CN103333233B - Agapanthus praecox auxin receptor protein TIR1 and coding gene and probe thereof - Google Patents

Abstract

The present invention relates to an agapanthus auxin receptor protein TIR1 and its coding gene and probe. The protein is the following protein (a) or (b): (a) composed of amino acids as shown in SEQ ID NO.4 (b) A protein derived from (a) in which the amino acid sequence shown in SEQ ID NO.4 has been substituted, deleted or added with one or several amino acids and has the activity of Agapanthus azalea auxin receptor protein. The present invention also provides a nucleic acid sequence encoding the above-mentioned protein, and a probe for detecting the above-mentioned nucleic acid sequence; the present invention uses genetic engineering technology to regulate the auxin signal transduction pathway of Agapanthus so as to control its growth and development and organ morphology. It provides a theoretical basis for molecular breeding and has great application value.

Description

Agapanthus abacifolia Auxin Receptor Protein TIR1 and Its Encoding Gene and Probe

technical field

The present invention relates to the key protein TIR1 and its coding gene and probe in the Agapanthus abacifolia auxin signal transduction pathway, and specifically relates to an Agapanthus agapanthus auxin receptor protein TIR1 and its coding gene and probe.

Background technique

Endogenous hormones play an important role in the growth and development of ornamental plants and the regulation of ornamental traits. Auxin is the only endogenous hormone with polar transport characteristics in plants. The content and distribution of auxin are related to the growth and development speed of plants and the polar structure and spatial form of various organs and tissues. The signal transduction pathway of auxin is relatively clear at present, in which auxin must bind to its receptor protein to regulate the downstream gene network and cause physiological responses, and TIR1 protein is the most important auxin receptor protein.

Somatic embryogenesis (somatic embryo) is the most effective technical system for plant molecular breeding and in vitro rapid propagation. Studies have shown that the induction of plant somatic embryos mainly depends on the regulation of exogenous auxin, and the exogenous auxin substance picloram has no effect on The somatic embryo induction of monocotyledonous bulbous flowers has special effects. Agapanthus is a tropical perennial flower with large flower volume, long flowering period, high ornamental value, and underground tuber tissue. Our previous establishment of Agapanthus somatic embryo system showed that auxin signal plays a decisive role in somatic embryo induction, somatic embryo morphology, somatic embryo number and somatic embryo seedling formation of Agapanthus lily. In addition, the use of exogenous regulatory substances to interrupt the polar transport of auxin has obvious regulatory effects on the flowering period, plant dwarfing, and inflorescence morphology of Agapanthus amanegsia. Therefore, auxin signaling plays a vital role in the industrial production and breeding improvement of flowers.

The coding gene of TIR1 has been cloned from various plants, including: Arabidopsis, rice, corn, grape, castor, etc. But for ornamental plants, especially flower bulbs, the cloning, expression pattern and protein sequence of TIR1 are still unclear. At present, there is no literature report related to Agapanthus TIR1 protein and its coding gene sequence.

Contents of the invention

The purpose of the present invention is to fill in the gaps in the cloning and expression pattern analysis of Agapanthus TIR1 gene and Agapanthus TIR1 protein, and provide a kind of Agapanthus TIR1 protein, and the present invention also provides a nucleic acid sequence encoding the above protein and detection of the Nucleic acid sequence probe; the present invention discloses the expression pattern of Agapanthus TIR1 protein and its nucleic acid sequence in the development of different organs and somatic embryos of Agapanthus in order to regulate the temporal and spatial characteristics of TIR1 gene expression by using genetic engineering technology in the future, thereby It provides a theoretical basis for somatic embryogenesis and plant type regulation, and has great application value.

On the one hand, the present invention provides Agapanthus growth hormone receptor protein, and described protein is the protein that is made up of aminoacid sequence as shown in SEQ ID NO.4; Or is substituted by the aminoacid sequence shown in SEQ ID NO.4 , a protein that lacks or adds one or several amino acids and has the activity of Agapanthus growth hormone receptor protein. The expression level of this protein was quite different in different somatic embryonic developmental stages and 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.4, 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.4 by amino acids with similar or similar properties.

In another aspect, the present invention provides a nucleic acid sequence encoding the above protein.

Preferably, the nucleic acid sequence is specifically: (a) the base sequence is as shown in the 1st to 1767th positions of SEQ ID NO.3; A sequence with at least 70% homology; or (c) a sequence that can hybridize with the nucleic acid shown in positions 1 to 1767 of SEQ ID NO.3.

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 1767 of SEQ ID NO.3, and 5' and/or 3' A sequence formed by adding 60 or less nucleotides at the end.

In addition, the present invention also provides a probe for detecting the above-mentioned nucleic acid sequence, the probe is a nucleic acid molecule having 8 to 100 consecutive nucleotides of the above-mentioned nucleic acid sequence, and the probe can be used to detect whether there is coded Apodida in a sample. Lotus TIR1-related nucleic acid molecules.

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 "Agapanthus TIR1 protein coding sequence" refers to a nucleotide sequence encoding a polypeptide having activity of Agapanthus TIR1 protein, such as the 1st to 1767th nucleotide sequence shown in SEQ ID NO.3 and its degenerate sequence. The degenerate sequence refers to the sequence generated after one or more codons are replaced by degenerate codons encoding the same amino acid in the 1st to 1767th nucleotides shown in SEQ ID NO.3. Due to the degeneracy of codons, a degenerate sequence with a homology as low as about 70% to the 1st to 1767th nucleotide sequence shown in SEQ ID NO.3 can also encode the sequence shown in SEQ ID NO.4 the sequence of. The term also includes a nucleotide sequence with at least 70% homology to the nucleotide sequence shown in SEQ ID NO.3.

The term also includes variants of the sequence shown in SEQ ID NO.3 that can encode the same function of the native Agapanthus TIR1 protein. These variations include (but are not limited to): deletions, insertions and/or substitutions of usually 1 to 90 nucleotides, and additions of less than 60 nucleotides at the 5' and/or 3' ends.

In the present invention, the term "Agapanthus TIR1 protein" refers to a polypeptide having the sequence shown in SEQ ID NO.4 having the activity of Agapanthus TIR1 protein. The term also includes variants of the sequence of SEQ ID NO.4 that have the same function as the natural Agapanthus TIR1 protein. 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 Agapanthus TIR1 protein.

Variations of the Agapanthus TIR1 protein of the present invention include: homologous sequences, conservative variants, allelic variants, natural mutants, induced mutants, hybridization with DNA related to Agapanthus TIR1 under high or low stringent conditions The protein encoded by the DNA, and the polypeptide or protein obtained by using the antiserum of Agapanthus TIR1 protein.

In the present invention, "Agapanthus TIR1 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.4. 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 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;Ala Leu

The invention also includes analogues of Agapanthus TIR1 protein or polypeptide. The difference between these analogs and Agapanthus TIR1-related polypeptides 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. This 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 Agapanthus TIR1 gene product can be analyzed by real-time fluorescence quantitative PCR method, that is, the presence and quantity of the mRNA transcript of Agapanthus TIR1 gene in cells can be analyzed.

The method for detecting whether there is Agapanthus TIR1-related nucleotide sequence in a sample of the present invention comprises using the above-mentioned probe to hybridize with the sample, and then detecting whether the probe is combined. The sample is a PCR-amplified product, wherein the PCR amplification primers correspond to the nucleotide coding sequence related to Agapanthus TIR1, and can be located on both sides or in the middle of the coding sequence. The primer length is generally 15-50 nucleotides.

In addition, according to the nucleotide sequence and amino acid sequence of Agapanthus TIR1 of the present invention, it is possible to screen Agapanthus TIR1-related homologous genes or homologous proteins on the basis of nucleic acid homology or homology of expressed proteins.

In order to obtain the array of genes related to Agapanthus TIR1, DNA probes can be used to screen the Agapanthus cDNA library. These probes are radioactively labeled with all or part of Agapanthus TIR1 with ³² P under low stringency conditions. Got it. A suitable cDNA library for screening is that from Agapanthus chinensis. Methods for constructing cDNA libraries from cells or tissues of interest are well known in the art of molecular biology. Additionally, 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 Agapanthus TIR1.

The Agapanthus TIR1-related nucleotide full-length sequence or its fragments of the present invention 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 the recombination method. 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.

By using the Agapanthus TIR1 protein of the present invention, substances, or inhibitors and antagonists, etc. that interact with the Agapanthus TIR1 protein can be screened out through various conventional screening methods.

Agapanthus has high ornamental value and is widely used. Its tall and straight scape is an excellent fresh-cut flower variety, and it is also the most expressive love flower besides roses. Its market demand is also increasing. The present invention clones for the first time the coding sequence of the key receptor protein TIR1 in the auxin signal transduction pathway in Agapanthus plant, and analyzes the expression pattern of TIR1 gene by using the method of fluorescence real-time quantitative PCR, so as to control the top growth advantage and plant type of plants in the future. It has important application prospects in regulating and reducing the ratio of somatic abnormal embryos, and also provides a theoretical basis for the selection of new varieties, which has great application value.

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 homologous comparison (GAP) result of the nucleotide sequence of Agapanthus TIR1 gene of the present invention and the nucleotide sequence of castor plant TIR1 gene mRNA;

Fig. 2 is the homologous comparison (FASTA) result of the amino acid sequences of Agapanthus TIR1 protein and grape TIR1 protein of the present invention, wherein the same amino acid is marked with a single amino acid character between the two sequences;

Figure 3 is a diagram of the expression level change of Agapanthus TIR1 gene under the regulation of exogenous IAA and NPA;

Fig. 4 is a graph showing changes in gene expression of Agapanthus TIR1 gene during somatic embryo development.

Detailed ways

Below in conjunction with specific embodiment, further illustrate the present invention. These examples are only for illustrating the present invention and are not intended to limit the 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 suggestion conditions of.

Embodiment 1 , the cloning of Agapanthus TIR1 gene

1. Acquisition of plant material

The leaf tissue of Agapanthus adult seedlings was taken for RNA extraction; Agapanthus was introduced into my country from South Africa in 2000, and the introduction experiment was carried out in Shanghai. In his master's thesis "Research on the Dwarfing of Agapanthus Potted Plants" published in 2011, Sun Bo introduced the basic biological characteristics and introduction history of Agapanthus in detail. The agapanthus material involved in this embodiment can be obtained through public channels.

2. Extraction of RNA

Total RNA (Trizol: Invitrogen) was extracted with "RNA prep pure plant total RNA extraction kit", 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 NANODROP1000Spectrophotometer) ;

3. Full-length cloning of genes

According to the protein function annotation results of Agapanthus japonica transcriptome sequencing (RNA-seq), the core fragment of Agapanthus TIR1 gene was obtained. The RACE method (SMARTer ^TM RACE cDNA Amplification Kit: Clonetech) was used for full-length cDNA cloning, which was carried out 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.),

Use the first-strand cDNA as a template and use primers for PCR:

TIR1F5'-ATGGGCAATCCCTAATCT-3' (SEQ ID NO.1)

and TIR1R5'-CTCGTGCTTTTCCCTGATG-3' (SEQ ID NO.2)

A 1476bp fragment was amplified, recovered and connected to the pMD18-T Simple vector vector, using RV-M and M13-47 as universal primers, using the method of terminator fluorescent labeling (Big-Dye, Perkin-Elmer, USA), in Sequencing was performed on the ABI377 sequencer (Perkin-Elmer, USA), and the sequencing results were compared with the existing database (GenBank) by performing BLAST on the NCBI website (http://blast.ncbi.nlm.nih.gov/). The nucleic acid sequence and encoded protein have a high homology with the known TIR1 genes of grape, Populus trichocarpa, and castor, and it is preliminarily considered to be a TIR1 gene;

(2) 3′ RACE

Two rounds of nested PCR complete the amplification of the 3' terminal sequence.

First round: UPM+3'-GSP1 (5'-TGTCCTTCTTCTTCGCCGCCTTTGGGT-3') (SEQ ID NO.5)

The second round: NUP+3'-GSP2 (5'-CAACGCCTTGCTGTTTCGGGTCTATT-3') (SEQ ID NO.6)

UPM and NUP are supplied with the kit. 3'RACE obtained the 3' end sequence (1887bp) of Agapanthus TIR1, recovered it, connected it to the pMD18-T Simple vector vector, used RV-M and M13-47 as universal primers, and used terminator fluorescent labeling (Big-Dye, Perkin-Elmer, USA) method, sequenced on the ABI377 sequencer (Perkin-Elmer, USA), and the sequencing results were compared by BLAST (http://blast.ncbi.nlm.nih.gov/) on the NCBI website The existing database (GenBank) shows that its nucleic acid sequence and encoded protein have a high homology with the known TIR1 genes of grape, Populus trichocarpa, and castor;

(3) 5′ RACE

Using 5′RACE ready cDNA as a template, the 5′ terminal sequence was amplified by two rounds of nested PCR.

The first round: UPM+5'-GSP1 (5'-ATCCATTGGCTCGTTGGTGAGGTAGTCG-3') (SEQ ID NO.7)

The second round: NUP+5'-GSP2 (5'-CCTTGCATACCAAAGACACGGCGCTAC-3') (SEQ ID NO.8)

UPM and NUP are supplied with the kit. 5'RACE obtained the 5' end sequence (1607bp) of Agapanthus TIR1 gene, recovered the connection and sequenced it with the same method as above, spliced the sequence results obtained by the above three methods, and submitted the spliced sequence to BLAST analysis , the results proved that the TIR1 gene newly obtained from Agapanthus is indeed an auxin receptor protein-related gene, and the sequencing results were combined with NCBI's ORF Finding (http://www.ncbi.nlm.nih.gov/gorf) to predict , found the start codon and stop codon of Agapanthus TIR1 gene, and designed specific primers from the start codon and stop codon respectively according to the obtained sequence,

ORF-F (5'-ATGGACGCGAAGAGGAAGAAAGA-3'), (SEQ ID NO.9)

ORF-R (5'-TCAGAGTGTGACAACAAAAGGAGGC-3'), (SEQ ID NO.10)

The full-length coding sequence of 1767bp Agapanthus TIR1 protein (SEQ ID NO.3) was amplified by PCR using the cDNA of Agapanthus abaciens as a template.

Example 2. Sequence information and homology analysis of Agapanthus TIR1 gene

The full-length open reading frame sequence of the new Agapanthus TIR1 gene of the present invention is 1767bp, and the detailed sequence is shown in the sequence shown in SEQ ID NO.3. According to the open reading frame sequence, the amino acid sequence of Agapanthus TIR1 protein was deduced, with a total of 588 amino acid residues, a molecular weight of 65.8kDa, and an isoelectric point (pI) of 5.83. For the detailed sequence, see the sequence shown in SEQ ID NO.4;

The open reading frame sequence of Agapanthus TIR1 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 Nucleic acid and protein homology search, and found that it has 72% identity with castor plant TIR1 gene (accession number: XM_002524640.1) at the nucleotide level, as shown in Figure 1 (Query: Agapanthus TIR1 Coding gene sequence; Sbjct: mRNA sequence of castor TIR1); at the amino acid level, it also has 78% identity and 87% similarity with grape TIR1 gene (accession number: XP_002271412.2), as shown in Figure 2 (Query: amino acid sequence of Agapanthus TIR1 protein; Sbjct: amino acid sequence of grape TIR1 protein). It can be seen that the Agapanthus TIR1 gene has high homology with TIR1 genes of other known species, both at the nucleic acid and protein levels.

Example 3. Expression differences of Agapanthus TIR1 gene at different developmental stages of somatic embryos and under exogenous regulation

1. Acquisition of materials: samples were taken from four different developmental stages of Agapanthus somatic embryos (callus; embryogenic callus; somatic embryos; somatic embryo seedlings); The 3-year-old plants of Agapanthus basilica regulate auxin signal. The exogenous regulatory substances are IAA (100ppm) and NPA (auxin polar transport inhibitor: 100ppm). After treatment, the control sample (no treatment), IAA treatment and NPA Leaves of treated plants were sampled. Wrap the samples with aluminum platinum paper and put them into liquid nitrogen immediately, and then transfer them to -80°C ultra-low temperature refrigerator for storage until use;

2. RNA extraction: use RNA prep pure plant total RNA extraction (Trizol: Invitrogen); extract total RNA from different sample tissues of Agapanthus lily;

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, and the maximum rRNA brightness in the electrophoresis band should be The brightness of the second rRNA is 1.5 to 2.0 times, otherwise it means that the rRNA sample is degraded; 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. Design specific primers for real-time fluorescent quantitative PCR analysis of gene expression in various organs and tissues. According to the obtained Agapanthus TIR1 gene sequence, use primer design software to design TIR1 gene quantitative analysis in Real-time PCR specific primer primers,

qT-F (5'-GATTGTGGCGAGGTGTAG-3'), (SEQ ID NO.11)

qT-R (5'-AGCGTGTTCAGGTTCTTG-3'). (SEQ ID NO.12)

The internal reference gene is Agapanthus Actin gene, and the primers are:

Actin-F (5′-CAGTGTCTGGATTGGAGG-3′), (SEQ ID NO.13)

Actin-R (5'-TAGAAGCACTTCCTGTG-3'). (SEQ ID NO.14)

6. Make the standard curve of the target gene and internal reference gene: use EASY Dilution (provided by the kit) to gradiently dilute the standard cDNA solution, then use the diluted cDNA solution as a template, and use specific primers for the target gene and internal reference gene Carry out Real-time PCR amplification, and draw the melting curve and standard curve after the reaction; analyze the melting curve, and judge whether the melting curve of the target gene and the internal reference gene has a single peak, so as to judge whether a single PCR amplification product can be obtained by using the primer ; Determine the appropriate dilution factor of the template cDNA by a standard curve;

7. Real-time fluorescent 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 are respectively amplified with specific primers for fluorescent quantitative analysis, and the Real-time PCR reaction is performed in BIO-RAD Chromo4 real-time quantitative instrument, the reaction system is 20μL, the reaction adopts a three-step method, denaturation at 94°C for 20s, followed by 40 cycles: 94°C for 15s; 57°C for 15s; 72°C for 25s; after each amplification, Melting curves were done to check whether the amplification product was specifically produced;

8. Using the 2- ^△△Ct method for relative quantitative analysis, the results show that the expression level of TIR1 gene in Agapanthus agapanthus TIR1 gene expression level in the leaves of exogenous IAA treatment is reduced (down 8%), while in the leaves of NPA treatment The expression of TIR1 gene increased by 7.7 times (Fig. 3), and the change trend of TIR1 gene expression between the two treatments was completely opposite. The above results indicate that there is a negative feedback regulation mechanism between auxin content and its receptor protein, that is, plants with higher auxin content than normal levels can reduce the level of auxin signal transduction by reducing the expression of its receptor protein, and vice versa. In addition, in the process of somatic embryogenesis of Agapanthus, the expression level of TIR1 gene was the lowest in the callus, and the expression level of TIR1 gene in the embryogenic callus increased by 2.27 times. The expression level of TIR1 in seedlings was the highest, which was 7.32 times that of calluses (Fig. 4). It shows that the TIR1 gene has obvious temporal and spatial differences in the development of Agapanthus somatic embryos. Therefore, the acquisition of Agapanthus TIR1 protein coding sequence has important application prospects and application value in regulating auxin signal to control plant apical growth advantage, plant type regulation and reducing the ratio of somatic abnormal embryos in the future.

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 (3)

1. the protein that aminoacid sequence as shown in SEQ ID NO.4 forms.

One kind coding claim 1 described in nucleic acid sequences to proteins.

3. nucleotide sequence as claimed in claim 2, is characterized in that, described nucleotide sequence is specifically as shown in 1st～1767 of SEQ ID NO.3.