Construction of cmv satellite rna biocontrol mutant and its application in tomato

Abstract

The application belongs to the field of plant protection, and particularly relates to a construction method of a CMV satellite RNA biocontrol mutant and application thereof to tomatoes. The application provides a construction method of a CMV satellite RNA biocontrol mutant: changing a key base in the satellite RNA which induces tomato necrosis, thereby reducing the risk of the satellite RNA changing into a necrosis type satellite RNA. Compared with a parent Yi strain, the CMV satellite RNA biocontrol mutant is mutated at least at any one of the following six sites: G328, G331, G337, C344, C349 and C354. The application also simultaneously provides application of the obtained CMV satellite RNA biocontrol mutant: reducing the pathogenic risk of plants.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of plant protection, and particularly relates to a construction method of a CMV satellite RNA biocontrol mutant and application thereof to tomatoes. BACKGROUND

[0002] Satellite RNA generally refers to a kind of parasitic long-chain non-coding RNA, which realizes its infection process and biological function by interacting with virus or host factors through its sequence or RNA structure. In nature, there are more than 20 kinds of plant viruses carrying satellite RNA. Among them, the plant virus with the highest satellite RNA occurrence rate is CMV. It is reported that more than 60 CMV isolates carry satellite RNA. CMV is a plant pathogen with important economic impact, which can infect more than 1200 plants including tomatoes, peppers and corn.

[0003] The replication of CMV satellite RNA not only depends on the replication enzyme of the helper virus, but also needs the non-translational template function of the viral genomic RNA to create optimal replication conditions for it. The genome length of CMV satellite RNA is between 330-405 nt, and there is no obvious sequence similarity with the helper virus genome. The infection of satellite RNA usually affects the symptom expression of CMV in host plants. In most cases, satellite RNA will reduce the pathogenicity of CMV in most host plants by reducing the replication and transcription levels of the virus, inactivating the suppressor activity of CMV 2b protein, etc. However, some satellite RNA strains carried by CMV will aggravate viral diseases in specific plant hosts, such as tobacco yellowing caused by Y strain (sat-Y) and tomato lethal necrosis caused by D strain (sat-D). The satellite RNA strain that causes tomato lethal necrosis has a certain universality. As early as 1977, Kaper and Waterworth reported that CMV satellite RNA caused tomato lethal necrosis disease in the field. Subsequently, the same tomato disease has occurred in France, Italy, Spain, Japan, China and other places. The necrotic satellite RNA strain infects tomatoes and causes the top of the plant to wilt and droop in a short period of time (about 10 days), rapidly leading to plant lethal necrosis.

[0004] Based on the characteristics of satellite RNA significantly reducing the pathogenicity of CMV, Tian et al. (Scientia Sinica 1986, 31(6): 574) used satellite RNA for biological control of CMV viral disease. Specifically, they purified CMV satellite RNA molecules from purified virus particles, mixed them with CMV to inoculate plants, and constructed and screened low pathogenicity CMV satellite RNA biocontrol agents S51 and S55 in plants. Greenhouse and field trials showed that the agents had good protective effects on CMV infection of crops such as peppers and tomatoes. In CN1654638A, satellite RNA was synthesized using in vitro transcription, and then a CMV-infected tobacco was inoculated to prepare a satellite RNA-containing attenuated strain. Harrison et al. (Nature 1987, 328(27): 799-802) synthesized satellite RNA 1-17N strains in transgenic tobacco, effectively reducing the replication level and pathogenicity of CMV. Tian's research group used transgenic technology to create CMV satellite RNA-1 transgenic tobacco and tomatoes, which showed resistance to CMV infection (Chinese Science B 1989, 948-956; Chinese Science B, 1990, 7: 708-713). In CN91104076, CMV coat protein (CP) and satellite RNA-1 were simultaneously transgenically expressed in tobacco to achieve resistance to CMV. Cao et al. (Plant Biotechnol J. 2019;17(10): 1856-1858) reported that injecting satellite RNA Ta-Tb into CMV-infected Nicotiana benthamiana plants could effectively reduce viral symptoms. The above indicates that wild-type satellite RNA can be used in various forms for the prevention and control of CMV viral disease.

[0005] Palukaitis and Roossinck found that weak satellite RNA strains were transferred between tobacco plants for multiple generations, which led to spontaneous mutations from weak to pathogenic mutant strains. In vitro mutation experiments found that necrotic satellite RNA strains had three necrosis-specific sites at their 3' end. Mutating the corresponding sites of weak satellite RNA strains to necrosis-specific bases caused the satellite RNA to exhibit lethal effects on tomatoes. Considering the high variability of satellite RNA, using wild-type satellite RNA for biocontrol applications on tomatoes has the potential risk of mutating into necrotic variants.

[0006] Tomato is an important economic crop, and its viral diseases are quite common, which has a serious impact on the production and quality of tomatoes. According to the virus prevalence survey, CMV is the most common virus of tomato viral diseases. Therefore, the prevention and control of CMV virus has important research significance for tomato planting production in China. The attenuated satellite RNA has a significant effect on the prevention and control of CMV disease on tomatoes, but there is a potential risk of spontaneous mutation into a lethal mutant for tomatoes, which restricts the biocontrol application of satellite RNA in tomato crops. SUMMARY

[0007] The problem to be solved by the present application is to provide a construction method of CMV satellite RNA biocontrol mutant and its application in tomatoes.

[0008] In order to solve the above problems, the present application provides a construction method of CMV satellite RNA biocontrol mutant: changing the key bases (which can be multiple key bases) in satellite RNA that induce tomato necrosis, thereby reducing the risk of satellite RNA turning into a necrosis type satellite RNA.

[0009] As an improvement of the construction method of CMV satellite RNA biocontrol mutant of the present application:

[0010] The parent strain of CMV satellite RNA biocontrol mutant is Yi strain;

[0011] Compared with the parent Yi strain, the CMV satellite RNA biocontrol mutant produces mutations in at least any one of the following 6 sites:

[0012] G328, G331, G337, C344, C349, C354.

[0013] As a further improvement of the construction method of CMV satellite RNA biocontrol mutant of the present application:

[0014] G328 is mutated to C, named G328C;

[0015] G331 is mutated to C, named G331C;

[0016] G337 is mutated to A, named G337A;

[0017] C344 is mutated to U, named C344U;

[0018] C349 is mutated to G, named C349G;

[0019] C354 is mutated to G, named C354G.

[0020] That is, the genetically engineered satellite RNA mutant provided by the application has a base sequence with the mutations as described above compared with the wild-type satellite RNA Yi strain (sat-Yi).

[0021] As a further improvement of the method for constructing the CMV satellite RNA biocontrol mutant of the application:

[0022] G328C and C354G are compensatory mutations, G331C and C349G are compensatory mutations, and G337A and C344U are compensatory mutations.

[0023] The CMV satellite RNA biocontrol mutant comprises at least any of the compensatory mutations described above.

[0024] As a further improvement of the method for constructing the CMV satellite RNA biocontrol mutant of the application:

[0025] The CMV satellite RNA biocontrol mutant is any of the following:

[0026] pCB301-Yim 331 C-G 349 Mutant plasmid (M2),

[0027] pCB301-Yim 331 C-G 349 & 328 C-G 354 Mutant plasmid (M3),

[0028] pCB301-Yi 337 A-U 344 Mutant plasmid (M4),

[0029] pCB301-Yi 337 C-G 344 Mutant plasmid (M5),

[0030] pCB301-Yi 337 A-U 344 & 331 C-G 349 Mutant plasmid (M6),

[0031] pCB301-Yi 337 A-U 344 & 331 C-G 349 & 328 C-G 354 Mutant plasmid (M7).

[0032] In the application, the CMV satellite RNA biocontrol mutant that can be selected is M2, M3, M4, M6, M7, and preferably M7.

[0033] The application also simultaneously provides the use of the CMV satellite RNA biocontrol mutant obtained by any of the above methods: reducing the pathogenic risk of plants.

[0034] As an improvement of the application, the plant is tomato.

[0035] The present application aims to provide a genetic modification strategy for satellite RNA, in order to solve the risk of wild-type satellite RNA mutating into a lethal mutant as a biocontrol agent. Specifically, under the premise of not affecting the replication and attenuation ability of satellite RNA, the satellite RNA is genetically modified by compensatory mutation method to change multiple functional sites related to tomato necrosis in the satellite RNA, so as to effectively overcome the risk of mutation into a lethal mutant.

[0036] Description: The lethal mutant refers to a satellite RNA originally having a biocontrol function (such as assisting virus attenuation or enhancing plant resistance), which, due to the base mutation of a single or multiple key sites (such as 3' end necrosis-related sites), causes a fundamental change in its biological function, from harmless or beneficial to the host (such as tomato) to a mutant satellite RNA that can induce systemic necrosis or death of the plant. This mutant may be naturally produced due to the high-frequency variation characteristics of satellite RNA, thereby posing a potential risk to the application of satellite RNA-based biological control.

[0037] The inventors found in the research that the satellite RNA contains multiple functional sites related to tomato necrosis, and verified by experiments that any change of these sites can effectively eliminate the ability of satellite RNA to induce tomato necrosis, and can well reduce the pathogenicity of CMV.

[0038] The present application takes the attenuated cucumber mosaic virus (CMV) satellite RNA as the research object, based on the predicted RNA structure, deeply mutates the functional sites in the CMV satellite RNA that cause tomato plant necrosis, but retains its attenuation ability, and constructs a satellite RNA biocontrol agent with low pathogenic risk.

[0039] In order to overcome or reduce the risk of satellite RNA mutating into a necrosis mutant, the present application selects the satellite RNA Yi strain (sat-Yi) with significant attenuation ability, mutates multiple necrosis-related sites, constructs a sat-Yi mutant, and reduces the risk of mutation into a necrosis mutant under the premise of not affecting the attenuation ability.

[0040] In the present application, unless otherwise specified, sat-Yi refers to the wild-type satellite RNA Yi strain without mutation. Specifically, the nucleotide sequence of sat-Yi is shown in SEQ ID No: 1.

[0041] In the present application, the infectious clone of sat-Yi is pCB301-sat-Yi. Specifically, the cDNA sequence of sat-Yi is inserted between the restriction sites of Stu I and Sma I in the pCB301 binary vector Figure 1 ).

[0042] In the embodiments of the present application, the helper virus for testing the activity of sat-Yi or its mutants is CMV Fny strain (hereinafter referred to as CMV), and its infectious clones are composed of pCB301-C1, pCB301-C2 and pCB301-C3. The Agrobacterium GV3101 containing the infectious clones of pCB301-C1, pCB301-C2 or pCB301-C3 is C1, C2 or C3, respectively.

[0043] According to the embodiments of the present application, the sat-Yi mutant of the present application refers to the derivative obtained by mutating one or more nucleotides based on the sat-Yi RNA.

[0044] In the present application, the site-directed mutagenesis PCR technique is used to construct the infectious clone of sat-Yi mutant; the mutant base is introduced by PCR primer, and the design of PCR primer follows the following principles:

[0045] The sequence part containing the mutation site is selected as the overlapping sequence of the upstream and downstream primers, and the Tm value of this sequence is ensured to be 55-60℃;

[0046] A sequence is selected on the vector, and the Tm value of this sequence needs to be 5-10℃ higher than that of the overlapping sequence, and the optimal temperature is 65℃.

[0047] In the embodiments of the present application, the plasmid pCB301-sat-Yi is used as the template, the site-directed mutagenesis PCR technique is used to replace the base G at the 342th position of sat-Yi sequence with base T, and the mutant clone M1: pCB301-Yim is obtained. The obtained mutant plasmid is transformed into Agrobacterium GV3101 to obtain Agrobacterium M1; the Agrobacterium M1 and the Agrobacterium carrying the Fny-CMV infectious clone are mixed at the same ratio for injection inoculation of tomato. After 21 days of inoculation, the tomato plants show severe systemic necrosis phenotype.

[0048] In the embodiments of the present application, the mutant clone M1 is used as the template, the site-directed mutagenesis PCR technique is used to mutate the base G at the 331th position of sat-Yi sequence and the base C at the 349th position to C and G (i.e. compensatory mutation), and the mutant clone M2: pCB301-Yim 331 C-G 349. Further site-directed mutagenesis was performed on the basis of M2, and the base G at position 328 and the base C at position 354 of sat-Yi sequence were mutated to compensate, to obtain mutant M3: pCB301-Yim 331 C-G 349 & 328 C-G 354 . M2 and M3 were mixed with Fny-CMV and inoculated to tomato plants, respectively, and both M2 and M3 mutants significantly attenuated the viral symptoms of CMV.

[0049] In an embodiment of the present application, the base G at position 337 and the base C at position 344 of sat-Yi sequence were mutated to base A and T (i.e., U), respectively, by site-directed mutagenesis PCR technology using pCB301-sat-Yi plasmid as a template, to obtain mutant clone M4: pCB301-Yi 337 A-U 344 . Meanwhile, the base G at position 337 and the base C at position 344 of sat-Yi sequence were mutated to compensate, to obtain mutant clone M5: pCB301-Yi 337 C-G 344 . Further site-directed mutagenesis was performed on the basis of M4, and the base G at position 331 and the base C at position 349 of sat-Yi sequence were mutated to compensate, to obtain mutant M6: pCB301-Yi 337 A-U 344 & 331 C-G 349 . Further site-directed mutagenesis was performed on the basis of M6, and the base G at position 328 and the base C at position 354 of sat-Yi sequence were mutated to compensate, to obtain mutant M7: pCB301-Yi 337 A-U 344 & 331 C-G 349 & 328 C-G 354 . M4, M5, M6, and M7 mutants were mixed with Fny-CMV and inoculated to tomato, respectively. Tomato inoculated with M5 showed severe systemic necrosis after 18 days of inoculation. Tomato inoculated with M4, M6, or M7 had lush leaves and thick stems, and had similar abilities to resist CMV.

[0050] In an embodiment of the present application, the accumulation levels of sat-Yi and its mutants were evaluated by the method of Northern hybridization, and the effects of the accumulation levels on the accumulation amount of CMV viral RNA were evaluated.

[0051] In an embodiment of the present application, total RNA for Northern hybridization was extracted from the top systemic leaves of tomato after 14 days of inoculation by Agrobacterium injection.

[0052] In an embodiment of the present application, the plants used for Agrobacterium infiltration inoculation are tomato seedlings at the two-leaf stage, and the variety is Hezuo 903.

[0053] In an embodiment of the present application, the tomato plants are grown in a plant growth chamber at 22-25°C with a light period of 16 hours.

[0054] In an embodiment of the present application, DNA sequencing is used to evaluate the genetic stability of sat-Yi mutants in vivo in tomato.

[0055] In the present application, the genetically engineered sat-Yi mutants M4, M6 and M7 can efficiently replicate and stably inherit in vivo in tomato plants, effectively controlling the viral disease of CMV on tomato plants. Among them, the mutant M7 is preferred as a tool for biocontrol of CMV, because it has the lowest risk of reverting to a necrotic satellite RNA, and has good biological safety and application prospects. Specifically, compared with the lethal mutant M1, M7 carries the most number of mutant sites. These mutations significantly reduce the possibility of reverting to a lethal type under the action of CMV replicase. Under the condition that the mutation frequency of CMV replicase is basically the same, the more and more dispersed the mutant sites are, the lower the probability of reverting to the original lethal type is. Therefore, M7 has higher safety while having the ability to inhibit CMV infection, and is more suitable as a preferred tool for biocontrol of CMV in tomato. BRIEF DESCRIPTION OF DRAWINGS

[0056] The specific embodiments of the present application will be further described in detail below with reference to the accompanying drawings.

[0057] Figure 1 is the plasmid map of pCB301-sat-Yi.

[0058] Figure 2 is the hairpin structure of sat-Yi and mutant necrotic sites in Example 1.

[0059] Figure 3 is the phenotype diagram of sat-Yi, M1, M2 and M3 mixed with CMV to inoculate tomato for 15 days in Example 2, respectively;

[0060] Figure 3 In the present application, the genetically engineered sat-Yi mutants M4, M6 and M7 can efficiently replicate and stably inherit in vivo in tomato plants, effectively controlling the viral disease of CMV on tomato plants. Among them, the mutant M7 is preferred as a tool for biocontrol of CMV, because it has the lowest risk of reverting to a necrotic satellite RNA, and has good biological safety and application prospects. Specifically, compared with the lethal mutant M1, M7 carries the most number of mutant sites. These mutations significantly reduce the possibility of reverting to a lethal type under the action of CMV replicase. Under the condition that the mutation frequency of CMV replicase is basically the same, the more and more dispersed the mutant sites are, the lower the probability of reverting to the original lethal type is. Therefore, M7 has higher safety while having the ability to inhibit CMV infection, and is more suitable as a preferred tool for biocontrol of CMV in tomato.

[0061] The upper row is a top view of the tomato plant, and the lower row is a side view of the tomato plant.

[0062] Mock is the inoculation infiltration buffer as a control, and the Vector group is inoculated with CMV and empty vector pCB301.

[0063] Figure 4Northern blotting of tomato inoculated with Agrobacterium sat-Yi, M1, M2, M3 and wild type CMV for 14 days in Example 3 of the present application;

[0064] Figure 4 In the above table, the following abbreviations are used:

[0065] Figure A is a Northern blot result chart for detecting the accumulation amount of Fny-CMV;

[0066] Figure B is a Northern blot result chart for detecting the accumulation amount of satRNA;

[0067] Figure C is a photo chart of RNA sample after formaldehyde agarose gel electrophoresis, and after electrophoresis at 65 V for 50 min, under ultraviolet light. The bands taken in the chart are 28S and 18S rRNA.

[0068] Figure 5 Figure for the overall phenotype and systemic leaf local phenotype of tomato inoculated with Agrobacterium sat-Yi, M4, M5, M6, M7 and wild type CMV for 18 days in Example 4 of the present application;

[0069] Figure 5 In the above table, the following abbreviations are used:

[0070] Figure A corresponds to the overall phenotype of tomato; and the upper row is a top view of tomato plant, and the lower row is a side view of tomato plant;

[0071] Figure B corresponds to the systemic leaf local phenotype;

[0072] Mock is inoculated with infiltration buffer as a control, and Vector group is inoculated with Fny-CMV and empty vector pCB301.

[0073] Note: Since M5 is dead, therefore Figure 5 the systemic leaf local phenotype corresponding to M5 is not shown in Figure B.

[0074] Figure 6 Northern blotting of tomato inoculated with Agrobacterium sat-Yi, M4, M5, M6, M7 and wild type Fny-CMV for 14 days in Example 3 of the present application;

[0075] Figure 6 In the above table, the following abbreviations are used:

[0076] Figure A is a Northern blot result chart for detecting the accumulation amount of Fny-CMV RNA3;

[0077] Figure B is a Northern blot result chart for detecting the accumulation amount of satRNA;

[0078] C is the photo of the RNA sample after formaldehyde agarose gel electrophoresis at 65 V for 50 min under ultraviolet light, and the bands of 28s and 18s rRNA are cut off in the figure. DETAILED DESCRIPTION

[0079] The present application is described in combination with the following specific examples, but the protection scope of the present application is not limited thereto.

[0080] In the present application, the common molecular biology experiments such as plasmid extraction and Northern blot are performed by using kit method or conventional molecular biology experiments, unless otherwise specified.

[0081] Example 1, construction of sat-Yi mutant clone

[0082] In the present application, seven mutant clones of sat-Yi, M1~M7, are constructed by using site-directed mutagenesis PCR technology, and the specific construction method is as follows:

[0083] (1) M1: construction of pCB301-Yim mutant plasmid

[0084] The plasmid map of pCB301-sat-Yi is shown in Figure 1 , and the sequence of sat-Yi is shown in SEQ ID NO: 1. Among them, the cDNA sequence of sat-Yi corresponds to the sequence of 768~1154 of pCB301-sat-Yi plasmid (SEQ ID NO: 9). The sequence of sat-Yi is from CMV satRNA isolate Yi (DQ412733) in NCBI. The cDNA sequence of sat-Yi is synthesized by Jin Si Rui Biotechnology Co., Ltd., and the company clones the synthesized cDNA fragment into pCB301 vector to obtain the infectious clone pCB301-sat-Yi of sat-Yi. The pCB301 vector is from the related literature (Plant Molecular Biology 1999, 40(4), 711-717).

[0085] By using mutagenesis PCR technology, the plasmid pCB301-sat-Yi is used as a template, and the mutant PCR amplification is carried out by using primers M1-F / M1-R to obtain the PCR product Yim.

[0086] The mutant PCR primer sequence used is as follows:

[0087] M1-F: 5'-TAAGGCTTATGCTATGCTGATCTCCGTGAATGTCTATCATTCC-3' (SEQ ID NO: 10);

[0088] M1-R: 5'-AGCATAGCATAAGCCTTAGCCTCTCCCTGCGTG-3' (SEQ ID NO: 11).

[0089] The PCR amplification system was as follows: 5x Q5 reaction buffer 5 μL, dNTP (2.5 mmol / L) 1 μL, primer M1-F / M1-R (10 μmol / L) 1 μL each, DMSO 1 μL, plasmid template pCB301-sat-Yi (10 ng / µL) 1 μL, Q5 polymerase (2 U / µL) 0.25 μL, ddH2O up to 25 μL; the PCR reaction program was as follows: 98 ℃ 3 min, 98 ℃ 20 s, 58 ℃ 25 s, 72 ℃ 4 min, 12 cycles, 46 ℃ 2 min, 72 ℃ 10 min.

[0090] 2 μL of the amplified PCR reaction product sample was taken for agarose gel electrophoresis detection, and the target band was detected under the ultraviolet system.

[0091] 2 μL of the amplified PCR reaction product sample was taken for agarose gel electrophoresis detection, and the target band was detected under the ultraviolet system. Dpn 1 μL of 10x Cutsmart buffer and 1 μL of I enzyme were added to the remaining 23 μL of PCR product, and the mixture was placed in a 37 ℃ water bath for overnight digestion to remove the template in the reaction system.

[0092] 2 μL of the digestion product was transformed into E. coli, and a sterile spreader was used to evenly spread it on a plate containing kan + , and placed in a 37 ℃ incubator for inverted culture for 12 h.

[0093] A sterile gun tip was used to randomly pick a single colony after culture, and the plasmid was extracted according to the Axygen mini-prepare kit method, and the plasmid was sent to GenScript Biotech (Shanghai) Co., Ltd. for sequencing. The sequencing results showed that the site had been successfully mutated. As described above, the 342th base G of sat-Yi was replaced with U. The replaced base is the red base in the figure. Figure 2

[0094] The sequence of M1 is shown as SEQ ID NO: 2.

[0095] (2) M2: pCB301-Yim 331 C-G 349 Construction of mutant plasmid

[0096] Referring to the construction method of the M1 mutant plasmid, the pCB301-Yim 331 C-G 349 ​Mutant plasmid. All procedures are similar, using plasmid M1 as template, using primers M2-F / M2-R for mutation PCR, primer sequences are as follows:

[0097] M2-F: 5'-GAGAGCCTAAGGCTTATGCTATGGTGATCTCCGTGAATGTCTATCATTC-3' (SEQ ID NO: 12)

[0098] M2-R: 5'-AGCCTTAGGCTCTCCCTGCGTGCGTCATGA-3' (SEQ ID NO: 13).

[0099] It is found that the prepared mutant plasmid M2 site has been mutated. As described, specifically, the 331st base G and the 349th base C of sat-Yi are replaced by C and G respectively. The replaced bases are the red bases in the figure. Figure 2

[0100] The sequence of M2 is shown in SEQ ID NO: 3.

[0101] (3) M3: pCB301-Yim 331 C-G 349 & 328 C-G 354 Construction of mutant plasmid

[0102] Referring to the construction method of M1 mutant plasmid, pCB301-Yim is constructed on the basis of plasmid M2 331 C-G 349 & 328 C-G 354 Mutant plasmid. All procedures are similar, using plasmid M2 as template, using primers M3-F / M3-R for mutation PCR, primer sequences are as follows:

[0103] M3-F: 5'-AGGGACAGCCTAAGGCTTATGCTATGGTGATGTCCGTGAATGTCTATCATTCCTC-3' (SEQ ID NO: 14),

[0104] M3-R: 5'-CTTAGGCTGTCCCTGCGTGCGTCATGACTCAT-3' (SEQ ID NO: 15).

[0105] It is found that the prepared mutant plasmid M3 site has been mutated. As described, specifically, the 328th base G and the 354th base C of sat-Yi are replaced by C and G respectively. The replaced bases are the red bases in the figure. Figure 2 ​​

[0106] The sequence of M3 is shown as SEQ ID NO: 4.

[0107] (4) M4: pCB301-Yi 337 A-U 344 Construction of mutant plasmid

[0108] Referring to the construction method of Ml mutant plasmid, pCB301-Yi was constructed based on plasmid pCB301-sat-Yi 337 A-U 344 Mutant plasmid. All procedures are identical, using plasmid pCB301-sat-Yi as a template, mutant PCR is performed using primers M4-F / M4-R, and the primer sequences are as follows:

[0109] M4-F: 5'-CTAAGACTTAGGTTATGCTGATCTCCGTGAATGTCTATCATTCC-3' (SEQ ID NO: 16)

[0110] M4-R: 5'-AGCATAACCTAAGTCTTAGCCTCTCCCTGCGTGC-3' (SEQ ID NO: 17).

[0111] It was found that the M4 site of the mutant plasmid prepared according to the above had been mutated. As described above, specifically, the 337th base G and the 344th base C of sat-Yi were replaced by A and U, respectively. The replaced bases are the red bases in the figure. Figure 2

[0112] The sequence of M4 is shown as SEQ ID NO: 5.

[0113] (5) M5: pCB301-Yi 337 C-G 344 Construction of mutant plasmid

[0114] Referring to the construction method of Ml mutant plasmid, pCB301-Yi was constructed based on plasmid pCB301-sat-Yi 337 C-G 344 Mutant plasmid. All procedures are identical, using plasmid pCB301-sat-Yi as a template, mutant PCR is performed using primers M5-F / M5-R, and the primer sequences are as follows:

[0115] M5-F: 5'-AAGCCTTAGGGTATGCTGATCTCCGTGAATGTCTATCATTC-3' (SEQ ID NO: 18),

[0116] ​M5-R: 5'-GCATACCCTAAGGCTTAGCCTCTCCCTGCGT-3' (SEQ ID NO: 19).

[0117] The results showed that the M5 site of the mutant plasmid prepared as described above had undergone a mutation. For example... Figure 2 Specifically, the 337th base G and the 344th base C of sat-Yi are replaced with C and G, respectively. The replaced bases are those marked in red in the figure.

[0118] The sequence of M5 is shown in SEQ ID NO:6.

[0119] (6) M6: pCB301-Yi 337 AU 344 & 331 CG 349 Construction of mutant plasmids

[0120] Following the construction method of the M1 mutant plasmid, pCB301-Yi was constructed based on plasmid M4. 337 AU 344 & 331 CG 349 Mutant plasmid. All procedures are similar, using plasmid M4 as a template and primers M6-F / M6-R for mutant PCR. The primer sequences are as follows:

[0121] M6-F: 5'-AGGGAGAGCCTAAGACTTAGGTTATGGTGATCTCCGTGAATGTCTATCATTC-3' (SEQID NO:20),

[0122] M6-R: 5'-TCTTAGGCTCTCCTGCGTGCGTCATGACTCA-3' (SEQ ID NO: 21).

[0123] The results showed that the M6 ​​site of the mutant plasmid prepared as described above had undergone a mutation. Figure 2 Specifically, the base G at position 331 and the base C at position 349 of sat-Yi are replaced with C and G, respectively. The replaced bases are those marked in red in the figure.

[0124] The sequence of M6 is shown in SEQ ID NO:7.

[0125] (7) M7: pCB301-Yi 337 AU 344 & 331 CG 349 & 328 CG 354 Construction of mutant plasmids

[0126] According to the construction method of the M1 mutant plasmid, the pCB301-Yi was constructed based on the plasmid M6. 337 A-U 344 & 331 C-G 349 & 328 C-G 354 The mutant plasmid. All procedures are the same, using plasmid M6 as a template, using primers M7-F / M7-R for mutation PCR, and the primer sequences are as follows:

[0127] M7-F: 5'-AGGGACAGCCTAAGACTTAGGTTATGGTGATGTCCGTGAATGTCTATCATTCCTC-3' (SEQ ID NO: 22),

[0128] M7-R: 5'-CTTAGGCTGTCCCTGCGTGCGTCATGACTCAT-3' (SEQ ID NO: 23).

[0129] It was found that the site of the mutant plasmid M7 prepared according to the above had been mutated. As described above, specifically, the 328th base G and the 354th base C of sat-Yi were replaced by C and G, respectively. The replaced bases are the red bases in the figure. Figure 2

[0130] The sequence of M7 is shown in SEQ ID NO: 8.

[0131] Example 2, Agrobacterium transformation, activation and expansion culture of sat-Yi related mutant vectors

[0132] Take 30 μL of Agrobacterium GV3101 competent cells, and add 2 μL of M1, M2, M3, M4, M5, M6, M7 plasmids obtained (sequencing success) in Example 1 respectively, mix gently, and ice for 30 min. Freeze in liquid nitrogen for 1 min, 42 ℃ water bath for 1 min, and then ice for 2 min. Add 500 μL of LB liquid medium without antibiotics; shake culture at 28 ℃, 220 r / min for 4 h.

[0133] ​After centrifugation (6000 rpm / 3 min) of the bacterial liquid, the supernatant is essentially LB liquid medium, and the precipitate is Agrobacterium (Agrobacterium is collected); in the clean bench, 400 μL of the supernatant is taken and discarded (so as to realize concentration); the remaining medium is blown to suspend Agrobacterium; the Agrobacterium suspension is added to LB solid medium containing three antibiotics (kanamycin, rifampicin and gentamicin, each at a concentration of 50 mg / mL) and uniformly coated with a coating rod, and cultured in a 28 ℃ incubator for 2 d. Thus, single colonies of M1, M2, M3, M4, M5, M6 and M7 are obtained, respectively.

[0134] In addition, the helper virus for testing the activity of sat-Yi or its mutants is CMV Fny strain (hereinafter referred to as CMV), and the infectious clone thereof is composed of pCB301-C1, pCB301-C2 and pCB301-C3. Agrobacterium GV3101 containing the infectious clone of pCB301-C1, pCB301-C2 or pCB301-C3 is named C1, C2 or C3, respectively. pCB301-C1, pCB301-C2 and pCB301-C3 are constructed by the applicant's laboratory, and the plasmid construction and Agrobacterium transformation methods have been described in the related literature (Viruses 2018; 10(11): 590).

[0135] The M1, M2, M3, M4, M5, M6 and M7 single colonies obtained above and 500 μL of Agrobacterium C1, C2, C3, pCB301-sat-Yi and empty vector pCB301 glycerol bacteria stored in a -80 ℃ refrigerator are respectively inoculated in 3 mL of LB liquid medium containing three antibiotics (kanamycin, rifampicin and gentamicin, each at a concentration of 50 μg / mL), and cultured at 28 ℃ on a shaker for 12-16 h.

[0136] 300 μL of each of the seven sat-Yi mutant vectors and C1, C2 and C3 Agrobacterium bacterial liquid is taken in 6 mL of LB liquid medium containing 50 μg / mL kanamycin and 50 μg / mL rifampicin, and 1.5 μL of 0.2 mol / L acetosyringone (with dimethyl sulfoxide as the solvent) and 300 μL of 0.5 mol / L morpholine ethanesulfonic acid (MES) buffer are added, and the mixture is cultured at 28 o C is cultured for 16 h, and the bacterial cells are collected by centrifugation at 6000 rpm / min for 3 min. The absorbance of the bacterial cells is measured by a spectrophotometer, and the concentration of Agrobacterium is adjusted using infiltration buffer so that the OD 600 value of the bacterial liquid is 0.5.

[0137] The specific formulation of the infiltration buffer is: 10 mL of 0.5 mol / L MES, 2.5 mL of 2 mol / L MgCl2, 0.2 mol / L acetyl-piperitone 500 μL, and double-distilled water to 500 mL.

[0138] The specific formulation of the 0.5 mol / L morpholine ethanesulfonic acid (MES) buffer is: dissolve 5.33 g of morpholine ethanesulfonic acid in 40 mL of double-distilled water, adjust the pH to 5.6 with 2 mol / L NaOH, and dilute to 50 mL.

[0139] Accordingly, the same concentration of Agrobacterium pCB301, sat-Yi, M1, M2, M3, M4, M5, M6, M7, and Agrobacterium C1, C2, C3 activated bacterial liquid is obtained.

[0140] Example 3, sat-Yi and its mutants M1, M2 and M3 co-infect tomato with CMV

[0141] Group Mock injects the infiltration buffer into tomato (903), group Vector injects CMV and pCB301 empty vector into tomato as a negative control, group sat-Yi injects sat-Yi and CMV into tomato as a positive control, and groups M1, M2 and M3 represent M1, M2 and M3 inoculating tomato with CMV as experimental groups.

[0142] Among them, Fny-CMV Agrobacterium, that is, a mixture of Agrobacterium C1, C2 and C3 with vectors pCB301-C1, pCB301-C2 and pCB301-C3 in a mixing ratio of 1:1:1 (volume ratio), so that it can form CMV, and the obtained is named Fny-CMV Agrobacterium mixed activated bacterial liquid.

[0143] Mix the Fny-CMV Agrobacterium mixed activated bacterial liquid with the activated bacterial liquid of pCB301, sat-Yi, M1, M2 and M3 at a ratio of 1:1 (V / V), and incubate in the dark for 4 h; obtain the incubated bacterial liquid.

[0144] Select tomato that has grown two true leaves, and use a needle to pierce the two cotyledons and one true leaf. Use a 1 mL needle-free syringe to inoculate the incubated bacterial liquid into the pierced leaf, and the leaf position of the inoculated leaves of the experimental and control groups needs to be consistent. All inoculated plants are placed in a 22-25°C plant growth chamber with a light duration of 16 h.

[0145] The phenotype of tomato plants infected with the virus was continuously observed after Agrobacterium infection. Phenotype recording was performed at different time points after inoculation, and the overall plant and local leaf symptoms were photographed at appropriate times. After taking the photos, the systemic leaf samples of tomato were collected, and the sampling leaf positions of each treatment group were ensured to be consistent to reduce experimental errors. As Figure 3 As described above, 15 days after inoculation, the tomato plants in the negative control group showed obvious symptoms of viral infection: plant dwarfing, deformed top leaves, and typical linearization. The tomato plants in the positive control group grew normally, with plant height and leaf density consistent with Mock, and no deformed leaves appeared at the top. In the experimental group, the tomato plants inoculated with M1 grew slowly, with the top leaves significantly drooping and even showing a significant systemic necrosis phenotype. However, the tomato plants inoculated with M2 and M3 had expanded leaves and thick stems, with no significant difference in plant height from Mock, indicating that these two mutations not only reversed the necrosis phenotype but also significantly reduced the impact of viral infection on the plant, effectively maintaining the normal growth phenotype of the tomato.

[0146] Total RNA was extracted from the samples to analyze the accumulation differences of the virus and satellite RNA in the plant. 0.1 g of leaf samples (14-day-old inoculated leaves) were weighed, cut into small pieces, and placed in a 2 mL centrifuge tube containing 2 small steel balls, and then the tube was placed in liquid nitrogen.

[0147] The samples in the 2 mL centrifuge tube were thoroughly ground using a grinder, and the specific RNA extraction method is described in Example 4 of the invention “Construction method and application of satellite RNA system expressing siRNA” of 202311809853.5.

[0148] The concentration and purity of RNA were determined at 260 nm using a microspectrophotometer.

[0149] According to the measured sample concentration, the RNA concentration of all groups was adjusted to 1000 ng / μL to ensure consistent loading amounts between different samples.

[0150] 10 μL of RNA loading and 1 μL of RNA sample were added to the PCR tube, mixed well, denatured at 65 ℃ for 10 min, and placed on ice for 3 min. Then, formaldehyde agarose gel electrophoresis was performed at 65 V for 50 min, and the RNA bands were observed under ultraviolet light. If the bands are clear and complete without degradation, continue electrophoresis at 65 V for 70 min.

[0151] The steps of CMV genome and satRNA transfer and Northern hybridization refer to the product instruction of digoxigenin labeling detection kit II (Roche, Switzerland).

[0152] The Northern Hybridization probe for detecting satRNA accumulation is complementary to the 5' nucleotide of sat-Yi and was synthesized by Shanghai Bioengineering Co., Ltd. The probe sequence for sat-Yi is as follows:

[0153] sat-Yi(+): 5'-gtgaaggatcatcacgcagatatatcccctctgcgcaact-DIG-3' (SEQ ID NO: 24).

[0154] Northern blotting probes for detecting the CMV genome are complementary to the shared nucleotide sequences of CMV RNA1, RNA2, and RNA3. Probes for detecting CMV RNA3 accumulation are complementary to the 3' nucleotides of CMV RNA3. These probes were synthesized by Shanghai Bioengineering Co., Ltd. The CMV genome and CMV RNA3 probe sequences are as follows:

[0155] CMV genome: 5'-GACTGACCATTTTAGCCGTAAGCTGGATGGACAACCCGTTC-DIG-3' (SEQ IDNO: 25);

[0156] CMV RNA3: 5'-CGGAGGGAGGATTCTGGGAACACGGAATCAGACTGG-DIG-3' (SEQ ID NO: 26).

[0157] Northern hybridization results are as follows Figure 4 As shown. Figure 4 The Northern blotting results showed that 14 days after inoculation, all satRNAs replicated normally with no significant difference in accumulation, indicating that mutations in different bases had no effect on satRNA replication. Simultaneously, the accumulation of CMV genome in the leaves of tomato systems inoculated with satRNA was reduced, mainly manifested as decreased accumulation of RNA1, RNA2, and RNA4A, suggesting that the modified satRNA could weaken viral replication within the plant.

[0158] Based on the above experimental results, 331 GC 349 and 328 GC 354 These two base pairs are directly involved in inducing the necrosis phenotype in tomatoes, so they were chosen as the target sites for modification in subsequent experiments.

[0159] right Figure 4 The following is a detailed explanation: Figure 4 In A: RNA1,2,RNA3,RNA4 and RNA4A represent the CMV genome of helper viruses composed of C1, C2 and C3.

[0160] wherein:

[0161] RNA1 and RNA2 encode proteins related to viral replication; the subgenomic RNA4A of RNA2 encodes the 2b protein;

[0162] RNA3 encodes the movement protein and coat protein, and the subgenomic RNA4 of RNA3 mainly expresses the coat protein;

[0163] Figure 4 The brightness of each group of bands in C is uniform, indicating that the loading amount of each group of RNA is consistent during the experiment, eliminating the expression deviation caused by the difference in loading amount, and ensuring the reliability of subsequent analysis.

[0164] Example 4, sat-Yi and its mutants M4, M5, M6 and M7 co-infect tomatoes with CMV

[0165] Mock group injects infiltration buffer into tomatoes (903), Vector group injects CMV and pCB301 empty vector into tomatoes as negative control, sat-Yi group injects sat-Yi and CMV into tomatoes as positive control, and M4, M5, M6 and M7 groups represent M4, M5, M6 and M7 respectively, which are used to infect tomatoes with CMV as experimental groups.

[0166] Among them, Fny-CMV Agrobacterium, that is, a mixture of Agrobacterium C1, C2 and C3 with vectors pCB301-C1, pCB301-C2 and pCB301-C3, the mixing ratio is 1:1:1 (volume ratio), so that it can form CMV; the obtained is named Fny-CMV Agrobacterium mixed activated bacteria liquid.

[0167] Mix the Fny-CMV Agrobacterium mixed activated bacteria liquid with the activated bacteria liquid of pCB301, sat-Yi, M4, M5, M6 and M7 at 1:1 (V / V) and incubate in the dark for 4 h.

[0168] The Agrobacterium infection process refers to Example 3.

[0169] After Agrobacterium infection, the plant phenotype of tomatoes infected with virus is continuously observed. The phenotype is recorded at different time points after inoculation, and the overall plant and local leaf symptoms are photographed at appropriate times. After taking pictures, the systemic leaf samples of tomatoes are collected to ensure that the sampling leaf positions of each treatment group are consistent to reduce experimental errors. For example Figure 5As stated, 18 days after inoculation, the tomato plants in the negative control group showed obvious symptoms of viral infection: stunted growth, deformed top leaves, and typical linearization characteristics. In contrast, the tomatoes in the positive control group had the same plant height and leaf density as the Mock group, and did not exhibit deformed top leaves. However, from the detailed images ( Figure 5 In B), mild symptoms such as yellowing leaf margins and blackening of the reticulate veins were observed in the lower systemic leaves, and these symptoms gradually spread upwards over time. In the experimental group, tomatoes inoculated with M5 exhibited a severe systemic necrosis phenotype. The plants completely withered and died by day 18 of infection. In contrast, tomato plants inoculated with M4, M6, and M7 had lush leaves and robust stems, and their overall phenotype was not significantly different from Mock, indicating that these three mutants could significantly reduce the symptoms of virus infection in plants. Further observation of leaf details revealed that the leaves of tomatoes inoculated with the three mutants did not show symptoms similar to those in the positive control group. Among them, the leaves of tomatoes inoculated with M4 were bright green and smooth, with no significant difference from the leaves inoculated with Mock. The leaves of tomatoes inoculated with M6 and M7 showed a slight wrinkled phenotype, and the phenotypic differences among the three groups were small.

[0170] The process of extracting total RNA from the sample and detecting the accumulation of Fny-CMV and satRNA by Northern hybridization is the same as in Example 3.

[0171] Northern hybridization results as follows Figure 6 As shown. Figure 6 The Northern blotting results showed that all satRNAs replicated normally 14 days after inoculation. Simultaneously, the accumulation of CMV RNA3 in the leaves of the tomato system inoculated with satRNA was slightly reduced, indicating that the modified satRNA can weaken viral replication within the plant.

[0172] Based on the above experimental results, among all mutant satRNAs, M7 was selected as the candidate line for satellite RNA biocontrol agents with low pathogenicity, considering both its resistance to CMV infection and replication stability.

[0173] Note: M4, M6, and M7 have similar resistance to CMV infection. Compared to the lethal mutant M1, the number of mutated bases gradually increases from M4 to M7, and the probability of the mutant satellite reverting to the lethal satellite continuously decreases. Therefore, M7 has higher safety while possessing the ability to inhibit CMV infection, making it a more suitable preferred tool for CMV biocontrol in tomatoes.

[0174] The above examples are only some specific examples of the present application, the protection scope of the present application is not limited to the above examples, and includes some variations. All variations that can be directly derived or inferred by those skilled in the art from the disclosure of the present application should be considered within the protection scope of the present application.

Claims

1. A CMV satellite RNA biocontrol mutant, characterized by: The nucleotide sequence of the CMV satellite RNA biocontrol mutant is shown in SEQ ID NO:

8.

2. A method for constructing CMV satellite RNA biocontrol mutants, characterized in that: The key bases in the satellite RNA that induce tomato necrosis were altered, thereby reducing the risk of the satellite RNA transforming into necrotic satellite RNA; the satellite RNA sequence was mutated as shown in SEQ ID NO:

8.

3. The application of the CMV satellite RNA biocontrol mutant as described in claim 1, characterized in that: This reduces the risk of CMV infection in plants, specifically tomatoes.

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