CN103756982B - Tulipa fosteriana flavonol synthase TfFLS protein and encoding gene thereof - Google Patents

Abstract

The invention discloses a kind of Tulipa fosteriana flavonol synthase TfFLS protein and encoding gene thereof; Does is described protein as SEQ? ID? shown in NO.2 aminoacid sequence composition protein or as SEQ? ID? aminoacid sequence shown in NO.2 passes through replacement, lacks or adds one or several amino acid and has the protein of turmeric flavonol synthase activity.Present invention also offers a kind of above-mentioned protein of encoding as SEQ? ID? nucleotide sequence shown in NO.1.Turmeric flavonol synthetic enzyme TfFLS gene of the present invention, expresses the flavonol synthetase albumen of restructuring in Bacillus coli cells, dihydrokaempferol can be made to generate kaempferol, show the function with flavonol synthetic enzyme.Utilize turmeric TfFLS gene of the present invention, by various conventional screening assays, can filter out, with TfFLS, interactional material, acceptor, inhibitor or antagonist etc. occur.

Description

Tulip flavonol synthase TfFLS protein and its coding gene

technical field

The invention belongs to the field of plant molecular biology, and relates to a key enzyme in the synthetic pathway of tulip flavonoids and its coding gene, in particular to a tulip flavonol synthase TfFLS protein and its coding gene.

Background technique

Flavonoids are a class of polyphenolic compounds that widely exist in nature. According to different modifications to the central C ring, they can be divided into major categories such as flavonoids, isoflavones, flavanones, flavonols, flavanols, and anthocyanins. Among them, flavonoid alcohols are the most common, accounting for about 1/3 of the total flavonoids. Flavonols are widely distributed in a variety of plants. They are inhibitors of several kinases related to intracellular signal transduction and cell transformation, and have a variety of biological activities, because they can selectively block intracellular signal transduction pathways , so flavonoids are considered as potential compounds for the treatment of tumors. Flavonol synthase (flavonol synthase, FLS) is a direct regulatory enzyme of flavonol synthesis in the flavonoid synthesis pathway. It catalyzes the hydroxylation of the C3 position in the flavonoid structure, thereby forming various flavonol substances, which determine the synthesis of flavonol. . Therefore, the FLS gene is one of the key genes in the study of the regulation of flavonoid metabolism.

Flavonoids are also a large class of anthocyanins that give flowers their full range of color from yellow to purple. Flavonols are generally yellow or colorless and can act as auxiliary pigments to affect flower color. The expression of FLS gene can directly affect the flower color of plants. In petunia, flavonol synthase can change the ratio of flavonol to anthocyanin by affecting co-pigment synthesis or substrate competition, thereby changing flower color (Mol J et al., 1998). The production of flavonols in Primulasinensis Sabine ex L. is controlled by gene B, and the flowers containing co-pigmentation are purple in color, and those lacking flavonols are purple in color (Forkman G, 1991). In petunia, increased expression of the flavonol synthase gene usually resulted in blue flowers, whereas antisense expression of FLS eDNA resulted in significantly lighter flower colors (Holton TA et al., 1993).

At present, people have cloned the FLS gene in plants such as petunia hybrida, potato (Solanum tuberosum), apple (Malus pumila), mandarin orange (citrus), grape (Vitis vinifera), ginkgo biloba (Ginkgo biloba), etc. The cloning and expression pattern of the flavonol synthase gene in tulip (Tulipa fosteriana) is still unclear. At present, there is no literature report related to tulip FLS protein and its coding gene sequence.

Contents of the invention

The object of the present invention is to provide a tulip flavonol synthase TfFLS protein and its coding gene. The invention discloses the expression pattern of tulip TfFLS coding gene in different organs and different development stages of tulip; the tulip TfFLS gene of the invention expresses recombinant flavonol synthase protein in Escherichia coli cells, which can make dihydrokaempferol produce kaempferol. Utilizing the tulip TfFLS gene of the present invention, substances, receptors, inhibitors or antagonists that interact with FLS can be screened out through various conventional screening methods.

The purpose of the present invention is achieved by the following technical solutions,

In a first aspect, the present invention relates to a protein having tulip flavonol synthase activity, said protein is a protein consisting of the amino acid sequence shown in SEQ ID NO.2; or the amino acid shown in SEQ ID NO.2 A protein derived from (a) whose sequence has undergone substitution, deletion or addition of one or several amino acids and has tulip flavonol synthase activity. There are large differences in the presence or absence and activity of the protein in different coloring stages of flower petals, in different organs.

Preferably, the protein is obtained by deletion, insertion and/or substitution of 1 to 50 amino acids in the amino acid sequence shown in SEQ ID NO.2, or by adding 1 to 20 amino acids at the C-terminal and/or N-terminal sequence.

Further preferably, the protein is a sequence formed by replacing 1 to 10 amino acids in the amino acid sequence shown in SEQ ID NO.2 by amino acids with similar or similar properties.

In a second aspect, the present invention relates to a nucleic acid sequence encoding the above-mentioned protein.

Preferably, the nucleic acid sequence is specifically:

(a) The base sequence is as shown in the 1st to 999th positions of SEQ ID NO.1;

or (b) a sequence having at least 70% homology with the nucleic acid shown in positions 1 to 999 of SEQ ID NO.1;

Or (c) a sequence capable of hybridizing to the nucleic acid shown in positions 1 to 999 of SEQ ID NO.1.

Preferably, the nucleic acid sequence is specifically the deletion, insertion and/or substitution of 1 to 90 nucleotides in the nucleic acid sequence shown in positions 1 to 999 of SEQ ID NO.1, and 5' and/or 3' A sequence formed by adding 60 or less nucleotides at the end.

In a third aspect, the present invention relates to the use of the above-mentioned nucleic acid sequence in preparing recombinant flavonol synthase.

Preferably, the preparation includes the following steps: constructing a prokaryotic expression vector containing the nucleic acid sequence, transforming the prokaryotic expression vector into Escherichia coli, inducing culture, and obtaining recombinant flavonol synthase

In the fourth aspect, the present invention relates to a recombinant flavonol synthase, which is prepared by the following method: constructing a prokaryotic expression vector containing the above nucleic acid sequence, and transforming the prokaryotic expression vector into the large intestine bacilli, induced and cultured to obtain the recombinant flavonol synthase.

The isolated DNA molecule provided by the present invention includes: a DNA molecule having a nucleotide sequence shown in SEQ ID NO.1; or a nucleotide sequence encoding a polypeptide having tulip TfFLS protein activity, and is identical to SEQ ID NO. .3 The sequence shown in 3 has at least 70% homology; or can hybridize with the nucleotide sequence of the sequence shown in SEQ ID NO.1.

In the present invention, "isolated DNA" and "purified DNA" mean that the DNA or fragment has been separated from the sequences on both sides of it in the natural state, and it also means that the DNA or fragment has been accompanied by the natural state. The components of the nucleic acid are separated and have been separated from the proteins that accompany them in the cell.

In the present invention, the term "tulip flavonol synthase TfFLS protein-encoding gene" refers to a nucleotide sequence encoding a polypeptide having tulip TfFLS protein activity, such as the nucleotide sequence shown in SEQ ID NO.1 and its degenerate sequence. The degenerate sequence refers to a sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid in the sequence shown in SEQ ID NO.1. Due to the degeneracy of codons, a degenerate sequence with as low as about 70% homology to the sequence shown in SEQ ID NO.1 can also encode the amino acid sequence shown in SEQ ID NO.2. The term also includes a nucleotide sequence having at least 70% homology with the nucleotide sequence from nucleotides 1 to 999 in the sequence shown in SEQ ID NO.1.

The term also includes variants of the sequence shown in SEQ ID NO. 1 that encodes a protein with the same function as native tulip TfFLS. These variations include (but are not limited to): deletions, insertions and/or substitutions of usually 1 to 90 nucleotides, and additions of up to 60 nucleotides at the 5' and/or 3' ends.

In the present invention, the term "tulip flavonol synthase TfFLS protein" refers to a polypeptide having a sequence shown in SEQ ID NO.2 having tulip TfFLS protein activity. The term also includes variant forms of the sequence shown in SEQ ID NO. 2 that have the same function as the natural tulip TfFLS. These variant forms include (but are not limited to): usually 1-50 amino acid deletions, insertions and/or substitutions, and addition of one or less than 20 amino acids at the C-terminal and/or N-terminal. For example, in the art, substitutions with amino acids with similar or similar properties generally do not change the function of the protein. As another example, adding one or several amino acids at the C-terminus and/or N-terminus usually does not change the function of the protein. The term also includes active fragments and active derivatives of the tulip TfFLS protein.

Variant forms of the tulip TfFLS polypeptide of the present invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, DNA capable of hybridizing with tulip TfFLS-related DNA under high or low stringent conditions The encoded protein, and the polypeptide or protein obtained by using the antiserum of tulip TfFLS polypeptide.

In the present invention, "Tulip TfFLS conservative variant polypeptide" refers to a polypeptide formed by replacing up to 10 amino acids with amino acids with similar or similar properties compared with the amino acid sequence shown in SEQ ID NO.2. These conservative variant polypeptides are preferably produced by substitutions according to Table 1.

Table 1

initial residue representative replacement preferred substitution Ala(A) Val; Leu; Ile Val

Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; ms; Lys; Arg Gln Asp(D) Glu Glu Cys(C) Ser Ser Gln(Q) Asn Asn Glu(E) Asp Asp Gly(G) Pro; Ala His(H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val; Met; Ala; Phc Leu Leu(L) Ile; Val; Met; Ala; Phc Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phc; Ile Leu Phc(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phc Tyr Tyr(Y) Trp; Phc; Thr; Ser Phc Val(V) Ile; Leu; Met; Phc; Ala Leu

The invention also includes analogs of tulip TfFLS proteins or polypeptides. The difference between these analogues and the TfFLS-related polypeptide of Tulip may be the difference in the amino acid sequence, or the difference in the modified form that does not affect the sequence, or both. These polypeptides include natural or induced genetic variants. Induced variants can be obtained by various techniques, such as random mutagenesis by radiation or exposure to mutagens, but also by site-directed mutagenesis or other techniques known in molecular biology. Analogs also include analogs with residues other than natural L-amino acids (eg, D-amino acids), and analogs with non-naturally occurring or synthetic amino acids (eg, β, γ-amino acids). It should be understood that the polypeptides of the present invention are not limited to the representative polypeptides listed above.

Modified (usually without altering primary structure) forms include: chemically derivatized forms of polypeptides such as acetylation or carboxylation, in vivo or in vitro. Modifications also include glycosylation, such as those resulting from polypeptides that are modified by glycosylation during synthesis and processing of the polypeptide or during further processing steps. Such modification can be accomplished by exposing the polypeptide to an enzyme that performs glycosylation, such as a mammalian glycosylase or deglycosylation enzyme. Modified forms also include sequences with phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, phosphothreonine). Also included are polypeptides that have been modified to increase their resistance to proteolysis or to optimize solubility.

In the present invention, the expression pattern of tulip TfFLS gene product can be analyzed by real-time fluorescence quantitative PCR method, that is, the presence or absence and quantity of mRNA transcript of TfFLS gene in cells can be analyzed.

In addition, according to the tulip TfFLS nucleotide sequence and amino acid sequence of the present invention, it is possible to screen tulip TfFLS related homologous genes or homologous proteins on the basis of nucleic acid homology or expressed protein homology.

In order to obtain the array of genes related to tulip TfFLS, DNA probes can be used to screen the tulip cDNA library. These probes are obtained by radioactively labeling all or part of tulip TfFLS with ³² P under low stringency conditions. A suitable cDNA library for screening is the library from Tulip. Methods for constructing cDNA libraries from cells or tissues of interest are well known in the art of molecular biology. In addition, many such cDNA libraries are commercially available, eg, from Clontech, Stratagene, Palo Alto, Cal. This screening method can identify the nucleotide sequences of gene families related to tulip TfFLS.

The full-length sequence of nucleotides related to tulip TfFLS of the present invention or its fragments can usually be obtained by PCR amplification, recombination or artificial synthesis. For the PCR amplification method, primers can be designed according to the relevant nucleotide sequences disclosed in the present invention, especially the open reading frame sequence, and the cDNA prepared by a commercially available cDNA library or a conventional method known to those skilled in the art can be used. The library is used as a template to amplify related sequences. When the sequence is long, it is often necessary to carry out two or more PCR amplifications, and then splice together the amplified fragments in the correct order.

After the relevant sequences are obtained, the relevant sequences can be obtained in large quantities by recombination. Usually, it is cloned into a vector, then transformed into a cell, and then the relevant sequence is isolated from the proliferated host cell by conventional methods.

In addition, mutations can also be introduced into the protein sequences of the invention by chemical synthesis.

In addition to recombinant production, fragments of the proteins of the invention can also be produced by direct synthesis of peptides using solid phase techniques (Stewart et al., (1969) Solid Phase Polypeptide Synthesis, WH Freeman Co., San FranciSco; Merrifield J. (1963) J. Am Chem. Soc 85:2149-2154). Protein synthesis in vitro can be performed manually or automatically. For example, peptides can be synthesized automatically using an Applied Biosystems Model 431A Peptide Synthesizer (Foster City, CA). Fragments of a protein of the invention can be chemically synthesized separately and then chemically linked to produce a full-length molecule.

The Escherichia coli DH5α and BL21 strains involved in the present invention have been published in "Sambrook J, Russell D W. Molecular Cloning Experiment Guide [M]. Huang Peitang, Wang Jiaxi, Zhu Houchu, etc. translation. 3rd edition. Beijing: Science Press , 2002" disclosed; Escherichia coli DH5α, pET-28a(+), BL21(DE3), etc. can be obtained through open commercial channels.

Tulip is one of the top ten cut flowers in the world. It has high ornamental value and is widely used. People's demand for new colors is also increasing. The beneficial effects of the present invention are: for the first time, the flavonol synthase TfFLS protein and its coding gene in the tulip petal flavonoid synthesis pathway are cloned, and TfFLS is transformed into Escherichia coli by constructing a prokaryotic expression vector, and it is found that the recombinant flavonol synthase can make Dihydrokaempferol produces kaempferol. The TfFLS provided by the present invention provides a way to regulate flower color by genetic engineering; using the tulip TfFLS protein of the present invention, through various conventional screening methods, substances that interact with tulip TfFLS, or receptors, inhibitors can be screened out or antagonists etc.

Description of drawings

Other characteristics, objects and advantages of the present invention will become more apparent by reading the detailed description of non-limiting embodiments made with reference to the following drawings:

Fig. 1 is the construction process flowchart of prokaryotic expression vector pET-28a (+)-TfFLS;

Fig. 2 is the high-performance liquid chromatography detection result of tulip TfFLS protein in vitro enzymatic activity reaction of the present invention; Wherein, a is the liquid chromatogram of dihydrokaempferol (dihydrokaempferol) standard substance; B is the in vitro enzymatic activity that uses TfFLS protein to carry out Reaction product; c is negative control reaction; d is the liquid chromatogram of kaempferol (Kaempferol) standard product.

Detailed ways

The present invention will be described in detail below in conjunction with the accompanying drawings and specific embodiments. The following examples will help those skilled in the art to further understand the present invention, but do not limit the present invention in any form. It should be noted that those skilled in the art can make several modifications and improvements without departing from the concept of the present invention. These all belong to the protection scope of the present invention. The experimental method that does not indicate specific conditions in the following examples is usually according to conventional conditions, such as molecular cloning such as Sambrook: the conditions described in the laboratory manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the manufacturer's instructions suggested conditions.

The cloning of embodiment 1, tulip TfFLS gene

1. Acquisition of plant material

Healthy, uniform-sized tulip bulbs (Tulipa fosteriana 'Shangnongzaoxia', approved by the Shanghai Crop Variety Approval Committee. Number: Shanghai Nongpin Huahua 2011 No. 004) were planted as usual and managed in the field until the flowers were fully open. Petal tissue was collected when the petals were fully colored for RNA extraction.

2. Extraction of RNA

Total RNA was extracted using the "RNA prep pure Plant Total RNA Extraction Kit" (RNA prep pure Plant Kit: Tiangen Biochemical Technology (Beijing) Co., Ltd.). The integrity of RNA was identified by formaldehyde denaturing gel electrophoresis, and then the purity and concentration of RNA were determined on a spectrophotometer (Thermo Scientific NANODROP1000 Spectrophotometer).

3. Full-length cloning of genes

According to the conserved amino acid sequences of FLS genes in other species, using the principle of homologous gene cloning, the RACE method (3'-Full RACE Core Set Vet.2.0: Treasure Bioengineering (Dalian) Co., Ltd., SMARTer ^TM RACE cDNA Amplification KIt: Clontech Laboratories, Inc.) for full-length cDNA cloning in three stages:

(1) RT-PCR to obtain the middle fragment of the gene

The extracted RNA was reverse-transcribed (Prime Script II lst Strand cDNA SynthesisKit; Treasure Bioengineering (Dalian) Co., Ltd.), using the first-strand cDNA as a template, using the amalgamative primer TfFLS-F (5'-ATYGAAGGNTAYGGMCANAARHTNC-3') (sequence shown in SEQ ID NO.3) and TfFLS-R (5'-WRAAHACNGGCCAYGACATNCK-3') (sequence shown in SEQ ID NO.4) carry out PCR, amplify the middle fragment, reclaim and connect to pMD18- On the T vector vector, use RV-M and M13-47 as universal primers, and use the method of terminator fluorescent labeling (Big-Dye, Perkin-Elmer, USA) to perform sequencing on the ABI377 sequencer (Perkin-Elmer, USA) . The sequencing results were compared with the existing database (GenBank) by BLAST (http://blast.ncbi.nlm.nih.gov/) on the NCBI website, and its nucleic acid sequence and encoded protein were compared with the known onion (Allium capa) The homology of the flavonol synthase gene is very high, so it is preliminarily considered to be a flavonol synthase gene.

(2) 3' RACE

The sequence at the 3' end was obtained by PCR amplification using the kit 3'-Full RACE Core Set Vet.2.0 (Bao Biological Engineering (Dalian) Co., Ltd.). , the upstream primer is TfFLS31 (5'-TGTGAGTGATGAGAGTCCTGCGAAA-3') (sequence shown in SEQ ID NO.5), and the downstream primer is the Outer primer provided by the kit;

The 3' end sequence of TfFLS obtained by 3' RACE was recovered, connected to the pMD18-T vector vector, and sent to Shanghai Invitrogen Company for sequencing with RV-M and M13-47 as primers. The sequencing results were compared with the existing database (GenBank) by BLAST (http://blast.ncbi.nlm.nih.gov/) on the NCBI website, and its nucleic acid sequence and encoded protein were compared with the known onion (Allium capa) The homology of the flavonol synthase gene is high.

(3) 5' RACE

The sequence at the 5' end was obtained by using the SMARTer ^TM RACE cDNA Amplification Kit, and the 5' end sequence of TfFLS was obtained by PCR using the 5' RACE ready cDNA as a template. The upstream primer is UPM provided for the kit, and the downstream primer is TFLS51 (5'-AAGTGAAACAAGTAATCCACCCACGCCT-3') (sequence shown in SEQ ID NO.6). The 5' end sequence of TfFLS obtained by 5' RACE amplification was recovered, ligated and sequenced. The sequencing results were compared with the existing database (GenBank) by BLAST (http://blast.ncbi.nlm.nih.gov/) on the NCBI website, and the nucleic acid sequence and encoded protein were compared with the known onion (Allium capa) The homology of the flavonol synthase gene is high.

The sequencing results of the sequences obtained by the above three methods were spliced, and the spliced sequences were submitted to BLAST analysis, and the results proved that the newly obtained TfFLS gene from Tulip was indeed a gene related to flavonol synthase. Combining the sequencing results with NCBI's ORF Finding (http://www.ncbi.nlm.nih.gov/gorf) prediction, the start codon and stop codon of the tulip TfFLS gene were found. Design specific primers ORF-F (5'-ATGGAGGTGGAAAGAGTGCAG-3') (sequence shown in SEQ ID NO.7) at the start codon and stop codon, ORF-R (5'-TTATTGTGGAAGCTTGTTAATCTTG-3') (sequence As shown in SEQ ID NO.8), with tulip cDNA as a template to carry out PCR, amplified to obtain the coding gene sequence (SEQ ID NO.1) of 999bp tulip TfFLS protein.

Example 2, Sequence Information and Homology Analysis of Tulip TfFLS Gene

The full-length CDS open reading frame sequence of tulip TfFLS is 999bp, and the detailed sequence is shown in SEQ ID NO.1; the amino acid sequence of tulip TfFLS is deduced according to the CDS open reading frame sequence, with a total of 332 amino acid residues and a molecular weight of 37672 Dalton, the isoelectric point (pI) is 5.39, the detailed sequence is shown in the sequence shown in SEQ ID NO.2.

The CDS open reading frame sequence of tulip TfFLS and the amino acid sequence of its encoded protein were nucleated in Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDS translations+PDB+SwissProt+Superdate+PIR databases using BLAST program Nucleotide and protein homology searches. Table 2 is the homologous comparison (GAP) result of the nucleotide sequence of tulip TfFLS gene of the present invention and onion (Allium cepa) FLS gene mRNA; As can be seen from Table 2, tulip TfFLS gene and onion (Allium cepa) FLS gene (GenBank login No. AY221247.1) has 71% identity at the nucleotide level; Table 3 is the homology comparison (FASTA) result of the amino acid sequence of the tulip TfFLS gene of the present invention and the onion (Allium cepa) FLS gene mRNA, wherein, The same amino acid is marked with an amino acid single character between the two sequences; as can be seen from Table 3, at the amino acid level, it also has 85% identity with the onion (Allium cepa) FLS gene (GenBank accession number AA063023.1) 74% similarity, as can be seen from Table 2 and Table 3, the tulip TfFLS gene and the onion FLS gene have high homology in terms of both nucleic acid and protein levels.

Table 2

table 3

Example 3, the expression difference of TfFLS gene of tulip in different developmental stages of flowers and in different tissues of tulip

1. Acquisition of materials

During the four different developmental stages of tulip flowers (buds, petals are not colored; buds, petals are beginning to color; flowers are partially open, petals are not fully colored; flowers are fully open, petals are fully colored), the bulbs, aboveground stems, leaves and For the petals (mixed samples of petals at each coloring stage), the samples were wrapped in aluminum platinum paper and immediately put into liquid nitrogen, and then transferred to a -80°C ultra-low temperature refrigerator for storage until use.

2. Extraction of RNA

RNA prep pure plant total RNA extraction kit (RNA prep pure Plant Kit: Tiangen Biochemical Technology (Beijing) Co., Ltd.) was used to extract the petals of tulip flowers at different developmental stages and the RNA in different tissues.

3. Determination of the integrity, purity and concentration of RNA

Use ordinary agarose gel electrophoresis (gel concentration 1.2%; 0.5×TBE electrophoresis buffer; 150v, 15min) to detect integrity; the maximum rRNA brightness in the electrophoresis band should be 1.5-2.0 times the brightness of the second rRNA, otherwise it means Degradation of rRNA samples. For RNA with better purity, A ₂₆₀ /A ₂₈₀ and A ₂₆₀ /A ₂₃₀ are about 2.0; use a spectrophotometer to measure the OD value and calculate the RNA content.

4. Acquisition of cDNA

Using 500ng of total RNA as a template, reverse transcription was performed according to the instructions of the TaKaRa PrimeScript ^TM RT reagent Kit Perfect Real Time kit from Treasure Biotech to obtain cDNA for future use.

5. Real-time fluorescent quantitative PCR analysis of the expression of TfFLS gene in various organs and tissues

According to the sequence of tulip TFLS gene that has been obtained, utilize primer design software primer premier5.0 to design the specific primer that is used for the quantitative analysis of tulip TfFLS gene in Real-time PCR: TfFLS-qF (5'-AGGAGGAGATT6C66CT6T6-3') (sequence As shown in SEQ ID NO.9) and TfFLS-qR (5'-TAGTCGGTAGGGTTCTT666-3') (sequence shown in SEQ ID NO.10), the internal reference gene is Actin (GenBank accession number AB456684), and its primer is Actin- F (5'-AGTCAGTCATACAGTGCCAATC-3') (sequence shown in SEQ ID NO.11), Actin-R (5'-TCATAAGAGAGTCGGTCAAATCC-3') (sequence shown in SEQ ID NO.12).

6. Make the standard curve of target gene and internal reference gene

Use EASY Dilution (provided by the kit) to serially dilute the standard cDNA solution, then use the diluted cDNA solution as a template to perform Real-time PCR amplification with specific primers for the target gene and the internal reference gene, and draw after the reaction Melting curve and standard curve; analyze the melting curve to determine whether the melting curve of the target gene and the internal reference gene has a single peak, so as to determine whether a single PCR amplification product can be obtained by using the primer; determine the appropriate dilution factor of the template cDNA through the standard curve .

7. Real-time fluorescence quantitative analysis of the target gene in the sample to be tested

Using the first strand of the synthesized cDNA as a template, the target gene and the internal reference gene were amplified with specific primers for fluorescence quantitative analysis. The Real-time PCR reaction was carried out on the BIO-RAD Chromo4 real-time quantitative instrument, and the reaction system was 20 μL , the reaction adopts a three-step method, denaturation at 94°C for 20s, followed by 41 cycles: 94°C for 15s; 56°C for 25s; .

The 2- ^△△Ct method was used for relative quantitative analysis. The results showed that the expression level of TfFLS gene decreased gradually with the development of flowers, and the expression level in the last developmental stage was 0.72 times of that in the first stage. Our previous studies showed that the total content of flavonols in petals gradually decreased during the flower opening process. Therefore, the expression change of TfFLS was consistent with the change trend of flavonols content, indicating that TfFLS was positively related to the synthesis of flavonols. TfFLS gene was expressed in bulbs, aerial stems, leaves, and petals, among which, the expression level of TfFLS gene was higher in petals and aerial stems, and the expression level was lowest in bulbs. The expression levels in petals are 1.4, 2.6, and 5.5 times higher than those in aboveground stems, leaves, and bulbs, which is consistent with the differences in flavonol content in these organs. It is further confirmed that TfFLS gene is positively correlated with flavonol synthesis , also showed that the expression of TfFLS has a certain spatial difference.

Embodiment 4, tulip TfFLS enzyme functional verification

1. Construction of pET-28a(+)-TfFLS prokaryotic expression vector

The construction process of pET-28a(+)-TfFLS prokaryotic expression vector is shown in Figure 1.

Sac I and Xho I enzyme cutting sites were respectively added at both ends of the upstream and downstream primers for amplifying the TfFLS gene ORF fragment. The upstream primer sequence is 5'-C GAGCTC ATGGAGGTGGAAAGAGTGCA6-3' (sequence shown in SEQ ID NO.13), and the downstream primer is 5'CC CTCGAG TTATTGTGGAAGCTTGTTAATCTT6-3' (sequence shown in SEQ ID NO.14). The underlines represent the restriction recognition sequences of Sac I and Xho I, respectively.

The ORF fragments of TfFLS cDNA were amplified by PCR, and the target bands were cut under ultraviolet light after electrophoresis, recovered with Sanprep column DNA gel recovery kit (Shanghai Sangong Bioengineering Co., Ltd.), connected to pMD18-Tvector, and constructed pMD18-TfFLS cloning vector, see the instructions for the connection system. Freeze-thaw method was used to transform Escherichia coli DH5α competent, and cultivate overnight at 37°C on LB solid plate medium containing 100 mg·l ^-1 ampicillin. The formula of LB medium is: tryptone 10g·l, yeast extract 5g·l ^-1 , sodium chloride 10g·l ^-1 . Adjust the pH to 7.0 and sterilize. The formula of LB solid medium is to add 15g·l ^-1 agar powder to LB liquid medium, and then sterilize. Pick a single colony for PCR identification, and send positive colonies for sequencing to confirm the correctness of the sequencing. The DH5α colony containing the pMD18-TfFLS vector with correct sequencing was added to 2ml LB liquid medium containing 100mg·1 ^-1 ampicillin and cultivated overnight until the _OD600 value was about 1.0. Use a plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd.) to extract the pMD18-TfFLS vector, and refer to the kit instructions for specific operations.

use Restriction endonucleases Sac I and Xho I were used to simultaneously digest the cloning vector pMD18-TfFLS and the prokaryotic expression vector pET-28a(+) (Novagen, USA) at 37°C for 15 minutes. For the enzyme digestion system, refer to the enzyme digestion instructions. Gel electrophoresis was performed on the digested product and recovered.

The digested TfFLS fragment and pET-28a(+) were ligated using a DNA Ligation Kit (TaKaRa, China). For the ligation method, see the kit instructions. The ligation product was transformed into Escherichia coli BL21 (DE3) competent. Cultivate overnight at 37°C on LB solid plate medium containing 50 mg·l ^-1 ammonia Bian. Pick a single colony and send it to sequencing to confirm that the TfFLS fragment is successfully connected to pET-28a(+) after positive PCR identification.

2. Fusion protein induction of TfFLS

Select a single clone of BL21(DE3) strain in good growth state, transfer it to 10ml LB liquid medium containing 50mg·l ^-1 ammonia Bian, and culture overnight at 37°C and 200rpm. Transfer to 300ml LB liquid culture containing 50mg·l ^-1 ammonia Bian at a ratio of 1:50 at 37°C and 200rpm and cultivate until OD ₆₀₀ is about 0.6. The culture was transferred to a shaker at 20° C. at 200 rpm, and shaken for 1 hour. Add 1ml of 1M IPTG (isopropyl-β-D-thiogalactopyranoside) (final concentration: 1mM), and continue to culture for 4-6h to induce fusion protein expression.

The overnight culture was centrifuged at 12,000 rpm for 10 min at 4°C to collect the cells, and the supernatant was discarded. Use PBS (140mM NaCl, 2.7mM KCl, 10mM Na ₂ HPO ₄ , 1.8mM KH ₂ PO ₄ ) at a ratio of 5:1 to dissolve and centrifuge to collect the bacteria, and ultrasonic (200w-300w) to disrupt the bacteria, ultrasound/interval=10sec /sec, 6 times. Use buffer B (8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl, the rest is distilled water, pH8.0) to resuspend the bacterial pellet (20-200ml cell culture). The amount of buffer B was 5ml·g ^-1 wet weight, and stirred at room temperature until the solution became translucent. Centrifuge at 10,000 rpm for 30 min at 4°C, discard the precipitate, and collect the supernatant for column purification.

Suspend the 50% Ni-NTA solution and pack it into a column to avoid air bubbles. The dosage of Ni-NTA is 5-10 mg protein per ml resin. Wait for the resin to settle naturally, wash the column with 5 times the column volume of ddH2O, and then add 5-10 times the column volume of 1×Ni-NTA buffer B to equilibrate the column. Put the sample on the column, wash the column with 5-10 times the volume of 1×Ni-NTA buffer B at a flow rate of 1ml.min ^-1 , and collect the effluent. Wash the column with 5-10 column volumes of 1×Ni-NTA buffer C (8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl, the rest is distilled water, pH6.3), and collect the effluent. Wash the column with 5 times the volume of 1×Ni-NTA buffer E (8M urea, 0.1M sodium phosphate buffer, 0.01M Tris-Cl, the rest is distilled water, pH 4.5) to elute the target protein.

The collected TfFLS fusion protein was incubated with the substrate. The substrate for incubation is Dihydrokaempferol. The volume of the incubation system is 360 μl, containing 4.5-22 μg TfFLS fusion protein extract, 100 μM substrate, 83 μM 2-oxoglutarate (2-oxoglutarate), 42 μM ammonium iron (II), 2.5 mM sodium ascorbate (Sodium ascorbate), 2 mg .ml ^-1 catalase (bovine) (bovine catalase). Incubation conditions: 37°C, pH 5.0, slight shaking for 5-10 minutes. The reaction was terminated with 15 μl of saturated aqueous EDTA. After the reaction was completed, it was extracted twice with 75 μl of ethyl acetate, and then analyzed by UPLC (Waters, USA). Two kinds of mobile phases were used for UPLC analysis, mobile phase A was 20% methanol solution, and B was 100% methanol solution for linear elution. Standard Dihydrokaempferol The standard product was purchased from SIGMA (USA), and the standard product kaempferol was purchased from the National Standard Materials Network. The product was detected at 350nm.

After the purified TfFLS protein was refolded, it was used in the in vitro enzyme activity experiment, and the protein produced by the recombinant plasmid strain induced by no IPTG was used as a negative control. The high performance liquid chromatography detection results shown in Figure 2 show that the TfFLS fusion protein makes dihydrokaempferol generate kaempferol, while the negative control reaction does not generate the corresponding product. This result indicated that TfFLS can generate flavonol from dihydroflavonol and has the activity of flavonol synthase.

Specific embodiments of the present invention have been described above. It should be understood that the present invention is not limited to the specific embodiments described above, and those skilled in the art may make various changes or modifications within the scope of the claims, which do not affect the essence of the present invention.

Claims (6)

1. the protein of one kind following (a):

A protein that () is made up of the such as aminoacid sequence shown in SEQ ID NO.2.

2. nucleic acid sequences to proteins described in a coding claim 1.

3. nucleotide sequence as claimed in claim 2, it is characterized in that, described nucleotide sequence is specially:

A () base sequence is as shown in SEQ ID NO.1 1st ~ 999.

4. the purposes of a nucleotide sequence as claimed in claim 2 in preparation restructuring flavonol synthetic enzyme.

5. purposes as claimed in claim 4, is characterized in that, described preparation comprises the steps: to build the prokaryotic expression carrier containing described nucleotide sequence, is transformed in intestinal bacteria, inducing culture by described prokaryotic expression carrier, obtains restructuring flavonol synthetic enzyme.

6. the preparation method of flavonol synthetic enzyme that recombinates, it is characterized in that, described preparation method comprises: build the prokaryotic expression carrier containing nucleotide sequence as claimed in claim 2, be transformed in intestinal bacteria by described prokaryotic expression carrier, inducing culture, obtains described restructuring flavonol synthetic enzyme.