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Salvia miltiorrhiza cucumber mosaic virus seedling stage injection inoculation method

A technology of cucumber mosaic virus, injection and inoculation, which is applied in the directions of microorganism-based methods, biochemical equipment and methods, and the determination/inspection of microorganisms to achieve the effects of controllable conditions, simplified experimental methods, and high inoculation efficiency

Pending Publication Date: 2022-05-31
ZHEJIANG CHINESE MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to provide a more efficient and operable salvia seedling in view of the lack of the main pathogen of salvia miltiorrhiza virus disease - the current situation of artificial inoculation system of cucumber mosaic virus on salvia miltiorrhiza, and the deficiency of traditional mechanical friction method Inoculation method

Method used

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  • Salvia miltiorrhiza cucumber mosaic virus seedling stage injection inoculation method
  • Salvia miltiorrhiza cucumber mosaic virus seedling stage injection inoculation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] The extraction of the total RNA of the host leaf plant tissue of embodiment 1 virus infection

[0020] 1. Propagation of Cucumber Mosaic Virus Virus Source: Sprinkle a small amount of 800-mesh carborundum evenly on the healthy leaves of Bunsen tobacco, take 1-2 pieces of Bunsen tobacco leaves infected by cucumber mosaic virus disease, buffer with 0.05M boric acid Add emery to the liquid and grind it thoroughly, and wear clean PE gloves on both hands to dip a small amount of diseased juice and rub it on the whole leaf. After 2 weeks, leaves with obvious symptoms of disease can be observed.

[0021] 2. Collect tobacco leaves (mosaic, shrunken and curly) with typical symptoms of cucumber mosaic virus disease and add liquid nitrogen to fully grind them into powder, quickly take 700 μl of plant powder with an Eppendorf tube, and add 350 μl of 80°C preheated Water-saturated phenol (pH5.2-6.0) and an equal volume of RNA extraction buffer (100mM Tris-HCl (pH8.0), 0.1mM LiCl, 10...

Embodiment 2

[0024] Example 2 Cucumber Mosaic Virus Injection Inoculation of Danshen Seedling Plants

[0025] 1. After disinfecting the seeds of Salvia miltiorrhiza in 3wt% sodium hypochlorite solution for 15 minutes, rinse them with sterile water for 3 times and dry them in the air. Northeast peat soil, vermiculite and perlite are mixed into a planting substrate in a ratio of 1:1:1, put into a seedling pot, and after the substrate is naturally washed with water, spread the seeds of salvia miltiorrhiza, cover the seeds with a little soil, and cover the seedling tray. After the second true leaf is produced, the seedlings can be transplanted and divided into one plant per pot, and the seedlings of Salvia miltiorrhiza can be inoculated when they grow to the 6-8 leaf stage.

[0026] 2. Use a 1ml sterile syringe to take 1ml of plant total RNA, use the needle on the syringe to pierce 3-5 holes on the back of the third and fourth leaves of Danshen without poking through the leaves, remove the nee...

Embodiment 3

[0028] The virus Western blot detection of embodiment 3 pathogenic salvia miltiorrhiza plants

[0029] 1. Obtain the salvia miltiorrhiza seedling leaves with obvious symptoms of cucumber mosaic virus infection from Example 2 for Western blot detection, use an Eppendorf tube to get the ground plant material powder in liquid nitrogen with a volume of 100 μl, add 100-150 μl 1X protein loading buffer solution (100mM Tris-HCl (pH 6.8), 20% glycerol, 4% SDS, 0.2% bromophenol blue, 5% β-mercaptoethanol), shaking, boiling water bath for 10min, immediately placed on ice for 2min, centrifuged at 12000rpm for 10min, Take the supernatant and put it in a new centrifuge tube for later use.

[0030] 2. Prepare PAGE gel (4.5% stacking gel, 12.5% ​​separating gel), take 20μl sample and load it on PAGE gel for electrophoresis, first 80V electrophoresis, after the sample enters the separating gel, increase the voltage to 120V, electrophoresis to bromophenol blue Just after running out, stop the...

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Abstract

The invention discloses a salvia miltiorrhiza cucumber mosaic virus seedling stage inoculation method, and belongs to the technical field of agricultural biology. According to the method, total RNA (Ribonucleic Acid) is extracted from tobacco leaves infected with the cucumber mosaic virus, and injection inoculation is carried out when a salvia miltiorrhiza plant is cultured to 6-8 leaf stages by adopting a needle tube injection mode, so that an infection system of the cucumber mosaic virus on the salvia miltiorrhiza plant is successfully established. The invention provides an ideal experimental model for researching the pathogenic mechanism between the cucumber mosaic virus and the salvia miltiorrhiza host, and has important theoretical value for revealing the interaction and virus evolution between the virus and the host plant. Compared with a friction inoculation method, the method has the advantages that the inoculation efficiency is greatly improved, and symptoms of salvia miltiorrhiza seedlings can be observed two weeks after inoculation. According to the method, efficient inoculation can be completed with a small amount of susceptible materials, the virus inoculation amount is controllable, the total RNA amount of injected leaves is small, and damage to the leaves is little and is closer to the field morbidity condition.

Description

technical field [0001] The invention relates to the field of plant virus inoculation, in particular to a method for seedling injection inoculation of salvia miltiorrhiza cucumber mosaic virus disease. Background technique [0002] Salvia miltiorrhiza is a commonly used bulk Chinese herbal medicine in my country, which is widely used in the treatment of cardiovascular and cerebrovascular diseases. In recent years, the demand for salvia miltiorrhiza is increasing day by day, and the planting area is also expanding. However, since Danshen is mainly produced through asexual reproduction, the problem of virus infection is serious, which limits the development of Danshen industry. Studies have shown that the main pathogen of salvia miltiorrhiza virus disease is cucumber mosaic virus (CMV), which can cause salvia miltiorrhiza to degenerate. Typical symptoms such as mottled, curled leaves, yellowing and dwarfing, the root system of the infected plants is small, the yield is greatl...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12Q1/18C12R1/94
CPCC12N15/8207C12Q1/18
Inventor 范慧艳开国银周杨
Owner ZHEJIANG CHINESE MEDICAL UNIVERSITY
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