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

Long double-stranded RNA expression vector for silencing whole genome or across genome

An expression vector and genome-wide technology, applied in the fields of RNA interference technology and functional genomics, can solve the problems of immature high-throughput RNA interference vectors and other issues

Inactive Publication Date: 2013-09-25
CHINA AGRI UNIV
View PDF1 Cites 2 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, high-throughput RNA interference vectors with these functions are still immature

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
  • Long double-stranded RNA expression vector for silencing whole genome or across genome
  • Long double-stranded RNA expression vector for silencing whole genome or across genome
  • Long double-stranded RNA expression vector for silencing whole genome or across genome

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1 Construction of Conjugated Promoter Plant RNAi Binary Expression Vector Containing Lox and attP Sites

[0018] 1. Mutation of Xho I restriction site of pTF101.1 vector

[0019] (1) Design the amplification primers as follows:

[0020] pTF 101.1FP: 5'-CTGGTCGACGTCCTTCTCAAATGAAATG-3'

[0021] pTF 101.1RP: 5'-GTCAATTGGAGCTCGGTACCCGGGGATC-3'

[0022] (2) Use the above primers to amplify the plasmid pTF101.1 as a template

[0023] The amplification system is as follows:

[0024] Pre-denaturation at 95°C for 3min, denaturation at 95°C for 30S, annealing at 52°C for 30S, extension at 72°C for 1min, 30 cycles, and finally extension at 72°C for 10min, and incubation at 4°C.

[0025] After recovering the target band amplified by PCR, it was digested with Sal I and Hind III to recover a 767bp fragment, which was connected between the Xho I and Hind III sites of the digested pTF101.1 vector to obtain a recombinant vector. Heat-shock transformed Escherichia coli DH5α ...

Embodiment 2

[0046] Example 2 Utilizing GCGS-1 Vector to Silence the Expression of GUS Gene in Transgenic Arabidopsis

[0047] 1. Obtaining transgenic Arabidopsis

[0048] (1) Add 0.1 μg of the constructed GCGS-1 carrier to 100 μL of Agrobacterium GV3301, transform by electric shock, add 900 μL of LB liquid medium, culture on a shaker at 28°C for 2 hours, centrifuge, remove 400 μL of the supernatant, and resuspend the bacteria The body was spread on LB solid medium containing 100mg / L Spe (specticomycin), 30mg / L Gen (gentamycin sulfate) and 100mg / L Rif (rifampicin), cultured at 28°C for 3 days, and screened Positive clone.

[0049] (2) Pick a single clone in 5mL liquid LB medium containing 100mg / L Spe, 30mg / LGen and 100mg / L Rif, culture it on a shaker at 28°C for 2 days, and inoculate it into LB liquid medium (containing 100mg / L Spe, 30mg / L Gen and 100mg / L Rif), shake culture at 28℃ until OD 600 When =0.5-0.8, the cells were collected by centrifugation, and resuspended in transformation ...

Embodiment 3

[0068] Example 3 Arabidopsis functional genome research based on long double-stranded RNA interference library

[0069] 1. Acquisition of cDNA library for functional genomics research

[0070] (1) Linearization of maize root cDNA library induced by low nitrogen

[0071] Construction of maize root cDNA library: Total RNA was extracted from maize roots treated with nitrogen deficiency for 2 days after 7 days of normal culture, and the mRNA was purified using the Poly(A) mRNA isolation and purification kit, and the full-length cDNA library construction kit ( Invitrogen, USA) constructed an entry clone library, and then used LR recombination to obtain a maize root cDNA library induced by low nitrogen.

[0072] Take 1 μL of the maize root cDNA library (1 μg / μL) induced by low nitrogen, add 1 μL of 10 × EcoRV buffer, 1 μL of EcoRV endonuclease, 6 μL of H 2 O, incubate at 37°C for 2h. Then add 40 μL H 2 O, 25 μL NaAc (3M), 187.5 μL absolute ethanol, store at -20°C overnight. Cen...

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 provides a long double-stranded RNA (ldsRNA) expression vector for silencing a whole genome or across the genome. The vector is a plant binary expression vector integrated with the following sequences: a terminator-a promoter-an LoxP site sequence-an attP1 site sequence-a ccdB gene-a CmR gene-an attP2 site sequence-the LoxP site sequence-the promoter-the terminator. The vector employs conjugated promoter sequences between T-DNA border sequences, the LoxP site sequence and the attP site sequences to integrate a plant or an insect cDNA library into the vector through an enzyme digestion reaction or a recombination reaction. Plant genes are silenced in a plant by an RNA interference (RNAi) or genes of diseases and insect pests are silenced by feeding, and thus the vector is applied to a plant functional genomic research for discovering new gene functions or screening target genes for host-delivered RNAi, and thereby cultivating broad-spectrum or specific new varieties with disease and pest resistance.

Description

technical field [0001] The invention relates to RNA interference technology and functional genomics, in particular to a long double-stranded RNA expression vector used for genome-wide or cross-genome silencing. Background technique [0002] Since the RNA interference (RNAi)-based gene knockout (knock out) or knockdown (knock down) was discovered in Caenorhabditis elegans, RNA interference technology has been widely used in various fields of life sciences, and has a great impact on revealing new Both the phenomenon of life and the invention of new technological methods are revolutionary. RNAi has been used to analyze the function of specific genes in microorganisms, nematodes, Drosophila, mammals and plants. In terms of high-throughput gene cloning and functional verification, RNAi was the first to use dsRNA synthesized in vitro to knock out or knock down the C.elegans gene on a large scale in order to analyze the gene function from the genome level. Although off-target phe...

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): C12N15/82A01H5/00
Inventor 刘培李天骄唐秀文
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