阅读说明：本技术 C-糖基转移酶及其在合成夏佛塔苷和异夏佛塔苷中的应用 (C-glycosyltransferase and application thereof in synthesis of schaftoside and isoschaftoside ) 是由 乔雪 叶敏 王子龙 王双 陈宽 于 2019-04-10 设计创作，主要内容包括：本发明公开了一种C-糖基转移酶及其在合成夏佛塔苷和异夏佛塔苷中的应用。本发明从黄芩、玉米、水稻、甘草、大薸、少根紫萍、一把伞南星中分离获得了2-羟基黄烷酮C-糖基转移酶基因(SbCGTa、GuCGTa、PsCGTa、LpCGTa、AeCGTa)和C-葡萄糖-2-羟基黄烷酮C-糖基转移酶基因(SbCGTb、ZmCGTb、OsCGTb1、OsCGTb2、GuCGTb、PsCGTb、LpCGTb、AeCGTb),首次发现这些基因编码的蛋白参与合成植物中植保素夏佛塔苷和异夏佛塔苷,从而为体外利用酶催化方法合成夏佛塔苷、异夏佛塔苷及其类似物提供了可行的方法。(The invention discloses C-glycosyltransferase and application thereof in synthesizing schaftoside and isoschaftoside. The invention separates 2-hydroxyflavanone C-glycosyltransferase genes (SbCGTa, GuCGTa, PsCGTa, LpCGTa, AeCGTa) and C-glucose-2-hydroxyflavanone C-glycosyltransferase genes (SbCGTb, ZmCGTb, OsCGTb1, OsCGTb2, GuCGTb, PsCGTb, LpCGTb and AeCGTb) from scutellaria baicalensis, corn, rice, liquorice, pistia stratiotes, duckweed and arisaema sylvestre, and discovers that proteins coded by the genes participate in the synthesis of phytoalexin schaftoside and isoschaftoside in plants for the first time, thereby providing a feasible method for synthesizing schaftoside, isoschaftoside and analogues thereof by an enzyme catalysis method in vitro.)

1. A method of synthesizing glucose and arabino-disaccharide carbon-glycoside compounds, comprising: catalyzing the compound shown in the formula I and uridine diphosphate glucose by using 2-hydroxyflavanone C-glycosyltransferase to generate a compound shown in the formula II, and catalyzing the compound shown in the formula II and uridine diphosphate arabinose by using C-glucose-2-hydroxyflavanone C-glycosyltransferase to generate a compound shown in the formula III:

wherein R is₁And R₂Independently of one another, R₁Is H, hydroxy or alkoxy, R₂Is H, alkyl, alkoxy, phenyl, substituted phenyl, phenylalkylene, substituted phenylalkylene, phenoxy, substituted phenoxy, phenylalkoxy, substituted phenylalkoxy, acetophenone, p-hydroxybenzophenone or 3 ', 4' -dihydroxyacetophenone; orIn formula I, R₁And R₂The mixture is subjected to ring formation,

the compound of formula I has the following structure:

the 2-hydroxyflavanone C-glycosyltransferase is selected from 2-hydroxyflavanone C-glycosyltransferase SbCGTa, GuCGTa, AeCGTa, LpCGTa and PsCGTa from scutellaria baicalensis, liquorice, arisaema cum bile, duckweed and pistia stratiotes, and the amino acid sequences of the 2-hydroxyflavanone C-glycosyltransferase are shown as SEQ ID No: 14. 18, 20, 22 and 24; the C-glucose-2-hydroxyflavanone C-glycosyltransferase is selected from C-glucose-2-hydroxyflavanone C-glycosyltransferase ZmCGTb, OsCGTb1, OsCGTb2, SbCGTb, GuCGTb, AeCGGTb, LpCGTb and PsCGTb from corn, rice, Scutellaria baicalensis, liquorice, Arisaema japonicum, duckweed and pistia stratiotes, and the amino acid sequences of the C-glucose-2-hydroxyflavanone C-glycosyltransferase are sequentially shown as SEQ ID No: 26. 16, 17, 15, 19, 21, 23 and 25.

2. The method of claim 1, wherein schaftoside, isoschaftoside, or an analog thereof is synthesized using 2-hydroxyflavanone C-glycosyltransferase and C-glucose-2-hydroxyflavanone C-glycosyltransferase, comprising the steps of:

1) 2-hydroxyflavanone C-glycosyltransferase catalyzes the reaction of 2-hydroxyflavanone and uridine diphosphate glucose to generate C-glucose-2-hydroxyflavanone;

2) the C-glucose-2-hydroxyflavanone C-glycosyltransferase catalyzes the reaction of the C-glucose-2-hydroxyflavanone and uridine diphosphate arabinose to generate C-arabinose-C-glucose-2-hydroxyflavanone;

3) dehydrating or spontaneously dehydrating the C-arabinose-C-glucose-2-hydroxyflavanone by HCl treatment to generate schaftoside, isoschaftoside or an analogue chrysin-6-C-alpha-L-arabinose-8-C-beta-D-glucose and chrysin-6-C-beta-D-glucose-8-C-alpha-L-arabinose;

in the reaction formula, R is OH or H; when R is OH, the product is schaftoside and isoschaftoside isomer;

when R is H, the product is chrysin-6-C-alpha-L-arabinose-8-C-beta-D-glucose and chrysin-6-C-beta-D-glucose-8-C-alpha-L-arabinose isomers.

3. The method of claim 1, wherein the synthetic product is 3 '-C-glucose-5' -C-arabinophloretin having the following formula:

the 2-hydroxyflavanone C-glycosyltransferase catalyzes the reaction of phloretin and uridine diphosphate glucose to generate 3 '-C-glucosylphloretin, and the C-glucose-2-hydroxyflavanone C-glycosyltransferase catalyzes the reaction of the 3' -C-glucosylphloretin and the uridine diphosphate arabinose to generate the 3 '-C-glucose-5' -C-arabinophloretin.

4. The method of claim 1, wherein the synthetic product is 3 '-C-glucose-5' -C-arabino-froloninone, and the reaction formula is as follows:

2-hydroxy flavanone C-glycosyl transferase catalyzes the reaction of the frolonine and uridine diphosphate glucose to generate 3 '-C-glucose frolonine, and the C-glucose-2-hydroxy flavanone C-glycosyl transferase catalyzes the reaction of the 3' -C-glucose frolonine and uridine diphosphate arabinose to generate 3 '-C-glucose-5' -C-arabinose frolonine.

5. The method of claim 1, wherein the synthetic product is methyl 2,4, 6-trihydroxy-3-C-glucose-5-C-arabinobenzoate, having the following reaction formula:

2-hydroxyflavanone C-glycosyltransferase catalyzes the reaction of methyl 2,4, 6-trihydroxybenzoate and uridine diphosphate glucose to produce methyl 2,4, 6-trihydroxy-3-C-glucosylbenzoate, and C-glucose-2-hydroxyflavanone C-glycosyltransferase catalyzes the reaction of methyl 2,4, 6-trihydroxy-3-C-glucosylbenzoate and uridine diphosphate arabinose to produce methyl 2,4, 6-trihydroxy-3-C-glucosylbenzoate.

6. A C-glycosyltransferase catalyzes glucose group transfer, which is characterized in that the C-glycosyltransferase is selected from 2-hydroxyflavanone C-glycosyltransferase SbCGTa, GuCGTa, AeCGta, LpCGTa and PsCGTa from scutellaria baicalensis, liquorice, arisaema cum bile, duckweed and pistia stratiotes, and the amino acid sequence of the C-glycosyltransferase is sequentially shown as SEQ ID No: 14. 18, 20, 22 and 24.

7. A C-glycosyltransferase gene encoding the C-glycosyltransferase of claim 6, selected from five 2-hydroxyflavanone C-glycosyltransferase genes SbCGTa, GuCGTa, AeCGTa, LpCGTa and PsCGTa isolated from Scutellaria baicalensis, Glycyrrhiza uralensis, Arisaema sylvestre, Pistacia stratiotes, and Pistia stratiotes, the nucleotide sequences of which are shown in sequence tables as SEQ ID No: 1. 5, 7, 9 and 11.

8. The C-glycosyltransferase is characterized by being selected from C-glucose-2-hydroxyflavanone C-glycosyltransferase ZmCGTb, OsCGTb1, OsCGTb2, SbCGTb, GuCGTb, AeCGTb, LpCGTb and PsCGTb from corn, rice, scutellaria baicalensis, liquorice, arisaema consanguineum schott, duckweed and pistia stratiotes, and the amino acid sequences of the C-glycosyltransferase ZmCGTb, the OsCGTb1, the LpCGTb and the PsCGTb are sequentially shown as SEQ ID No: 26. 16, 17, 15, 19, 21, 23 and 25.

9. A C-glycosyltransferase gene encoding the C-glycosyltransferase of claim 8, selected from the group consisting of eight C-glucose-2-hydroxyflavanone C-glycosyltransferase genes ZmCGTb, osctb 1, osctb 2, SbCGTb, GuCGTb, aecggtb, lpcggtb, and PsCGTb isolated from maize, rice, scutellaria, glycyrrhiza, arisaema japonicum, duckweed, and pistia stratiotes, and having the nucleotide sequences as set forth in SEQ ID nos: 13. 3, 4, 2, 6, 8, 10 and 12.

10. A vector, expression cassette, host bacterium or transgenic cell line comprising the C-glycosyltransferase gene of claim 7 or 9.

Technical Field

The invention relates to the field of molecular biology, in particular to a C-glycosyltransferase (CGT) gene and an encoding protein thereof and application thereof in synthesizing schaftoside and isoschaftoside.

Background

Schaftoside and isoschaftoside are flavone dicarboglycosides with different glycosyl groups, and are distributed in various plants as plant protection elements. This group is currently contained in 19 plants of the family 39, including aquatic and terrestrial plants, monocotyledonous and dicotyledonous plants, and vegetation and shrub plants¹. Schaftoside and isoschaftoside have obvious physiological activity and pharmacological activity. In herba Antenoronis Neofiliformis, schaftoside as allelopathy inhibitor can inhibit growth of striga asiatica^2a-c. Within the phloem of rice, schaftoside acts as an antifeedant. Schaftoside and isoschaftoside in herba Artemisiae Annuae tuber have nematocidal activity. In addition, schaftoside and isoschaftoside also have various pharmacological activities. Schaftoside and isoschaftoside in fructus Colocasiae Esculentae, scleroderma plant (Scleropyrum), cane molasses and Scutellariae radix show antioxidant activity^3a-d. Schaftoside in herba Desmodii Styracifolii as angiotensin converting enzyme inhibitor with antihypertensive effect⁴. Isoschaftoside in Heguo taro can reduce renal Na⁺,K⁺-ATPase Activity exhibits antihypertensive Activity⁵. Schaftoside and isoschaftoside in leaf of plant (Costus spiralis) of genus Costus exhibit anti-diabetic function by inhibiting alpha-glucosidase⁶. Isoschaftoside in Jatropha curcas leaves shows activity of resisting malignant cell proliferation on A375 cells⁷. The schaftoside in Rhododendron isolobae and Eleusine indica has effects of resisting hepatotoxicity and inhibiting mouse pneumonia activity induced by LPS^8a-b. However, the biosynthetic process of schaftoside and isoschaftoside in plants is unclear.

In recent years, biosynthetic pathways of flavonoid carbon glycosides in plants have been reported, and there are three main pathways: 1) c-glycosyltransferase (OsCGT) in rice catalyzes 2-hydroxyflavanone to generate C-glucose-2-hydroxyflavanone, and dehydratase selectively catalyzes to generate flavone-6-C-glucose, such as isovitexin and isoorientin⁹. Furthermore, corn C-glycosyltransferase (UGT708A6)¹⁰C-sugar in buckwheatTransferase (FeCGTa and FeCGTb)¹¹Soybean medium C-glycosyltransferase (UGT708D1)¹²The catalytic function of (a) is similar to that of OsCGT. The above C-glycosyltransferases have a certain similarity in amino acid sequence, except for the similarity in catalytic function, and all contain a DPFF conserved region. 2) C-glycosyltransferase (GtUF6CGT1) in radix Gentianae directly catalyzes apigenin to generate isovitexin, which is a reaction for directly catalyzing flavone mono-glucoside on flavone parent nucleus¹³. GtUF6CGT1 selectively carries out carbon glycosidation reaction at C-6 position of flavone. Similarly, C-glycosyltransferase (PlugT43) in Pueraria lobata directly catalyzes daidzein to produce puerarin¹⁴. PlugT43 selectively undergoes a glycosidation reaction at the C-8 position of the isoflavone. 3) For the biosynthesis of the flavone diglucoside, only 2C-glycosyltransferases (FcCGT and CuCGT) were found in Citrus¹⁵. FcCGT and CuCGT firstly catalyze 2-hydroxy flavanone to generate C-glucose-2-hydroxy flavanone, then further catalyze to generate 2C-glucose-2-hydroxy flavanone, and dehydrate to generate New Zealand vitexin. FcCGT and CuCGT share the same DPFF conserved region as C-glycosyltransferase in most plants. However, the biosynthesis process of flavobidesmosides having different sugar groups, such as schaftoside and isoschaftoside, is not clear. Therefore, the elucidation of the biosynthesis process of the phytoalexin schaftoside and isoschaftoside in plants is of great significance.

Disclosure of Invention

The invention aims to clarify the biosynthesis process of schaftoside and isoschaftoside and establish a method for synthesizing schaftoside, isoschaftoside and analogues thereof by an in vitro enzyme method.

In order to achieve the technical purpose, the invention screens out possible C-glycosyltransferase gene sequences from seven plants, namely scutellaria baicalensis (Scutellaria baicalensis), corn (Zea mays), rice (Oryza sativa), liquorice (Glycyrrhiza uralensis), Pistia stratiotes (Pistia stratiotes), duckweed (Landolia puncinate) and Arisaema sylvestre (Arisaema esculentum) by a transcriptome analysis method.

Firstly, a possible C-glycosyltransferase gene is amplified by adopting an RT-PCR method, a recombinant vector is constructed by an improved quick-change method or a seamless splicing method, the recombinant vector is expressed in escherichia coli, and a corresponding C-glycosyltransferase protein is obtained after purification.

Through functional characterization, 13C-glycosyltransferases, namely five 2-hydroxyflavanone C-glycosyltransferases (SbCGTa, GuCGTa, PsCGTa, LpCGTa, AeCGTa) and eight C-glucose-2-hydroxyflavanone C-glycosyltransferases (SbCGTb, ZmCGTb, OsCGTb1, OsCGTb2, GuCGTb, PsCGTb, LpCGTb and AeCGTb) are found in scutellaria baicalensis, corn, rice, liquorice, pistia stratiotes, duckweed and arisaema sylvestre.

Five 2-hydroxyflavanone C-glycosyltransferase genes separated from scutellaria baicalensis, corn, rice, liquorice, pistia stratiotes, duckweed and arisaema consanguineum are named as SbCGTa, GuCGTa, PsCGTa, LpCGTa and AeCGTa respectively, and the nucleotide sequences of the genes are shown as SEQ ID No: 1. 5, 11, 9 and 7; the eight C-glucose-2-hydroxyflavanone C-glycosyltransferase genes are respectively named as SbCGTb, ZmCGTb, OsCGTb1, OsCGTb2, GuCGTb, PsCGTb, LpCGTb and AeCGTb, and the nucleotide sequences of the eight C-glucose-2-hydroxyflavanone C-glycosyltransferase genes are respectively shown as SEQ ID No: 2. 13, 3, 4,6, 12, 10 and 8.

Vectors, expression cassettes, host bacteria and transgenic cell lines containing the above-described 2-hydroxyflavanone C-glycosyltransferase gene or C-glucose-2-hydroxyflavanone C-glycosyltransferase gene are also within the scope of the present invention.

The amino acid sequences of the proteins SbCGTa, GuCGTa, AeCGTa, LpCGTa and PsCGTa coded by the 2-hydroxyflavanone C-glycosyltransferase gene are respectively shown as SEQ ID No: 14. 18, 20, 22 and 24. The amino acid sequences of the proteins ZmCGTb, OsCGTb1, OsCGTb2, SbCGTb, GuCGTb, AeCGTb, LpCGTb and PsCGTb coded by the C-glucose-2-hydroxyflavanone C-glycosyltransferase gene are respectively shown as SEQ ID No: 26. 16, 17, 15, 19, 21, 23 and 25.

The invention discovers that the C-glycosyltransferase participates in the biosynthesis process of the phytoalexin schaftoside and isoschaftoside in plants. The 2-hydroxy flavanone C-glycosyl transferase and the C-glucose-2-hydroxy flavanone C-glycosyl transferase can be used for realizing the synthesis of analogs such as schaftoside, isoschaftoside, chrysin-6-C-alpha-L-arabinose-8-C-beta-D-glucose, chrysin-6-C-beta-D-glucose-8-C-alpha-L-arabinose, 3 '-C-glucose-5' -C-arabinophloretin and the like by an in vitro enzyme method.

Thus, the present invention provides a method for synthesizing glucose and arabino-disaccharide carbon-glycoside compounds, comprising: catalyzing the compound shown in the formula I and uridine diphosphate glucose by using 2-hydroxyflavanone C-glycosyltransferase to generate a compound shown in the formula II, and catalyzing the compound shown in the formula II and uridine diphosphate arabinose by using C-glucose-2-hydroxyflavanone C-glycosyltransferase to generate a compound shown in the formula III:

wherein R is₁And R₂May be independent of each other or R₁And R₂Looping;

when R is₁And R₂Independently of each other, R₁Is H, hydroxy or alkoxy, R₂Is H, alkyl, alkoxy, phenyl, substituted phenyl, phenylalkylene, substituted phenylalkylene, phenoxy, substituted phenoxy, phenylalkoxy, substituted phenylalkoxy, acetophenone, p-hydroxybenzophenone, 3 ', 4' -dihydroxyacetophenone;

or, in formula I, R₁And R₂Cyclizing, wherein the compound of formula I has the following structure:

in the above formula, R is H or OH; the compound of formula II is obtained accordingly and has the following structure:

in the above formula, R is H or OH; the compound of formula III is correspondingly obtained as follows:

when R is₁When it is an alkoxy group, the alkoxy group is preferably a C1-C6 alkoxy group, such as methoxy (-OCH)₃) Ethoxy (-OCH)₂CH₃) And propoxy group (-OCH)₂CH₂CH₃) And the like.

When R is₂When the alkyl group is an alkyl group, the alkyl group is preferably a C1-C10 alkyl group, more preferably a C1-C6 alkyl group, such as a methyl group, an ethyl group, a propyl group, an isopropyl group, etc.

When R is₂When it is an alkoxy group, the alkoxy group is preferably a C1-C6 alkoxy group, such as methoxy (-OCH)₃) Ethoxy (-OCH)₂CH₃) And propoxy group (-OCH)₂CH₂CH₃) And the like.

When R is₂In the case of substituted phenyl, the substituent on the phenyl group may be one or more, and these substituents may be in the para, meta or ortho position, and the substituents are preferably hydroxy, C1-C6 alkyl, C1-C6 alkoxy, such as methyl, ethyl, propyl, methoxy, ethoxy, propoxy, etc.

When R is₂When it is a phenylalkylene group, it may be represented by- (CH)₂)_n-ph, for example, phenylmethylene (i.e., benzyl), phenylethylene, and the like; when R is₂When substituted phenylalkylene, it may be represented by- (CH)₂)_nSubstituted phenyl, which may have one or more substituents in the para, meta or ortho position, preferably hydroxy, C1-C6 alkyl, C1-C6 alkoxy, such as methyl, ethyl, propyl, methoxy, ethoxy, propoxy, etc. Wherein n is a positive integer, preferably a positive integer of 1-6.

When R is₂The structure of the compound is shown as follows when the compound is phenoxy, substituted phenoxy, phenylalkoxy, substituted phenylalkoxy, acetophenone group, p-hydroxybenzophenone group or 3 ', 4' -dihydroxyacetophenone group:

r in phenyl in the above-mentioned substituted phenoxy group₃Represents one or more substituents, which may be in the para, meta or ortho position, preferably hydroxy, C1-C6 alkyl, C1-C6 alkoxy, such as methyl, ethyl, propyl, methoxy, ethoxy, propoxy and the like.

R on phenyl in the above-mentioned substituted phenylalkoxy group₄Represents one or more substituents, which may be in the para, meta or ortho position, preferably hydroxy, C1-C6 alkyl, C1-C6 alkoxy, such as methyl, ethyl, propyl, methoxy, ethoxy, propoxy and the like.

N in the structural formulas of the phenylalkoxy and the substituted phenylalkoxy represents a positive integer, and preferably represents a positive integer of 1-6.

Further preferably, R₁Is hydrogen, hydroxy or methoxy; r₂Selected from one of the following groups:

referring to fig. 1, the in vitro enzymatic reaction for synthesizing schaftoside, isoschaftoside or analogs thereof is as follows: 2-hydroxyflavanone C-glycosyltransferase (CGTa) takes 2-hydroxyflavanone and uridine diphosphate glucose (UDPG) as raw materials to catalyze and generate C-glucose-2-hydroxyflavanone; the C-glucose-2-hydroxyflavanone C-glycosyltransferase (CGTb) takes C-glucose-2-hydroxyflavanone and uridine diphosphate arabinose (UDP-ara) as raw materials to catalyze and generate C-arabinose-C-glucose-2-hydroxyflavanone; the C-arabinose-C-glucose-2-hydroxy flavanone is dehydrated or spontaneously dehydrated by HCl to generate schaftoside, isoschaftoside, chrysin-6-C-alpha-L-arabinose-8-C-beta-D-glucose, chrysin-6-C-beta-D-glucose-8-C-alpha-L-arabinose and other analogue isomers.

The invention has the beneficial effects that:

the invention discovers 13C-glycosyltransferases in scutellaria baicalensis, corn, rice, liquorice, pistia stratiotes, duckweed and arisaema cum bile for the first time. These enzymes are involved in the synthesis of the phytoalexins schaftoside and isoschaftoside in plants, and elucidate the biosynthesis process. The invention discloses an in vitro enzyme catalysis method, which provides a feasible method for synthesizing schaftoside, isoschaftoside and analogues thereof.

Drawings

FIG. 1 is a reaction formula for in vitro enzymatic synthesis of schaftoside and isoschaftoside and their analogs using 2-hydroxyflavanone C-glycosyltransferase and C-glucose-2-hydroxyflavanone C-glycosyltransferase of the present invention.

FIG. 2 shows the reaction formulae of SbCGTa and SbCGTb for catalyzing 2-hydroxy pinocembrin (1) to generate chrysin-6-C-alpha-L-arabinose-8-C-beta-D-glucose (5a) and chrysin-6-C-beta-D-glucose-8-C-alpha-L-arabinose (5b) in sequence, and SbCGTa and SbCGTb for catalyzing 2-hydroxy naringenin (2) to generate schaftoside (6b) and isoschaftoside (6a) in sequence and the liquid chromatogram of each compound.

FIG. 3 is a liquid chromatogram of each compound and a reaction formula in which SbCGTa and SbCGTb catalyze the formation of 3 '-C-glucose-5' -C-arabinosylphloretin (9) from phloretin (7).

FIG. 4 shows the reaction formula of SbCGTa and SbCGTb for catalyzing frolonic acid (10) to generate 3 '-C-glucose-5' -C-arabinose frolonic acid (14), and SbCGTa and SbCGTb for catalyzing methyl 2,4, 6-trihydroxy-3-C-glucose-5-C-arabinose benzoate (15), and the liquid chromatogram of each compound.

FIG. 5 functional characterization experimental results for 2-hydroxyflavanone C-glycosyltransferase (CGTa) and C-glucose-2-hydroxyflavanone C-glycosyltransferase (CGTb) in maize, rice, licorice, pistia stratiotes, duckweed, Arisaema sylvestre, wherein:

(A) CGTa catalyzes 2-hydroxy naringenin to generate C-glucose-2-hydroxy naringenin, and CGTa catalyzes 2-hydroxy pinocembrin to generate C-glucose-2-hydroxy pinocembrin;

(B) CGTb catalyzes C-glucose-2-hydroxycinnulin to generate C-arabinose-C-glucose-2-hydroxycinnulin, and CGTb catalyzes C-glucose-2-hydroxycinnulin to generate C-arabinose-C-glucose-2-hydroxycinnulin.

Detailed Description

The experimental procedures in the following examples are conventional unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemicals, unless otherwise specified.