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

Construction method of fusarium graminearum single-stranded circular DNA (Deoxyribonucleic Acid) virus FgGMTV1/HB58 infectious cloning

A technology of DNA virus, Fusarium graminearum, which is applied in the field of constructing the invasive clone of DNA virus of Fusarium graminearum, can solve the problems of hysteresis, mycoviruses are not paid enough attention, and it is not easy for scholars to discover and pay attention to, etc. Achieve the effect of simplifying cloning and transfection steps

Active Publication Date: 2019-05-28
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
View PDF0 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the study of mycoviruses has not received enough attention, and has shown a certain lag
The main reasons include, first, fungal virus infection does not show obvious symptoms, and is not easy to be discovered and valued by scholars, and most mycologists often ignore that it is caused by fungal virus infection when they come into contact with strains with abnormal colonies

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
  • Construction method of fusarium graminearum single-stranded circular DNA (Deoxyribonucleic Acid) virus FgGMTV1/HB58 infectious cloning
  • Construction method of fusarium graminearum single-stranded circular DNA (Deoxyribonucleic Acid) virus FgGMTV1/HB58 infectious cloning
  • Construction method of fusarium graminearum single-stranded circular DNA (Deoxyribonucleic Acid) virus FgGMTV1/HB58 infectious cloning

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Virion extraction and observation in the Fusarium graminearum HB58 bacterial strain of embodiment 1

[0036] The Fusarium graminearum strain HB58 was isolated from wheat ears with head blight in Hebei Province. The Fusarium graminearum strain HB58 carrying FgGMTV1 was cultured in PDB medium for 1 week; Grind to powder in nitrogen; add buffer according to the ratio of 5mL sodium phosphate buffer (0.1M): 1g mycelium dry powder, and incubate on a shaker for 1–2h (100rpm, 4°C) to release virus particles; centrifuge at low speed Remove the bulky impurities for 20min (5000rpm, 4°C), take the supernatant into a new centrifuge tube, add an equal volume of chloroform to the mixture, mix gently, and centrifuge for 20min (12000rpm, 4°C); take the supernatant and add the supernatant In a centrifuge tube (35mL), balance each centrifuge tube and ultracentrifuge for 3h (32000rpm, 4°C); pour off the supernatant carefully, add 1mL sodium phosphate buffer (0.01M) to the bottom precipitat...

Embodiment 2

[0042] Extraction and observation of nucleic acid in virus particles in the Fusarium graminearum HB58 bacterial strain of embodiment 2

[0043]Take an appropriate amount of virus purification solution 20–100 μL, add sterilized distilled water to 200 μL; add an equal volume (200 μL) of extraction buffer (20mM Tris-HCl pH8.0; 1% SDS; 200mM NaCl; 5mM EDTA), add an equal volume of saturated Phenol / chloroform (400μL), shake slightly for 2–5 minutes, centrifuge at 12000rpm for 2 minutes, take the supernatant aqueous phase, add an equal volume of saturated phenol / chloroform, mix and extract, centrifuge at 12000rpm for 2 minutes, add an equal volume of chloroform / isoamyl Extract once with alcohol (24:1), take the supernatant water phase and add 2.5 times the volume of anhydrous cold ethanol, mix well, put it at -80°C for 1 hour, centrifuge at 12000rpm for 15 minutes, pour off the ethanol, and use 70% cold ethanol (0.5 mL) and washed twice, vacuum-dried for 2–5 minutes, the precipitate...

Embodiment 3

[0045] Nuclease digestion and observation of nucleic acid in virion in embodiment 3 Fusarium graminearum HB58 bacterial strain

[0046] Select DNase I, S1 nuclease, exonuclease I and exonuclease III to treat 2–5 μg virion nucleic acid extracts respectively, and the reaction system and reaction time are carried out according to the instructions of each tool enzyme.

[0047] Results: In order to further characterize the nucleic acid in the virions, DNase I, S1 nuclease, exonuclease I and exonuclease III were used for treatment ( Figure 4 ). As a result, there was no DNA band in the swimming lane treated with DNase I, indicating that the nucleic acid was DNA; DNA exonuclease III was a specific exonuclease for processing double-stranded nucleic acids, and the DNA band treated with this enzyme did not change, proving that the Nucleic acid is not double-stranded linear DNA; S1 nuclease is a single-stranded specific endonuclease, and the DNA bands treated with this enzyme disappear...

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 construction method of fusarium graminearum single-stranded circular DNA (Deoxyribonucleic Acid) virus FgGMTV1 / HB58 infectious cloning. A virus genome contains two single-stranded circular DNA molecules including DNA-A and DNA-B, and genome sequences of the DNA-A and the DNA-B are shown as SEQ ID NO: 1 to 2; the virus genome does not contain a single-stranded circular DNAmolecule DNA-C and the genome sequence of the DNA-C is shown as SED ID NO: 3. The construction method comprises the following steps: constructing DNA-A infectious cloning, constructing DNA-B infectious cloning and constructing DNA-C infectious cloning. When the infectious cloning is constructed, only one bacterium cloning vector pBluescript II SK(+) is needed and fungi can be cloned and transfected successfully; cloning and transfection steps are simplified and a plurality of times of PCR (Polymerase Chain Reaction) cloning are not needed; a plurality of cloning vectors and transfection vectors are not needed, and the transfection can be finished, without the need of cloning the fungi to a binary expression vector.

Description

technical field [0001] The invention relates to the technical field of Fusarium graminearum control technology, in particular to a method for constructing an infectious clone of Fusarium graminearum DNA virus. Background technique [0002] Fusarium graminearum Schw. is an important species of Fusarium, which can cause diseases of grass crops in the field. If there is wheat head blight. After infecting wheat kernels or corn, white flocculent or fluffy hyphae are produced, and then white to rose, white to pink or white to brick red. The sexual form is Gibberella zeae Schw.Petch is a fungus belonging to the subphylum Deuteromycota. Amorphous fungi belonging to the subphylum Ascomycota. In the culture medium, the white aerial hyphae are dense, and the hyphae in the base secrete reddish-brown pigment. Life history includes both sexual and asexual generations. The sexual generation produces ascus shells, which are pear-shaped with a mouth. Club-shaped ascus tufted with ascus...

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
IPC IPC(8): C12N15/66C12N15/80
Inventor 郭立华李鹏飞王双超邱德文杨秀芬曾洪梅李广悦袁京京任杰
Owner INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
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