Plant expression vector for synthesizing taxadiene, strain DZGGPPSTS, dioscorea zingiberensis and preparation method therefor

A plant expression vector, taxadiene technology, applied in the field of plant expression vectors for the synthesis of taxadiene, strain DZGGPPSTS and Dioscorea scutellaria and its preparation, can solve the problem of unfavorable discovery and separation of new taxanes, taxanes low olefin content

Inactive Publication Date: 2020-03-24
HUBEI UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the taxadiene content in the reported transgenic plants is very low, such as 0.025 mg/kg (dry weight) for Arabidopsis, ...

Method used

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  • Plant expression vector for synthesizing taxadiene, strain DZGGPPSTS, dioscorea zingiberensis and preparation method therefor
  • Plant expression vector for synthesizing taxadiene, strain DZGGPPSTS, dioscorea zingiberensis and preparation method therefor
  • Plant expression vector for synthesizing taxadiene, strain DZGGPPSTS, dioscorea zingiberensis and preparation method therefor

Examples

Experimental program
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Effect test

Embodiment 1

[0033]Example 1 Construction of taxadiene-expressing vector pCAMBIA-GGPPS-TS

[0034] According to the cauliflower mosaic virus 35S gene promoter sequence (CaMV35S) (nucleotide sequence shown in SEQ ID NO. 1), the Canadian yew (Taxus canadensis) geranyl geranium pyrophosphate synthase gene GGPPS sequence ( Genbank accession: AF081514) (in which the plastid signal peptide sequence encoding the N-terminal 98 amino acids in the original sequence was removed, and the initiation codon ATG was added) (the nucleotide sequence is shown in SEQ ID NO. 2) and Agrobacterium nopaline synthase gene terminator sequence (Tnos) (nucleotide sequence shown in SEQ ID NO. 3) was combined into a GGPPS gene expression cassette CaMV35S-GGPPS-Tnos (S1).

[0035] (2) According to the cauliflower mosaic virus 35S gene promoter sequence (CaMV35S2) (the nucleotide sequence is shown in SEQ ID NO. 4) containing repeat enhancers, Taxus brevifolia taxadiene synthase Gene TS sequence (Genbank accession: U4879...

Embodiment 2

[0037] Example 2 Preparation method of transgenic Dioscorea officinalis plant for synthesizing taxadiene

[0038] 1. The vector pCAMBIA-GGPPS-TS prepared in Example 1 was transformed into Agrobacterium tumefaciens EHA105 by freeze-thaw method to obtain the genetically engineered strain DZGGPPSTS. The strain is preserved in the China Center for Type Culture Collection (Wuhan University), and the preservation number is CCTCC NO: M2019685.

[0039] 2. Take the young stems of Dioscorea officinalis, wash them with tap water, sterilize them in a sodium hypochlorite solution containing ~1% available chlorine concentration for 15 minutes, rinse with sterile water for 3 times, cut them into small pieces, and inoculate them in 1 / 2MS medium + 20g / L sucrose+7g / L agar+2mg / LBA+1mg / L NAA induced callus ( figure 2 a), after callus formation, cut into small pieces and propagate on 1 / 2MS medium+20g / L sucrose+7g / L agar+1mg / L BA+0.5mg / L NAA. The culture conditions are temperature 25±1℃, light ...

Embodiment 3

[0050] Example 3 Detection and Analysis of Synthesis of Taxadiene by Transgenic Dioscorea Shields Plants

[0051] 1. Extract the leaf and rhizome samples of transgenic and non-transgenic plants according to the methods reported in the literature, and analyze them by gas chromatography-mass spectrometry (GC-MS). image 3 GC-MS analysis results of synthetic taxadiene for transgenic Dioscorea scutellariae plants; (a) GC profile of leaves of non-transgenic plants; (b) GC profile of leaves of transgenic plant L8, with an obvious peak at 13.35 min; ( c) The GC profile of the rhizome of the transgenic plant L8, there is an obvious peak at 13.52min; (d) MS analysis of the peak 2 in (b), m / z 272, 257, 229, 122, 121 and Fragments characteristic of taxadiene cleavage such as 107. image 3 The analysis results of the present invention prove that the transgenic plants of the present invention can synthesize taxadiene.

[0052] 2. Table 1 shows the content of taxadiene in leaves and rhizo...

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Abstract

The invention provides a plant expression vector for synthesizing taxadiene, a strain DZGGPPSTS, dioscorea zingiberensis and a preparation method therefor. The vector contains a taxus chinensis geranylpyrophosphate synthase gene GGPPS expression cassette CaMV35S-GGPPS-Tnos and a taxus chinensis taxadiene synthase gene TS expression cassette CaMV35S2-TS-CaMV 3' UTR. The strain DZGGPPSTS is obtainedthrough transducing the vector into agrobacterium tumefaciens and has a collection number of CCTCC NO: M2019685; and a transgenic plant capable of synthesizing the taxadiene is obtained through integrating the vector into a genome of dioscorea zingiberensis through strain DZGGPPSTS mediating and carrying out regenerating. A gene GGPPS and a gene TS are over-expressed simultaneously, the dioscoreazingiberensis serves as an acceptor plant, and thus, the transgenic plant with high taxadiene synthesizing capability is obtained.

Description

technical field [0001] The invention relates to the technical field of plant genetic engineering, in particular to a plant expression vector for synthesizing taxadiene, a strain DZGGPPTSS and Dioscorea serrata and a preparation method thereof. Background technique [0002] Taxol derived from Taxus is by far the most important plant anticancer drug. It has been the first-line drug for the clinical treatment of breast cancer, uterine cancer, lung cancer and head and neck cancer since the 1990s. The treatment of other tumors is still under study. middle. In addition, other taxane anticancer drugs (such as Taxotere, Abraxane) have also been developed and applied clinically, and the research on taxane anticancer drugs has become one of the hot spots in drug research and development. The discovery of new taxanes is key to the development of new drugs. There are 11 species of yew in the world with limited resources. There are 4 species and 1 variety in my country, all of which gr...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/54C12N15/60C12N1/21A01H5/00A01H4/00A01H6/00
CPCA01H4/001A01H4/008C12N9/1085C12N9/88C12N15/8243C12Y205/01029C12Y402/03017
Inventor 陈永勤沈君豪杨之帆
Owner HUBEI UNIV
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