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Rhizoma gastrodiae glucosyltransferase gene and application

A technology of glucosyltransferase and gene, which is applied in the field of molecular biology and plant genetic engineering, can solve the problems of long growth cycle of Gastrodia elata, lack of sight, and the total amount of wild Gastrodia elata resources.

Active Publication Date: 2020-10-13
KUNMING UNIV OF SCI & TECH
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0002] Gastrodia elata is a rare and precious Chinese medicinal material. It used to rely mainly on wild resources, but the total amount of wild Gastrodia elata resources is small, which is far from meeting people's demand for it.
Gastrodia elata began to be cultivated artificially in the 1960s, and with more and more researches on the living habits of Gastrodia elata, the technology of artificially cultivating Gastrodia elata has been continuously improved, but the artificial cultivation of Gastrodia elata still faces many factors such as long growth cycle and great influence from the external environment Gastrodia elata still can not meet people's needs
[0006] There are no bibliographical reports relevant to the technical solution of the present invention

Method used

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  • Rhizoma gastrodiae glucosyltransferase gene and application
  • Rhizoma gastrodiae glucosyltransferase gene and application
  • Rhizoma gastrodiae glucosyltransferase gene and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1: Gastrodia elata glucosyltransferase gene ( UGT gene) acquisition

[0041] 1. Gastrodia elata cDNA acquisition

[0042] Select Zhaotong Wutianma ( G. elata Bl. f. glauca S. Chow ) as the extraction material, put 0.1g Gastrodia elata into liquid nitrogen and store it in a -80°C refrigerator for subsequent experiments. Trizol Reagent (Invitrogen) was used to extract the RNA of Gastrodia elata; put 0.1g sample into liquid nitrogen to pre-cool After grinding, add 1 mL of Trizol extract and continue to grind in the mortar until the grinding liquid is red and transparent. After standing at room temperature for 5 minutes, transfer it to a 1.5 mL centrifuge tube and add 0.2 mL of chloroform to shake and mix well; Centrifuge at 12,000 rpm and 4°C for 15 minutes, then pipette the supernatant into a new centrifuge tube; then add 0.25 mL of isopropanol, 0.25 mL of sodium chloride and sodium citrate to mix the high-salt solution, mix well and place at -20°C for 30 mi...

Embodiment 2

[0073] Example 2: UGT Construction and Identification of Gene Prokaryotic Expression Vector

[0074] Use DNAman to analyze the restriction sites of the full-length UGT gene, use this template to design specific primers with restriction sites for PCR amplification, obtain cDNA fragments with restriction sites, and connect them to the high-copy pMD- 18T linear vector, transformed into Escherichia coli competent DH5α; using EcoRI and NotⅠ double enzyme digestion detection, the results are shown in image 3 a, After successful cutting, the target fragment was recovered by gel, and connected to pGEX-4T to obtain pGEX4T- UGT ; The recombinant plasmid pGEX4T- was extracted from DH5α UGT Do enzyme digestion detection ( image 3 b), cutting out a fragment of about 1700 bp indicates that the amplified UGT fragment has been inserted into the vector pGEX-4T; the plasmid pGEX4T- UGT The results of the sequencing comparison were 100% consistent, and the correct UGT prokaryotic expressio...

Embodiment 3

[0075] Example 3: UGT Induction Analysis of Gene Prokaryotic Expressed Proteins

[0076] Proteins were induced using the IPTG method. Picking was successfully transferred into pGEX4T -UGTThe BL21 strain was placed in LB liquid medium and shaken on a shaker at 37°C until the OD of the bacterial solution was 0.6-0.8. Then IPTG was added to make the final concentrations of IPTG in the bacterial solution 0.5mM and 1mM respectively, put into a shaker at 28°C, and about 1.5mL of the bacterial solution at 0, 2, 4, 6, and 8 hours were taken to extract total protein for SDS-PAGE analysis.

[0077] The protein size of glucosyltransferase is about 65kDa, the pGEX-4T vector contains GST tag, the size is about 25kDa, so pGEX4T- UGT The expressed protein content is about 90kDa; the results show that when the inducer concentration is 0.5mM and the induction time is 6h, the induction effect is better, the results are shown in Figure 4 .

[0078] Add IPTG to 500mLOD of 0.6-0.8 bacterial...

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Abstract

The invention discloses a rhizoma gastrodiae glucosyltransferase gene. The nucleotide sequence of the rhizoma gastrodiae glucosyltransferase gene is shown as SEQID NO:1, and an amino acid sequence asshown in SEQID NO:2 is coded. A glucosyltransferase gene sequence is screened out according to a CDS sequence provided by a rhizoma gastrodiae transcriptome, a nest type PCR primer is designed, and through amplification and splicing, an overall length sequence of the glucosyltransferase gene is obtained; a prokaryotic expression vector pGEX4T-UGT is constructed, proteins are obtained through expression and purification, a eukaryotic expression vector is constructed, subcellular localization is performed on expression of a UGT gene, glucosyltransferase is transferred into monokaryon honey mushroom mycelia by an agrobacterium tumefaciens infection method for the constructed eukaryotic expression vector pH2GW7.0 35S-UGT, transgene honey mushrooms are obtained, biosynthetic gastrodin is used,the rhizoma gastrodiae is artificially cultured by the transgene honey mushrooms, the content of gastrodin in the rhizoma gastrodiae is increased, and besides, the gastrodin can also be produced through culturing genetic engineering honey mushrooms.

Description

technical field [0001] The invention belongs to the fields of molecular biology and plant genetic engineering, and in particular relates to a glucosyltransferase gene of Gastrodia elata, and the gene is transferred into Armillaria monokaryotes, and 4-hydroxyphenylcarbinol is synthesized by a biosynthetic method. converted into gastrodin. Background technique [0002] Gastrodia elata is a rare and precious Chinese medicinal material. It used to rely mainly on wild resources, but the total amount of wild Gastrodia elata resources is so small that it is far from meeting people's demand for it. Gastrodia elata began to be cultivated artificially in the 1960s, and with more and more researches on the living habits of Gastrodia elata, the technology of artificially cultivating Gastrodia elata has been continuously improved, but the artificial cultivation of Gastrodia elata still faces many factors such as long growth cycle and great influence from the external environment Make Ga...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/10C12N15/54C12N15/80C12N1/15C12P19/44C12R1/645
CPCC12N9/1051C12N15/80C12P19/44
Inventor 李昆志李珩珺刘云霞周春艳徐慧妮
Owner KUNMING UNIV OF SCI & TECH
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