阅读说明：本技术 苯乳酸尿苷二磷酸葡萄糖基转移酶的应用 (Application of phenyllactic acid uridine diphosphate glucosyltransferase ) 是由 廖志华 陈敏 杨春贤 曾俊岚 邱飞 于 2019-09-30 设计创作，主要内容包括：本发明公开了苯乳酸尿苷二磷酸葡萄糖基转移酶(Phenyllactate UDP-Glycosyltransferase,PLA-UGT)基因的应用,苯乳酸尿苷二磷酸葡萄糖基转移酶PLA-UGT的氨基酸序列如SEQ ID NO.4所示；编码苯乳酸尿苷二磷酸葡萄糖基转移酶PLA-UGT基因的核苷酸序列如SEQ ID NO.3所示；本发明首次发现苯乳酸尿苷二磷酸葡萄糖基转移酶PLA-UGT以尿苷二磷酸葡萄糖为糖基供体,将苯乳酸糖基化形成苯乳酰葡萄糖酯,对于提高药用植物中托品烷生物碱含量和发展托品烷生物碱合成生物学具有重要的意义。(The invention discloses an application of phenyllactic acid uridine diphosphate glucosyltransferase (PLA-UGT) gene, wherein the amino acid sequence of the phenyllactic acid uridine diphosphate glucosyltransferase (PLA-UGT) is shown in SEQ ID No. 4; the nucleotide sequence of the PLA-UGT gene for encoding the phenyllactic acid uridine diphosphate glucosyltransferase is shown as SEQ ID NO. 3; the invention discovers for the first time that the phenyllactic acid uridine diphosphate glucosyltransferase PLA-UGT takes uridine diphosphate glucose as a glycosyl donor, glycosylates the phenyllactic acid to form phenyllactyl glucose ester, and has important significance for improving the content of tropane alkaloid in medicinal plants and developing the synthetic biology of tropane alkaloid.)

1. Application of phenyllactic acid uridine diphosphate glucosyltransferase in catalyzing phenyllactic acid reaction in vivo or in vitro to generate phenyllactyl glucose.

2. Use according to claim 1, characterized in that: the amino acid sequence of the phenyllactic acid uridine diphosphate glucosyltransferase is shown as SEQ ID NO. 4; or an amino acid sequence which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid residues in SEQ ID NO.4 and has the function of phenyllactic acid uridine diphosphate glucosyltransferase.

3. Use according to claim 1, characterized in that: the nucleotide sequence of the phenyllactic acid uridine diphosphate glucosyltransferase is shown as SEQ ID NO. 3; or nucleotide sequence which is obtained by substituting and/or deleting and/or adding one or more bases of the nucleotide in SEQ ID NO.3 and has the function of phenyllactic acid uridine diphosphate glucosyltransferase.

4. Use according to any one of claims 1 to 3, characterized in that: the catalytic reaction system of phenyllactic acid uridine diphosphate glucosyltransferase takes phenyllactic acid and uridine diphosphate glucose as substrates, and phenyllactic acid uridine diphosphate glucosyltransferase protein as a catalyst.

5. Use according to claim 4, characterized in that: the reaction system also contains MnCl₂And a reaction buffer solution, wherein the reaction buffer solution is Tris-HCl with the pH value of 7.0 and the concentration of 2 mM.

6. Application of phenyllactic acid uridine diphosphate glucosyltransferase in improving alkaloid content of solanaceae TAs resource plants.

7. Use according to claim 6, characterized in that: the alkaloid is echinosporine, hyoscyamine, anisodamine or scopolamine.

8. Use according to claim 6, characterized in that: the Solanaceae TAs resource plant is belladonna, stramonium or scopolia.

9. The application of phenyllactic acid uridine diphosphate glucosyltransferase in a metabolic pathway for reconstructing phenyllactic acid reaction to generate phenyllactyl glucose in plants or microorganisms is characterized in that: the amino acid sequence of the phenyllactic acid uridine diphosphate glucosyltransferase is shown as SEQ ID NO. 4; or an amino acid sequence which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid residues in SEQ ID NO.4 and has the function of phenyllactic acid uridine diphosphate glucosyltransferase.

10. Use according to claim 9, characterized in that: the microorganism is a bacterium or a fungus.

Technical Field

The invention relates to the technical field of biology, in particular to an application of phenyllactic acid uridine diphosphate glucosyltransferase gene.

Background

Tropane Alkaloids (TAs) are a class of anticholinergic drugs with great medical value, widely used for anesthesia, analgesia, cough relief, asthma relief and motion sickness resistance, and also used for controlling stiffness and tremor of parkinson's disease. The tropine alkaloids commonly used in clinic are hyoscyamine (hyoscyamine) and scopolamine (scopolamine), and the market demands of the hyoscyamine and scopolamine are quite large. Currently, TAs are extracted from a few plant sources of TAs in Solanaceae, including belladonna (Atropabelladonna), stramonium (Datura stramnonium) and hyoscyamine (Hyoscyamus niger), wherein belladonna is the main commercial cultivation drug source of scopolamine and hyoscyamine, and is also a plant source of TAs in pharmacopoeia. The weight fraction of the hyoscyamine in the wild belladonna plant is 0.02-0.17% (dry weight), and the content of the scopolamine is very low, which is only 0.01-0.08% of the dry weight. Therefore, the cultivation of belladonna with high tropane alkaloid yield has been a long sought goal in the industry.

The phenyllactyl glucose is an intermediate product in a tropine alkaloid biosynthesis pathway, the phenyllactyl glucose and tropine are subjected to esterification condensation to form conchioline, and the conchioline is an important precursor for hyoscyamine biosynthesis. Therefore, the biosynthesis of phenyllactyl glucose is crucial for the biosynthesis of tropane alkaloids. No relevant research reports exist, and the development of the tropine alkaloid synthesis biology is greatly hindered.

Disclosure of Invention

In view of the above, the present invention aims to provide a glycosyltransferase PLA-UGT using uridine diphosphate glucose as glycosyl donor, and esterifying phenyl lactic acid to form phenyllactyl glucose, which is of great significance for increasing the content of plant medicinal components in TAs resources and developing TAs synthetic biology, and the reaction process is as follows:

the invention also aims to provide the application of phenyllactic acid uridine diphosphate glucosyltransferase in improving the alkaloid content of solanaceae TAs resource plants; the invention also aims to provide the application of the phenyllactic acid uridine diphosphate glucosyltransferase in a metabolic pathway for reconstructing phenyllactic acid reaction to generate phenyllactyl glucose in plants or microorganisms.

In order to achieve the above purpose, the invention provides the following technical scheme:

1. application of phenyllactic acid uridine diphosphate glucosyltransferase in catalyzing phenyllactic acid reaction in vivo or in vitro to generate phenyllactyl glucose.

In the invention, the amino acid sequence of the phenyllactic acid uridine diphosphate glucosyltransferase is shown as SEQ ID NO. 4; or an amino acid sequence which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid residues in SEQ ID NO.4 and has the function of phenyllactic acid uridine diphosphate glucosyltransferase.

Preferably, the nucleotide sequence encoding the phenyllactic acid uridine diphosphate glucosyltransferase is shown in SEQ ID No. 3; or nucleotide sequence which is obtained by substituting and/or deleting and/or adding one or more bases of the nucleotide in SEQ ID NO.3 and has the function of phenyllactic acid uridine diphosphate glucosyltransferase.

In the invention, the catalytic reaction system of the phenyllactic acid uridine diphosphate glucosyltransferase takes phenyllactic acid and uridine diphosphate glucosyltransferase as substrates and phenyllactic acid uridine diphosphate glucosyltransferase protein as a catalyst.

Preferably, the reaction system also contains MnCl₂And a reaction buffer solution, wherein the reaction buffer solution is Tris-HCl with the pH value of 7.0 and the concentration of 2 mM.

2. Application of phenyllactic acid uridine diphosphate glucosyltransferase in improving alkaloid content of solanaceae TAs resource plants.

Preferably, the alkaloid is conchioline, hyoscyamine, anisodamine or scopolamine.

Preferably, the Solanaceae TAs resource plant is belladonna, stramonium or scopolia.

3. Use of a uridine-diphosphate glucosyltransferase in a plant or a microorganism for reconstituting a phenyllactic reaction to produce a phenyllactyl glucose metabolic pathway, said uridine-diphosphate glucosyltransferase having an amino acid sequence as shown in SEQ ID No. 4; or an amino acid sequence which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid residues in SEQ ID NO.4 and has the function of phenyllactic acid uridine diphosphate glucosyltransferase.

Preferably, the microorganism is a bacterium or a fungus.

The invention has the beneficial effects that: the invention discovers for the first time that the phenyllactic acid uridine diphosphate glucosyltransferase PLA-UGT takes uridine diphosphate glucose as a glycosyl donor, glycosylates phenyllactic acid to form phenyllactyl glucose ester, finds that the expression level of the PLA-UGT is inhibited through interference expression, and reduces the synthesis of belladonna mesospiramine, hyoscyamine, anisodamine and scopolamine, thereby indicating that the PLA-UGT participates in the biosynthesis of tropane alkaloid, so that the content of the tropane alkaloid can be regulated and controlled by regulating and controlling the expression level of the PLA-UGT, and the invention has important significance for regulating and controlling the content of medicinal ingredients of TAs resource plants and developing TAs synthetic biology.

Drawings

In order to make the object, technical scheme and beneficial effect of the invention more clear, the invention provides the following drawings for explanation:

FIG. 1 shows the process of obtaining hairy root and the results of molecular detection (a: explant induced hairy root, b: single culture of hairy root, C: liquid culture of hairy root, d: detection results of hairy root genes rolB and rolC, e: detection result of resistance gene (NPTII), f: 35S primer and PLA-UGT-RNAi downstream primer, M: Marker, P: positive control, N: negative control, C: empty plasmid control, 1-5: transgenic hairy root);

FIG. 2 is a graph of qPCR to detect relative expression levels of genes in PLA-UGT resistant hairy roots, respectively;

FIG. 3 shows that UPLC-MS/MS determines the content of tropane alkaloid in PLA-UGT transgenic interfering hairy roots (A: conchioline, B: hyoscyamine, C: anisodamine, D: scopolamine).

FIG. 4 shows the detection and catalytic activity analysis of the recombinant PLA-UGT protein of Escherichia coli (a: the analysis of PLA-UGT protein by SDS-PAGE gel; b: the reaction result of adding heat-inactivated PLA-UGT to the enzyme catalysis system; c: the addition of PLA-UGT to the enzyme catalysis system; d: the extraction of ion chromatography result).

Detailed Description

The present invention is further described with reference to the following drawings and specific examples so that those skilled in the art can better understand the present invention and can practice the present invention, but the examples are not intended to limit the present invention.