Cotton GhUGT71 gene and application thereof in phlorizin synthesis

Abstract

The invention discloses a cotton GhUGT71 gene and application thereof in phlorizin synthesis, and belongs to the field of gene engineering. The invention provides a GhUGT71 gene for regulating and controlling the synthesis amount of phlorizin of cotton. The nucleotide sequence of the GhUGT71 gene is shown as SEQ ID NO. 1. The GhUGT71 gene is a coding gene of UDP-glucosyltransferase GhUGT71, and an in-vitro enzyme reaction experiment proves that the GhUGT71 protein can efficiently catalyze phloretin to generate phlorizin. Transgenic experiments prove that the phlorizin content of the cotton can be remarkably increased by overexpressing the GhUGT71 gene in the cotton. The GhUGT71 gene can be used for preparing transgenic cotton varieties with high phlorizin content, and has great significance in enriching cotton germplasm resources and efficiently producing phlorizin.

Description

Technical Field

[0001] The invention relates to the field of genetic engineering, in particular to a cotton GhUGT71 gene and an application thereof in phlorizin synthesis. Background Art

[0002] Cotton is the field crop with the longest industrial chain in the world. In addition to the fiber as the raw material of the textile industry, the by-products such as cottonseed oil, cottonseed meal, and cottonseed hulls produced by processing are also closely related to human life. Developing and improving the value of cotton products is of great significance to the cotton industry. The high-value utilization of cotton products is the driving force for researchers to identify and evaluate bioactive substances in cottonseeds and study the molecular mechanism of their accumulation.

[0003] Flavonoids are secondary metabolites widely found in plants and play an important role in human health, with anti-inflammatory, anti-mutagenic, anti-cancer and anti-bacterial properties. Phlorizin and phlorizin are the main members of the dihydrochalcone class of compounds. Studies have shown that phlorizin lowers blood sugar by inhibiting sodium-dependent glucose transporter 2, which is involved in the absorption of glucose in the human intestine. Currently, several synthetic analogs of phlorizin have been approved for clinical anti-diabetic treatment. Although the effects of phlorizin on human health, especially diabetes and membrane permeability, have been reported in the literature, the understanding of the physiological activity of phlorizin in plants and the key enzymes in the biosynthetic pathway is still limited.

[0004] Studies have shown that phlorizin is a glycoside formed by the combination of phlorizin and glycosides as glucoside, and is one of the "few flavonoids" in nature. Its biosynthesis is mainly catalyzed by glycosyltransferase to transfer a UDP-glucose to the -OH group of phlorizin. Depending on the binding -OH site, different substances are generated. For example, binding to the 2'-OH generates phlorizin, and the 4'-OH generates trilobatin. Mining and identifying the phlorizin synthesis gene in cotton can provide phlorizin synthesis tool enzymes for metabolic engineering, provide data support for the functional analysis of the cotton UGT family, and provide excellent genes and germplasm resources for the high added value of cotton products. Summary of the invention

[0005] The purpose of the present invention is to provide a cotton GhUGT71 gene and its application in the synthesis of phlorizin to solve the problems existing in the above-mentioned prior art. Overexpression of the GhUGT71 gene in cotton can significantly increase the phlorizin content of cotton.

[0006] To achieve the above object, the present invention provides the following solutions:

[0007] The present invention provides the use of cotton GhUGT71 gene in any of the following:

[0008] (1) Application in regulating the synthesis of plant phlorizin;

[0009] (2) Application in cultivating transgenic plants with high phlorizin content;

[0010] The nucleotide sequence of the cotton GhUGT71 gene is shown in SEQ ID NO.1.

[0011] The present invention also provides the use of the recombinant vector comprising the cotton GhUGT71 gene in any of the following items:

[0012] (1) Application in regulating the synthesis of plant phlorizin;

[0013] (2) Application in the cultivation of transgenic plants with high phlorizin content.

[0014] The present invention also provides the use of the engineered bacteria comprising the recombinant vector in any of the following:

[0015] (1) Application in regulating the synthesis of plant phlorizin;

[0016] (2) Application in the cultivation of transgenic plants with high phlorizin content.

[0017] The present invention also provides the use of cotton GhUGT71 protein in any of the following:

[0018] (1) Application in regulating the synthesis of plant phlorizin;

[0019] (2) Application in cultivating transgenic plants with high phlorizin content;

[0020] The amino acid sequence of the cotton GhUGT71 protein is shown in SEQ ID NO.2.

[0021] Preferably, the expression level of the cotton GhUGT71 gene is up-regulated to increase the phlorizin content of the plant;

[0022] The plants include cotton.

[0023] The present invention also provides a method for increasing the content of phlorizin in a plant, comprising the steps of overexpressing the cotton GhUGT71 gene in a plant to increase the content of phlorizin in the plant;

[0024] The nucleotide sequence of the cotton GhUGT71 gene is shown in SEQ ID NO.1.

[0025] The present invention also provides a method for cultivating a transgenic plant with a high phlorizin content, comprising the following steps:

[0026] Overexpressing the cotton GhUGT71 gene in plant cells, then cultivating the plant cells, and using the plant cells to regenerate plants, thereby obtaining transgenic plants with high phlorizin content;

[0027] The nucleotide sequence of the cotton GhUGT71 gene is shown in SEQ ID NO.1.

[0028] Preferably, the plant comprises cotton.

[0029] The present invention also provides a method for preparing phlorizin, comprising the steps of using the protein expressed by the cotton GhUGT71 gene to perform an enzyme reaction;

[0030] The enzyme reaction system includes phloretin and UDP-glucose;

[0031] The amino acid sequence of the protein expressed by the cotton GhUGT71 gene is shown in SEQ ID NO.2.

[0032] Preferably, the temperature of the enzyme reaction is 37°C and the pH is 8.0.

[0033] The present invention discloses the following technical effects:

[0034] The present invention provides a GhUGT71 gene for regulating the synthesis amount of cotton phlorizin, and its nucleotide sequence is shown in SEQ ID NO.1. The GhUGT71 gene is a coding gene for UDP-glucose transferase GhUGT71, and in vitro enzyme reaction experiments have shown that the GhUGT71 protein can efficiently catalyze phlorizin to produce phlorizin. The present invention also proves through transgenic experiments that overexpressing the GhUGT71 gene in cotton can significantly increase the phlorizin content of cotton. The GhUGT71 gene can be used to prepare transgenic cotton with a high phlorizin content, which is of great significance for enriching cotton germplasm resources and efficiently producing phlorizin. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 This is the SDS-PAGE electrophoresis diagram for detecting the expression of pMAL-GhUGT71 recombinant protein;

[0037] Figure 2 It is the HPLC spectrum of the enzyme reaction experiment; A is the blank control group pMAL, and B is pMAL-GhUGT71;

[0038] Figure 3 The catalytic activity statistics of GhUGT71 protein under different environmental conditions; A represents different pH values, and B represents different temperatures;

[0039] Figure 4 Statistical graph of phlorizin content in the control (ZMS24) and GhUGT71 overexpression strain (OE-GhUGT71); *** indicates P < 0.001 compared with the control. DETAILED DESCRIPTION

[0040] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0041] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0042] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0043] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] The Column Buffer used in the present invention has the following formula: 1L Column Buffer includes 20mL 1M Tris-HCl (pH 7.4), 11.7g NaCl, 2.0mL 0.5M EDTA and 0.7mL β-mercaptoethanol.

[0046] Example 1

[0047] 1. Acquisition of GhUGT71 sequence

[0048] A candidate glycosyltransferase gene (GhUGT71) for phlorizin synthesis was screened using transcriptome analysis, and the gene was subsequently cloned and sequenced. The results showed that the gene was 1458 bp in length, with a CDS length of 1458 bp, no introns, and encoded 485 amino acids. Protein prediction software analysis revealed that the protein molecular weight was 54.085 kDa and the isoelectric point was 5.531.

[0049] 2. GhUGT71 in vitro enzyme reaction verification

[0050] In order to quickly verify the function of the GhUGT71 gene, the CDS sequence of the GhUGT71 gene was constructed on the pMAL-c5x protein expression vector, and the recombinant protein was purified. Using phlorizin as a substrate, UDP-glucose, recombinant protein, Tris-HCl (pH 7.4), MgCl2 and water were added, and the reaction was carried out at 37°C for 1 h. Subsequently, high performance liquid chromatography (HPLC) was used to detect whether phlorizin was produced.

[0051] The specific steps are as follows:

[0052] 2.1 Construction of pMAL-GhUGT71 protein expression vector

[0053] Using the cDNA of cotton ZMS24 (China Cotton Research Institute 24) as a template, primers pMAL-UGT71-F and pMAL-UGT71-R were used to clone the CDS sequence of the GhUGT71 gene. Through homologous recombination reaction, the CDS fragment of the GhUGT71 gene was cloned into the pMAL-c5x vector to construct the pMAL-GhUGT71 protein expression vector. The correctly sequenced plasmid was transformed into Escherichia coli BL21 (DE3) competent cells for protein induced expression.

[0054] pMAL-UGT71-F: 5'-ATCGAGGGAAGGATTTCACATATGATGAAGAAAGTAGAGCTGA TATTC-3' (SEQ ID NO. 3);

[0055] pMAL-UGT71-R: 5'-ATTACCTGCAGGGAATTCGGATCCTCAGGTGTTGGCTAGCATAT CCT-3' (SEQ ID NO. 4).

[0056] 2.2 Purification of pMAL-GhUGT71 protein by induction

[0057] a. First, pick the blank control pMAL and pMAL-GhUGT71 E. coli BL21 (DE3) monoclonal strains and add them to 5 mL LB medium. Culture them at 37°C and 200 rpm for 16 hours. Add 5 mL of the cultured bacterial solution to 250 mL LB medium and culture for 2-3 hours until the OD 600 =0.4-0.6.

[0058] b. Cool the bacterial solution on ice for 30 min, add IPTG to a final concentration of 0.5 mM, and culture with shaking at 16°C for 16 h.

[0059] c. Centrifuge at 4℃, 5000rpm for 10min, discard the supernatant, collect the cells, and add 20mL Column Buffer to resuspend the cells.

[0060] d. Ultrasonic cell disruptor was used to disrupt the bacteria (ultrasonication for 3 s, pause for 3 s, ultrasonication for 180 times; power 200 W).

[0061] e. Centrifuge at 4°C and 10,000 rpm, take the supernatant, add 0.5 mL of Amylose resin, and let it bind overnight, shaking and mixing 3 times.

[0062] f. Centrifuge at 4°C, 5000 rpm, then add 10-20 mL of Column Buffer and wash 4-5 times

[0063] g. Add 2 mL of Elution Buffer (add maltose to the column buffer to a final concentration of 10 mM) to elute the target protein and collect it in a 2 mL centrifuge tube. Run the purified protein and crude protein on a protein gel to verify whether the protein extraction is successful. The results are as follows: Figure 1 As shown, the target protein was successfully purified and recovered. The protein was set aside for use and then subjected to in vitro enzyme reaction experiments.

[0064] 2.3 pMAL-GhUGT71 in vitro enzyme reaction experiment

[0065] The purified proteins obtained in the above steps were added with substrate, Tris-HCl (pH 7.4), MgCl2, and UDP-glucose, and incubated at 37°C for 1 hour. The reaction solution was then centrifuged and tested on a microfluidic machine to see if phlorizin was generated. The reaction substances required for a 200 μL reaction system are shown in Table 1:

[0066] Table 1200 μL in vitro enzyme reaction system

[0067] Reaction material Dosage Purification of proteins 50μL Tris-HCl (1M, pH 7.4) 40μL <![CDATA[MgCl2(1M)]]> 2μL UDP-Glucose (100 mM) 7.5μL Substrate (10 mM) 10μL water 90.5μL

[0068] 2.4 Analysis of GhUGT71 in vitro enzyme reaction solution by high performance liquid chromatography (HPLC)

[0069] The blank control and GhUGT71 in vitro enzyme reaction solution were respectively detected by high performance liquid chromatography.

[0070] Flow rate: 0.5 mL / min; sample volume: 10 μL; chromatographic column: Agilent ZORBAX RX-SIL (2.1×150 mm, 5 μm), column temperature 30°C.

[0071] The mobile phases were 0.2% formic acid aqueous solution (aqueous phase) and methanol (organic phase). The gradient elution conditions were: 0 min, 5% organic phase, 95% aqueous phase; 5 min, 5% organic phase, 95% aqueous phase; 30 min, 100% organic phase; 75 min, 100% organic phase.

[0072] The results are as follows Figure 2 As shown, the pMAL blank control cannot produce a new substance phlorizin, while the GhUGT71 purified protein can produce a new substance phlorizin, that is, the GhUGT71 protein can catalyze phlorizin to produce phlorizin.

[0073] 3. Study on GhUGT71 enzyme kinetics

[0074] In order to study the optimal catalytic reaction conditions of GhUGT71 protein, we carried out enzyme kinetics study on the purified protein. We selected Tris-HCl with different pH values ​​(pH = 5.0, 6.0, 7.4, 8.0, 9.0, 10.0) and different reaction temperatures (4°C, 10°C, 16°C, 30°C, 37°C, 40°C, 50°C, 65°C), and used the peak area of ​​phlorizin generated as a measure of enzyme activity. The results are shown in Figure 2. Figure 3 As shown, GhUGT71 protein exhibited higher catalytic activity under the conditions of 37°C and pH 8.0.

[0075] 4. Creation and identification of GhUGT71 overexpressing cotton and determination of phlorizin content

[0076] The GhUGT71 gene was constructed into an overexpression vector, which was then transformed into cotton through Agrobacterium-mediated method to obtain a GhUGT71 overexpression strain.

[0077] The steps are as follows: clone the CDS sequence of GhUGT71 from cotton Zhongmiao 24, and clone the CDS fragment of GhUGT71 (as shown in SEQ ID NO.1, and the amino acid sequence of the expressed GhUGT71 protein is shown in SEQ ID NO.2) into the target vector pCAMBIA2300 through homologous recombination reaction to obtain the pCAMBIA2300-GhUGT71 expression vector.

[0078] The primer sequence of pCAMBIA2300-GhUGT71 is:

[0079] OE-GhUGT71-F:5'- CTGTACAAGGGTACC ATGAAGAAAGTAGAGCTGA-3' (SEQ ID NO.5);

[0080] OE-GhUGT71-R:5'- CTGCTCAGGCGCGCC TCAGGTGTTGGCTAGC-3' (SEQ ID NO. 6).

[0081] SEQ ID NO.1:

[0082]

[0083] SEQ ID NO.2:

[0084] MKKVELIFVPTPGAGHLAPTVEFVKRLIHHDDRILVTILSIEWHSSGFADAYTESLATLQ SDRIQVIQVSRVDPPPLHLWTLAPEAFVYALIQSYIPHVRNAVTNILSMKSTSDSTRVAGLVLDLFCSPMVDVATELGLSSYIYLTSNASFFGLVLYLQALHRQIS SEFEYPNPENPSKDLGHLIPGFANPVPSCVLPSFMFNKHGSYTAFVKIAERFKDAKGIVINTFEELETYALSCFVNGQNPPIYPVGPVIHLDSLPHPELDQLQRDRI MKWLDNQPESSVVFLCFGSHGCHGPPQVKEIALGLEQSGQRFLWSLHMPETPLNDAAGAVHYKNPEEMLPEGFLERIQERGMICRWAPQVDVLGHKAVGGFVSHCG WNSILESLWHGVPIVTWPIYAEQQLNAYMMKEVGLAVELRLDYRGGTSDIVEAEEIEEGVRQVMDAGSEVRKKVKEMAEMARKAVMNGGSSFNSIGRFIEDMLANT.

[0085] The pCAMBIA2300-GhUGT71 plasmid was transferred into the Agrobacterium tumefaciens strain LBA4404, and stable transgenic overexpression cotton seedlings were obtained by Agrobacterium-mediated method. Total RNA was extracted from the leaves of transgenic seedlings, and the expression of GhUGT71 in the overexpression strain and the wild-type strain was compared by real-time fluorescence quantitative method to screen the overexpression strain. Subsequently, the fresh leaves were ground with liquid nitrogen, 100 ml of which was extracted by adding 1 ml of 70% methanol, and the supernatant was then measured by liquid chromatography-mass spectrometry (LC-MS) to determine the amount of phlorizin in the overexpression strain and the wild-type strain. The results are shown in Figure 2. Figure 4 As shown, the phlorizin content in the GhUGT71 overexpression strains (OE-GhUGT71-1, OE-GhUGT71-2 and OE-GhUGT71-3) was significantly higher than that in the control wild type (ZMS24).

[0086] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of cotton GhUGT71 gene in any of the following: (1) Application in regulating the synthesis of plant phlorizin; (2) Application in cultivating transgenic plants with high phlorizin content; The nucleotide sequence of the cotton GhUGT71 gene is shown in SEQ ID NO.

1.

2. Use of the recombinant vector comprising the cotton GhUGT71 gene according to claim 1 in any of the following: (1) Application in regulating the synthesis of plant phlorizin; (2) Application in the cultivation of transgenic plants with high phlorizin content.

3. Use of the engineered bacteria comprising the recombinant vector described in claim 2 in any of the following: (1) Application in regulating the synthesis of plant phlorizin; (2) Application in the cultivation of transgenic plants with high phlorizin content.

4. Application of cotton GhUGT71 protein in any of the following: (1) Application in regulating the synthesis of plant phlorizin; (2) Application in cultivating transgenic plants with high phlorizin content; The amino acid sequence of the cotton GhUGT71 protein is shown in SEQ ID NO.

2.

5. The use according to any one of claims 1 to 4, characterized in that: Up-regulating the expression level of the cotton GhUGT71 gene to increase the phlorizin content of the plant; The plants include cotton.

6. A method for increasing the content of phlorizin in plants, characterized in that: The method comprises the steps of overexpressing the cotton GhUGT71 gene in the plant to increase the phlorizin content of the plant; The nucleotide sequence of the cotton GhUGT71 gene is shown in SEQ ID NO.

1.

7. A method for cultivating transgenic plants with high phlorizin content, characterized in that: The following steps are involved: Overexpressing the cotton GhUGT71 gene in plant cells, then cultivating the plant cells, and using the plant cells to regenerate plants, thereby obtaining transgenic plants with high phlorizin content; The nucleotide sequence of the cotton GhUGT71 gene is shown in SEQ ID NO.

1.

8. The method according to claim 6 or 7, characterized in that The plants include cotton.

9. A method for preparing phlorizin, characterized in that: The method comprises the steps of using the protein expressed by the cotton GhUGT71 gene to perform an enzyme reaction; The enzyme reaction system includes phloretin and UDP-glucose; The amino acid sequence of the protein expressed by the cotton GhUGT71 gene is shown in SEQ ID NO.

2.

10. The preparation method according to claim 9, characterized in that: The temperature of the enzyme reaction was 37°C and the pH was 8.0.

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