CN113462703A - Plant anthocyanin metabolism related gene Rd3GTs and encoding protein and application thereof - Google Patents

Abstract

The invention discloses a Rhododendron delavayi anthocyanin metabolism related gene Rd3GTs, and belongs to the technical field of biological engineering. The nucleotide sequence of the Rd3GTs gene is shown in SEQ ID NO. 1 or SEQ ID NO. 2. The invention also discloses a Rd3GTs protein expressed and recombined by the Rd3GTs gene in an escherichia coli cell, which can catalyze cyanidin reaction in vitro to generate cyanidin 3-O-glucoside and has the function of flavonoid 3-O-glycosyltransferase. The invention also discloses application of the Rd3GTs gene in transgenic plants, and the Rd3GTs gene is transferred into an arabidopsis mutant and tobacco, so that the anthocyanin phenotype of the arabidopsis mutant is successfully recovered, and the color of tobacco petals is deepened. The Rd3GTs gene has potential application value in the aspects of flower color modification of transgenic plants and medicinal plant medicinal components improvement.

Description

Plant anthocyanin metabolism related gene Rd3GTs and encoding protein and application thereof

Technical Field

The invention relates to the technical field of bioengineering, in particular to a plant anthocyanin metabolism related gene Rd3GTs and a coding protein and application thereof.

Background

Anthocyanin is one of the most important pigment substances which influence the color presentation of plants, and can endow the plants with a series of different colors from red to purple. Research shows that anthocyanin has many important functions besides imparting color to tissues such as flowers and fruits. It can protect plant cells from being damaged by ultraviolet rays, resist pathogens and herbivores, serve as signal molecules to promote interaction between plants and microorganisms, and influence growth and development of pollen, transport of hormones in plants, and the like. In addition, a large number of experiments prove that the anthocyanin has close relation with human health, has biological activities of oxidation resistance, virus resistance, cell proliferation resistance and the like, is used for treating diseases such as arteriosclerosis, cardiovascular and cerebrovascular diseases and the like, and is one of secondary metabolites which are focused on by researchers at present.

The anthocyanin is synthesized under the control of a structural gene for coding the synthesis of the anthocyanin, and the flavonoid 3-O-glycosyltransferase 3GT is the last key enzyme in the anthocyanin synthesis pathway and is an enzyme necessary for the biosynthesis of various anthocyanins of plants. The glycosylation catalyzed by 3GT transfers the activated sugar molecule in the glycosyl donor to the C-3 position of the anthocyanidin to form anthocyanin, which can enhance the stability and water solubility of anthocyanin. Studies have shown that mutations in the 3GT gene or inactivation of the enzyme can affect and alter the color or anthocyanin content of the target plant. For example, after the morning glory Ip3GT gene is mutated, the flower color is changed from dark color to light color or spot color; after the expression of the 3GT gene of the phalaenopsis is down regulated by RNA interference, the petal color of the transgenic phalaenopsis is lightened to different degrees. Thus, 3GT is an important key enzyme influencing plant anthocyanin synthesis, and is also a target enzyme for improving plant color phenotype and beneficial health-care components (anthocyanin).

The 3GT gene was originally isolated from maize in 1977, and has been cloned from petunia, purple sweet potato, alfalfa, pear, butterfly orchid, grape, strawberry, litchi and other plants. With the continuous improvement of cDNA cloning technology, researchers in China clone cDNA or genomic DNA sequences of 3GT genes from a plurality of plants, but no report is found on the cloning and function research of the 3GT genes of rhododendron delavayi, so that the evolution research of 3GT is greatly limited, and meanwhile, the utilization of 3GT of rhododendron plants and the regulation and improvement of anthocyanin biosynthesis are also limited.

Disclosure of Invention

In order to solve the problems in the prior art, the invention aims to provide a plant anthocyanin metabolism related gene Rd3GTs which can be used for anthocyanin improved transgenic plants.

The invention provides a plant anthocyanin metabolism related gene Rd3GTs, wherein the cDNA nucleotide sequence of the gene is shown as SEQ ID NO. 1 or SEQ ID NO. 2.

Preferably, the plant is Rhododendron delavayi Franch.

The invention also provides Rd3GTs protein expressed by the gene, and the protein amino acid sequence coded by the SEQ ID NO. 1 nucleotide sequence is shown as SEQ ID NO. 3; the amino acid sequence of the protein coded by the SEQ ID NO. 2 nucleotide sequence is shown as SEQ ID NO. 4.

The invention also provides a recombinant expression vector containing the gene.

Preferably, the vector plasmid is pET 32.

The invention also provides a binary expression vector containing the gene.

Preferably, the vector plasmid is pBI 121.

The invention also provides a recombinant host cell containing the expression vector.

The invention also provides application of the gene or the protein or the expression vector in transgenic plants with improved anthocyanin.

Preferably, the plant is arabidopsis thaliana or tobacco.

Compared with the prior art, the technical scheme of the invention has the following beneficial effects:

the invention uses total RNA of Rhododendron delavayi Franch as a template to clone and obtain genes Rd3GTs related to anthocyanin metabolism of Rhododendron delavayi Franch, can be used for constructing an expression vector, can express recombinant Rd3GTs protein in recombinant cells, has the function of flavonoid 3-O-glycosyltransferase, and can catalyze cyanidin to react to generate cyanidin 3-O-glucoside in-vitro enzyme activity reaction.

The invention transfers the Rd3GTs gene into the arabidopsis mutant, and the obtained transgenic plant can successfully recover the anthocyanin phenotype of the arabidopsis mutant, and can recover the synthesis of anthocyanin in the mutant cotyledon and hypocotyl of arabidopsis. The Rd3GTs gene is transferred into a tobacco plant, and the obtained transgenic plant can deepen the color of tobacco petals.

The invention uses total RNA of Rhododendron delavayi Franch as a template to clone and obtain genes Rd3GTs related to anthocyanin metabolism of Rhododendron delavayi Franch, which can be used for obtaining transgenic plants, regulating and improving anthocyanin biosynthesis of the transgenic plants, and achieving the effects of flower color modification of the transgenic plants and improvement of medicinal components of the transgenic medicinal plants.

Drawings

FIG. 1: multiple sequence alignment analysis chart of Rd3 GTs;

FIG. 2: a phylogenetic analysis diagram of Rd3 GTs;

FIG. 3: graphs of amplification and validation results for Rd3GT1 and Rd3GT 6;

FIG. 4: coli growth profile, a: growth curve of pET32a (+) -Rd3GT1-BL21, B: the growth curve of pET32a (+) -Rd3GT6-BL 21;

FIG. 5: rhododendron delavayi 3GT protein electrophoresis diagram, A: rd3GT1 protein, B: rd3GT6 protein;

FIG. 6: detection of Rd3GT1 in vitro enzyme activity product, a: cyanidin standard, B: cyanidin 3-O-glucoside standard, C: pET32a (+) -BL21 empty carrier protein negative control, D: rd3GT1 enzyme activity reaction;

FIG. 7: detection of Rd3GT6 in vitro enzyme activity product, a: cyanidin standard, B: cyanidin 3-O-glucoside standard, C: pET32a (+) -BL21 empty carrier protein negative control, D: rd3GT6 enzyme activity reaction;

FIG. 8: phenotype change and RT-PCR detection of Rd3GTs transgenic Arabidopsis plants, A: phenotypic comparison of different arabidopsis plants, B: comparing the RT-PCR results of different Arabidopsis plants;

FIG. 9: analyzing metabolites of transgenic arabidopsis;

FIG. 10: analyzing anthocyanin content of transgenic arabidopsis;

FIG. 11: phenotype change and RT-PCR detection of Rd3GTs transgenic tobacco plants, A: phenotypic comparison of different tobacco plants, B: comparing the RT-PCR results of different tobacco plants;

FIG. 12: analyzing anthocyanin components of the transgenic tobacco petals;

FIG. 13: and (4) analyzing the anthocyanin content of the transgenic tobacco.

Detailed Description

The invention provides a plant anthocyanin metabolism related gene Rd3GTs, which comprises genes Rd3GT1 and Rd3GT6, wherein the cDNA nucleotide sequence of the gene Rd3GT1 is shown in SEQ ID NO. 1; the cDNA nucleotide sequence of the gene Rd3GT6 is shown in SEQ ID NO 2.

The Rd3GTs gene is obtained by taking total RNA in Rhododendron delavayi Franch as a template, designing related primers and carrying out PCR amplification. In the invention, a gene Rd3GT1 is obtained by cloning a primer pair 3GT-1F1 and 3GT-1F 2; the gene Rd3GT6 was cloned by primer pair 3GT-6F1 and 3GT-6F 2. The nucleotide sequence of the primer 3GT-1F1 in the invention is shown in SEQ ID NO. 5; the nucleotide sequence of the primer 3GT-1F2 is shown in SEQ ID NO. 6; the nucleotide sequence of the primer 3GT-6F1 is shown in SEQ ID NO. 7; the nucleotide sequence of primer 3GT-6F2 is shown in SEQ ID NO. 8.

The genes Rd3GT1 and Rd3GT6 obtained by the invention can be respectively coded to obtain Rd3GTs protein, the gene Rd3GT1 is coded to obtain the protein Rd3GT1, and the amino acid sequence is shown as SEQ ID NO. 3; the gene Rd3GT6 is coded to obtain protein Rd3GT6, and the amino acid sequence of the protein is shown as SEQ ID NO. 4.

The invention utilizes DNAMAN to carry out multi-sequence alignment analysis on the obtained proteins Rd3GT1 and Rd3GT6, the glycosyltransferase (UGT78D2) with the known function of Arabidopsis thaliana and the glycosyltransferase (UGT78G1) with the known function of alfalfa, and the comparison result is shown in figure 1. As can be seen, Rd3GT1 and Rd3GT6 are highly similar to the conserved regions of UGT78D2 and UGT78G1, which indicates that the Rd3GT1 and the Rd3GT6 obtained by the invention have the active sites commonly existing in flavonoid 3-O-glycosyltransferase and PSPGbox specific to glycosyltransferase.

According to the invention, glycosyltransferase amino acid sequences of different plant sources are downloaded from NCBI, multi-sequence comparison is completed by utilizing Clustalw, then the construction of a phylogenetic tree is carried out by utilizing MEGA6.0, the obtained Rd3GT1 and Rd3GT6 are subjected to phylogenetic analysis, and the analysis result is shown in figure 2. As can be seen, Rd3GT1 and Rd3GT6 both belong to flavonoid 3-O-glycosyltransferase, which indicates that the Rd3GTs protein encoded by the Rd3GTs gene of Rhododendron delavayi may have similar functions to flavonoid 3-O-glycosyltransferase.

The invention aims at constructing a prokaryotic expression vector aiming at the Rd3GTs gene. As an optional implementation mode, the gene fragment of Rd3GT1 or Rd3GT6 obtained by amplification is cloned on pET32 to obtain a recombinant plasmid pET32-Rd3GT1 or pET32-Rd3GT6, and the recombinant plasmid is introduced into Escherichia coli BL21 cells, so that the obtained recombinant bacteria can generate target protein Rd3GT1 or Rd3GT6 by induction. The present invention is not limited to specific prokaryotic expression vectors.

In vitro enzyme activity experiments are carried out on the obtained target protein Rd3GT1 or Rd3GT6, and both Rd3GT1 and Rd3GT6 can catalyze cyanidin to react to generate cyanidin 3-O-glucoside, so that the Rhododendron delavayi Rd3GT1 and Rd3GT6 obtained by the invention have flavonoid 3-O-glycosyltransferase activity.

The invention aims at constructing a binary expression vector for Rd3GTs genes. As an optional implementation mode, the invention clones the Rd3GT1 or Rd3GT6 gene segment obtained by amplification to pMD18-T, transforms to escherichia coli JM109 competent cells, cultures and screens strains, and extracts positive plasmids; connecting the obtained recombinant plasmid T + Rd3GT1(X + B) or T + Rd3GT6(X + B) with a eukaryotic expression vector plasmid pBI121, transforming into escherichia coli JM109 competent cells, culturing and screening strains, and extracting to obtain a recombinant plasmid pBI121-Rd3GT1 or pBI121-Rd3GT 6; the recombinant plasmid is introduced into agrobacterium GV3101 cell and may be used in plant genetic transformation. The present invention is not limited to a specific binary expression vector, and is not limited to a specific transformation method.

The Rd3GTs gene, the protein obtained by coding the gene or the expression vector containing the Rd3GTs gene can be applied to improvement of anthocyanin of transgenic plants. In the present invention, the plant host to be transformed may be any anthocyanin-containing plant.

In the invention, arabidopsis thaliana is infected by agrobacterium GV3101 containing genes Rd3GT1 and Rd3GT6 respectively, and the anthocyanin in cotyledon and hypocotyl of an arabidopsis transgenic plant is successfully recovered compared with a mutant plant. Meanwhile, total RNAs of the wild type, the mutant and the transgenic plant are respectively extracted, and RT-PCR verification shows that the total RNAs are successfully detected in the transgenic plant, namely Rd3GT1 and Rd3GT6, but not detected in the wild type and the mutant, and the result is shown in a figure 8-B. The Rd3GTs gene is shown to be controllable in arabidopsis thaliana and improve the synthesis of anthocyanin.

The invention uses agrobacterium GV3101 containing genes Rd3GT1 and Rd3GT6 to infect tobacco, obtains transgenic tobacco plants through injection method and tissue culture, transplants the tobacco into soil for continuous culture after the tobacco successfully roots, observes petal phenotype after blooming, finds that the color of the tobacco petals is deepened compared with wild plants. Meanwhile, total RNA of the wild type and the transgenic plant is respectively extracted, and RT-PCR verification shows that Rd3GT1 and Rd3GT6 are successfully detected in the transgenic plant, but not detected in the wild type, and the result is shown in figure 11-B. The Rd3GTs gene is shown to regulate and improve the synthesis of anthocyanin in tobacco.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1

In this example, the Rhododendron delavayi Rd3GTs gene was cloned.

Extracting total RNA of Rhododendron delavayi Franch, sequencing by transcriptome, and designing two sets of primers according to sequencing result of transcriptome, wherein the sequences of the primers are shown in Table 1.

TABLE 1 Rhododendron delavayi Rd3GTs Gene cloning primer sequences

The nucleotide sequence of the primer 3GT-1F1 in Table 1 is shown in SEQ ID NO. 5; the nucleotide sequence of the primer 3GT-1F2 is shown in SEQ ID NO. 6; the nucleotide sequence of the primer 3GT-6F1 is shown in SEQ ID NO. 7; the nucleotide sequence of primer 3GT-6F2 is shown in SEQ ID NO. 8.

PCR amplification is carried out by taking total RNA of Rhododendron delavayi Franch as a template, an amplified fragment of 1425bp can be obtained by cloning 3GT-1F1 and 3GT-1F2 by utilizing a primer pair, and a coding gene Rd3GT1 of Rhododendron delavayi Franch Rd3GT1 is obtained by ORFFinder software, wherein the cDNA nucleotide sequence of the gene is shown as SEQ ID NO. 1; the 1465bp amplified fragment can be cloned by using a primer pair 3GT-6F1 and 3GT-6F2, and the coding gene Rd3GT6 of Rhododendron delavayi Rd3GT6 is obtained by ORFFinder software, and the cDNA nucleotide sequence of the gene is shown as SEQ ID NO. 2.

The PCR procedure was: 8min at 94 ℃; 30s at 94 ℃, 1.5min at 53 ℃, 8min at 72 ℃ and 30 cycles.

In this embodiment, the amplification steps and verification methods of Rd3GT1 and Rd3GT6 are conventional in the art, and the amplification and verification results are shown in fig. 3. The fragment of Rd3GT1 obtained by amplification in the embodiment can be encoded to obtain Rd3GT1, and the amino acid sequence is shown as SEQ ID NO. 3; the obtained Rd3GT6 fragment can be coded to obtain the Rd3GT6, and the amino acid sequence is shown as SEQ ID NO. 4.

Example 2

In the embodiment, the enzyme activity of Rhododendron delavayi Rd3GTs is detected.

1. Construction of prokaryotic expression vector

(1) The genes Rd3GT1 and Rd3GT6 obtained by cloning are used as templates, a primer pair DJ-3GT1-32/28F, DJ-3GT1-32/28R with EcoRI enzyme cutting sites and a primer pair DJ-3GT6-32/28F, DJ-3GT6-32/28R with BamH I and HindIII enzyme cutting sites are respectively adopted for PCR amplification, and the primer sequences are shown in Table 2.

TABLE 2 Rhododendron delavayi Rd3GTs Gene amplification primer sequences

The nucleotide sequence of the primer DJ-3GT1-32/28F in Table 2 is shown as SEQ ID NO. 9; the nucleotide sequence of the primer DJ-3GT1-32/28R is shown as SEQ ID NO. 10; the nucleotide sequence of the primer DJ-3GT6-32/28F is shown as SEQ ID NO. 11; the nucleotide sequence of the primer DJ-3GT6-32/28R is shown as SEQ ID NO. 12.

PCR program is 94 ℃ for 8 min; 30s at 94 ℃, 1.5min at 54 ℃, 8min at 72 ℃ and 30 cycles.

(2) Respectively obtaining a Rd3GT1 product with EcoRI enzyme cutting sites at the 5 'end and the 3' end and a Rd3GT6 product with BamHI and HindIII enzyme cutting sites at the 5 'end and the 3' end by PCR amplification, respectively carrying out gel recovery, carrying T loading, transforming JM109 competent cells, bacteria liquid PCR and enzyme cutting identification on the products. The corresponding plasmids were then sequenced, and the sequencing results showed successful introduction of cleavage sites at both ends of Rd3GT1 and Rd3GT 6.

(3) Inoculating the bacterial liquid with correct sequencing, extracting a large amount of plasmids, carrying out enzyme digestion, and verifying the size and the brightness of the enzyme digestion product. After verification, the vector is connected with a pET32a (+) vector, the transformed JM109 competent cells are transferred, cloning is randomly selected on a transformation plate the next day, bacterial liquid PCR and enzyme digestion verification are carried out, and the size of the band is found to be in accordance with the expectation. Sequencing the positive clones, wherein the sequencing result is consistent with the original sequence, and the prokaryotic expression vector is successfully constructed.

(4) The recombinant plasmids obtained by construction are respectively named pET32-Rd3GT1 and pET32-Rd3GT6, and are introduced into Escherichia coli BL21 cells to prepare a large amount of soluble recombinant protein.

2. Induction and expression of soluble recombinant proteins

(1) The Escherichia coli is streaked and inoculated in LB solid culture medium containing Amp (100 mu g/ml), and is inversely cultured for 12-16h at 37 ℃ in a constant temperature incubator. Clones were picked and cultured overnight.

Inoculating the bacterial liquid into 5ml test tube containing LB liquid culture medium according to 1% of inoculation amount the next day, culturing at 200rpm and 37 ℃ with shaking, taking out one bacterial liquid every half an hour, and temporarily storing at 4 ℃. One of the tubes was not inoculated with bacteria and used as a blank control. 2ml of the suspension was taken out at each time and OD was measured₆₀₀And (3) repeatedly measuring for three times at each time point, drawing a growth curve, and obtaining a result shown in figure 4. As can be seen from figure 4, after 2h, the escherichia coli enters a logarithmic phase of growth, and when 2.5h, the growth speed is fastest, and the state of the bacteria is optimal.

(2) IPTG induction was carried out 2.5h after selection on the strains pET32-Rd3GT1 and pET32-Rd3GT6, respectively. Through the exploration of different IPTG concentrations and different induction times, the optimal induction conditions of the Rd3GT1 protein are finally determined as follows: carrying out shake culture at 15 ℃ and 200rpm for 2.5h, adding IPTG (isopropyl thiogalactoside) to make the final concentration be 0.2mM, and continuing induction culture in a constant-temperature shaking table for 24 h; the optimal induction conditions for the Rd3GT6 protein were 15 deg.C, shaking at 200rpm for 2.5h, adding IPTG to a final concentration of 1mM, and continuing induction culture in a constant temperature shaker for 60 h.

By the induction expression, the proteins of Rd3GT1 and Rd3GT6 can be prepared in a large scale.

3. Separation and purification of recombinant protein

(1) Separation and purification:

a. column assembling: the filling solution was slowly added to the Ni-NTA pre-packed column while compacting with 20% ethanol, and when the filling volume was about 2ml, further compacting with 10ml 20% ethanol, and finally the column was equilibrated with 20mM PBS buffer and stored at 4 ℃ until use.

Mass preparation of Rd3GTs recombinant proteins

Inoculating and shaking according to the inoculation amount of 1% under the optimal induction condition, and preparing a large amount of escherichia coli cultures; subpackaging the escherichia coli culture into 50ml centrifuge tubes, placing the centrifuge tubes in a high-speed centrifuge for centrifugation, and setting the temperature as follows: 4 ℃, rotation speed: centrifuging for 10min at 5000rmp, pouring off the supernatant, and collecting thalli; adding 5ml of 20mMPBS suspension thallus into every 50ml of bacterial liquid, inserting into ice, and carrying out ice bath for 30 min; ultrasonic: setting the ultrasonic power to be 45%, stopping for 5s after 5s, setting for 10min, ultrasonically breaking cells, and repeating the ultrasonic treatment once after the bacterial liquid is cooled; and (3) centrifuging the bacterial liquid after wall breaking in a high-speed centrifuge at the set temperature of: centrifuging at 4 deg.C and 6000rpm for 15min to obtain supernatant as crude protein extractive solution.

c. Loading: the collected supernatant was repeated 3 times on a Ni column and equilibrated with 3 volumes of PBS.

d. Imidazole elution: after equilibration, elution was carried out with elution buffers having imidazole concentrations of 10mM, 20mM, 50mM, 100mM, 200mM, and 500mM in this order, and 5 tubes of eluents, 2 ml/tube, were collected for each concentration gradient.

e. And (3) column washing: washing the column with 20mM PBS buffer, washing, soaking in 20% ethanol, and storing at 4 deg.C.

SDS-PAGE electrophoretic validation: and (4) carrying out SDS-PAGE electrophoresis on each tube of eluate collected after column chromatography, and analyzing elution and purification conditions of the recombinant protein.

(2) And (3) dialysis and concentration:

a. treatment of the dialysis bag: cutting the dialysis bag to 15-20 cm in 2% (w/v) Na₂HCO₃Solution (containing 1mM EDTA-ana)₂pH of 8.0) for 10min, cleaning with deionized water, and placing in 1mM EDTA₂Boiling for 10min (pH is 8.0), cleaning dialysis bag with deionized water, soaking in deionized water, and storing at 4 deg.C. The old dialysis bag can be reused after checking no leakage and boiling with distilled water for 30 min.

b. According to the result of SDS-PAGE electrophoresis, protein eluates with single bands and good concentration are collected and put into a treated dialysis bag, and 2L of dialyzates I, II, III and IV are dialyzed in sequence in a refrigerated chromatography cabinet at 4 ℃ for about 10 hours.

Dialysate I: 20mM PBS, 500mM NaCl (pH 7.4); and (3) dialysate II: 20mM PBS, 300mM NaCl (pH 7.4); dialysate III: 20mM PBS, 100mM NaCl (pH 7.4); dialysate IV: 20mM PBS (pH7.4).

c. The protein filled into the dialysis bag is embedded into precooled allochroic silica gel, the mixture is concentrated in a refrigerator at 4 ℃ to about 1-2 ml, a small amount of sample is taken to carry out SDS-PAGE electrophoresis to verify the concentration condition, and the electrophoresis result is shown in figure 5 (lane 1: empty vector bacteria; lane 2: IPTG induction is not added; lane 3: unpurified protein; lane 4: purified protein). The remaining sample was stored at-80 ℃ until use.

4. Activity characterization of Rd3GTs

The enzyme activity of the proteins Rd3GT1 and Rd3GT6 in vitro is detected by taking cyanidin as a substrate and UDP-glucose as a glycosyl donor, and the configuration of an enzyme activity reaction system is carried out according to the table 3.

TABLE 3 recombinant protease activity reaction systems

After the reaction system is mixed, the mixture is placed in a water bath kettle at 30 ℃ for reaction for 5min, after the reaction is finished, 50 mu l of 5% hydrochloric acid is used for stopping the reaction, the mixture is centrifuged at 12000rpm for 5min, the supernatant is transferred to a brown sample application bottle, and the reaction product is detected by HPLC, and the result is shown in figures 6-7. As shown in FIGS. 6-7, both Rd3GT1 and Rd3GT6 are capable of catalyzing the reaction of cyanidin to form cyanidin 3-O-glucoside, demonstrating that Rhododendron delavayi Rd3GT1 and Rd3GT6 proteins have flavonoid 3-O-glycosyltransferase activity.

Example 3

This example investigates the effect of Rd3GTs on arabidopsis and tobacco anthocyanin synthesis.

1. Construction of binary expression vectors

(1) PCR was performed using the cloned Rd3GT1 and Rd3GT6 genes as templates with primers having BamHI and XbaI cleavage sites, respectively, and the primer sequences are shown in Table 4.

TABLE 4 Rhododendron delavayi Rd3GTs Gene amplification primer sequences

The nucleotide sequence of the primer DJ-3GT1-121F in Table 4 is shown in SEQ ID NO. 13; the nucleotide sequence of the primer DJ-3GT1-121R is shown as SEQ ID NO. 14; the nucleotide sequence of the primer DJ-3GT6-121F is shown as SEQ ID NO. 15; the nucleotide sequence of the primer DJ-3GT6-121R is shown in SEQ ID NO 16.

PCR program is 94 ℃ for 8 min; 30s at 94 ℃, 1.5min at 54 ℃, 8min at 72 ℃ and 30 cycles.

(2) A large number of gene fragments were obtained by PCR amplification, and after detection by 0.8% agarose gel electrophoresis, the Rd3GT1 product and the Rd3GT6 product having XbaI and BamHI cleavage sites were recovered and verified.

(3) And (3) connecting the gel recovery product which is verified to be correct with a pMD18-T cloning vector overnight at 16 ℃, transforming to escherichia coli JM109 competent cells the next day, and randomly selecting a single clone to perform bacterial liquid PCR after the clone grows out of the transformation plate. And (3) inoculating the screened positive clone, extracting plasmids, carrying out XbaI and BamHI double enzyme digestion verification, and sequencing the plasmids which are verified to be positive.

(4) And (3) carrying out double enzyme digestion on the recombinant plasmid T + Rd3GT1(X + B)/T + Rd3GT6(X + B) obtained in the step (3) and the eukaryotic expression vector plasmid pBI121 by using XbaI and BamHI restriction enzymes at the same time. And verifying the enzyme digestion product by 0.8 percent agarose gel electrophoresis, cutting out the target fragment and the vector fragment with correct sizes, and recovering and verifying the target fragment and the vector fragment. And (4) verifying that the carrier fragment and the target fragment are correct according to the molar ratio of 1: 10 mix and ligate overnight at 16 ℃.

(5) All the ligation products were transformed into E.coli JM109 competent cells and plated on Kan⁺A resistant LB solid medium surface; and selecting a single clone for carrying out PCR identification on the bacterial liquid the next day, carrying out inoculation on the positive clone, extracting a recombinant plasmid, carrying out double enzyme digestion identification on XbaI and BamHI, successfully carrying out sequencing, and introducing the recombinant plasmid with the correct sequencing result, namely pBI121-Rd3GT1/pBI121-Rd3GT6, into an agrobacterium GV3101 cell to prepare the genetic transformation of arabidopsis and tobacco.

2. Transgenic Arabidopsis plants acquisition and result analysis

(1) Respectively and uniformly spreading a proper amount of arabidopsis thaliana 3GT mutant and wild seeds in a flowerpot filled with moist soil, covering a preservative film, ventilating the preservative film by using toothpick jacks, placing the preservative film in a constant-temperature illumination culture chamber at 25 ℃, and watering once every 2-3 days; after sprouting, uncovering the film and thinning the seedlings, wherein 5-8 seedlings are planted in each pot; removing pods and reserving flowers when the plants grow to the full-bloom stage, and using the pods and reserving flowers as materials for agrobacterium infection.

(2) pBI-Rd3GT1(GV3101)/pBI-Rd3GT6(GV3101) were inoculated into 5ml of a solution containing Rif (final concentration 50mg/L) and Kan, respectively⁺The mixture was cultured in LB liquid medium (final concentration: 50mg/L) at 30 ℃ and 200rpm with shaking for 48 hours.

(3) The whole amount of the above-mentioned bacterial suspension was inoculated into 250ml of a suspension containing Rif (final concentration: 50mg/L) and Kan⁺(final concentration: 50mg/L) in LB liquid medium and then was shaken to OD₆₀₀The value is 1.0 or more.

(4) Transferring the cultured fresh bacterial liquid into 50ml centrifuge tubes respectively for centrifugation, and setting the temperature as follows: 4 ℃, rotation speed: centrifuging at 5000rpm for 15min, pouring off waste liquid, and retaining precipitate.

(5) After a small amount of 5% sucrose solution was taken to resuspend the cells, the corresponding volume of 5% sucrose solution was added, and 60. mu.l of silwet-77 (surfactant) was added per 100ml of the suspension, which was stirred on a magnetic stirrer.

(6) After the stirring is stopped, a pot of arabidopsis thaliana is taken and inversely buckled in the bacterial liquid, so that the flowers are completely soaked in the bacterial liquid and kept for 5 min. And stirring the bacterial liquid uniformly again, and then continuously infecting the next pot of arabidopsis thaliana. The arabidopsis thaliana taken out is horizontally placed in a tray, a small amount of distilled water is added for covering, the arabidopsis thaliana is treated overnight at room temperature in a dark environment, the next day, the arabidopsis thaliana is transferred to an illumination culture shelf for normal culture, and the arabidopsis thaliana is watered and sprayed with nutrient solution regularly.

(7) Collecting seeds of T1 generation after the transgenic arabidopsis grows to the mature period, respectively naming the seeds as pBI-Rd3GT1-T1 and pBI-Rd3GT6-T1, and screening the seeds of T1 generation:

appropriate transgenic T1 generation seeds were placed in 1.5ml ep tubes at a rate of 1: adding pasteurization liquid in a ratio of 8 (pasteurization liquid: water), and shaking up and down for 15min to wash the seeds; after washing, 12000rpm, centrifuging for 1min at room temperature; the seeds were sown on 1/2MS screening medium containing 1% sucrose after washing three times with sterile water on an ultraclean bench (Kan)⁺The final concentration is 50mg/L, and the final concentration of Carben is 100 mg/L); vernalizing at 4 ℃ for 2d, and culturing on a light culture shelf; and transplanting the screened green resistant seedlings into soil after the seeds germinate.

(8) At the moment, the petiole base of part of transgenic arabidopsis plants is restored to be purple, the plants with the restored colors are cultured to be mature, and T2 seeds are harvested from each plant.

Appropriate amounts of wild type, 3GT mutant and T2 generation Rd3GT1 and Rd3GT6 transgenic seeds were sown in 3% sucrose-containing 1/2MS medium (anthocyanin induction medium) in a clean bench, and the color difference of seedlings was observed under a body microscope after culture for about 5 days after vernalization, and the results are shown in FIG. 8-A. As can be seen from FIG. 8-A, anthocyanin in cotyledon and hypocotyl of transgenic plant was successfully recovered compared with mutant plant, which indicates that Rd3GTs can regulate and improve synthesis of anthocyanin in Arabidopsis.

Total RNA of wild type, 3GT mutant and Rd3GTs transgenic Arabidopsis seedlings with recovered phenotype are respectively extracted, and whether the recombinant vector is successfully transferred into the Arabidopsis mutant or not is verified by using an RT-PCR method, and the result is shown in figure 8-B. As can be seen from FIG. 8-B, Rd3GTs were successfully detected in the transgenic plants, but not in the wild type and the mutant, indicating that the synthesis of anthocyanin can be regulated and improved after Rd3GTs gene is transferred in Arabidopsis thaliana.

Collecting wild type, 3GT mutant and Rd3GTs transgenic Arabidopsis seedlings with recovered phenotype to extract anthocyanin, and performing qualitative and quantitative analysis, wherein the results are shown in FIGS. 9-10. As can be seen from FIG. 9, various anthocyanins (peak 1-4) can be detected in wild type seedlings, only one anthocyanin (peak 2) can be detected in 3GT mutant seedlings, and the number of anthocyanin species in 3GT mutant seedlings is remarkably reduced. And various anthocyanin peaks (peak 1-4) can be detected in the Rd3GTs transgenic arabidopsis seedlings, which indicates that the anthocyanin species in the Rd3GTs transgenic arabidopsis seedlings are recovered. Meanwhile, as can be seen from FIG. 10, the anthocyanin content in the Rd3GTs transgenic Arabidopsis is significantly higher than that of the wild type and 3GT mutant, the Rd3GT1 gene is transferred into the Arabidopsis, and the anthocyanin content can reach 50.89 mu g/g at most; when the gene is transferred into the Rd3GT6 gene, the highest anthocyanin content can reach 73.91 mu g/g. Shows that after the Rd3GTs gene is transferred into arabidopsis thaliana, the synthesis of anthocyanin can be regulated and improved, and the content of anthocyanin is increased.

3. Transgenic tobacco plant acquisition and result analysis

(1) Uniformly scattering a proper amount of tobacco K326 seeds in a flowerpot filled with moist soil, covering a preservative film, inserting a toothpick into a hole on the preservative film for ventilation, placing the preservative film in a constant-temperature illumination culture chamber at 25 ℃, and watering every 2-3 days; after sprouting, film uncovering and thinning, 2 plants/pot; when the plant grows to the seven-leaf stage, the plant is used as an agrobacterium infection material.

a. Suspending the cultured bacterial liquid by using the instantaneous tobacco resuspension, and detecting the OD600 value of the bacterial liquid by using an ultraviolet spectrophotometer to enable the final concentration to reach 0.06-0.08.

b. And lightly scratching a little wound on the back of the proper leaf by using an injection needle, sucking the diluted bacterial liquid by using a disposable injector, and injecting the bacterial liquid into the tobacco leaf in a way of aiming at the wound, wherein the injector needs to be replaced by a new injector when different bacterial liquids are injected.

c. The front surface of the leaf is marked, and the leaf can be detoxified after being cultivated in a greenhouse for about 4 days.

d. Shearing the leaves with the bacterial solution, completely soaking the leaves in a detoxifying solution (150ml of sterile water +150ml of sodium hypochlorite + 0.1% Tween20), timing for 3min, observing the back of the leaves within 3min, and taking out the leaves and sequentially rinsing the leaves in sterile water when small black spots appear.

e. Cutting the rinsed tobacco leaves into pieces of 5 × 5mm2, inoculating to MS differentiation culture medium, culturing, changing the culture medium every month, observing whether contamination occurs every day, and timely changing bottles.

f. Culturing until callus is differentiated from the explant and adventitious buds with the length of 2-3 cm grow, cutting the adventitious buds, transferring the adventitious buds into an MS rooting culture medium, replacing the culture medium every month, and stopping replacing the rooting culture medium when the adventitious buds are differentiated to root.

g. Transplanting the seedlings to soil after the root system grows, and culturing under constant temperature of 25 ℃.

h. And after the tobacco blooms, observing the phenotype of the flower, collecting petals of the discolored flower, extracting total RNA, and verifying whether the recombinant vector is successfully transferred into the tobacco by using an RT-PCR method.

After the tobacco successfully rooted, the tobacco was transplanted to soil for continuous culture, and the phenotypic change of petals was observed after blooming, and the results are shown in FIG. 11-A. As can be seen from FIG. 11-A, the color of the petals of the transgenic tobacco is darker than that of the wild type plant, indicating that Rd3GTs can regulate and improve the synthesis of anthocyanin in tobacco.

Total RNA of wild type and Rd3GTs transgenic tobacco is respectively extracted, whether the recombinant vector is successfully transferred into a tobacco plant is verified by using an RT-PCR method, and the result is shown in figure 11-B. As can be seen from FIG. 11-B, Rd3GTs were successfully detected in the transgenic plants, but not in the wild type, indicating that the synthesis of anthocyanin can be regulated and improved after Rd3GTs gene is transferred into tobacco plants.

Anthocyanin in wild type and Rd3GTs transgenic tobacco plants is respectively extracted for qualitative and quantitative analysis, and the results are shown in figures 12-13. As can be seen from FIG. 12, various anthocyanins (Peak 1-5) can be detected in the Rd3GTs transgenic tobacco plants, and the types of the anthocyanins are increased compared with those in wild tobacco plants. As can be seen from FIG. 13, the anthocyanin content in the Rd3GTs transgenic tobacco plant is significantly higher than that of the wild type, the Rd3GT1 gene is transferred into the tobacco plant, and the anthocyanin content can reach 56.35 mu g/g to the highest extent; when the gene is transferred into the Rd3GT6 gene, the highest anthocyanin content can reach 77.05 mu g/g. Shows that after Rd3GTs gene is transferred into tobacco, the synthesis of anthocyanin can be regulated and controlled, and the content of anthocyanin is improved.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of Guizhou Master

<120> plant anthocyanin metabolism related gene Rd3GTs, and coding protein and application thereof

<160> 16

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1395

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atgaccaaaa atatctcaag ggaccgtcat gtggccgtct taccgttccc tttctccagc 60

cacgccggcc gcctcctcac cctcgtccgc cgcctcgccg ccgccgcccc caacgtcact 120

ttctccttct acagcacccc taaatccatc gagtccttgt tctcgccggc ggagagagtc 180

cccggcaacg tgaggccgta cgcggtgccg gacggggtgc cggaggggca cgtgttctcc 240

ggggagcccg tggagcacgt taacttgtac cttacggcgg tgggggaggg ggagagcctc 300

aggggagtgt tgaaggcggc cgaggcggag acagggcgga ggatcgggtg tgttatgtcg 360

gatgcgttta tgtggttcgc cggcgatttg gcggaggaga tgggggtccc gtgggtcccg 420

ttcatggccg ggggtgctaa ctctataact gcgcattttt acaccgatct gatcagggaa 480

actgttggaa tgcatgacat tgttgggcgg gagaacgaca tcgtgaaatt tatcccagga 540

ttctcggagc tacgcctcgg ggacttgccg acgggagtcc tgttcgggaa cttggaatct 600

ccgttcgcaa tcatgctaca caaaatgggg cgtgctctgc ccaaagcaac ggccattgct 660

atcaactcct tcgaagaact agaccctgat atcatccaag atctcaagtc caagttcaaa 720

atgattctca acgtcagccc gtttagcgca atatcgttgc cttcttcgcc gccgccgccc 780

cccacctcgt acacggatga gtacggatgc atgccgtggt tggacaatcg caaagccgcc 840

tctgtcgcct atatcggctt tggaactttg gctacgccac caccggttga gattgcggca 900

ttagctgaag cattagaagc tagcggcact ccgtttctct ggtctctcaa ggataatttc 960

aaagagtttt tcccggaagg attcattaag agaactagcg agcgagggaa aattgtgccg 1020

tgggcacctc aggaacaagt tctggcacat ggttcagttg gagttttcgt gactcactgc 1080

gggtggaact cggcactgga gagcatcgcg gcaggtgtgc cgcttatagg gaggccgttc 1140

ttcggcgatc atcagctaaa cgcgtggttg gtggaaaatg tgtggaaaat tggtgtgagg 1200

gtggagggtg gagttttcac aaagagtggc acaatgtctg cccttgaact ggttttgact 1260

catgaaaagg ggaaggaact gagggcgcga gttgaaatgt ttaaaaagct tgctttgaag 1320

gctgtcggac ccgaagggag ctcaactcgc aatctccata ctttgttgga gatagtagca 1380

gggtacaatc tttag 1395

<210> 2

<211> 1365

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgaccaatt cctcaaaagg ccgacacgtc gccgttttgc cattcccatt ctccacacac 60

gccgccccaa tcctcagcat catccgccgc ctcgcctccg ccgctcccga cgtcacattc 120

tccttcttca gcactcctca atccatccaa accctgttcc catccgaaaa tcccgagaac 180

aacataagac ctcacgccat atcggacggc gttcccgagg ggttcgtgtt ctcggggaag 240

caccatgagg acatcaactt gttcctggcg gcagggaagg agagcttcga ggccgggatg 300

aaggcggccg aggcggagac cgggcggagg attgattgtg tggtgtcgga tgcgtttatg 360

tggttctcat gcgagttggc agaggagatg ggggtgccgt gggtcacgct gtgggtgtcg 420

ggggcatgct cacttgcggc tcattgttat actgacctta tcagggaaac tgttggaatg 480

catgatactg ctggacgcga agacgaaatc gtgaaattcg tcccgggatt ttcggaggta 540

cgactcgggg acttgcccag cggggtcgtg tatggaaact tagaatcacc cttctcaatg 600

atgctataca aaatgggcca ggttttgcac aaagcggccg cagttgccat aaactccttc 660

gacgaactgg aacctgaacc cgtgaaagtt ctcgcgtcga agttgaagct cctcacctgt 720

ggcccgttca acccgatatc accaccgccg tcgtccaact tggatgagta cggctgtatc 780

ccgtggttgg accggcgtaa agcagcttca gtggcgtaca tcggctttgg aaccgtggct 840

acaccgccac cggttgagct agcggcgtta gctgaagcac tagaagctag tagcacaccg 900

tttctctggt ctctcagaga caatttcaaa caacatttac cggaaggatt cctgaagaga 960

acgagtgagc tagggaaaat cgtgccgtgg gcgcctcagg cgcaagtttt agcgcatcgc 1020

tcagtcgggg ttttcataaa ccactgcggc tggaattcgg tggtggagag cgtcgaggcc 1080

ggtgtgccaa tcatcggtag gccgttcttc ggggatcatc aggtggacgc gtggatggtg 1140

gagaatgtgt ggaagattgg cgtcagggtg gagggtgcag tcttcacaaa aggtatcaca 1200

atgtctgccc ttgaactggt tttgtcccat gatcagaagg ggaaggaatt gagggagcaa 1260

gttggaaagt acaaagagtt tgctttgaag gcttttggac ccaaagggag atcaacgcaa 1320

aatttgagca cattgctgga gatagtagca gggtacaacc tttag 1365

<210> 3

<211> 464

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 3

Met Thr Lys Asn Ile Ser Arg Asp Arg His Val Ala Val Leu Pro Phe

1 5 10 15

Pro Phe Ser Ser His Ala Gly Arg Leu Leu Thr Leu Val Arg Arg Leu

20 25 30

Ala Ala Ala Ala Pro Asn Val Thr Phe Ser Phe Tyr Ser Thr Pro Lys

35 40 45

Ser Ile Glu Ser Leu Phe Ser Pro Ala Glu Arg Val Pro Gly Asn Val

50 55 60

Arg Pro Tyr Ala Val Pro Asp Gly Val Pro Glu Gly His Val Phe Ser

65 70 75 80

Gly Glu Pro Val Glu His Val Asn Leu Tyr Leu Thr Ala Val Gly Glu

85 90 95

Gly Glu Ser Leu Arg Gly Val Leu Lys Ala Ala Glu Ala Glu Thr Gly

100 105 110

Arg Arg Ile Gly Cys Val Met Ser Asp Ala Phe Met Trp Phe Ala Gly

115 120 125

Asp Leu Ala Glu Glu Met Gly Val Pro Trp Val Pro Phe Met Ala Gly

130 135 140

Gly Ala Asn Ser Ile Thr Ala His Phe Tyr Thr Asp Leu Ile Arg Glu

145 150 155 160

Thr Val Gly Met His Asp Ile Val Gly Arg Glu Asn Asp Ile Val Lys

165 170 175

Phe Ile Pro Gly Phe Ser Glu Leu Arg Leu Gly Asp Leu Pro Thr Gly

180 185 190

Val Leu Phe Gly Asn Leu Glu Ser Pro Phe Ala Ile Met Leu His Lys

195 200 205

Met Gly Arg Ala Leu Pro Lys Ala Thr Ala Ile Ala Ile Asn Ser Phe

210 215 220

Glu Glu Leu Asp Pro Asp Ile Ile Gln Asp Leu Lys Ser Lys Phe Lys

225 230 235 240

Met Ile Leu Asn Val Ser Pro Phe Ser Ala Ile Ser Leu Pro Ser Ser

245 250 255

Pro Pro Pro Pro Pro Thr Ser Tyr Thr Asp Glu Tyr Gly Cys Met Pro

260 265 270

Trp Leu Asp Asn Arg Lys Ala Ala Ser Val Ala Tyr Ile Gly Phe Gly

275 280 285

Thr Leu Ala Thr Pro Pro Pro Val Glu Ile Ala Ala Leu Ala Glu Ala

290 295 300

Leu Glu Ala Ser Gly Thr Pro Phe Leu Trp Ser Leu Lys Asp Asn Phe

305 310 315 320

Lys Glu Phe Phe Pro Glu Gly Phe Ile Lys Arg Thr Ser Glu Arg Gly

325 330 335

Lys Ile Val Pro Trp Ala Pro Gln Glu Gln Val Leu Ala His Gly Ser

340 345 350

Val Gly Val Phe Val Thr His Cys Gly Trp Asn Ser Ala Leu Glu Ser

355 360 365

Ile Ala Ala Gly Val Pro Leu Ile Gly Arg Pro Phe Phe Gly Asp His

370 375 380

Gln Leu Asn Ala Trp Leu Val Glu Asn Val Trp Lys Ile Gly Val Arg

385 390 395 400

Val Glu Gly Gly Val Phe Thr Lys Ser Gly Thr Met Ser Ala Leu Glu

405 410 415

Leu Val Leu Thr His Glu Lys Gly Lys Glu Leu Arg Ala Arg Val Glu

420 425 430

Met Phe Lys Lys Leu Ala Leu Lys Ala Val Gly Pro Glu Gly Ser Ser

435 440 445

Thr Arg Asn Leu His Thr Leu Leu Glu Ile Val Ala Gly Tyr Asn Leu

450 455 460

<210> 4

<211> 454

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 4

Met Thr Asn Ser Ser Lys Gly Arg His Val Ala Val Leu Pro Phe Pro

1 5 10 15

Phe Ser Thr His Ala Ala Pro Ile Leu Ser Ile Ile Arg Arg Leu Ala

20 25 30

Ser Ala Ala Pro Asp Val Thr Phe Ser Phe Phe Ser Thr Pro Gln Ser

35 40 45

Ile Gln Thr Leu Phe Pro Ser Glu Asn Pro Glu Asn Asn Ile Arg Pro

50 55 60

His Ala Ile Ser Asp Gly Val Pro Glu Gly Phe Val Phe Ser Gly Lys

65 70 75 80

His His Glu Asp Ile Asn Leu Phe Leu Ala Ala Gly Lys Glu Ser Phe

85 90 95

Glu Ala Gly Met Lys Ala Ala Glu Ala Glu Thr Gly Arg Arg Ile Asp

100 105 110

Cys Val Val Ser Asp Ala Phe Met Trp Phe Ser Cys Glu Leu Ala Glu

115 120 125

Glu Met Gly Val Pro Trp Val Thr Leu Trp Val Ser Gly Ala Cys Ser

130 135 140

Leu Ala Ala His Cys Tyr Thr Asp Leu Ile Arg Glu Thr Val Gly Met

145 150 155 160

His Asp Thr Ala Gly Arg Glu Asp Glu Ile Val Lys Phe Val Pro Gly

165 170 175

Phe Ser Glu Val Arg Leu Gly Asp Leu Pro Ser Gly Val Val Tyr Gly

180 185 190

Asn Leu Glu Ser Pro Phe Ser Met Met Leu Tyr Lys Met Gly Gln Val

195 200 205

Leu His Lys Ala Ala Ala Val Ala Ile Asn Ser Phe Asp Glu Leu Glu

210 215 220

Pro Glu Pro Val Lys Val Leu Ala Ser Lys Leu Lys Leu Leu Thr Cys

225 230 235 240

Gly Pro Phe Asn Pro Ile Ser Pro Pro Pro Ser Ser Asn Leu Asp Glu

245 250 255

Tyr Gly Cys Ile Pro Trp Leu Asp Arg Arg Lys Ala Ala Ser Val Ala

260 265 270

Tyr Ile Gly Phe Gly Thr Val Ala Thr Pro Pro Pro Val Glu Leu Ala

275 280 285

Ala Leu Ala Glu Ala Leu Glu Ala Ser Ser Thr Pro Phe Leu Trp Ser

290 295 300

Leu Arg Asp Asn Phe Lys Gln His Leu Pro Glu Gly Phe Leu Lys Arg

305 310 315 320

Thr Ser Glu Leu Gly Lys Ile Val Pro Trp Ala Pro Gln Ala Gln Val

325 330 335

Leu Ala His Arg Ser Val Gly Val Phe Ile Asn His Cys Gly Trp Asn

340 345 350

Ser Val Val Glu Ser Val Glu Ala Gly Val Pro Ile Ile Gly Arg Pro

355 360 365

Phe Phe Gly Asp His Gln Val Asp Ala Trp Met Val Glu Asn Val Trp

370 375 380

Lys Ile Gly Val Arg Val Glu Gly Ala Val Phe Thr Lys Gly Ile Thr

385 390 395 400

Met Ser Ala Leu Glu Leu Val Leu Ser His Asp Gln Lys Gly Lys Glu

405 410 415

Leu Arg Glu Gln Val Gly Lys Tyr Lys Glu Phe Ala Leu Lys Ala Phe

420 425 430

Gly Pro Lys Gly Arg Ser Thr Gln Asn Leu Ser Thr Leu Leu Glu Ile

435 440 445

Val Ala Gly Tyr Asn Leu

450

<210> 5

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

accaaacaaa tactgtaata at 22

<210> 6

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

aactgtacgt ctctctcact cg 22

<210> 7

<211> 19

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

acaaacccca gagccatca 19

<210> 8

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cagagccatc acaaaaacaa 20

<210> 9

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

cggaattcat gaccaaaaat atctca 26

<210> 10

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

cggaattcct aaagattgta ccctgc 26

<210> 11

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

cgggatccat gaccaattcc tcaaaa 26

<210> 12

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

cccaagcttc taaaggttgt accctgc 27

<210> 13

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

gctctagaat gaccaaaaat atctca 26

<210> 14

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

cgggatccct aaagattgta ccctgc 26

<210> 15

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 15

gctctagaat gaccaattcc tcaaaa 26

<210> 16

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 16

cgggatccct aaaggttgta ccctgc 26

Claims (10)

1. A plant anthocyanin metabolism related gene Rd3GTs is characterized in that: the cDNA nucleotide sequence of the gene is shown as SEQ ID NO. 1 or SEQ ID NO. 2.

2. The gene of claim 1, wherein: the plant is Rhododendron delavayi Franch.

3. An Rd3GTs protein expressed by the gene of any one of claims 1 to 2, wherein: the protein amino acid sequence coded by the SEQ ID NO. 1 nucleotide sequence is shown as SEQ ID NO. 3; the amino acid sequence of the protein coded by the SEQ ID NO. 2 nucleotide sequence is shown as SEQ ID NO. 4.

4. A recombinant expression vector comprising the gene according to any one of claims 1 to 2.

5. The recombinant expression vector of claim 4, wherein: the vector plasmid was pET 32.

6. A binary expression vector comprising the gene of any one of claims 1 to 2.

7. The binary expression vector of claim 6, wherein: the vector plasmid is pBI 121.

8. A recombinant host cell comprising the expression vector of any one of claims 4 to 7.

9. Use of the gene according to any one of claims 1 to 2 or the protein according to claim 3 or the expression vector according to any one of claims 4 to 7 in transgenic plants with improved anthocyanin.

10. The use of claim 9, wherein: the plant is Arabidopsis thaliana or tobacco.

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