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Atropa belladonna WRKY transcription factor gene and recombinant plant expression vector and application thereof

A technology for plant expression vectors and transcription factors, which can be used in the improvement of belladonna alkaloids. In the field of recombinant plant expression vectors, it can solve the problems of inability to achieve chemical total synthesis, unclear biosynthesis pathways of secondary metabolites, and high costs.

Active Publication Date: 2018-04-20
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

[0003] The content of most plant secondary metabolites in natural plants is extremely low, but using chemical synthesis method, the process is complicated, the cost is too high, and the biosynthesis pathway of many plant secondary metabolites is not clear and cannot be realized total chemical synthesis

Method used

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  • Atropa belladonna WRKY transcription factor gene and recombinant plant expression vector and application thereof
  • Atropa belladonna WRKY transcription factor gene and recombinant plant expression vector and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Embodiment 1, the cloning of belladonna AbWRKY1 gene

[0024] (1) Extraction of total RNA from belladonna fibrous roots

[0025] Take an appropriate amount of belladonna fibrous root tissue, grind it in liquid nitrogen, add it to a 1.5mL Eppendorf (EP) centrifuge tube filled with lysate, shake it fully, and extract total RNA according to the instructions of the TIANGEN kit. The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and the RNA concentration was determined on a spectrophotometer.

[0026] (2) Cloning of belladonna AbWRKY1 gene

[0027] Using the extracted total RNA as a template, cDNA was synthesized according to the instructions of the Tiangen FastKing cDNA First Strand Synthesis Kit; gene-specific primers were designed according to the sequence of the AbWRKY1 gene, and the specific primers were as follows:

[0028] AbWRKY1-F: 5'-atgcattataactcaacgtt-3' (SEQ ID NO.1);

[0029] AbWRKY1-R: 5'-ttagaatctagagagaaact-3' (SEQ ID...

Embodiment 2

[0032] Embodiment 2, the construction of the recombinant plant expression vector containing AbWRKY1 gene

[0033] In order to study the effect of AbWRKY1 gene on tropine alkaloids in belladonna, the overexpression vector pBI121-AbWRKY1 of AbWRKY1 was constructed. In the vector construction, the restriction site of BamH1 was introduced into the forward primer, and the restriction site of Sac1 was introduced into the reverse primer. The primers are shown in Table 1;

[0034] Table 1, pBI121-AbWRKY1 vector construction primer sequence

[0035] Primer name

Primer sequence (5'→3')

pBI121-AbWRKY1-F (SEQ ID NO.5)

cgcggatccatgcattataactcaacgtt

pBI121-AbWRKY1-R (SEQ ID NO.6)

cgcgagctcttagaatctagagagaaact

[0036] Then, using the correctly sequenced pMD19-T vector containing the AbWRKY1 gene as a template, and the sequences shown in SEQ ID NO: 5 and SEQ ID NO: 6 as primers, KOD plus from TOYOBO Company was used for PCR amplification. The PCR amplif...

Embodiment 3

[0038] Example 3, Agrobacterium rhizogenes-mediated AbWRKY1 overcarrier genetic transformation of belladonna to obtain transgenic belladonna hairy roots

[0039] (1) Acquisition of Agrobacterium rhizogenes Engineering Bacteria Containing pBI121-AbWRKY1 Expression Vector

[0040] The pBI121-AbWRKY1 expression vector in Example 2 was transformed into Agrobacterium rhizogenes (such as C58C1) by the freeze-thaw method, and PCR verification was performed. The results showed that the pBI121-AbWRKY1 expression vector had been successfully constructed into the strain of Agrobacterium rhizogenes.

[0041] (2) Agrobacterium rhizogenes mediates AbWRKY1 gene transformation of belladonna

[0042] a. Belladonna explant preparation

[0043] Belladonna seeds were soaked in ethanol with a volume fraction of 75% for 1 min, then soaked with NaClO with a mass fraction of 50% for 20 min, rinsed with sterile water for 3-4 times, blotted the surface moisture with sterile absorbent paper, and inocu...

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Abstract

The invention relates to an atropa belladonna WRKY transcription factor gene and a recombinant plant expression vector and application thereof. The gene has a specific gene promoter activation role for synthesis of scopolamine, wherein the nucleotide sequence thereof is as shown in SEQ ID NO.3, and the coded amino acid sequence is as shown in SEQ ID NO.4. The AbWRKY1 gene is overexpressed in atropa belladonna, so that the content of scopolamine in atropa belladonna hairy root is increased, therefore, the gene can be applied to improvement of quality of the atropa belladonna, and can increase the content of scopolamine in the atropa belladonna.

Description

technical field [0001] The invention belongs to the field of biotechnology, relates to belladonna WRKY transcription factor, and also relates to a recombinant plant expression vector containing belladonna WRKY transcription factor gene and its application in increasing belladonna alkaloids. Background technique [0002] Tropane alkaloids (TAs) are a class of anticholinergic drugs with great medical value, widely used in anesthesia, analgesia, cough relief, asthma and anti-motion sickness, and also used to control stiffness in Parkinson's disease and tremors. Hyoscyamine and scopolamine are commonly used clinically, and the market demand for both is huge. Among them, scopolamine has weaker side effects, stronger drug effect, and more expensive price. At present, TAs are extracted from a few TAs resource plants of Solanaceae, including Atropa belladonna, Datura stramonium and Hyoscyamus niger. , is also a TAs drug source plant included in the Pharmacopoeia. The mass fractio...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/82A01H5/06A01H6/82
CPCC07K14/415C12N15/8243
Inventor 廖志华陈敏杨春贤邱飞强玮
Owner 成都上交致远生物科技有限公司
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