CN103695406A - Tulip phenylalanine ammonialyase TfPAL protein and coding gene thereof - Google Patents

Abstract

The invention discloses a tulip phenylalanine ammonia-lyase TfPAL protein and its coding gene, the protein is composed of the amino acid sequence shown in SEQ ID NO.2 or the amino acid shown in SEQ ID NO.2 A protein whose sequence has undergone substitution, deletion or addition of one or several amino acids and has tulip phenylalanine ammonia lyase activity. The present invention also provides a nucleic acid sequence as shown in SEQ ID NO.1 encoding the above protein. The invention provides a theoretical basis for regulating the spatiotemporal expression characteristics of the tulip TfPAL gene by genetic engineering technology in the future, thereby regulating the flower color, and has practical application value.

Description

Tulip phenylalanine ammonia lyase TfPAL 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 anthocyanins and its encoding gene, in particular to a tulip phenylalanine ammonia-lyase TfPAL protein and its encoding gene.

Background technique

Phenylalanine ammonia-lyase (PAL) is a key enzyme in the metabolic pathway of phenylpropanoids in plants (Okada et al., 2008), and is the link connecting primary metabolism and secondary metabolism. Alcohol, anthocyanin, tannin, isoflavone, flavanone, lignin, coumarin, sinapinate and salicylic acid play an important role in the synthesis of secondary metabolites, which are involved in plant growth and development, resistance Insect resistance, signal transduction, UV protection, and pollen fertility (Dixon et al., 2002), so PAL has a very close relationship with plants, and it has become a research hotspot in recent years.

In recent years, researchers have studied the PAL activity of various plants including impatiens, amaranth, stevia, sweet potato, corn, cotton and peach. The results showed that: (1) The activity of PAL is related to Anthocyanin content was significantly positively correlated. (2) Strong light, hormones, low temperature, mechanical damage and infection by pests and diseases can all induce the increase of PAL activity. (3) PAL activity was negatively correlated with nitrogen content in tissues. Specific to the relationship between PAL activity and anthocyanin synthesis, Arakawa believes that PAL whose activity is regulated by light is the key enzyme for anthocyanin synthesis (Arakawa O, 1988). The increase of PAL activity in apple peel was accompanied by the increase of anthocyanin content, but it may not be the only key enzyme controlling anthocyanin (Zhou Aiqin et al., 1997). Another study confirmed that ethylene can increase anthocyanin synthesis by inducing PAL activity (Cheng G and Breen P, 1991)

PAL genes exist as multigene families in plants, with at least 2 gene members in raspberry, 4 members in Arabidopsis, at least 26 members in tomato, and more in potato (Solanum tuberosum), estimated There are 40-50 (Chang et al. 2008; Joos and Hahlbrock, 1992; Kumar and Ellis, 2001). PAL exists in all green plants, and has been isolated and purified from various plants such as rice, wheat, and corn, and has also been found in fungal and algal cells (Heilmann and Merfort, 1998). But the cloning and expression pattern of the PAL gene in tulip (Tulipa fosteriana) is still unclear. At present, there is no literature report related to tulip PAL protein and its coding gene sequence.

Contents of the invention

The object of the present invention is to provide a tulip phenylalanine ammonia lyase TfPAL protein and its coding gene. The invention provides a tulip TfPAL protein sequence, and a nucleic acid sequence encoding the protein; discloses the expression patterns of the tulip TfPAL protein and its nucleotide sequence in different organs and different developmental stages of tulip; Regulating the spatio-temporal expression characteristics of the tulip TfPAL gene, thereby providing a theoretical basis for the main regulation of flower color, has a certain application value.

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 phenylalanine ammonia lyase activity, said protein is a protein consisting of the amino acid sequence shown in SEQ ID NO.2; or represented by SEQ ID NO.2 A protein derived from (a) that has substituted, deleted or added one or several amino acids in the amino acid sequence shown and has tulip phenylalanine ammonia lyase 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 2136th positions of SEQ ID NO.1;

or (b) a sequence having at least 70% homology with the nucleic acid shown in positions 1 to 2136 of SEQ ID NO.1;

Or (c) a sequence capable of hybridizing to the nucleic acid shown in positions 1 to 2136 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 2136 of SEQ ID NO.1, and 5' and/or 3' A sequence formed by adding 60 or less nucleotides at the end.

The isolated DNA molecule provided by the present invention comprises: a DNA molecule having a nucleotide sequence shown in SEQ ID NO.1; or a nucleotide sequence encoding a polypeptide having tulip TfPAL protein activity, and is identical to SEQ ID NO. .1 The sequence shown in 1 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 phenylalanine ammonia-lyase TfPAL protein coding gene" refers to the nucleotide sequence encoding a polypeptide having tulip TfPAL protein activity, such as the nucleotide sequence shown in SEQ ID NO.1 and its abbreviation and 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 with at least 70% homology to the nucleotide sequence from nucleotides 1 to 2136 in the sequence shown in SEQ ID NO.1.

The term also includes variants of the sequence shown in SEQ ID NO. 1 that can encode a protein with the same function as native tulip TfPAL. 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 phenylalanine ammonia-lyase TfPAL protein" refers to a polypeptide having the sequence shown in SEQ ID NO.2 having tulip TfPAL protein activity. The term also includes variants of the sequence shown in SEQ ID NO.2 that have the same function as the natural tulip TfPAL. 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 TfPAL protein.

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

In the present invention, "Tulip TfPAL conservative variant polypeptide" means that compared with the amino acid sequence shown in SEQ ID NO.2, at most 10 amino acids are replaced by amino acids with similar or similar properties to form a polypeptide. 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; His; 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; Phe Leu Leu(L) Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Leu; Val; Ile; Ala; Tyr Leu Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Leu

The invention also includes analogs of tulip TfPAL protein or polypeptide. The difference between these analogues and the TfPAL-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 TfPAL gene product can be analyzed by real-time fluorescence quantitative PCR method, that is, the presence or quantity of the mRNA transcript of TfPAL gene in cells can be analyzed.

In addition, according to the TfPAL nucleotide sequence and amino acid sequence of the present invention, the TfPAL-related homologous genes or proteins of Tulip can be screened on the basis of nucleic acid homology or homology of expressed proteins.

In order to obtain the array of genes related to tulip TfPAL, the cDNA library of tulip can be screened with DNA probes, which are obtained by radioactively labeling all or part of tulip TfPAL 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 TfPAL.

The full-length sequence of TfPAL-related nucleotides 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.

Tulip is one of the top ten cut flowers in the world. It has high ornamental value and is widely used. Enriching flower colors is one of the main purposes of tulip breeding. The beneficial effect of the present invention is that: for the first time, the key enzyme phenylalanine ammonia lyase TfPAL protein and its coding gene for the synthesis of anthocyanin in tulip petals are disclosed, so as to use genetic engineering technology to regulate the temporal and spatial expression characteristics of tulip TfPAL gene in the future, thereby regulating flower color It provides a theoretical basis and has certain application value.

Detailed ways

The present invention will be described in detail below in conjunction with 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, generally 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 suggestion conditions of.

Embodiment 1, the cloning of tulip TfPAL 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 Recognition Flowers 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 PlantKit: 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 NANODROP 1000 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 Ver.2.0: Treasure Bioengineering (Dalian) Co., Ltd., SMARTer ^TM RACEcDNA 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 1st Strand cDNA Synthesis Kit: Treasure Bioengineering (Dalian) Co., Ltd.), using the first-strand cDNA as a template, using primer TfPAL-F (5'-AACACCAYCCWGGYCARATT-3') (sequence as shown in SEQ ID NO.3) and TfPAL-R (5'-GAGCAGTAWGAYGCCATK-3') (sequence as shown in SEQ ID NO.4) for PCR to amplify the middle fragment, recovery and connection 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 known to be consistent with the known lily (Lilium hybrida) benzene The homology of alanine ammonia lyase is very high, so it is preliminarily considered to be a phenylalanine ammonia lyase.

(2) 3' RACE

The sequence at the 3' end was obtained by nested PCR amplification using the kit 3'-Full RACE Core Set Ver.2.0 (Bao Bioengineering (Dalian) Co., Ltd.). The upstream primer of the first round of PCR is TfPAL31 (5'-AAGGTGCTGACAACAGGTGCTAACG-3') (sequence shown in SEQ ID NO.5), and the downstream primer is the Outer primer provided by the kit, which is diluted 50 times with the first round of PCR products, and taken 1 μl was used as a template for the second round of PCR. The upstream primer of the second round of PCR was TfPAL32 (5'-CGGGGAGAAGGAAAAGGATGTGAGT-3') (sequence shown in SEQ ID NO.6), and the downstream primer was the inner primer provided by the kit.

The 3' end sequence of TfPAL obtained by 3'RACE was recovered, connected to the pMD18-T vector vector, and RV-M and M13-47 were used as primers, and sent to Shanghai Invitrogen Company for sequencing. 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 known to be similar to those of the known lily (Lilium hybrida) The homology of phenylalanine ammonia lyase 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' RACEready cDNA was used as a template for nested PCR amplification. The upstream primer of the first round of PCR is the UPM provided by the kit, and the downstream primer is TfPAL51 (5'-AGCCCTGACCCAACAGCAGTGCCATTTA-3') (sequence shown in SEQID NO.7), and the second round of PCR is carried out using the first round of PCR product as a template . The upstream primer of the second round of PCR is the NUP provided by the kit, and the downstream primer is TfPAL52 (5'-TTGGTGTTGAGGAGACTGGTGATGGCTT-3') (sequence shown in SEQ ID NO.8) to amplify the 5' RACE to obtain the 5' end of TfPAL Sequences were 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 its nucleic acid sequence and encoded protein were known to be similar to those of the known lily (Lilium hybrida) The homology of phenylalanine ammonia lyase 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. The results proved that the TfPAL gene newly obtained from tulip was indeed a gene related to phenylalanine ammonia-lyase. 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 TfPAL gene were found. Specific primers ORF-F (5'-ATGGAGCAGAACGGCAATGGAGTCG-3') (sequence shown in SEQ ID NO.9), ORF-R (5'-TTAGCTGATTGGAAGAGGAGCACC-3') (sequence As shown in SEQ ID NO.10), with tulip cDNA as a template to carry out PCR, amplified to obtain the full-length coding gene sequence (SEQ ID NO.1) of 2136bp tulip TfPAL protein.

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

The full-length CDS open reading frame sequence of tulip TfPAL is 2136bp, and the detailed sequence is shown in SEQ ID NO.1; the amino acid sequence of tulip TfPAL is deduced according to the CDS open reading frame sequence, with a total of 711 amino acid residues and a molecular weight of 77098.9 Dalton, the isoelectric point (pI) is 6.20, the detailed sequence is shown in the sequence shown in SEQ ID NO.2.

The CDS open reading frame sequence of tulip TfPAL and the amino acid sequence of its encoded protein were analyzed by BLAST program in the databases of Non-redundant GenBank+EMBL+DDBJ+PDB and Non-redundant GenBank CDStranslations+PDB+SwissProt+Superdate+PIR Acid and protein homology searches. Table 2 is the homologous comparison (GAP) result of the nucleotide sequence of tulip TfPAL gene of the present invention and the nucleotide sequence of lily (Lilium hybrida) PAL gene mRNA; As can be seen from table 2, it and lily (Lilium hybrida) phenylalanine ammonia solution Enzyme (GenBank accession number AB699156.1) has 82% similarity at nucleotide level; Table 3 is the homologous comparison (FASTA) of the amino acid sequence of tulip TfPAL gene of the present invention and lily (Lilium hybrida) PAL gene mRNA As a result, 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 is also related to lily (Lilium hybrida) phenylalanine ammonia lyase (GenBank accession number BAM28966 .1) also have 94% identity and 89% similarity; As can be seen from Table 2 and Table 3, tulip TfPAL gene and lily (Lilium hybrida) phenylalanine ammonia-lyase all exist comparatively no matter from nucleic acid or protein level high homology.

Table 2

table 3

Embodiment 3, Tulip TFPAL gene expression difference in different developmental stages of flowers and in different tissues of tulip opposite sex

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 good 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 500 ng of total RNA as a template, reverse transcription was performed according to the instructions of the TaKaRa PrimeScript ^TM RT reagent KitPerfect Real Time kit from Baobio Company to obtain cDNA for future use.

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

According to the sequence of tulip TFLS gene that has been obtained, utilize primer design software Primer premier 5.0 to design the specific primer that is used for the quantitative analysis of tulip TfPAL gene in Real-time PCR: TfPAL-qF (5'-AGTACGTATTTGCCTACGCCGA-3') (sequence such as Shown in SEQ ID NO.11) and TfPAL-qR (5'-CTCACATCCTTTTCCTTCTCCC-3') (sequence shown in SEQ ID NO.12), the internal reference gene is Actin (GenBank accession number AB711684), and its primer is Actin-F (5'-AGTCAGTCATACAGTGCCAATC-3') (sequence shown in SEQ ID NO.13), Actin-R (5'-TCATAAGAGAGTCGGTCAAATCC-3') (sequence shown in SEQ ID NO.14).

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 respectively amplified with specific primers for fluorescence quantitative analysis. The Real-time PCR reaction was carried out on the BIO-RAD Chromo 4 real-time quantitative instrument. The reaction system was 20μL, the reaction adopts a three-step method, denaturation at 94°C for 20s, followed by 41 cycles: 94°C for 15s; 60°C for 25s; 72°C for 20s; produce.

Using 2 ^-ΔΔCt method for relative quantitative analysis, the results showed that the expression level of TfPAL gene increased with the development of flowers. The expression level in the last developmental stage was 11.2 times that of the first stage, showing the same trend as the accumulation of anthocyanins, indicating that TfPAL was positively correlated with the synthesis of anthocyanins. TfPAL gene was expressed in bulbs, aerial stems, leaves, and petals, with the highest expression level in flowers, followed by aerial stems, and the lowest expression level in bulbs. The expression levels in petals were 1.6, 2.3, and 2.6 times higher than those in aboveground stems, leaves, and bulbs, which indicated that the expression of TfPAL gene had certain tissue specificity.

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. A protein that is (a) or (b): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO.2; (b) The amino acid sequence shown in SEQ ID NO.2 is substituted, deleted or added with one or several amino acids and has tulip phenylalanine ammonia-lyase activity derived from (a). 2. The protein according to claim 1, characterized in that, the protein is the amino acid sequence shown in SEQ ID NO.2 after 1 to 50 amino acid deletions, insertions and/or substitutions, or at the C-terminal and/or A sequence obtained by adding within 1 to 20 amino acids to the N-terminus. 3. The protein according to claim 2, characterized in that, 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. 4. A nucleic acid sequence encoding the protein of claim 1. 5. The nucleic acid sequence according to claim 4, wherein the nucleic acid sequence is specifically: (a) The base sequence is as shown in the 1st to 2136th positions of SEQ ID NO.1; or (b) a sequence having at least 70% homology with the nucleic acid shown in positions 1 to 2136 of SEQ ID NO.1; Or (c) a sequence capable of hybridizing to the nucleic acid shown in positions 1 to 2136 of SEQ ID NO.1. 6. The nucleic acid sequence according to claim 4, wherein the nucleic acid sequence is specifically the deletion, insertion and deletion of 1 to 90 nucleotides in the nucleic acid sequence shown in the 1st to 2136th positions of SEQ ID NO.1. /or substitution, or addition of a sequence within 60 nucleotides at the 5' and/or 3' end.