Combined expression vector for expressing trans-chrysanthemic acid and application of combined expression vector in regulating tomato VI type gland hair to synthesize trans-chrysanthemic acid

A technology of joint expression and plant expression vectors, applied in the field of regulating the synthesis of trans chrysanthemic acid in tomato type VI glandular hairs, can solve the problems of high cost, limited sources, harmful environment, etc., and achieve endogenous defense capabilities Improved effect

Active Publication Date: 2022-03-01
CHONGQING UNIV
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  • Abstract
  • Description
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  • Application Information

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Problems solved by technology

As an important precursor of pyrethrins, trans-chrysanthemum acid has been only chemically synthesized, but this method is costly, the source is limited and most of them are harmful to the environment

Method used

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  • Combined expression vector for expressing trans-chrysanthemic acid and application of combined expression vector in regulating tomato VI type gland hair to synthesize trans-chrysanthemic acid
  • Combined expression vector for expressing trans-chrysanthemic acid and application of combined expression vector in regulating tomato VI type gland hair to synthesize trans-chrysanthemic acid
  • Combined expression vector for expressing trans-chrysanthemic acid and application of combined expression vector in regulating tomato VI type gland hair to synthesize trans-chrysanthemic acid

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1 fusion gene vector construction

[0034] The complete open reading frames (ORFs) of pyrethrum TcCDS, TcADH2, and TcALDH1 were synthesized using previously published sequences (Xu, H., Moghe, G.D., Wiegert-Rininger, K., Schilmiller, A.L., Barry, C.S., Last, R.L. ,Pichersky,E.,2018.Coexpression Analysis Identifies Two OxidoreductasesInvolved in the Biosynthesis of the Monoterpene Acid Moiety of NaturalPyrethrin Insecticides in Tanacetum cinerariifolium.Plant Physiol.176,524-537;Yang,T.,Gao,L.,Hu , Stoopen, G., Wang, C., Jongsma, M.A., 2014. Chrysanthemyldiphosphate synthase operates inplanta as abifunctional enzyme with chrysanthemol synthase activity. JBiol Chem. 289, 36325-35.). The ORF sequences of TcCDS, TcADH2 and TcALDH1 are shown in SEQ ID NO.1-3 in sequence.

[0035] Synthesize the 924bp genomic sequence upstream of the start codon of the gene encoding the metallocarboxypeptidase inhibitor of tomato S.lycopersium cvM82, and name it the metallocarboxy...

Embodiment 2

[0051] Example 2 Reconstruction of trans chrysanthemic acid synthesis pathway in cultivated tomato VI glandular trichomes

[0052] 1. Construction of pPZP212-CDS and pPZP212-CAA

[0053] In this example, based on the binary vector pPZP212, a vector (pPZP212-CAA) containing an MCPI promoter-driven expression cassette for TcCDS and an MCPI promoter-driven expression cassette for ADH2 / ALDH1 (pPZP212-CAA) was constructed, which is used to construct a stable expression cassette under the control of an MCPI promoter. A transgenic tomato line expressing TcCDS+ADH2 / ALDH1, figure 2 is a schematic diagram of the structure of the constructs for the synthesis of the expression cassettes for the synthesis of chrysanthemum alcohol (pPZP212-CDS) and trans-chrysanthemum acid (pPZP212-CAA). Concrete construction steps are as follows (PCR amplification reaction system and amplification procedure in the present embodiment are the same as embodiment 1):

[0054] 1. Construction of pPZP212-CDS ...

Embodiment 3

[0102] Example 3 Detection of trans chrysanthemic acid and its related compounds produced by TcCDS+ fusion gene ADH2 / ALDH1 transgenic tomato

[0103] For the extraction and determination of trans chrysanthemic acid, the leaves, petioles, and stems of plants about seven weeks old were collected according to the following steps:

[0104] 1. Extraction method: Take 300 mg of tissue and grind it with liquid nitrogen, and take the supernatant. The supernatant was treated with acetic acid to make it weakly acidic, and then fully mixed with 300 μl methyl tert-butyl ether (MTBE) containing 0.01 ng / μl internal standard tetradecane, and incubated for 10 min, which is the free trans Chrysanthemum acid extraction method: the supernatant was treated with 4N NAOH for 10min, immediately added to 4N HCL to neutralize, then fully mixed with 300μl methyl tert-butyl ether (MTBE) containing 0.01ng / μl internal standard tetradecane, and incubated 10min, this is the total trans chrysanthemic acid e...

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Abstract

The invention discloses a combined expression vector for expressing trans-chrysanthemic acid and a construction method and application of the combined expression vector, the combined expression vector is an expression cassette with a plant expression vector as a skeleton and containing an MCPI promoter driving TcCDS gene and an expression cassette containing an MCPI promoter driving fusion gene ADH2 / ALDH1, the TcCDS gene is a gene for coding inulin diphosphate synthase, and the fusion gene ADH2 / ALDH1 is a gene for coding trans-chrysanthemic acid. The fusion gene ADH2 / ALDH1 is a fusion gene formed by a gene TcADH2 for coding alcohol dehydrogenase 2 and a gene TcALDH1 for coding aldehyde dehydrogenase 1 through a DNA (Deoxyribose Nucleic Acid) linker, and the MCPI promoter is a glandular hair specific metal carboxypeptidase inhibitor promoter. According to the invention, a trans-chrysanthemic acid biosynthesis pathway is reconstructed in the tomato type VI glandular hair, a large amount of trans-chrysanthemic acid is generated, and the result shows that the tomato type VI glandular hair is an efficient platform for biosynthesis of trans-chrysanthemic acid through metabolic engineering.

Description

technical field [0001] The invention relates to the field of plant molecular biology, in particular to a combined expression vector for synthesizing trans chrysanthemic acid, a construction method and its application in regulating the synthesis of trans chrysanthemic acid by glandular hairs of tomato type VI glands. Background technique [0002] Natural pyrethrin is a natural high-efficiency insecticide derived from the Compositae plant Pyrethrum (Tanacetum cinerariifolium), which has broad-spectrum and strong insecticidal and repellent effects, and is less toxic to mammals than chemically synthesized analogues , without environmental hazards, is one of the best choices of biopesticides, and has broad application prospects. At present, the production of pyrethrins is mainly extracted naturally from pyrethrum plants. However, the planting of pyrethrums is deeply affected by altitude, soil and temperature. The global annual production is far from meeting the society's demand f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82C12N15/62C12N15/52A01H5/00A01H6/82C12P7/40
CPCC12N15/8243C12N9/0008C12N9/0006C12P7/40
Inventor 徐海洋王莹陈静文静王楚
Owner CHONGQING UNIV
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