A method for efficient and stable inoculation of tomato with cucumber mosaic virus

Abstract

The application provides a method for efficiently and stably inoculating tomatoes with cucumber mosaic virus, and relates to the technical field of crop disease-resistant breeding.The method for efficiently and stably inoculating tomatoes with cucumber mosaic virus comprises the following specific steps: S1, propagation: propagating CMV virus by using dcl 2 / 4i n.benthamiana; S2, extraction: extracting CMV virions; S3, inoculation: rubbing the tomatoes seedlings with the CMV virions in S2; and S4, cultivation: cultivating the tomatoes seedlings. The method significantly improves the efficiency and stability of CMV infection of tomatoes by using virions to infect the tomatoes seedlings. Furthermore, the dcl 2 / 4i n.benthamiana is used to propagate CMV virions, thereby shortening the propagation cycle of CMV virions, significantly improving the yield of CMV virions, and enabling repeated large-scale inoculation, which is conducive to efficient screening of CMV-resistant tomato varieties.

Description

TECHNICAL FIELD

[0001] The application relates to the technical field of crop disease-resistant breeding, in particular to a method for efficiently and stably inoculating tomatoes with cucumber mosaic virus. BACKGROUND

[0002] CMV is a typical member of the Cucumovirus genus of the Bromoviridae family, and the virus particle is an isometric icosahedral sphere with an axial symmetry, with a diameter of about 29 nm. The CMV genome comprises three positive single-stranded RNAs, namely RNA1, RNA2 and RNA3. The size of RNA1 is 3390-3410 nt, the size of RNA2 is 3035-3350 nt, and the size of RNA3 is 2190-2200 nt. The CMV genome encodes five open reading frames, wherein the ORF located in RNA1 encodes a 1a protein with a molecular weight of 111-112 kDa, which has methyltransferase and helicase activities; RNA2 has two ORFs, which encode a 2a protein with a molecular weight of 11-13 kDa and a 2b protein with a molecular weight of 13 kDa, wherein the 2a protein is a CMV replicase and the 2b protein is a CMV virus silencing suppressor; RNA3 also has two ORFs, which encode a 3a protein with a molecular weight of 31 kDa and a 24-26 kDa coat protein, wherein the 3a protein is a CMV movement protein. CMV can infect more than 1,300 species of plants belonging to more than 500 genera and more than 100 families, including important economic crops such as vegetables, oil plants, fruit trees and flowers. According to the survey from 2013 to 2017, CMV has become a dominant virus that damages vegetable crops in China, and is distributed in 31 provinces (municipalities and autonomous regions). CMV often co-infects Solanaceae, Cucurbitaceae, Leguminosae and Cruciferae vegetables with other viruses, and causes serious damage to vegetables in China.

[0003] However, the immaturity of the CMV inoculation system brings difficulties to the screening of CMV-resistant varieties of various crops and CMV-related research work. The commonly used method for inoculation is the method of rubbing the virus juice on the host. This method has a relatively good inoculation efficiency on tobacco, but has a low inoculation efficiency on crops such as tomatoes, cucumbers and peppers. In addition, there are great differences in the incidence rate and disease severity between different batches, which brings great obstacles to the evaluation of disease resistance. The use of aphids for virus transmission requires facilities and skills for culturing aphids, cannot control the consistency of the initial inoculation amount, is difficult to work, has poor repeatability between different batches, and has a great risk of losing control of aphids. Once out of control, it will cause devastating damage to the test materials and even non-test materials.

[0004] Therefore, a new method for efficiently and stably inoculating tomatoes with cucumber mosaic virus has been developed. SUMMARY

[0005] (I) Technical problems solved

[0006] In view of the deficiencies of the prior art, the application provides a method for efficiently and stably inoculating tomatoes with cucumber mosaic virus, which solves the problem of low infection efficiency of virus juice rubbing inoculation on tomatoes, cucumbers, peppers and other crops, and great differences in disease incidence and disease severity between different batches, which greatly hinders the evaluation of disease resistance.

[0007] (II) Technical scheme

[0008] To achieve the above object, the application is implemented by the following technical scheme: a method for efficiently and stably inoculating tomatoes with cucumber mosaic virus, comprising the following specific steps:

[0009] S1, propagation: propagating CMV virus by using dcl2 / 4i nicotiana benthamiana;

[0010] S2, extraction: extracting CMV virions;

[0011] S3, inoculation: inoculating tomato seedlings by rubbing with CMV virions in S2;

[0012] S4, cultivation: cultivating the tomato seedlings.

[0013] Preferably, the S1 propagates CMV virus by using dcl2 / 4i nicotiana benthamiana, and further comprises the following specific steps:

[0014] S101: first, uniformly spray diatomite on dcl2 / 4i nicotiana benthamiana plants of 4-5 leaf stage;

[0015] S102: add the leaves infected and caused by CMV to Buffer C, grind into homogenate by using a mortar, then pour the ground homogenate onto the leaves of dcl2 / 4i nicotiana benthamiana, and gently rub to form micro-wounds;

[0016] S103: after rubbing for 3-5 minutes, spray water on the rubbed leaves, then place them in a 24℃ greenhouse for dark culture for 24 hours, then uncover them under the conditions of 24℃ and 10h light / 14h darkness, and then normally culture for 14-21 days.

[0017] Preferably, the extraction of CMV virions in S2 further comprises the following specific steps:

[0018] S201: put 50g of leaves infected and caused by CMV, 75mL of Buffer A, 375μL of TGA and 75mL of chloroform into a cell wall breaking machine, and crush the leaves into homogenate;

[0019] S202: Stir the homogenate in S201 using magnetic stirring at 4°C for 1-2 h, then evenly distribute the stirred homogenate into 4 50 mL centrifuge tubes, and centrifuge the homogenate in the centrifuge tubes at 8000 rcf for 15 min;

[0020] S203: Take the supernatant in the centrifuge tubes in S202 and transfer it to a tube for ultracentrifugation, with a content of 27 mL per tube, and add 5 mL of a mixture of Buffer A and 10% sucrose buffer, then centrifuge the mixture at 37000 rpm for 1.5 h.

[0021] S204: Pour out the supernatant after centrifugation for 1.5 h in S203, and add 1 mL of Buffer C solution to each tube, then combine them into a 50 mL tube and store at 4°C overnight;

[0022] S205: Centrifuge the 50 mL tube stored overnight in S204 at 8000 rcf for 10 min, then take the supernatant, and measure A260 using a spectrophotometer and calculate the concentration;

[0023] S206: Add glycerol with a concentration of 10% at a volume ratio of 1:1 to the supernatant taken in S205, and calculate the final concentration, then distribute the extracted virus particles into 1.5 mL centrifuge tubes and store them at -80°C.

[0024] Preferably, the rubbing inoculation of tomato seedlings with CMV virus particles in S3 further comprises the following specific steps:

[0025] S301: First, dilute the virus particles to 30 ng / μL using Buffer C solution;

[0026] S302: Then, evenly spray 100-mesh diatomite on the tomato seedlings with only 2 cotyledons using a duster;

[0027] S303: Then, use a pipette to spot 15 μL of virus particles (30 ng / μL) on each leaf, with 30 μL per tomato plant;

[0028] S304: Then, gently rub the leaves in S303 3-5 times until there are micro-wounds, and after rubbing for 3-5 minutes, spray water on the rubbed leaves;

[0029] S305: Cover the infected tomato seedlings with a lid, and incubate them in the dark at room temperature of 24°C for 24 hours, then uncover them.

[0030] Preferably, the temperature culture condition of the tomato seedling in S4 is 24 DEG C, the light culture condition of the tomato seedling in S4 is 8000 lux of light intensity, and the light culture condition of the tomato seedling in S4 is 10h light / 14h darkness.

[0031] Preferably, the rotor model of the matching tube of the ultracentrifuge in 203 is P70AT, and the specification of the matching tube is 40mL*2.

[0032] (III) Beneficial Effects

[0033] The application provides a method for efficiently and stably inoculating tomatoes with cucumber mosaic virus.

[0034] 1. The method for efficiently and stably inoculating tomatoes with cucumber mosaic virus, which uses virus particles to infect tomato seedlings, significantly improves the efficiency and stability of CMV infection of tomatoes, has obvious symptoms, is repeatable, can be inoculated on a large scale, and is beneficial to efficient screening of CMV-resistant tomato varieties.

[0035] 2. The method for efficiently and stably inoculating tomatoes with cucumber mosaic virus, which uses two cotyledon-sized tomato seedlings as inoculation materials, and typical symptoms can be observed after two weeks of inoculation, greatly shortening the experimental period and significantly improving the screening period of CMV-resistant germplasm and the experimental period of related research work.

[0036] 3. The method for efficiently and stably inoculating tomatoes with cucumber mosaic virus, which uses dcl2 / 4i Nicotiana benthamiana to proliferate CMV virus particles, shortens the proliferation period of CMV virus particles, and significantly improves the yield of CMV virus particles.

[0037] 4. The method for efficiently and stably inoculating tomatoes with cucumber mosaic virus, which uses tomato seedlings with two cotyledons just unfolded after germination for 7 days as inoculation materials, while other CMV inoculation methods require plants to be sown for 3-4 weeks, the method shortens the inoculation period, accelerates breeding speed, and more quickly breeds tomato varieties with strong CMV resistance, and secondly, reduces the evaluation period, which means faster understanding of the reaction of plants to CMV, and is beneficial to timely adjustment of breeding strategies and improvement of breeding efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0038] Figure 1 The process flowchart of the application is shown in the figure;

[0039] Figure 2 The figure is a CMV virus particle proliferation and detection diagram of the application;

[0040] Figure 3 The figure is a symptom manifestation diagram of the application after CMV inoculation of tomatoes;

[0041] Figure 4 Figure of detection result after the tomato of the application is inoculated with CMV. DETAILED DESCRIPTION

[0042] The technical solutions in the embodiments of the application will be clearly and completely described below with reference to the drawings in the embodiments of the application. Obviously, the described embodiments are only part of the embodiments of the application, rather than all the embodiments of the application. Based on the embodiments in the application, all other embodiments obtained by a person of ordinary skill in the art without creative work are within the protection scope of the application.

[0043] Embodiment one:

[0044] As shown in the drawings, Figure 1 The embodiment of the application provides a method for efficiently and stably inoculating tomatoes with cucumber mosaic virus, S1 of which uses dcl2 / 4i Nicotiana benthamiana to multiply CMV virus, including the following specific steps:

[0045] Step one: 4-5 leaf stage dcl2 / 4i Nicotiana benthamiana 110 is selected, and diatomite is uniformly sprayed on the two leaf blades under the central leaf;

[0046] Step two: the CMV-infected and pathogenic leaf blades in step one are added with Buffer C, and are ground into homogenate in a mortar, and then the homogenate is taken to the leaf blades;

[0047] Step three: then the inoculated plants are placed in a 24℃ greenhouse, and are covered with a cover for dark culture;

[0048] Step four: after 24 hours, the cover is removed, and the plants are normally cultured under the condition of 24℃, 10h light / 14h darkness.

[0049] CMV virus particle extraction in S2 includes the following specific steps:

[0050] Step one: 50g of CMV-infected and pathogenic dcl2 / 4i Nicotiana benthamiana leaf blades, 50mL of Buffer A, 250μL of TGA, and 50mL of chloroform are put into a cell wall breaking machine, and the leaf blades are broken into homogenate;

[0051] Step two: the homogenate in step one is transferred to a beaker, and the cell wall breaking machine cavity is washed with 25mL of Buffer A, 125μL of TGA, and 25mL of chloroform, and the liquid is again transferred to the beaker;

[0052] Step three: the beaker is placed on a magnetic stirrer, and the homogenate is stirred at 4℃ for 1-2h, and the homogenate is evenly divided into four 50mL centrifuge tubes;

[0053] Step four: put the centrifuge tube in step three into the centrifuge, centrifuge at 8000rcf for 15min;

[0054] Step five: then suck the supernatant and transfer it into the ultracentrifuge tube, 27mL per tube, and add 5mL of Buffer A and 10% sucrose buffer mixture, centrifuge at 37000rpm for 1.5h;

[0055] Step six: pour out the supernatant, add 1mL of Buffer C solution in each tube, and pour them into a 50mL tube, and store at 4℃ overnight;

[0056] Step seven: put the 50mL tube into the centrifuge again, centrifuge at 8000rcf for 10min;

[0057] Step eight: suck the supernatant in step seven, measure A260 by spectrophotometer, A260 is 18.800, calculate the concentration as C=A260 / 5, the concentration is 3.760μg / μL;

[0058] Step nine: add 10% glycerol according to the volume ratio of 1:1 in step eight, so that the final concentration is 1.880μg / μL, and store the extracted virus particles in 1.5mL centrifuge tubes at-80℃.

[0059] In S3, tomato seedlings are inoculated by rubbing with CMV virus particles, including the following specific steps:

[0060] Step one: select 36 strains of AC tomato and Micro-Tom tomato with only 2 cotyledon sizes, and evenly spray 100 mesh silica on the tomato seedlings with only 2 cotyledon sizes with a duster;

[0061] Step two: dilute the virus particles to 30ng / μL with Buffer C solution, and then use a pipette to spot 15μL of virus particles on each leaf, 30μ per tomato;

[0062] Step three: gently rub each leaf 3-5 times until a small wound is produced, and the negative control is inoculated with the same volume of Buffer C solution, 24 strains of Micro-Tom and AC tomato, and 12 strains of negative control;

[0063] Step four: 3-5 minutes after inoculation, spray water on the rubbed leaves;

[0064] Step five: cover the infected tomato seedlings with a lid and incubate them in the dark at 24℃;

[0065] Step six: uncovering after 24 hours, and normal culture for 14-21 days under the condition of 24°C, 10h light / 14h dark.

[0066] The specific experimental steps are as follows:

[0067] Total RNA extraction:

[0068] Put 3mL Extration Buffer and 3mL RNA extraction phenol into a 15mL centrifuge tube, preheat at 65°C. Put 1g tomato leaves into a mortar containing liquid nitrogen, grind into powder, and then quickly transfer to the preheated 15mL centrifuge tube, mix well. Add 2.25mL chloroform, mix well, then centrifuge at 4°C, 4000r / min for 25min. Absorb the supernatant, and transfer the supernatant to a centrifuge tube containing 4mL isopropanol, mix gently, and stand at -20°C overnight. After standing overnight, centrifuge the solution at 4°C, 4000rpm for 25min, and transfer the precipitate to a new sterile 1.5mL centrifuge tube. Add 1mL 75% alcohol, centrifuge at 4°C, 12000rpm for 5min, discard the supernatant, and repeat the step. Let the precipitate stand at room temperature for 15min, and after the alcohol in the tube evaporates, add 300μL TE Buffer to dissolve the precipitate. Add 1mL Trizol to the dissolved RNA solution, mix well, and stand at room temperature for 10min. Add 200μL chloroform, mix well, stand for 10min, then centrifuge at 4°C, 12000rpm for 15min. Absorb the supernatant, and transfer the supernatant to a centrifuge tube containing 700μL isopropanol, mix gently, stand at -20°C for 30min, then centrifuge at 4°C, 12000rpm for 15min. Discard the supernatant, add 1mL 75% alcohol, centrifuge at 4°C, 12000rpm for 5min, discard the supernatant, and repeat the step. Let the precipitate stand at room temperature for 15min, and after the alcohol in the tube evaporates, add 150μL TE Buffer or ddH2O to dissolve the precipitate. Measure the concentration of the dissolved RNA, which is used for subsequent RT-PCR or Northern blot analysis.

[0069] RT-PCR detection:

[0070] RT-PCR detection of extracted tomato total RNA after removing the genome, using II cDNA Synthesis Kit (Nanjing Novogene Bioinformatics Technology Co., Ltd.) kit to remove the genome and carry out RT-PCR. Genome removal system and reaction conditions: add 1 μg tomato total RNA in a PCR tube, add ddH2O to make up 5.5 μL, react at 65℃ for 5 min and immediately place on ice, then add 2 μL 4×gDNA wiper Mix, react at 42℃ for 2 min. Reverse transcription system and reaction conditions: add 1 μL 10×RT Mix, 1 μL HiScript II Enzyme Mix and 0.5 μL Fny-2b-R primer to the PCR tube after genome removal, react at 50℃ for 45 min, then inactivate at 85℃ for 2 min. PCR system and reaction conditions: add 5 μL 10×PCR Mix, 0.5 μL Fny-2b-F, 0.5 μL Fny-2b-R and 0.5 μL reverse transcription product in a PCR tube, add ddH2O to make up 10 μL. Reaction conditions are: 95℃ for 2 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, 28-30 cycles; 72℃ for 5 min, 12℃ for 5 min.

[0071] High molecular weight Northern blot detection:

[0072] Total tomato RNA (10 μg) was denatured at 65 °C for 5 min and separated by electrophoresis on a 1.5% (w / v) agarose gel containing formaldehyde (37%, 12.3 mol / L). RNA was transferred to a Hybond N+ membrane using a capillary method and cross-linked to both sides of the membrane for 2 min each at 1200 Lux UV light. The cross-linked membrane was stained with methylene blue and photographed to quantify the amount of tomato RNA loaded. The photographed membrane was placed in a hybridization tube, and an appropriate amount of Church Buffer (1% BSA, 1 mmol / L EDTA, 7% SDS, 0.5 mol / L Na2HPO4) was added for pre-hybridization at 65 °C for 1 h. After pre-hybridization, a CMV-specific probe was added, and hybridization was performed at 65 °C for 16 h. Probe preparation system and reaction conditions: 0.8 μL of 250 μM dNTP (A / C / G Mix), 1.35 μL of 100 μM dTTP, 0.65 μL of 100 μM Biotin-11-dUTP, 1 μg DNA template, 0.2 μL of TaKaRa LA Taq, 2 μL of 10×LA Taq Bufffer, and 0.8 μL each of primers Fny-2b-F and Fny-2b-R. Reaction conditions: 95℃ for 2 min; 95℃ for 30 s, 58℃ for 30 s, 72℃ for 30 s, 34 cycles; 72℃ for 5 min, 12℃ for 5 min. After hybridization, wash with 1×SSC (containing 0.1% SDS) at 65℃ for 10 min, then with 0.2×SSC (containing 0.1% SDS) at 65℃ for 10 min, and finally with 0.1×SSC (containing 0.1% SDS) at 65℃ for 5 min. Then, develop the color using a chemiluminescent nucleic acid detection module kit (Thermo Scientific™, catalog number 89880) according to the product instructions. The signal was acquired using a Tanon 5200 (Shanghai Tianneng).

[0073] Comparison of symptoms after inoculation of Micro-Tom tomatoes and AC tomatoes:

[0074] Symptoms were visible on Micro-Tom tomatoes 7 days after inoculation, and were most pronounced 17-20 days after inoculation, including typical mosaic patterns, narrow and elongated leaves, and severe stunting. Figure 3 As shown in A;

[0075] Similarly, AC tomatoes began to show symptoms 7 days after inoculation, with the most obvious symptoms appearing 15 days after inoculation. Typical symptoms included mosaic patterns, elongated or even linearized leaves, and severe stunting of the plant. Figure 3 As shown in B;

[0076] RT-PCR using specific primers amplified a specific viral band of 584 bp in all inoculated tomatoes, with an infection efficiency of 100%. Figure 4 As shown in A;

[0077] The RNA of the treated and control tomatoes is extracted by mixing every 3 plants into one sample, and the accumulation of CMV virus in the tomatoes is detected by Northern blot, and it is found that a large amount of CMV virus RNA accumulates in the Micro-Tom tomatoes after 17 days of inoculation and in the AC tomatoes after 15 days of inoculation, as shown in B and C of FIG. 1. Figure 4

[0078] In conclusion, the virus particle is used to infect the tomato seedlings, the efficiency and stability of the CMV infection of the tomatoes are significantly improved, the disease symptoms are obvious, the repeatability is good, the large-scale inoculation is available, and the efficient screening of the CMV-resistant tomato varieties is facilitated.

[0079] Although the embodiments of the present application have been shown and described, it will be understood by those of ordinary skill in the art that various changes, modifications, substitutions and alterations can be made thereto without departing from the principles and spirit of the present application, the scope of which is defined by the appended claims and their equivalents.​

Claims

1. A method for efficient and stable inoculation of tomato plants with Cucumber mosaic virus, characterized in that, The method comprises the following specific steps: S1, propagation: propagating CMV virus by using dcl2 / 4i N.benthamiana; S2, extraction: extracting CMV virions; S3, inoculation: inoculating tomato seedlings by rubbing CMV virions in S2; S4, cultivation: cultivating tomato seedlings; The step S1 of propagating CMV virus by using dcl2 / 4i N.benthamiana further comprises the following specific steps: S101: first, uniformly spray diatomite on dcl2 / 4i N.benthamiana plants at the size of 4-5 leaf stages; S102: add the leaves infected and caused by CMV to Buffer C, grind into homogenate by using a mortar, and then pour the ground homogenate into the leaves of dcl2 / 4i N.benthamiana plants and gently rub to cause micro-wounds; S103: after rubbing for 3-5 minutes, spray water on the rubbed leaves, and then place them in a 24℃ greenhouse for dark culture for 24 hours, and then uncover them under the condition of 24℃ and 10 h light / 14 h darkness, and then normally culture for 14-21 days; The step S2 of extracting CMV virions further comprises the following specific steps: S201: put 50 g of leaves infected and caused by CMV, 75 mL of Buffer A, 375 μL of TGA and 75 mL of chloroform into a cell wall breaking machine, and crush the leaves into homogenate; S202: use magnetic stirring at 4℃ to stir the homogenate in S201 for 1-2 h, then evenly divide the stirred homogenate into four 50 mL centrifuge tubes, and then centrifuge the homogenate in the centrifuge tubes at 8000 rcf for 15 min; S203: take the supernatant in the centrifuge tubes in S202, transfer it to a centrifuge tube matched with an ultracentrifuge, the content of each tube is 27 mL, and then add 5 mL of a mixture of Buffer A and 10% sucrose buffer, and then centrifuge the mixture at 37000 rpm for 1.5 h; S204: pour out the supernatant after centrifugation for 1.5 h in S203, add 1 mL of Buffer C solution to each tube, and then collect them into a 50 mL tube, and place it at 4℃ overnight; S205: centrifuge the 50 mL tube placed overnight in S204 at 8000 rcf for 10 min, then take the supernatant, and then measure A260 by using a spectrophotometer and calculate the concentration; S206: add 10% glycerol at a volume ratio of 1:1 to the taken supernatant in S205, calculate the final concentration, and then divide the extracted virions into 1.5 mL centrifuge tubes and store them at -80℃.

2. A method for efficient and stable inoculation of tomato plants with Cucumber Mosaic Virus according to claim 1, characterized in that: The step S3 of inoculating tomato seedlings by rubbing CMV virions further comprises the following specific steps: S301: first, dilute the virions to 30 ng / μL by using Buffer C solution; S302: uniformly spray diatomite with a mesh size of 100 on tomato seedlings with only 2 cotyledons by using a powder sprayer; S303: then use a pipette to point 15 μL of virions on each leaf, and inoculate 30 μL per tomato plant; S304: Then, rub the leaves in S303 gently for 3-5 times until micro-wounds are generated, and spray water on the rubbed leaves after 3-5 minutes of rubbing; S305: Cover the tomato seedlings after the infection is completed with a cover, and uncover the cover after dark culture at a room temperature of 24℃ for 24 hours.

3. A method for efficient and stable inoculation of tomato plants with Cucumber Mosaic Virus according to claim 1, characterized in that: The temperature culture condition of the tomato seedlings in S4 is 24℃, the light culture condition of the tomato seedlings in S4 is a light intensity of 8000 lux, and the light culture condition of the tomato seedlings in S4 is 10h light / 14h darkness.

4. A method for efficient and stable inoculation of tomato plants with Cucumber Mosaic Virus according to claim 1, characterized in that: The rotor model of the matching pipe of the ultracentrifuge in S203 is P70AT, and the specification of the matching pipe is 40 mL*2.

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