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CN-121975840-A - Method for improving content of linarin in chrysanthemum through transferring gene OlOMTs of schizophragma integrifolium

CN121975840ACN 121975840 ACN121975840 ACN 121975840ACN-121975840-A

Abstract

The invention discloses a method for improving the linarin content of cultivated chrysanthemum by a transgenic technology, which comprises cloning two key O-methyltransferase genes OlOMT and OlOMT310 related to linarin synthesis in the long-split-leaf chrysanthemum, constructing the genes into a plant super-expression vector, transforming cultivated chrysanthemum by an agrobacterium-mediated method, screening transgenic plants and detecting the linarin content. Experiments prove that the content of the linarin in the chrysanthemum plants transformed with the genes is obviously improved compared with that of wild chrysanthemum plants, and the potential standard of medicinal development is reached. The invention identifies and verifies the two key genes responsible for the biosynthesis of the linarin in the long-split-leaf chrysanthemum for the first time, introduces the functions of the two key genes into the cultivated chrysanthemum through a genetic engineering means, creates a new chrysanthemum germplasm with high linarin content on the premise of not directly using endangered species, and provides a safe and efficient raw material source for developing chrysanthemum source medicaments or health care products with anti-inflammatory and antioxidant effects.

Inventors

  • JIANG JIAFU
  • Jing Ruyue
  • FANG WEIMIN
  • WANG ZHENXING
  • SONG AIPING
  • WANG HAIBIN
  • SU JIANGSHUO
  • CHEN FADAI

Assignees

  • 南京农业大学

Dates

Publication Date
20260505
Application Date
20260120

Claims (10)

  1. 1. A method for increasing the content of linarin in cultivated chrysanthemum, which is characterized by comprising the following steps: a. Cloning coding regions (CDS) of OlOMT (chr5.1g020250) and OlOMT310 (chr9.1g057310) genes associated with linarin synthesis in echinacea (Opisthopappus longilobus); b. c, constructing the genes cloned in the step a into a plant over-expression vector; c. C, transforming the super-expression vector constructed in the step b into cultivated chrysanthemum by an agrobacterium-mediated method; d. Screening and identifying the obtained transgenic chrysanthemum plants; e. And detecting the linarin content of the transgenic chrysanthemum plants, and screening out the plant lines with the linarin content increased by more than 2 times compared with the cultivated chrysanthemum.
  2. 2. The method according to claim 1, wherein the OlOMT gene in step a has a DNA coding sequence shown in SEQ ID NO. 1 and the OlOMT gene 310 has a DNA coding sequence shown in SEQ ID NO. 2.
  3. 3. The method of claim 1, wherein the plant over-expression vector in step b is pORE-R4-35AA, and the gene expression is driven by a 35S promoter.
  4. 4. The method of claim 1, wherein the cultivar of chrysanthemum in step c is 'nannong Beijing jade'.
  5. 5. The method according to claim 1, wherein the agrobacterium-mediated transformation method in step c is a leaf disc method, comprising cutting sterile leaves of cultivated chrysanthemum into leaf discs, pre-culturing, infecting the leaf discs with agrobacterium liquid containing the overexpression vector, co-culturing the infected leaf discs, transferring the co-cultured leaf discs into a culture medium containing a screening antibiotic for induction and screening of resistant buds, and transferring the resistant buds into a rooting culture medium to obtain complete transgenic plants.
  6. 6. The method of claim 1, wherein the detection of the linarin content in step e uses ultra-high performance liquid chromatography tandem mass spectrometry (UPLC-MS/MS).
  7. 7. A transgenic chrysanthemum plant with high content of linarin obtained by the method of any one of claims 1-6.
  8. 8. The use of the transgenic chrysanthemum plant of claim 7 in the preparation of a medicament or health-care product with anti-inflammatory and antioxidant effects.
  9. 9. A combination of genes for increasing the linarin content of cultivated chrysanthemum, characterized in that said combination comprises OlOMT250,250 and OlOMT310,310 genes of aster ageratoides.
  10. 10. Use of the gene combination of claim 9 in plant genetic engineering.

Description

Method for improving content of linarin in chrysanthemum through transferring gene OlOMTs of schizophragma integrifolium Technical Field The invention relates to a plant genetic engineering technology, in particular to a method for improving the content of chrysanthemum linarin by transferring the OlOMTs gene of the schizophragma integrifolium. Background The linarin (Linarin) is a flavonoid compound with various biological activities such as anti-inflammatory, antioxidant and immunoregulation, and is listed as the quality control index (more than or equal to 8.00 mg/g) of wild chrysanthemum medicinal materials by the pharmacopoeia of the people's republic of China. However, the current widely cultivated chrysanthemum varieties (such as 'nan nong Beijing jade') have extremely low content of linarin, which severely limits the development of medicinal value. The longhairy antenoron flower (Opisthopappus longilobus) is a special rare or endangered plant in the tai mountain of China, the content of the linarin is up to 30.66 mg/g, the standards of pharmacopoeia are far exceeded, and the longhairy antenoron flower is an excellent natural source of linarin. Studies show that biosynthesis of linarin requires 4' -methylation modification of precursor apigenin to produce farnesoid, which is a key step in linarin synthesis. O-methyltransferase (OMTs) responsible for this methylation reaction in Echinacea longifolia is the core molecular basis for its high content of montanin. Although some studies have revealed the function of plant OMTs, the related studies on the plant, the key genes for flavonoid methylation and their relation to linarin synthesis, have been limited. In addition, the long-split aster is not recorded by pharmacopoeia yet, and safety concerns exist when the long-split aster is directly taken as a medicine. Therefore, how to introduce the high linarin synthesis capability of the long-split aster into cultivated chrysanthemums by a genetic engineering means and create new germplasm of the high linarin chrysanthemums becomes a technical problem to be solved urgently. Disclosure of Invention The invention aims to provide a method for efficiently and accurately improving the content of linarin in chrysanthemum by transferring the gene OlOMTs of the schizophragma integrifolium, which obviously improves the content of linarin in the chrysanthemum and provides a new technical approach and germplasm resource for improving the medicinal value of the chrysanthemum. The method for improving the content of the linarin in the cultivated chrysanthemum comprises the following steps of: a. Cloning coding regions (CDS) of OlOMT (chr5.1g020250) and OlOMT310 (chr9.1g057310) genes associated with linarin synthesis in echinacea (Opisthopappus longilobus); b. c, constructing the genes cloned in the step a into a plant over-expression vector; c. C, transforming the super-expression vector constructed in the step b into cultivated chrysanthemum by an agrobacterium-mediated method; d. Screening and identifying the obtained transgenic chrysanthemum plants; e. detecting the linarin content of the transgenic chrysanthemum plants, and screening out the plant lines with the linarin content increased by more than 2 times compared with the cultivated chrysanthemum, preferably 2-5 times. Preferably, the DNA coding sequence of OlOMT gene in step a is shown as SEQ ID NO.1, and the DNA coding sequence of OlOMT310 gene is shown as SEQ ID NO. 2. Prokaryotic expression and enzyme activity analysis, namely constructing cloned genes into a pCold II prokaryotic expression vector, inducing expression and purifying recombinant proteins. In vitro enzyme activity experiments prove that OlOMT250,250 and OlOMT310,310 proteins can efficiently catalyze apigenin to be converted into farnesoid (a direct precursor of linarin). Preferably, the plant over-expression vector in the step b is pORE-R4-35AA, and the 35S promoter drives gene expression. Preferably, the cultivar of the chrysanthemum described in step c is 'nan nong Beijing jade'. Preferably, the agrobacterium-mediated transformation method in the step c is a leaf disc method, and comprises the steps of cutting sterile seedling leaves of cultivated chrysanthemums into leaf discs, pre-culturing, infecting the leaf discs with agrobacterium liquid containing the overexpression vector, co-culturing the infected leaf discs, transferring the co-cultured leaf discs into a culture medium containing screening antibiotics for induction and screening of resistant buds, and transferring the resistant buds into a rooting culture medium to obtain complete transgenic plants. Preferably, the linarin content detection in step e is performed by high-performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS). Molecular identification, namely screening out positive transgenic lines through PCR detection. The transgenic chrysanthemum plant with high content of the montan glycoside obtained by the method