CN116376941A - EpGT6 gene of anthocyanin glycosyltransferase of epimedium herb and application thereof - Google Patents
EpGT6 gene of anthocyanin glycosyltransferase of epimedium herb and application thereof Download PDFInfo
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Abstract
The invention discloses an epimedium herb anthocyanin glycosyltransferase gene EpGT6 and application thereof, belonging to the technical field of epimedium herb anthocyanin transferase gene screening, wherein the nucleotide sequence of the EpGT6 gene is shown as SEQ ID NO.1; or a sequence having identity of 90% or more to the nucleotide sequence shown in SEQ ID NO.1; or a sequence obtained by substitution, deletion or addition of a nucleotide from the sequence shown in SEQ ID NO.1; or a different transcript or homologous gene sequence is generated from the nucleotide sequence shown in SEQ ID NO.1; the anthocyanin is synthesized by catalyzing anthocyanin and glycosyl by the gene, and a foundation is provided for breeding of epimedium lanuginosum.
Description
Technical Field
The invention relates to the technical field of screening of epimedium anthocyanin transferase genes, in particular to an epimedium anthocyanin glycosyltransferase gene EpGT6 and application thereof.
Background
Herba Epimedii is a traditional Chinese medicine, and has effects of nourishing liver and kidney, dispelling pathogenic wind and dampness, and strengthening tendons and bones. In addition, the flowers and leaves of the epimedium herb contain rich anthocyanin, have peculiar color and are abnormal and beautiful, and are also popular ornamental plants for gardens.
In anthocyanin biosynthesis, glycosylation is the most downstream step, and is mainly catalyzed by Glycosyltransferases (GTs) to bind anthocyanin to various glycosyl groups to form glycoside. The modification of the anthocyanidin plays a vital role in maintaining the stability of anthocyanin, and the disaccharide anthocyanin is more stable than the monosaccharide anthocyanin. However, the mechanism of anthocyanin components and glycosylation modification in epimedium is not completely clear at present. The epimedium has rich anthocyanin, has higher antioxidant activity, can regulate the body health of people, is often used as an effective component of various health products, and has great economic value, so that cloning and identifying the epimedium anthocyanin glycosyltransferase gene with special catalytic function, substrate specificity and biological activity has important theoretical and application values.
Therefore, screening and applying the gene of the epimedium lanuginosum anthocyanin glycosyltransferase is a problem which needs to be solved by the person skilled in the art.
Disclosure of Invention
In view of the above, the invention provides an epimedium herb anthocyanin glycosyltransferase gene EpGT6, discovers a catalytic formation mechanism of anthocyanin in the epimedium herb, and lays a theoretical foundation for breeding the epimedium herb with good quality and strong antioxidant activity.
In order to achieve the above purpose, the present invention adopts the following technical scheme:
the epimedium lanuginosum anthocyanin glycosyltransferase gene EpGT6, and the nucleotide sequence of the EpGT6 gene is shown as SEQ ID NO.1; or a sequence having identity of 90% or more to the nucleotide sequence shown in SEQ ID NO.1; or a sequence obtained by substitution, deletion or addition of a nucleotide from the sequence shown in SEQ ID NO.1; or a different transcript or homologous gene sequence is produced from the nucleotide sequence shown in SEQ ID NO. 1.
As the same invention concept as the technical scheme, the invention also claims the protein coded by the anthocyanin glycosyltransferase gene EpGT6, and the amino acid sequence is shown as SEQ ID NO.2.
As the same inventive concept as the technical scheme, the invention also claims the application of anthocyanin glycosyltransferase gene EpGT6 in catalyzing anthocyanin to combine with various glycosyl groups to form anthocyanin.
Preferably, the anthocyanin comprises cyanidin-3-O-rhamnoside.
As the invention conception same as the technical scheme, the invention also claims the application of the anthocyanin glycosyltransferase gene EpGT6 in breeding of the epimedium herb varieties with high anthocyanin content, strong antioxidant activity and high quality.
As the same invention concept as the technical scheme, the invention also claims a recombinant vector, an expression cassette, a recombinant bacterium and a recombinant cell containing the anthocyanin glycosyltransferase gene EpGT 6.
As the invention concept same as the technical scheme, the invention also claims the application of the recombinant vector, the expression cassette, the recombinant bacterium and the recombinant cell in catalyzing anthocyanin and various glycosyl to form anthocyanin.
According to the technical scheme, compared with the prior art, the glycosyltransferase gene EpGT6 is a key enzyme gene involved in modification of the epimedium anthocyanin. The gene can be applied to heterologously synthesizing and producing anthocyanin by using synthetic biology or metabolic engineering technology, and provides theoretical support for molecular breeding of epimedium.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only embodiments of the present invention, and that other drawings can be obtained according to the provided drawings without inventive effort for a person skilled in the art.
FIG. 1 is a graph of the analysis of the evolutionary tree of the sequence of EpGT6 protein of Epimedium herb anthocyanin glycosyltransferase and other plant glycosyltransferases.
FIG. 2 is a graph showing the analysis of the expression level of the EpGT6 gene of the epimedium herb anthocyanin glycosyltransferase in different tissues.
FIG. 3 is an SDS-PAGE electrophoresis of recombinant protein EpGT 6. M: protein maker;1: unpurified post-induction recombinant protein EpGT6;2: unpurified post-induction recombinant protein EpGT6 repeat; 3: the protein GT6 was purified.
FIG. 4 is a graph of the major enzymatic activity product of the recombinant protein EpGT6 identified by UPLC technology according to the invention.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely with reference to the accompanying drawings, in which it is apparent that the embodiments described are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Materials, reagents and instruments used in the examples of the present invention are conventional in the art and are commercially available unless otherwise specified.
Plant material and growth environment in the present invention: herba Epimedii is planted in the Shawan region of Leshan city, sichuan province, and grows under a shading net with a shading degree of 75%. Picking newly-unfolded leaves, washing with double distilled water, wrapping with tinfoil paper, and immediately quick-freezing in liquid nitrogen. The obtained product is stored in a refrigerator at-80deg.C for subsequent RNA extraction. Taking different tissues (roots, stems, leaves, flowers and fruits) of epimedium lanuginosum, washing with double distilled water, wrapping with tinfoil paper, immediately quick-freezing in liquid nitrogen, and then sending to Beijing Xingbmai technology Co.
Anthocyanin standards were purchased from Jiangsu Yongjiang medical science and technology Co. The glycosyl donor UDP-Glc was purchased from Shanghai Seiyaka Biotechnology Co. The E.coli expression vector pMAL-c2X was from the laboratory of the institute of medical and plant science of the national academy of medical science.
Example 1
Cloning of EpGT6 gene of Epimedium lanuginosum
(1) Screening of candidate genes
122 UGTs of Arabidopsis thaliana (Arabidopsis thaliana) are used as query terms, and the BLAST algorithm is adopted to search UGT genes in the genome of the epimedium lanuginosum. The presence of the retention domains in the UGT protein sequence was further confirmed by Hidden Markov Models (HMM) in the Pfam database (http:// Pfam. Xfa m. Org /) and NCBI CDD database (http:// www.omicsclass.com/arc/310). Wherein the UGT accession number of Pfam is PF00201. Information on UGTs (including ORFs, amino acid length) is obtained from genomic data.
(2) Sequence characteristics and evolutionary relationship analysis of epimedium lanosum anthocyanin glycosyltransferase gene EpGT6
The invention screens a candidate glycosyltransferase gene sequence (named as EpGT6 according to general rule) from the genome data of epimedium lanuginosum, has a length of 1383bp, contains a complete ORF, and predicts a polypeptide with a length of 461 amino acids. The proteins encoded by EpGT6 were found to have a higher similarity to these proteins compared to sequences of other plant anthocyanin glycosyltransferases with known functions. The invention uses Clustal X2 software to compare EpGT6 with other known functional plant glycosyltransferase protein sequences, and then uses the maximum likelihood method to construct a phylogenetic tree in MEGA 6.0, and the bootstrap value is 1000 (figure 1). From FIG. 1, it is clear that EpGT6 forms a branch with known glycosyltransferases modifying anthocyanin glycosyl, such as morning glory and Arabidopsis thaliana, and the function is possibly related to the modification of anthocyanin with sugar.
(3) Total RNA extraction and reverse transcription of plants
Fresh epimedium herb leaves are taken to be placed into liquid nitrogen for quick freezing, then the leaves are rapidly put into a grinding bowl added with the liquid nitrogen for grinding, and the liquid nitrogen is timely supplemented in the grinding process to prevent tissue from melting until the tissue is completely ground into powder. By using
The Super total RNA extraction kit is used for extracting plant total RNA, and the obtained RNA solution is used for detecting the integrity, purity and concentration. mu.L of 6×loading buffer was added to the 2. Mu.LR NA solution, and the mixture was mixed well, and the mixture was put into 1.2% agarose gel wells, followed by gel electrophoresis (180V, 10 min). The gel block was then placed under a fully automated gel imager to observe the strip. If two very distinct bands (28S rRNA and 18S rRNA) are present, the intensity of the 28S rRNA band is 1.5-2.0 times that of the 18S rRNA band, indicating that the RNA is more complete. Detection of total RNA concentration, OD Using a Nanodrop 2000 ultraviolet Spectrophotometer 260 /OD 280 Value, OD 260 /OD 230 Values. If OD 260 /OD 280 The value of (2) is 1.8-2.1, and OD 260 /OD 230 And the total RNA quality is high and is higher than 2.0, and the total RNA can be used for the subsequent reverse transcription experiment. Reverse transcription experiments were performed using FastKing gDNA Dispe lling RT SuperMix and the cDNA obtained by reverse transcription was stored in a-20℃refrigerator for use.
(4) Nest PCR primer for designing candidate glycosyltransferase gene EpGT6
Nest type primer is designed according to the EpGT6 gene sequence, and the primer sequence is as follows:
first round:
forward primer: epGT6Lf1:5'-AAGCTAGTCAATCCTATTCCTAA-3', as shown in SEQ ID NO. 3;
reverse primer: epGT6Lr1:5'-CAAGACAAAACATAAAGAATTTAAAC-3', as shown in SEQ ID NO. 4;
a second wheel:
forward primer: epGT6Lf2:5'-ATGGCCGACACCAATGCCG-3', as shown in SEQ ID NO. 5;
reverse primer: epGT6Lr2:5'-ACGAAGCATCCCTTTCAGCT-3', as shown in SEQ ID NO. 6.
The primers were synthesized by Beijing Sanbo polygala tenuifolia Biotechnology Limited liability company.
(5) Amplification of full-Length sequence of EpGT6 Gene of EpGT6, an Epimedium lanuginosum glycosyltransferase
Using
high fidelity DNA Polymerases (NewEnglandBioLabs, MA, USA) for nested PCR amplification. The PCR procedure was set as follows: first round: using cDNA as a template, and carrying out PCR amplification by using a first round of primers, wherein the pre-denaturation temperature is 98 ℃ for 30s; denaturation temperature 98 ℃,10s, annealing temperature 55 ℃,30s, extension temperature 72 ℃,1min 20s, denaturation-annealing-extension for 35 cycles; finally, the temperature is 72 ℃ and the time is 5min. After the first round of PCR amplification is completed, the PCR product is used as a template, and a second round of PCR amplification is carried out by using a second round of primers under the same PCR condition. The PCR product was purified by AxyPrep DNA gel recovery kit (Corning, NY, USA), then cloned into pTOPO-Blunt simple vector (LANY, china, beijing)), transformed into E.coli competent cell Trans1-T1 (TransGen Biotech, china, beijing), positive clones were selected for sequencing (Beijing Sanbo polygala Biotechnology Co., ltd.) and EpGT6 gene clones with correct sequences were selected and saved for the construction of subsequent expression vectors.
The length of the full-length Open Reading Frame (ORF) of the epimedium herb glycosyltransferase gene EpGT6 obtained by sequencing is 1383bp, and the nucleotide sequence is shown as SEQ ID NO.1; encoding 461 amino acids, and the amino acid sequence is shown as SEQ ID NO.2.
Prokaryotic expression and functional analysis of epimedium lanuginose glycosyltransferase EpGT6
1) Expression of EpGT6 in different tissues of Epimedium
Through analysis of transcriptome data of different tissues of epimedium lanuginosum, the expression level of EpGT6 in leaves is highest. It was therefore assumed that EpGT6 is involved in the modification of anthocyanins in leaves (fig. 2).
2) Construction and transformation of escherichia coli expression vector
(1) The vector and fragment were ligated using a seamless cloning kit. Sequentially, 5. Mu.L of CutSmartBuffer, 1. Mu.L of EcoRI/XbaI, 10. Mu.L of vector plasmid and 33. Mu.L of Nuclease-Free Water were added to each reagent in a 0.2mL PCR cuvette, and reacted in a Water bath at 37℃for 30 minutes.
(2) mu.L of the ligation product was pipetted into 40. Mu.L of Trans1-T1 E.coli competent cells freshly thawed on ice, gently flicked, mixed and then allowed to stand on ice for 30min.
(3) The mixture was heat-shocked in a water bath at 42℃for 60s and then immediately placed on ice for 2min.
(4) 300. Mu.L of LB liquid medium without antibiotics equilibrated to room temperature, 200rpm at 37℃and shaking culture for 1h.
(5) The shaken bacterial solution is evenly spread on an LB screening plate (carbenicillin resistance). After the bacterial liquid is completely absorbed by the culture medium, the culture dish is inverted and placed in a constant temperature incubator at 37 ℃ for overnight culture (12-16 h).
(6) And (3) identifying and screening positive clones by bacterial liquid PCR, and sending the positive clones to the CTABO biotechnology limited liability company for sequencing and verifying whether the positive clones are correctly connected with the pMAL-c2X vector.
(7) The plasmid carrying the EpGT6 gene (pMAL-c 2X-EpGT 6) was transferred into the E.coli expression strain (BL 21), and the method was the same as that of Trans1-T1 E.coli.
Induction purification of recombinant proteins and detection of enzymatic Activity
(1) BL 21-expressing strains containing pMAL-c2X-UGTs were isolated and cultured in 2mL of LB (containing carbenicillin 50 mg/L) liquid medium with shaking overnight at 37℃at 200rpm.
(2) 1.5mL of the overnight cultured bacterial liquid is taken and cultured in 300mL of fresh LB medium (containing 50mg/L of carbenicillin and 0.4% (W/V) glucose by filtration sterilization) by shaking in a shaking table at 37 ℃ until the bacterial liquid reaches OD 600 About 0.5, and the rotation speed was 200rpm.
(3) Adding 0.3-0.5 mM IPTG (isopropyl-beta-D-thiogalactoside) into 300mL bacterial liquid, shaking and culturing at 16 ℃ for 24h with the rotating speed of 200rpm.
(4) The cells were collected by centrifugation at 8,000rpm at 4℃for 3 min.
(5) Purifying and concentrating the target recombinant protein of the collected thalli. The crude bacterial proteins containing recombinant UGTs were purified according to MBP fusion protein purification systems manual from new england biological laboratories, inc (New EnglandBioLab), and the eluted crude proteins were concentrated in protein solution using Millipore (30 KDa) and finally replaced with enzyme activity reaction buffer. After SDS-PAGE electrophoresis, the recombinant proteins were stained with Coomassie blue dye solution to confirm the size of the recombinant proteins (FIG. 3).
(6) The enzyme activity was determined using the concentrated fusion protein. The UGT enzyme activity reaction system is 100 mu L: comprising 2mM acceptor (cyanidin-3-O-rhamnoside), 100mM donor (UDP-Glc), and 10-20ng of fusion protein. After reacting for 0.5h at 30 ℃, adding methanol with the same volume to stop the reaction, and mixing vigorously. Centrifugal at 13000rpm for 10min, 140. Mu.L was taken and checked for product formation by UPLC to confirm UGT activity.
The product peak was detected in the reaction with cyanidin-3-O-rhamnoside, whereas in the blank (reaction with boiling fusion protein, other conditions unchanged) no peak was detected (FIG. 4). This result suggests that EpGT6 is an anthocyanin glycosyltransferase with cyanidin-3-O-rhamnoside as a specific substrate.
UPLC Condition
Detection was performed using UPLC. The sample compounds were first isolated using UPLC (Agilent) with a separation column model RRHD Eclipse Plus C (1.8 μm, 100X 2.1mm i.d.; agilent). Mobile phase: phase A is 0.1% formic acid aqueous solution and phase B is acetonitrile. Elution gradient: (0-1.5 min, 21-24% of B phase, 1.5-3 min, 24-25% of B phase, 3-4min, 25-29% of B phase, 4-5min, 29% of B phase, 5-6.5 min, 29-32% of B phase, 6.5-7 min, 32-44% of B phase, 7-8 min, 44-45% of B phase, 8-9 min, 45-46% of B phase, 9-11 min and 46' -95% of B phase). The flow rate is 0.6 mL/min, the column temperature is 35 ℃, the sample injection amount is 5 mu L, and the PDA detection wavelength is 340nm.
In the present specification, each embodiment is described in a progressive manner, and each embodiment is mainly described in a different point from other embodiments, and identical and similar parts between the embodiments are all enough to refer to each other. For the device disclosed in the embodiment, since it corresponds to the method disclosed in the embodiment, the description is relatively simple, and the relevant points refer to the description of the method section.
The previous description of the disclosed embodiments is provided to enable any person skilled in the art to make or use the present invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other embodiments without departing from the spirit or scope of the invention. Thus, the present invention is not intended to be limited to the embodiments shown herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (7)
1. The epimedium lanuginosum anthocyanin glycosyltransferase gene EpGT6 is characterized in that the nucleotide sequence of the EpGT6 gene is shown as SEQ ID NO.1; or a sequence having identity of 90% or more to the nucleotide sequence shown in SEQ ID NO.1; or a sequence obtained by substitution, deletion or addition of a nucleotide from the sequence shown in SEQ ID NO.1; or a different transcript or homologous gene sequence is produced from the nucleotide sequence shown in SEQ ID NO. 1.
2. The protein encoded by anthocyanin glycosyltransferase gene EpGT6 according to claim 1, wherein the amino acid sequence is shown in SEQ ID NO.2.
3. Use of the anthocyanin glycosyltransferase gene EpGT6 of claim 1 to catalyze the binding of anthocyanin to various glycosyls to form anthocyanin.
4. The use according to claim 3, wherein the anthocyanin comprises cyanidin-3-O-rhamnoside.
5. The application of over-expressing the anthocyanin glycosyltransferase gene EpGT6 in breeding of a variety of epimedium lanuginosum with high anthocyanin content, strong antioxidant activity and high quality in claim 1.
6. A recombinant vector, an expression cassette, a recombinant bacterium, a recombinant cell comprising the anthocyanin glycosyltransferase gene EpGT6 of claim 1.
7. The recombinant vector, the expression cassette, the recombinant bacterium and the recombinant cell of claim 5, which are used for catalyzing anthocyanin and various glycosyl to combine to form anthocyanin.
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