CN114736921B - Method for obtaining genetically transformed plant by utilizing iris DFR gene - Google Patents

Abstract

The invention provides a recombinant expression vector for expressing iris DFR genes, a host cell containing the recombinant expression vector, a plant cell and a plant transformed by genes, and application of the recombinant expression vector and the plant cell and the plant transformed by genes in the aspect of cultivating plants for synthesizing blue anthocyanin, particularly application in the aspect of cultivating plant varieties with new colors or new fruit colors, and particularly application in the aspect of cultivating blue flower varieties.

Description

A method for obtaining genetically transformed plants using the Malin DFR gene

Technical field

The present invention relates to a recombinant expression vector expressing the DFR gene of Malin, as well as host cells, genetically transformed plant cells and plants containing the recombinant expression vector, and their application in cultivating plants that synthesize blue anthocyanins, especially It is an application in cultivating plant varieties with new flower colors or new fruit colors, especially in cultivating blue flower varieties.

Background technique

Iris lactea Pall.var.chinensis (Fisch.) Koidz, hereafter referred to as I.lacteavar.chinensis) is a perennial herbaceous root plant of the genus Iris in the family Iris. It is a variant of the white Iris, also known as Malan flower, Malian flower, etc. It is widely distributed in Northeast my country, North China, Northwest China and other places, and is the city flower of Yinchuan City, Ningxia.

The petals of horsetail are mainly blue, light blue or bluish-purple. As we all know, anthocyanins are one of the main pigments that contribute to the color of flower petals. However, there are currently few reports on the anthocyanin biosynthetic pathway and flower color mechanism of Malinia chinensis.

Generally speaking, Dihydroflavonol 4-reductase (DFR) is the key enzyme in the anthocyanin biosynthetic pathway; DFR can catalyze dihydroflavonol ( Three substrates: Dihydroquercetin (DHQ), Dihydrokaempferol (DHK) and Dihydromyricetin (DHM), generate the corresponding colorless cyanidin, colorless geranium pigment and colorless delphinium Pigments (three colorless proanthocyanidins); Anthocyanidin 4-synthase (ANS) then converts these three colorless proanthocyanidins into anthocyanins directly related to flower color production, namely cyanidin (magenta) ), geranium pigment (red) and delphinium pigment (blue-purple); among them, delphinium pigment is also called blue anthocyanin. The flower and fruit colors of plants are the result of the combination of these three pigments.

In order to reveal the anthocyanin biosynthetic pathway of Malus chinensis, the laboratory of the inventor of the present application used Malin chinensis petals as test materials, and cloned the Malin chinensis DFR gene using PCR technology through transcriptome sequencing and obtained its bioinformatics characteristics ( See the literature: Hujuan et al., Cloning and bioinformatics characteristics analysis of the DFR gene of Malinus chinensis, Northern Horticulture 2017(24): 109-115).

The protein amino acid number encoded by the DFR gene of most species is between 300 and 400, and the protein encoded by DFR has at least two classic domains, one is a highly conserved domain that binds to NADPH, and the other is a domain that binds to the substrate. The protein-binding domain is roughly located near amino acids 132 to 157 of the protein encoded by DFR.

However, different species of DFR have differences in their selection of three substrates, DHM, DHK and DHQ, in anthocyanin biosynthesis, such as substrate preference and catalytic efficiency. Because of this, the synthesis in different plants Different types of anthocyanins give different colors. For example, the DFR of gerbera can use DHM, DHK and DHQ as substrates; the DFR of petunia mainly catalyzes DHM and has no catalytic effect on DHK, so petunia lacks the orange geranium pigment. In addition, the expression levels of DFR genes in different species are also different at different growth stages and in different parts. For example, three DFR genes (DFRA, DFRB and DFRC) were isolated from petunia, but only the DFRA gene was transcribed and expressed in flowers. There is only trace expression in ovules and stems.

At present, although the DFR gene of Malin chinensis has been successfully cloned, the substrate specific preference of Malin chinensis DFR and the mechanism behind how it changes flower color are not clear, which limits the specific application of the DFR gene of Malin chinensis, for example. Application in cultivating plants that synthesize blue anthocyanins, especially in cultivating plant varieties with new flower colors or new fruit colors.

Contents of the invention

In view of the above problems and/or other problems of related technologies, the first aspect of the present invention provides a recombinant expression vector, wherein the recombinant expression vector includes a first gene expression cassette and a second gene expression cassette;

The first gene expression cassette is an expression cassette expressing the DFR gene; the first gene expression cassette contains a gene sequence encoding DFR; the gene sequence encoding DFR is shown in SEQ ID No: 1 Or have more than 80% sequence homology with the sequence shown in SEQ ID No: 1;

The second gene expression cassette is an expression cassette expressing the F3'5'H gene of Lycium barbarum or the expression cassette expressing the F3'5'H gene of Petunia.

Preferably, the second gene expression cassette is an expression cassette expressing the F3'5'H gene of Lycium barbarum L.; the second gene expression cassette contains a gene sequence encoding Lycium barbarum F3'5'H; the encoding F3'5'H gene of Lycium barbarum L. The gene sequence of Lycium barbarum F3'5'H is as shown in SEQ ID No: 2 or has more than 80% sequence homology with the sequence shown in SEQ ID No: 2; or,

The second gene expression cassette is an expression cassette expressing the petunia F3'5'H gene; the second gene expression cassette includes a gene sequence encoding the petunia F3'5'H gene; the encoding petunia F3'5'H gene The gene sequence of the F3'5'H gene is as shown in SEQ ID No:7 or has more than 80% sequence homology with the sequence shown in SEQ ID No:7.

More preferably, the first gene expression cassette, from the 5' end to the 3' end, sequentially includes the first promoter, the gene sequence encoding the DFR and the first terminator;

The sequence of the first promoter is shown in SEQ ID No: 3;

The sequence of the first terminator is shown in SEQ ID No: 4;

The second gene expression cassette, from the 5' end to the 3' end, sequentially includes the second promoter, the gene sequence encoding Lycium barbarum or Petunia F3'5'H, and the second terminator;

The sequence of the second promoter is shown in SEQ ID No: 5;

The sequence of the second terminator is shown in SEQ ID No: 6.

A second aspect of the invention provides a host cell comprising the above recombinant expression vector.

A third aspect of the present invention provides a genetically transformed plant cell, wherein the genetically transformed plant cell contains the above-mentioned recombinant expression vector.

The fourth aspect of the present invention provides a genetically transformed plant, wherein the genetically transformed plant contains the above-mentioned genetically transformed plant cells.

The fifth aspect of the present invention provides the use of the above-mentioned recombinant expression vector, the above-mentioned host cell, the above-mentioned gene-transformed plant cell, and the above-mentioned gene-transformed plant in cultivating plants that synthesize blue anthocyanins.

Preferably, the application is an application in cultivating plant varieties with new flower colors or new fruit colors.

Preferably, the application is an application in cultivating blue flower varieties.

The sixth aspect of the present invention provides a method for obtaining genetically transformed plants using the DFR gene of Malinia chinensis, wherein the method includes the following steps:

Step 1): Construct the above recombinant expression vector;

Step 2): Transfer the recombinant expression vector constructed in step 1) into plant explants to form plant callus, and further culture to obtain gene-transformed regenerated plants containing the Malin DFR gene and the F3'5'H gene; The F3'5'H gene is the F3'5'H gene of Lycium barbarum or petunia.

The invention provides a recombinant expression vector expressing the DFR gene of Malin, as well as host cells containing the recombinant expression vector, genetically transformed plant cells and genetically transformed plants, as well as their application in cultivating plants that synthesize blue anthocyanins. Applications, especially applications in breeding plant varieties with new flower colors or new fruit colors, especially applications in breeding blue flower varieties.

Description of drawings

The upper part of Figure 1 shows the HPLC results of the DHQ standard, and the lower part shows the HPLC results of the DHQ substrate after the enzymatic reaction; the ordinate represents the electrical signal response value (AU) of the detector, and the abscissa represents time; All the following are the same;

The upper part of Figure 2 shows the HPLC results of the DHK standard, and the lower part shows the HPLC results of the DHK substrate after enzymatic reaction;

The upper part of Figure 3 shows the HPLC results of the DHM standard, and the lower part shows the HPLC results of the DHM substrate after enzymatic reaction.

Detailed ways

The present invention will be further described below through specific embodiments, but the present invention is not limited to these specific embodiments. Materials, reagents, etc. used in the following specific embodiments can be obtained from commercial sources unless otherwise specified. If specific techniques or conditions are not specified, the techniques or conditions described in literature in the field shall be followed (for example, refer to the third edition of "Molecular Cloning Experiment Guide" translated by J. Sambrook et al., Huang Peitang et al., Science Press ) or follow the product instructions.

1. Cloning and identification of the DFR gene of Malin

See the literature: Hujuan et al., Cloning and bioinformatics characteristics analysis of the DFR gene of Malinus chinensis, Northern Horticulture 2017(24): 109-115.

The full length of the Malin DFR gene is 1427 bp, encoding a total of 357 amino acids (the inventor's laboratory named the gene IlDFR, and the GenBank accession number is KY907171). This gene has high homology with the DFR of various plants at the amino acid level, and is closely related to the DFR of the same genus I.hollandica in terms of phylogeny; its relative molecular mass of the protein is predicted to be 39.99kDa, and it is isoelectric. Point 5.89, mainly composed of α-helices and β-sheets, is located in the cytoplasm, and is an acidic, hydrophilic, unstable protein without a signal peptide. The Amino Acid DFR gene has an amino acid sequence that is very similar to the relatively conserved NADPH-binding domain unique to DFR in most species, with only four amino acid residues differing.

2. Study on the substrate binding characteristics of Malin DFR

1. Construction of prokaryotic expression vector of Malin DFR gene

According to the codon preference of prokaryotes, the Malin DFR gene sequence cloned and identified in the first part was sent to GenScript Biotechnology Co., Ltd. for optimization and synthesis.

The pTrc-CKS plasmid (purchased from General Biosystems (Anhui) Co., Ltd.) and the above-mentioned synthesized Malin DFR optimized sequence were double-digested by BamHI and Nhe I respectively, recovered by agarose gel electrophoresis, and then ligated. Transformed into JM109 competent cells. Pick the positive clones and use colony PCR to identify the obtained pTrc-CKS-DFR positive recombinants. After agarose gel electrophoresis analysis, the size of the detected target band is consistent with the expected; and then verified by sequencing. The result is correct, indicating that the recombinant plasmid pTrc-CKS-DFR has been successfully constructed; JM109 carrying the recombinant plasmid pTrc-CKS-DFR was obtained.

2. Prokaryotic expression, purification and identification of Malin DFR

The JM109 strain carrying the recombinant plasmid pTrc-CKS-DFR obtained above was inoculated into 5 mL (containing 25 mg/L ampicillin) liquid LB medium, cultured at 37°C and 220 rpm to the logarithmic phase, and the bacterial liquid was mixed at 1:100. Ratio transfer to 1L fresh liquid LB medium (containing 25mg/L ampicillin), culture to the logarithmic phase, add IPTG to a final concentration of 0.5mM, induce culture at 16℃, 220rpm for 15h, 4℃, 6,000× centrifuge for 10 minutes, collect the cells, add PBS buffer to resuspend the cells, break with ultrasonic, collect the supernatant and precipitate after centrifugation, and conduct 12% SDS-PAGE detection and Western Blot identification. The results show that the obtained recombinant protein (horse DFR) is expressed in a soluble form, and the expressed protein is approximately 95kDa.

The recombinant protein was purified using nickel agarose gel packed column affinity chromatography. After measuring its concentration with a protein quantitative detection instrument, the protein concentration was adjusted to 0.5 mg/mL with 100 mM Tris-HCL (pH 7.0).

3. Analysis of substrate binding characteristics of Malin DFR

Add an equal amount of Malin DFR (the recombinant protease obtained above) to the enzymatic reaction system containing equal amounts of DHQ, DHK and DHM substrates (containing 100mM Tris-HCl, pH 7.0, 20mM NADPH) respectively; react at 30°C After 30 minutes, the reaction was terminated. The reaction solution was filtered, and 10 μL was taken for analysis by waters2695 high performance liquid chromatography. The results are shown in Figure 1-3.

Refer to Figure 1. The upper part is the HPLC result of DHQ standard substance, and the lower part is the HPLC result of DHQ substrate after enzymatic reaction. Comparing the two results, it can be seen that after the contact between Malin DFR and DHQ, no new The product shows that Malin DFR does not catalyze DHQ substrate.

Refer to Figure 2. The upper part is the HPLC result of the DHK standard substance, and the lower part is the HPLC result of the DHK substrate after the enzymatic reaction. Comparing the two results, it can be seen that after the DHK substrate is enzymatically reacted, the DHK substrate is The peak area of the product decreased significantly, and there were new products (retention time was about 3.625 min).

Refer to Figure 3. The upper part is the HPLC result of the DHM standard substance, and the lower part is the HPLC result of the DHM substrate after the enzymatic reaction. Comparing the two results, it can be seen that after the DHM substrate is enzymatically reacted, the DHM substrate is The peak area of the product decreased significantly, and there were new products (retention time was about 3.625 min).

Based on the in vitro enzyme activity analysis results in Figures 1 to 3, it can be inferred that the DFR protein, under the action of NADPH cofactor, can catalyze DHM first, followed by DHK, and cannot catalyze DHQ.

Based on the above-mentioned research results on the substrate binding properties of Malin DFR and the currently known anthocyanin biosynthetic pathway theory, the inventor of the present application believes that the speculation that can be made is: the substrate specificity of Malin DFR protein for DHM Preferences, including the inability to catalyze DHQ substrates, may be the main reason why the flower colors of horsetail are light blue and blue-violet, but not red.

3. The formation mechanism and application of blue petals and blue fruits of plants

1. Flavonoid 3’5’-hydroxylase F3’5’H gene

There are many flower colors in nature, but some important flowers such as carnations, roses (or roses), chrysanthemums, narcissus, etc. do not have blue color due to the lack of enzymes (such as F3'5'H) related to the biosynthesis of delphinium pigment (blue-purple). and purple strains; the same is true for the fruit color; this is also a problem that cannot be solved using traditional cross-breeding methods. Gene transformation technology is used to introduce the F3’5’H gene of certain plants into specific plant culture cells, which can change and regulate the anthocyanin biosynthetic pathway. This is also a key step in cultivating new flower or fruit color plant varieties.

2. Dihydroflavonol-4-reductase DFR gene

However, simply introducing the F3’5’H gene of other plants, or simultaneously introducing the DFR gene of general plants, may not necessarily lead to the formation of blue petals and blue fruits. This is because the DFR of general plants, if there is no specific preference for the binding of DHQ, DHK and DHM substrates, will produce cyanidin (magenta), geranium pigment (red) and delphinium pigment (blue-violet) There are three types of anthocyanins, and the flower and fruit colors presented are a mixture of these three pigments.

Therefore, utilizing the substrate-specific preference of DFR gene for DHM is the key to using gene transformation to cultivate new blue anthocyanin varieties (technical solution of this application), especially for cultivating new flower colors or new fruit colors. Plant varieties, especially those used to cultivate new varieties with blue flowers.

Based on the research results of the substrate binding properties of Malin DFR, the inventor of the present application has made many attempts and found that the recombinant expression vector combining the F3'5'H gene of black wolfberry or petunia and the Malin DFR gene can successfully Obtain new genetically transformed varieties.

4. Methods for obtaining genetically transformed plants using the Malin DFR gene

In a specific embodiment of the present invention, a recombinant expression vector is provided, wherein the recombinant expression vector includes a first gene expression cassette and a second gene expression cassette;

The first gene expression cassette is an expression cassette expressing the DFR gene; the first gene expression cassette contains a gene sequence encoding DFR; the gene sequence encoding DFR is shown in SEQ ID No: 1 Or have more than 80% sequence homology with the sequence shown in SEQ ID No: 1;

The second gene expression cassette is an expression cassette expressing the F3'5'H gene of Lycium barbarum or the expression cassette expressing the F3'5'H gene of Petunia.

In a preferred embodiment of the present invention, the second gene expression cassette is an expression cassette expressing the Lycium barbarum F3'5'H gene; the second gene expression cassette contains the gene encoding Lycium barbarum F3'5'H. Gene sequence; the gene sequence encoding Lycium barbarum F3'5'H is as shown in SEQ ID No: 2 or has more than 80% sequence homology with the sequence shown in SEQ ID No: 2; or,

The second gene expression cassette is an expression cassette expressing the petunia F3'5'H gene; the second gene expression cassette includes a gene sequence encoding the petunia F3'5'H gene; the encoding petunia F3'5'H gene The gene sequence of the F3'5'H gene is as shown in SEQ ID No:7 or has more than 80% sequence homology with the sequence shown in SEQ ID No:7.

The percentage of "sequence homology" with respect to nucleic acid sequences is determined by determining the number of nucleotides present in the two sequences to produce the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and dividing the result Multiply by 100 to yield the percent homology of the sequence. Sequence homology, also known as sequence identity.

In a specific embodiment of the present invention, the gene sequence encoding Lycium barbarum DFR in the above-mentioned first gene expression cassette, the gene sequence encoding Lycium barbarum F3'5'H in the above-mentioned second gene expression cassette, and the gene sequence encoding Lycium barbarum F3'5'H in the above-mentioned second gene expression cassette. The gene sequence of bovine F3'5'H can be deleted, inserted or mutated with a small number of nucleotides based on the sequences shown in SEQ ID NO.1, SEQ ID NO.2 and SEQ ID NO.7 respectively. , to obtain nucleotide sequences with more than 80% homology. Substitutions of a small number of nucleotides (deletions or insertions, or nucleotide mutations), especially variants obtained by substitutions of conserved regions, can still encode Hematin DFR protein and Lycium barbarum F3 with the same or basically the same functions. '5'H protein, petunia F3'5'H protein, then these variants are within the protection scope of the scheme of this application.

The following Example 1 takes the recombinant expression vector containing the expression cassette of the DFR gene of Malin and the expression cassette of F3'5'H gene of Lycium barbarum as an example for detailed explanation.

Example 1

1. Construct recombinant expression vector

The recombinant expression vector of Example 1 includes a first gene expression cassette and a second gene expression cassette.

Among them, the first gene expression cassette, from the 5' end to the 3' end, contains the first promoter, the gene sequence encoding the DFR and the first terminator in sequence.

The gene sequence encoding Malin DFR is shown in SEQ ID No: 1.

The first promoter is the Arabidopsis ACT2 (actin 2) promoter, whose sequence is shown in SEQ ID No: 3; the first terminator is the Arabidopsis HSP 18.2 terminator, whose sequence is shown in SEQ ID No: 4 .

Among them, the second gene expression cassette, from the 5' end to the 3' end, contains the second promoter, the gene sequence encoding Lycium barbarum F3'5'H, and the second terminator.

The gene sequence encoding Lycium barbarum F3’5’H is shown in SEQ ID No: 2.

The second promoter is the Arabidopsis UBQ (Ubiquitin) promoter, and its sequence is shown in SEQ ID No: 5; the second terminator is the Arabidopsis UBQ terminator, and its sequence is shown in SEQ ID No: 6.

The construction process of recombinant expression vector is as follows:

(1) Synthesize LrF3’5’H DNA (SEQ ID No: 2) as fragment a; synthesize IlDFR DNA (SEQ ID No: 1) as fragment b;

(2) Digest the pNULPGE600 vector (15004bp) with Pac I and PmaC I restriction endonucleases, separate it by gel electrophoresis (6bp, 14998bp), and recover the 14998bp fragment as fragment c.

(3) The synthesized LrF3'5'H DNA fragment a is also digested with Pac I and PmaC I restriction enzymes, and then ligated with the fragment c recovered from the pNULPGE600 vector to form a pNULPGE0601 transition vector d (16537 bp) .

(4) Digest the transition vector d with Spe I and Srf I restriction enzymes, and then separate it by gel electrophoresis (9 bp, 16528 bp), and recover the 16528 bp fragment e.

(5) The synthesized IlDFR DNA fragment b was also digested with Spe I and Srf I restriction endonucleases, and then ligated with the 16528 bp fragment e recovered after digestion with the transition vector to form a multi-gene expression vector, named pNULPGE0602 (17617 bp ).

(6) After transforming the pNULPGE0602 vector into E. coli, sequence the inserted fragment to verify it.

2. Gene transformation and culture to obtain genetically transformed plants

The recombinant expression vector (pNULPGE0602 vector) obtained by the above construction was transferred into plant explants to form plant callus tissue, and further cultured to obtain transformed regenerated plants containing the Malin DFR gene and the Lycium barbarum LrF3’5’H gene.

Regarding the plant explants to be transformed, petunia leaf explants were used in this example; specifically, sterile petunia leaves were cut to a size of 0.5×0.5cm on a clean workbench to obtain petunia leaves. Petunias leaf explants serve as receptors for gene transformation.

The preparation of the Agrobacterium infection solution is as follows: Add the plasmid vector (the recombinant expression vector pNULPGE0602 vector obtained above) to the Agrobacterium strain EHA105 competent cells, mix gently; then quickly put it into liquid nitrogen for 2 minutes; then quickly put it into 37°C metal bath for 5 minutes (mix the bacteria once every 2 minutes); keep on ice for 5 minutes, mixing twice during this period; add 500 μL of preheated 28°C LB culture medium, mix thoroughly; culture on a 28°C shaker (200rpm) for 2 -4 hours, take 100 μL of the cultured bacterial liquid, spread it evenly into the solid LB medium containing kanamycin that has been preheated to 28°C, and culture it at 28°C overnight; then pick the resistant colonies until they contain kanamycin. In the LB liquid medium of namycin, culture on a shaker (200rpm) at 28°C until OD550=0.8-1.0; centrifuge at 4000rpm for 10 minutes, remove the supernatant, and resuspend the centrifuged Agrobacterium bacteria in the infection solution (4.41g /L MS+30g/L sucrose+0.2mg/L naphthylacetic acid+1mg/L 6-benzyladenine+20mg/L acetosyringone, pH value 5.8), prepare the Agrobacterium infection solution of this embodiment.

Regarding the process of Agrobacterium-mediated gene transformation: Soak the above-mentioned petunia leaf explants in the Agrobacterium infection solution prepared above for 3 minutes, and then place them in the co-culture medium (4.41g/L MS+30g/ L sucrose + 0.2 mg/L naphthalene acetic acid + 1 mg/L 6-benzyl adenine + 20 mg/L acetosyringone + agar 0.7 g/L, pH value 5.8), and cultured in the dark at 22°C for 3 days; then, after infection The explants were transferred to the selection medium (4.41g/L MS+30g/L sucrose+0.2mg/L naphthylacetic acid+1mg/L 6-benzyladenine+Agar 0.7g/L+500mg/L carbenicillin+ 25 mg/L kanamycin, pH 5.8), culture in an incubator at 25°C and 16 hours of light until adventitious buds grow; cut off the adventitious buds and move them to rooting medium (4.41 g/L MS +20g/L sucrose+Agar 0.7g/L+250mg/L carbenicillin, pH 5.8) until the genetically transformed petunia plants grow into roots. Genetically transformed petunia plants are planted in the soil. After flowering, the flower color will be blue.

Regarding the identification of the obtained gene-transformed petunia plants: the CTAB method was used to extract the genomic DNA of the gene-transformed petunia plants, and after PCR amplification and identification (PCR amplification product 360 bp), the gene-transformed petunia obtained by transformation in this example The plant contains the Malin DFR gene.

In addition, the inventor of the present application also applied the substrates DHM and/or DHK to the incision of the cut adventitious buds, and then transferred them to the rooting medium for culture. The callus in the incision showed blue.

Example 2

The recombinant expression vector of Example 2 includes a first gene expression cassette and a second gene expression cassette.

The first gene expression cassette in Example 2 is the same as the first gene expression cassette in Example 1, and the details will not be repeated.

The second gene expression cassette of Example 2, from the 5' end to the 3' end, sequentially includes a second promoter, a gene sequence encoding petunia F3'5'H, and a second terminator.

The gene sequence encoding petunia F3'5'H is shown in SEQ ID No: 7.

The second promoter is the promoter of Arabidopsis thaliana UBQ, and its sequence is shown in SEQ ID No: 5; the second terminator is the Arabidopsis thaliana UBQ terminator, and its sequence is shown in SEQ ID No: 6.

The vector construction process in step 1) is basically the same as that in Embodiment 1, and will not be described in details.

The process of gene transformation and culture in step 2) to obtain gene-transformed plants is basically the same as in Example 1, and will not be described in detail.

The genetically transformed petunia plants obtained in Example 2 were planted in the soil. After flowering, the flower color also turned blue.

In addition, similarly, the inventor of the present application also applied the substrates DNM and/or DHK to the incision part of the adventitious buds in Example 2, and then transferred it to the rooting medium for culture. The callus tissue in the incision part showed blue.

It should be understood that although this specification is described in terms of implementations, not each implementation only contains an independent technical solution. This description of the specification is only for the sake of clarity. Persons skilled in the art should take the specification as a whole and understand each individual solution. The technical solutions in the embodiments can also be appropriately combined to form other embodiments that can be understood by those skilled in the art.

The series of detailed descriptions listed above are only specific descriptions of feasible implementations of the present invention. They are not intended to limit the protection scope of the present invention. Any equivalent implementations or implementations that do not deviate from the technical spirit of the present invention are not intended to limit the protection scope of the present invention. All changes should be included in the protection scope of the present invention.

Claims (6)

1. A recombinant expression vector, characterized in that:

the recombinant expression vector comprises a first gene expression cassette and a second gene expression cassette;

the first gene expression cassette is an expression cassette for expressing the iris DFR gene; the first gene expression cassette comprises a gene sequence encoding iris lactea DFR; the gene sequence of the code iris lactea DFR is shown as SEQ ID No. 1;

the second gene expression cassette is an expression cassette for expressing the F3'5' H gene of lycium ruthenicum or an expression cassette for expressing the F3'5' H gene of petunia;

the second gene expression cassette is an expression cassette for expressing F3'5' H genes of lycium ruthenicum; the second gene expression cassette comprises a gene sequence encoding lycium ruthenicum F3'5' h; the gene sequence of the coded lycium ruthenicum F3'5' H is shown in SEQ ID No. 2; or,

the second gene expression cassette is an expression cassette for expressing petunia F3'5' H genes; the second gene expression cassette comprises a gene sequence encoding a petunia F3'5' h gene; the gene sequence of the gene for coding the petunia F3'5' H is shown as SEQ ID No. 7.

2. The recombinant expression vector of claim 1, wherein:

the first gene expression cassette sequentially comprises a first promoter, the gene sequence for encoding iris lactea DFR and a first terminator from a 5 'end to a 3' end;

the sequence of the first promoter is shown as SEQ ID No. 3;

the sequence of the first terminator is shown as SEQ ID No. 4;

the second gene expression cassette sequentially comprises a second promoter, the gene sequence for encoding the lycium ruthenicum or the petunia F3'5' H and a second terminator from the 5 'end to the 3' end;

the sequence of the second promoter is shown as SEQ ID No. 5;

the sequence of the second terminator is shown as SEQ ID No. 6.

3. Use of a recombinant expression vector according to any one of claims 1 to 2 for growing plants that synthesize blue anthocyanin.

4. A use according to claim 3, wherein: the application is the application in the aspect of cultivating plant varieties with new flower colors or new fruit colors.

5. The use according to claim 4, wherein: the application is the application in the aspect of cultivating blue flower varieties.

6. A method for obtaining a genetically transformed plant by utilizing a iris DFR gene is characterized in that:

the method comprises the steps of,

step 1): constructing the recombinant expression vector of any one of claims 1 to 2;

step 2): transferring the recombinant expression vector constructed in the step 1) into a plant explant to obtain a gene conversion regeneration plant containing the iris DFR gene and the F3'5' H gene;

the F3'5' H gene is F3'5' H gene of Lycium ruthenicum or petunia.