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Application of Pn3-32-i5 protein and coding gene thereof in production of notoginsenoside R1
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A technology of Panax notoginseng saponins and encoding genes, which is applied in the biological field and can solve the problems of long plant growth cycle, incomplete excavation of notoginseng saponins R1 synthesis pathway, and difficulty in product purification.
Active Publication Date: 2021-05-28
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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Problems solved by technology
Notoginseng saponin R1 is mainly isolated and extracted from Panax notoginseng, but the separation and extraction method has many disadvantages, such as low content, large difference, difficult product purification, long plantgrowth cycle, and serious damage to biological resources, especially wild resources, etc.
At present, relevant progress has been made in the research on the biosynthetic pathway of ginsenosides, including some key enzymes in the synthetic pathway of ginsenosides, such as squalene synthase (Squalene synthase), which catalyzes the isoprenoid pathway towards sterol and triterpene saponins. , SS), squalene epoxidase (Squaleneepoxidase, SE) that catalyzes the generation of 2,3-oxidized squalene, dammarene diol synthase (DS) that catalyzes the generation of dammarene diol, and responsible for hydroxyl The synthetic cytochrome P450 enzyme, etc., but the synthetic pathway of notoginseng saponin R1 has not been fully excavated
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[0125] 2. After completing step 1, take the total RNA of Panax notoginseng leaves, and use a reverse transcription kit to perform reverse transcription to obtain the cDNA of Panax notoginseng.
[0126] The reaction system is 15 μL, including 3 μL Panax notoginseng cDNA, 1 μL Oligo(dT)18 primer (10 μM concentration), 1 μL dNTP Mix (dATP, dTTP, dGTP and dCTP concentration are all 10 mM), 4 μL 5×RT Buffer, 1 μL Maxima HMinus Enzyme Mix and 10 μL RNase-free water.
[0127] Reaction program: 50°C for 50min, 85°C for 5min, 4°C for heat preservation.
[0128] 3. Using the cDNA of Panax notoginseng as a template, using the primer SexA1-Pn3-32-i5: 5'-GCG ACCTGGT ATGGATAACCAAGAAGCTAGAATCAG-3' (the underline is the recognition site of restriction endonuclease SexAI) and primer Pn3-32-i5-Asc1: 5'-GC GGCGCGCC CTATTGTGCATCT...
Embodiment 2
[0134] Example 2, the application of Pn3-32-i5 protein in the preparation of notoginseng saponin R1
[0135] 1. Construction of recombinant plasmid pM3-SynAtUGD1, recombinant plasmid pM9-SynAtUXS3, recombinant plasmid pM16-UGTPg101 and recombinant plasmid pRS425-LEU2-PTEF1-Pn3-32-i5-TCYC1
[0136] 1. Construction of recombinant plasmid p-SynAtUXS3, recombinant plasmid p-SynAtUGD1 and recombinant plasmid p-UGTPg101
[0137] The SynAtUXS3 gene shown in SEQ ID NO: 2, the SynAtUGD1 gene shown in SEQ ID NO: 3 and the UGTPg101 gene shown in SEQ ID NO: 4 were respectively synthesized by GenScript Biotechnology Co., Ltd.
[0138] The SynAtUXS3 gene was inserted into the multiple cloning site of the pUC57 vector to obtain the recombinant plasmid p-SynAtUXS3.
[0139] The SynAtUGD1 gene was inserted into the multiple cloning site of the pUC57 vector to obtain the recombinant plasmid p-SynAtUGD1.
[0140] The UGTPg101 gene was inserted into the multiple cloning site of the pUC57 vector t...
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Abstract
The invention discloses an application of a Pn3-32-i5 protein and a coding gene thereof in production of notoginsenoside R1. The amino acid sequence of the Pn3-32-i5 protein is as shown in SEQ ID NO: 5. According to the present invention, the coding gene of phosphoglucose mutase 1, the coding gene of alpha-phosphoglucose mutase, the coding gene of uridine diphosphate glucose pyrophosphorylase, the coding gene of arabidopsis thaliana UDP-glucose dehydrogenase 1, the coding gene of arabidopsis thaliana UDP-glucuronic acid decarboxylase 3, the coding gene of ginseng UDP-glycosyltransferase 101 and a Pn3-32-i5 gene are introduced into BY-PPD-PPT, so as to obtain the recombinant saccharomycetes Rg1-XM+Pn3-32-i5. The recombinant saccharomycetes Rg1-XM+Pn3-3-i5 can be used for simultaneously producing notoginsenoside R1 and ginsenoside Rg1, and has a relatively good industrial application prospect. The product has an important application value.
Description
technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to the application of Pn3-32-i5 protein and its coding gene in the production of notoginseng saponin R1. Background technique [0002] Notoginseng saponin R1 (chemical formula shown in formula (I)) is a dammarane-type triterpenesaponin compound, which is mainly distributed in Araliaceae plants such as ginseng, notoginseng, American ginseng, etc. Apoptosis, promoting blood circulation and removing blood stasis, etc., is a chemical substance with great medicinal value. Notoginseng saponin R1 is mainly isolated and extracted from Panax notoginseng, but the separation and extraction method has many disadvantages, such as low content, large difference, difficult product purification, long plantgrowth cycle, and serious damage to biological resources, especially wild resources. [0003] [0004] At present, using the principle of synthetic biology to design and transform micr...
Claims
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Application Information
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