Sugarcane yellow leaf virus infectious cloning vector, construction method and application
By constructing a recombinant plasmid containing the complete genome of sugarcane yellow leaf virus and exogenous genes, and using Agrobacterium-mediated infection of plants, the cloning problem of sugarcane yellow leaf virus was solved, achieving efficient infection of sugarcane virus and expression of exogenous proteins, thus promoting sugarcane breeding and variety improvement.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INST OF MICROBIOLOGY CHINESE ACAD OF SCI
- Filing Date
- 2024-10-11
- Publication Date
- 2026-04-14
AI Technical Summary
Existing technologies make it difficult to effectively construct infectious clones of sugarcane yellow leaf virus, hindering in-depth research into the pathogenic mechanism of sugarcane yellow leaf disease and affecting the progress of sugarcane breeding and variety improvement.
Recombinant plasmids and recombinant expression plasmids containing the complete genome and exogenous genes of sugarcane yellow leaf virus (SCYLV) were constructed. Agrobacterium-mediated infection was used to infect plants, resulting in symptoms of leaf curling, wrinkling and yellowing. The specific methods included the preparation of recombinant plasmids and plant infection.
Successful infection of Nicotiana Bunsenata and sugarcane enabled efficient expression of exogenous proteins, providing a tool for studying the infection mechanism of sugarcane yellow leaf virus and improving sugarcane varieties.
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Abstract
Description
Technical Field
[0001] This invention belongs to the field of biotechnology, specifically relating to the infectious cloning vector of sugarcane yellow leaf virus, its construction method, and its application. Background Technology
[0002] Sugarcane is a tropical cash crop used in sugar production, biofuel production, pulp and fiber products, generating significant economic benefits. However, sugarcane mosaic virus (SPV), a viral disease, has a significant negative impact on sugarcane quality and yield, causing substantial losses to the sugarcane industry. Consequently, sugarcane yellow leaf disease and SPV have become widespread in recent years. The traditional method of asexual reproduction via tubers has become a major driving force behind the rapid cross-regional spread of the virus. How to utilize modern biological methods for sugarcane variety selection has become a crucial issue in sugarcane breeding. Previous studies have demonstrated that plant virus expression vectors have great potential in exploring plant gene function, expressing exogenous genes, and editing plant genes. Traditional transgenic methods typically require expensive chemicals or complex cell culture processes to introduce genes, while viral vectors can introduce genes into plant cells in a more natural and efficient manner. As a natural gene vector, plant viruses have great development potential. Through viral modification and optimization, more efficient and precise gene delivery and expression can be achieved. Therefore, if new sugarcane virus vectors can be developed and applied using sugarcane viruses, it can greatly promote the progress of sugarcane gene function research and sugarcane variety improvement.
[0003] Common viral diseases of sugarcane mainly include two types: sugarcane yellow leaf disease and sugarcane mosaic virus. Sugarcane yellow leaf disease is caused by sugarcane yellow leaf virus (SCYLV) and sugarcane baculovirus. SCYLV, the main pathogen of sugarcane yellow leaf disease, belongs to the genus *Potato Leaf Roll Virus* of the family Southern Bean Mosaic Virology. The pathogenicity of SCYLV has been controversial in the past because the symptoms are not very specific, and the presence of the virus is not strictly correlated with the symptoms. Late-stage SCYLV infection leads to severe yellowing of the midrib, extending to the leaf, followed by tissue necrosis from the leaf tip to the leaf base, but the virus is difficult to detect in the veins. Symptoms typically include increased respiration, decreased photosynthesis, changes in the hexose to sucrose ratio, and increased starch content. SCYLV can only reproduce through insects or cuttings and cannot be transmitted through friction or seed propagation. Sugarcane yellow leaf disease can be propagated by sugarcane aphids and sorghum aphids in a recurrent, semi-persistent manner. For many years, an infectious clone of SCYLV has not been achieved. Therefore, substantial breakthroughs in the specific research on the pathogenic mechanism of SCYLV have been difficult to achieve. Summary of the Invention
[0004] The technical problem to be solved by this invention is the infectious cloning of sugarcane yellow leaf virus and its construction method. The technical problem to be solved is not limited to the described technical subject matter; other technical subjects not mentioned herein will be clearly understood by those skilled in the art through the following description.
[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solutions: The present invention provides a recombinant plasmid containing the complete genome of sugarcane yellow leaf virus (SCYLV), wherein the nucleotide sequence of the complete genome is nucleotides 771-6676 of SEQ ID No. 1.
[0006] Furthermore, the nucleotide sequence of the above recombinant plasmid is SEQ ID No. 1.
[0007] The present invention also provides a recombinant expression plasmid, wherein the recombinant expression plasmid is a recombinant expression vector obtained by inserting a foreign gene between nucleotides 4418-4419 of a recombinant plasmid with the nucleotide sequence of SEQ ID No. 1.
[0008] The recombinant expression vector can express the exogenous gene.
[0009] In a specific embodiment of the present invention, the exogenous gene is EGFP, whose encoding gene is position 31-747 of SEQ ID No. 2 or position 226-942 of SEQ ID No. 3.
[0010] The present invention also provides a recombinant microorganism containing the aforementioned recombinant plasmid.
[0011] The present invention also provides another recombinant microorganism containing the aforementioned recombinant expression plasmid.
[0012] The aforementioned microorganisms can be prokaryotic microorganisms, such as Escherichia coli or Agrobacterium. Specifically, it could be Agrobacterium tumefaciens GV3101.
[0013] The application of the aforementioned recombinant plasmids, recombinant expression plasmids, recombinant microorganisms, or the whole genome of SCYLV in the preparation of plants with curled and / or wrinkled and / or yellowed leaves.
[0014] The aforementioned recombinant plasmids or recombinant expression plasmids or recombinant microorganisms or the whole genome of SCYLV are used in the preparation of products with curled and / or wrinkled leaves and / or yellowed leaves.
[0015] The present invention also provides a method for preparing plants with curled and / or wrinkled and / or yellowed leaves, comprising the following steps: infecting a recipient plant with the aforementioned recombinant plasmid or the aforementioned recombinant expression plasmid through Agrobacterium-mediated infection to obtain plants with curled and / or wrinkled and / or yellowed leaves.
[0016] The present invention also provides a method for causing plant leaves to curl and / or wrinkle and / or turn yellow, the method comprising infecting a target plant with the aforementioned recombinant plasmid or the aforementioned plant via Agrobacterium-mediated infection, thereby causing the leaves of the target plant to curl and / or wrinkle and / or turn yellow.
[0017] The above-mentioned plant is any one of the following: M1) Dicotyledons or monocotyledons; M2) Plants of the Solanales or Poales; M3) Plants of the Solanaceae family or the Poaceae family; M4) Plants of the genus *Nicotiana* or *Saccharum*; M5) Tobacco or sugarcane.
[0018] The recombinant plasmid of the present invention is obtained by inserting the pCB301 vector as a backbone into the whole genome of SCYLV. The recombinant Agrobacterium (pCB301-SCYLV infectious clone) prepared with the recombinant plasmid can successfully infect Nicotiana benthamiana and has a high infection rate. Furthermore, the recombinant expression plasmid infectious clone prepared with the aforementioned recombinant plasmid can successfully infect Nicotiana benthamiana and sugarcane and has a high infection rate and can express exogenous proteins. Attached Figure Description
[0019] Figure 1 A schematic diagram illustrating the construction of an infectious clone.
[0020] Figure 2 Symptoms of infection with invasive clones (without EGFP) in *Nicotiana spp.*
[0021] Figure 3 Detection of infectious clones (without EGFP) infecting Nicotiana spp.
[0022] Figure 4 Symptoms of infection with invasive clones (containing EGFP) in Nicotiana spp.
[0023] Figure 5 Detection of infectious clones (containing EGFP) infecting Nicotiana spp.
[0024] Figure 6 For the expression and detection of exogenous proteins.
[0025] Figure 7 These are the results observed using a confocal microscope.
[0026] Figure 8 Symptoms of sugarcane inoculated with plants expressing EGFP protein are shown in the left image, which is taken under ultraviolet light, and the right image is taken under white light.
[0027] Figure 9 The results of RT-PCR detection of pCB301-SCYLV-EGFP in sugarcane seedlings.
[0028] Figure 10 This study describes the immunoblotting detection of pCB301-SCYLV-EGFP in sugarcane seedlings. NS is a non-specific binder. Detailed Implementation
[0029] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.
[0030] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.
[0031] Example 1: Synthesis and Determination of the Complete Genome Sequence of Sugarcane Yellow Leaf Virus 1. Using the Sugarcane yellow leaf virus isolate PI 157033 (Sequence ID: MN097766.1, URL: https: / / www.ncbi.nlm.nih.gov / nuccore / MN097766.1 / ) and Sugarcane yellow leaf virus complete genome (Sequence ID: NC_000874.1, URL: https: / / www.ncbi.nlm.nih.gov / nuccore / NC_000874.1 / ) from the NCBI website as references, perform sequence assembly. Combine the assembled reference sequences to form the full-length sequence, obtaining the target sequence (with Stu I and Sma I restriction enzyme sites).
[0032] 2. The obtained target sequence and pCB301 vector were double-digested with Stu I and Sma I, respectively. Then, the digestion products of the target sequence and pCB301 vector were recombined into single fragments to obtain the recombinant product. The recombinant product was transformed into Escherichia coli strain DH5α. Positive clones were screened by PCR using primers SCYLV-1F and SCYLV-2R (Table 1). The plasmid containing the target clone was extracted to obtain the infectious cloning vector pCB301-SCYLV of sugarcane yellow leaf virus SCYLV, and the target sequence was sequenced.
[0033] The PCR amplification system consisted of 3 μL template DNA, 1 μL each of forward and reverse primers (10 μM), 2 μL 10 μM dNTPs, 1 μL Phanta Super-Fidelity DNA Polymerase, 10 μL 5×SF Buffer, and 32 μL ddH2O. The total reaction volume was 50 μL. The overall reaction conditions were: 95℃ for 2 min; 95℃ for 30 sec, 55℃ for 30 sec, 72℃ for 3 min, for 30 cycles; 72℃ for 10 min.
[0034] The recombination reaction was performed in a 10 μL system: 5 μL of 2×ClonExpress Mix (Vazyme C115-01-AA); 1 μL of linearized cloning vector pCB301 digested with Sal I and Eco RI; 1.5 μL of insert 1; 1.5 μL of insert 2; and 1.5 μL of insert 3. Recombination was carried out at 55℃ for 30 min.
[0035] Sequencing results showed that the nucleotide sequence of pCB301-SCYLV is SEQ ID No.1, totaling 10500 bp. Nucleotides 1-770 and 6677-10500 are the pCB301 empty vector sequence (nucleotides 770 and 6677 linked together form the pCB301 empty vector sequence), while nucleotides 771-6676 are the complete SCYLV genome sequence (totaling 5906 bp).
[0036] Table 1 Primer Information
[0037] Example 2: Inoculation and Detection of pCB301-SCYLV Infectious Clones I. Preparation of Infection Solution Infection medium of Agrobacterium containing pCB301-SCYLV: pCB301-SCYLV was electroporated into Agrobacterium tumefaciens strain GV3101. After colony PCR verification, single colonies were picked and inoculated into LB medium containing kanamycin (50 mg / L) and rifampin (25 mg / L) resistance. The culture was carried out overnight at 28°C with shaking. After centrifugation at 4000 rpm for 10 min, the bacterial cells were collected to obtain Agrobacterium containing pCB301-SCYLV. The Agrobacterium containing pCB301-SCYLV was resuspended in Agrobacterium suspension buffer (10 mM MgCl2, 10 mM MES (pH=5.6) and 150 mM acetylsyringone in ddH2O solution). The bacterial concentration was adjusted to its OD value using a spectrophotometer. 600nm =1.0, and let stand at room temperature in the dark for 3 h to obtain an infection solution of Agrobacterium containing pCB301-SCYLV.
[0038] Infection solution of Agrobacterium containing empty vector pCB301: replace pCB301-SCYLV with empty vector pCB301, and perform the other operations as with Agrobacterium containing pCB301-SCYLV to obtain the infection solution of Agrobacterium containing empty vector pCB301.
[0039] II. Infection of Nicotiana spp. plants Test plants: Nicotiana benthamiana plants at the 4-5 leaf stage.
[0040] Experimental group (i.e., pCB301-SCYLV): The bottom leaves of the test plants were injected using a 1 ml syringe, with each leaf receiving 0.3 ml of the infection solution. After inoculation, the plants were placed in a greenhouse at 25°C for cultivation (14 hours of light / 10 hours of darkness alternation). The experiment was repeated 3 times, with 6 plants of Nicotiana Bunsenata injected each time.
[0041] Empty vector group (i.e. pCB301): The infection solution of Agrobacterium containing pCB301-SCYLV was replaced with the infection solution of Agrobacterium containing empty vector pCB301, and the rest of the operation was the same as the experimental group.
[0042] II. Testing 1. Symptom observation Eight days after infection, the systemically infected leaves of *Nicotiana bungeana* began to show obvious downward curling of new leaves after infiltration with *Agrobacterium*. More pronounced downward curling of new leaves could be observed at 16 dpi and 26 dpi. Figure 2 ).
[0043] 2. RT-PCR detection of SCYLV in infected plants Seven days after inoculation, total RNA was extracted from Nicotiana bungeana in each group of tested plants using the above-mentioned trizol method, and RT-PCR was performed using virus-specific primers SCYLV-TF / SCYLV-TR.
[0044] SCYLV-TF: 5'-CAGAAGACGCGCTAACCGTC-3'; SCYLV-TR: 5'-CTATTTGGGATTCTGGAAAAGGC-3'.
[0045] The results are as follows Figure 3 As shown, M is the molecular weight marker, and numbers 2-6 represent the five replicate plants in the experimental group, compared with the empty vector group (i.e., Figure 3 Compared to pCB301 in the experimental group, the four duplicate plants (numbered 2, 3, 4, and 5) were able to amplify the viral genome fragment after inoculation with the virus. Figure 3 ).
[0046] Example 3: Modification and application of the pCB301-SCYLV exogenous protein expression vector I. Construction of the pCB301-SCYLV exogenous protein expression vector pCB301-SCYLV-EGFP The primers used in the following experiments are listed in Table 1.
[0047] The subgenome promoter sequence before SCYLV ORF3-5 was repetitively constructed, and EGFP was inserted to both sides of the SCYLV subgenome (including P3a), named SCYLV-EGFP-1 and SCYLV-EGFP-2 respectively.
[0048] The construction method of pCB301-SCYLV-EGFP is as follows: Using pCB301-SCYLV as the amplification template, fragment 1-1 is amplified using SCYLV-1F and SCYLV-ORF5R-2, fragment 1-2 is amplified using SCYLV-2U-2-F and SCYLV-2UTB-R, and fragment 1-4 is amplified using SCYLV-3UTR-2-F and SCYLV-2R; using EGFP as the amplification template, fragment 1-3 is amplified using EGFP-F-TYCZ-SCYLV and EGFP-R-TYCZ2-SCYLV. Fragments 1-1, 1-2, 1-3, and 1-4 are ligated to the double-digested pCB301 using homologous recombination. The infectious cloning vector is obtained according to the method in Example 1 and sequenced. The results showed that pCB301-SCYLV-EGFP-2 was obtained by inserting SEQ ID No. 3 between bases 6448-6449 of pCB301-SCYLV (SEQ ID No. 1).
[0049] II. Inoculation and Detection of Infectious Clones Using the pCB301-SCYLV Exogenous Protein Expression Vector pCB301-SCYLV-EGFP 1. Preparation of the infiltration solution Infection solution of Agrobacterium containing pCB301-SCYLV-EGFP: pCB301-SCYLV-EGFP was electroporated into Agrobacterium strain EHA105. After colony PCR verification, single colonies were picked and inoculated into LB medium containing kanamycin (50 mg / L) and rifampin (25 mg / L) resistance. The culture was carried out overnight at 28°C with shaking. After centrifugation at 4000 rpm for 10 min, the bacterial cells were collected to obtain Agrobacterium containing pCB301-SCYLV-EGFP-1. Agrobacterium containing pCB301-SCYLV-EGFP-1 was resuspended in Agrobacterium suspension buffer (10 mM MgCl2, 10 mM MES (pH=5.6) and 150 mM acetylsyl syringone in ddH2O solution). The bacterial concentration was adjusted to its OD value using a spectrophotometer. 600nm =1.0, and let stand at room temperature in the dark for 3 h to obtain an Agrobacterium infection solution containing pCB301-SCYLV-EGFP-1.
[0050] Infection solution of Agrobacterium containing empty vector pCB301: replace pCB301-SCYLV with empty vector pCB301, and perform the other operations as with Agrobacterium containing pCB301-SCYLV to obtain the infection solution of Agrobacterium containing empty vector pCB301.
[0051] 2. Infecting Nicotiana bungeana plants Test plants: Nicotiana benthamiana plants at the 4-5 leaf stage.
[0052] pCB301-SCYLV-EGFP-1 group: The bottom leaves of the test plants were injected using a 1 ml syringe, with each leaf receiving 0.3 ml of the infection solution. After inoculation, the plants were placed in a greenhouse at 25°C (14 hours light / 10 hours dark alternation). The experiment was repeated 3 times, with 10 plants of Nicotiana benthamiana injected each time.
[0053] pCB301-SCYLV group: The infection solution of Agrobacterium containing pCB301-SCYLV was replaced with the infection solution of Agrobacterium containing pCB301-SCYLV-EGFP, and the rest of the operation was the same as that of pCB301-SCYLV-EGFP group.
[0054] Empty vector group (i.e. pCB301): Agrobacterium containing pCB301-SCYLV-EGFP was replaced with Agrobacterium containing empty vector pCB301, and the rest of the operation was the same as the pCB301-SCYLV-EGFP group.
[0055] 3. Testing (1) RT-PCR detection Seven days after inoculation, RT-PCR was used for testing. The primers for the test are as follows: F: 5'-GCGATCACATGGTCCTGCTG-3'; R: 5'-GATGCCATGCCGACCCATTTCGGTGACTAGGATATACGGGAGG-3'.
[0056] The results are as follows Figure 4 As shown, the target band could be detected in the leaves of Nicotiana bungeana in the pCB301-SCYLV-EGFP group, indicating that pCB301-SCYLV-EGFP has infection activity.
[0057] (2) Symptom observation The phenotype of plants 8 days after inoculation with pCB301-SCYLV-EGFP is as follows Figure 5 As shown. By Figure 5 It can be seen that the leaf surface shows slight wrinkling symptoms.
[0058] (3) WB detection Proteins were extracted from leaves of plants in the pCB301-SCYLV-EGFP group and the empty vector group 8 days after inoculation and detected by Western blotting. The antibody α-GFP used in the detection was purchased from Beijing TransGen Biotech Co., Ltd., catalog number HT801-01. The internal control was rbcL, obtained directly by Coomassie Brilliant Blue staining.
[0059] The results are as follows Figure 6 As shown, EGFP can be detected in tobacco leaves inoculated with Agrobacterium containing pCB301-SCYLV-EGFP.
[0060] (4) Observation using a confocal microscope Observation of the leaves in the above groups using confocal microscopy revealed that EGFP can be injected into the epidermal cells of Nicotiana Bunsenata leaves ( Figure 7 epidermal leaf cell) and stem ( Figure 7 EGFP is expressed in phloem cells (the part in red box is EGFP).
[0061] In summary, the pCB301-SCYLV-EGFP infectious clone is biologically active, successfully infecting Nicotiana spp. and expressing exogenous proteins.
[0062] Example 4: Inoculation and Detection of SCYLV-EGFP Sugarcane The pCB301-SCYLV-EGFP used in this experiment is the same pCB301-SCYLV-EGFP as described in the above examples. The experiment was repeated three times.
[0063] Experimental group (pCB301-SCYLV-EGFP): Agrobacterium containing the pCB301-SCYLV-EGFP plasmid was activated, and the OD was adjusted using MMA buffer (10 mM MES (SIGMA, catalog number: 69892-500G), 10 mM MgCl2, 200 μM Acetosyringone). 600nm The concentration was adjusted to 0.1 to obtain a mixture. The roots of eight tuber-propagated, virus-free sugarcane seedlings (Badila) were immersed in the mixture and placed at room temperature in the dark for 8 hours. After immersion, the roots were rinsed with clean water and placed in soil for growth at room temperature. After 27 days, the sugarcane seedlings were photographed, and RT-PCR and Western blot analyses were performed. The detection methods were the same as in Example 3.
[0064] Control group: Agrobacterium was replaced with the empty vector pCB301 instead of the pCB301-SCYLV-EGFP plasmid, and the other steps were the same as the experimental group.
[0065] Results of photography under ultraviolet light are as follows Figure 8 As shown, SCYLV-EGFP expresses EGFP protein in sugarcane leaves.
[0066] Both RT-PCR and Western blot assays demonstrated that SCYLV-EGFP possesses strong infectivity and EGFP protein expression ability in sugarcane. Figure 9 and Figure 10 ).
[0067] pCB301-SCYLV (SEQ ID No. 1):
[0068] EGFP (SEQ ID No. 2, 954bp, where positions 31-747 are the coding gene for the EGFP protein) 5'-cgaatacgggcgctaaccgctcacgaaggaATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGtaaacaaaataaatacggaggggcatcccgagccaccatataggttgcaagagtggaacgggaagtcctatagataagccgcgcgattctggattacaaacttctagcgggagttctcatcggtgttctcatcgcaatccccctagtgatcatagcggcttacctgatattcctcaaaatcagtgcacacatccgagcgatagtgaa-3'。
[0069] SEQ ID No.3 (945bp, where positions 226-942 are the encoding gene for EGFP protein) 5'-atacggaggggcatcccgagccaccatataggttgcaagagtggaacgggaagtcctatagataagccgcgcgattctggattacaaacttctagcgggagttctcatcggtgttctcatcgcaatccccctagtgatcatagcggcttacctgatattcctcaaaatcagtgcacacatccgagcgatagtgaacgaatacgggcgctaaccgctcacgaaggaATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGACCTACGGCGTGCAGTGCTTCAGCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAACGTCTATATCATGGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCACAACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGtaa-3'。
[0070] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.
Claims
1. A recombinant plasmid characterized in that, The recombinant plasmid contains the complete genome of sugarcane yellow leaf virus, and the nucleotide sequence of the complete genome is nucleotides 771-6676 of SEQ ID No.
1.
2. The recombinant plasmid of claim 1, wherein, The nucleotide sequence of the recombinant plasmid is SEQ ID No.
1.
3. A recombinant expression plasmid characterized in that, The recombinant expression plasmid is a recombinant expression vector obtained by inserting a foreign gene between nucleotides 4418-4419 of the recombinant plasmid with the nucleotide sequence SEQ ID No.
1.
4. A recombinant microorganism, characterized in that, The recombinant microorganism contains the recombinant plasmid as described in claim 1 or 2.
5. A recombinant microorganism, characterized in that, The recombinant microorganism contains the recombinant expression plasmid as described in claim 3.
6. The use of the recombinant plasmid of claim 1 or 2, the recombinant expression plasmid of claim 3, the recombinant microorganism of claim 5, or the whole genome of the sugarcane yellow leaf virus of claim 1 in the preparation of plants with curled and / or wrinkled and / or yellowed leaves.
7. The use of the recombinant plasmid of claim 1 or 2, the recombinant expression plasmid of claim 3, the recombinant microorganism of claim 5, or the whole genome of the sugarcane yellow leaf virus of claim 1 in the preparation of products from plants with curled and / or wrinkled and / or yellowed leaves.
8. A method for preparing plants with curled and / or wrinkled and / or yellowed leaves, comprising the following steps: infecting a recipient plant with the recombinant plasmid of claim 1 or 2 or the recombinant expression plasmid of claim 3 via Agrobacterium-mediated infection to obtain plants with curled and / or wrinkled and / or yellowed leaves.
9. A method for causing the leaves of a plant to curl and / or wrinkle and / or turn yellow, the method comprising infecting a target plant with the recombinant plasmid of claim 1 or 2 or the recombinant expression plasmid of claim 3 via Agrobacterium tumefaciens, thereby causing the leaves of the target plant to curl and / or wrinkle and / or turn yellow.
10. The method according to claim 8 or 9, characterized in that, The plant is any one of the following: M1) Dicotyledons or monocotyledons; M2) Plants of the Solanales or Poales; M3) Plants of the Solanaceae family or the Poaceae family; M4) Plants of the genus *Nicotiana* or *Saccharum*; M5) Tobacco or sugarcane.