CN-121518510-B - Chalcone isopentenyl transferase gene GiPT, giPT protein, amplification primer set and application

CN121518510BCN 121518510 BCN121518510 BCN 121518510BCN-121518510-B

Abstract

The invention provides chalcone isopentenyl transferase genes GiPT, giPT protein, an amplification primer set and application, and belongs to the technical field of biological genes. The invention successfully digs and functionally identifies the key aromatic prenyltransferase-GiPT participating in prenylation modification of chalcone active ingredients based on the whole genome sequencing data of Glycyrrhiza uralensis (Glycyrrhiza inflata) by adopting a reverse genetics strategy. The enzyme is proved to be capable of specifically catalyzing biosynthesis of psoralen and licochalcone C by taking DMAPP as a donor, is a first characterized chalcone isopentenyl transferase in liquorice, fills a gap of key enzyme research of the species in a chalcone isopentenyl metabolic pathway, and provides an important basis for deeply analyzing molecular mechanisms formed by quality of liquorice medicinal materials.

Inventors

XIANG LI
Mi Yaolei
JI ZUEN
LUO HENGLEI
ZHANG HUJUAN
GAO RANRAN
SUN XIAOTONG
CAO XUE
SHI YUHUA
YIN QINGGANG
WANG MENGYUE
CHU YANG
WU LAN

Assignees

中国中医科学院中药研究所

Dates

Publication Date: 20260512
Application Date: 20260116

Claims (5)

1. The GiPT protein expressed by the chalcone isopentenyl transferase gene GiPT is characterized in that the amino acid sequence of the GiPT protein is shown as SEQ ID NO. 2.
2. A chalcone isopentenyl transferase gene GiPT has a nucleotide sequence shown in SEQ ID NO. 1.
3. Use of the chalcone isopentenyl transferase GiPT protein of claim 1 as a chalcone isopentenyl transferase.
4. Use of the GiPT protein of claim 1 for catalyzing the production of psoralen from the substrate isoliquiritigenin.
5. Use of the GiPT protein of claim 1 for catalyzing the formation of licochalcone C from the substrate licochalcone.

Description

Chalcone isopentenyl transferase gene GiPT, giPT protein, amplification primer set and application Technical Field The invention relates to the technical field of biological genes, in particular to chalcone isopentenyl transferase genes GiPT, giPT protein, an amplification primer group and application. Background Licochalcone (Licochalcone) is a characteristic secondary metabolite mainly existing in Glycyrrhiza glabra, structurally forms a 'trans' -chalcone skeleton by connecting two aromatic rings through alpha, beta-unsaturated ketone, and often has isopentenyl modification. Wherein, the components such as licochalcone A, B, C and the like show remarkable biological activities such as anti-tumor, anti-virus (such as respiratory syncytial virus inhibition) and the like, and the structural derivative also has good patent medicine potential. However, the current method for obtaining licochalcone components still highly depends on plant extraction, so that not only is the efficiency low and the cost high, but also the pressure on wild licorice resources is increased, and the requirements of high-value development and industrialization are difficult to meet. Research shows that prenylation modification of licochalcone components in licorice is a key step for enhancing structural diversity and biological activity. This type of reaction is mainly catalyzed in plants by the UbiA superfamily aromatic prenyl transferase (PHB-type UbiA Prenyltransferases, PT). Such membrane bound enzymes are capable of transferring isopentenyl groups onto electron rich aromatic acceptors with the aid of Mg 2+ using dimethylallyl pyrophosphate (DMAPP) or the like as a donor. Although UbiA-PT has been studied for its metabolic pathways such as flavones and coumarins, its role in the prenylation of chalcones, particularly licochalcone, has not been reported. Notably, ubiA-PT generally exhibits a high degree of substrate or donor specificity and functions vary significantly from species to species. For example, hpPT px in Hypericum perforatum recognizes only xanthone substrates and specifically utilizes DMAPP, whereas two closely related PTs in Murraya koenigii exhibit distinct substrate preferences. These properties make functional predictions based on homology alignment less reliable and must be validated one by experimental means. UbiA-PT is more challenging to screen genes and identify functions than glycosyltransferases or methyltransferases due to membrane localization, difficult expression and complex functions, and the related studies are still very limited. Therefore, in order to achieve sustainable, efficient production of licochalcone components, there is a need to systematically mine and identify key UbiA-PT genes involved in their prenylation modifications. The method is not only helpful for elucidating the biosynthesis mechanism of licochalcone, but also lays a foundation for realizing the green manufacture of the high-value active ingredients by a subsequent synthetic biological means (such as microbial heterologous synthesis or plant metabolic engineering), and has important significance for protecting wild resources and promoting the modernization of traditional Chinese medicines. Disclosure of Invention The invention aims to provide chalcone isopentenyl transferase genes GiPT and GiPT proteins, an amplification primer set and application thereof, and fills the terminal blank of licochalcone biosynthesis paths in liquorice. In order to achieve the above object, the present invention provides the following technical solutions: the invention provides a chalcone isopentenyl transferase gene GiPT, wherein the chalcone isopentenyl transferase gene GiPT comprises one or more of the following sequences: (1) A DNA molecule having a gene sequence shown in SEQ ID NO. 1; (2) A DNA molecule having a homology of 80% or more with the gene sequence shown in SEQ ID NO. 1; (3) A DNA molecule which hybridizes with the gene sequence shown in SEQ ID NO. 1. The invention also provides GiPT protein expressed by the chalcone isopentenyl transferase gene GiPT, wherein the GiPT protein comprises one or more of the following sequences: (1) A protein having an amino acid sequence shown in SEQ ID NO. 2; (2) Has more than 80 percent of sequence similarity with the amino acid sequence shown in SEQ ID NO.2, and has the protein which is synthesized by catalyzing chalcone compounds and is derived from (1). The invention also provides an application of the chalcone isopentenyl transferase GiPT protein as an isopentenyl transferase. The invention also provides an application of the chalcone isopentenyl transferase gene GiPT or GiPT protein in catalyzing substrate isoliquiritigenin to generate psoralen. The invention also provides an application of the chalcone isopentenyl transferase gene GiPT or GiPT protein in catalyzing substrate licarpa chalcone to generate licochalcone C. The invention also provides a primer pair for amplifying the chalcone isopente