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

Agrobacterium tumefaciens mediated RNA (Ribonucleic Acid) interfering method for filamentous fungi genes

A technology of Agrobacterium tumefaciens and RNA interference, which is applied in the field of RNA interference, can solve the problems of limited application range of interference vectors, low interference efficiency, and limited application range, and achieve the effects of stable transformants, easy operation, and good application prospects

Inactive Publication Date: 2013-05-08
张振颖 +2
View PDF3 Cites 6 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there are still many problems when RNAi technology is used in the research of filamentous fungi: most of the fungal cells are polyploid, and the cell wall contains more polysaccharides and polysaccharide proteins, so the effect of RNA silencing is not as good as that in native , Ideal for animal cells, but has defects such as low interference efficiency and off-target effects
In addition, the scope of application of interference vectors reported in the current literature is mostly limited, which is species-specific and only suitable for dermatophyte / penicillium / aspergillus, which greatly limits its application scope

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
  • Agrobacterium tumefaciens mediated RNA (Ribonucleic Acid) interfering method for filamentous fungi genes
  • Agrobacterium tumefaciens mediated RNA (Ribonucleic Acid) interfering method for filamentous fungi genes
  • Agrobacterium tumefaciens mediated RNA (Ribonucleic Acid) interfering method for filamentous fungi genes

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0047] 1. Preparation of competent cells: For specific experimental steps, refer to "Molecular Cloning Experiment Guide" (third edition).

[0048] 2. Transform competent cells: For specific experimental steps, refer to "Molecular Cloning Experiment Guide" (third edition).

[0049] 3. Enzyme digestion method: for parameters such as the amount of restriction endonuclease used, the choice of buffer, and the enzyme digestion system, refer to the instruction manual attached to Dalian Takara Company;

[0050] 4. Plasmid extraction method: refer to the instructions attached to the plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd.)

[0051] 5. The method of purifying PCR products: refer to the instructions attached to the DNA purification and recovery kit (Tiangen Biochemical Technology Co., Ltd.)

[0052] 6. Ligation of PCR products and vector fragments: For the usage of DNA ligase and Taq enzyme, refer to the instructions attached to Dalian Takara Company.

[0053]...

Embodiment 1

[0065] The construction of embodiment 1 PCB309

[0066] 1. Construction of pCB298: Using pBI121 as a template, use primers PCB298-F and PCB298-R to amplify the RK2 replication region and nptⅡ, with a length of 4566bp. The reaction conditions were pre-denaturation at 94°C for 1 min; denaturation at 94°C for 30 sec, annealing at 55°C for 45 sec, extension at 72°C for 4 min, 30 cycles; 10 min at 72°C. The PCR fragment was purified and recovered, digested with XbaI, purified and recovered after digestion. The purified fragments after enzyme digestion were ligated, and the single fragments were ligated into circular plasmids. The ligation product was transformed into Escherichia coli DH5α, a single colony was picked, the plasmid was extracted, and verified by XbaI single enzyme digestion.

[0067] Recombinant vector pCB289 enzyme digestion identification picture figure 1 : Among them, 1 is marker: DL4500; 2 is the 4566bp PCB289 digestion product; the digestion product is consist...

Embodiment 2

[0116] Example 2. Construction of RNA interference vector pCB309-pfgrt

[0117] 1. Construction of pUC-pgt

[0118] Using the pSilent-1 plasmid as a template, primers PUT-F and PUT-R were used to amplify the sequences of the Ptrpc promoter, Gus spacer sequence and Ttrpc terminator, and the fragment length was about 2.2 kb. The reaction conditions were pre-denaturation at 94°C for 1 min; denaturation at 94°C for 30 sec, annealing at 55°C for 45 sec, extension at 72°C for 3 min, 30 cycles; 10 min at 72°C. The purified PCR fragment was connected to the Simple T vector, and connected into a circular plasmid to obtain pUC-pgt. The ligation product was transformed into Escherichia coli DH5α, a single colony was picked, the plasmid was extracted, PCR was verified with primers PUT-F and PUT-R, and the positive clone was named as the recombinant vector pUC-pgt.

[0119] PCR identification map of recombinant vector pUC-pgt; Figure 5 : Among them, 1 is marker: DL4500; 2 is the PUC-pg...

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 agrobacterium tumefaciens mediated RNA (Ribonucleic Acid) interfering method suitable for various filamentous fungi gene study. A recombinant vector PCB309-pfgrt suitable for transfecting agrobacterium tumefaciens is obtained through a genetic recombination and Interference system screening method, and the transformation system of agrobacterium tumefaciens is optimized. According to the method, the problem that the RNA interference vector of filamentous fungi is hard to construct and the interference efficiency is low is solved. The method has wide application prospect in gene function and genetic transformation of filamentous fungi.

Description

technical field [0001] The invention belongs to the technical field of RNA interference. It specifically relates to a recombinant vector PCB309-pfgrt, Agrobacterium tumefaciens used for transfection, and a method for RNA interference of filamentous fungal genes mediated by Agrobacterium tumefaciens. Background technique [0002] RNA interference is a relatively conservative gene silencing mechanism in eukaryotes. It mediates specific degradation or translation inhibition of intracellular mRNA through endogenous or exogenous double-stranded RNA, thereby leading to the expression silencing of target genes and producing corresponding Loss of functional phenotype. In 1992, after Macino first discovered RNAi in fungi in Neurospora crassa, RNAi technology has gradually become a new method for genetic modification of fungi. RNAi technology has special advantages in filamentous fungi: it is not interfered by the multinucleation, heterokaryon and non-homologous recombination freque...

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): C12N15/63C12N1/21C12N1/14C12R1/01C12R1/645
Inventor 张振颖侯彬彬刘晓明
Owner 张振颖
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