Sugarcane scbs protein and key domain deletion mutant thereof

Abstract

The application belongs to the field of new application of functional proteins and mutants thereof, and discloses application of sugarcane ScCBS protein and key domain deletion mutants thereof. The nucleotide sequence of the sugarcane ScCBS protein is shown as SEQ ID NO:1, and the amino acid sequence is shown as SEQ ID NO:2. The key domain deletion mutants of the ScCBS protein are ScCBS△motif1, ScCBS△motif3, ScCBS△motif4 and ScCBS△motif9, which respectively lack 28, 50, 49 and 29 amino acids. The application proves that the ScCBS protein can interact with SCSMV P1 and inhibit the RNA silencing suppressor activity by means of yeast two-hybrid, two-molecular fluorescence complementary experiment and agrobacterium transient expression system; the four mutants all lose the interaction and inhibition activity. Therefore, the ScCBS protein and the key domain deletion mutants thereof can be used for cultivating excellent SCSMV-resistant materials, and have wide application prospects in sugarcane disease-resistant molecular breeding.

Description

Technical Field

[0001] This invention patent belongs to the field of new applications of functional proteins and their mutants, specifically involving the application of sugarcane ScCBS protein and its key domain deletion mutants. Background Technology

[0003] SCSMV is a member of the genus *Poacevirus* in the family Potyviridae. Its encoded serine protease P1 protein has been confirmed as a viral RNA silencing repressor (RSS), playing a crucial role in viral infection of the host. The P1 protein of the *Poacevirus* genus has been verified as an RNA silencing repressor, playing a vital role in resisting host RNA silencing defenses. Studies have shown that the SCSMV P1 protein plays a functional role in inhibiting RNA silencing, and some conserved motifs and positively selected sites in the P1 protein are associated with RSS activity and protein stability.

[0004] Cystathione-β-synthase (CBS) domain proteins (CDCPs) are an evolutionarily conserved superfamily of proteins containing varying numbers of CBS domains. CBS domains were first discovered in the archaea *Methanococcus jannaschii*. Each CBS domain contains approximately 60 amino acid residues, forming two α-helices and three β-chains, and typically exists as tandem repeats, particularly in pairs or quadrupedal pairs within polypeptides. CBS domains are present in cytoplasmic and membrane proteins (metabolic enzymes, kinases, and transsulfurization channels) performing various functions, as well as in the proteomes of archaea, prokaryotes, and eukaryotes. CBS domains are integrated as highly efficient regulatory elements into proteins with diverse functions, and their enhancement or deactivation of protein activity is related to the binding of different ligands. The enzymatic activity of CBS domain-associated enzymes and transporter domains is regulated by the CBS domains by mediating the binding of adenosine-based molecules such as adenine nucleotide (AMP), adenosine triphosphate (ATP), or S-adenosylmethionine (ASM). Furthermore, in AMP-dependent protein kinases (AMPKs), signals can be transmitted remotely between different subunits of AMPK via the CBS domain, and the conformational regulation induced by the CBS domain can be integrated into more complex regulatory mechanisms.

[0005] CDCP family genes play important roles in regulating plant growth and development, environmental stress, and pathogen infection. In Arabidopsis thaliana, some CDCP genes respond to drought, salt, and damage stress in root and stem tissues; AtCBSX1 regulates development by modulating the thioredoxin system in chloroplasts; AtCBSX3 participates in plant development and redox systems by regulating the production of reactive oxygen species (ROS) in mitochondria; and CBSX regulates the activity of plastid TRXm through energy sensing, thereby regulating cyclic electron transport in chloroplasts and ultimately regulating ATP homeostasis to achieve optimal growth. In soybean, transgenic Arabidopsis plants overexpressing GmCBS21 and GmCBSDUF3 exhibit enhanced tolerance to low nitrogen stress as well as drought and salt stress. In rice, several genes encoding CDCPs have been found to participate in responses to various stresses. Overexpression of OsCBSX4 resulted in strong resistance to salt stress in tobacco plants. OsCBSX3 plays a positive regulatory role in the synergistic regulation of rice resistance to rice blast fungus through salicylic acid and jasmonic acid-mediated signal transduction pathways. Both OsCBSX9 and OsCBSCBS4 showed significant upregulation under salt and drought stress conditions in rice plants. In conclusion, CBSs play an important role in regulating plant responses to biotic and abiotic stresses.

[0006] Studies have shown that sugarcane CBS interacts with SSCMV P1 and affects the RSS activity of the P1 protein. However, the molecular mechanism by which ScCBS exerts its disease resistance through other host protein components remains unclear. This invention constructs conserved domain deletion mutants of ScCBS and utilizes Y2H, BiFC experiments, and an Agrobacterium-mediated transient expression system. It was discovered that four ScCBS motif deletion mutants (ScCBS△motif1, ScCBS△motif3, ScCBS△motif4, and ScCBS△motif9) can alter the interaction with SSCMV P1 and inhibit SSCMV P1 RSS activity. This has significant implications and broad application prospects for modern sugarcane genetic breeding technology. Summary of the Invention

[0007] Based on this, the purpose of this invention is to provide the application of sugarcane ScCBS protein and its key domain deletion mutants.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] The ScCBS protein, the amino acid sequence of which is shown in SEQ ID NO: 2, and the nucleotide sequence of its encoding gene, which is shown in SEQ ID NO: 1.

[0010] The application of the aforementioned ScCBS protein in its interaction with SSCMV P1.

[0011] The above-mentioned ScCBS protein is used to inhibit the activity of the SSCMV P1 protein RNA silencing repressor.

[0012] The ScCBS protein key domain deletion mutant ScCBS△motif1 has 28 consecutive amino acids deleted from position 2 to position 29 relative to the amino acid sequence shown in SEQ ID NO: 2. The nucleotide sequence of the deleted portion is shown in SEQ ID NO: 3. The nucleotide sequence encoding the mutant ScCBS△motif1 is shown in SEQ ID NO: 4.

[0013] The ScCBS protein key domain deletion mutant ScCBS△motif3 has 50 consecutive amino acids deleted from position 66 to 115 relative to the amino acid sequence shown in SEQ ID NO: 2. The nucleotide sequence of the deleted portion is shown in SEQ ID NO: 5. The nucleotide sequence encoding the mutant ScCBS△motif3 is shown in SEQ ID NO: 6.

[0014] The ScCBS protein key domain deletion mutant ScCBS△motif4 has 49 consecutive amino acids deleted from position 82 to 130 relative to the amino acid sequence shown in SEQ ID NO: 2. The nucleotide sequence of the deleted portion is shown in SEQ ID NO: 7. The nucleotide sequence encoding the mutant ScCBS△motif4 is shown in SEQ ID NO: 8.

[0015] The ScCBS protein key domain deletion mutant ScCBS△motif9 has 29 consecutive amino acids deleted from position 201 to 229 relative to the amino acid sequence shown in SEQ ID NO: 2. The nucleotide sequence of the deleted portion is shown in SEQ ID NO: 9. The nucleotide sequence encoding the mutant ScCBS△motif9 is shown in SEQ ID NO: 10.

[0016] The application of the aforementioned ScCBS protein key domain deletion mutant in regulating the interaction between ScCBS protein and SSCMV P1 protein.

[0017] The above-mentioned ScCBS protein key domain deletion mutants are used to regulate the ScCBS protein to inhibit the activity of the SSCMV P1 protein RNA silencing repressor.

[0018] The application of the aforementioned ScCBS protein and the aforementioned ScCBS protein key domain deletion mutant in sugarcane molecular breeding.

[0019] The beneficial effects of this invention are as follows:

[0020] This invention provides applications of the ScCBS protein and its key domain deletion mutants. First, yeast two-hybrid (Y2H) and bimolecular fluorescence complementation (BiFC) assays revealed that ScCBS interacts with the SSCMV P1 protein, while four ScCBS protein key domain deletion mutants failed to interact with SSCMV P1, indicating that the motifs motif1, motif3, motif4, and motif9 of the sugarcane ScCBS protein are key domains for the interaction between ScCBS and SSCMV P1. Second, using an Agrobacterium-mediated transient expression system, it was found that ScCBS weakens the RSS activity of SSCMV P1, while the inhibitory effect of the mutants ScCBS△motif1, ScCBS△motif3, ScCBS△motif4, and ScCBS△motif9 on the RSS activity of SSCMV P1 protein was relieved, meaning that ScCBS can inhibit SSCMV P1. The RSS activity of the P1 protein depends on four key domains: Motif1 (2-29 aa), Motif3 (66-115 aa), Motif4 (82-130 aa), and Motif9 (201-229 aa). Attached Figure Description

[0021] Figure 1 This is a schematic diagram of a mutant ScCBS protein with a missing key domain.

[0022] Figure 2 To verify the interaction between the ScCBS protein and its key domain deletion mutants and the SSCMV P1 protein Y2H.

[0023] Figure 3 To verify the interaction between the ScCBS protein and its key domain deletion mutants and the SSCMV P1 protein BiFC.

[0024] Figure 4 The effect of ScCBS protein and its key domain deletion mutant on the RSS activity of SSCMV P1 protein. a: GFP fluorescence expression after co-injection of ScCBS protein and its key domain deletion mutant with SSCMV P1 protein. b and c: Transcriptional expression levels of GFP and SSCMV P1 5 days after co-injection of ScCBS protein and its key domain deletion mutant with SSCMV P1 protein. All values ​​are expressed as mean ± standard error (n=3). Different letters represent significant differences (p<0.05). Detailed Implementation

[0025] The technical solution of the present invention will be specifically described below with reference to specific embodiments and accompanying drawings. The embodiments described below should be understood as further detailed descriptions of certain aspects, characteristics, and implementations of the present invention, but should not be considered as limitations on the present invention. The present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. Rather, these embodiments are provided to enable those skilled in the art to have a more thorough and comprehensive understanding of the disclosure of the present invention.

[0026] Unless otherwise specified, the experimental methods used in the following embodiments are conventional experimental methods or methods recommended in the corresponding manufacturer's instructions; the materials and reagents used are commercially available unless otherwise specified.

[0027] Example 1: Obtaining the ScCBS gene and key domain deletion mutants

[0028] 1. Obtaining the ScCBS gene

[0029] (1) Experimental materials

[0030] Leaf samples were collected from sugarcane variety LCP85-384, disinfected with 75% alcohol, and used for RNA extraction.

[0031] (2) Total RNA extraction and cDNA synthesis from sugarcane leaves

[0032] Total RNA was extracted from sugarcane leaves using the TRIzol kit. After determining the RNA concentration using a full-function microplate reader, the RNA was reverse transcribed into cDNA according to the instructions of the HiScript II 1st Strand cDNA Synthesis kit, which served as a template for subsequent gene amplification.

[0033] (3) Cloning of the ScCBS gene

[0034] The cloning primer pair ScCBS-F / R (Table 1) was designed using Primer Primer 6 software for cloning the ScCBS gene, with a target fragment size of 705 bp. Using cDNA as a template, PCR amplification was performed using the LA Taq polymerase kit. The reaction system is shown in Table 2, and the reaction procedure is shown in Table 3. The PCR product was detected by 1.5% agarose gel electrophoresis and then purified using a gel extraction kit. The purified fragment was ligated into the pMD19-T vector using seamless cloning technology and transformed into *E. coli* DH5α competent cells. After colony PCR identification and sequencing verification, the intermediate vector plasmid of the ScCBS gene was obtained. Sequencing results showed that the nucleotide sequence of the cloned ScCBS gene is shown in SEQ ID NO: 1, and the amino acid sequence of the encoded protein is shown in SEQ ID NO: 2. The plasmid was extracted and preserved for subsequent construction of ScCBS protein key domain deletion mutants.

[0035] 2. Obtaining mutants with deletion of key structural domains in the ScCBS protein

[0036] The distribution of conserved motifs in the ScCBS protein was analyzed using the MEM online website tool. Deletion mutations were performed on three conserved motifs (motif 1, 3, and 9) and one CBS domain region (motif 4) of the ScCBS protein. The locations of the deleted amino acids are shown in [reference needed]. Figure 1 Using the ScCBS gene intermediate vector plasmid as a template, amplification was performed using the LA Taq polymerase kit and 2× GC Buffer I. The upstream fragment was amplified using ScCBS-F paired with the reverse primers for each mutant, and the downstream fragment was amplified using the forward primers for each mutant paired with ScCBS-R. Primer sequences are shown in Table 1, PCR reaction systems in Table 2, and PCR reaction procedures in Table 3. PCR products were detected by 1.5% agarose gel electrophoresis and purified by gel extraction. Subsequently, using the recovered upstream and downstream fragments as a mixed template, the two fragments were spliced ​​using the DNA Assembly Mix Plus seamless cloning kit to obtain the deletion mutant gene fragment. After gel purification, it was ligated into the pMD19-T vector. The ligation product was transformed into *E. coli* DH5α competent cells, plated on LB solid medium containing 50 μg / mL Amp, and incubated upside down at 37°C for 14 h. Single colonies were picked for colony PCR detection, and positive clones were sent for sequencing verification. After the sequencing results were correctly aligned, plasmids were extracted, and intermediate vector plasmids of ScCBS△motif1, ScCBS△motif3, ScCBS△motif4, and ScCBS△motif9 deletion mutants were finally obtained.

[0037] Sequencing results confirmed the sequences of each deletion mutant as follows:

[0038] ScCBS△motif1: 28 amino acids are deleted starting from the second amino acid. The nucleotide sequence of the deleted fragment is shown in SEQ ID NO: 3, and the full-length nucleotide sequence of the mutant is shown in SEQ ID NO: 4;

[0039] ScCBS△motif3: 50 amino acids are deleted starting from amino acid position 66. The nucleotide sequence of the deleted fragment is shown in SEQ ID NO: 5, and the full-length nucleotide sequence of the mutant is shown in SEQ ID NO: 6;

[0040] ScCBS△motif4: 49 amino acids are deleted starting from amino acid position 82. The nucleotide sequence of the deleted fragment is shown in SEQ ID NO: 7, and the full-length nucleotide sequence of the mutant is shown in SEQ ID NO: 8;

[0041] ScCBS△motif9: 29 amino acids are deleted starting from amino acid position 201. The nucleotide sequence of the deleted fragment is shown in SEQ ID NO: 9, and the full-length nucleotide sequence of the mutant is shown in SEQ ID NO: 10.

[0042] Table 1 Primers for sugarcane ScCBS protein and key domain deletion mutants

[0043]

[0044] Table 2 PCR reaction system

[0045]

[0046] Table 3 PCR reaction procedure

[0047]

[0048] Example 2: Validation of the interaction between the ScCBS protein key domain deletion mutant and SSCMV P1

[0049] 1. Yeast two-hybrid experiment (Y2H)

[0050] (1) Construction of yeast expression vector

[0051] A yeast expression vector was constructed using a seamless cloning ligation method. The specific steps are as follows:

[0052] (i) Linearization of expression vectors. Yeast expression vectors pGBKT7(BD) and pGADT7(AD) were double-digested with restriction endonucleases EcoRI and BamHI. The reaction mixture consisted of 5 µL 10× FastDigest Green Buffer, 3 µL EcoRI, 3 µL BamHI, and 3 µg of the target plasmid, brought to a final volume of 50 µL with nuclease-free sterile water. The reaction mixture was incubated at 37 °C for 1 h. After detection by 1.5% agarose gel electrophoresis, the linearized vector was recovered and purified, and stored at -20 °C for later use. (ii) PCR amplification of the target gene. Given that the 5' and 3' sequences of ScCBS and its various deletion mutants are essentially identical, universal primers AD-ScCBS(△)-F / R were used for amplification. The ScCBS△motif1 mutant, due to its deletion location near the 5' end, was amplified using a specific upstream primer AD-ScCBS(△motif1)-F paired with a universal downstream primer; the ScCBS△motif9 mutant, due to its deletion location near the 3' end, was amplified using a universal upstream primer paired with a specific downstream primer AD-ScCBS(△motif9)-R. The SCSMV P1 gene (SEQ ID NO. 11) was amplified using independently designed specific primer pairs. Primer sequences are detailed in Table 4. Using the intermediate vector plasmids (ScCBS, ScCBS△motif1, ScCBS△motif3, ScCBS△motif4, ScCBS△motif9) and the SCSMV P1 plasmid successfully constructed in Example 1 as templates, PCR amplification was performed using high-fidelity enzymes. The reaction system is shown in Table 5, and the reaction procedure is shown in Table 6. The PCR products were purified using a gel extraction kit and stored at -20°C for later use.

[0053] (iii) Recombinant Ligation. The target gene fragment was constructed into a yeast expression vector using the ClonExpress II One Step Cloning Kit. The ligation reaction system consisted of 2 µL 5× CE II Buffer, 1 µL Exnase II, 3 µL linearized vector, and 2 µL gel-recovered product, which was then brought to a final volume of 10 µL with nuclease-free sterile water. After incubating the reaction solution at 37 °C for 35 min, it was transformed into *E. coli* DH5α competent cells and plated on LB agar containing 50 μg / mL Amp and 50 μg / mL Kan, and cultured overnight. Positive clones were picked for sequencing verification. After successful sequencing, plasmids were extracted to obtain recombinant plasmids AD-ScCBS, AD-ScCBS△motif1, AD-ScCBS△motif3, AD-ScCBS△motif4, AD-ScCBS△motif9, and BD-SCSMV P1, which were used for subsequent yeast transformation experiments.

[0054] Table 4 Primer sequences used for constructing yeast two-hybrid vectors

[0055]

[0056] Table 5. PCR reaction system for constructing yeast two-hybrid vectors

[0057]

[0058] Table 6. PCR reaction procedure for constructing yeast two-hybrid vectors

[0059]

[0060] (2) Yeast transformation

[0061] Salmon sperm carrier DNA was placed in a 100°C water bath for 7 min, then quickly transferred to an ice-water bath for cooling. This step was repeated once, and the mixture was then kept on ice for later use. 1–2 µg of the previously constructed AD recombinant plasmid (AD-ScCBS, AD-ScCBS△motif1, AD-ScCBS△motif3, AD-ScCBS△motif4, or AD-ScCBS△motif9) and BD recombinant plasmid (BD-SCSMV P1) were co-transformed into 50 µL of Y2H Gold yeast competent cells, and the mixture was gently tapped to mix. Then, 10 µL of pretreated carrier DNA and 500 µL of PEG / LiAC solution (purchased from Clontech) were added, and the mixture was pipetted and mixed. The mixture was incubated in a 30°C water bath for 30 min, inverting and mixing 7–9 times every 10 min. Subsequently, it was transferred to a 42°C water bath for heat shock for 15 min, inverting and mixing 7–9 times every 7.5 min. After heat shock, centrifuge at 5000 rpm for 1 min, discard the supernatant, and resuspend the bacterial cells in 1 mL of nuclease-free sterile water. Centrifuge at 1000 rpm for 1 min, discard the supernatant, and resuspend the bacterial cells in 400 µL of nuclease-free sterile water. Spread 100 µL of the bacterial suspension onto SD / -Leu-Trp (DDO) solid medium and incubate upside down at 28–30 °C for 2–3 days. Select successfully transformed yeast colonies.

[0062] (3) Mutual verification

[0063] Single colonies from DDO solid medium were picked and propagated. The correctness of plasmid transformation was verified by colony PCR. The verified yeast culture was centrifuged at 2000 rpm for 1 min, the supernatant was discarded, and the cells were resuspended in 1 mL of nuclease-free sterile water. The centrifugation and washing were repeated once; the supernatant was discarded again, and the cells were resuspended in 600 µL of nuclease-free sterile water. The bacterial resuspension was diluted 10-fold and plated onto SD / -Leu-Trp / X-α-Gal (DDO / X) and SD / -Leu / -Trp / -His / -Ade / X-α-Gal / AbA (QDO / X / A) solid media, respectively. The cultures were incubated upside down at 30℃ for 3–5 days, and the growth morphology and color changes of the yeast colonies were observed. AD-T / BD-53 was used as a positive control, and AD-T / BD-Lam as a negative control.

[0064] (4) Verification results

[0065] The Y2H test results are as follows: Figure 2 As shown, yeast co-transformed with ScCBS and SCSMV P1 grew normally on QDO / X / A plates; however, yeast co-transformed with ScCBS△motif1, ScCBS△motif3, ScCBS△motif4, or ScCBS△motif9 and SCSMV P1 failed to grow normally on QDO / X / A plates. These results indicate that the ScCBS protein can interact with the SCSMV P1 protein, and the four ScCBS protein key domain deletion mutants all lost their ability to interact with the SCSMV P1 protein.

[0066] 2. Bimolecular fluorescence complementation assay (BiFC)

[0067] (1) Construction of BiFC expression vector

[0068] BiFC expression vectors were constructed using a seamless cloning ligation method. The BiFC expression vectors pSuper1300-nYC (nYC) and pSuper1300-nYN (nYN) were double-digested with restriction endonucleases XbaI and KpnI. The digestion products were purified using a gel extraction kit for later use. Using the candidate gene intermediate vector plasmids (ScCBS, ScCBS△motif1, ScCBS△motif3, ScCBS△motif4, ScCBS△motif9, SSCMV P1) constructed in Example 1 as templates, PCR amplification was performed using the primers listed in Table 7: sequences homologous to the ends of the pSuper-nYC vector were introduced at both ends of the ScCBS and its deletion mutant genes, and sequences homologous to the ends of the pSuper-nYN vector were introduced at both ends of the SSCMV P1 gene. After gel extraction and purification of the PCR products, the ScCBS gene and its key domain deletion mutant gene were directionally constructed into the pSuper-nYC vector using a seamless cloning ligation method to obtain recombinant vectors nYC-ScCBS, nYC-ScCBS△motif1, nYC-ScCBS△motif3, nYC-ScCBS△motif4, and nYC-ScCBS△motif9; the SSCMV P1 gene was directionally constructed into the pSuper-nYN vector to obtain the recombinant vector nYN-SCSMV P1.

[0069] Table 7 Primer sequences used for BiFC vector construction

[0070]

[0071] (2) Agricultural Stalk Conversion

[0072] Take 1 µg of the recombinant vector plasmid constructed above, add it to 50 µL of GV3101 Agrobacterium competent cells, gently mix, and incubate on ice for 15 min. Then, flash freeze in liquid ammonia for 5 min, and immediately place in a 37°C water bath for 5 min. Add 400 µL of LB liquid medium and incubate in a shaker at 28–30°C for 4 h. Take 50–100 µL of the bacterial culture and spread it on LB solid medium containing 50 μg / mL Kan and 50 μg / mL LRif, and incubate upside down at 28–30°C for 2–3 days. Select positive Agrobacterium colonies.

[0073] (3) Mutual verification

[0074] Single colonies of positive Agrobacterium were picked and inoculated into LB liquid medium, and cultured at 28°C and 200 rpm in the dark for 12 h. The cultured bacterial suspension was centrifuged at 5000g for 10 min to collect the bacterial cells; the cells were washed and resuspended in MES buffer (containing 100 μM MES, 10 mM MgCl2, and 200 μM acetylsyl syringone; pH 5.4–5.8), and the OD of the bacterial suspension was adjusted. 600 The concentration was adjusted to 0.5, and the mixture was incubated in the dark at room temperature for 2 hours. The bacterial suspension was drawn up with a 1 mL disposable sterile syringe and injected into the underside of 4-6 year old Nicotiana benthamiana leaves. nYN-Nb-SKP1 / nYC-BRA-P0 was set as a positive control, and the empty vector nYN / nYC was set as a negative control.

[0075] (4) Verification results

[0076] BiFC test results are as follows Figure 3 As shown, a significant YFP fluorescence signal was observed in the nuclei of *Tobacco Benedictine arvense* leaf cells co-injected with the recombinant vectors ScCBS and SSCMV P1; however, no YFP fluorescence signal was observed in *Tobacco Benedictine arvense* leaf cells co-injected with the recombinant vectors ScCBSΔmotif1, ScCBSΔmotif3, ScCBSΔmotif4, or ScCBSΔmotif9, respectively, along with SSCMV P1. The BiFC assay results were consistent with the Y2H assay results, further demonstrating that the motif1, motif3, motif4, and motif9 motifs of the sugarcane ScCBS protein are key domains mediating its interaction with the SSCMV P1 protein.

[0077] Example 3: Effects of ScCBS and its key domain deletion mutants on the RSS activity of SSCMV P1 protein

[0078] The effect of the ScCBS key domain on the activity of the SSCMV P1 protein RNA silencing suppressor (RSS) was verified using an Agrobacterium-mediated transient expression system.

[0079] 1. Construction of Transient Expression Carriers

[0080] Transient expression vectors were constructed using a seamless cloning ligation method. The transient expression vector PGD was double-digested with restriction endonucleases NcoI and SalI, and the digestion products were purified by gel extraction for later use. Using the intermediate vector plasmids constructed in Example 1 (ScCBS, ScCBS△motif1, ScCBS△motif3, ScCBS△motif4, ScCBS△motif9, SCSMVP1) as templates, PCR amplification was performed using the primers listed in Table 8, introducing sequences homologous to the ends of the pGD vector at both ends of the target gene. After PCR products were purified by gel extraction, each target gene was constructed into the pGD vector using a seamless cloning ligation method to obtain recombinant plasmids pGD-HA-SCSMV P1, pGD-HA-ScCBS and its four mutant plasmids (pGD-HA-ScCBS△motif1, pGD-HA-ScCBS△motif3, pGD-HA-ScCBS△motif4 and pGD-HA-ScCBS△motif9).

[0081] Table 8 Primer sequences used for construction of transient expression vectors

[0082]

[0083] 2. Agrobacterium-mediated transformation

[0084] The recombinant vector plasmid was transformed into Agrobacterium strain GV3101 competent cells and plated on LB solid medium containing 50 μg / mL Kan and 50 μg / mL Rif. The cells were then incubated upside down at 28–30 °C for 2–3 days. Single colonies were picked and propagated for identification by culture PCR, following the Agrobacterium transformation steps in the BiFC experiment of Example 2.

[0085] 3. Agrobacterium-mediated leaf injection

[0086] Positive single colonies were picked and inoculated into LB liquid medium and cultured at 28°C and 200 rpm in the dark for 12 h. The cultured bacterial suspension was centrifuged at 5000g for 10 min to collect the bacterial cells; the cells were washed and resuspended with MES buffer (containing 100 μM MES, 10 mM MgCl2, and 200 μM acetylsyl syringone; pH 5.4–5.8), and the OD of the bacterial suspension was adjusted. 600 The bacterial solution was adjusted to 0.5 and incubated in the dark at room temperature for 2 hours. The bacterial solution was then drawn up with a 1 mL disposable sterile syringe and injected into the back of 4-6 leaf-aged tobacco leaves.

[0087] 4. Co-expression experiment

[0088] The reporter gene vector pCHF3-GFP, empty vector PGD, PGD-HA-SCSMV P1, and recombinant vectors of the ScCBS gene and its mutants were transformed into Agrobacterium GV3101, respectively. The bacterial cultures were mixed in equal volumes to establish the following infection combinations:

[0089] Negative control: pGD+pGD+pCHF3-GFP

[0090] Positive control: pGD-HA-SCSMV P1+pGD+pCHF3-GFP

[0091] Experimental group 1: pGD-HA-SCSMV P1+pGD-HA-ScCBS+pCHF3-GFP

[0092] Experimental group 2: pGD-HA-SCSMV P1 + pGD-HA-ScCBS mutant (ScCBS△motif1, ScCBS△motif3, ScCBS△motif4 or ScCBS△motif9) + pCHF3-GFP

[0093] The above-mentioned mixed bacterial suspension was injected into leaves of wild-type Nicotiana benthamiana. GFP fluorescence expression in the leaves was observed and photographed under a 365 nm UV lamp at 3, 5, and 7 days post-inoculation, and samples were collected for quantitative detection of transcriptional levels. Three biological replicates were set up for each group.

[0094] 5. RT-qPCR quantitative detection

[0095] (1) Design of fluorescent quantitative primers

[0096] qPCR primers for GFP, SSCMV P1 and internal reference genes were designed using the NCBI Primer-BLAST online tool. The primer sequences are shown in Table 9.

[0097] (2) RNA extraction and cDNA synthesis

[0098] Total RNA was extracted from tobacco leaves using the TRIzol kit. After determining the RNA concentration and purity using a full-function microplate reader, the RNA was reverse transcribed into cDNA according to the instructions of the HiScript II 1st Strand cDNA Synthesis kit.

[0099] (3) qPCR detection and data analysis

[0100] Using cDNA as a template, real-time quantitative PCR was performed using 2×ChamQ SYBR qPCR Master Mix reagent. The reaction system is shown in Table 10, and the reaction procedure is shown in Table 11. 2 -△△CTThe relative expression levels of genes were calculated using the method, and analysis of variance and significance of differences were performed using SPSS 23.0 software. Plotting software was used to create graphs.

[0101] Table 9 Primers for quantitative fluorescence detection

[0102]

[0103] Table 10. Real-time PCR reaction system

[0104]

[0105] Table 11 Quantitative Real-Time PCR Reaction Procedure

[0106]

[0107] 6. Experimental Results

[0108] like Figure 4 As shown in Figure a, when SSCMV P1 and ScCBS were co-injected into tobacco for 3 days, the GFP fluorescence intensity was significantly weaker than that of the positive control group (pGD-HA-SCSMV P1+pGD+pCHF3-GFP), and the GFP fluorescence intensity gradually decreased with the extension of infection time, indicating that ScCBS can weaken the RSS activity of SSCMV P1. However, when SSCMV P1 was co-expressed with four ScCBS mutants, the GFP fluorescence intensity was comparable to that of the positive control group within 3-7 days, indicating that ScCBS△motif1, ScCBS△motif3, ScCBS△motif4, and ScCBS△motif9 all lost the ability to inhibit the RSS activity of SSCMV P1 protein.

[0109] like Figure 4 As shown in b and 4c, 5 days post-infection, in the SCSMV P1 and ScCBS co-expression group, the transcription levels of the reporter genes GFP and SCSMVP1 were significantly downregulated compared to the positive control group, decreasing to 53.9% and 45.0% of the positive control group, respectively. However, in the SCSMVP1 co-expression group with each ScCBS mutant, the transcription levels of GFP and SCSMV P1 were not significantly different from those in the positive control group.

[0110] The above results indicate that ScCBS can inhibit the RSS activity of SSCMV P1 protein, and this inhibition depends on the key domains of motif1, motif3, motif4, and motif9.

[0111] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention should be considered equivalent substitutions. Various modifications and improvements made to the technical solutions of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. ScCBS protein, characterized by: The amino acid sequence of the ScCBS protein is shown in SEQ ID NO: 2, and the nucleotide sequence of its encoding gene is shown in SEQ ID NO:

1.

2. The application of the ScCBS protein as described in claim 1 in its interaction with SCSMV P1.

3. The application of the ScCBS protein as described in claim 1 in inhibiting the activity of the SSCMV P1 protein RNA silencing repressor.

4. The ScCBS△motif1 mutant, which lacks a key structural domain of the ScCBS protein, is characterized by: The mutant ScCBS△motif1 has 28 consecutive amino acids missing from position 2 to position 29 relative to the amino acid sequence shown in SEQ ID NO:

2. The nucleotide sequence of the missing part is shown in SEQ ID NO:

3. The nucleotide sequence encoding the mutant ScCBS△motif1 is shown in SEQ ID NO:

4.

5. The ScCBS△motif3 mutant, which lacks a key structural domain of the ScCBS protein, is characterized by: The mutant ScCBS△motif3 has 50 consecutive amino acids from position 66 to position 115 missing from the amino acid sequence shown in SEQ ID NO:

2. The nucleotide sequence of the missing part is shown in SEQ ID NO:

5. The nucleotide sequence encoding the mutant ScCBS△motif3 is shown in SEQ ID NO:

6.

6. The ScCBS△motif4 mutant, which lacks a key structural domain of the ScCBS protein, is characterized by: The mutant ScCBS△motif4 has 49 consecutive amino acids from position 82 to position 130 missing from the amino acid sequence shown in SEQ ID NO:

2. The nucleotide sequence of the missing part is shown in SEQ ID NO:

7. The nucleotide sequence encoding the mutant ScCBS△motif4 is shown in SEQ ID NO:

8.

7. The ScCBS△motif9 mutant, which lacks a key structural domain of the ScCBS protein, is characterized by: The mutant ScCBS△motif9 has 29 consecutive amino acids, from position 201 to position 229, missing from the amino acid sequence shown in SEQ ID NO:

2. The nucleotide sequence of the missing portion is shown in SEQ ID NO:

9. The nucleotide sequence encoding the mutant ScCBS△motif9 is shown in SEQ ID NO:

10.

8. The application of the ScCBS protein key domain deletion mutant as described in any one of claims 5 to 7 in regulating the interaction between ScCBS protein and SSCMV P1 protein.

9. The use of the ScCBS protein key domain deletion mutant as described in any one of claims 5 to 7 in regulating the ScCBS protein to inhibit the activity of the SSCMV P1 protein RNA silencing repressor.

10. The application of the ScCBS protein as described in claim 1, or the ScCBS protein key domain deletion mutant as described in any one of claims 5 to 7, in sugarcane molecular breeding.

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