P-coumaroyl ester 3'-hydroxylase gene LjC3'H in lonicera japonica thumb. and application thereof

A technology of coumaryl ester and honeysuckle, applied in application, genetic engineering, plant gene improvement, etc., can solve the problems of unreported research on the relationship with chlorogenic acid, cloning, and functional verification.

Inactive Publication Date: 2013-04-17
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0010] Although the p-coumaryl ester 3'-hydroxylase gene has been cloned in many species, the cloning and functional verification of the gene for Lonicera Lonicerae has not been reported, and the relationship between the gene and chlorogenic acid There is also no report on the relationship

Method used

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  • P-coumaroyl ester 3'-hydroxylase gene LjC3'H in lonicera japonica thumb. and application thereof
  • P-coumaroyl ester 3'-hydroxylase gene LjC3'H in lonicera japonica thumb. and application thereof
  • P-coumaroyl ester 3'-hydroxylase gene LjC3'H in lonicera japonica thumb. and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Cloning of embodiment 1, LjC3'H

[0027] 1.1 Extraction of honeysuckle total RNA

[0028] (1) Put the honeysuckle material into a pre-cooled mortar, add liquid nitrogen and quickly grind it into a uniform powder. Pay attention to ensure that the material is immersed in liquid nitrogen during the grinding process.

[0029] (2) After the liquid nitrogen volatilizes, quickly transfer the material to a pre-cooled centrifuge tube, add 1ml TRIZOL solution for every 50-100mg of tissue material, mix well, place it at room temperature for 5min, centrifuge at 12000r / min, 2-8℃ for 10min precipitation.

[0030] (3) Add 0.2ml chloroform to every 1ml TRIZOL solution, oscillate for 15sec to mix thoroughly, place at room temperature for 5min, centrifuge at 12000r / min, 2-8°C for 15min.

[0031] (4) Take the supernatant, add 0.5ml of isopropanol to every 1ml of TRIZOL, mix well, and place at room temperature for 10-20min.

[0032] (5) Centrifuge at 12,000 r / min at 2-8°C for 10 minute...

Embodiment 2

[0109] Example 2, Functional verification of honeysuckle p-coumaroyl ester 3'-hydroxylase gene LjC3'H

[0110] 2.1 Construction of prokaryotic expression vector

[0111] 2.1.1 Obtaining the target gene

[0112] (1) Primer design: Design a pair of LjC3'H genes with EcoRI and XhoI restriction sites for PCR amplification.

[0113] P2:5'-G AATTCA TGGCATTCGCTCTCCTC-3'

[0114] EcoRI

[0115] P3:5'-C TCGAGC TAGTAATCCACTGGGACACG-3'

[0116] wxya

[0117] (2) PCR reaction system:

[0118]

[0119] (3) The PCR reaction conditions are

[0120] 94°C for 5min; 94°C for 30sec, 60°C for 30sec, 72°C for 2min, 35 cycles; finally 72°C extension for 7min. The PCR products were recovered by electrophoresis and used in subsequent reactions. PCR product electrophoresis as figure 1 shown.

[0121] 2.1.2 Construction steps of prokaryotic expression vector

[0122] The prokaryotic expression vector pET28a (purchased from Treasure Bioengineering Co., Ltd.) was introduced, and the vec...

Embodiment 3

[0149] Example 3, LjC3'H protein induction expression and protein purification of Lonicera japonica

[0150] 3.1 Induced expression of target protein

[0151] Escherichia coli BL21 single clones containing recombinant expression plasmids pETLjC3'H and pET28a were respectively inserted into 5 mL LB medium containing 50 μg / mL kanamycin, cultured overnight at 37 ° C, diluted 100 times the next day and continued to culture to OD 600 When it was 0.6, IPTG (Isopropyl-beta-D-thio galactoside) with a final concentration of 0.5mmol / L was added, and the expression was induced at 28°C for 4h. Bacterial cultures were harvested by centrifugation, resuspended in phosphate buffer (50mmol / L PBS, pH 7.2), crushed on ice by ultrasonication, and detected by SDS-PAGE for total and soluble proteins.

[0152] 3.2 Purification of target protein

[0153] Pass the supernatant over chelated Ni 2+ Nickel Sepharose column (5ml), and then reduce the concentration of urea by gradient (so that the concen...

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PUM

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Abstract

The invention discloses a p-coumaroyl ester 3'-hydroxylase gene LjC3'H in lonicera japonica thumb. and a prokaryotic expression vector containing the gene LjC3'H, and also discloses application of the p-coumaroyl ester 3'-hydroxylase gene LjC3'H in lonicera japonica thumb. in preparation of chlorogenic acid. Experiments prove that coumaroyl quinic acid and coumaroyl shikimic acid can be respectively catalyzed into chlorogenic acid and caffeoyl shikimic acid by adding an LjC3'H protein purified in vitro into an enzymatic reaction system, so that a theoretical foundation and a practical basis for developing, producing and increasing the content of a target product namely chlorogenic acid are provided.

Description

technical field [0001] The invention belongs to the technical field of biogenetic engineering, and in particular relates to a honeysuckle p-coumaroyl ester 3'-hydroxylase gene LjC3'H and an application thereof. Background technique [0002] Plant secondary metabolism refers to the process that some organisms use certain primary metabolites as "raw materials" to form some special chemical substances under the catalysis of a series of enzymes. These special chemical substances are secondary metabolites, such as Alkaloids, flavonoids, terpenoids, organic acids, lignin, etc., are a large class of small molecular organic compounds that are not necessary for plant growth, but play an irreplaceable role in the survival and development of plants in complex environments. role. [0003] Plant secondary metabolites have important economic value. Many clinically applied drugs are derived from secondary metabolites of plants. About 25% of western clinical medicines are secondary metab...

Claims

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Application Information

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IPC IPC(8): C12N15/53C12N15/70C12N15/74C12P7/62
Inventor 向凤宁蒲高斌王鹏周冰谦
Owner SHANDONG UNIV
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