Virulence auxiliary vector for agrobacterium tumefaciens-mediated high molecular weight T-DNA (Transfer-Deoxyribonucleic Acid) transformation and preparation method as well as application thereof
An Agrobacterium-mediated and auxiliary vector technology, applied in the field of genetic engineering, can solve the problems of fragmentation, loss, and incomplete integration of large molecular weight T-DNA, and achieve the effect of promoting genetic transformation.
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0050] Construction of recombinant binary expression vector
[0051] Construction of a1.pVCT2020 vector
[0052] Design primers according to the pBIN19 vector sequence (GenBank accession No: U09365), the upstream primer is shown in SEQ ID NO: 1, the sequence is: 5'-cg gaattc agggagtcacgttatgac-3' (the underline is the EcoR I site), the downstream primer is shown in SEQ ID NO: 2, and the sequence is: 5'-cg ctcgag tcccgctcagaagaac-3' (the Xho I site is underlined). Using pBIN19 vector as template, PfuDNA polymerase, PCR amplification, PCR amplification conditions are 94°C pre-denaturation for 3 minutes, 94°C denaturation for 20 seconds, 50°C annealing for 20 seconds, 72°C extension for 2 minutes and 10 seconds, 3 cycles , anneal at 60°C for 20 seconds, extend at 72°C for 2 minutes and 10 seconds, 27 cycles, extend at 72°C for 10 minutes, and recover a 1116 bp fragment by agarose electrophoresis, which contains the nopaline synthase gene promoter Pnos and kanamycin The prime...
Embodiment 2
[0063] Construction of Basic Vector Containing virE-virG Chimeric Operator
[0064] Construction of b1.pVCT1213 vector
[0065] According to the reported pTiC58 vector sequence of Agrobacterium strain C58 (GenBank accession: NC_003308), design specific PCR primers containing promoter Promoter, VirE1 gene coding region and VirE2 gene coding region (referred to as Promoter-VirE1-VirE2), where the upstream primer As shown in SEQ ID NO: 3, the sequence is 5'-gaattacattcacacggcacc-3', and the downstream primer is shown in SEQ ID NO: 4, and the sequence is 5'-tcacacgtggtggtggtggtggtgcagactgtttacggttgg-3'. Using the pTiC58 vector of Agrobacterium strain C58 as a template, PCR amplification was performed using PrimeStar HS DNA polymerase. The reaction conditions were: 98°C pre-denaturation for 1 minute, 98°C denaturation for 10 seconds, 56°C annealing for 15 seconds, and 72°C extension for 2 minutes for 30 seconds, 28 cycles, 72°C extension for 10 minutes. The PCR product was identi...
Embodiment 3
[0070] Construction of toxic helper vector
[0071] Construction of c1.pVCT2270 vector
[0072] as attached Figure 5 As shown, the obtained pVCT2268 vector was single-digested with the restriction endonuclease Nru I, and was identified by agarose gel electrophoresis to obtain the backbone fragment of the pVCT2268 vector; at the same time, it was three-digested with the restriction endonucleases Spe I, Xba I, and Ema1105 I The vector pVCT1244 was identified by agarose gel electrophoresis, and a 3465bp fragment containing the virE-virG chimeric operon was recovered, and the virE-virG chimeric operon was ligated with the backbone fragment of the pVCT2268 vector under the action of T4 ligase overnight at 16°C. A recombinant vector was obtained, named pVCT2270, as attached Figure 8 shown.
[0073] Construction of c2.pVCT2272 vector
[0074] Digest the pVCT2270 vector with restriction endonuclease Sal I, excise the T-DNA region on the pVCT2270 vector, and use 1% agarose gel el...
PUM
Abstract
Description
Claims
Application Information
- R&D Engineer
- R&D Manager
- IP Professional
- Industry Leading Data Capabilities
- Powerful AI technology
- Patent DNA Extraction
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