Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

Method for transforming plant protoplast by RNA (Ribonucleic Acid) virus

An RNA virus and protoplast technology is applied in the field of RNA virus transformation of plant protoplasts, which can solve the problems of restricting the popularization of BNYVV transfection protoplast method and high requirements for instruments.

Inactive Publication Date: 2014-08-06
CHINA AGRI UNIV
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In addition, because the electric shock transformation method has higher requirements on the instruments used in the experiment, which limits the promotion of the BNYVV transfection protoplast method

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for transforming plant protoplast by RNA (Ribonucleic Acid) virus
  • Method for transforming plant protoplast by RNA (Ribonucleic Acid) virus
  • Method for transforming plant protoplast by RNA (Ribonucleic Acid) virus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1, the extraction of the total RNA of the host leaf plant tissue of virus infection

[0044] In this example, Tetragonia expansa was used as the plant to be infected, and it was infected with a polymorphic RNA virus Beet necrotic yellow vein virus (BNYVV), and the total RNA of the infected Tetragonia leaf tissue was extracted , ready for the transformation of protoplasts. details as follows:

[0045] (1) Juice mechanical friction inoculation of host apricot BNYVV: Sprinkle a small amount of carborundum evenly on the leaves of the apricot infected with BNYVV, take the diseased leaves or separate the diseased spots, grind them thoroughly with 0.05M PB buffer, and wear clean hands Latex gloves are dipped in a small amount of disease juice and rubbed to smear the whole leaf. After 5-7 days, yellow circular lesions can be seen on the leaves.

[0046] (2) Collect diseased apricot leaves (the average number of spots per diseased leaf is 60-90), add about 8-10g of ...

Embodiment 2

[0050] Embodiment 2, the preparation of Ben Sheng tobacco protoplast

[0051] In this example, protoplasts for transformation were prepared from Nicotiana benthamiana. details as follows:

[0052] (1) Select the first and second full-length leaves of Tobacco benthamiana at the 6-8 leaf stage cultivated in the greenhouse (24°C, 12h light, 12h dark) for protoplast preparation, and chop them as much as possible (leaf Area less than or equal to 0.5mm 2 ), quickly put the chopped leaves into the enzymatic hydrolysis solution (recipe above). The ratio of leaves to enzymatic hydrolysis solution is 3g of leaves (equivalent to about 20 complete leaves) and 5ml of enzymatic hydrolysis solution.

[0053] (2) Stand for enzymolysis in a dark place at 24°C for 3-4 hours, observe under a microscope that most of the cells are spherical to stop the enzymolysis, centrifuge at 80rpm for 1min, and release the enzymolyzed cells.

[0054] (3) Use a 200-mesh nylon filter to filter the enzymatic ...

Embodiment 3

[0060] Embodiment 3, PEG-mediated tobacco protoplast transformation

[0061] In this example, the plant total RNA extracted from the diseased leaves of Apricot apricot infected with BNYVV obtained in Example 1 will be transformed into the protoplasts of Nicotiana benthamiana prepared in Example 2 through a PEG-mediated method, with the aim of constructing BNYVV The recombinant N. benthamiana protoplasts were used to study the pathogenic mechanism between BNYVV and N. benthamiana host. details as follows:

[0062] (1) Add the appropriate amount of total plant RNA extracted from the diseased leaves of Apricot apricot infected by BNYVV obtained in Example 1 to a 2ml centrifuge tube (treated with DEPC), and add 200 μl of the protoplasts of Nicotiana benthamiana prepared in Example 2 solution (concentration of 1 × 10 6 pcs / ml), make the protoplasts and plant total RNA fully mixed, then add 220μl PEG4000 solution (recipe above), mix well (the action must be gentle), and place at r...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention discloses a method for transforming a plant protoplast by an RNA virus. The method for transforming plant protoplast by the RNA virus in the invention comprises a step of transferring a total RNA extracted from a plant infected by the RNA virus into a plant protoplast, wherein the RNA virus is a multi-component RNA virus, such as a beet necrotic yellow vein virus; and the plant protoplast is a tobacco protoplast, such as a nicotiana benthamiana protoplast. According to the invention, nicotiana benthamiana tobacco leaves are used as raw materials for preparing the protoplast; the plant RNA of an infected leaf infected by the beet necrotic yellow vein virus is inoculated in the nicotiana benthamiana protoplast by a polyethylene glycol mediator, so as to successfully establish infection of the beet necrotic yellow vein virus on the protoplast. The method disclosed by the invention provides an ideal model for researching a pathogenic mechanism between the beet necrotic yellow vein virus and a nicotiana benthamiana host, and has an important theoretical value for discovering interaction between the virus and the host plant and virus evolution.

Description

technical field [0001] The invention relates to a method for transforming plant protoplasts with RNA viruses. Background technique [0002] Beet bushy root disease is an important disease that harms sugar beet, and has become a devastating disease in all sugar beet producing areas in the world, especially affecting sugar beet yield and sugar content greatly. In recent years, the disease has occurred to varying degrees in major sugar beet producing areas such as Inner Mongolia, Gansu, Xinjiang and Heilongjiang, seriously affecting the development of sugar beet sugar industry in China. The causative agent of the disease is beet necrotic yellow vein virus (Beet necrotic yellow vein virus, BNYVV), which is a representative virus of the genus Benyvirus and spread by Polymyxa beta (Tamada, T and Baba, T.Beet necrotic yellow vein virus from rhizomania-affected sugar beet in Japan. Ann Phytopathol Soc Japan. 1973, 39:325-332). BNYVV is a positive-sense RNA polypartite virus, and i...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/87
Inventor 韩成贵范慧艳李大伟于嘉林王颖张永亮武文琦王晓辉
Owner CHINA AGRI UNIV
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com