UvScd1, an effector protein of rice false smut fungus, and its application

Abstract

This invention discloses a rice false smut fungus effector protein, UvScd1, and its application. The target gene UvScd1 was introduced into the secretory expression vector pBE-S. A strain of Bacillus subtilis capable of secreting the target protein UvScd1 was obtained. Rice leaves were then treated with the bacterial solution of this strain and inoculated with rice sheath blight and bacterial leaf blight. The results showed that rice treated with the bacterial solution exhibited enhanced resistance to rice sheath blight and bacterial leaf blight. Therefore, this invention provides a possibility for researching and developing biocontrol strains of Bacillus subtilis for the prevention and control of rice sheath blight and bacterial leaf blight.

Description

Technical Field

[0001] This invention relates to the field of protein application technology, specifically to a rice false smut fungus effector protein UvScd1 and its applications. Background Technology

[0002] Rice is a vital food crop globally, and with the world's growing population, the demand for rice production is increasing daily. Rice production is threatened by various diseases, particularly rice sheath blight and bacterial leaf blight. These diseases not only significantly reduce rice yield and quality but also cause substantial economic losses to farmers. To ensure food security, mitigating the impact of rice diseases has become a key task in agricultural research. Currently, traditional control measures mainly rely on chemical pesticides, but their use is often limited by environmental protection and food safety requirements.

[0003] In recent years, with the advancements in molecular biology and genetic engineering technologies, researchers have begun to explore more innovative disease control strategies. Among these, the use of effector proteins for disease resistance research has gradually attracted attention. Although effector protein-based control strategies have shown good results in the laboratory stage, they still face challenges in practical applications. First, obtaining specific induced resistance proteins usually requires gene cloning. This involves multiple steps, including extracting genes from pathogens, rice, or other sources, constructing expression vectors, and transforming host cells. In traditional laboratory operations, this process requires significant time and experimental resources, and demands high skill levels from the operators. Second, after obtaining the target protein, fermentation culture is usually required in host cells (such as E. coli or fungi). Optimizing the fermentation process is complex, requiring consideration of various conditions such as nutrients, temperature, and pH to achieve the optimal protein expression level. After fermentation, protein extraction and purification are also relatively cumbersome, involving multiple centrifugations, filtrations, and chromatography. The entire process is time-consuming and prone to protein degradation or denaturation. Furthermore, during the extraction and purification process, inducer proteins are often susceptible to changes in structure and function due to environmental conditions such as temperature, pH, and ionic strength. Many proteins are prone to denaturation or aggregation under normal temperature or inappropriate storage conditions, thereby losing their biological activity, which directly affects their effectiveness in practical applications. Therefore, overcoming the problems of cumbersome production and poor stability of effector proteins in practical applications are issues that need to be addressed in current research and applications. Summary of the Invention

[0004] To address the shortcomings of existing technologies, the present invention aims to provide a rice blast fungus effector protein UvScd1 and its application, thereby solving the technical problems of cumbersome production steps and poor stability when UvScd1 is applied in practice.

[0005] To achieve the above objectives, the present invention adopts the following technical solution:

[0006] This invention proposes a rice blast fungus effector protein UvScd1, the amino acid sequence of which is shown in SEQ ID NO.1 and its nucleotide sequence is shown in SEQ ID NO.2.

[0007] This invention also proposes the application of UvScd1, an effector protein of rice false smut, in rice disease resistance.

[0008] Preferred for use in the prevention and control of rice sheath blight pathogen.

[0009] The preferred method is to prevent and control bacterial blight of rice.

[0010] This invention discovered that the effector protein UvScd1 of rice blast fungus can produce lesion-like spots when overexpressed in rice, indicating that the UvScd1 gene is likely related to plant disease resistance. To test the potential of this protein for disease control, and considering the cumbersome process and instability of protein acquisition, this experiment introduced the target gene UvScd1 into the secretory expression vector pBE-S, and obtained a strain of Bacillus subtilis capable of secreting the target protein UvScd1.

[0011] The present invention further proposes the application of a recombinant vector containing the UvScd1 gene, an effector protein of rice false smut, as described above, in regulating rice disease resistance.

[0012] Preferably, the method for preparing the recombinant vector includes the following steps: cloning the sequence fragment of the designal peptide of the rice false smut effector protein UvScd1 gene into the pBE-S Bacillus subtilis secretory expression vector to obtain the recombinant vector pBE-S-UvScd1.

[0013] Compared with the prior art, the present invention has the following beneficial effects:

[0014] This invention introduces the target gene UvScd1 into the secretory expression vector pBE-S, and obtains a strain of Bacillus subtilis capable of secreting the target protein UvScd1. Rice leaves are then treated with the bacterial solution of this strain and inoculated with rice sheath blight and bacterial leaf blight pathogens. The results show that rice treated with the bacterial solution exhibits enhanced resistance to both rice sheath blight and bacterial leaf blight. Therefore, this invention provides a possibility for researching and developing biocontrol strains of Bacillus subtilis for the prevention and control of rice sheath blight and bacterial leaf blight. Attached Figure Description

[0015] Figure 1The image shows the plasmid map of the Bacillus subtilis secretory vector pBE-S and the amino acid sequence of the UvScd1 introduced fragment. A: Plasmid map of pBE-S, full length 5926 bp; B: Amino acid sequence of the introduced fragment in the pBE-S vector.

[0016] Figure 2 Figure showing the enhanced resistance of rice to rice sheath blight and bacterial blight by Bacillus subtilis containing UVScd1. Detailed Implementation

[0017] The present invention will be further described in detail below through specific preferred embodiments, but the present invention is not limited to the following embodiments.

[0018] It should be noted that, unless otherwise specified, all chemical reagents involved in this invention were purchased through commercial channels.

[0019] The wild-type strain JS60-2 of *Strombus oryzae*, the causal agent of rice false smut, was preserved in our laboratory and exhibits good pathogenicity to rice. It was cultured at 27°C on PSA solid medium or PSB liquid medium. It was also stored long-term at -70°C on PSB liquid medium containing 50% glycerol.

[0020] LB medium: 10g tryptone, 5g yeast extract, 10g sodium chloride (NaCl), add ddH2O to 1L. If preparing LB solid medium, add 15g agar powder. Sterilize at 121℃ for 20min.

[0021] Example 1: Construction of effector protein genes and expression vectors

[0022] (1) Wild-type strains of *Strombus oryzae*, the causal agent of rice false smut, were cultured in PSB liquid medium for 5-6 days, and mycelia were collected by filtration. Total RNA was extracted from the mycelia using an RNA extraction kit (Takara), and the purity and concentration of total RNA were detected using a Nanodrop 2000 spectrophotometer (ThermoScientific).

[0023] (2) cDNA synthesis was performed using the TransScript One-Step gDNA Removal and cDNA Synthesis Supermix kit from Beijing TransGen Biotech Co., Ltd. The specific steps were as follows, following the manufacturer's instructions: Add 1 μg total RNA, 1 μL Random Primer (0.1 μg / μL), and 10 μL 2×TS Reaction Mix sequentially to an RNase-free PCR tube. 1 μL of RT / RI Enzyme Mix, 1 μL of gDNA Remover, and RNase-free ddH2O were added to a final volume of 20 μL. The solution was mixed thoroughly and incubated at 25°C for 10 min, then at 42°C for 15 min, and finally heated at 85°C for 5 sec. The resulting cDNA was stored at -20°C. Using the cDNA as a template, PCR amplification was performed using primers: F, CGGC CGGTGCACATATGATGAACGCCGTCGTCATCAACAG (SEQ ID NO.3); R, TGGTGATGATGGTGATGTCTAGCTTGCAGAGCTGGGCAGTCA (SEQ ID NO.4). The reagents used for PCR were... PCR Master Mix (Yisheng Biotechnology) was used, with a reaction volume of 50 μL. The reaction conditions were: 95℃ for 3 min; 95℃ for 30 sec, 58℃ for 30 sec, 72℃ for 30 sec, 35 cycles; 72℃ for 5 min. PCR amplification yielded a 441 bp fragment, of which the introduced fragment encoded 133 amino acids.

[0024] (3) After recovering the fragment using the direct purification method of PCR product, the Bacillus subtilis secretory vector pBE-S (its vector map is shown in Figure 1) was purified using Sac I and Xba I. Figure 1 As shown, since this vector has its own unique signal peptide, the predicted signal peptide in the amino acid sequence of UvScd1 was removed, and the amino acid sequence of the inserted fragment (as shown in SEQ ID NO.5) was double-digested at 37℃ for 30 min. After digestion, the vector fragment was recovered, and the digested vector was ligated to the purified target fragment using a One Step Cloning Kit (Novizan). The ligation system was: Exnase II 1 μL, 5×CE II 2 μL, UvScd1 gene fragment 2 μL (200 ng), pBE-S vector 3 μL (50 ng), and sterile water 2 μL; reaction was carried out at 37℃ for 30 min.

[0025] (4) All ligation products were collected and transformed into *E. coli* DH5α competent cells using the heat shock method. The transformation products were plated on LB agar plates (200 mL containing 40 μL Kana). Single-colony PCR verification was performed using primers pBE-S-FGTCTGTACGTTCCTAAACTAG (SEQ ID NO. 6) and PBE-SR, ACCAGCCTATGCCTACA (SEQ ID NO. 7). Two positive colonies were selected for sequencing confirmation. The sequencing results showed that the insert fragment was 399 base pairs in size, and the encoded protein had 133 amino acid residues, as shown in SEQ ID NO. 5. Plasmids were extracted from single colonies with correct sequencing results and transformed into *Bacillus subtilis* SCK6 competent cells using electroporation to obtain *Bacillus subtilis* SCK6 strains containing the target gene. The transformation products were plated on LB agar plates (200 mL containing 40 μL Kana). Incubate overnight at 37°C, select 10 positive single clones for colony PCR verification, and store the colonies of the selected positive clones at -70°C.

[0026] SEQ ID NO.1:

[0027] MKTSVVALAALLGLASAQNAVVINSCSETVYVQSFPYNGGAPGPLTTLQPGQSFSEQFRPSGSTIKIAKTKTLNRPLFFGYSFSSNPDYAYYEFSTEWGNPFAGNRNVLSPGSGCEAFDCQANDGQCYSTPAHKKVYGCPRPVNLTAQLCKSEQ ID NO.2:

[0028] ATGAAGACCTCTGTTGTCGCTCTCGCTGCCCTCCTGGGTCTCGCCTCTGCCCAGAACGCCGTCGTCATCAACAGCTGCTCTGAAACAGTCTACGTGCAGTCTTTCCCGTACAACGGAGGTGCCCCCGGCCCTCTTACCACCCTCCAGCCTGGACAGTCCTTTTCCGAGCAATTCAGACCCTCTGGCTCGACATTAAGATTGCCAAGACCAAGACCCTGAACAGGCCG TTGTTCTTTGGATACTCGTTCAGTTCAAACCCGGACTACGCCTACTACGAGTTCAGCACGGAATGGGGGAATCCGTTTGCCGGAAACCGTAATGTCTTGTCTCCTGGCAGTGGCTGCGAGGCGTTTGATTGCCAGGCCAACGATGGTCAATGCTACAGCACGCCTGCGCACAAGAAGGTTTATGGTTGCCCCCGGCCGGTCAACCTGACTGCCCAGCTCTGCAAGTAA

[0029] SEQ ID NO.5:

[0030] NAVVINSCSETVYVQSFPYNGGAPGPLTTLQPGQSFSEQFRPSGSTIKIAKTKTLNRPLFFGYSFSSNPDYAYYEFSTEWGNPFAGNRNVLSPGSGCEAFDCQANDGQCYSTPAHKKVYGCPRPVNLTAQLCK

[0031] Example 2: Identification of resistance to rice sheath blight and bacterial blight by Bacillus subtilis containing UvScd1 protein.

[0032] 1. Treatment of rice leaves with Bacillus subtilis

[0033] (1) Bacillus subtilis containing empty vectors pBE-S and pBE-S-UvScd1 stored at -70℃ was activated by streaking on LB solid plates (containing 0.02% Kana by mass / volume, i.e. 0.02g Kana in 100ml of water) and incubated at 37℃ for 12h.

[0034] (2) Bacillus subtilis was cultured overnight at 29°C and 200 r / min in 5 mL LB liquid medium (containing 0.02% Kana).

[0035] (3) Take 1-2 mL of the well-cultured bacterial solution and place it in 30 mL of LB liquid medium (containing 0.02% Kana), and incubate at 29℃ and 200 r / min until OD. 600 =0.8-1.0;

[0036] (4) Centrifuge at 5000 r / min for 10 min, collect the precipitate, wash the precipitate twice with ddH2O, and finally suspend the precipitate in 30 mL of ddH2O and adjust the OD. 600 =0.8;

[0037] (5) Add Tween 20 to the Bacillus subtilis suspension to adjust the final mass concentration to 0.2%, and shake well to increase the adhesion of the bacterial solution;

[0038] (6) Spray the leaves of three-week-old Nipponbare rice with Bacillus subtilis suspension, using ddH2O and bacterial suspension containing empty vector pBE-S as controls;

[0039] 2. Rice leaves were inoculated with rice sheath blight pathogens and bacterial blight pathogens.

[0040] Rice leaves were treated with Bacillus subtilis UvScd1 for 48 hours, and then inoculated with Rhizoctonia solani, the rice sheath blight pathogen, via mycelial blocks. Disease incidence was assessed and lesion area calculated 2 days after inoculation. Figure 2 AB: A diagram illustrating the enhanced resistance of rice to rice sheath blight in plants containing UvScd1 Bacillus subtilis. After treating rice leaves with the bacterial solution for 48 hours, scissors were used to dip into the solution of Xanthomonas oryzae pv. oryzae, and approximately 2 cm of the leaf tip was cut off. The incidence of rice bacterial blight was investigated 21 days later, and the length of lesions was recorded. Figure 2 CD: A graph showing the enhancement of rice resistance to bacterial blight in rice containing Bacillus subtilis var. uvScd1. The experiment was repeated three times. "a,b" indicates a significant difference, p<0.05.

[0041] Finally, it should be noted that the above embodiments do not limit the present invention in any way. Those skilled in the art can make modifications and improvements based on the present invention. Therefore, any modifications or improvements made without departing from the spirit of the present invention are within the scope of protection claimed by the present invention.

Claims

1. The application of a rice false smut effector protein UvScd1 in enhancing resistance to rice sheath blight and bacterial blight, wherein the amino acid sequence of the rice false smut effector protein UvScd1 is shown in SEQ ID NO.

1.

2. A protein containing rice false smut effector proteins UvScd1 The application of recombinant vectors in enhancing resistance to rice sheath blight and bacterial blight, the gene sequence encoding the rice false smut effector protein UvScd1 is shown in SEQ ID NO.

2.

3. The application according to claim 2, characterized in that, The method for preparing the recombinant vector includes the following steps: cloning the sequence fragment of the designal peptide of the rice false smut effector protein UvScd1 gene into the pBE-S Bacillus subtilis secretory expression vector to obtain the recombinant vector pBE-S-UvScd1.

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