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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

Active Publication Date: 2020-11-17
GUIZHOU UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the rol gene can promote the synthesis of secondary metabolites in the root, it has the side effect of inducing plant deformity in the aboveground part. Ooms et al. (Ooms G, 1985) found that although the Brassica napus transformed with the Ri plasmid can Flowers, but does not produce seeds

Method used

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  • Method for increasing content of tropane alkaloid in atropa belladonna
  • Method for increasing content of tropane alkaloid in atropa belladonna
  • Method for increasing content of tropane alkaloid in atropa belladonna

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Effect test

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...

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Abstract

The invention discloses a method for increasing the content of tropane alkaloid in atropa belladonna. The gene expression quantity of a TAs alkaloid synthesis pathway is effectively increased by expressing a gene rolC or rolB in the atropa belladonna, and the content of hyoscyamine and anisodamine in the atropa belladonna is increased, but variation influence is caused on plant phenotype; and thegene rolC or rolB expressed by H6H-p tissue localization does not influence plant phenotypic growth while the alkaloid content is increased. By selective control of the gene rolC or rolB, a foundationis laid for further cultivating a high-alkaloid-yield atropa belladonna strain in the medicinal field of the atropa belladonna, and a new method is provided for further developing TAs alkaloid gene engineering metabolism research of the atropa belladonna.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for increasing the content of tropine alkaloids in belladonna. Background technique [0002] Tropane alkaloids (Tropane alkaloids, TAs) are a class of anticholinergic drugs commonly used in clinical practice, and have good effects in the treatment of analgesia, anesthesia, anti-motion sickness, detoxification, Parkinson's disease, and pesticide poisoning. At present, tropane alkaloids are mainly extracted from natural plants such as Atropa belladonna, Datura stramonium, and Anisodus tanguticus. The market demand is huge, and the cost of industrial synthesis is relatively high. Belladonna is a commercial drug source plant that is included in the Chinese Pharmacopoeia and mainly provides tropane alkaloids. The 2015 edition of the Pharmacopoeia requires that the content of scopolamine in belladonna hay should not be less than 0.3%, while the content of scopolamine in wild bell...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84C12N15/31A01H5/00A01H6/82
CPCC12N15/8243C07K14/195
Inventor 强玮廖志华张明生付维路星星
Owner GUIZHOU UNIV
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