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Glycyrrhiza uralensis chalcone synthetase, encoding gene and application thereof

A chalcone synthase and gene technology, applied in the field of plant molecular biology, to achieve the effects of enhanced gene expression, great economic value and far-reaching significance

Inactive Publication Date: 2008-07-16
BEIJING WEIMING KAITUO CROP DESIGN CENT COMPANYLIMITED
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In licorice, the cloning of chalcone synthase gene has not been reported

Method used

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  • Glycyrrhiza uralensis chalcone synthetase, encoding gene and application thereof
  • Glycyrrhiza uralensis chalcone synthetase, encoding gene and application thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0030] Embodiment 1, the separation of chalcone synthase gene CHS7G cDNA

[0031] 1. Preparation of plant material and isolation of total RNA

[0032] The mature seeds of Glycyrrhiza uralensis were sterilized and germinated on 1 / 2MS medium, and cultured aseptically for about 20 days in a culture room with a constant temperature of 25°C and a light cycle of 16 hours. The plants grew robustly and were ready for use in total RNA extract.

[0033] Invitrogen’s Trizol reagent was used to extract total RNA from the material. The entire operation process was guaranteed to be free of RNase contamination and strictly followed the RNA extraction process instructions of Trizol reagent. The extracted RNA was divided into small portions and frozen at -80°C for later use.

[0034] 2. Acquisition of CHS7G gene sequence:

[0035] Primers were designed according to the sequence of soybean chalcone synthase gene CHS7 (GenBank Accession No.M98871):

[0036] Gmchs7F: 5'-AGGAAAGATGGTTAGCGTAGC-3...

Embodiment 2

[0059] Example 2. Expression vector construction and transformation of licorice hairy roots mediated by Agrobacterium rhizogenes A4.

[0060] 1. Construction of plant expression vector pXQ-35s-CHS7G

[0061] The licochalcone synthase gene CHS7G was excised from the T vector using the enzyme cutting site XbaI / SacI, and after the plant expression vector pXQ-35s with the 35s promoter and nos terminator was digested with XbaI / SacI ( pCambia1301 obtained after knocking out the GUS gene) was connected to construct the expression vector pXQ-35s-CHS7G (as shown in Figure 1).

[0062] 2. Transform the recombinant plasmid pXQ-35s-CHS7G into Agrobacterium rhizogenes A4 by freeze-thaw method

[0063] A. Take 2-3 μl plasmid (concentration 1 μg / μl) to transform the freshly prepared Agrobacterium rhizogenes A4 competent cells, and place on ice for 30 minutes;

[0064] B. Immerse in liquid nitrogen for 5 minutes, 37 ℃ water bath for 5 minutes, add 500 μl of empty YEB (beef extract 5g / L, yeast...

Embodiment 3

[0070] Embodiment 3, molecular detection of transgenic hairy root system

[0071] Randomly select 7 licorice hairy root lines obtained after resistance screening in Example 2, and use the CTAB method to extract total DNA (with reference to "Molecular Cloning Experiment Guide"); use the DIG DNA Labeling and Detection kit of Roche Company to extract Octin labeled CHS7G gene fragment was used as a probe for Southern hybridization. All operations were carried out in strict accordance with the instructions of the kit. The hybridization results are shown in Figure 2. In Fig. 2, M is the DNA molecular weight standard (λDNA / HindIII); + is the positive control recombinant plasmid pXQ-35s-CHS7G; fx1 is the wild-type hairy root of the negative control non-transformed exogenous gene; others are obtained after resistance screening 7 licorice hairy root strains. The hybridization results showed that all 7 of the selected transgenic hairy root systems were positive, and all of them were m...

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Abstract

The invention discloses a chalcone synthase which is derived from a Ural licorice and the coding gene thereof. The experiments show that after the licorice chalcone synthase gene is transformed in a licorice hairy root, the expression of the chalcone synthase gene is strengthened, and compared with the hairy root which is not transformed, the total flavonoid content in the corresponding licorice hairy root can be improved by about 2.5 times. The chalcone synthase is a first key enzyme in the metabolism path of the flavonoid compounds in plants; the licorice chalcone synthase gene which is provided by the invention provides an effective technical means for the synthesis of the flavonoid compounds in the genetically engineered plants, which has broad application prospect and great economic value, and the invention can lead the licorice to obtain more broad application in the fields of medicine, health care products, food and cosmetics.

Description

technical field [0001] The invention relates to a chalcone synthase gene and application thereof, in particular to a chalcone synthase gene in Glycyrrhizauralensis Fisch and its application in increasing the total flavonoid content of plants, and belongs to the field of plant molecular biology. Background technique [0002] Flavonoids are widely distributed in the plant kingdom, and most of them are combined with sugars to form glycosides or carbon sugar groups in plants, and some exist in free forms. The core of natural flavonoids often contains substituents such as hydroxyl, methoxy, alkoxy, isopentenyloxy and the like. Due to the existence of these auxiliary chromophores, the compounds are mostly yellow. [0003] Flavonoids have a wide range of important physiological functions, and are the main active ingredients of many Chinese herbal medicines. They are widely used in clinical treatment and health food with flavonoids as functional factors. In terms of pharmacy, ther...

Claims

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Application Information

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IPC IPC(8): C12N9/00C12N15/52C12N15/82C12N15/65A01H1/00
Inventor 周骅刘敬梅张海超夏勉李毅
Owner BEIJING WEIMING KAITUO CROP DESIGN CENT COMPANYLIMITED
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