Method for carrying out cotransformation on double genes of AaPMT and AaTRI to improve content of tropine alkaloid in anisodus acutangulus hairy roots

A co-transformation and alkaloid technology, applied in the field of bioengineering, can solve the shortage of wild resources and other problems, and achieve the effect of alleviating the shortage problem

Inactive Publication Date: 2011-10-12
SHANGHAI NORMAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
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AI Technical Summary

Problems solved by technology

Sanfensan has a long history of 300-400 years as a Chinese herbal medicine for antispasmodic and analgesic in Yunnan. However, due to the large amount of picking, wild resources are very scarce
So far, there are few reports at home and abroad on isolating and cloning the double key enzyme gene from Sanfensan and increasing the content of tropane alkaloids in the hairy roots of Sanfensan through co-transformation strategy

Method used

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  • Method for carrying out cotransformation on double genes of AaPMT and AaTRI to improve content of tropine alkaloid in anisodus acutangulus hairy roots
  • Method for carrying out cotransformation on double genes of AaPMT and AaTRI to improve content of tropine alkaloid in anisodus acutangulus hairy roots
  • Method for carrying out cotransformation on double genes of AaPMT and AaTRI to improve content of tropine alkaloid in anisodus acutangulus hairy roots

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] 1.1. Extraction of total RNA from three-point hairy root and synthesis of cDNA first strand

[0038] The RNA prep pure plant kit provided by TIANGEN was used to extract total RNA from the three-point transgenic hairy roots (refer to the instructions in the kit for the extraction steps). The fresh weight of the three-thirds transgenic hairy root used to extract total RNA is about 0.1g-0.15g, and the DNA in the sample has been removed with DNase working solution during the extraction process. Measure the relative absorbance value of the extracted RNA on a spectrophotometer, and calculate the purity and concentration of the extracted RNA. Finally, 1 μg of RNA was used as the initial amount for each sample, and the first-strand cDNA was synthesized with reverse transcriptase XL (AMV) (for the operation steps, refer to the relevant instructions provided by Promega).

[0039] 1.2. Design of AaPMT and AaTRI coding sequence-specific primers and acquisition of target gene fragm...

Embodiment 2

[0045] Example 2 Construction of plant expression vectors containing three-thirds AaPMT and / or AaTRI genes

[0046] 2.1. Intermediate vector pCAMBIA1304 + build (see figure 1 )

[0047] Using pBI121 and pCAMBIA1304 as materials, construct the plant expression vector pCAMBIA1304 + . Specifically, pBI121 and pCAMBIA1304 were digested with HindIII / EcoRI; the pBI121-GUS expression cassette and the large pCAMBIA1304 fragment were recovered; ligation transformation was carried out, and single-clonal colonies were picked to extract plasmids for digestion verification. The results showed that the plant expression vector pCAMBIA1304 + The build was successful. like figure 1 shown.

[0048] 2.2. Plant expression vector pCAMBIA1304 + - Construction of AaPMT

[0049] The successful pCAMBIA1304 constructed above + Basically, replace the GFP+GUS gene with the AaPMT gene cloned from Sanfensan. Specifically, BglII / BstEII double enzyme cut pMD18T-AaPMT and pCAMBIA1304 + ; Recover A...

Embodiment 3

[0054] Example 3 Agrobacterium rhizogenes mediates the genetic transformation of AaPMT and AaTRI genes to obtain three-thirds of transgenic hairy roots

[0055] 3.1. Containing plant expression vector pCAMBIA1304 + -Acquisition of AaPMT-AaTRI Agrobacterium rhizogenes engineering bacteria

[0056] The plant bivalent expression vector body pCAMBIA1304 containing AaPMT and AaTRI gene in embodiment 2 + -AaPMT-AaTRI was transformed into Agrobacterium rhizogenes C58C1, and a single clone colony was picked for PCR verification. The results showed that the plant expression vector containing AaPMT and AaTRI genes had been successfully constructed in Agrobacterium rhizogenes strain C58C1.

[0057] 3.2. Agrobacterium rhizogenes mediates genetic transformation of AaPMT and AaTRI genes

[0058] 3.2.1. Preculture of explants

[0059] Three-thirds sterile seedling leaves (1cm×1cm) and stems (1cm) explants were cut and cultured on MS solid medium (the medium was in a transparent plastic c...

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Abstract

The invention relates to a method for carrying out cotransformation on double genes of AaPMT and AaTRI to improve the content of tropine alkaloid in anisodus acutangulus hairy roots, and the method comprises the following steps: constructing a plant diplobivalent efficient expression carrier containing AaPMT and AaTRI genes by utilizing encoding frame sequence of rate-limiting enzyme AaPMT and AaTRI genes cloned from the medicinal plant anisodus acutangulus roots, mediating through agrobacterium rhizogene, and carrying out double-gene transformation on the anisodus acutangulus plant, thus obtaining the anisodus acutangulus hairy roots containing transgene AaPMT and AaTRI, wherein the average content of the tropine alkaloid is obviously improved. The invention provides a method for improving the content of the tropine alkaloid in the trisection hairy roots, thus providing more resources and tremendous commercial demands on tropine alkaloid clinically needed.

Description

technical field [0001] The invention relates to a method in the technical field of bioengineering, in particular to a method for increasing the content of tropine alkaloids in three-thirds hairy roots by utilizing a double-key enzyme gene co-transformation metabolic engineering strategy. Background technique [0002] Tropane alkaloids, also known as tropane alkaloids, include: hyoscyamine, anisodamine, scopolamine, and anisodine. Tropine alkaloids are more and more widely used in various clinical subjects. It is a class of anticholinergic drugs acting on the parasympathetic nervous system, and has the functions of anesthesia, spasmolysis, pain relief and anti-shock. In addition, it also has the effect of improving microcirculation, and can be used clinically to treat diseases of microcirculation disorders; it can also be used to treat glaucoma, etc. Anisodamine and anisodine act in addition to the parasympathetic nervous system. In rats, it also has a protective effect on ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/06
Inventor 开国银杨胜周文涛罗秀芹刘源云
Owner SHANGHAI NORMAL UNIVERSITY
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