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

Soybean yellow leaf curl virus infectious cloning vector as well as construction method and application thereof

A technology of cloning vectors and construction methods, which is applied in the field of genetic engineering, can solve problems such as the unknown function of virus transmission mediator proteins, and achieve the effect of simple operation and good repeatability

Active Publication Date: 2022-02-08
INST OF PLANT PROTECTION CHINESE ACAD OF AGRI SCI
View PDF2 Cites 1 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

For soybean yellow leaf curl virus, it is not known which soybean varieties are resistant to the virus, and the function of the virus's vector and encoded protein is unknown

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
  • Soybean yellow leaf curl virus infectious cloning vector as well as construction method and application thereof
  • Soybean yellow leaf curl virus infectious cloning vector as well as construction method and application thereof
  • Soybean yellow leaf curl virus infectious cloning vector as well as construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Cloning and determination of the full genome sequence of soybean yellow leaf curl virus of embodiment 1

[0037] (1) Collection of soybean samples suspected of being infected with geminivirus

[0038] (2) Extraction of total plant DNA

[0039] The total DNA of the diseased leaves will be extracted by the CTAB method, and the specific operation is as follows: Weigh 0.1 g of fresh leaves of the plant and put them into a tube, freeze them in liquid nitrogen and grind them for 1 minute in a sample grinder (frequency set to 40 Hz) to powder. Add 700 μL of 65°C preheated CTAB extract solution (add 2% v / v β-mercaptoethanol when used), heat at 65°C for 1 hour, and shake the centrifuge tube every 10-20 minutes. Add an equal volume of 24:1 chloroform:isoamyl alcohol, mix up and down for extraction, and centrifuge at 10,000 rpm for 5 min at 4°C. Carefully transfer the supernatant to a new 1.5 mL centrifuge tube, add 1 / 10 volume of 3M NaAc and an equal volume of -20°C pre-cooled ...

Embodiment 2

[0042] The construction of embodiment 2 soybean yellow leaf curl virus infective clone pBinPLUS-SbYLCV (such as figure 1 )

[0043] According to the full sequence obtained in Example 1, primers SbYLCV-insert1 / SalI-F, SbYLCV-insert1-R, SbYLCV-insert2-F, SbYLCV-insert2 / EcoRI-R were designed, and primers were used to SbYLCV-insert1 / SalI-F, SbYLCV-insert1-R, obtain a DNA fragment of about 3 kb, that is, insert 1, the size of which is 1 copy of the genome; use the primer pair SbYLCV-insert2-F, SbYLCV-insert2 / EcoRI-R to obtain a DNA fragment of about 3 kb, namely Insertion fragment 2, the size is 1 copy of the genome; after purification of the amplified PCR product, two complete nucleotide sequences of SbYLCV containing different enzyme cutting sites were respectively obtained.

[0044] Among them, the PCR amplification system is 16μL ddH 2 O, 5 μL 5×TransStart FastPfu Buffer, 2 μL 2.5 μM dNTPs, 0.4 μL each of upstream and downstream primers (10 μM), 1 μL template DNA, 0.2 μL TransS...

Embodiment 3

[0047] Example 3 Inoculation of pBinPLUS-SbYLCV Infectious Clones

[0048] Electric shock transformation of pBinPLUS-SbYLCV was introduced into Agrobacterium strain EHA105, and after colony PCR verification, a single spot was picked and inoculated in YEP medium containing kanamycin (50mg / L) and rifampicin (50mg / L) resistance Shake the culture overnight at 28°C, when the OD of the cultured bacteria 600 When =1.0-1.5, 4000rpm, centrifugal 10min, discard supernatant culture medium, collect thalline, then inoculate buffer (containing 10mM MgCl 2 , ddH of 10mM MES (PH5.6) and 100mM acetosyringone 2 O solution) to resuspend the bacteria, adjust the concentration of the bacteria solution to make it OD 600 =1.0 or so, stand at room temperature in the dark for 2 hours. Select Nicotiana benthamiana at the 4-5 leaf stage. When injecting, lightly prick a few small holes on the back of the plant leaves, and slowly inoculate the bacterial solution into the leaf tissue through the small h...

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 soybean yellow leaf curl virus infectious cloning vector as well as a construction method and application thereof. The method specifically comprises the following steps: on the basis of obtaining the complete sequence of the genome of the soybean yellow leaf curl virus, constructing two forward repeated genomes of the soybean yellow leaf curl virus on a binary expression vector pBinPLUS by a gene recombination method; transforming the recombinant vector into an agrobacterium strain EHA105 through electric shock, so that the obtained virus vector can successfully infect plants such as soybeans and nicotiana benthamiana. The invention provides a mature system for researching the genome structure and function of the virus and the interaction between the virus and the host.

Description

technical field [0001] The invention relates to the field of genetic engineering, in particular to the construction of a plant DNA virus-Geminiviridae soybean yellow leaf curl virus (Soybean yellow leaf curl virus, SbYLCV) invasive cloning vector. Background technique [0002] Geminiviruses are a class of plant single-stranded DNA viruses that widely exist around the world. The size is about 18nm×30nm. The virus particles have a double icosahedral structure and no envelope. Geminivirus genomes are divided into single-component and double-component, and the genome size is generally about 2.5-3.0Kb. [0003] Soybean yellow leaf curl virus (SbYLCV) belongs to the Geminiviridae single-component geminivirus, with a genome length of 2782bp. The virus contains six open reading frames, in which the viral strands encode V1 and V2, and the complementary strands encode C1, C2, C3 and C4. The virus seriously threatens the production of leguminous crops, especially soybean crops. After...

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): C12N15/84C12N15/74C12N15/34C12N1/21A01H5/12A01H6/54A01H6/82C12R1/41
CPCC12N15/8283C12N15/8205C07K14/005C12N15/743C12N2750/12022Y02A50/30
Inventor 杨秀玲周雪平杜敏陈诚
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