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

Preparation of strain for traceless genetic transformation

A purpose, Fusarium oxysporum technology, applied in the field of molecular biology, can solve problems affecting yield and quality, crop yield reduction, etc., and achieve the effect of genetic transformation

Pending Publication Date: 2022-07-08
TIANJIN AGRICULTURE COLLEGE
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Fusarium oxysporum is the main pathogen that causes plant wilt and root rot. It invades from the root of the host plant and infects upwards along the vascular bundle, causing large-scale crop yield reduction and seriously affecting yield and quality.

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
  • Preparation of strain for traceless genetic transformation
  • Preparation of strain for traceless genetic transformation
  • Preparation of strain for traceless genetic transformation

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] To prepare a Fusarium oxysporum ura3 deletion mutant, the steps are as follows:

[0025] (1) Cloning of upstream and downstream fragments of Fusarium oxysporum ura3 gene

[0026] The genomic DNA of Fusarium oxysporum was extracted using the UNIQ-10 Column Fungal Genome Extraction Kit and used as the template for this step. Amplification of 5 of the Fusarium oxysporum ura3 gene (SEQ ID NO: 1) using primer pairs up-F / R (SEQ ID NO: 4 and 5), down-F / R (SEQ ID NO: 6 and 7), respectively 'Upstream fragment (up fragment, SEQ ID NO:2) and 3' downstream fragment (down fragment, SEQ ID NO:3). PCR system: template 2μL, dNTPs 4μL, primers up-F / R and down-F / R 1μL each, 5×buffer 10μL, ddH 2 O 31 μL, fastpfu enzyme 1 μL. PCR program: pre-denaturation at 95 °C for 2 min, denaturation at 95 °C for 20 s, annealing at 57 °C for 20 s, extension at 72 °C for 2 min, repeat 35 cycles of denaturation to extension, and a final extension at 72 °C for 5 min. The PCR products were subjected to...

Embodiment 2

[0039] Embodiment 2 Traceless genetic transformation application

[0040] Use the Fusarium oxysporum ura3 deletion mutant as the starting strain to knock out the AMT gene (SEQ ID NO: 14), the primers used and the genetic transformation position are as follows Figure 5 shown. The AMT gene is a gene encoding a methyltransferase. Methyltransferase, also known as transmethylase, is an important enzyme ubiquitous in organisms. Its receptors range from DNA, RNA to proteins, lipids, etc., and play a very important role in fungal morphology and physiological metabolism. , which can make fungi adapt to different environments and improve their survivability.

[0041] Using the DNA of Fusarium oxysporum as a template, primer pairs ura3-F / R, A-up-F / R (SEQ ID NO: 15 and 16) and A-down-F / R (SEQ ID NO: 17) were used, respectively and 18) were amplified to obtain the ura3 gene fragment, the upstream fragment (A-up, SEQ ID NO: 19) and the downstream fragment (A-down, SEQ ID NO: 20) of the ...

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 preparation of a strain capable of being used for traceless genetic transformation, and belongs to the technical field of molecular biology. The fusarium oxysporum ura3 deletion mutant capable of being used for traceless genetic transformation is constructed by virtue of PEG-mediated fungal protoplast transformation and homologous replacement of a transformed fragment and a target gene fragment in a genome under the condition that other resistance genes influencing original genetic characters of fusarium oxysporum are not introduced, so that the fusarium oxysporum ura3 deletion mutant can be used for the traceless genetic transformation; and a new tool is provided for research on genetic characteristics of fusarium oxysporum.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, in particular to the preparation of a strain that can be used for traceless genetic transformation. Background technique [0002] Orotate 5′-phosphate decarboxylase (OMP decarboxylase) is a key enzyme involved in pyrimidine biosynthesis, catalyzing the decarboxylation of orotate monophosphate (OMP) to form uridine monophosphate (UMP). In Fusarium oxysporum, the gene for synthesizing OMP decarboxylase is ura3. The mutants lacking the ura3 gene show uracil auxotrophy, which requires exogenous uridine and uracil for normal growth. Meanwhile, 5-fluoroorotic acid (5-FOA) can serve as a substrate for OMP decarboxylase and be converted into the toxic intermediate 5-fluoro-UMP, a pyrimidine analog that can be incorporated into nucleic acid chains in place of urine Pyrimidines, or thymines, interfere with the processing of RNA and DNA, affecting their function and ultimately leading to cell de...

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 Applications(China)
IPC IPC(8): C12N15/87C12N15/60C12N1/15C12R1/77
CPCC12N15/87C12N9/88C12Y401/01023
Inventor 李二峰尉婧王鑫磊王碧香李诗瑶
Owner TIANJIN AGRICULTURE COLLEGE
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