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Belladonna wrky transcription factor gene and its recombinant plant expression vector and application

A plant expression vector and transcription factor technology, applied in the field of improving the application of belladonna alkaloids and recombinant plant expression vector, can solve the problems of inability to achieve full chemical synthesis, high cost, and unclear biosynthetic pathways of secondary metabolites.

Active Publication Date: 2019-03-29
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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  • Belladonna wrky transcription factor gene and its recombinant plant expression vector and application
  • Belladonna wrky transcription factor gene and its recombinant plant expression vector and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1. Cloning of the belladonna AbWRKY1 gene

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

[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 containing lysis buffer, and after sufficient shaking, extract total RNA according to the instructions of the TIANGEN kit. The total NA mass was identified by formaldehyde denaturing gel electrophoresis, and the RNA concentration was determined on a spectrophotometer.

[0026] (2) Cloning of AbWRKY1 gene of belladonna

[0027] Using the extracted total RNA as a template, synthesize cDNA according to the instructions of Tiangen FastKing cDNA first-strand synthesis kit; design gene-specific primers according to the sequence of the AbWRKY1 gene, and the specific primers are as follows:

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

[0029] AbWRKY1-R: 5'-ttagaatctagagagaaact-3' (SEQ ID NO. 2); ...

Embodiment 2

[0032] Example 2. Construction of recombinant plant expression vector containing AbWRKY1 gene

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

[0034] Table 1. Primer sequences for pBI121-AbWRKY1 vector construction

[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, PCR amplification was carried out using KOD plus of TOYOBO Company, and the PCR ampli...

Embodiment 3

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

[0039] (1) Obtainment of Agrobacterium rhizogenes engineering bacteria containing pBI121-AbWRKY1 expression vector

[0040] The pBI121-AbWRKY1 expression vector in Example 2 was transferred into Agrobacterium rhizogenes (such as C58C1) by freeze-thaw method, and verified by PCR. The results show that the pBI121-AbWRKY1 expression vector has been successfully constructed into the Agrobacterium rhizogenes strain.

[0041] (2) Agrobacterium rhizogenes-mediated transformation of belladonna with AbWRKY1 gene

[0042] a. Belladonna explant preparation

[0043] Belladonna seeds were soaked in ethanol with a volume fraction of 75% for 1 min, then soaked in NaClO with a mass fraction of 50% for 20 min, rinsed with sterile water for 3-4 times, dried with sterile absorbent paper, and inoculated in hormone-free 1 / 2MS (Murashige and Skoog,...

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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 a belladonna WRKY type transcription factor, and also relates to a recombinant plant expression vector containing a belladonna WRKY type transcription factor gene and an application in improving belladonna alkaloids. Background technique [0002] Tropine alkaloids (tropane alkaloids, TAs) are a class of anticholinergic drugs with great medical value, widely used in anesthesia, analgesia, cough, asthma and anti-motion sickness, also used to control the stiffness of Parkinson's disease and tremors. Hyoscyamine and scopolamine are commonly used clinically, both of which have huge market demands. Among them, scopolamine has weaker side effects, stronger efficacy and higher price. At present, TAs are extracted from a few Solanaceae TAs resource plants, including Atropa belladonna, Datura stramonium and Hyoscyamus niger. Belladonna is the main commercial cultivated medicinal source of scopolami...

Claims

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Application Information

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Patent Type & Authority Patents(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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