Cassava mebcsl gene for resisting cassava mosaic disease and application thereof

Abstract

This invention discloses a cassava variety resistant to cassava mosaic virus. MeRBCS1 Genes and their applications, the aforementioned MeRBCS1 The nucleotide sequence of the gene is shown in SEQ ID NO:1, and the amino acid sequence of the protein is shown in SEQ ID NO:2. This invention demonstrates the interaction between MerBCS1 and the motility protein BC1 of Sri Lanka cassava mosaic virus. CRISPR / Cas9 technology was used to transform cassava FEC via Agrobacterium-mediated transformation, editing cassava... MeRBCS1 Gene mutation was obtained, and compared with the control, the mutant... merbcs1 The disease resistance was significantly improved, and the viral expression level was significantly lower than that of the control.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering, specifically to a cassava variety resistant to cassava mosaic virus. MeRBCS1 Genes and their applications. Background Technology

[0002] Cassava (Manihot esculenta Crantz) belongs to the Euphorbiaceae family and the genus Manihot. It originated in Latin America and has been widely cultivated in tropical regions of Africa, the Americas and Asia due to its drought and poor soil tolerance. It has become an important food and economic crop in these regions.

[0003] Against the backdrop of global warming and intensified extreme weather, cassava faces serious threats from pests and diseases, among which cassava mosaic disease (CMD), transmitted by the whitefly (Bemisia tabaci), is the most widespread and severe. Field symptoms mainly manifest as mottled and mosaic-like leaves, curled and deformed young leaves, and severe cases result in significant yield reduction.

[0004] Cassava mosaic disease (CMD) is caused by various viruses belonging to the genus Begomovirus in the family Geminiviridae. The typical genome is a two-component single-stranded DNA (DNA-A / DNA-B). The virus can be transmitted through two main routes:

[0005] 1) Vector transmission: Whiteflies migrate and spread the virus after feeding on infected plants;

[0006] 2) Asexual propagation: Virus-carrying stems (cuttings) can spread over long distances and rapidly during cultivation;

[0007] Therefore, CMD carries the dual risks of "rapid spread in the field" and "amplification of the reproductive system".

[0008] Sri Lankan cassava mosaic virus (SLCMV) is a highly destructive cassava mosaic virus. Major cassava varieties cultivated in my country lack effective resistance to SLCMV. Given cassava's reliance on asexual reproduction, the efficient spread of whiteflies, and cross-border trade, failure to control SLCMV will result in significant yield reductions and pose a serious threat to the safety of the supply chain.

[0009] Therefore, breeding SLCMV-resistant cassava varieties is of great significance. It can solve the serious problem of the lack of SLCMV-resistant cassava varieties in my country, and can also provide high-quality parents for future SLCMV-resistant cassava breeding, ensuring the healthy and sustainable development of my country's cassava industry. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a cassava resistant to cassava mosaic disease. MeRBCS1 This invention relates to genes and their applications. The motility protein BC1 of Sri Lanka mosaic virus, a highly pathogenic cassava mosaic virus, was selected as bait. A cassava cDNA yeast double-hybrid library was used to screen for cassava proteins that interact with BC1. The results showed that the cassava MeRBCS1 protein interacts with BC1. CRISPR / Cas9 technology was used to transform cassava FECs via Agrobacterium-mediated transformation, thus editing cassava FECs. MeRBCS1 Gene gain mutant merbcs1 After inoculation with the virus, compared with the control, the mutant... merbcs1 The disease resistance was significantly improved, and the viral expression level was significantly lower than that of the control.

[0011] To achieve the above objectives, the technical solution designed by the present invention is as follows:

[0012] This invention provides a cassava resistant to cassava mosaic virus. MeRBCS1 Genes, the ones mentioned MeRBCS1 The CDS sequence of the gene is shown in SEQ ID NO:1.

[0013] The above-mentioned cassava MeRBCS1 The gene-encoded MeRBCS1 protein, the amino acid sequence of which is shown in SEQ ID NO:2.

[0014] Furthermore, the MeRBCS1 protein interacts with Sri Lanka mosaic virus motility protein BC1, wherein the amino acid sequence of motility protein BC1 is shown in SEQ ID NO:3.

[0015] This invention also provides a CRISPR / Cas9 gene editing recombinant vector pCAMBIA1301-Cas9-sgRNA- MeRBCS1 The recombinant vector pCAMBIA1301-Cas9-sgRNA- MeRBCS1 For cassava containing the above-mentioned ingredients MeRBCS1 The CRISPR / Cas9 gene editing vector is pCAMBIA1301-Cas9-sgRNA.

[0016] The present invention also provides a recombinant vector pCAMBIA1301-Cas9-sgRNA containing the above-mentioned recombinant vector pCAMBIA1301-Cas9-sgRNA- MeRBCS1 Agrobacterium, wherein the Agrobacterium cell is LAB4404.

[0017] The application of one of the following in improving cassava's resistance to mosaic virus, including:

[0018] (1) The above-mentioned cassava MeRBCS1 Gene;

[0019] (2) The above-mentioned recombinant vector pCAMBIA1301-Cas9-sgRNA- MeRBCS1 ;

[0020] (3) Agrobacterium mentioned above.

[0021] Furthermore, the mosaic disease is a viral disease caused by cassava mosaic virus, specifically Sri Lanka mosaic virus.

[0022] The use of any of the following in the development of transgenic cassava varieties resistant to cassava mosaic virus, including:

[0023] (1) The above-mentioned cassava MeRBCS1 Gene;

[0024] (2) The above-mentioned recombinant vector pCAMBIA1301-Cas9-sgRNA- MeRBCS1 ;

[0025] (3) Agrobacterium mentioned above.

[0026] This invention also provides a method for breeding transgenic cassava varieties resistant to cassava mosaic virus, comprising the following steps:

[0027] 1) Constructing the CRISPR / Cas9 gene editing recombinant vector pCAMBIA1301-Cas9-sgRNA- MeRBCS1,

[0028] 2) Construct a vector containing pCAMBIA1301-Cas9-sgRNA- MeRBCS1 Agrobacterium,

[0029] 3) Using Agrobacterium-mediated transformation, the recombinant vector pCAMBIA1301-Cas9-sgRNA- MeRBCS1 Agrobacterium was introduced into cassava, and the bacteria were screened to obtain... MeRBCS1 Gene knockout mutants are genetically modified cassava varieties.

[0030] The beneficial effects of this invention are:

[0031] 1. This invention is the first to demonstrate a specific interaction between the cassava MeRBCS1 protein and the Sri Lanka cassava mosaic virus (SLCMV) motility protein BC1, revealing a key host factor for virus intercellular / systemic movement dependence and providing a verifiable functional target for molecular design of resistance breeding. By editing MeRBCS1 to disrupt its interaction interface with BC1, the viral motility process is blocked from the host side, reducing viral spread and accumulation within the plant. The source of resistance is clear and reproducible.

[0032] 2. This invention combines yeast double-hybrid screening with CRISPR / Cas9 site-directed editing to rapidly create the merbcs1 mutant based on the cassava FEC system. The process is simple, efficient, and suitable for standardized and large-scale application. Under artificial inoculation conditions, the merbcs1 mutant showed a significantly lower disease index and a significantly downregulated viral expression / accumulation compared to the control (WT), demonstrating a stable and reliable disease-resistant phenotype.

[0033] In summary, this invention demonstrates the interaction between the MerBCS1 protein of cassava and the Sri Lanka cassava mosaic virus motility protein BC1, and creates a new type of cassava virus using CRISPR / Cas9 technology. merbcs1 Mutant cassava, merbcs1 The mutant cassava showed significantly enhanced resistance to cassava mosaic virus compared to the control WT. The method provided by this invention is of great significance for breeding cassava resistant to mosaic virus, as it can not only reduce the damage caused by cassava mosaic virus and increase cassava yield, but also reduce pesticide use, thereby reducing environmental pollution and achieving good environmental and social benefits. Attached Figure Description

[0034] Figure 1 This is a schematic diagram of the yeast two-hybrid point-to-point verification of the MeRBCS1 protein and the BC1 protein in Example 1;

[0035] Figure 2 This is a schematic diagram illustrating the BiFC interaction verification between the MerBCS1 protein and the BC1 protein in Example 1;

[0036] Figure 3 This is a schematic diagram illustrating the Co-IP interaction verification between the MeRBCS1 protein and the BC1 protein in Example 1;

[0037] Figure 4 This is a schematic diagram illustrating the GST pull-dwon interaction verification between the MeRBCS1 protein and the BC1 protein in Example 1;

[0038] Figure 5 This is a schematic diagram of PCR detection of the merbcs1 mutant cassava in Example 2.

[0039] In the diagram, M: DNA maker; Mock: non-gene-edited SC8; KO1-8: gene-edited cassava lines 1-8;

[0040] Figure 6 This is a Sanger sequencing peak diagram of the target genes of the merbcs1 mutant cassava in Example 2.

[0041] In the figure, Mock: non-gene-edited SC8; KO1-3: gene-edited cassava lines 1-3. The red box represents the PAM region, and the underline represents the target sequence.

[0042] Figure 7 This is a diagram showing the Hi-TOM analysis results of the target gene of the merbcs1 mutant cassava in Example 2.

[0043] In the figure, Mock: non-gene-edited SC8; KO1-3: gene-edited cassava lines 1-3. The yellow area represents the PAM region, and the underline represents the target sequence; red bases represent inserted bases, and - represents deleted bases; WT: wild type, D: deletion, I: insertion;

[0044] Figure 8 This is a schematic diagram of tissue culture seedlings of cassava WT and mutant merbcs1 cassava in Example 2;

[0045] Figure 9 This is a schematic diagram showing the amount of virus accumulated in mutant cassava and control cassava after inoculation with SLCMV in Example 3. Detailed Implementation

[0046] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0047] In the examples, techniques or conditions not specifically specified were performed under standard experimental conditions. Reagents or instruments not explicitly named as manufacturers were all commercially available, standard products. Viral clones, strains, and plasmid biological materials used in the following examples were all provided by the applicant's laboratory.

[0048] Example 1: Screening of MeRBCS1 protein and interaction between MeRBCS1 and BC1 proteins

[0049] 1. Yeast library screening

[0050] First, design primers with homologous arms:

[0051] BD-BC1-F:

[0052] CTGCATATGGCCATGGAGGCCGAAATGGAGAATAATAGTAGCAAT,

[0053] BD-BC1-F:

[0054] TATGCGGCCGCTGCAGGTCGACGTTATACATTTTTGGATACAT;

[0055] PCR amplification was performed using primers BD-BC1-F / R, with total DNA from infected SC8 cassava leaves as a template. A high-fidelity enzyme was used to amplify the target fragment containing the vector homologous sequence. After the PCR program was completed, 5 μL of the PCR product was aspirated and analyzed using a 1% agarose gel to observe whether a band of the expected size was amplified. If the band size met expectations, the next step was performed. PCR products were recovered using the Gel Extract Kit from Kangwei Century Biotechnology; specific operating procedures were followed according to the kit's instructions.

[0056] The purified target fragment was ligated with the linearized pGBKT7 vector after enzyme digestion using homologous recombinase. The ligation product was transformed into E. coli, and the colonies were screened using plates containing Kana resistance. The colonies that grew were selected for PCR detection. Positive colonies were sent for testing, and plasmids were extracted from the colonies that were correctly sequenced, thus obtaining the yeast two-hybrid bait vector pGBKT7-BC1.

[0057] AH109 yeast containing the bait vector pGBKT7-BC1 was inoculated into 50 mL of SD / -Trp liquid medium for small-scale culture and incubated at 30°C and 200 rpm until the OD600 value reached 1.5. 50 mL of the yeast culture was then collected at 3000 rpm. The culture was then transferred to 300 mL of liquid YPDA medium and incubated at 30°C and 200 rpm until the OD600 value reached 1.5. 600 The value is 0.6.

[0058] Collect the bacterial cells by centrifugation at 4000 rpm for 10 min, discard the supernatant, and resuspend the cells in 1×TE. Centrifuge again to collect the cells, and repeat the washing process once more. Resuspend the cells in 1.5 mL of 1×TE / LiAc to obtain the competent yeast cells for the bait vector.

[0059] Add 100 μg of cassava total cDNA library plasmid and 200 μL of boiled and chilled salmon sperm DNA to 1 mL of bait vector competent cells and mix well. Add 6 mL of PEG / LiAc and shake vigorously to increase transformation efficiency. Incubate at 30℃ and 200 rpm for 30 min.

[0060] Add 700 μL of dimethyl sulfoxide, mix well, and place in a 42°C water bath for heat shock for 15 minutes, inverting once during the process to mix thoroughly. Immediately after heat shock treatment, immerse in ice and cool for approximately 2 minutes.

[0061] Centrifuge at 2500 rpm for 5 min at room temperature, remove the supernatant with a cut pipette tip, and resuspend the cells in 10 mL of 1×TE medium. Take 10 μL of the resuspended yeast and dilute it 10-fold, 100-fold, and 1000-fold with sterile water, then plate it onto SD / -Trp-Leu medium and calculate the transformation efficiency. Next, plate the remaining resuspended yeast onto SD / -Trp-Leu-His medium, 150 μL per plate. Incubate the plates upside down at 30°C for 3–5 days.

[0062] Larger colonies (greater than 2 mm) grown on SD / -Trp-Leu-His medium were transferred to 30 μL of sterile water, mixed thoroughly, and then spotted onto SD / -Trp-Leu, SD / -Trp-Leu-His-Ade, and SD / -Trp-Leu-His-Ade+X-α-gal media. The plates were inverted and incubated at 30°C for 3–5 days. The yeast growth was then observed.

[0063] 2. Yeast two-hybrid point-to-point verification

[0064] Based on the sequence information of the MeRBCS1 gene, the CDS region was identified, and primers with homologous arms of the AD vector were designed.

[0065] AD-RBCS1-F:acgtaccagattacgctcatATGATGGCTACCTCTATGCTTT,

[0066] AD-RBCS1-R: cagctcgagctcgatggatccTTAATAATCGGTGCCGGGAGG.

[0067] Using total cDNA from SC8 cassava as a template, the target fragment containing the vector homologous sequence was amplified using a high-fidelity enzyme.

[0068] After the PCR program is complete, aspirate 5 μL of the PCR product and perform a 1% agarose gel electrophoresis to observe whether a band of the expected size has been amplified. If the band size is as expected, proceed to the next step. Use the Gel Extract Kit from Kangwei Century Biotechnology to recover the PCR product; refer to the kit's instructions for specific operating procedures.

[0069] The purified target fragment was ligated with the enzyme-digested linear pGADT7 vector using homologous recombinase. The ligation product was transformed into *E. coli*, and the colonies were screened using plates containing Amp resistance. Positive colonies were selected for PCR detection. Sequencing results showed that... MeRBCS1 The CDS sequence (SEQ ID NO.1) of the gene is 861 bp in length. MeRBCS1The amino acid sequence encoded by the gene (SEQ ID NO.2) contains 286 amino acids.

[0070] MeRBCS1 The nucleotide sequence of the gene (SEQ ID NO.1) is as follows:

[0071] ;

[0072] MeRBCS1 The amino acid sequence (SEQ ID NO.2) of the MeRBCS1 protein encoded by the gene is as follows:

[0073] MATSMLSTATVASINRASPAQASMVAPFTGLKSTSAFPATTKTSADITSLASNGGRVQCMQVWPTRGKKKFETLSYLPPLSREQLASEIDYLLRSGWIPCLEFELEHGFVYRAHGSLPGYYDGRYWTMWKLPMFGCTDSSQVLKELDELIKAHPDGFARIIGFDNVRQVQCISFIAYKPPGTDY.

[0074] Two BD vectors, pGBKT7-BC1 (BD-BC1) and pGBKT7 (BD-T7), and two AD vectors, pGADT7-MeRBCS1 (AD-MeRBCS1) and pGADT7 (AD-T7), were transduced pairwise into AH109 yeast competent cells. The transformed yeast cells were plated on SD / -Trp-Leu medium and incubated at 30°C for 3 days. Single colonies were picked for plaque PCR detection. Successfully detected plaques were inoculated into SD / -Trp-Leu liquid medium and incubated at 30°C and 200 rpm until OD500. 600 The value was 1.0. The above yeast was spotted onto SD / -Trp-Leu, SD / -Trp-Leu-His-Ade, and SD / -Trp-Leu-His-Ade+X-α-gal plates, respectively, and incubated at 30℃ for 3-4 days. Yeast growth was observed. The results showed ( Figure 1 All yeast strains were able to grow on the two-deficient SD / -Trp-Leu medium. Only AD-MeRBCS1 / BD-BC1 and the positive control could grow on the four-deficient SD / -Trp-Leu-His-Ade medium, and they could also grow and turn blue on the SD / -Trp-Leu-His-Ade+X-α-Gal medium. This indicates that MeRBCS1 interacts with the bait protein BC1 in yeast.

[0075] 3. Verification of bimolecular fluorescence complementarity (BiFC) between MerBCS1 and BC1 proteins

[0076] The nucleic acid sequences of BC1 and MerBCS1 proteins were ligated into SPYncE and SPYcE vectors, respectively, and the interaction between the two proteins was verified by bimolecular fluorescence complementation assay.

[0077] according to BC1 Based on the gene sequence information, primers with homologous arms of the SPYncE vector were designed.

[0078] SPYncE-BC1-F: caggcctggcgcgccactagtATGGAGAATAATAGTAGCAATGCAG,

[0079] SPYncE-BC1-R: cccgggagcggtaccctcgagTACATTTTTGGATACATGGCTCTGC.

[0080] Using a 100-fold diluted BD-BC1 plasmid as a template, the target fragment containing the vector homologous sequence was amplified using a high-fidelity enzyme. The purified target fragment was ligated with the enzyme-digested linear SPYncE vector using a homologous recombinase. The ligation product was transformed into *E. coli*, and plaques were selected using Kana-resistant plates for PCR detection. Positive plaques were sent for testing, and successfully tested plaques were preserved. The successfully tested bacteria were cultured on a large scale, plasmids were extracted, and transformed into *Agrobacterium* EHA105 for subsequent experiments.

[0081] according to MeRBCS1 Based on the gene sequence information, primers with homologous arms of the SPYcE vector were designed.

[0082] SPYcE-RBCS1-F:gcctggcgcgccactagtgATGGCTACCTCTATGCTTC;

[0083] SPYcE-RBCS1-R: cccgggagcggtaccctcgaATAATCGGTGCCGGGAGGC.

[0084] Using a 100-fold diluted AD-RBCS1 plasmid as a template, the target fragment containing the vector homologous sequence was amplified using a high-fidelity enzyme. The purified target fragment was ligated with the enzyme-digested linear SPYcE vector using a homologous recombinase. The ligation product was transformed into *E. coli*, and plaques were selected using Kana-resistant plates for PCR detection. Positive plaques were sent for testing, and successfully tested plaques were preserved. The successfully tested bacteria were cultured on a large scale, plasmids were extracted, and transformed into *Agrobacterium* EHA105 for subsequent experiments.

[0085] Agrobacterium EHA105 containing SPYcE, SPYncE, SPYcE-RBCS1 and SPYncE-BC1 vectors respectively was streaked on solid LB medium (containing the corresponding antibiotics) for activation. Single colonies of Agrobacterium were picked and added to 4 mL of liquid LB medium (containing the corresponding antibiotics) and cultured in a shaker at 30°C (200 rpm) for 12 h.

[0086] Remove the Agrobacterium culture medium, centrifuge at 4000 rpm for 10 min, discard the supernatant LB medium, resuspend the Agrobacterium in the suspension, and adjust the OD. 600 The concentration is 0.8~1.0, and the solution is left to stand at room temperature for 3 hours.

[0087] The four bacterial cultures were mixed in equal proportions: SPYcE+SPYncE-BC1, SPYncE+SPYcE-RBCS1, SPYcE-RBCS1+SPYncE-BC1, and SPYcE+SPYncE.

[0088] Using a 1mL disposable medical syringe with the needle removed, place the needle hole against the back of a tobacco leaf and inject the mixed suspension into the leaf. After 48 hours, cut the leaf near the inoculation hole into a square with sides of approximately 3mm.

[0089] Add ddH2O to a glass slide, place the square leaf with its back side facing up, and gently cover it with a coverslip. Observe the YFP fluorescence (RFP fluorescence is nuclear localization) under a fluorescence confocal microscope. The excitation light for the YFP fluorescence channel is 514 nm, and the excitation light for the RFP fluorescence channel is 543 nm.

[0090] The results show that ( Figure 2 After injection of SPYncE-BC1 (nYFP-BC1) and SPYcE-RBCS1 (cYFP-MeRBCS1), fluorescence appeared in the YFP channel, while the other three negative controls did not show fluorescence, proving that there is an interaction between the two.

[0091] 4. Verification of MeRBCS1 and BC1 proteins by co-immunoprecipitation (Co-IP)

[0092] The nucleic acid sequences of BC1 and MeRBCS1 proteins were ligated into pAB002-MYC and pRR002-GFP vectors, respectively, and BC1-MYC and MeRBCS1-GFP were expressed in tobacco leaves. The interaction between the two proteins was verified by Co-IP.

[0093] according to BC1 Based on the gene sequence information, primers containing the homologous arm of the pAB002-MYC vector were designed.

[0094] pAB002-BC1-F:ttcatttggagaggacacgcATGGAGAATAATAGTAGCAATGCAG;

[0095] PAB002-BC1-R:gatattggatccttctagaTACATTTTTGGATACATGGCTCT.

[0096] Using a 100-fold diluted BD-BC1 plasmid as a template, the target fragment containing the vector homologous sequence was amplified with a high-fidelity enzyme. The purified target fragment was ligated with the enzyme-digested linear pAB002-MYC vector using a homologous recombinase. The ligation product was transformed into *E. coli*, and the colonies were screened using Kana-resistant plates. Positive colonies were then selected for PCR detection. The successfully tested colonies were sent for testing, and the results were preserved. The successfully tested bacteria were cultured on a large scale, plasmids were extracted, and the colonies were transformed into *Agrobacterium* EHA105 for subsequent experiments.

[0097] according to MeRBCS1Gene sequence information; design primers containing homologous arms of the pRR002-GFP vector;

[0098] pRR002-RBCS1-F:tttcatttggagaggacacgcATGGCTACCTCTATGCTTTCA;

[0099] pRR002-RBCS1-R:ctcgcccttgctcaccattctagaATAATCGGTGCCGGGAGG.

[0100] Using a 100-fold diluted AD-RBCS1 plasmid as a template, the target fragment containing the vector homologous sequence was amplified with a high-fidelity enzyme. The purified target fragment was ligated with the enzyme-digested linear pRR002-GFP vector using a homologous recombinase. The ligation product was transformed into *E. coli*, and plaques were selected using Kana-resistant plates for PCR detection. Positive plaques were sent for testing, and successfully tested plaques were preserved. The successfully tested bacteria were cultured on a large scale, plasmids were extracted, and transformed into *Agrobacterium* EHA105 for subsequent experiments.

[0101] Agrobacterium EHA105 containing the vectors pAB002-BC1-MYC, pRR002-Flag-GFP, and pRR002-MeRBCS1-GFP were streaked on solid LB medium (containing the corresponding antibiotics) for activation. Single colonies of Agrobacterium were picked and added to 4 mL of liquid LB medium (containing the corresponding antibiotics) and cultured in a shaker at 30°C (200 rpm) for 12 h.

[0102] Remove the Agrobacterium culture medium, centrifuge at 4000 rpm for 10 min, discard the supernatant LB medium, resuspend the Agrobacterium in the suspension, and adjust the OD. 600 The concentration is 0.8~1.0, and the solution is left to stand at room temperature for 3 hours.

[0103] The three bacterial cultures were mixed in equal proportions in pairs: pAB002-BC1-MYC+pRR002-Flag-GFP and pAB002-BC1-MYC+pRR002-MeRBCS1-GFP.

[0104] Using a 1mL disposable medical syringe with the needle removed, place the needle hole against the back of a tobacco leaf and inject the mixed suspension into the leaf. After 48 hours, cut off the leaves near the inoculation well, wrap them in aluminum foil, and quickly place them in liquid nitrogen. Grind them thoroughly into powder using a mortar (pre-cooled with liquid nitrogen). Weigh 1g of the sample and place it in a 2mL centrifuge tube (pre-cooled with liquid nitrogen). Add 1mL of IP buffer (40mM Tris-HCl pH 7.5, 150mM NaCl, 5mM MgCl2, 5mM DTT, 2mM EDTA pH 8.0, 0.1% Triton X-100, 2% glycerol, 1% protease inhibitor cocktail), and vortex vigorously until completely mixed. After incubating on ice for 30 minutes, centrifuge at 12000rpm for 10 minutes at 4°C. Collect the supernatant (repeat this step 2-3 times until the solution is free of impurities), and take 60μL as input for Western blot analysis.

[0105] After mixing the Anti-GFP@M2 Magnetic Beads by pipetting, transfer 40 μL to a 1.5 mL sterile centrifuge tube and wash three times with 1 mL of IP buffer. Add the washed GFP beads to the extracted protein and incubate on a rotary shaker at 4 °C for 2 h. Adsorb the GFP beads with a magnetic rack and remove the supernatant. Add 1 mL of IP buffer and incubate on a rotary shaker at 4 °C for 10 min. Repeat 6–8 times.

[0106] After the final incubation, aspirate the supernatant, add 16 μL of 5×Dual Color Protein Loading Buffer and 64 μL of IP buffer, and then perform Western blot analysis. Western blot results show ( Figure 3 BC1-MYC can only be specifically detected when MeRBCS1-GFP is present, while BC1-MYC cannot be detected in the control, indicating that BC1 and MeRBCS1 interact in the plant.

[0107] 5. GST pull-down validation of the interaction between MerBCS1 and BC1 proteins

[0108] The nucleic acid sequences of BC1 and MeRBCS1 proteins were ligated into pMAL-c5x and pGEX-6P-1 vectors, respectively, and BC1-MBP and MeRBCS1-GST were expressed in E. coli DL21, respectively. The interaction between the two proteins was verified by GST pull-down.

[0109] according to BC1Based on the gene sequence information, primers with homologous arms of the pMAL-c5x vector were designed.

[0110] pMAL-c5x-BC1-F:ggccgcgatatcgtcgacggaATGGAGAATAATAGTAGCAAT;

[0111] pMAL-c5x-BC1-R:ttatttaattacctgcagggaattaTACATTTTTGGATACATGG.

[0112] Using a 100-fold diluted BD-BC1 plasmid as a template, the target fragment containing the vector homologous sequence was amplified using a high-fidelity enzyme. The purified target fragment was ligated with the enzyme-digested linear pMAL-c5x vector using a homologous recombinase. The ligation product was transformed into *E. coli*, and plaques were selected using Kana-resistant plates for PCR detection. Positive plaques were sent for testing, and successfully tested plaques were preserved. The successfully tested bacteria were cultured on a large scale, plasmids were extracted, and transformed into *E. coli* DL21 for subsequent experiments.

[0113] according to MeRBCS1 Based on the gene sequence information, primers with homologous arms of the pGEX-6P-1 vector were designed.

[0114] pGEX-6P-1-RBCS1-F: ctgttccaggggcccctgggaATGGCTACCTCTATGCTTTCA;

[0115] pGEX-6P-1-RBCS1-R: cggccgctcgagtcgacccgggaaATAATCGGTGCCGGGAGG.

[0116] Using the AD-RBCS1 plasmid diluted 100-fold as a template, the target fragment containing the vector homologous sequence was amplified with a high-fidelity enzyme. The purified target fragment was ligated with the linearized pGEX-6P-1 vector after enzyme digestion using a homologous recombinase. The ligation product was transformed into *E. coli*, and the colonies were screened using plates containing Kana antibody. Positive colonies were selected for PCR detection. Positive colonies were sent for testing, and successfully tested colonies were preserved. The successfully tested bacteria were cultured on a large scale, plasmids were extracted, and transformed into *E. coli* DL21 for subsequent experiments.

[0117] The selected expression strains were cultured in liquid culture until OD... 600 The concentration was set to 0.4, and then 10 mL was transferred to a new 1 L LB liquid medium containing the corresponding antibiotic for large-scale incubation until the OD value reached 0.4. 600The concentration was set to 0.4. Then, 1 mL of 1 M IPTG was added to the bacterial culture, and the mixture was incubated at 180 rpm with shaking at 18°C ​​for 20 h. The cells were then collected by centrifugation at 6000 rpm for 10 min at 4°C, and resuspended in 40 mL of protein resuspension buffer, then placed on ice. 40 µL each of 1 M PMSF, 1 M DTT, and Triton-X100 were added to the cells, mixed thoroughly, and the cells were sonicated on ice (4 min, 3 s interval, 6 s rest). The mixture was then centrifuged at 20000 rpm for 30 min at 4°C, and the protein supernatant was collected.

[0118] The supernatant containing MeRBCS1 protein was purified using a GST column. The protein supernatant was added to a regenerated GST column, and the column was circulated three times using a constant flow pump at a flow rate of approximately 1 mL / min. After column chromatography, contaminating proteins were washed with protein extraction buffer, followed by elution with protein buffer containing 20 mM reduced glutathione, repeated five times (one column volume each time). The eluted protein was collected and subjected to SDS-PAGE electrophoresis as described above to assess the purification effect.

[0119] The supernatant containing BC1 protein was purified using an MBP column. The protein supernatant was added to a regenerated MBP column, and the column was circulated three times using a constant flow pump at a flow rate of approximately 1 mL / min. After column chromatography, contaminating proteins were washed with protein extraction buffer, followed by elution with protein buffer containing 20 mM maltose, repeated five times (one column volume each time). The eluted proteins were collected and subjected to SDS-PAGE electrophoresis as described above to assess the purification effect.

[0120] The purified BC1-MBP was mixed with MerBCS1-GST and GFP-GST proteins and incubated. Washed GST beads were added to the purified protein and incubated on a rotary shaker at 4°C for 2 hours. The GFP beads were centrifuged at 700g, the supernatant was removed, 1 mL of IP buffer was added, and the mixture was incubated on a rotary shaker at 4°C for 10 minutes. This process was repeated 6–8 times.

[0121] After the final incubation, aspirate the supernatant, add 16 μL of 5×Dual Color Protein Loading Buffer and 64 μL of IP buffer, and then perform Western blot detection.

[0122] Western blot results showed that ( Figure 4 ): After BC1-MBP binds to MeRBCS1-GST, elution can specifically detect BC1-MBP, while the control cannot detect it, indicating that BC1-MBP and MeRBCS1-GST interact in vitro.

[0123] Example 2 MeRBCS1 Obtaining gene-edited cassava

[0124] 1. Construction of CRISPR / Cas9 gene editing recombinant vectors

[0125] The software used for gene editing target selection was CRISPR-P 2.0, developed by Huazhong Agricultural University. The target selection website contains a cassava database and can be found at http: / / crispr.hzau.edu.cn / CRISPR2 / . For sgRNA, the promoter was selected as U6, and for PAM, NGG was chosen. After target selection, primers were synthesized. If the first base of the target sequence is G, primer 5 was used at the target site. End-connector sequence GATT, lower primer 5 Add adapter sequence AAAC to the upper primer; if it is not G, add adapter GATTG to the lower primer and 5. End-connector sequence AAAC, 3 Add C to the end. Target primer:

[0126] RBCS1-sgRNA-F:GATTGCCACCATGCTGGCTTGAGCA,

[0127] RBCS1-sgRNA-R: AAACTGCTCAAGCCAGCATGGTGGC;

[0128] Dilute the primers above and below the target to the working solution concentration, and mix them in equal volumes, as shown in Table 1 below. After thorough mixing, place the mixture in a 98°C water bath for 3 minutes, then turn off the power and allow it to cool naturally to room temperature. Store at -20°C for later use.

[0129] Table 1 Target Primer Annealing System

[0130]

[0131] The gene editing vector pCAMBIA1301-Cas9-sgRNA was digested with the BsaI-HFv2 restriction enzyme from NEB, following the enzyme's instructions. Electrophoresis was performed after digestion to check for complete cleavage; undigested vectors served as controls. The digested products were then recovered using a gel extraction kit from Kangwei Century, following the kit's instructions. The concentration of the recovered linear vector was determined, and ligation was performed using T4 ligase at a linear vector to target primer molar ratio of 5:1, following the manufacturer's instructions.

[0132] The ligation product was transformed into *E. coli* DH5α, and the colonies were screened using plates containing Kana resistance. Growing colonies were selected for PCR detection. Positive colonies were sent for testing, and successfully tested colonies were preserved. The successfully tested bacteria were cultured on a large scale, and the CRISPR / Cas9 gene-editing recombinant vector pCAMBIA1301-Cas9-sgRNA was extracted. MeRBCS1 .

[0133] pCAMBIA1301-Cas9-sgRNA- MeRBCS1 It contains the hygromycin (Hyg) resistance gene. pCAMBIA1301-Cas9-sgRNA- MeRBCS1 The cells were transformed into Agrobacterium LBA4404 and, after testing, used for cassava genetic modification.

[0134] 2. Agrobacterium-mediated CRISPR / Cas9 gene editing vector transformation of cassava

[0135] Containing pCAMBIA1301-Cas9-sgRNA- MeRBCS1 Agrobacterium-mediated transformation was performed on cassava FECs. The genetically transformed FECs were then transferred to MSN medium (containing 250 mg / L carbenicillin and 15 mg / L hygromycin), and this process was repeated every 14 days until cotyledon formation was induced. The newly grown cotyledons were then transferred to CEM solid medium (with the carbenicillin concentration maintained) and cultured under light (28°C). The medium was changed every 14 days, removing excess tissue each time to avoid affecting cotyledon growth. Once the cotyledons had induced shoots, they were transferred to MS solid medium (hygromycin concentration 15 mg / L) and cultured under light (28°C) for root selection.

[0136] 3. Identification of the transgenic effect of cassava-resistant seedlings

[0137] The selected transgenic cassava was initially subjected to molecular testing to confirm its transgenic nature. DNA was extracted from leaves of normally rooting cassava seedlings, and PCR detection was performed using a standard PCR enzyme. The primers were:

[0138] Target detection - F: CAGTGAGCGCAACGCAATTA;

[0139] Target Detection - R: GCCTCTTCGCTATTACGCCA,

[0140] Electrophoresis was performed. The electrophoresis results revealed ( Figure 5 All nine transgenic lines were positive.

[0141] The gene editing target identification methods used were Sanger sequencing and Hi-TOM assay, with the following primers:

[0142] RBCS1-HT-F:GGAGTGAGTACGGTGTGCGGCTACCTCTATGCTTTCAACTG,

[0143] RBCS1-HT-R: GAGTTGGATGCTGGATGGAGGGAGGTGATATCAGCGGAG;

[0144] The PCR products that were successfully detected by electrophoresis were sent to Shanghai Sangon Biotech for sequencing and to the Rice Research Institute of the Chinese Academy of Agricultural Sciences.

[0145] Sanger sequencing results showed that ( Figure 6 The presence of heterogeneous peaks at the target sequence indicates that the transgenic line... MeRBCS1 Gene target generation and editing. Hi-TOM test results show ( Figure 7 The three gene-edited lines tested all underwent gene editing, with KO1 and KO3 showing identical editing patterns. All three gene-edited lines were homozygous mutations, and all experienced frameshifts leading to mutations in the encoded protein. Subsequent experiments selected KO1 and KO2 lines (…). Figure 8 ).

[0146] Example 3 merbcs1 Disease resistance testing of mutant cassava

[0147] 1. Grafting and inoculation with viruses

[0148] Pick merbcs1 For both mutant cassava tissue culture seedlings (i.e., KO1 and KO2 lines) and non-transgenic SC8 tissue culture seedlings, select stems of uniform size, using each stem segment (containing one bud) as the scion, with the lower end of the stem segment cut into a cone shape. Use SC8 stem segments of uniform thickness as rootstocks, each approximately 2 cm long and containing about two or three buds. Cut a V-shaped notch at the upper end of the SC8 stem segment, insert the scion into the V-shaped notch of the rootstock, and cover the graft union with a sterilized silicone tube (with an inner diameter 2 mm longer than the stem diameter and a length of approximately 5 mm), ensuring a close and compact fit. Transplant the micrografts into fresh MS medium. Culture under light for 7-10 days. After the scion develops new leaves, surgically remove the buds or lateral buds of the rootstock.

[0149] 2. Disease resistance analysis

[0150] Fifteen days after grafting, cassava leaves were harvested, and DNA was extracted from the scion using the CTAB method. The virus content was then detected using real-time quantitative PCR. The scion DNA was used as a template for quantitative PCR to detect the virus content. Primers for SLCMV quantification:

[0151] F: ACGCCAGGTCTGAGGCTGTA

[0152] R: GTTCAACAGGCCGTGGGACA.

[0153] For quantitative experiments, 2-3 technical replicates should be set up for each sample. After obtaining the Ct value, the average value should be calculated to avoid large errors caused by individual operations. The relative expression level of the virus can be calculated.

[0154] The results showed that, compared with the control, merbcs1 The viral accumulation in the mutant strain was significantly reduced. Figure 9 ).

[0155] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for knocking out cassava MeRBCS1 The application of genes in improving cassava resistance to mosaic virus or in breeding transgenic cassava varieties resistant to cassava mosaic virus is characterized by: The mosaic disease is a viral disease caused by cassava Sri Lanka mosaic virus. MeRBCS1 The CDS sequence of the gene is shown in SEQ ID NO:

1.

2. The application according to claim 1, characterized in that: The knockout of cassava MeRBCS1 The gene was generated using the CRISPR / Cas9 gene editing recombinant vector pCAMBIA1301-Cas9-sgRNA- MeRBCS1 The recombinant vector pCAMBIA1301-Cas9-sgRNA- MeRBCS1 For cassava containing targeted MeRBCS1 CRISPR / Cas9 gene editing vector for the sgRNA of the gene.

3. The application according to claim 2, characterized in that: The knockout of cassava MeRBCS1 Agrobacterium containing the above-mentioned recombinant vector pCAMBIA1301-Cas9-sgRNA- MeRBCS1 Agrobacterium, wherein the Agrobacterium cell is LAB4404.

4. A method for breeding transgenic cassava varieties resistant to cassava mosaic virus, characterized in that: including the following steps: 1) Constructing the CRISPR / Cas9 gene editing recombinant vector pCAMBIA1301-Cas9-sgRNA- MeRBCS1 , 2) Construct a vector containing pCAMBIA1301-Cas9-sgRNA- MeRBCS1 Agrobacterium, 3) Using Agrobacterium-mediated transformation, the recombinant vector pCAMBIA1301-Cas9-sgRNA- MeRBCS1 Agrobacterium was introduced into cassava, and the bacteria were screened to obtain... MeRBCS1 Gene knockout mutants are genetically modified cassava varieties; wherein, the mosaic disease is a viral disease caused by cassava Sri Lanka mosaic virus. MeRBCS1 The CDS sequence of the gene is shown in SEQ ID NO:1.

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