Xanthohumol-related protein and application thereof in stabilizing products in xanthohumol synthesis pathway

A synthesis route and a technology for fulvicol, applied in the biological field, can solve the problems of low content of picric acid and fulvicol, uneconomical and the like, and achieve the effect of wide application prospect

Active Publication Date: 2019-08-13
INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The problem is that the content of picric acid and xanthohumol in beer is very low (every 500 milliliters of beer contains 0.31 mg of xanthohumol), and in order to achieve health care, adults need to ingest at least 150 mg per day, so h

Method used

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  • Xanthohumol-related protein and application thereof in stabilizing products in xanthohumol synthesis pathway
  • Xanthohumol-related protein and application thereof in stabilizing products in xanthohumol synthesis pathway
  • Xanthohumol-related protein and application thereof in stabilizing products in xanthohumol synthesis pathway

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0069] Application of embodiment 1, XNRP1 and XNRP2 in xanthohumol synthetic pathway

[0070] This embodiment provides two kinds of proteins from hops (Freshops Company), whose names are XNRP1 and XNRP2 respectively, the amino acid sequence of XNRP1 is sequence 1 in the sequence listing, and its coding gene in hops is the sequence in the sequence listing The DNA molecule shown in 2 (this DNA molecule is recorded as XNRP1 gene); The amino acid sequence of XNRP2 is sequence 3 in the sequence listing, and its coding gene in hops is the DNA molecule shown in sequence 4 in the sequence listing (the DNA The molecule is denoted as XNRP2 gene).

[0071] 1. In this example, the xanthohumol synthesis pathway was reorganized in yeast cells to detect the function of XNRP1 and XNRP2 in the synthesis of xanthohumol. The specific method is as follows:

[0072] 1. Construction of yeast expression vector

[0073] Replace the DNA fragment between the Not I and Pac I recognition sequences of t...

Embodiment 2

[0141] Enzyme activity of chalcone isomerase of embodiment 2, XNRP1 and XNRP2

[0142] Using the XNRP1-His and XNRP2-His obtained in Example 1 to detect whether XNRP1 and XNRP2 have the enzyme activity of chalcone isomerase, the 500 μL chalcone isomerase enzyme activity detection system is as follows:

[0143] To the reaction system obtained by adding corresponding substances to 50mM Tris-HCl (pH is 6.4 or 7.5, two kinds of pH values ​​are set in total), each substance and its final concentration added in the reaction system are respectively: 5% (v / v ) ethanol, 50 μ M substrate (naringenin chalcone or isoliquiritigenin, a substrate for each reaction system, naringenin chalcone (ChromaDex company, item number ASB-00014207-010 10mg); isoliquiritigenin ( sigma company, product number I3766-10MG)), 0.1 mg / mL BSA and 1 μg XNRP1-His or XNRP2-His, one protein for each reaction system, and a system without adding XNRP1-His and XNRP2-His was used as a control.

[0144] Incubate the ab...

Embodiment 3

[0146] Example 3, Analysis of the expression of XNRP1 and XNRP2 genes in various tissues

[0147] Seven tissues of hop root, stem, young leaf, old leaf, bract, cone and glandular hair were selected, RNA was extracted with RNA extraction kit, and after reverse transcription into cDNA, fluorescent quantitative PCR method was used, with GAPDH as Internal reference genes, to determine the relative expression levels of XNRP1 gene and XNRP2 gene transcription level in each tissue.

[0148] The primers used for the XNRP1 gene were: 5'-GGCAACTGCCCTCAACTC, 3'-CCTTAATTCCCATGCCAAGT.

[0149] The primers used for the XNRP2 gene were: 5'-ACTTGGACTCCGATGTTGTGA, 3'-CCGTATTGGGACCCTTTGA.

[0150] The primers used for the GAPDH gene were: 5'-GCACTGGAGCTGCTAAGGCTG, 3'-CCTGCTGTCACCAATGAAGTCG.

[0151] The results showed that both XNRP1 and XNRP2 were specifically highly expressed in glandular trichomes ( Figure 5 )

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Abstract

The invention discloses xanthohumol-related protein and application thereof in stabilizing products in a xanthohumol synthesis pathway. The disclosed xanthohumol-related protein is the following A1) or A2) or A3), wherein A1) is a protein with the amino acid sequence shown as a sequence 1; A2) is a functionally identical protein obtained after substitution and/or deletion and/or addition of one ormore amino acid residues of the amino acid sequence shown as the sequence 1 in a sequence table; A3) is a fusion protein obtained by linking the N end or/and the C end of A1) or A2) with a label. Experiments show that the xanthohumol-related protein has the function of stabilizing the products in the xanthosole synthesis pathway and has a broad application prospect in the fields of flavonoid compounds, demethyl xanthohumol synthetic biology application, human health care and the like.

Description

technical field [0001] The invention relates to a xanthohumol-related protein and its application in stabilizing a product in a xanthohumol synthesis pathway in the field of biotechnology. Background technique [0002] Hops (also known as Hubu, hops, etc., Humulus lupulus L., is diploid, 2n=2x=20) belongs to the genus Humulus of the family Cannabis in the order Rosaceae, is a perennial climbing herb, dioecious, and the female flowers are made of The bracts are arranged in a cone-like inflorescence, 3 to 4 cm long, with a large number of yellow glandular hairs growing under the inner bracts. The female flower is mainly used in brewing beer, and is the main source of the unique aroma and bitterness of beer, and is considered to be the "green gold" of beer brewing. In 2011, the global beer sales revenue was more than 150 billion US dollars. At present, China has become the world's largest beer production and consumption country. In 2011, China's beer market sales revenue was ...

Claims

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Application Information

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IPC IPC(8): C07K19/00C12N9/90C12P7/26C12P17/06
CPCC12N9/90C12Y505/01006C12P7/26C12P17/06C07K2319/00
Inventor 王国栋班兆男
Owner INST OF GENETICS & DEVELOPMENTAL BIOLOGY CHINESE ACAD OF SCI
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