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Atropa belladonna calmodulin AbCaM1 gene and recombinant plant expression vector and applications thereof

A plant expression vector, calmodulin technology, applied in the application of belladonna calmodulin AbCaM1 gene in increasing the alkaloid content of belladonna, recombinant plant expression vector field, to achieve the effect of stabilizing new drug sources

Active Publication Date: 2019-06-04
成都上交致远生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CaM can target a large number of transcription factors to regulate its activity, but whether overexpression of CaM can increase the content of scopolamine and scopolamine in belladonna has not been reported

Method used

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  • Atropa belladonna calmodulin AbCaM1 gene and recombinant plant expression vector and applications thereof
  • Atropa belladonna calmodulin AbCaM1 gene and recombinant plant expression vector and applications thereof
  • Atropa belladonna calmodulin AbCaM1 gene and recombinant plant expression vector and applications thereof

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] Embodiment 1, the cloning of belladonna AbCaM1 gene

[0023] (1) Extraction of total RNA from belladonna genome

[0024] The total RNA of belladonna genome was extracted using the RNA kit from TIANGEN company. The specific steps are as follows: take 50-100 mg belladonna fibrous roots and grind them into powder quickly in liquid nitrogen, add 550 μL lysate RL (mercaptoethanol has been added), and vortex vigorously Shake and mix, centrifuge at 9800rpm for 5min, transfer 450μL to filter column CS, place CS in a collection tube, centrifuge at 12000rpm for 5min, carefully draw 400μL of the supernatant in the collection tube into an RNase-free centrifuge tube, slowly add 0.5 Double the volume of supernatant with absolute ethanol, mix well, transfer to adsorption column CR3, centrifuge at 12,000 rpm for 1 min, discard 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 wa...

Embodiment 2

[0030]Embodiment 2, construct the recombinant plant expression vector and engineering bacterium containing AbCaM1 gene

[0031] The amplified AbCaM1 gene (its DNA sequence is shown in SEQ ID NO.1) was connected to the PJET vector by ligase to obtain the intermediate vector pJET-AbCaM1, which was sequenced by Shanghai Sunny Biotechnology Co., Ltd. to confirm the correctness of the gene .

[0032] Then the intermediate vector PJET-AbCaM1 and the expression vector pBI121 were digested with BamH I and Sac I, the AbCaM1 gene fragment and the large fragment of the pBI121 vector backbone were recovered, ligated and transformed into Escherichia coli DH5α, positive clones were screened, and then the plasmid was extracted for PCR detection and analysis. After digestion and verification, a recombinant plant expression vector containing the AbCaM1 gene was obtained, which was named pBI121-AbCaM1 vector.

[0033] Transform the pBI121-AbCaM1 vector into Agrobacterium tumefaciens (such as E...

Embodiment 3

[0034] Embodiment 3, Agrobacterium tumefaciens mediates AbCaM1 gene to transform belladonna

[0035] Pre-cultivation of explants: Soak belladonna seeds overnight in 1g / L GA3 solution, rinse with tap water for a while, then disinfect with 70% ethanol for 1min, and then use 50% mass fraction of Sodium hypochlorite solution was sterilized for 10 minutes, then washed several times with sterile water, inoculated on MS + 200mg / L Cef solid medium without adding hormones; cultured at 25°C, 16h / 8h (light / dark) under light conditions for about At about 15 days, two cotyledons germinated from the seeds, and the hypocotyls were taken out, about 1 cm in length, for genetic transformation.

[0036] Co-cultivation of Agrobacterium and explants: pick Agrobacterium EHA105-pBI121-AbCaM1 single clone from the streak culture plate, inoculate in 15mL containing Rif(20mg / L)+Str(50mg / L+Kan(50mg / L) L) in YEP liquid medium, 28°C, 200r / min shaking culture for 24-48h; 5000rpm centrifugation for 6min to...

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Abstract

The invention relates to an atropa belladonna calmodulin AbCaM1 gene and a recombinant plant expression vector and applications thereof. The nucleotide sequence of the AbCaM1 gene is as shown in SEQ ID NO.1. A gene engineering method is adopted, and the AbCaM1 gene is transferred into atropa belladonna plants through the mediation of agrobacterium tumefaciens. Compared with wild atropa belladonna,the contents of hyoscyamine and scopolamine in the trans-AbCaM1 gene atropa belladonna are obviously improved. The establishment of the method provides a new idea for reducing the production cost andefficiently producing the hyoscyamine and the scopolamine.

Description

technical field [0001] The invention belongs to the field of plant metabolism engineering, and relates to a belladonna calmodulin AbCaM1 gene, a recombinant plant expression vector containing the gene, and an application of the belladonna calmodulin AbCaM1 gene in increasing the alkaloid content of belladonna. 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. Duboisia hybrid and Atropa belladonna are the two most important TAs resource plants, among which belladonna is a TAs source plant included in the Chinese Pharmacopoeia (Chinese Pharmacopoeia, 2015). However, hyoscyamine (or its racemate atropine) and scopolamine are commonly used in modern clinical practice, including anisodamine, which has a slightly lower drug effect, and they all act on parasympathetic nerves. System...

Claims

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Application Information

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IPC IPC(8): C12N15/29C12N15/82C07K14/415
Inventor 廖志华陈敏杨春贤赵腾飞强玮
Owner 成都上交致远生物科技有限公司
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