CN114561424B - Method for injection inoculation of red sage root cucumber mosaic virus at seedling stage - Google Patents

Method for injection inoculation of red sage root cucumber mosaic virus at seedling stage Download PDF

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CN114561424B
CN114561424B CN202210300333.0A CN202210300333A CN114561424B CN 114561424 B CN114561424 B CN 114561424B CN 202210300333 A CN202210300333 A CN 202210300333A CN 114561424 B CN114561424 B CN 114561424B
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CN114561424A (en
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范慧艳
开国银
周杨
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Zhejiang Chinese Medicine University ZCMU
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Abstract

The invention discloses a method for inoculating a red-rooted salvia root cucumber mosaic virus in a seedling stage, and belongs to the technical field of agricultural biology. The method extracts total RNA from tobacco leaves infected with cucumber mosaic virus, and adopts a needle tube injection mode to perform injection inoculation in a period from the cultivation of a red sage root plant to 6-8 leaves, so as to successfully establish an infection system of the cucumber mosaic virus on the red sage root plant. Provides an ideal experimental model for researching the pathogenic mechanism between cucumber mosaic virus and red sage root host, and has important theoretical value for revealing the interaction between the virus and host plant and virus evolution. Compared with the friction inoculation method, the method has the advantages that the inoculation efficiency is greatly improved, and the symptoms of the red sage root seedlings can be observed after two weeks of inoculation. The invention can complete high-efficiency inoculation with a small amount of disease-sensing materials, the virus inoculation amount is controllable, the total RNA amount of the injected leaves is small, and the damage to the leaves is less and is more similar to the field disease condition.

Description

Method for injection inoculation of red sage root cucumber mosaic virus at seedling stage
Technical Field
The invention relates to the field of plant virus inoculation, in particular to a method for injecting and inoculating a red-rooted salvia root cucumber mosaic virus in a seedling stage.
Background
The red sage root is a common bulk Chinese medicinal material in China and is widely applied to the treatment of cardiovascular and cerebrovascular diseases. In recent years, the demand of red sage root is increasing, and the planting area is expanding. However, the problem of virus infection is serious because the radix salviae miltiorrhizae is mainly propagated asexually in production, and the development of the radix salviae miltiorrhizae industry is limited. Research shows that the main pathogen of the red sage virus disease is cucumber mosaic virus (Cucumber mosaic virus, CMV), which can cause red sage degeneration, the incidence rate in fields is high, and the incidence rate of some fields is up to 60% -80%, which is represented by typical symptoms such as flower leaves, mottle, leaf rolling, yellowing, dwarfing and the like, the root system of infected plants is tiny, the yield is greatly reduced, the content of medicinal components such as tanshinone II is sharply reduced, and the quality and quality of red sage root are seriously influenced.
The artificial inoculation method of plant virus is established, and the plant virus is inoculated to relevant host plants, which is the premise and the basis for researching the pathogenicity and the harm of the virus and screening the resistant varieties of the host plants. The mechanical friction inoculation method is a plant virus inoculation method commonly used at present, but the mechanical friction inoculation needs more infectious materials, and has certain requirements on the host range of the infectious materials, and some plants can be successfully infected only by the sap friction of the same genus or same species of infectious plants. In addition, mechanical tribological inoculation requires that the leaves be sprayed with silicon carbide in advance, resulting in micro-wounds that provide conditions for viral infection. However, the amount of the carborundum is not well controlled, and the carborundum is time-consuming and labor-consuming and has a certain technical difficulty. At present, no report on successful infection of the root of red-rooted salvia by plant viruses through mechanical friction inoculation exists. Regarding the research of the red sage virus disease, mainly adopts a field natural disease mode, the method obtains disease sensing materials by creating and utilizing natural disease conditions, however, factors influencing the disease in the field, such as temperature, humidity, pathogenic microorganism number and the like, need to establish a disease nursery, have long identification period, high labor cost, unstable results and poor experimental batch repeatability. In addition, the red sage virus disease is caused by various plant viruses, the influence on the inoculation result is complex, and if the natural disease in the field cannot clearly play the role of the main pathogen, the consistency, objectivity and reliability of the result are influenced. The establishment of a single pathogen inoculation method is the key for researching the pathogenic mechanism of the red-rooted salvia virus disease.
Disclosure of Invention
The invention aims at providing a more efficient and highly operable seedling stage inoculation method of the red sage root, aiming at the current situation of lack of an artificial inoculation system of the cucumber mosaic virus on the red sage root, which is a main pathogen of the red sage root virus, and aiming at the defects of the traditional mechanical friction method. The method not only realizes rapid identification of virus disease resistance of the red sage root material in seedling stage, but also provides an ideal model for researching pathogenic mechanism between cucumber mosaic virus and red sage root, and has important theoretical value for revealing interaction between modified virus and host plant and virus evolution.
The technical scheme adopted by the invention is as follows: the tobacco leaf infected with the cucumber mosaic virus is used as a material of a virus source, total plant RNA in the leaf is extracted, and the total plant RNA is inoculated into the red sage root seedling in an injection inoculation mode, so that the infection of the cucumber mosaic virus on the red sage root plant is successfully established.
The technical scheme adopted by the invention is as follows:
a method for inoculating the cucumber mosaic virus of red-rooted salvia in seedling stage includes such steps as extracting the total RNA with concentration greater than or equal to 0.5 mu g/mu l from tobacco leaf infected with cucumber mosaic virus, and injection inoculating to the back of leaf cultured to 6-8 leaf stage by needle tube injection.
Further, tobacco leaves infected with cucumber mosaic virus were propagated by the following method:
tobacco leaves infected with cucumber mosaic virus were ground with 0.05M PB buffer, and virus sap was inoculated onto healthy tobacco leaves by mechanical abrasion to propagate the virus source.
Further, the tobacco is a raw tobacco Nicotiana benthamiana or a common tobacco Nicotiana tabacum.
Further, injection inoculation is specifically:
the red sage plant is cultivated to 6-8 leaf stage, the needle of the injector is used to spread the back of the leaf to 3-5 pinholes and unable to penetrate the leaf to cause micro wound, and the total RNA is injected slowly from the back of the leaf to complete inoculation.
Further, the total RNA amount per leaf of the inoculated plant is 200. Mu.l or more at the time of injection inoculation.
Further, the red sage seedling after inoculation is placed in an artificial greenhouse for culture management, the day and night temperature is kept at 20-22 ℃, and the temperature is regulated to 25 ℃ day and night after 3 days.
Compared with the field natural disease method adopted in the current production, the method for artificially inoculating the red-rooted salvia root cucumber mosaic virus in the seedling stage has the advantages of pure inoculation pathogen, high inoculation efficiency, good repeatability and the like. The method can be carried out indoors, has controllable conditions, is not influenced by seasons and external environmental conditions, can identify virus infection conditions only by about two weeks, and is suitable for screening disease-resistant breeding of the red sage root. Meanwhile, the total plant RNA extracted from the disease leaves is injected into the inoculated plant leaves, instead of the conventional viral RNA extracted from virions or the agrobacterium of virus infectious clone, so that the experimental method is greatly simplified, the experimental cost is saved, and the possibility of researching the virus is provided for a laboratory without the virus infectious clone. The traditional mechanical friction inoculation method damages the cell wall by the treatment of spraying silicon carbide, so that the virus enters the cytoplasm through the exposed membrane, and the technology can not quantify and synchronously infect, so that the experimental treatment is difficult to carry out reliable measurement. The injection inoculation method can control the virus inoculation amount and lock smaller leaves to be closer to the disease condition in the field.
Drawings
The invention is further described below with reference to the drawings and examples;
FIG. 1 is a graph showing symptoms of plants after 2 weeks of inoculation of total RNA of leaf plants infected with cucumber mosaic virus with Salvia Miltiorrhiza seedlings.
FIG. 2 is a graph showing the results of detection of cucumber mosaic virus coat protein expression on diseased leaves of the Salvia Miltiorrhiza system.
Detailed Description
EXAMPLE 1 extraction of Total RNA from virus-infected host leaf plant tissue
1. Expanding propagation of cucumber mosaic virus sources: a small amount of 800-mesh carborundum is uniformly scattered on healthy leaf blades of the raw tobacco, 1-2 leaf blades of the raw tobacco infected by cucumber mosaic virus are taken, carborundum is added into 0.05M boric acid buffer solution for full grinding, the two hands are dipped with clean PE gloves, a small amount of disease juice is rubbed and smeared on the whole leaf blades, and the leaf blades with obvious disease symptoms can be observed after 2 weeks.
2. Collecting leaf pieces (flowers, shrinkage and curling) of the leaf of the present tobacco, adding liquid nitrogen, fully grinding into powder, rapidly taking 700 μl plant powder by using an Eppendorf tube, adding 350 μl water saturated phenol (pH 5.2-6.0) preheated at 80deg.C and equal volume of RNA extraction buffer (100 mM Tris-HCl (pH 8.0), 0.1mMLiCl,10mM EDTA,1.0%SDS), and shaking for 15sec; standing for 5min. 350ml of chloroform was added thereto, the mixture was shaken for 15sec, left for 5min, and centrifuged at 12000rpm for 15min.
3. Adding the supernatant into new RNase-free Eppendorf tube, adding equal volume of 4M LiCl solution, mixing, and standing at-20deg.C for more than 5 hr. Then centrifuged at 12000rpm at 4℃for 15min. The supernatant was transferred to a new centrifuge tube. And (5) drying the precipitate at room temperature or drying the precipitate by a super clean bench.
(4) The precipitate is washed once with 70vol% ethanol, and can be washed again with absolute ethanol, dried at room temperature or dried by a super clean bench. Preserving the dry powder at-20deg.C, adding appropriate amount of DEPC ddH before inoculating injection 2 O dissolves and precipitates to obtain RNA solution, the concentration of total RNA is more than or equal to 0.5 mug/mu l, and the total RNA solution is placed in ice bath for standby.
Example 2 cucumber mosaic Virus injection inoculation of Salvia Miltiorrhiza Miq plant
1. Sterilizing Saviae Miltiorrhizae radix seed in 3wt% sodium hypochlorite solution for 15min, washing with sterile water for 3 times, and air drying. Mixing northeast peat soil, vermiculite and perlite according to the ratio of 1:1:1 to form a planting matrix, loading the planting matrix into a seedling raising basin, naturally washing the matrix, then spreading red sage root seeds, slightly covering soil on the seeds, covering a seedling raising tray cover, transplanting and seedling separation after the second true leaves are generated, and inoculating the red sage root seedlings after each basin is separated into one plant and the red sage root seedlings grow to 6-8 leaf periods.
2. Taking 1ml of total RNA of a plant by using a 1ml sterile injector, pricking 3-5 holes on the back of the 3 rd and 4 th leaves of the red sage root by using a needle head on the injector, not pricking the leaves, removing the needle head, slowly injecting the total RNA of the plant on the back of the leaves of the red sage root, infiltrating the whole leaves as much as possible, and injecting about 200 mu l of total RNA of the plant into each leaf. The day and night temperature is kept at 20-22 ℃ after inoculation, the temperature is adjusted to 25 ℃ after 3 days, and the seedlings are normally grown and managed.
3. The results of observation of the symptoms of the red sage seedlings after 2 weeks of inoculation are shown in fig. 1, and because the inoculation wound surface is small, the red sage seedlings after injection inoculation grow well, the red sage roots subjected to aseptic water control injection inoculation are asymptomatic, and the systematic leaves of the red sage seedlings subjected to cucumber mosaic leaf plant total RNA injection obviously show the symptoms of downward curling of flowers and leaves.
EXAMPLE 3 viral Western blot detection of disease-causing Danshen plants
1. From example 2, leaves of Salvia Miltiorrhiza Miq seedling with obvious infection symptoms of cucumber mosaic virus were obtained for Western blot detection, 100. Mu.l of plant material powder ground in liquid nitrogen was taken out by an Eppendorf tube, 100-150. Mu.l of 1 Xprotein loading buffer (100 mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS,0.2% bromophenol blue, 5% beta-mercaptoethanol) was added, shaking, boiling water bath for 10min, immediately placed on ice for 2min, and centrifuged at 12000rpm for 10min, and the supernatant was placed in a new centrifuge tube for standby.
2. Preparing PAGE gel (4.5% concentrated gel and 12.5% separating gel), taking 20 μl sample, loading to the PAGE gel, performing electrophoresis at 80V, increasing voltage to 120V after the sample enters the separating gel, stopping electrophoresis until bromophenol blue just runs out, and transferring protein onto nitrocellulose membrane by electrotransfer method (200 mA,80 min). The transferred nitrocellulose membrane was rinsed in TBST buffer and transferred to 10ml of blocking solution (TBST+5% nonfat milk powder). Blocking at 37deg.C for at least 2hr or overnight at 4deg.C.
3. After the sealing is completed, a certain volume of cucumber mosaic virus CP specific antiserum is directly added into the sealing liquid for reaction at 37 ℃ for at least 1hr. The nitrocellulose membrane was rinsed 3 times in TBST buffer for 10min each. Adding the nitrocellulose membrane into an AP-A secondary antibody diluted by TBST (1:5000), and reacting for 30-60min at 37 ℃. The membrane was then washed 3 times with TBST for 10min each. The nitrocellulose membrane was developed in alkaline phosphatase buffer (AP) containing 330. Mu.g/ml NBT and 165. Mu.g/ml BCIP under dark conditions until the bands were clear. As a result, as shown in FIG. 2, the CP expression of cucumber mosaic virus was detected in all 6 samples of total RNA of the leaf plant, whereas the CP expression of cucumber mosaic virus was not detected in the Salvia Miltiorrhiza plant injected with the sterile water control. Two weeks after injection, cucumber mosaic virus infects the roots of red-rooted salvia to the system leaves and begins CP protein expression. Repeated inoculation experiment results show that the inoculation success rate of the method is up to 70-80%.
The method can realize the artificial inoculation of cucumber mosaic virus and red sage root plants, and can carry out systematic infection after 2 weeks. The injection inoculation liquid is the total plant RNA extracted from tobacco (benthonic tobacco or common tobacco) leaves, and is not the conventional virus infectious clone, thereby greatly simplifying the experimental method and providing possibility for researching the virus for laboratories without the virus infectious clone. In addition, the injection inoculation can also control the single plant inoculation amount, has less damage to leaves and is more similar to the field disease condition, and is suitable for developing the research on the pathogenic mechanism between cucumber mosaic virus and red sage root hosts and the screening of germplasm resources of antiviral red sage root.
The above embodiments are intended to illustrate the present invention, not to limit it, and any modifications and changes made to the present invention within the spirit of the present invention and the scope of the appended claims fall within the scope of the present invention.

Claims (4)

1. A method for inoculating the cucumber mosaic virus of red sage root in seedling stage is characterized by thatCucumber mosaic virus) The total RNA with the extraction concentration of more than or equal to 0.5 mug/mul in tobacco leaves is used as an inoculation liquid, injection inoculation is carried out on the back of the leaves of the red sage root plant cultivated to 6-8 leaf stage by adopting a needle tube injection mode, and the total RNA of the plant is injected and inoculated per leaf when the injection inoculation is carried out, the total RNA amount is more than 200 mul; the tobacco is the primary tobaccoNicotiana benthamiana) Or (b)Nicotiana tabacum。
2. The method according to claim 1, characterized in that the cucumber mosaic virus infected tobacco leaves are propagated by the following method:
tobacco leaves infected with cucumber mosaic virus were ground with 0.05M PB buffer, and virus sap was inoculated onto healthy tobacco leaves to propagate the virus source.
3. The method according to claim 1, characterized in that the injection vaccination is in particular: 3-5 pinholes are marked on the back of the third and fourth leaves of the red sage root cultivated to the 6-8 stage by using the needle of the syringe, and the leaves cannot be marked, so that micro wounds are caused, and total RNA is injected from the back of the leaves to finish inoculation.
4. The method according to claim 1, characterized in that: the red sage seedling after inoculation is placed in an artificial greenhouse for culture management, the day and night temperature is kept at 20-22 ℃, and the temperature is regulated to 25 ℃ day and night after 3 days.
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