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Protein UGT146 as well as coding genes and application thereof

A technology of UGT146 and protein, applied in application, genetic engineering, plant gene improvement, etc., can solve the problems of unidentified coding genes, limited application, low content, etc.

Active Publication Date: 2019-03-15
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Lignans and their glycosides (mainly larch resinol-4-O-β-D-glucoside and straight iron clematis Ning B) are one of the important antiviral active substance bases of Isatis indigo Lignans. and its glycosides are relatively low in Isatis indigo, and the highest level of clematis Ning B is only 0.900mg / g, which greatly limits its clinical application
Most of the key enzyme genes in the synthetic pathway of lignans and their glycosides have been identified, but the genes encoding glycosyltransferases that catalyze key glycosylation modifications have not yet been identified

Method used

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  • Protein UGT146 as well as coding genes and application thereof
  • Protein UGT146 as well as coding genes and application thereof
  • Protein UGT146 as well as coding genes and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0091] Embodiment 1, the preparation of recombinant glycosyltransferase

[0092] One, the acquisition of the coding gene (UGT146 gene) of the glycosyltransferase derived from Isatis indigo

[0093] The inventors of the present invention discovered the coding gene (UGT146 gene) of glycosyltransferase in Isatis sativa through a large number of experiments. The nucleotide sequence of the UGT146 gene is shown as sequence 1 in the sequence listing. The UGT146 gene encodes the protein UGT146, and the amino acid sequence of the protein UGT146 is shown in Sequence 2 in the sequence listing.

[0094] The amino acid sequence of protein UGT146 was searched for homology in Non-redundantGenBank CDS translation+PDB+Swissprot database using BLAST program in NCBI database.

[0095] UGT phylogenetic tree (neighbor-joining method) see figure 1 . The results showed that the protein UGT146 had high homology with UGT in other species at the amino acid level, and had a typical active site domai...

Embodiment 2

[0116] Example 2. Recombinant glycosyltransferase converts larch resinol to generate larch resinol monoglucoside and larch resinol diglucoside

[0117] The solution to be tested was the crude enzyme solution of recombinant bacteria A (induced by IPTG) or the crude enzyme solution of recombinant bacteria B prepared in Example 1.

[0118] 1. Prepare the reaction system. The reaction system consisted of 300 μL of the solution to be tested, 1.5 μL of larch resin alcohol solution (solvent in methanol, concentration 40 mM) and 3 μL uridine diphosphate-β-O-D-glucose solution (solvent in ultrapure water, concentration 40 mM).

[0119] 2. Take the reaction system prepared in step 1 and put it in a water bath at 30°C for 12 hours.

[0120] 3. Take the system that has completed step 2, add 2 times the volume of pure methanol (for the purpose of terminating the reaction), and shake to extract.

[0121] 4. After completing step 3, centrifuge at 13000rpm for 15min and collect the supernat...

Embodiment 3

[0136] Embodiment 3, the acquisition and identification of transgenic UGT146 hairy roots

[0137] 1. Construction of recombinant plasmid pCAMBIA1300-super-UGT146

[0138] The schematic diagram of the construction of the recombinant plasmid pCAMBIA1300-super-UGT146 is shown in Figure 5 .

[0139] 1. Using the PCR amplification product obtained in step 2 of Example 1 as a template, use primer 2F: 5'-GCTTCTGCAGGGGCCCGGGGATGAAACAGGAGCTGGTTTTC and primer 2R: 5'-GGATCCACTAGTTTTAAATgAGAGATATTCGAAGTGACATC to perform PCR amplification to obtain a PCR amplification product.

[0140] 2. Take the PCR amplification product obtained in step 1, perform agarose gel electrophoresis, and then use a DNA gel recovery kit to recover a DNA fragment of about 1500 bp.

[0141] 3. Take the pCAMBIA1300-super plant expression vector, cut it with the restriction endonuclease SalI, and recover the vector skeleton of about 10kb.

[0142] 4. Ligate the DNA fragment obtained in step 2 and the vector back...

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Abstract

The invention discloses a protein UGT146 as well as coding genes and application thereof. The protein UGT146 is a protein of which the amino acid sequence is shown as a sequence 2 in a sequence table.Experiments prove that the protein UGT146 has glycosyl transferase activity, and is capable of efficiently transforming lariciresinol to produce lariciresinol-4-O-beta-D-glucoside and / or lariciresinol-4'-O-beta-D-glucoside and / or clemastanin B. The gene UGT146 is introduced into hairy roots of isatis tinctoria, and UGT146 transgenic hairy roots are obtained; compared with hairy roots of isatis tinctoria without the introduced gene UGT146, the content of the lariciresinol-4-O-beta-D-glucoside, lariciresinol-4'-O-beta-D-glucoside and clemastanin B produced from lariciresinol in the UGT146 transgenic hairy roots is obviously increased. The protein UGT146 disclosed by the invention has an important application value.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to protein UGT146 and its coding gene and application. Background technique [0002] Woad (Isatis indigotica Fort.) is a biennial herb of the genus Isatis in the family Cruciferae. Isatis indigo root is Radix Isatidis, and its leaves are one of the sources of Daqingye. They are commonly used traditional Chinese medicines. They have the effects of clearing away heat and detoxification, cooling blood and relieving throat. They are often used clinically for influenza, mumps, and Japanese encephalitis. , acute and chronic hepatitis, herpes zoster and other diseases. Lignans and their glycosides (mainly larch resinol-4-O-β-D-glucoside and straight iron clematis Ning B) are one of the important antiviral active substance bases of Isatis indigo Lignans. The content of clematis and its glycosides in Isatis indica is relatively low, and the highest level of clematisin B is only 0.90...

Claims

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Application Information

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IPC IPC(8): C12N9/10C12N15/54C12N15/62C12N15/82C07K19/00A01H5/06A01H6/20C12P19/44
CPCC07K2319/43C12N9/1048C12N15/8243C12P19/44
Inventor 黄璐琦唐金富杨健郭娟谭宇萍
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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