Virus vector for expressing exogenous gene in monocotyledon C4 plant and application

Abstract

The invention discloses a viral vector for expressing an exogenous gene in a monocotyledon C4 plant and an application of the viral vector. The method comprises the following steps: by taking genome RNA (Ribonucleic Acid) of a sugarcane mosaic virus isolate SCMV-mDu which is accidentally found in a large-scale field virus disease survey and infects corn to only cause slight mosaic symptoms as a template, amplifying to obtain full-length cDNA (Complementary Deoxyribose Nucleic Acid), and constructing the full-length cDNA to a plant binary expression vector to obtain an infectious clone pSCmDu; an expected expression gene coding region sequence of which the two sides are fused with single restriction enzyme cutting sites is inserted into a pSCmDu virus genome to obtain a virus vector, agrobacterium is converted to obtain an SCMV-mDu VOX system, the system is suitable for efficiently expressing protein and polypeptide in monocotyledonous C4 plants such as corn and sorghum, the symptoms caused by SCMV-mDu infected plants are slight, and the SCMV-mDu VOX system can be used for efficiently expressing protein and polypeptide in the monocotyledonous C4 plants. The method can be used for efficient and systematic expression of genes, and has a wide application prospect in the fields of improvement of germplasm resources of important monocotyledonous C4 crops and genetic breeding.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and in particular, relates to a virus vector for expressing foreign genes in monocotyledonous C4 plants and its application. Background Art

[0002] Globally, corn ( Zea mays L.) and sorghum [ Sorghum bicolor (L.) Moench] is a widely planted C4 crop of the Gramineae family. They are not only important food crops, but also important raw materials for producing livestock feed and making biofuels. The current genetic transformation technology for corn and sorghum has problems such as long cycle, high cost and low efficiency, which limits the large-scale and rapid research on their gene functions; at the same time, this technical limitation also hinders the rapid and high-throughput expression of heterologous functional proteins in C4 plants such as corn and sorghum, such as resistance-related proteins, small peptides, antigenic determinants and antibodies. Therefore, it is necessary to develop alternative methods for stable transgenic technology.

[0003] Studies have shown that virus-mediated overexpression (VOX) technology can be used to express proteins in plants in a high-throughput, transient and rapid manner, thereby facilitating the study of plant gene function (Gleba, Klimyuk, & Marillonnet, 2007). Currently, seven viruses have been reported to be used as VOX vectors in corn. These viruses include barley stripe mosaic virus (Cheuk & Houde, 2018), barley yellow striate mosaic virus (Gao et al., 2019), foxtail mosaic virus (FoMV) (Bouton et al., 2018), maize dwarf mosaic virus (MDMV) (Xie et al., 2021), maize mosaic virus (Kanakala et al., 2023), wheat streak mosaic virus (WSMV) (Tatineni et al., 2011) and sugarcane mosaic virus (SCMV) (Mei et al., 2019), but these viruses all cause severe mosaic symptoms and even necrosis, which is very unfavorable for studying gene function. Sorghum has a wide range of adaptability and good stress resistance. With global warming and frequent extreme climates, rapid screening and mining of stress resistance genes in sorghum will be of guiding significance for genetic improvement and molecular breeding of grass crops. Although the sorghum genome has been sequenced, there is still no VOX vector for studying the gene function of the sequenced sorghum variety BTx623.

[0004] Potyviruses (potyviruses, classified as Potyvirus Genus, PotyviridaeThe largest known class of RNA viruses that infect plants is the family Viridae, which has a wide range of natural hosts, some of which infect only monocots, including crops such as corn, sugarcane and sorghum. The genome of this type of virus is positive-sense single-stranded RNA, which is assembled with the virus-encoded coat protein to form a long linear virion. Potyviruses have the following advantages as VOX vectors: (1) Compared with spherical virus particles, curved, elongated virus particles can load larger exogenous insertion fragments in the genomic RNA by appropriately increasing the particle length; (2) Since potyviruses only have one RNA genome, when constructing a VOX vector, it is only necessary to consider inserting the heterologous gene coding region into the coding frame of the viral RNA; (3) Studies have shown that there are at least six different positions in the genome of potyviruses that can be used to express heterologous proteins, among which the sites at the P1 / HC-Pro and NIb / CP junctions are the most commonly used insertion sites for designing exogenous genes, which also makes it possible to express multiple proteins through one vector (Mei et al., 2019; Xie et al., 2021).

[0005] Given these advantages of potyviruses as VOX vectors, although a variety of viral vectors have been developed for gene function analysis in dicots, such as turnip mosaic virus, tobacco etch virus, plum pox virus, and tobacco vein mosaic virus (Dolja et al., 1992; Fernández-Fernández et al., 2001; Beauchemin et al., 2005; Gao et al., 2012; Xie et al., 2021), potyvirus vectors that can be used in monocots are rarely reported. Recently, although SCMV-MDMV-B isolates can be transformed into VOX vectors in maize, SCMV-MDMV-B VOX vectors need to be inoculated into maize seedlings with the gene gun bombardment technology (Mei et al., 2019). However, the gene gun bombardment technique requires expensive gold particles and specific precision equipment, and has low inoculation efficiency. Moreover, most SCMV isolates, including SCMV-MDMV-B, cause severe mosaic and plant dwarfing symptoms in corn, which is not conducive to the observation and determination of plant phenotypes after overexpression of genes mediated by them. Therefore, identifying and modifying SCMV isolates that cause mild mosaic symptoms and getting rid of the limitations of gene gun bombardment technology will be more suitable for the development of VOX vectors and the study of gene functions in corn and sorghum. Summary of the invention

[0006] The purpose of the present invention is to provide a virus vector for expressing foreign genes in monocotyledonous C4 plants and its application.

[0007] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a viral vector for expressing foreign genes in monocotyledonous C4 plants, and the construction method thereof comprises the following steps: (1) Using the genomic RNA of the sugarcane mosaic virus isolate SCMV-mDu (NCBI accession number OQ948124), which was accidentally discovered during a large-scale field virus disease survey and only caused mild mosaic symptoms in corn, as a template, the full-length cDNA of its genomic RNA was amplified by RT-PCR and then cloned into the pCB301 vector to construct the infectious clone pSCmDu; (2) The nucleotide sequence corresponding to the protease cleavage site between NIb (nuclear inclusion protein b) and CP (capsid protein) at nucleotides 10153-10179 in the infectious clone pSCmDu was doubled, and a nucleotide with Billion I and Stu I single restriction site ( Billion I. Stu I can be Sister I, Afl II, Kpn I, Pac I, Good morning I, Stu 1) to obtain a viral vector for expressing foreign genes in monocotyledonous C4 plants.

[0008] Preferably, the reporter gene is GFP The viral vector constructed for expressing foreign genes in monocotyledonous C4 plants is pSCmDu::GFP.

[0009] Furthermore, the full sequence of the viral vector of the present invention is formed by connecting SEQ ID NO: 1~2 in sequence.

[0010] In a second aspect, the present invention provides a biological material containing the viral vector, wherein the biological material includes an expression vector, an engineered bacterium or a host cell.

[0011] In a third aspect, the present invention provides a monocotyledonous C4 plant exogenous gene expression system (SCMV-mDu VOX system), wherein the coding region of the exogenous gene is inserted into the Billion I and Stu I linearized the viral vector and transformed Agrobacterium to obtain.

[0012] In a fourth aspect, the present invention provides any of the following applications of the viral vector, the biological material or the expression system: 1) For gene expression; 2) Used to study the functions of genes, proteins, and peptides; 3) Used to construct transgenic plants.

[0013] Furthermore, the plant is a monocotyledonous plant.

[0014] Monocotyledonous C4 plants, such as corn and sorghum, are preferred.

[0015] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects: The SCMV-mDu VOX system constructed by the present invention is suitable for efficiently expressing proteins in monocotyledonous C4 plants (such as corn and sorghum), and the symptoms caused by SCMV-mDu infection of corn and sorghum are mild, and the phenotype of plants after overexpressing proteins is little disturbed. When the SCMV-mDu VOX system is used, it only needs to insert the coding region of the gene to be studied into the vector for efficient gene expression. The method of the present invention is simple, rapid, efficient, low-cost, and does not require genetic transformation. It has good market application prospects in the field of plant germplasm resource improvement and genetic breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 The corresponding vector maps in Examples 1 and 2 of the present invention; wherein, A: map of SCMV-mDu infectious cloning plasmid pSCmDu; B: vector map of pSCmDu::GFP and the restriction endonuclease sites artificially inserted are marked.

[0017] Figure 2 This is a schematic diagram of expressing GFP using pSCmDu::GFP in Example 3 of the present invention.

[0018] Figure 3 The phenotypes of plants infected with pSCmDu and pSCmDu::GFP in Examples 1 and 3 of the present invention are shown; wherein A: symptoms of pSCmDu infecting corn and sorghum leaves; B: GFP fluorescence results observed under a confocal microscope of pSCmDu::GFP-infiltrated Nicotiana benthamiana leaves; C: symptoms of pSCmDu::GFP-infected corn and sorghum leaves and GFP fluorescence results under UV light. DETAILED DESCRIPTION

[0019] The present invention provides a VOX vector suitable for gene function research and protein expression of monocotyledonous C4 plants such as corn and sorghum, and a method for using the same.

[0020] The present invention adopts the following technical solution: The invention provides a gene expression system mediated by a mild isolate of sugarcane mosaic virus. The plasmid pSCmDu::GFP is prepared by using RNA of a sugarcane mosaic virus isolate SCMV-mDu that infects corn and causes only mild mosaic symptoms as a template, amplifying the full-length cDNA of its genomic RNA by RT-PCR, and then cloning it into a pCB301 vector to construct an intermediate plasmid pSCmDu, and the nucleotide sequence corresponding to the protease cleavage site between NIb / CP corresponding to nucleotides 10153-10179 in pSCmDu is doubled (the sequence is doubled so that both ends of the polypeptide corresponding to the exogenous inserted fragment have protease cleavage sites, so that the polypeptide can be cut out from the polyprotein of the virus, otherwise it will form a fusion protein with the virus protein), and a nucleotide sequence with Billion I and Stu I single enzyme digestion GFP The coding region sequence was sequenced to obtain the plasmid pSCmDu::GFP; this plasmid can be used as a control vector in the experiment, and on the other hand, it can be used to Billion I and Stu I double enzyme digestion GFP Linearize and connect to the exogenous gene coding region.

[0021] In the aforementioned gene expression system SCMV-mDu VOX, the nucleotide sequence of the plasmid pSCmDu::GFP is formed by sequentially connecting SEQ ID NOs: 1 to 2.

[0022] The present invention also provides the application of the gene expression system SCMV-mDu VOX in gene expression in plants. For example, first, Agrobacterium containing a pSCmDu:: gene coding region to be studied (without the stop codon) vector is infiltrated into Nicotiana benthamiana. After 5 to 7 days, crude extracts of Nicotiana benthamiana inoculated leaves are taken and rubbed to inoculate monocotyledonous plant seedlings such as corn and sorghum. The seedlings are cultured for 5 to 10 days under the conditions of 16h / 24°C during the day and 8h / 22°C at night. The expression of the target gene can be observed in the upper leaves of the inoculated plants, and the expression of the target gene is detected by using methods such as RT-PCR and immunoblotting.

[0023] The plants involved in the present invention include monocotyledonous plants, including but not limited to corn and sorghum.

[0024] The present invention uses the SCMV-mDu isolate, which was first isolated from a corn production field in my country and naturally infected corn and only caused mild mosaic symptoms, as material to construct its infectious cDNA clone, and through modification to obtain a VOX vector that can be used for the study of gene functions in a variety of plants, providing a new system and method for studying plant gene functions and expressing heterologous proteins (such as resistance-related proteins, antigenic determinants and antibodies) and small peptides.

[0025] The present invention also provides a method for studying the gene function of monocotyledonous plants such as corn and sorghum based on the SCMV-mDu VOX system, comprising the following steps: 1. Construction of SCMV-mDu infectious cDNA clone: ​​Using diseased corn leaf samples naturally infected by SCMV-mDu isolates as materials, the full-length cDNA of its genomic RNA was amplified by RT-PCR and connected into plant binary expression vectors such as pCB301. After transformation into Agrobacterium, it became infectious, and the SCMV-mDu infectious cDNA clone pSCmDu was successfully constructed.

[0026] 2. Construction of pSCmDu::GFP vector: Double the protease cleavage site between the NIb and CP proteins in pSCmDu, and directly insert the doubled cleavage site sequence with Billion I and Stu I single restriction site GFP The coding region sequence was obtained and the transformed pSCmDu was named pSCmDu::GFP.

[0027] 3. Amplify the coding region of the plant gene to be studied (remove the stop codon) and connect it to Billion I and Stu I double-digested pSCmDu::GFP linearized vector and transformed into Agrobacterium. The Agrobacterium (OD 600 = 1.2), infiltrated the upper expanded leaves of 4-5 week-old Nicotiana benthamiana; 5-7 days later, crude extracts of the inoculated Nicotiana benthamiana leaves were taken and rubbed into monocot seedlings such as corn and sorghum. The target gene expression was observed in the upper leaves of the inoculated plants under the conditions of 16h / 24℃ during the day and 8h / 22℃ at night for 5-10 days, and RT-PCR and Western blot were used to detect the target gene expression.

[0028] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0029] Example 1 Construction of SCMV-mDu infectious cDNA clone

[0030] In this example, the SCMV-mDu isolate, which was first isolated from a corn production field in my country and naturally infected corn and caused only mild mosaic symptoms, was used as the material to construct an infectious cDNA clone. The backbone vector of the infectious clone was pCB301 (the vector pCB301 was kindly donated by Professor Li Zhenghe of Zhejiang University, see Wang, Q.; Ma, X.; Qian, S.; Zhou, X.; Sun, K.; Chen, X.; Zhou, X.; Jackson, AO; Li, Z. Rescue of a plant negative-strand RNA virus from cloned cdna: insights into enveloped plantvirus movement and morphogenesis. PLoS Pathog. 2015, 11, e1005223).

[0031] The specific implementation methods are as follows: (1) Method for extracting total RNA from diseased corn leaves: a. Grind the diseased corn leaves stored in an ultra-low temperature refrigerator into powder in liquid nitrogen and add 1 mL of TransZol (Beijing Quanshijin Co., Ltd.), mix well, and keep at room temperature for 5 min; b. Add 200 µL of chloroform, mix well, and centrifuge at 12,000 g for 15 min at 4°C. c. Aspirate the upper aqueous phase, add an equal volume of isopropanol, mix well, and let stand at room temperature for 30 min; d. Centrifuge at 12,000 g for 10 min, wash the precipitate with 1 mL 75% ethanol, and dry at room temperature for 3 min; e. The obtained RNA was dissolved in 30 µL DEPC-treated ddH2O and heated at -80 o C Save for later use.

[0032] (2) Amplification of the complete SCMV-SDm genome cDNA sequence by RT-PCR in two sections

[0033] a. Reverse transcription

[0034] The reaction system is as follows (total volume 25 μL):

[0035] The reaction was carried out in a 42℃ water bath for 1 h. The reverse transcription product was stored at -20℃ for future use.

[0036] b. Using the above cDNA as a template, amplify the full-length fragment of the SCMV-SDm genome, and use the DNA polymerase: TransStart ® FastPfu DNA Polymerase( TransGene , UK).

[0037] The PCR reaction system is as follows (total volume 50 μL):

[0038] The PCR program is: 95 o C, 2 min; 95 o C, 20 s, 54 o C, 20 s, 72 o C, 2 min 30 s (if the target amplified fragment exceeds 1 kb, the extension time is calculated based on an amplification rate of 2-4 kb / min), 33 cycles; 72 o C extension for 5 min.

[0039] F0 / R4264 and F4264 / R9574 amplified the first half and the second half of the SCMV full-length genome, respectively, and there was a 20 bp overlap between the two fragments; F-pCB301 / R-pCB301 (reverse PCR) linearized the pCB301 vector and added poly(A) and SCMV-mDu homology arm sequences.

[0040] c. The amplified fragments were subjected to 1.5% agarose gel electrophoresis, the gel was cut, recovered and eluted into 30 μL ddH2O, and then the three fragments were homologously recombined.

[0041] The connection system is as follows: Fragment 1 (150-200 ng) 2 μL

[0042] Fragment 2 (150-200 ng) 2 μL

[0043] Linearized pCB301 vector (about 100 ng) 1 μL

[0044] 2 × Basic Assembly Mix 5 μL

[0045] 50 o C, reaction time 30 min.

[0046] d. The ligation product was transformed into E. coli competent cells (DH5α), and the recombinant plasmid was identified and the positive clones were screened.

[0047] The specific E. coli transformation method (heat shock method) is as follows: Thaw 100 μL of E. coli DH5α competent cells on ice, add 5-10 μL of ligation product, mix gently and place on ice for 30 min. o After heat shock at 37 °C for 90 s, the cells were immediately placed on ice for 5 min, and 800 μL of LB liquid medium without antibiotics was added. o C, 180 rpm for 45 min, 5,000 rpm, centrifuge for 3 min, discard 800 μL of the upper layer, mix the remaining 200 μL of bacterial solution by pipetting and spreading on LB plates containing the corresponding antibiotics, and incubate at 37 o C and incubate for 10-12 h.

[0048] e. Extract plasmids from the screened positive clones, transform the plasmids into Agrobacterium C58C1, culture the identified positive Agrobacterium colonies, and infiltrate Nicotiana benthamiana leaves. After infiltration for at least 5 days, collect the infiltrated leaves and rub them to inoculate corn seedlings. Determine whether the infectious clone is infectious by observing the disease occurrence of corn.

[0049] The method for extracting plasmids from E. coli is as follows: Pick a single colony and inoculate it into 3-5 mL of LB liquid medium containing the corresponding antibiotics (containing 50 μg / mL Kan + ) in 37 o C, 180 rpm for 8-12 h, collect the bacteria, and use the Tiangen Plasmid Extraction Kit to extract plasmid DNA according to the instructions. The specific steps are as follows: After fully suspending the bacteria with 250 μL solution P1, add 250 μL solution P2, mix gently, then add 350 μL solution P3, immediately mix gently and centrifuge at 12,000 rpm for 10 min. Transfer the supernatant to the balanced adsorption column, centrifuge at 12,000 rpm for 1 min, add 500 μL protein removal solution PD, centrifuge at 12,000 rpm for 1 min, then add 600 μL rinse solution PW, centrifuge at 12,000 rpm for 1 min, repeat the wash once, and centrifuge at 12,000 rpm for 2 min to completely remove the rinse solution. Place the adsorption column in a new centrifuge tube and add 50 μL 65 o C preheated ddH2O, let it stand for 2 minutes, and then centrifuge at 12,000 rpm for 2 minutes to obtain plasmid DNA.

[0050] The Agrobacterium transformation method (liquid nitrogen freeze-thaw method) is as follows: Take 100 μL competent cells, add 1 μg plasmid DNA, put it in ice bath for 30 min, freeze it in liquid nitrogen for 1 min, and freeze it at 37 oC water bath for 5 min, then add 800 μL LB liquid medium (without antibiotics) and o After 4-6 h of culture at 180 rpm, the plate was spread on a plate containing the corresponding antibiotic (50 μg / mL Kan + and 50 μg / mL Rif + ) on solid plate medium, 28 o C for about 48h.

[0051] The method of inoculating tobacco with Agrobacterium is as follows: First, pick the corresponding Agrobacterium cells and place them in 2 mL LB liquid medium (containing 50 μg / ml Kan + and 50 μg / ml Rif + ),28 o C, shake at 180 rpm for 24 h. Then take the activated bacterial solution and inoculate it into LB liquid medium (containing 50 μg / ml Kan + and 50 μg / ml Rif + ) in 28 o C, shake at 180 rpm overnight. Centrifuge at 5,000 rpm for 5 min to collect the cells, discard the culture medium, suspend with resuspension solution (10 mmol / L MgCl2, 10 mmol / L MES, 100 μmol / L AS), dilute to an appropriate multiple, and measure OD with a UV spectrophotometer. 600 Use the resuspension solution to adjust the OD of the pSCmDu bacterial solution to 600 The OD of the bacterial solution expressing P22 is 1.2-1.4. 600 The induction was set to 0.3 and allowed to stand at room temperature for at least 3 h. The tobacco leaves were infiltrated with a syringe without a needle.

[0052] The primers used for the construction of SCMV-mDu infectious clones are shown in Table 1: Table 1 Primers used in the construction of SCMV-mDu infectious clones

[0053] Example 2 Construction of pSCmDu::GFP vector

[0054] F0-SCMV / R-NIb amplifies the first half of SCMV-mDu; F-IN-GFP / R-IN-GFP amplifies the GFP (Use primers to introduce single restriction sites at both ends of GFP Billion I and Stu I); IR-PCB301 / F-CP amplification includes CPThe SCMV-mDu sequence at the beginning and the full length of the pCB301 vector were connected by homologous recombination.

[0055] The transformed pSCmDu was named pSCmDu::GFP (plasmid map as shown in Figure 1 As shown in the figure), the full sequence of the vector is formed by connecting SEQ ID NO: 1~2 in sequence.

[0056] The primers used for constructing the pSCmDu::GFP vector are shown in Table 2: Table 2 Primers used in the construction of pSCmDu::GFP vector

[0057] Example 3 Application of pSCmDu::GFP Agrobacterium Infiltration and VOX System in Maize and Sorghum

[0058] (1) pSCmDu::GFP VOX Agrobacterium infiltration

[0059] The pSCmDu::GFP infectious clone cloned under the double 35S promoter of the pCB301 vector was injected into Nicotiana benthamiana leaves by Agrobacterium infiltration. Fluorescent cells could be observed in the infiltrated area after 5 days.

[0060] The specific implementation method is: a. The correctly sequenced pSCmDu::GFP plasmid was transformed into Agrobacterium C58C1 competent cells, and the operation method was the same as in Example 1.

[0061] b. Pick the corresponding Agrobacterium cells and place them in 2 mL LB liquid medium (containing 50 μg / mL Kan + and 50 μg / mL Rif + ),28 o C, shake at 180 rpm for 24 h. Then take the activated bacterial solution and inoculate it into LB liquid medium (containing 50 μg / mL Kan + and 50 μg / mL Rif + ) in 28 o C, shake at 180 rpm overnight; centrifuge at 5,000 rpm for 5 min to collect the cells, discard the culture medium, suspend with resuspension solution (10 mmol / L MgCl2, 10 mmol / L MES, 100 μmol / L AS), dilute appropriately, and measure OD with a UV spectrophotometer. 600 , adjust the OD with resuspension solution 600 The induction temperature was 1.4 and the mixture was allowed to stand at room temperature for 4 to 6 hours.

[0062] (2) 5 to 14 days after the infiltration of Nicotiana benthamiana with Agrobacterium, take the inoculated leaves, add 3 volumes (w / v) of 0.01 M phosphate buffer (pH 7.0), grind, transfer to a 1.5 ml centrifuge tube, and grind for 4 h. o C, centrifuge at 4500 g for 3 min, take the supernatant and rub it on the leaves of corn or sorghum seedlings about 8 days after germination and transplanting, and culture under 16 h / 24℃ daytime and 8 h / 22℃ nighttime conditions. Green fluorescent spots can be observed in the systemic leaves of corn 4 to 5 days after inoculation (under UV light), and green fluorescent spots can be seen in the newly grown leaves of sorghum 10 days after inoculation.

[0063] Seven days after inoculation, the first systemically infected leaf in corn could be observed to have green fluorescence throughout the leaf through a handheld UV lamp, and photographed and recorded; about 21 days after inoculation, clear fluorescence could be observed in the newly grown leaves of sorghum; these results indicate that the pSCmDu::GFP VOX system can efficiently and systemically express GFP protein in corn and sorghum leaves.

[0064] Schematic diagram of GFP expression using pSCmDu::GFP is shown in Figure 2 .

[0065] The phenotypes of plants infected with pSCmDu and pSCmDu::GFP in Examples 1 and 3 are shown in Figure 3 .

[0066] It can also be seen from this example that the pSCmDu::GFP VOX system can be used to study plant gene functions and express exogenous proteins, polypeptides, etc.

[0067] The SCMV-mDu VOX system of the present invention is an innovative mild sugarcane mosaic virus-mediated gene expression platform with significant advantages and broad application prospects. The system utilizes mild virus isolates accidentally discovered during large-scale field surveys, and through carefully designed cloning and vector construction strategies, it achieves efficient and systematic exogenous gene expression in monocotyledonous C4 crops such as corn and sorghum. Compared with other viral vectors, the symptoms caused by the SCMV-mDu VOX system are extremely mild, which reduces the interference with the normal physiology of the plant, making the research results more accurate. At the same time, it has the advantages of simple operation, low cost, and no need for complex genetic transformation processes, and is easy to apply. In addition, the SCMV-mDu VOX system has high design flexibility, can be easily inserted into the coding region of exogenous genes, and achieves rapid expression. It is the first mild virus vector successfully used in corn and sorghum, and provides an important tool for plant gene function research and crop genetic improvement. At the same time, the system's efficient expression capability provides a powerful platform for studying exogenous proteins, peptides and other biologically active molecules, helping to accelerate crop variety improvement and new variety development, meet the growing demand for food and bioenergy, and is of great significance to promoting sustainable agricultural development.

[0068] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

[0069] References

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Claims

1. A viral vector for expressing foreign genes in monocotyledonous C4 plants, characterized in that: The method for constructing the viral vector comprises: (1) Using the genomic RNA of the sugarcane mosaic virus isolate SCMV-mDu, which was accidentally discovered during a large-scale field virus disease survey and only caused mild mosaic symptoms in corn, as a template, the full-length cDNA of its genomic RNA was amplified by RT-PCR and then cloned into the pCB301 vector to construct the infectious clone pSCmDu; (2) The nucleotide sequence corresponding to the protease cleavage site between NIb and CP corresponding to nucleotides 10153-10179 in the infectious clone pSCmDu was doubled, and a nucleotide with Bln I and Stu I single restriction site of the expected expression gene or reporter gene coding region sequence, that is, the virus vector expressing foreign genes in monocotyledonous C4 plants is obtained; The accession number of SCMV-mDu in NCBI is OQ948124.

2. The viral vector according to claim 1, characterized in that The reporter gene is GFP The viral vector constructed for expressing foreign genes in monocotyledonous C4 plants is pSCmDu::GFP.

3. The viral vector according to claim 2, characterized in that The full sequence of the viral vector is formed by connecting SEQ ID NO: 1 to 2 in sequence.

4. A biological material containing the viral vector according to any one of claims 1 to 3, wherein the biological material is an expression vector, an engineered bacterium or a host cell.

5. A monocotyledonous C4 plant exogenous gene expression system, characterized in that: The expression system is to insert the coding region of the foreign gene into Bln I and Stu I linearized virus vector according to any one of claims 1 to 3, and transformed into Agrobacterium.

6. Any of the following uses of the viral vector according to any one of claims 1 to 3, the biological material according to claim 4, or the expression system according to claim 5: 1) For gene expression; 2) Used to study the functions of genes, proteins, and peptides; 3) Used to construct transgenic plants.

7. The use according to claim 6, characterized in that: The plant is a monocotyledonous plant.

8. The use according to claim 7, characterized in that: The monocotyledonous plant is a monocotyledonous C4 plant.

9. The use according to claim 8, characterized in that: The monocotyledonous C4 plants include corn and sorghum.

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