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
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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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