Method for increasing content of tropane alkaloid in atropa belladonna
A technology of tropane and alkaloids, which is applied in the field of increasing the content of tropane alkaloids in belladonna, can solve problems such as inability to produce seeds, and achieve the effect of increasing gene expression
- Summary
- Abstract
- Description
- Claims
- Application Information
AI Technical Summary
Problems solved by technology
Method used
Image
Examples
Embodiment 1
[0034]Cloning of embodiment 1, H6H-p promoter and rolC gene fragment
[0035] Using belladonna DNA as a template, two synthetic specific primers 121-H6H-F and 121-H6H-R were used for PCR. The upstream primer contained Hind III, and the downstream contained a BamH I restriction site. The specific primer sequences are as follows:
[0036] F-H6H-121: 5'-gaccatgattacgccaagcttagtcgctttaactatcttttgatg-3' (SEQ ID NO. 1);
[0037] R-H6H-121: 5'-ggactgaccacccggggatccggagtaaaagtctcaaataagaaag-3' (SEQ ID NO. 2).
[0038] PCR amplification conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 94°C for 30 s, annealing at 56°C for 30 s, extension at 72°C for 1 min; post-extension at 72°C for 2 min.
[0039] The amplified product was subjected to agarose gel electrophoresis, and the target fragment H6H-p promoter was recovered. The full length of H6H-p is 1100bp, and the specific sequence is shown in SEQ ID NO.7.
[0040]
[0041] Using C58C1 bacterial liquid a...
Embodiment 2
[0049] Embodiment 2, pCaMV35S-rolC, pH6H-p-rolC expression vector construction
[0050] Using pBI121 as the plant expression vector, pBI121-pH6H-p-GUS was obtained by linking Hind III and BamH I on the pBI121 vector with the pMD19-T-H6H-p target fragment H6H-p that had been cut with the same enzyme. Then pMD19-T-rolC was digested with BamHI and Sac I, and then ligated with pBI121 to obtain the pCaMV35S-rolC expression vector. The expression vector was transformed into DH5α Escherichia coli competent cells, spread on the LB medium containing kan resistance, cultured overnight at 37°C, single colony was activated for about 4-6 hours, and PCR detection of bacterial liquid was performed. The correctly sequenced DH5α-pCaMV35S-rolC can be directly expanded and cultivated, and the extracted plasmid can be transformed into EHA105 Agrobacterium competent cells. The correctly sequenced DH5α-pH6H-p-GUS was expanded and cultivated, the plasmid was extracted, and the two restriction sites...
Embodiment 3
[0051] Embodiment 3, transformation of engineering bacteria and transformation of belladonna
[0052] Take an appropriate amount of sample bacteria liquid that has been successfully sequenced for activation, extract plasmids, transform EHA105 Agrobacterium competent cells, spread them on plates containing the three resistances corresponding to Str, Rif, and Kan, and culture at 28°C. After 48 hours, pick a single colony for activation, and use PCR for positive identification. The rolC gene band was detected at about 600 bp, and all detected single colonies had bands. The transformation was successful, and the transformed Agrobacterium liquid can be used for the next experiment .
[0053] Belladonna cotyledons and hypocotyls were used as transformation objects, and EHA105 was used as a mediator to infect belladonna cotyledon leaves and hypocotyls, and complete belladonna was obtained through co-cultivation, sterilization (long clustered buds), and rooting culture. Transgenic pl...
PUM
Login to view more Abstract
Description
Claims
Application Information
Login to view more - 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.
© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap