Application of atropa-belladonna ornithine decarboxylase to cultivating high-yield hyoscyamine and anisodamine atropa-belladonna varieties

A technology of belladonna ornithine decarboxylase and nightshade ornithine decarboxylase, applied in the field of application of belladonna ornithine decarboxylase in cultivating high-yielding hyoscyamine and anisodamine belladonna varieties, can solve the problem of undeveloped enzymes Kinetic experiments, research needs to be in-depth and other issues, to achieve the effect of increasing TAs content and deepening understanding

Pending Publication Date: 2019-07-12
SOUTHWEST UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In Solanaceae producing tropane alkaloid plants, only the ornithine decarboxylase gene (DsODC) of Datura datura has been cloned, although Michael et al expressed the recombinant protein of DsODC in E. coli in 1996, but did not No related enzyme kinetic experiments were carried out
Therefore, the current research on plant ODC, especially the ODC gene of Solanaceae tropane alkaloid-producing plants needs to be further studied

Method used

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  • Application of atropa-belladonna ornithine decarboxylase to cultivating high-yield hyoscyamine and anisodamine atropa-belladonna varieties
  • Application of atropa-belladonna ornithine decarboxylase to cultivating high-yield hyoscyamine and anisodamine atropa-belladonna varieties
  • Application of atropa-belladonna ornithine decarboxylase to cultivating high-yield hyoscyamine and anisodamine atropa-belladonna varieties

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Embodiment 1, belladonna AbODC gene cloning

[0030] According to the full-length cDNA sequence of the belladonna AbODC reported on NCBI, the primers for cloning the AbODC coding region were designed. The specific primer sequences are as follows:

[0031] AbODC-F1: 5'-atggccggccaaacagtcatcg-3' (SEQ ID NO.1);

[0032] AbODC-R1: 5'-tcagcttggataagcataagcaagg-3' (SEQ ID NO.2);

[0033] Then, using the belladonna cDNA as a template and the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 as primers, high-fidelity enzyme KOD plus DNA polymerase was used for PCR amplification. The PCR reaction conditions were: pre-denaturation at 94°C for 3 minutes ; 30 cycles (denaturation at 94°C for 30s→annealing at 56°C for 30s→extension at 68°C for 1.5min), and extension at 68°C for 8min; after the PCR product was subjected to agarose gel electrophoresis and gel recovery and purification, the pJET vector was connected and sequenced. Amplified to obtain the sequence shown in SEQ ID NO.3, a...

Embodiment 2

[0034] Example 2, construction and induced expression of AbODC prokaryotic expression vector

[0035] According to the sequence of the sequenced AbODC coding region, analyze the restriction site contained in the sequence, combine the restriction site carried by the multi-cloning site of the prokaryotic expression vector pET28a(+), design the relevant primers for prokaryotic expression construction, the specific primers are as follows:

[0036] pAbODC-F1:5'-gc ggatcc atggccggccaaacagtcatcg-3' (SEQ ID NO. 4);

[0037] pAbODC-R1:5'-cgc gtcgac gcttggataagcataagcaagg-3' (SEQ ID NO. 5).

[0038] Use the above primers to amplify the AbODC coding region by PCR. Since the upstream primer introduces the BamH I restriction site and the downstream primer introduces the Sac I restriction site, the AbODC coding region is connected into the prokaryotic expression through the BamH I and Sac I restriction sites. Vector pET28a (+) (Geng C, Zhao T, Yang C, et al. Metabolic characterization...

Embodiment 3

[0056] Embodiment 3, construction and transformation of AbODC plant overexpression vector

[0057] According to the sequence of the coding region of the AbODC cloned in Example 1, analyze the restriction sites contained in the sequence, combine with the overexpression vector 1305 + The restriction site carried by the cloning site of the vector, and the relevant primers for overexpression construction are designed, and the specific primers are as follows:

[0058] oeAbODC-F1:5'-gc agatct gatggccggccaaacagtcatcg-3' (SEQ ID NO. 6);

[0059] oeAbODC-R: 5'-gcg gtcacc tcagcttggataagcataagcaagg-3' (SEQ ID NO. 7);

[0060]Use the sequences shown in SEQ ID NO.6 and SEQ ID NO.7 as primers to introduce corresponding enzyme cutting sites in the upstream and downstream of the coding region through PCR amplification. The restriction sites introduced upstream and downstream of the forward fragment are: the upstream introduction of the BglII restriction site and the downstream introduct...

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Abstract

The invention discloses application of atropa-belladonna ornithine decarboxylase to cultivating high-yield hyoscyamine and anisodamine atropa-belladonna varieties. The amino acid sequence coded by theatropa-belladonna ornithine decarboxylase is shown as SEQ ID NO.12, and as the ornithine decarboxylase is over expressed in atropa belladonna, rooting of the atropa belladonna can be improved, and the content of putrescine, N-methyl putrescine and TAs in plants can be increased; by researching AbODC, functions of the AbODC in polyamine and tropane-alkaloid biosynthesis are definite, understandingof biosynthetic pathways of tropane alkaloids can be deepened, and the AbODC has important theoretical value; related results of researching also indicate that the AbODC has the important applicationvalue in the aspects of improving the TAs biosynthetic pathways with a metabolic engineering technology and increasing the TAs content.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to the application of belladonna ornithine decarboxylase in cultivating high-yielding hyoscyamine and anisodamine belladonna varieties. Background technique [0002] Tropine alkaloids are a class of natural alkaline organic compounds with significant physiological activity derived from a small number of Solanaceae plants. They are widely used clinically as anticholinergic drugs for anesthesia, analgesia, cough relief, hypnosis, anti-motion sickness and treatment Parkinson et al. Hyoscyamine, anisodamine, and scopolamine are commonly used clinically. These TAs mainly rely on the extraction from a few TAs medicinal plants of Solanaceae, but the content of TAs in wild tropane-producing plants is generally low, and the content of scopolamine and scopolamine is extremely low, resulting in high commercial production costs of TAs. Therefore, cultivating high-yield plant materials of TAs is t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/66C12N9/88C12P13/00A01H5/00A01H6/82
CPCC12N9/88C12N15/66C12N15/8243C12P13/00C12Y401/01017
Inventor 廖志华陈敏杨春贤曾令江赵腾飞
Owner SOUTHWEST UNIV
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