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Hemsleya amabilis triterpene synthetase HcOSC6 gene and engineering bacteria thereof as well as application to preparation of gourd dienol

A technology of cucurbitadienol and transgenic engineering, applied in the biological field, can solve the problems of unclear gene function, influence the progress of the biosynthesis of snow bile, etc., and achieve the effect of enriching raw materials

Active Publication Date: 2020-08-11
YUNNAN AGRICULTURAL UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the biosynthetic pathway of eucholantin in plants of the genus eucalyptus has not been reported, especially the gene function of OSCs responsible for forming the carbon skeleton of euchocholin is not clear, which affects the advancement of the biosynthesis of eucholin.

Method used

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  • Hemsleya amabilis triterpene synthetase HcOSC6 gene and engineering bacteria thereof as well as application to preparation of gourd dienol
  • Hemsleya amabilis triterpene synthetase HcOSC6 gene and engineering bacteria thereof as well as application to preparation of gourd dienol
  • Hemsleya amabilis triterpene synthetase HcOSC6 gene and engineering bacteria thereof as well as application to preparation of gourd dienol

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1 Gene identification

[0042] (1) Candidate gene screening

[0043]Based on the basic function annotation information of the transcriptome Unigene, OSCs candidate genes were screened in the sequencing annotation results. At the same time, cucurbitadienol synthase (CBS) identified in Cucurbitaceae plants, mainly identified in Luo Han Guo (Siraitiagrosvenorii) SgCBS, CsBi identified in cucumber (Cucumis sativus), CsBi identified in melon (Cucumismelo) and cpCPQ identified in zucchini (Cucurbita pepo) were used as clues, and the sequence local BLAST analysis was performed, and then the screening results were sorted out and analyzed, and finally found that Six oxidative squalene cyclases (Oxidosqualenecyclases, OSCs) genes were named as HcOSC1, HcOSC2, HcOSC3, HcOSC4, HcOSC5 and HcOSC6 respectively. The function of HcOSC6 is annotated as cucurbitadienol synthase (CBS). Finally, according to the ID number corresponding to the Unigene, the nucleotide sequence of the...

Embodiment 2

[0053] Example 2 Recombinant plasmid construction

[0054] (1) Recombinant plasmid construction

[0055] Firstly, the vector pYES2 was linearized, and the linearized vector was obtained by single enzyme digestion with BamH I enzyme. Use the EasyPure Quick Gel Extraction Kit to recover, measure its concentration after recovery, and finally store it in a -20°C refrigerator for later use; then perform homologous recombination, and assemble according to the operating instructions of the homologous recombinase kit for homologous recombination , and then calculate the amount of each component according to the concentration of the insert and the vector according to the recombination instructions; finally add each component to the PCR reaction tube on ice. Carry out reorganization operation according to the system of following table 1:

[0056] Table 1 Recombination reaction system

[0057]

[0058]

[0059] Wherein, X=(0.02×HcOSC6 base log number) ng / concentration of lineari...

Embodiment 3

[0063] Embodiment 3 constructs genetically modified engineered bacterium

[0064] (1) Competent preparation of GIL77 yeast strain (lanosterol synthase-deficient type)

[0065] Pick the GIL77 strain on the YPD plate and inoculate it into 100 mL of YPD medium supplemented with ergosterol (20 μg / mL), heme (13 μg / mL) and Tween 80 (5 mg / mL), and culture at 30 °C and 220 rpm To OD=1.3-1.5, ice-bath for 30 minutes; centrifuge to collect yeast cells.

[0066] Yeast cells were washed repeatedly with 25 mL of water, 2 mL of 1M sorbitol, 2 mL of 0.1M lithium acetate, and 2 mL of sorbitol. Finally, the cells were dispersed in 250 μL of 1M sorbitol, and 100 μL was dispensed into pre-cooled 1.5 ml sterile centrifuge tubes.

[0067] (2) GIL77 yeast transformation

[0068] Add 2 μg of recombinant yeast plasmid DNA to 100 μL of yeast competent cells, mix gently; and transfer to a pre-cooled 0.2 cm electroporation cuvette, ice bath for 10 min, use the GenePulser electroporation system (BioRa...

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Abstract

The invention discloses a hemsleya amabilis triterpene synthetase HcOSC6 gene and engineering bacteria thereof as well as an application to preparation of gourd dienol. The hemsleya amabilis triterpene synthetase HcOSC6 gene has a nucleotide sequence shown in SEQ ID No.1, and the engineering bacteria can express hemsleya amabilis triterpene synthetase HcOSC6. In a culture medium taking D-glucose and D-galactose as carbon sources, the genetically modified engineering bacteria can express hemsleya amabilis triterpene synthetase HcOSC6 in cells, under the catalysis of hemsleya amabilis triterpenesynthetase HcOSC6, an endogenous substrate squalene-2,3-oxide of yeast can be subjected to a cyclization reaction, and gourd dienol is produced and has the yield about 5.5 mg / L. An important way forproducing the gourd dienol is provided, and rich raw materials are provided for biological synthesis of cucurbitacin such as hemsleyadin and the like.

Description

technical field [0001] The invention belongs to the field of biological technology, and in particular relates to a gene of genus triterpene synthase HcOSC6 and its engineering bacteria and its application in preparing cucurbitacinol. Background technique [0002] Cucurbitaceae plants contain many secondary metabolites that play an important role in human production and life, such as: (1) cucurbitacins, which are bitter substances isolated from Cucurbitaceae plants at first, and later found that cucurbitacins also exist In plants such as Brassicaceae, on the one hand, cucurbitacin has significant anticancer activity and is a potential new drug candidate compound, the most representative of which is the cucurbitacin isolated and extracted from plants of the genus Elephant. IIa (25-acetoxy-23,24-dihydrocucurbitacin F) and cucurbitacin IIb (23,24-Dihydrocucurbitacin F), both of which are the most effective new antibacterial drugs in the development and utilization of cucurbitaci...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/52C12N9/00C12N15/81C12N1/19C12P33/00C12R1/865
CPCC12N9/93C12N15/81C12P33/00Y02A50/30
Inventor 张广辉陈庚杨生超赵艳段绍凤刘冠泽郭兆宽李莹
Owner YUNNAN AGRICULTURAL UNIVERSITY
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