阅读说明：本技术 与日本蛇根草花色苷合成相关的dfr酶、编码基因、表达载体、双元表达载体及其应用 (DFR enzyme related to synthesis of japonicas anthocyanin, coding gene, expression vector, binary expression vector and application thereof ) 是由 鞠志刚 孔德静 施洪喜 于 2021-09-06 设计创作，主要内容包括：本发明涉及生物工程技术领域,特别是涉及与日本蛇根草花色苷合成相关的DFR酶、编码基因、表达载体、双元表达载体及其应用。本发明所述DFR酶的氨基酸序列如SEQ ID NO.1所示。本发明通过对日本蛇根草花瓣组织的转录组进行测序,根据测序结果设计引物,扩增得到控制日本蛇根草花色苷合成的OjDFR1基因的cDNA,通过表达载体得到所述DFR酶,进而验证其具有DFR酶的催化活性,从而证明了本发明提供的DFR酶可以控制日本蛇根草花色苷的合成,编码该DFR酶的基因可以用于植物花色苷的改良。(The invention relates to the technical field of bioengineering, in particular to DFR enzyme related to synthesis of japonicas anthocyanin, a coding gene, an expression vector, a binary expression vector and application thereof. The amino acid sequence of the DFR enzyme is shown in SEQ ID NO. 1. The invention verifies that the DFR enzyme has the catalytic activity of the DFR enzyme by sequencing the transcriptome of the petal tissue of the Japanese snakeroot, designing a primer according to the sequencing result, amplifying to obtain the cDNA of the OjDFR1 gene for controlling the synthesis of the anthocyanin of the Japanese snakeroot and obtaining the DFR enzyme through an expression vector, thereby proving that the DFR enzyme provided by the invention can control the synthesis of the anthocyanin of the Japanese snakeroot and the gene for coding the DFR enzyme can be used for improving the plant anthocyanin.)

1. A DFR enzyme involved in the synthesis of anthocyanin from Japanese serpentium, characterized in that the amino acid sequence of the DFR enzyme is shown in SEQ ID NO. 1.

2. A gene OjDFR1 encoding the DFR enzyme of claim 1, wherein the cDNA of the gene OjDFR1 has the sequence shown in SEQ ID No. 2.

3. A primer set for amplifying the gene OjDFR1 of claim 2, wherein the primer set comprises OjDFR1F1 and OjDFR1R 1; the nucleotide sequence of the OjDFR1F1 is shown in SEQ ID NO. 3; the nucleotide sequence of the OjDFR1R1 is shown in SEQ ID NO. 4.

4. An expression vector for expressing the DFR enzyme of claim 1, wherein the expression vector comprises the gene OjDFR1 of claim 2 and a base vector; the base vector includes the pET-32a (+) vector.

5. The expression vector of claim 4, wherein the cDNA sequence of the gene OjDFR1 is located between the EcoR I and HindIII cleavage sites of the base vector.

6. A binary expression vector comprising the gene OjDFR1 of claim 2 and a base vector; the base vector includes the pBI121 vector.

7. The binary expression vector of claim 6, wherein the cDNA sequence of the gene OjDFR1 is located between BamH I and Xba I cleavage sites of the basic vector.

8. Use of the gene OjDFR1 according to claim 2, the primer pair according to claim 3, or the binary expression vector according to claim 6 or 7 for plant anthocyanin improvement.

9. Use according to claim 8, wherein the plant comprises Japanese hop, tobacco or Arabidopsis thaliana.

10. A method of altering plant anthocyanins comprising: introducing the binary expression vector of claim 6 or 7 into Agrobacterium, infecting a plant with said Agrobacterium to obtain an anthocyanin-altered plant.

Technical Field

The invention relates to the technical field of bioengineering, in particular to DFR enzyme related to synthesis of japonicas anthocyanin, a coding gene, an expression vector, a binary expression vector 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. Anthocyanin is synthesized under the control of a structural gene for coding the synthesis of anthocyanin, and flavanonol 4-reductase DFR is an important regulation point in an anthocyanin synthesis path and is an enzyme necessary for the biosynthesis of various plant anthocyanins. Through the catalysis of DFR, flavanonol can react to generate corresponding colorless anthocyanidin, and then corresponding anthocyanin is generated under the catalysis of other enzymes. In conclusion, DFR plays an important role in determining anthocyanin content and anthocyanin type, and has important significance in the aspects of improving plant stress resistance, promoting fruit ripening and the like as an important regulation and control point for changing plant color.

The DFR gene is firstly separated from corn in 1985, and is cloned from plants such as poplar, peony, cabbage, Chinese cabbage, sweet osmanthus, strawberry, freesia, mango, grapevine and the like. With the continuous improvement of cDNA cloning technology, researchers in China clone cDNA or genomic DNA sequences of DFR genes from a plurality of plants, but no report is found on functional analysis research of DFR genes of Japan snakeweed, which is a madder plant, so that the evolution research of DFR is greatly limited, and the utilization of DFR of madder plants and the regulation and improvement of anthocyanin biosynthesis are also limited.

Disclosure of Invention

In order to solve the above problems, the present invention provides a DFR enzyme, a coding gene, an expression vector, a binary expression vector and applications thereof, which are involved in the synthesis of Japan snakeroot anthocyanin. The DFR enzyme related to the synthesis of the Japan snakeroot anthocyanin provided by the invention can control the synthesis of the Japan snakeroot anthocyanin, and the gene coding the DFR enzyme can be used for improving plant anthocyanin.

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

the invention provides a DFR enzyme related to the synthesis of japonicas anthocyanin, and the amino acid sequence of the DFR enzyme is shown in SEQ ID NO. 1.

The invention also provides a gene OjDFR1 for coding the DFR enzyme as claimed in claim 1, wherein the sequence of cDNA of the gene OjDFR1 is shown as SEQ ID NO. 2.

The invention also provides a primer pair for amplifying the gene OjDFR1, wherein the primer pair comprises OjDFR1F1 and OjDFR1R 1; the nucleotide sequence of the OjDFR1F1 is shown in SEQ ID NO. 3; the nucleotide sequence of the OjDFR1R1 is shown in SEQ ID NO. 4.

The invention also provides an expression vector for expressing the DFR enzyme, wherein the expression vector comprises the gene OjDFR1 and a basic vector; the base vector includes the pET-32a (+) vector.

Preferably, the cDNA sequence of the gene OjDFR1 is located between the EcoR I and Hind III cleavage sites of the base vector.

The present invention also provides a binary expression vector comprising the gene OjDFR1 of claim 2 and a base vector; the base vector includes the pBI121 vector.

Preferably, the cDNA sequence of the gene OjDFR1 is located between the BamH I and Xba I cleavage sites of the basic vector.

The invention also provides the application of the gene OjDFR1 or the primer pair or the binary expression vector in improvement of plant anthocyanin.

Preferably, the plant comprises Japanese ophiorrhiza, tobacco or Arabidopsis thaliana.

The invention also provides a method for changing plant anthocyanin, which comprises the following steps: and (3) introducing the binary expression vector into agrobacterium, and infecting plants by using the agrobacterium to obtain plants with modified anthocyanin.

Has the advantages that:

the invention provides a DFR enzyme related to the synthesis of japonicas anthocyanin, and the amino acid sequence of the DFR enzyme is shown in SEQ ID NO. 1. The invention verifies that the DFR enzyme has the catalytic activity of the DFR enzyme by sequencing the transcriptome of the petal tissue of the Japanese snakeroot, designing a primer according to the sequencing result, amplifying to obtain the cDNA of the OjDFR1 gene for controlling the synthesis of the anthocyanin of the Japanese snakeroot and obtaining the DFR enzyme through an expression vector, thereby proving that the DFR enzyme provided by the invention can control the synthesis of the anthocyanin of the Japanese snakeroot and the gene for coding the DFR enzyme can be used for improving the plant anthocyanin.

Drawings

FIG. 1 is a diagram showing the multi-sequence alignment analysis of OjDFR1 protein; wherein AtDFR is the DFR of Arabidopsis thaliana, NtDFR is the DFR of tobacco, OjDFR1 is the DFR of serpentium chinense, OjANR is the anthocyanidin reductase of serpentium chinense, OjFR is the flavonoid reductase of serpentium chinense;

FIG. 2 is a phylogenetic analysis of the OjDFR1 protein;

FIG. 3 shows purification and enzyme activity detection analysis of OjDFR1 protein, wherein A is separation and purification of OjDFR1 soluble recombinant protein, 1-5 respectively represent 1: maker, 2: soluble empty carrier protein, 3: OjDFR1 soluble recombinant protein was not induced with IPTG, 4: after 24 hours induction of the OjDFR1 soluble recombinant protein; 5: purified OjDFR1 soluble recombinant protein; b is enzyme activity detection by using dihydroquercetin as a substrate; c is enzyme activity detection by taking dihydromyricetin as a substrate; d is enzyme activity detection by taking dihydrokaempferol as a substrate;

FIG. 4 shows phenotypic changes and RT-PCR detection of OjDFR1 transgenic Arabidopsis plants, where Wild Type is Wild Type, Mutant is Mutant, OjDFR1-3 is transgenic Arabidopsis plant 3, and OjDFR1-5 is transgenic Arabidopsis plant 5; in the right figure, OjDFR1 is a target gene, and beta-actin is an internal reference gene;

FIG. 5 shows phenotypic changes of OjDFR1 transgenic tobacco petals and RT-PCR detection, wherein Wild Type is Wild Type, OjDFR1-4 is transgenic tobacco plant 4, and OjDFR1-5 is transgenic tobacco plant 5; in the right figure, OjDFR1 is the target gene, and NtTub1 is the reference gene.

Detailed Description

The invention provides a DFR enzyme related to the synthesis of japonicas anthocyanin, wherein the amino acid sequence of the DFR enzyme is shown in SEQ ID NO. 1: MGVEDATAAAAATKAGTVCVTGAGGFIGSWLVMRLLERDYIVRATVRNPGDTKKVKHLLELPKASTNLTLWKADMTEEGSFDEAIQDCDGVFHVATPMDFESKDPENEVIKPTIDGILNIIRSCVKAKTVKRLVYTSSAGTVNVQEHQRPVYDENDESDLDFIYSKKMTGWMYFASKLLAEKEAREASKENNIDFISIIPTLVVGPFITPTFPPSLITALSLITGNEAHYSIIKQGQFVHLDDLCEAHIFLYENPKAEGRYICSNYDGTIHDLAKIMREKWPEYYIPDELKGIDKNIPVVSFCSKKLTGMGFQYKYNLDDMFKGAIDTCRQKGLLPHSTQILENGQENGLIPESQQK are provided. The DFR enzyme can catalyze the reaction of dihydroquercetin and dihydromyricetin to generate corresponding colorless anthocyanidin.

The invention provides a gene OjDFR1 for coding the DFR enzyme, wherein the sequence of cDNA of the gene OjDFR1 is shown as SEQ ID NO. 2: ATGGGAGTGGAGGATGCAACTGCTGCTGCTGCTGCGACAAAGGCCGGCACAGTGTGTGTGACCGGAGCTGGTGGATTTATAGGATCATGGCTTGTTATGAGACTCCTTGAACGTGACTATATTGTTCGTGCCACTGTCCGGAATCCAGGGGATACAAAGAAAGTGAAACATCTTCTTGAGTTGCCAAAAGCCAGCACGAATTTGACCCTTTGGAAAGCCGATATGACTGAAGAAGGAAGTTTTGATGAGGCCATTCAAGATTGTGATGGGGTTTTTCATGTTGCCACACCTATGGATTTTGAATCTAAAGACCCTGAGAATGAAGTGATCAAGCCAACAATTGATGGGATTTTGAACATCATAAGATCATGCGTCAAGGCCAAAACAGTGAAGAGGCTGGTTTACACTTCATCAGCTGGAACAGTCAATGTTCAAGAACACCAACGGCCTGTCTATGACGAGAACGACGAGAGTGATTTGGATTTCATCTATTCCAAGAAGATGACAGGATGGATGTATTTTGCTTCAAAACTTTTGGCTGAGAAAGAAGCACGAGAAGCATCCAAAGAGAACAATATTGATTTCATCAGTATTATACCAACGCTAGTCGTAGGTCCATTCATCACGCCAACATTCCCACCAAGCCTAATAACTGCACTTTCATTGATAACTGGGAATGAAGCACATTATTCAATCATTAAGCAAGGTCAATTCGTGCATTTGGATGATCTGTGTGAAGCCCATATATTCTTGTACGAGAATCCCAAAGCCGAGGGAAGATACATTTGCTCCAATTATGATGGAACTATTCATGATTTGGCCAAAATTATGAGAGAGAAATGGCCAGAATACTATATCCCTGATGAGTTGAAGGGAATAGACAAGAACATACCTGTGGTGTCCTTTTGTTCCAAGAAATTGACAGGCATGGGTTTCCAATATAAGTACAATTTGGATGACATGTTCAAGGGAGCCATTGATACGTGCCGTCAAAAGGGACTACTACCCCATTCAACCCAAATCCTTGAAAACGGCCAAGAGAATGGATTAATCCCAGAATCCCAGCAAAAATAG are provided. The invention verifies that the DFR enzyme has the catalytic activity of the DFR enzyme by sequencing the transcriptome of the petal tissue of the Japanese snakeroot, designing a primer according to the sequencing result, amplifying to obtain the cDNA of the OjDFR1 gene for controlling the synthesis of the anthocyanin of the Japanese snakeroot, obtaining the DFR enzyme through an expression vector, thereby proving that the DFR enzyme provided by the invention can control the synthesis of the anthocyanin of the Japanese snakeroot, and the gene for coding the DFR enzyme can be used for improving the plant anthocyanin.

The invention also provides a primer pair for amplifying the gene OjDFR1, wherein the primer pair comprises OjDFR1F1 and OjDFR1R 1; the nucleotide sequence of the OjDFR1F1 is shown in SEQ ID NO. 3: CGATTCTCACATTCCATCTTCA, respectively; the nucleotide sequence of the OjDFR1R1 is shown in SEQ ID NO. 4: GGGAAGACATTTACGCAT are provided.

The present invention provides an expression vector for expressing the DFR enzyme of claim 1, said expression vector comprising the above gene OjDFR1 and a base vector; the base vector includes the pET-32a (+) vector. The source of the basic vector is not particularly limited in the present invention, and commercially available products known to those skilled in the art may be used.

In the present invention, the cDNA sequence of the gene OjDFR1 is preferably located between the EcoR I and Hind III cleavage sites of the base vector; the primer pair for amplifying the cDNA sequence of the gene OjDFR1 comprises OjDFR1F2 and OjDFR1R 2; the nucleotide sequence of the OjDFR1F2 is shown in SEQ ID NO. 5: CGGAATTCATGGGAGTGGAGGATGCA, respectively; the nucleotide sequence of the OjDFR1R2 is shown in SEQ ID NO. 6: CCCAAGCTTCTATTTTTGCTGGGATTC are provided.

The invention also provides a binary expression vector, which comprises the gene OjDFR1 and a basic vector; the base vector includes the pBI121 vector. In the present invention, the cDNA sequence of the gene OjDFR1 is preferably located between the BamH I and Xba I cleavage sites of the basic vector; the primer pair for amplifying the cDNA sequence of the gene OjDFR1 comprises OjDFR1F3 and OjDFR1R 3; the nucleotide sequence of the OjDFR1F3 is shown as SEQ ID NO. 7: GCTCTAGAATGGGAGTGGAGGATGCA; the nucleotide sequence of OJDFR1R3 is shown in SEQ ID NO.8, namely CGGGATCCCTATTTTTGCTGGGATTC. The source of the basic vector is not particularly limited in the present invention, and commercially available products known to those skilled in the art may be used.

The invention also provides the application of the gene OjDFR1 or the primer pair or the binary expression vector in improvement of plant anthocyanin. In the present invention, the plant preferably includes Japanese ophiorrhiza, tobacco or Arabidopsis thaliana. The binary expression vector containing the gene OjDFR1 provided by the invention can recover the synthesis of anthocyanin in the mutant cotyledon and hypocotyl of arabidopsis thaliana and deepen the flower color of tobacco; the gene OjDFR1 provided by the invention has potential application value in the aspects of flower color modification of transgenic plants and improvement of medicinal components of medicinal plants.

The invention also provides a method for changing plant anthocyanin, which comprises the following steps: and (3) introducing the binary expression vector into agrobacterium, infecting plants by using the agrobacterium to obtain plants with modified anthocyanin. In the present invention, the Agrobacterium preferably comprises a GV310 Agrobacterium. The present invention does not require any particular manner of introduction, and may employ any manner of introduction known to those skilled in the art.

In order to further illustrate the present invention, the DFR enzyme, encoding gene, expression vector, binary expression vector and applications thereof related to the synthesis of Japan serpentium anthocyanin provided in the present invention will be described in detail with reference to the following examples, which should not be construed as limiting the scope of the present invention.

Example 1

Cloning of OjDFR1 Gene of Japanese Snake root

Designing primers OjDFR1F1 (shown in SEQ ID NO. 3) and OjDFR1R1 (shown in SEQ ID NO. 4) according to a transcriptome sequencing result of a petal tissue of the Japanese snakegourd, amplifying cDNA of an OjDFR1 gene for controlling the synthesis of anthocyanin of the Japanese snakegourd, and carrying out PCR amplification to obtain an 1074bp amplification fragment (shown in SEQ ID NO. 2) and encode 357 amino acids (shown in SEQ ID NO. 1); the reaction system for the PCR amplification is shown in Table 1:

TABLE 1 PCR amplification reaction System

Composition (I)	Dosage (mu l)
		Template (cDNA of Japanese snake root grass petal)	1
OjDFR1F1	1
		OjDFR1R1	1
dNTP(2.5mM)	1.6
		MgCl2(25mM)	2
Taq enzyme (2.5U)	0.4
		10×Taq Buffer	2

The reaction process of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 8 min; denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 90s, and extension at 72 ℃ for 8min for 30 cycles.

The results of comparison analysis of the cDNA of the OjDFR1 gene obtained by the above amplification and the DFR of Arabidopsis thaliana revealed that the OjDFR1 gene has an active site and a conserved domain which are ubiquitous in dihydroflavonol 4-reductase (FIG. 1).

Subsequently, the amino acid sequences of DFR derived from various plants and other NADPH-dependent reductases were aligned with the amino acid sequence encoded by cDNA of OjDFR1 gene (OjDFR1 protein) in multiple sequences to complete the construction of phylogenetic tree, and the result showed that the OjDFR1 protein was classified into DFR family, which was consistent with the amino acid alignment (fig. 2). From the results of the above bioinformatics analysis, it is presumed that the DFR1 gene of Japanese snakeweed has a function similar to that of flavanonol 4-reductase.

Example 2

Enzyme activity detection of OjDFR1 of Japanese snakegourd

Construction of prokaryotic expression vector

In order to verify the enzyme activity of OjDFR1 protein, the full-length OjDFR1 gene obtained by cloning is used as a template, and the PCR amplification of the OjDFR1 open reading frame is carried out by using primers OjDFR1F2 (shown in SEQ ID NO. 5) and OjDFR1R2 (shown in SEQ ID NO. 6) with EcoR I and Hind III enzyme cutting sites respectively, wherein the PCR reaction system is shown in Table 2, and the reaction process of the PCR amplification is as follows: pre-denaturation at 94 ℃ for 8 min; denaturation at 94 ℃ for 30s, annealing at 53 ℃ for 90s, and extension at 72 ℃ for 8min for 30 cycles. The DNA fragments obtained by the above amplification were digested with restriction enzymes EcoR I and Hind III, respectively, and cloned into pET-32a (+), and the recombinant plasmid thus constructed was named pET32-OjDFR1 and introduced into E.coli BL21 cells to prepare a large amount of soluble recombinant protein (for the experimental procedures, see the following documents: Sun W, Shen H, Xu H, et al. Chalcone Isomerase a Key Enzyme for Biosynthesis of antisense in Biosynthesis in Ophiorhiza japonica data _ Sheet _1.doc [ J ]. Frontiers in Plant Science,2019, 10).

TABLE 2 PCR amplification reaction System

Composition (I)	Dosage (mu l)
		Template (T vector plasmid containing OjDFR1 gene 1 ng/. mu.l)	1
Primer 1	1
		Primer 2	1
dNTP(2.5mM)	1.6
		MgCl2(25mM)	2
Taq enzyme (2.5U)	0.4
		10×Taq Buffer	2

Induction and expression of soluble recombinant proteins

Inoculating the escherichia coli in an LB solid culture medium containing Amp (the concentration is 0.1mg/mL) by streaking, and carrying out inverted culture in a constant-temperature incubator at 37 ℃ for 12-16 h; selecting clone, culturing at 37 ℃ at 200rpm for 14 h; inoculating 1ml of culture in 5ml of test tube containing LB liquid culture medium at 200rpm and 37 ℃, performing shake culture, taking out one culture 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, wherein the result shows that the escherichia coli enters a growth logarithmic phase after 2 hours, the growth speed is fastest and the state of the bacterium is optimal after 2.5 hours, so that the bacterium is induced by IPTG after 2.5 hours. Through tests of different IPTG concentrations and different induction times, the optimal induction condition of the protein is finally determined to be 15 ℃, and the protein is induced for 24 hours under the IPTG concentration of 0.2 mM.

Preparing a large amount of protein according to the conditions, and separating and purifying the target protein by eluting with nickel column and imidazole, wherein the elution process is as follows:

a. column assembling: slowly adding filling liquid into Ni-NTA pre-packed column, continuously compacting with 20% ethanol, when the filling volume is about 2ml, further compacting with 10ml 20% ethanol, balancing column with 20mM PBS buffer solution, and storing at 4 deg.C;

b. mass production of recombinant proteins

(1) Preparing a large amount of escherichia coli culture under the optimal induction conditions;

(2) 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;

(3) adding 5ml of 20mM PBS suspension thallus into every 50ml of bacterial liquid, inserting the thallus into ice, and carrying out ice bath for 30 min;

(4) ultrasonic: outputting for 5s or more, stopping for 5s, outputting at 400-600W, setting for 10min, ultrasonically breaking cells, and repeating the ultrasonic treatment once after the bacterial liquid is cooled;

(5) 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 solution was repeatedly applied to the Ni column 3 times and equilibrated with 3 volumes of PBS;

d. imidazole elution: after equilibration, elution is carried out by using elution buffers with imidazole concentration of 10mM, 20mM, 50mM, 100mM, 200mM and 500mM in sequence, and 5 tubes of eluent with 2ml per tube are 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.

Collecting a large amount of eluted protein, dialyzing, concentrating the target protein with allochroic silica gel at low temperature, storing the target protein in a-80 deg.C refrigerator, and detecting enzyme activity (A in FIG. 3).

Activity identification of OjDFR1 protein

Three kinds of flavanonols (DHQ, DHM and DHK) were used as substrates, and the reaction system was 35. mu.g of DFR1 protein, 10. mu.l of flavanonol (10mg/ml), 40. mu.l of 100mM TrisHCl (pH7.0), 50. mu.l of 20mM NADPH, and after 30min of reaction at 30 ℃, the reaction product was detected by HPLC. The results are shown in FIGS. 3B to D.

As is clear from the results B to D in FIG. 3, it was found that the OjDFR1 protein catalyzes the reaction of DHQ and DHM to produce the corresponding leucoanthocyanidin, and it was confirmed that OjDFR1, Japanese hop, has flavanonol 4-reductase activity.

Example 3

Effect of OjDFR1 Gene on anthocyanin Synthesis in Arabidopsis and tobacco

Construction of binary expression vectors

In order to verify the influence of the OjDFR1 gene on the synthesis of Arabidopsis anthocyanin, the full-length OjDFR1 gene obtained by cloning was used as a template, and the primers OjDFR1F3 (shown in SEQ ID NO. 7) and OjDFR1R3 (shown in SEQ ID NO. 8) with BamH I and Xba I restriction sites were used to perform PCR amplification of the OjDFR1 open reading frame, and the system and reaction process of the PCR amplification were the same as those of example 2. The DNA fragment amplified above was digested with two restriction enzymes, BamH I and Xba I, respectively, and cloned into pBI121 (cloning procedure similar to that of example 2 except that plasmid pET-32a (+) was replaced with plasmid pBI 121). The recombinant plasmid thus constructed was named pBI121-OjDFR1 and introduced into Agrobacterium GV3101 cells to prepare for genetic transformation of Arabidopsis thaliana by the following specific introduction methods:

(1) adding 10 mul of recombinant plasmid into 200 mul of agrobacterium GV3101 competent cells, mixing evenly and gently, and carrying out ice bath for 30 min;

(2) putting the mixture into a water bath kettle at 42 ℃, and thermally shocking for 90 s;

(3) taking out the mixture, and immediately carrying out ice bath for 2 min;

(4) adding 500 μ l of non-resistant LB liquid culture medium into a sterile operating platform, performing constant temperature shaking culture at 30 deg.C and 200rpm for 1.5h, centrifuging at 5000rpm for 3 min;

(5) mu.l of the supernatant suspension was taken out from the sterile console and applied to LB solid medium (50mg/LKan +50mg/LRif), and cultured in an inverted state at 30 ℃ for 48 hours.

Phenotypic analysis of transgenic plants

Since tissue culture and plant regeneration systems of japanese snakehead are being established, first, the OjDFR1 gene is introduced into an arabidopsis mutant to perform preliminary functional verification.

And infecting arabidopsis thaliana by using the constructed eukaryotic expression vector through an agrobacterium infection solution, obtaining a receptor material DFR mutant plant, and obtaining an overexpression transgenic plant 3 and a transgenic plant 5 through Kan resistance screening. Transgenic plants were grown individually and seeds were harvested and the harvested seeds were plated on anthocyanin-inducing medium containing resistance and observed for phenotypic changes in seedlings at the T2 generation (see, for specific experimental procedures, Wei S, Meng X, Liang L, et al, molecular and Biochemical Analysis of Chalcone Synthase from free fresh idea in Biosynthetic Pathway J. os One, Pl 2015,10(3): e 0119054), the results are shown in FIG. 4.

Meanwhile, a transgenic tobacco plant 4 and a transgenic plant 5 are obtained by an injection method and tissue culture, the transgenic tobacco is transplanted into soil for continuous culture after the tobacco successfully roots, and the phenotypic change of petals is observed after flowering (the specific experimental process is referred to as following documents: spark I A, runons J, Kearns A, et al Rapid, transformed expression of fluorescent expression proteins and generation of stable transformation polypeptides, [ J ]. Nature Protocols,2006,1(4):2019-25.), and the result is shown in figure 5.

As can be seen from FIG. 4, compared with the mutant plants, the anthocyanin in the cotyledon and hypocotyl of the transgenic plants is successfully recovered, and meanwhile, the total RNA of the wild type, the mutant and the transgenic plants is respectively extracted, and the RT-PCR verification shows that OjDFR1 is successfully detected in the transgenic plants, but not detected in the wild type and the mutant.

As can be seen from FIG. 5, the color of tobacco petals was deepened compared with that of the wild type plant, and it was confirmed by RT-PCR that OjDFR1 was successfully detected in the transgenic plant, but not in the wild type.

Enzyme activity detection analysis shows that OjDFR1 can catalyze flavanonol to react to generate colorless anthocyanidin, and proves that OjDFR1 obtained by cloning is really flavanonol 4-reductase. Meanwhile, after the OjDFR1 gene is transferred into the Arabidopsis mutant, the synthesis of anthocyanin in cotyledons and hypocotyls of the Arabidopsis mutant can be recovered, and the flower color of tobacco is deepened.

In conclusion, the OjDFR1 gene of the Japanese snakeroot provided by the invention can control the synthesis of anthocyanin of the Japanese snakeroot and can be applied to the improvement of anthocyanin of other plants; has potential application value in the aspects of flower color modification of transgenic plants and improvement of medicinal plant medicinal components.

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.

Sequence listing

<110> Guizhou university of traditional Chinese medicine

<120> DFR enzyme related to synthesis of anthocyanin in Japanese snake root, encoding gene, expression vector, binary expression vector and application thereof

<160> 8

<170> SIPOSequenceListing 1.0

<210> 1

<211> 357

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

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Met Gly Val Glu Asp Ala Thr Ala Ala Ala Ala Ala Thr Lys Ala Gly

1 5 10 15

Thr Val Cys Val Thr Gly Ala Gly Gly Phe Ile Gly Ser Trp Leu Val

20 25 30

Met Arg Leu Leu Glu Arg Asp Tyr Ile Val Arg Ala Thr Val Arg Asn

35 40 45

Pro Gly Asp Thr Lys Lys Val Lys His Leu Leu Glu Leu Pro Lys Ala

50 55 60

Ser Thr Asn Leu Thr Leu Trp Lys Ala Asp Met Thr Glu Glu Gly Ser

65 70 75 80

Phe Asp Glu Ala Ile Gln Asp Cys Asp Gly Val Phe His Val Ala Thr

85 90 95

Pro Met Asp Phe Glu Ser Lys Asp Pro Glu Asn Glu Val Ile Lys Pro

100 105 110

Thr Ile Asp Gly Ile Leu Asn Ile Ile Arg Ser Cys Val Lys Ala Lys

115 120 125

Thr Val Lys Arg Leu Val Tyr Thr Ser Ser Ala Gly Thr Val Asn Val

130 135 140

Gln Glu His Gln Arg Pro Val Tyr Asp Glu Asn Asp Glu Ser Asp Leu

145 150 155 160

Asp Phe Ile Tyr Ser Lys Lys Met Thr Gly Trp Met Tyr Phe Ala Ser

165 170 175

Lys Leu Leu Ala Glu Lys Glu Ala Arg Glu Ala Ser Lys Glu Asn Asn

180 185 190

Ile Asp Phe Ile Ser Ile Ile Pro Thr Leu Val Val Gly Pro Phe Ile

195 200 205

Thr Pro Thr Phe Pro Pro Ser Leu Ile Thr Ala Leu Ser Leu Ile Thr

210 215 220

Gly Asn Glu Ala His Tyr Ser Ile Ile Lys Gln Gly Gln Phe Val His

225 230 235 240

Leu Asp Asp Leu Cys Glu Ala His Ile Phe Leu Tyr Glu Asn Pro Lys

245 250 255

Ala Glu Gly Arg Tyr Ile Cys Ser Asn Tyr Asp Gly Thr Ile His Asp

260 265 270

Leu Ala Lys Ile Met Arg Glu Lys Trp Pro Glu Tyr Tyr Ile Pro Asp

275 280 285

Glu Leu Lys Gly Ile Asp Lys Asn Ile Pro Val Val Ser Phe Cys Ser

290 295 300

Lys Lys Leu Thr Gly Met Gly Phe Gln Tyr Lys Tyr Asn Leu Asp Asp

305 310 315 320

Met Phe Lys Gly Ala Ile Asp Thr Cys Arg Gln Lys Gly Leu Leu Pro

325 330 335

His Ser Thr Gln Ile Leu Glu Asn Gly Gln Glu Asn Gly Leu Ile Pro

340 345 350

Glu Ser Gln Gln Lys

355

<210> 2

<211> 1074

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atgggagtgg aggatgcaac tgctgctgct gctgcgacaa aggccggcac agtgtgtgtg 60

accggagctg gtggatttat aggatcatgg cttgttatga gactccttga acgtgactat 120

attgttcgtg ccactgtccg gaatccaggg gatacaaaga aagtgaaaca tcttcttgag 180

ttgccaaaag ccagcacgaa tttgaccctt tggaaagccg atatgactga agaaggaagt 240

tttgatgagg ccattcaaga ttgtgatggg gtttttcatg ttgccacacc tatggatttt 300

gaatctaaag accctgagaa tgaagtgatc aagccaacaa ttgatgggat tttgaacatc 360

ataagatcat gcgtcaaggc caaaacagtg aagaggctgg tttacacttc atcagctgga 420

acagtcaatg ttcaagaaca ccaacggcct gtctatgacg agaacgacga gagtgatttg 480

gatttcatct attccaagaa gatgacagga tggatgtatt ttgcttcaaa acttttggct 540

gagaaagaag cacgagaagc atccaaagag aacaatattg atttcatcag tattatacca 600

acgctagtcg taggtccatt catcacgcca acattcccac caagcctaat aactgcactt 660

tcattgataa ctgggaatga agcacattat tcaatcatta agcaaggtca attcgtgcat 720

ttggatgatc tgtgtgaagc ccatatattc ttgtacgaga atcccaaagc cgagggaaga 780

tacatttgct ccaattatga tggaactatt catgatttgg ccaaaattat gagagagaaa 840

tggccagaat actatatccc tgatgagttg aagggaatag acaagaacat acctgtggtg 900

tccttttgtt ccaagaaatt gacaggcatg ggtttccaat ataagtacaa tttggatgac 960

atgttcaagg gagccattga tacgtgccgt caaaagggac tactacccca ttcaacccaa 1020

atccttgaaa acggccaaga gaatggatta atcccagaat cccagcaaaa atag 1074

<210> 3

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

cgattctcac attccatctt ca 22

<210> 4

<211> 18

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gggaagacat ttacgcat 18

<210> 5

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

cggaattcat gggagtggag gatgca 26

<210> 6

<211> 27

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

cccaagcttc tatttttgct gggattc 27

<210> 7

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

gctctagaat gggagtggag gatgca 26

<210> 8

<211> 26

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

cgggatccct atttttgctg ggattc 26