CN110835367A - Pear flowering regulating transcription factor PbrSPL15 and application thereof - Google Patents

Abstract

The invention discloses a pear flowering regulation transcription factor PbrSPL15. The pear regulation flowering transcription factor PbrSPL15 has a nucleotide sequence as shown in SEQ ID No.1, or has more than 90% homology with SEQ ID No.1 , and encodes DNA molecules that regulate plant flowering-related proteins. The pear flowering regulation transcription factor PbrSPL15 provided by the invention can regulate the early flowering of plants, shorten the childhood period, provide new gene resources, help to accelerate the breeding process, reduce agricultural production costs and improve economic benefits.

Description

Pear regulation of flowering transcription factor PbrSPL15 and its application

technical field

The invention belongs to the technical field of plant genetic engineering, and relates to the construction of a pear flowering regulation transcription factor PbrSPL15 and its recombinant vector construction and application in regulating the flowering ability of plants.

Background technique

Plant integrity is divided into two stages: vegetative growth and reproductive growth. Higher plants need to undergo a certain period of vegetative growth after germination before they can enter reproductive growth. Flowering is a sign that plants change from vegetative growth to reproductive growth. For most plants, proper flowering time is crucial to their survival and normal reproduction, and is directly related to crop yield and economic benefits. As an important trait in plant life history, flowering time plays a central role in plant production and evolution (Luo Rui and Guo Jianjun, 2010).

Due to differences in their own characteristics, different plants experience different lengths of childhood before flowering. The childhood of perennial woody plants is longer than that of annual herbaceous plants, ranging from several years to several decades. Fruit trees, as a class of woody plants, have a long childhood, which not only limits directional breeding, but also has a great impact on the economic value of fruits.

There are many kinds of plants in nature, and different plants bloom in different seasons, and even in different time periods of the same day, the flowering times of plants are also different. The regulation of plant flowering time is a very complex process. There are many factors that induce plant flowering, including photoperiod, temperature, hormones, and age. Changes in these conditions will affect plant flowering to varying degrees. At present, the regulation of plant flowering time is mainly studied in the model plant Arabidopsis thaliana. By changing the external environmental conditions and any internal conditions, the generated signals are pooled, and the main flowering integration genes such as SOC1, FT, and LFY are used to realize the regulation of the flowering time of Arabidopsis (Sun Changhui et al., 2007; Zhou Chuanmiao, 2013). So far, six flowering pathways have been discovered, namely vernalization, photoperiod, and temperature pathways that sense exogenous environmental signals; autonomic, gibberellin, and age pathways that sense endogenous signals (Srikanth and Schmid, 2011; Zhou Chuanmiao, 2013).

As a plant-specific transcription factor, SPL is involved in many physiological and biochemical processes such as plant morphogenesis, developmental stage transition, sporogenesis, flower and fruit development, fertility, and gibberellin response, and plays an important role in plant growth and development. regulation (Gou et al., 2011). When entering the reproductive growth stage, SPL is abundantly expressed in shoot tips and inflorescences, suggesting that SPL may play an important role in flower induction and flowering (Cardon et al., 1997; Schmid et al., 2003; Zimmerman et al. , 2004; Shikata et al., 2009). However, studies on SPL regulation of flowering in pears have not been reported.

As the third largest fruit in the world, pear has a wide variety of cultivated varieties and a wide area in my country. Pear trees generally bloom in spring, but the temperature in spring is extremely unstable, which is prone to freezing damage, resulting in a lower fruit set rate. At the same time, as a perennial woody plant, pear trees have a long childhood (at least 3 years). Therefore, this study carried out gene cloning and functional research on shortening fruit tree childhood. The economic benefits of fruit trees are of great significance.

references:

S,Nettesheim K,Saedler H,Huijser P.Functionalanalysis of the Arabidopsis thaliana SBP-box gene SPL3:a novel gene involvedin the floral transition[J]. Plant Journal,2010,12(2):367-377.Cardon GH,

S,Nettesheim K,Saedler H, Huijser P.Functional analysis of the Arabidopsis thaliana SBP-box gene SPL3: a novel gene involved in the floral transition[J]. Plant Journal,2010,12(2):367-377.

Gou JY, Felippes F, Liu CJ, Weigel D, Wang JW.Negative regulation ofanthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPLtranscription factor[J].Plant Cell,2011, 23(4):1512-1522.

Shikata M, Koyama T, Mitsuda N, Ohme-Takagi M. Arabidopsis SBP-Box genesSPL10, SPL11and SPL2 control morphological change in association with shootmaturation in the reproductive phase[J].Plant Cell Physiol,2009,50(12):2133-2145 .

Srikanth A,Schmid M.Regulation of flowering time:all roads lead toRome[J].Cell Mol Life Sci,2011,68(12):2013-2037.

Zimmerman P, Hirsch-Hoffmann M, Hennig L, Gruissem W. GENEVESTIGATOR. Arabidopsis microarray database and analysis toolbox[J]. Plant Physiology, 2004, 136(1): 2621-2632.

Luo Rui, Guo Jianjun. Plant flowering time: natural variation and genetic differentiation. Acta Botany[J].2010,45(1):109-118.

Sun Changhui, Deng Xiaojian, Fang Jun, Chu Chengcai. Research progress on flowering induction in higher plants[J]. Hereditary, 2007,29(10): 1182-1190.

Zhou Chuanmiao, Wang Jiawei.Molecular mechanism of flowering in perennial herbs[J].Chinese Journal of Cell Biology,2013(8):1073-1076.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the flowering regulation transcription factor PbrSPL15 in pear and its application in regulating plant flowering.

In order to achieve the above object of the invention, the present invention provides the following technical solutions: pear regulates flowering transcription factor PbrSPL15, and the pear regulates flowering transcription factor PbrSPL15, the nucleotide sequence of which is shown in SEQ ID No. 1 has more than 90% homology, and encodes a DNA molecule that regulates plant flowering-related proteins.

The present invention also provides the amino acid sequence encoded by the pear flowering regulation transcription factor PbrSPL15, the amino acid sequence shown in SEQ ID No. 2, or the amino acid sequence of SEQ ID NO. 2 through one or several amino acid residues A protein derived from SEQ ID NO. 2 related to the substitution of the base and related to the regulation of plant flowering.

The present invention also provides a recombinant expression vector, expression kit, transgenic strain or recombinant bacteria containing the transcription factor PbrSPL15.

The recombinant expression vector containing the transcription factor PbrSPL15 of the present invention also belongs to the protection scope of the present invention, and the recombinant expression vector containing the gene can be constructed by using the existing plant expression vector.

A primer pair for amplifying the full length or any fragment of the transcription factor PbrSPL15 also belongs to the protection scope of the present invention, and the primer pair is preferably SEQ ID No.3/SEQ ID No.4.

The present invention also provides the application of at least one of the gene, the protein, the recombinant expression vector, the expression kit, the transgenic line or the recombinant bacteria in plant breeding.

The present invention also provides the application of at least one of the gene, the protein, the recombinant expression vector, the expression kit, the transgenic line or the recombinant bacteria in regulating the flowering ability of plants. The regulation of the present invention can be Including early or late, when applied to early flowering, the expression of transcription factor PbrSPL15 can be promoted in plants by transgenic, and when delayed, it can be achieved by inhibiting the expression of this gene. Both transgenic and inhibiting genes can be commonly used in the prior art. method to achieve.

Preferably, the plant is Arabidopsis thaliana.

Preferably, when the plant is Arabidopsis, the application comprises the following steps:

1) providing the pear-regulated flowering transcription factor PbrSPL15;

2) connecting the pear-regulated flowering transcription factor PbrSPL15 with a vector to obtain a recombinant vector;

3) transferring the recombinant vector into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens;

4) Infecting Arabidopsis thaliana with the recombinant Agrobacterium tumefaciens, and transforming unflowered wild-type Arabidopsis thaliana plants using the inflorescence dip method to obtain homozygous Arabidopsis thaliana that overexpresses the pear-regulated flowering transcription factor PbrSPL15.

Preferably, step 1) is: using pear phloem cDNA as a template, performing PCR amplification to obtain pear regulating flowering transcription factor PbrSPL15; the amplified specific primer pair includes forward primer F1 and reverse primer R1; the forward The sequence of primer F1 is shown in SEQ ID No.3; the sequence of the reverse primer R1 is shown in SEQ ID No.4.

Preferably, the pear is Dangshansu pear.

Beneficial effects of the present invention: The pear flowering regulation transcription factor PbrSPL15 provided by the present invention has the effect of promoting early flowering after biological function verification, and over-expression of the PbrSPL15 can significantly shorten the time from the date of receiving light to the first flower of the plant. Elapsed time, thus earlier the plant blooms. The PbrSPL15 provided by the present invention provides new gene resources for regulating early flowering and shortening the childhood period of plants, which is beneficial to accelerate the breeding process, reduce the agricultural production cost and improve the economic benefit. The pear PbrSPL15 can be applied to the cultivation of new plant varieties. According to the description in the Examples section, the transgenic lines overexpressing PbrSPL15 in Arabidopsis thaliana flower significantly earlier than the wild type. The expression levels of the regulated downstream flowering integration factor genes SOC1 and FT were higher than those of the wild type.

Description of drawings

Fig. 1 is the amplification electrophoresis diagram of the transcription factor PbrSPL15 regulating flowering in pear of the present invention;

Figure 2 is a schematic flow chart of the cloning, isolation and functional verification of the pear-regulated flowering transcription factor PbrSPL15 of the present invention;

Fig. 3 is the construction process and the vector structure diagram of the expression recombinant vector in Example 4, wherein Fig. 3A is the construction process of the recombinant expression vector, Fig. 3B is the pENTR-SD-D-TOPO vector structure diagram, Fig. 3C is the pMDC83 vector structure diagram ;

Fig. 4 is the expression pattern diagram of the pear regulation and flowering transcription factor PbrSPL15 of the present invention responding to the age mechanism in Example 2;

5 is a subcellular localization map of the pear-regulated flowering transcription factor PbrSPL15 of the present invention in Example 3;

Fig. 6 is the identification diagram and phenotype statistics diagram of the transgenic PbrSPL15 gene line in Example 5, wherein Fig. 6A is the PCR identification diagram of the PbrSPL15 gene transgenic Arabidopsis plant, Fig. 6B is the flowering phenotype diagram, and Fig. 6C is the time required for plant flowering ( From the day when the plant receives light to the time required for the plant to open the first flower) statistic diagram, Figure 6D is a statistic diagram of the number of rosette leaves of the plant;

Figure 7 shows the relative expression of flowering integration factor in the PbrSPL15 gene-transformed line in Example 6, wherein the right figure is the relative expression of the downstream flowering integration factor gene SOC1, and the left figure is the relative expression of another flowering integration factor FT in the plant .

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

The present invention provides a pear flowering regulation transcription factor PbrSPL15, and the nucleotide sequence of the pear flowering regulation transcription factor PbrSPL15 is shown in SEQ ID No.1. In the present invention, the pear flowering regulation transcription factor PbrSPL15 is preferably derived from the phloem of Dangshansu pear branches, and the pear flowering regulation transcription factor PbrSPL15 comprises an open reading frame of 492 bp.

In the present invention, the pear regulating flowering transcription factor PbrSPL15 is obtained by the following methods: extracting the RNA of Dangshansu pear from the phloem of Dangshansu pear branches; reverse transcribing the RNA to obtain cDNA; using the cDNA as a template, using The specific primer F1 and the reverse specific primer R1 were amplified to obtain the pear-regulated flowering transcription factor PbrSPL15. In the present invention, the sequence of the forward primer F1 is shown in SEQ ID No.3, specifically 5'-CACC GAATTC ATGGAGGGATGAGTAAATTG-3';

The sequence of the reverse primer R1 is shown in SEQ ID No. 4, specifically 5'- CTCGAG TTTGATTTGGAAGTGCTTGT-3'.

In the present invention, the branches of the Dangshansu pear are preferably strong annual branches, and the RNA of the Dangshansu pear is extracted by the CTAB method, and there is no other special limitation.

In the present invention, after obtaining Dangshansu pear RNA, reverse transcription is performed to obtain cDNA; in the present invention, the reverse transcription can be carried out by a conventional method in the field, preferably using Thermo reverse transcription kit (United States), the specific method The steps refer to the instructions of the kit.

In the present invention, after the cNDA is obtained, the obtained cDNA is used as a template, and forward primer F1 and reverse primer R1 are used to amplify the gene sequence of the pear flowering regulation transcription factor PbrSPL15. In the present invention, the sequence of the forward primer F1 is shown in SEQ ID No.3; the sequence of the reverse primer R1 is shown in SEQ ID No.4. In the specific implementation of the present invention, the amplification system is preferably a 50 μL system, and the amplification system includes 100 ng of template DNA, I-5 ^TM 2×High-Fidelity Master Mix, 10.0 μM forward primer and 10.0 μM reverse to the primer. The I-5 ^™ 2 x High-Fidelity Master Mix described in the present invention is preferably purchased from Molecular Cloning Labotratones. The procedure of the amplification reaction described in the present invention is preferably: pre-denaturation at 98°C for 2 min; 35 amplification cycles, including denaturation at 98°C for 10s, annealing at 58°C for 15s, and extension at 72°C for 1 min; It was then incubated at 20°C.

In the present invention, after amplifying and obtaining the pear-regulated flowering transcription factor PbrSPL15, the amplified product is preferably subjected to 1% agarose gel electrophoresis, and after recovery, sequencing is verified to obtain the nucleosides of the pear-regulated flowering-regulated transcription factor PbrSPL15 acid sequence (shown in Figures 1 and 2).

The present invention also provides the protein encoded by the pear flowering regulation transcription factor PbrSPL15, the amino acid sequence of the protein is shown in SEQ ID No. 2, and the protein encoded by the PbrSPL15 includes 163 amino acids. The protein of the invention is located in the nucleus and belongs to the nuclear protein; the protein can significantly reduce the number of rosette leaves before bolting, shorten the time from the date of receiving light to the first flower, thereby promoting early flowering of the plant.

The present invention provides the application of the pear-regulated flowering transcription factor PbrSPL15 or the protein in the early flowering ability of plants.

In the present invention, the plant is preferably Arabidopsis thaliana. When the plant of the present invention is Arabidopsis thaliana, the following steps are included: 1) providing the pear flowering-regulating transcription factor PbrSPL15; 2) connecting the pear-regulating flowering-regulating transcription factor PbrSPL15 with a vector to obtain a recombinant vector; 3) adding the The recombinant vector is transformed into Agrobacterium tumefaciens to obtain recombinant Agrobacterium tumefaciens; 4) Arabidopsis thaliana is transformed with the recombinant Agrobacterium tumefaciens by inflorescence dipping method to obtain an Arabidopsis thaliana that overexpresses the pear-regulated flowering transcription factor PbrSPL15.

In the present invention, step 1) is preferably as follows: using Dangshansu pear branch phloem cDNA as a template, performing PCR amplification to obtain the pear-regulated flowering transcription factor PbrSPL15; in the present invention, PCR amplification to obtain the pear-regulated flowering transcription factor PbrSPL15 For specific methods and steps, please refer to the above-mentioned method for obtaining the pear flowering-regulating transcription factor PbrSPL15, which will not be repeated here.

In the present invention, the pear flowering regulation transcription factor PbrSPL15 is obtained, and the pear flowering regulation transcription factor PbrSPL15 is connected with a vector to obtain a recombinant vector. In the present invention, the vector preferably TOPO vector as an intermediate vector, pMDC83 vector as an expression vector, in the present invention The construction method of the recombinant vector described in specifically comprises the following steps:

The obtained pear regulation and flowering transcription factor PbrSPL15 gene comprising the EcoRI and XhoI restriction sites is connected with the entry vector TOPO to obtain a recombinant vector, and in the specific implementation process of the present invention, the TOPO cloning kit produced by Thermo Fisher is preferably used for carrying out. , and the specific method steps refer to the instructions of the kit.

In the present invention, after the recombinant vector is obtained, the recombinant vector is preferably transferred into Escherichia coli strain DH5α, and colony PCR sequencing is performed to verify whether the recombinant expression vector is successfully constructed; Colony PCR sequencing verification method conventional in the field; after PCR verification is successful in the present invention, the bacterial liquid is verified by Sanger sequencing.

In the present invention, after the entry vector is successfully constructed, the entry vector plasmid is cut into linear fragments by MluI enzyme, and the LR reaction is performed with the target vector plasmid using the Gateway system to construct a recombinant expression vector.

In the present invention, after the recombinant vector is obtained, the recombinant vector is preferably transformed into Escherichia coli strain DH5α, and colony PCR sequencing is performed to verify whether the recombinant expression vector is successfully constructed; the method for colony PCR sequencing verification in the present invention adopts the method in the art Conventional colony PCR sequencing verification method; in the present invention, after the PCR verification is successful, the bacterial liquid is verified by Sanger sequencing.

In the present invention, after the recombinant vector is obtained, the recombinant vector is transferred into Agrobacterium tumefaciens GV3101 to obtain recombinant Agrobacterium tumefaciens; in the present invention, the method for transferring the recombinant vector into Agrobacterium tumefaciens is preferably freezing and thawing In the present invention, the specific freeze-thaw method method steps are shown in "Molecular Cloning Experiment Guide" (Sambrook, Huang Peitang translation, "Molecular Cloning Experiment Guide" third edition, Science Press, 2002).

In the present invention, after the recombinant Agrobacterium tumefaciens is obtained, the recombinant Agrobacterium tumefaciens is infected with the Arabidopsis thaliana by the flower soaking method to obtain the homozygous Arabidopsis thaliana that overexpresses the transcription factor PbrSPL15. In the present invention, the method for infecting Arabidopsis with the recombinant Agrobacterium tumefaciens is as follows: dissolving the recombinant Agrobacterium tumefaciens in the induction solution, and immersing the unopened inflorescences of Arabidopsis in the bacterial solution In , the Arabidopsis thaliana overexpressing the transcription factor PbrSPL15 was obtained by infecting by vacuum method. In the present invention, the recombinant Agrobacterium tumefaciens is subjected to liquid expansion and culture to obtain bacterial liquid, and the OD ₆₀₀ of the bacterial liquid is preferably 0.6-0.8, more preferably 0.7. In the present invention, the time for vacuum infection is preferably 5-10 min, more preferably 7 min. In the present invention, the amount of bacterial liquid during vacuum infection is preferably such that the entire plant can be completely submerged.

In the present invention, the induction solution preferably contains MS, sucrose, 6-BA and Silwet L-77; the addition amount of MS in the induction solution in the present invention is preferably 4.747g/L, and the addition of sucrose The amount is preferably 50g/L, the addition amount of 6-BA is preferably 10μg/L, and KOH needs to be used to adjust the pH to 5.70, and the addition amount of the Silwet L-77 is preferably 250～400μl/L , more preferably 350 μl/L. After the infection, it is preferable to cultivate in the dark for 24h, followed by normal light cultivation for 16h/8h. The incubation temperature after the infection is 24-26°C.

In the present invention, after the infection, the Arabidopsis thaliana screening for overexpressing the pear-regulated flowering transcription factor PbrSPL15 is preferably performed. The screening described in the present invention is preferably carried out by using the screening medium supplemented with hygromycin, and the Arabidopsis thaliana that can grow in the screening medium supplemented with hygromycin is the Arabidopsis thaliana that overexpresses the pear-regulated flowering transcription factor PbrSPL15 .

In the present invention, after obtaining the Arabidopsis thaliana line that overexpresses the pear-regulated flowering transcription factor PbrSPL15, it is screened successively to the T3 generation homozygote, and preferably, the detection and verification of each generation of transgenic positive plants is carried out. In the present invention, the detection is preferably PCR amplification detection using specific primers for pear regulation of flowering transcription factor PbrSPL15. The reaction procedure for PCR amplification detection in the present invention is preferably 94°C for 3 min of pre-denaturation; 35 cycles of denaturation at 94°C for 30s, 58°C annealing for 30s, and 72°C extension for 1min10s, after the cycle is completed, 72°C for 10min extension, and then at 20°C for 35 cycles. ℃ insulation. The 20 μL system is preferably: 15.2 μL ddH ₂ O, 1.6 μL 2.5×dNTP, 1 μL primer F1, 1 μL primer R2, 1 μL template cDNA, 0.2 μL Taq enzyme (200U). In the obtained Arabidopsis thaliana strains, fragments of the expected size can be amplified, indicating positive transgenic strains. The planting and transplanting management of Arabidopsis described in the present invention may adopt conventional methods in the art.

In the present invention, it is preferable to record the number of rosette leaves of each transgenic Arabidopsis thaliana in each generation and the time from the date of receiving the light to the first flower of the plant. of statistics. The planting and transplanting management of Arabidopsis described in the present invention may adopt conventional methods in the art.

After obtaining the Arabidopsis thaliana T3 homozygous line of the transgenic pear regulating flowering transcription factor PbrSPL15, it is preferable to measure the expression levels of the transcription factor PbrSPL15 regulating the downstream flowering integration factor SOC1 and another plant flowering integration factor FT, See Figure 6A, Figure 6B.

The pear flowering regulation transcription factor PbrSPL15 provided by the present invention and its application in early flowering ability are described in detail below with reference to specific examples, but they should not be construed as limiting the protection scope of the present invention.

Example 1

Cloning of full-length cDNA of pear PbrSPL15 gene

A pear full-length cNDA library was screened to screen a transcription factor PbrSPL15 that regulates flowering. According to the recombination characteristics of the Gateway system and the sequence of the PbrSPL15 gene, primers were designed with Primer premier 5.0, and the phloem cDNA of 'Dangshansuli' was used as a template to amplify the whole long. The detailed steps are as follows:

The research material Dangshansu pear was planted in the National Pear Engineering Center of Nanjing Agricultural University, and its seedling age was 3 to 5 years. The annual branches of Dangshansu pear with robust growth vigor were selected, 500 mg samples were randomly weighed, and immediately frozen with liquid nitrogen. Total RNA was extracted by CTAB method. Prepare RNA-free blue, yellow, white and 1.5ml centrifuge tubes before the experiment; Freeze with liquid nitrogen. CTAB extraction buffer: 2% CTAB, 2% PVPK-30, 10 mM Tris-HCl (pH 8.0), 25 mM EDTA, 2 M NaCl, 0.5 g/L spermidine. After the extraction was completed, the quality of the extracted RNA was detected by 1% agarose gel electrophoresis, and the concentration and quality of RNA were detected by NanoDrop 2000 spectrophotometer.

Synthesis of the first strand of cDNA was performed according to the manual of the Thermo Scientific RevertAid First Strand cDNASynthesis Kit. The resulting first-strand cDNA was used for amplification of the PbrSPL15 gene. PCR was performed as follows: pre-denaturation at 98°C for 2 min; 35 amplification cycles including denaturation at 98°C for 10 s, annealing at 58°C for 15 s, extension at 72°C for 1 min, and extension at 72°C for 10 min after cycling, followed by incubation at 20°C. After the amplification was completed, the PCR product of a single target band was detected by 1% agarose gel electrophoresis, and the specific target band was recovered according to the extraction steps of the gel recovery kit (purchased from Kangwei Century, China).

Example 2

qRT-PCR analysis of pear-regulated transcription factor PbrSPL15 gene in Dangshansu pear branches of different ages

In order to analyze the response pattern of PbrSPL15 gene in Dangshansu pear to age, the expression pattern of PbrSPL15 gene was analyzed using Real-time PCR technology. According to the sequence of the coding region of the PbrSPL15 gene, the upstream and downstream PCR primers for amplifying the entire coding region of the gene were designed with Primer primier 5.0 software according to the general principle of primer design. RNA was extracted using the kit, and the first strand of cDNA was synthesized according to the operation manual of the Thermo Scientific RevertAid First Strand cDNA Synthesis Kit. The 20μL reaction system includes: 10μL SYBR Green, 5μL sterile ultrapure water, 1μL cDNA, 2μL forward primer, F2: 5'-GTCGTAGGCGTTTGTCAGGA-3' (SEQ ID No. 5), 2μL reverse primer, R2: 5 '-AGCCTTCCACAGCATGAGAC-3' (SEQ ID No. 6). (with UBQ as an internal reference, the sequence is as follows: UBQ-F: 5'-CCCTTCACTTGGTTCTCCGT-3' (SEQ ID No.7);

UBQ-R: 5'-TAATCAGCAAGCGTGCGACC-3' (SEQ ID No. 8)).

The qRT-PCR procedure was as follows: pre-denaturation at 94°C for 5 min; denaturation at 94°C for 3 s, annealing at 60°C for 10 s, extension at 72°C for 30 s, 55 cycles; extension at 72°C for 3 min, and incubation at 40°C for 30 s.

Using the phloem of Dangshansu pear branches at different ages as materials, with the increase of branch age, the relative expression of PbrSPL15 gene gradually decreased, as shown in Figure 4. The mechanism of PbrSPL15 gene response to age is indicated.

Example 3

Subcellular localization of pear-regulated flowering transcription factor PbrSPL15 protein

According to the nucleotide sequence of PbrSPL15 gene and the map of TOPO vector and pMDC83-GFP vector, EcoRI and XhoI restriction sites were added before and after the gene sequence. The sequence of the restriction site is as follows:

EcoRI: GAATTC

XhoI: CTCGAG

The cDNA of the phloem of Dangshansu pear branches was used as the template, and the primers added the restriction sites were used for amplification. The PCR program used was: pre-denaturation at 98°C for 2 min; Cycle; 10min extension at 72°C. The primer sequences for the restriction sites are as follows:

F1: 5'-CACC GAATTC ATGGAGGGGATGAGTAAATTG-3' (SEQ ID No. 3)

R1: 5'- CTCGAG TTTGATTTGGAAGGGCTTGT-3' (SEQ ID No. 4)

The stop codon TAG was removed from the gene 3' in order to allow the gene to be fused to GFP. After the PCR product was electrophoresed on a 1% agarose gel, the target band was recovered using a gel kit. The recovered and purified amplified fragment was ligated with TOPO entry vector, and transformed into E. coli competent DH5α by heat shock method. The transformed bacterial liquid was detected by PCR, and the positive bacterial liquid identified by PCR was sequenced.

Extract the correct sequencing result and the plasmid of pMDC83-GFP vector. Use MluI to cut the TOPO vector plasmid into a linear fragment, and fuse it with the pMDC83-GFP vector plasmid, so that the two are subjected to LR reaction, the two are connected by LR recombination, and transferred into Escherichia coli competent DH5α by heat shock method. The bacterial liquid was detected by PCR, and the positive bacterial liquid identified by PCR was sequenced to obtain the correct recombinant target vector named pMDC83-GFP-PbrSPL15. The recombinant vector pMDC83-PbrSPL15 was introduced into Agrobacterium GV3101 by freeze-thaw method (refer to Sambrook, translated by Huang Peitang, "Molecular Cloning Experiment Guide" 3rd Edition, Science Press, 2002).

The instantaneous transformation method of tobacco leaves is adopted, and the induction solution is prepared as follows:

Table 1 Preparation method of induction liquid in transient transformation of tobacco leaves

Specific operation method:

1) Take out the preserved Agrobacterium solution from the -80°C refrigerator, streak the LB solid medium with the corresponding resistance (usually kana and rifampicin), place it upside down in a 30°C incubator, and activate the bacterial solution. ;

2) To grow a single clone, expand the culture in the LB liquid medium of kana and rifampicin antibiotics, shake at 28～30°C, 220～240rpm, shake and culture overnight;

3) Collect bacterial liquid, generally use a 10mL centrifuge tube to collect twice, depending on the amount of bacteria at the bottom of the tube, more can be collected;

4) Pour off the supernatant, drain the liquid, and add 2 mL of induction solution;

5) Vortex and mix, and shake up and down on a shaker for induction for more than 3 hours;

6) Infected tobacco by injection, injected from the back of Bunchen tobacco; 2-3 days later, the fluorescence is observed by confocal scanning microscope, and the leaves are evacuated with a vacuum pump or syringe before observation for clearer observation.

The localization of PbrSPL15 protein was determined by detecting the location of GFP fluorescence in leaf epidermal cells by injecting PbrSPL15-GFP and control plasmids with empty 35S-GFP into tobacco epidermal cells. The results are shown in Figure 5. Figure 5A shows the localization of the control empty vector, and the green fluorescence is all over the cell membrane and nucleus; Figure 5B shows the transient expression of PbrSPL15-GFP in tobacco epidermal cells, and the green fluorescence is distributed in the nucleus, but no green fluorescence is found elsewhere. The results showed that the PbrSPL15 protein was located in the nucleus and was a nuclear localized protein.

Example 4

Application of pear-regulated flowering transcription factor PbrSPL15 in early flowering in Arabidopsis

1. Plant transformation vector construction

As shown in Figure 3, according to the recombination characteristics of the Gateway system and the sequence of the PbrSPL15 gene, the stop codon TAG was removed from the 3' of the gene, in order to fuse the gene with GFP. After the PCR product was electrophoresed on a 1% agarose gel, the target band was recovered using a gel kit. The recovered and purified amplified fragment was ligated with TOPO entry vector, and transformed into E. coli competent DH5α by heat shock method. The transformed bacterial liquid was detected by PCR, and the positive bacterial liquid identified by PCR was sent for sequencing.

Extract the correct sequencing result and the plasmid of pMDC83-GFP vector. The successfully constructed TOPO entry vector was cut into linear fragments, and the PCR product was subjected to 1% agarose gel electrophoresis, and the target band was recovered using a gel kit. LR reaction was carried out with the plasmid of pMDC83-GFP vector, the two were connected by LR recombination, and the heat shock method was used to transform E. coli competent DH5α, and the transformed bacterial solution was detected by PCR, and the positive bacterial solution was identified by PCR. Sent for sequencing, the correct recombinant destination vector was obtained and named pMDC83-GFP-PbrSPL15. The recombinant vector pMDC83-PbrSPL15 was introduced into Agrobacterium GV3101 by freeze-thaw method (refer to Sambrook, translated by Huang Peitang, "Molecular Cloning Experiment Guide" 3rd Edition, Science Press, 2002).

2. The steps of Agrobacterium-mediated Arabidopsis genetic transformation are as follows:

(1) Agrobacterium culture: Take the Agrobacterium tumefaciens liquid stored in the ultra-low temperature refrigerator (-80°C), and streak it on the LB plate medium supplemented with kanamycin 50mg/L and rifampicin 50mg/L Line, pick a single clone, shake culture in LB liquid medium containing kanamycin 50mg/L and rifampicin 50mg/L, 28-30 ℃, 220-240rpm/min shaking culture for 16-24 hours.

(2) When the OD ₆₀₀ reaches 0.6-0.8, the cells are collected, centrifuged at 5000 rpm/min for 20 min.

(3) prepare induced transformation liquid, as follows:

Table 2 The preparation method of Arabidopsis thaliana flower soaking induction solution

(4) The bacteria were resuspended in the same volume of transformation medium.

(5) The siliques and flowers that have been opened are subtracted from the Arabidopsis to be transformed.

(6) Put the Arabidopsis thaliana upside down into a 100ml beaker, soak the entire inflorescence in the infection solution, and vacuum to 380mmHg, and the infection time is 5-10min, preferably 7min.

(7) After the infection, it is preferable to culture in dark for 24 hours, and then perform normal light culture for 16h/8h. The post-infection culture temperature is 22-25° C., normal management is performed, and Arabidopsis thaliana seeds are finally collected.

3. Screening of transgenic positive seedlings

The PbrSPL15 gene T ₀ generation Arabidopsis thaliana seeds were obtained according to the above method, with hygromycin resistance (20 μg/mL) as the phenotypic marker, and containing antibiotics carboxybenzidine (100 μg/mL), timentin (200 μg/mL) ) to suppress the contamination of miscellaneous bacteria in MS medium to screen seeds, and screen to T ₃ generation homozygous overexpression plants.

3.1 Planting of transgenic Arabidopsis

(1) The preparation method of MS medium is as follows:

Table 3 MS medium preparation method

After 121 ℃, high temperature sterilization for 20 minutes, when the temperature of the medium is cooled to 40 ℃ (not hot to touch), add antibiotics, the concentration is as follows:

Table 4 MS medium antibiotic preparation method

(2) Arabidopsis planting

Arabidopsis seeds were sterilized with 70% alcohol for 1 min, and washed with sterile water for 5 times; 8.33% of sodium hypochlorite was disinfected for 5 min, washed with sterile water for 5 times, and evenly sown in hygromycin (20 μg/ml), carboxybian (100 μg/ml) ml) and Timentin (200 μg/ml) resistant MS solid medium surface. Vernalization at 4°C in the dark for 48h, normal light for 16h/8h, and when the screened resistant seedlings grow two true leaves, transplant them in a nutrient pot, at 22-25°C, under 16h/8h light. The formula of nutrient soil is nutrient soil: vermiculite=1:1.5. After transplanting, cover with a plastic transparent cover to keep warm and moisturizing. After 2 to 3 days of slowing the seedlings, remove the plastic transparent cover and cultivate normally.

3.2 RNA extraction from Arabidopsis leaves

The leaves of transgenic Arabidopsis plants were used as materials, and RNA was extracted by CTAB method. The above description is for detailed description, and will not be described here.

Synthesis of the first strand of cDNA was performed according to the manual of the Thermo Scientific RevertAid First Strand cDNASynthesis Kit. The above description is for detailed description, and will not be described here.

Example 5

Detection of early flowering function of PbrSPL15 transgenic positive plants

1. Detection of transgenic positive plants

PCR amplification was performed using gene-specific forward primer F1: 5'-CACCGAATTCATGGAGGGGATGAGTAAATTG-3' (SEQ ID No. 3) and vector-specific primer; R3: 5'-CGTCGTCCTTGAAGAAGATG-3' (SEQ ID No. 9). The reaction system was 20 μL: Taq mix 10 μL, ddH ₂ O 7 μL, F1 primer 1 μL, R2 primer 1 μL, cDNA 1 μL; PCR program: 94°C 3min, 94°C 30s, 58°C 30s, 72°C 2min, 72°C 10min, 20 ℃5min. The PCR products were detected by 1% agarose gel electrophoresis. In the selected transgenic lines, fragments of the expected size could be amplified, indicating that they were positive transgenic lines, as shown in Figure 5A.

2. Transgenic phenotype statistics

In order to identify whether Arabidopsis thaliana infected with PbrSPL15 gene is related to flowering, the control line (wild-type Arabidopsis thaliana WT) and Arabidopsis thaliana infected with PbrSPL15 gene are normally managed in a culture room, and the temperature of the culture room is preferably 22-25 ℃, the light is preferably 16h/8h, record the number of rosette leaves before the plant bolts and the time when the plant only blooms the first flower from the day it receives the light, and take pictures when the plant blooms.

Under normal management conditions, the phenotype statistics of the growth of Arabidopsis plants (OE1 and OE2) and wild-type plants (WT) transformed with the PbrSPL15 gene are shown in Figure 6 . Among them: Figure 6A is the PCR identification diagram of PbrSPL15 transgenic Arabidopsis plants, and Figure 6B is the phenotype of PbrSPL15 transgenic plants and wild-type WT plants under normal growth conditions. Figure 6C is a statistical graph of the time required for the plant to open the first flower under normal growth conditions. Figure 6D is a statistical diagram of the number of rosette leaves before bolting of plants under normal growth conditions. The results showed that under normal growth conditions, the PbrSPL15 transgenic lines (OE1 and OE2) exhibited significantly earlier flowering than the wild type (WT) (Fig. 6).

Example 6

Expression of flowering integrators in PbrSPL15 transgene-positive homozygous plants

In the transgenic Arabidopsis lines, the reduced number of rosette leaves before bolting and the shortened time the plants experienced from the date of light exposure to the first flower suggested that they may have the ability to flower earlier than WT. Therefore, it is necessary to identify the expression level of Kaihua integrin gene in plants. In the T3 generation transgenic plants, the homozygotes were identified, and the RNA of the transgenic homozygous positive seedlings was extracted and reverse transcribed into cDNA. Using Actin of Arabidopsis thaliana as an internal reference, the expression levels of flowering integration factors SOC1 and FT in the plants were subjected to qRT. - PCR analysis as shown in Figure 7. The results showed that under the same long-day conditions, the expression levels of SOC1 and FT in the transgenic lines (OE1, OE2) were higher than those in the wild type (WT) (Fig. 7). Furthermore, it was shown that the overexpressed PbrSPL15 gene can effectively regulate the downstream flowering integrators and enhance the expression of the flowering integrin genes in transgenic plants, thereby promoting the flowering of plants.

The nucleotide sequences of the Actin primers are shown below:

Forward primer Actin-F: 5'-AGCTACATGACGCCATTTCC-3' (SEQ ID No. 10)

Reverse primer Actin-R: 5'-CCCTGTAAAGCAGCACCTTC-3' (SEQ ID No. 11)

The nucleotide sequence of the SOC1 primer is shown below:

Forward primer SOC1-F: 5'-AGCTACATGACGCCATTTCC-3' (SEQ ID No. 12)

Reverse primer SOC1-R: 5'-CCCTGTAAAGCAGCACCTTC-3' (SEQ ID No. 13)

The nucleotide sequences of the FT primers are shown below:

Forward primer FT-F: 5'-AGCTACATGACGCCATTTCC-3' (SEQ ID No. 14)

Reverse primer FT-R: 5'-CCCTGTAAAGCAGCACCTTC-3' (SEQ ID No. 15)

It can be seen from the above examples that the pear flowering regulation transcription factor PbrSPL15 provided by the present invention has the effect of early flowering ability of plants through biological function verification, and overexpression of the flowering regulation transcription factor PbrSPL15 can effectively reduce the rosette before the flowering of tree bolting. The number of leaves, thus earlier the flowering of the plant. Compared with the control wild type, the transgenic overexpression lines of Arabidopsis thaliana had a much earlier flowering time, and the number of rosette leaves in the transgenic overexpression lines of Arabidopsis thaliana was less, and the plants bloomed from the date of light exposure to the first bloom. The time spent by the flower is shorter than that of the wild type, and the plants bloom earlier.

The above are only the preferred embodiments of the present invention. It should be pointed out that for those skilled in the art, without departing from the principles of the present invention, several improvements and modifications can be made. It should be regarded as the protection scope of the present invention.

sequence listing

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Claims (10)

1. The pear flowering regulating transcription factor PbrSPL15 is characterized in that the nucleotide sequence is shown in SEQ ID No.1, or the pear flowering regulating transcription factor PbrSPL15 has homology of more than 90% with SEQ ID No.1 and encodes DNA molecules of proteins related to plant flowering regulation.

2. The pear of claim 1, regulating flowering transcription factor PbrSPL 15.

3. The protein according to claim 2, characterized in that the amino acid sequence is shown as SEQ ID No.2, or the protein derived from SEQ ID No.2 is obtained by substituting the amino acid sequence of SEQ ID No.2 by one or more amino acid residues and is related to plant flowering regulation.

4. A recombinant expression vector, an expression kit, a transgenic strain or a recombinant bacterium containing the transcription factor PbrSPL15 of claim 1.

5. Also within the scope of the present invention is a primer pair for amplifying the full length or any fragment of the transcription factor PbrSPL15 according to claim 1, preferably SEQ ID No.3/SEQ ID No. 4.

6. The use of at least one of the gene of claim 1, the protein of claim 2 or 3, the recombinant expression vector of claim 4, an expression kit, a transgenic line or a recombinant bacterium in plant breeding.

7. Use of at least one of the gene of claim 1, the protein of claim 2 or 3, the recombinant expression vector of claim 4, an expression kit, a transgenic line or a recombinant bacterium for regulating the flowering performance of a plant.

8. Use according to claim 7, wherein the plant is Arabidopsis thaliana.

9. Use according to claim 8, characterized in that it comprises the following steps:

1) providing the pear flowering regulating transcription factor PbrSPL15 of claim 1;

2) connecting the pear flowering regulating transcription factor PbrSPL15 with a vector to obtain a recombinant vector;

3) transferring the recombinant vector into agrobacterium tumefaciens to obtain recombinant agrobacterium tumefaciens;

4) and infecting arabidopsis with the recombinant agrobacterium tumefaciens to obtain the arabidopsis with the pear over-expression flowering transcription factor PbrSPL15 regulated.

10. The use according to claim 9, wherein step 1) is: PCR amplification is carried out by taking phloem cDNA of pear stems as a template to obtain a pear flowering regulating transcription factor PbrSPL 15; the amplification specific primer pair comprises a forward primer F1 and a reverse primer R1; the sequence of the forward primer F1 is shown in SEQ ID No. 3; the sequence of the reverse primer R1 is shown in SEQ ID No. 4.

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