smut fungus are hepatotoxic and carcinogenic, and contaminated rice can harm human and livestock health through the food chain. The occurrence of rice false smut is closely related to climatic conditions; high temperatures of 25℃~30℃, continuous rainy and humid environments during the heading stage, and excessive application of nitrogen fertilizer all exacerbate the disease's spread. The rice false smut fungus is spread by wind and rain and spreads rapidly under suitable temperature and humidity conditions, posing a significant threat to rice production, leading to decreased grain filling rate and reduced thousand-grain weight, with yield losses reaching 20%~30% in severe cases. In recent years, with climate warming and changes in planting patterns, the occurrence of rice false smut has shown an increasing trend. Currently, the main methods for controlling rice false smut in agricultural production are agricultural control and chemical control. Agricultural control primarily relies on resistant varieties; however, variations in pathogen virulence often lead to the loss of varietal resistance. Chemical control, using triazole fungicides sprayed during the heading stage, can effectively suppress the pathogen, but long-term use may lead to resistance. Therefore, establishing a new control system and developing specific drug targets against the key pathogenic factors of rice false smut is urgently needed. Summary of the Invention

[0003] The purpose of this invention is to provide a UvMCM1 The application of the gene in inhibiting smuttoxin biosynthesis and / or controlling rice false smut, the aforementioned UvMCM1 Genes are beneficial for the prevention and control of rice false smut and provide a basis for the development of novel drug targets.

[0004] The technical solution adopted in this invention is: This invention provides UvMCM1 The application of the gene in inhibiting smuttoxin biosynthesis and / or controlling rice false smut, the aforementioned Uvmcm1 The base sequence of the gene is shown in SEQ ID NO.1.

[0005] Preferably, by constructing the UvMCM1 CRISPR recombinant vectors were used to prepare knockout mutants to inhibit the biosynthesis of smutin in rice false smut fungus, thereby further preventing the occurrence of rice false smut.

[0006] Preferably, the CRISPR recombinant vector is prepared as follows: The spacer fragment was synthesized by annealing using the primers shown in SEQ ID NO.3 and SEQ ID NO.4. The sequence of the spacer fragment is shown in SEQ ID NO.2. The spacer fragment and the linearized CRISPR vector are linked to obtain the CRISPR recombination vector.

[0007] Preferably, the CRISPR vector is pmCAS9.

[0008] Preferably, the restriction endonuclease used to digest the CRISPR vector is... Bsm BI.

[0009] Preferably, the annealing conditions for synthesizing the spacer fragment are 95℃ for 5 min and 25℃ for 10 min.

[0010] Preferably, the method for preparing the knockout mutant is as follows: The CRISPR recombinant vector and the Soeing fragment were co-transformed into rice false smut pathogen and cultured to obtain the knockout mutant; the base sequence of the Soeing fragment is shown in SEQ ID NO.26.

[0011] Preferably, the mass ratio of the CRISPR recombinant vector to the Soeing fragment is 1:1.

[0012] Preferably, the method for synthesizing the Soeing fragment is as follows: S1. Using rice false smut fungus as a template, amplification was performed using the following three pairs of primers: 1) Fragment 1 was obtained by amplification using SEQ ID NO.5 and SEQ ID NO.6; 2) Fragment 2 was obtained by amplification using SEQ ID NO.7 and SEQ ID NO.8; 3) Fragment 3 was obtained by amplification using SEQ ID NO. 9 and SEQ ID NO. 10; S2. Fragment 1, fragment 2 and fragment 3 are mixed and used as a template, and then amplified using SEQ ID NO.5 and SEQ ID NO.10 to obtain the Soeing fragment.

[0013] Compared with the prior art, the beneficial effects of the present invention are: This invention provides UvMCM1 The application of the gene in inhibiting smuttoxin biosynthesis and / or controlling rice false smut, the aforementioned Uvmcm1The gene's base sequence is shown in SEQ ID NO.1. This invention uses CRISPR / Cas9 technology to knock out the gene. UvMCM1 , to obtain the knockout mutant ∆ Uvmcm1 Strains. Detection of the biosynthesis and pathogenicity of *Ustilago maydis*, the causal agent of rice false smut, revealed that compared to the wild-type strain, ∆ Uvmcm1 The mutant strain showed significantly reduced smutin biosynthesis and pathogenicity; the complement strain was able to essentially restore the pathogenicity and smutin synthesis levels of the wild-type strain. Based on UvMCM1 This invention provides a novel control system for rice false smut, offering a basis for specific drug targets for rice false smut. Attached Figure Description

[0014] Figure 1 For PCR verification UvMCM1 A schematic diagram of gene knockout primer design.

[0015] Figure 2 Verification of ∆ for Southern blot Uvmcm1 Knockout mutant design and results: A: Knockout mutant design; B: Southern blot validation results.

[0016] Figure 3 For WT wild type, ∆ Uvmcm1 With C∆ Uvmcm1 Results of in-field inoculation of complement strains.

[0017] Figure 4 For WT wild type, ∆ Uvmcm1 With C∆ Uvmcm1 Statistics on the number of rice blast bacteria in the field of complement strains.

[0018] Figure 5 For high performance liquid chromatography detection of WT wild type, ∆ Uvmcm1 With C∆ Uvmcm1 The synthesis of smutin in complement strains. Detailed Implementation

[0019] The present invention will be further illustrated below with specific embodiments, but these embodiments do not limit the scope of the invention. Modifications or substitutions to the details and form of the technical solutions of the present invention may be made without departing from the spirit and scope of the invention, but all such modifications or substitutions fall within the protection scope of the present invention.

[0020] The inventive concept of this invention is as follows: Although relatively few in number (typically only 1-5 per genome) in fungi, MADS-box transcription factors play crucial roles in regulating morphogenesis, stress response, and pathogenicity. Compared to plants and animals, fungal MADS-box proteins generally have simpler structures but highly diverse functions, playing a key regulatory role, especially in the host infection process of pathogenic fungi. Fungal MADS-box proteins are predominantly of the SRF type, characterized by: a conserved MADS domain—responsible for DNA binding; lack of a K domain; and a variable C-terminal region—involved in transcriptional regulation or protein-protein interactions, determining functional specificity. *Spodoptera extinctae* contains only two MADS-box transcription factors, among which... UvMCM1 The relationship between genes and the biosynthesis and pathogenicity of smut in rice false smut has not been reported.

[0021] In view of the above-mentioned shortcomings in the prior art, the purpose of the present invention is to provide... UvMCM1 The application of genes and their encoded proteins in regulating the biosynthesis and pathogenicity of smutin in rice false smut fungus, knocking out the aforementioned Uvmcm1 The mutant obtained from the gene significantly reduced the biosynthesis and pathogenicity of smutin in rice false smut, through overexpression of the aforementioned gene. UvMCM1 The mutant obtained from the gene significantly increased the biosynthesis of smutin and pathogenicity in rice false smut fungus, therefore the aforementioned UvMCM1 Genes can positively regulate the biosynthesis of smutin and pathogenicity in rice false smut fungus.

[0022] To enable those skilled in the art to better understand and implement the technical solutions of this invention, the invention will be further described below with reference to specific embodiments. Unless otherwise specified, all reagents used in this invention are commercially available, and all methods used are conventional techniques in the art.

[0023] Example 1 UvMCM1 The application of genes in inhibiting smuttoxin biosynthesis and / or controlling rice false smut is as follows: 1. Validation of knockout strains of rice false smut.

[0024] 1.1 Construction of CRISPR vectors.

[0025] (1) Design of sgRNA primers: The sgRNA sequence of the target gene was designed using the CRISPR-Cas9 system. The CDS sequence of the target gene was obtained from the rice false smut database, and the 20bp sgRNA sequence with the highest score was selected using http: / / www.rgenome.net / cas-designer / .

[0026] UvMCM1 The CDS sequence is shown in SEQ ID NO.1.

[0027] SEQ ID NO.1:

[0028] (2) Synthesis of spacer fragment: Mix 5 μL each of 100 μM forward and reverse primers, incubate at 95℃ for 5 min and 25℃ for 10 min. Primer sequences are shown in SEQ ID NO.3 and SEQ ID NO.4.

[0029] The sequence of the spacer fragment is shown in SEQ ID NO.2.

[0030] SEQ ID NO.2: TGAGCTCGACGAGTCACAGTACTCGAGCTGCTCAGTGTCA.

[0031] (3) The CRISPR vector was digested with enzymes to prepare a linearized CRISPR vector. The system is shown in Table 1.

[0032] Table 1. CRISPR vector digestion system After preparing the CRISPR vector according to Table 1, digest it in a water bath at 55°C for 4 hours to obtain the linearized CRISPR vector.

[0033] (4) The linearized CRISPR vector was linked to the spacer fragment. The linking system is shown in Table 2.

[0034] Table 2 Connection System After preparing the solution according to Table 2, connect it at 16°C overnight.

[0035] (5) Transformation and sequencing: 10 μL of the ligation product was transformed into DH5α competent cells, and a single colony was taken for shaking culture and sequencing to obtain the CRISPR recombinant vector.

[0036] 1.2 Homologous fragment amplification.

[0037] (1) Primer design: Primers were obtained from the rice false smut pathogen database. UvMCM1 The genomic sequences of the target gene were obtained, 2 kb upstream and downstream of the target gene, along with the hygromycin gene sequence. The hygromycin gene was then used to replace the target gene. Primer S1F was designed 1 kb upstream of the target gene; primers S2R and S3F were designed 20 bp upstream of the target gene and 20 bp upstream of the target gene; primers S4R and S5F were designed 20 bp downstream of the target gene and 20 bp upstream of the target gene; and primer S6R was designed 1 kb downstream of the target gene. S2R and S3F were reverse complementary sequences; S4R and S5F were reverse complementary sequences.

[0038] (2) Amplification of homologous fragments: The upstream fragment of the gene was amplified by S1F and S2R, the hygromycin gene fragment was amplified by S3F and S4R, and the downstream fragment of the gene was amplified by S5F and S6R. Then, the upstream, downstream and hygromycin gene fragments were connected by S1F and S6R primers to obtain the Soeing fragment. The base sequence of the Soeing fragment is shown in SEQ ID NO.26.

[0039] SEQ ID NO.26:

[0040] 1.3 Preparation and transformation of rice false smut protoplasts.

[0041] (1) The wild-type strain of rice false smut preserved in the ultra-low temperature freezer was activated on PSA plate, cultured at 28℃ for 14 days, and an appropriate amount of mycelial blocks were cut into 50mL PSB liquid medium and cultured at 28℃ and 140rpm for 7 days to obtain rice false smut culture medium that produces spores.

[0042] The wild-type strain of *Ustilaginoidea* virens is disclosed in the reference: Zheng, X., Fang A., Qiu S., Zhao G., Wang J., Wang S., Wei J., Gao H., Yang J., Mou B., Cui F., Zhang J., Liu J., and Sun W. 2022. *Ustilaginoidea* virens secretes a family of phosphatases that stabilize the negative immune regulator OsMPK6 and suppress plant immunity. *Plant Cell*, 34: 3088-3109. doi: 10.1093 / plcell / koac154.

[0043] (2) Take the culture medium of rice false spore-producing fungus, filter it through sterile three-layer filter paper, collect it into a sterile 50 mL Corning tube, and centrifuge at 3500 rpm for 10 min to collect spores. Discard the supernatant, resuspend the precipitate in 50 mL of fresh PSB medium containing Amp, transfer it to a sterile Erlenmeyer flask, and incubate at 28℃ and 150 rpm for 24 h for germination. Filter the newly germinated bacterial solution and collect the fresh mycelium for later use.

[0044] (3) Prepare 20 mL of enzyme hydrolysate. The enzyme hydrolysate formula is shown in Table 3.

[0045] Table 3 Enzyme hydrolysate formulation After configuration, centrifuge at 120 rpm for 30 min at room temperature, then centrifuge at 3500 rpm for 6 min, and filter the supernatant using a bacterial filter.

[0046] Driselase was purchased from Sigma, D9515; Lysing enzyme was purchased from Yuan Ye, S10107, with an enzyme activity of 200 U / mg.

[0047] (4) Enzymatic hydrolysis of mycelium: Mix fresh mycelium and filtered enzymatic hydrolysate in a 50 mL Corning tube and enzymatically hydrolyze at 28°C and 90 rpm for 3 h.

[0048] (5) Protoplast collection and purification: Filter the enzymatically digested mixture through sterile 3-layer filter paper and collect the protoplasts at room temperature, 3500 rpm, for 10 min; remove the supernatant, rinse the protoplasts with 20 mL of 1.2 M KCl, and reflux at room temperature, 3500 rpm, for 10 min; remove the supernatant, suspend the protoplasts in 20 mL of 1×STC, and reflux at room temperature, 3500 rpm, for 10 min; remove the supernatant, reflux the protoplasts again with 20 mL of 1×STC, and reflux at room temperature, 3500 rpm, for 10 min; remove the supernatant, reflux the protoplasts a third time with 1×STC and adjust the concentration to 1×10⁻⁶ STC as needed. 8 per mL.

[0049] The STC formula is as follows: 100g sucrose, 50mL 0.5M Tris-Cl (pH 8), and 500mL ultrapure water; sterilize at 121℃ for 20min.

[0050] (6) Protoplast transformation: Take 150 μL of prepared protoplasts into a 50 mL Corning tube, add 5 μg of the constructed CRISPR recombinant vector and 5 μg of the amplified Soeing fragment, and let stand at room temperature for 20 min. Add 1 mL v / v 40% PEG8000, mix well, and let stand for 20 min. Continue to add 5 mL of TB3 medium containing Amp, and incubate at 28℃ and 90 rpm for 36 h. After 36 h of incubation, add 45 mL of Bottom TB3 medium, invert to mix well, pour evenly into 5 9 cm culture dishes, seal, and incubate overnight at 28℃. After overnight incubation, prepare TOP TB3 medium containing 200 μg / mL hygromycin, 10 mL per dish, seal, and incubate at 28℃ for about 20 days.

[0051] (7) Select transformants and culture them on a culture dish containing PSA for 10 days to verify the knockout.

[0052] PSA: Potato Sucrose Agar.

[0053] (8) The mycelium of the transformant was used to extract DNA crudely, and then PCR verification was performed. The primers used for PCR verification were: SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24 and SEQ ID NO.25.

[0054] (9) Reculture the transformants that have been verified by PCR, extract DNA from the mycelium, and perform Southern Blot verification. See the schematic diagram of the primer design for PCR verification of gene knockout. Figure 1 The Southern Blot validation results are shown below. Figure 2 .

[0055] 2. Verification of complement strains of rice false smut.

[0056] 2.1 Construction of the replenishment carrier.

[0057] (1) Primer design: Primers were selected at the multiple cloning sites of the laboratory-modified pY2P102-8266pro-3Flag vector sequence. Eco RV and Pst Two restriction enzyme sites were selected. Forward primers were designed 20 bp upstream of the restriction sites and 20 bp upstream of the GFP gene sequence, with the sequence SEQ ID NO. 17. Primers were then applied to the ends of the GFP gene sequence. UvMCM1 A reverse primer was designed at the front end of the gene, with the sequence SEQ ID NO.18; and its reverse complementary sequence SEQ ID NO.19; a reverse primer was designed at 20 bp from the end of the gene sequence and 20 bp downstream of another restriction site, SEQ ID NO.20.

[0058] (2) Amplification of homologous recombination fragments: Using the genome of rice false smut fungus P1FZ and a plasmid containing the GFP gene as templates, the target gene fragments were amplified using primers SEQ ID NO.17 and SEQ ID NO.20.

[0059] (3) Ligation, transformation and sequencing are the same as above.

[0060] 2.2 Replenishment and transformation.

[0061] The replacement transformation method is the same as the knockout transformation, except that protoplasts need to be prepared from the conidia of the knockout strain, and neomycin is used as a selection marker at a concentration of 875 μg / mL.

[0062] 2.3 Verification of the complement transformant.

[0063] Complementary transformants cultured on PSA medium for 10 days were collected, and mycelia were scraped into 2 mL centrifuge tubes containing sterile steel beads. After freezing in liquid nitrogen, the tubes were ground, 1×SDS loading buffer was added, and the tubes were placed in a dry bath at 100°C to denature the protein for 10 min. After cooling, the tubes were centrifuged at 12,000 rpm for 10 min. The results were then validated using Western blotting.

[0064] 3. Determination of smuttin.

[0065] Sterilize the cellophane three times in ultrapure water at 121°C for 20 minutes each time. Then, place the cellophane on a PSA plate using sterilized tweezers, taking care to avoid air bubbles. Next, punch holes in wild-type, mutant, and complementary strains of the same age using a 5 mm diameter punch. Place the bacterial cakes on the PSA medium lined with cellophane and incubate in the dark at 28°C for 28 days.

[0066] Scrape the mycelium cultured for 28 days into a new 50 mL EP tube and weigh the mycelium. Then add 30 mL of ethyl acetate and incubate for 1 day at 28 °C and 150 rpm in a shaker.

[0067] The solution was rotary evaporated at 45°C, then acetonitrile was added at a ratio of 1:1000. After thorough mixing, the solution was filtered through a 0.22 μm organic phase filter and then subjected to high-performance liquid chromatography (HPLC). The HPLC system was an LC-20A equipped with a UV detector with a detection wavelength of 280 nm and a ZORBAX SB-C18 column. Mobile phase B consisted of acetonitrile + 0.1% formic acid, and mobile phase A consisted of water + 0.1% formic acid. Flow rate: 1 mL / min; column temperature: 30°C; injection volume: 10 μL. ZORBAX SB-C18 column: 4.6 mm × 150 mm, 5 µm.

[0068] The gradient elution program for high performance liquid chromatography is shown in Table 4.

[0069] Table 4 Gradient elution program for high performance liquid chromatography 4. Experimental results.

[0070] The gene was knocked out using CRISPR / Cas9 technology. UvMCM1 Southern blot analysis validated transformants 7 and 17, yielding the knockout mutant strain ∆. Uvmcm1 That is, transformant strain No. 17, the results are shown in Figure 2 B.

[0071] The obtained mutant strain ∆ Uvmcm1、 complement strain C∆ Uvmcm1 Field inoculation was conducted simultaneously with the wild-type strain, and the incidence of rice false smut was statistically analyzed. The results are shown in […]. Figure 3 and Figure 4 This indicates that, relative to the wild type and complement, ∆ Uvmcm1 The pathogenicity of the mutant strain was significantly reduced.

[0072] High performance liquid chromatography results showed that ∆ Uvmcm1 The mutant strain showed a significantly reduced level of smutin biosynthesis, while the complement strain was able to revert to the accumulation level of the wild type. Results are shown below. Figure 5 .

[0073] The primer sequences involved in this invention are shown in Table 5.

[0074] Table 5 Primer sequence list The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0075] The embodiments described above are merely examples of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention.

Claims

1. UvMCM1 The use of a gene in the inhibition of smut element biosynthesis and / or the control of rice smut, characterized in that, The Uvmcm1 The base sequence of the gene is shown as SEQ ID NO.

1.

2. Use according to claim 1, wherein By constructing the UvMCM1 CRISPR recombinant vectors were used to prepare knockout mutants to inhibit the biosynthesis of smutin in rice false smut fungus, thereby further preventing the occurrence of rice false smut.

3. Use according to claim 2, wherein the compound is ###0002### The preparation method of the CRISPR recombination carrier is as follows: The spacer fragment is synthesized by annealing the primers shown in SEQ ID NO. 3 and SEQ ID NO. 4, and the sequence of the spacer fragment is shown in SEQ ID NO.

2. The spacer fragment and the linearized CRISPR carrier are connected to obtain the CRISPR recombination carrier.

4. The use according to claim 3, wherein the compound is ###0002### The CRISPR carrier is pmCAS9.

5. The use according to claim 4, wherein the compound is ###0002### The restriction enzyme used to cut the CRISPR vector is Bsm BI.

6. The use according to claim 3, wherein the compound is ###0002### The annealing conditions for synthesizing the spacer fragment are 95℃ for 5min and 25℃ for 10min.

7. The use according to claim 2, wherein the compound is ###00003### 2 The preparation method of the knockout mutant is as follows: The CRISPR recombination carrier and the Soeing fragment are co-transformed into the rice smut fungus, and the knockout mutant is obtained after culture; the base sequence of the Soeing fragment is shown in SEQ ID NO.

26.

8. Use according to claim 7, wherein the compound is ###0002### The mass ratio of the CRISPR recombination carrier and the Soeing fragment is 1:

1.

9. The use according to claim 7, wherein the compound is ###0002### The method for synthesizing the Soeing fragment is as follows: S1, taking the rice smut fungus as a template, the following three pairs of primers are used for amplification respectively: 1) amplification with SEQ ID NO. 5 and SEQ ID NO. 6 to obtain fragment 1; 2) amplification with SEQ ID NO. 7 and SEQ ID NO. 8 to obtain fragment 2; 3) amplification with SEQ ID NO. 9 and SEQ ID NO. 10 to obtain fragment 3; S2, taking the mixture of fragment 1, fragment 2 and fragment 3 as a template, amplification is carried out with SEQ ID NO. 5 and SEQ ID NO. 10 to obtain the Soeing fragment.

Images