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Anisodus luridus ornithine decarboxylase ALODC gene as well as recombinant expression vector and application thereof

A technology of ornithine decarboxylase and expression vector, applied in the field of vector and application containing the gene

Active Publication Date: 2018-12-18
TIBET AGRI & ANIMAL HUSBANDRY COLLEGE +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no related report about the overexpression of endogenous ODC gene in Lingdangzi

Method used

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  • Anisodus luridus ornithine decarboxylase ALODC gene as well as recombinant expression vector and application thereof
  • Anisodus luridus ornithine decarboxylase ALODC gene as well as recombinant expression vector and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0022] Embodiment 1, the cloning of bell zipper ornithine decarboxylase ALODC gene

[0023] (1) Extraction of the total RNA of the bell subgenome

[0024] The total RNA of Bell subgenome was extracted using RNA kit from TIANGEN Company. Take 50-100 mg of plant fibrous roots and grind them into powder quickly in liquid nitrogen, add 550 μL of lysis solution RL (mercaptoethanol has been added), and vortex vigorously to mix. Centrifuge at 9800rpm for 5min, transfer 450μL to filter column CS, place CS in a collection tube, centrifuge at 12000rpm for 5 minutes, carefully draw 400μL of the supernatant in the collection tube into an RNase-free centrifuge tube, slowly add 0.5 times the volume of the supernatant mixed with absolute ethanol, transferred to the adsorption column CR3, centrifuged at 12,000 rpm for 1 min, discarded the waste liquid, and put CR3 back into the collection tube. Add 350 μL protein-removing solution RW1 to CR3, centrifuge at 12,000 rpm for 1 min, discard the ...

Embodiment 2

[0030] Embodiment 2, construction contains the plant expression vector of AlODC gene and construction of engineering bacterium

[0031] First, the ALODC gene cloned in Example 1 was connected to the PJET carrier (Dalian Takara Company) to construct the intermediate carrier PJET-AlODC; then the intermediate carrier PJET-AlODC was digested with (BamH I / Sac I), and simultaneously The expression vector pBI121 was digested with restriction enzymes, the AlODC gene fragment and the large fragment of the pBI121 vector were recovered, ligated and transformed, single clones were picked, and the plasmid was extracted for PCR detection and enzyme digestion verification to obtain a plant expression vector containing the AlODC gene, referred to as AlODC-pBI121.

[0032] The recombinant expression vector AlODC-pBI121 was transformed into Agrobacterium tumefaciens (EHA105), and positive clones were screened and verified by PCR.

Embodiment 3

[0033] Embodiment 3, Agrobacterium tumefaciens mediates ALODC gene transformation plant and transgene screening

[0034] (1) Pre-cultivation of explants; the GA3 solution of 1g / L of the seeds of the bell seed was soaked overnight, rinsed slightly with tap water, and then sterilized with 70% ethanol for 1min, and then decontaminated with 50% sodium hypochlorite solution. Bacteria for 10 minutes, rinsed with sterile water several times, and inoculated on MS+200mg / L Cef solid medium without adding hormones. 25C, 16h / 8h (light / dark) light conditions and culture for about 15 days, the seeds germinate two cotyledons, the hypocotyl is about 1cm long, and can be used for genetic transformation.

[0035] (2) Co-cultivation of Agrobacterium activation and explants: Pick a single clone of Agrobacterium EHA105-pBI121-AlODC from the streaked plate, inoculate it in 15mL YEP (Rif+Str+Kan) liquid medium, 28°C, 200r / min shaking culture for 24-48h; 5000rpm centrifuge for 6min to collect the ba...

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Abstract

The invention relates to an anisodus luridus ornithine decarboxylase ALODC gene as well as a recombinant expression vector and application thereof. By cloning the anisodus luridus ornithine decarboxylase ALODC gene, through an agrobacterium tumefaciens mediation method, the gene is transferred into anisodus luridus, a transgenic plant is obtained, after ALODC over-expression is conducted, the contents of putrescine and tropane alkaloids in the anisodus luridus plant can be significantly improved, and an ideal method is provided for effectively producing hyoscyamine and scopolamine by means ofa transgenic technology.

Description

technical field [0001] The invention belongs to the field of biological technology, and relates to the gene of ornithine decarboxylase ALODC of bell pepper, and also relates to the carrier containing the gene and its application. Background technique [0002] Tropane alkaloids (TAs) are a class of secondary metabolites derived from a few Solanaceae plants. Because of their significant pharmacological activity, they have been used as traditional medicines for nearly 3000 years. Hyoscyamine (or its racemate atropine) and scopolamine are commonly used in modern clinical practice, including anisodamine, which is less effective. They are all anticholinergic drugs that act on the parasympathetic nervous system Drugs, mainly used for analgesia, anesthesia, anti-motion sickness drugs, treatment of Parkinson's disease, improvement of microcirculation, detoxification, treatment of pesticide poisoning, etc., the market demand is very huge, among which scopolamine has stronger efficacy,...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/60C12N15/70C12N15/82A01H5/00A01H6/82C12N9/88
CPCC12N9/88C12N15/8243C12Y401/01017
Inventor 兰小中廖志华陈敏赵腾飞
Owner TIBET AGRI & ANIMAL HUSBANDRY COLLEGE
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