CN110835367B

CN110835367B - Pear flowering regulating transcription factor PbrSPL15 and application thereof

Info

Publication number: CN110835367B
Application number: CN201810923281.6A
Authority: CN
Inventors: 吴巨友; 曹鹏; 王鹏; 张绍铃; 刘哲; 吴潇; 孔佳君; 黄智�; 齐开杰; 谢智华
Original assignee: Nanjing Agricultural University
Current assignee: Nanjing Agricultural University
Priority date: 2018-08-14
Filing date: 2018-08-14
Publication date: 2022-04-29
Anticipated expiration: 2038-08-14
Also published as: CN110835367A

Abstract

The invention discloses a pear flowering regulating transcription factor PbrSPL15, wherein the nucleotide sequence of the pear flowering regulating transcription factor PbrSPL15 is shown as SEQ ID No.1, or has homology of more than 90% with SEQ ID No.1, and encodes DNA molecules of proteins related to plant flowering regulation. The pear flowering regulating transcription factor PbrSPL15 provided by the invention can regulate plants to bloom in advance and shorten the childhood period, provides new gene resources, is beneficial to accelerating the breeding process, reduces the agricultural production cost and improves the economic benefit.

Description

Pear flowering regulating transcription factor PbrSPL15 and application thereof

Technical Field

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

Background

The plant is intact and divided into two stages: vegetative growth and reproductive growth. Higher plants require a period of vegetative growth after germination before entering reproductive growth. Flowering is a hallmark of the transition of plants from vegetative to reproductive growth, and for most plants, proper flowering time is critical for 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 (rhoui and guo Jianjun, 2010).

Due to the difference of the characteristics of the plants, different plants experience different growth periods before flowering. Perennial woody plants have a relatively long childhood compared to annual herbaceous plants, varying from years to decades. As a woody plant, the fruit tree has a long juvenile period, which not only limits the directional breeding, but also has a great influence on the economic value of the fruit.

The plants in nature are various in variety, different plants can bloom in different seasons, and even in different time periods of the same day, the flowering time of the plants is different. The control of plant flowering time is a very complex process, and factors inducing plant flowering are many, including photoperiod, temperature, hormone, age and the like, and the change of the conditions can affect the plant flowering to different degrees. At present, the regulation and control of plant flowering time is mainly researched in the model plant Arabidopsis thaliana. The generated signals are collected by changing external environmental conditions and any internal conditions, and the flowering time of arabidopsis is regulated and controlled by using main flowering integration genes such as SOC1, FT, LFY and the like (Sunchonghui et al, 2007; Zhou Chuan Miao, 2013). So far, 6 flowering approaches are found in total, namely vernalization, photoperiod and temperature approaches for sensing external environment signals; autonomous, gibberellin, and age pathways for the perception of endogenous signals (Srikanth and Schmid, 2011; Zhou Chuan Miao, 2013).

As a specific transcription factor of plants, SPL participates in a plurality of physiological and biochemical processes such as plant morphogenesis, development stage conversion, sporulation, flower and fruit development, fertility, gibberellin response and the like, and plays an important role in plant growth and development (Gou et al, 2011). When entering the reproductive growth stage, SPL is expressed in large amounts in the shoot tips and inflorescences, suggesting that SPL may play an important role in flower development induction and flowering (Cardon et al, 1997; Schmid et al, 2003; Zimmerman et al, 2004; Shikata et al, 2009). However, no studies have been reported on SPL regulated flowering in pears.

The pear is used as the third fruit in the world, and has various cultivated varieties and wide area in China. The pear trees usually bloom in spring, but the temperature is extremely unstable in spring, so that the frost damage is easy to occur, and the fruit setting rate is reduced. Meanwhile, as perennial woody plants, the juvenile period of the pear trees is long (at least 3 years), so that gene cloning and function research on shortening the juvenile period of the pear trees is carried out, and the research result lays a foundation for regulating and controlling pear tree blossoming and has important significance for improving economic benefits of the pear trees.

Reference documents:

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 of anthocyanin biosynthesis in Arabidopsis by a miR156-targeted SPL transcription factor[J].Plant Cell,2011, 23(4):1512-1522.

Shikata M,Koyama T,Mitsuda N,Ohme-Takagi M.Arabidopsis SBP-Box genes SPL10, SPL11and SPL2control morphological change in association with shoot maturation in the reproductive phase[J].Plant Cell Physiol,2009,50(12):2133-2145.

Srikanth A,Schmid M.Regulation of flowering time:all roads lead to Rome[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.

rui, Guojiangjun, plant flowering time natural variation and genetic differentiation. plant science [ J ] 2010,45(1):109-118.

Higher plant flowering induction research progress [ J ] inheritance, 2007,29(10): 1182-1190.

Zhou Chuan Miao, Wang Jia Wei, molecular mechanism of perennial herb blossoming [ J ] Chinese report of cell biology, 2013(8):1073-1076.

Disclosure of Invention

The invention aims to provide a flowering transcription factor PbrSPL15 in pear and application thereof in regulating flowering of plants.

In order to achieve the above object, the present invention provides the following technical solutions: the pear controls flowering transcription factor PbrSPL15, the pear controls flowering transcription factor PbrSPL15, the nucleotide sequence of the pear controls flowering transcription factor PbrSPL15 is shown as SEQ ID No.1, or has more than 90% homology with SEQ ID No.1, and the pear controls DNA molecules of proteins related to plant flowering.

The invention also provides an amino acid sequence coded by the pear flowering regulating transcription factor PbrSPL15, wherein the amino acid sequence is shown as SEQ ID No.2, or the amino acid sequence of the SEQ ID No.2 is substituted by one or more amino acid residues, and the protein is related to plant flowering regulation and is derived from the SEQ ID No. 2.

The invention also provides a recombinant expression vector, an expression kit, a transgenic strain or a recombinant bacterium containing the transcription factor PbrSPL 15.

The recombinant expression vector containing the transcription factor PbrSPL15 also belongs to the protection scope of the invention, and the existing plant expression vector can be used for constructing the recombinant expression vector containing the gene.

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

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

The 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 bacterium in regulating the flowering capability of plants, the regulation can be early or delayed, when the gene is applied to early flowering, the transcription factor PbrSPL15 can be promoted to be expressed in the plants through the transgene, when the gene is delayed, the expression of the gene can be inhibited, and the transgene and the inhibiting gene can be realized through a common method in the prior art.

Preferably, the plant is arabidopsis thaliana.

Preferably, when the plant is arabidopsis thaliana, the application comprises the following steps:

1) providing the pear flowering regulating transcription factor PbrSPL 15;

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, and transforming a non-flowering wild type arabidopsis plant by using a floral dip method to obtain homozygote arabidopsis of the overexpression pear flowering regulating transcription factor PbrSPL 15.

Preferably, step 1) is: performing PCR amplification by taking the pear phloem cDNA as a template to obtain a pear flowering regulating transcription factor PbrSPL 15; the specific primer pair for amplification 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.

Preferably, the pear is Dangshan pear.

The invention has the beneficial effects that: the pear flowering regulating transcription factor PbrSPL15 has the effect of promoting early flowering through biological function verification, and the over-expression of PbrSPL15 can obviously shorten the time from the date of receiving light to the first flower of the plant, so that the plant flowers early. The PbrSPL15 provided by the invention provides new gene resources for regulating and controlling early flowering and shortening of childhood period of plants, is beneficial to accelerating breeding process, reduces agricultural production cost and improves economic benefit. The pear PbrSPL15 can be applied to the cultivation of new plant varieties, according to the records in the embodiment section, the transgenic line of Arabidopsis over-expressing PbrSPL15 has obviously early flowers compared with the wild type, and the expression levels of the downstream flowering integration factor genes SOC1 and FT regulated by PbrSPL15 in the transgenic line of Arabidopsis are higher than those of the wild type.

Drawings

FIG. 1 is an electrophoresis diagram of amplification of a pear flowering-regulating transcription factor PbrSPL 15;

FIG. 2 is a schematic flow chart of cloning, isolation and functional verification of a pear flowering regulating transcription factor PbrSPL 15;

FIG. 3 is a construction flow of the recombinant expression vector and a structure diagram of the vector in example 4, wherein FIG. 3A is a construction flow of the recombinant expression vector, FIG. 3B is a structure diagram of a pENTR-SD-D-TOPO vector, and FIG. 3C is a structure diagram of a pMDC83 vector;

FIG. 4 is a graph showing the expression pattern of the pear flowering-regulating transcription factor PbrSPL15 responding to age mechanism in example 2;

FIG. 5 is the subcellular localization map of the pear flowering-regulating transcription factor PbrSPL15 in example 3;

FIG. 6 is an identification chart and a phenotype statistic chart of strains transformed with PbrSPL15 gene in example 5, in which FIG. 6A is a PCR identification chart of a plant transformed with PbrSPL15 gene Arabidopsis thaliana, FIG. 6B is a flowering phenotype chart, FIG. 6C is a statistic chart of the time required for the plant to flower (the time required for the plant to flower the first flower from the day the plant receives light), and FIG. 6D is a statistic chart of the number of rosette leaves of the plant;

FIG. 7 shows the relative expression levels of flowering-integration factors in the PbrSPL15 transgenic line of example 6, wherein the right graph shows the relative expression level of the downstream flowering-integration factor gene SOC1, and the left graph shows the relative expression level of another flowering-integration factor FT in plants.

Detailed Description

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

The invention provides a pear flowering regulating transcription factor PbrSPL15, wherein the nucleotide sequence of the pear flowering regulating transcription factor PbrSPL15 is shown as SEQ ID No. 1. In the invention, the pear flowering transcription factor PbrSPL15 is preferably derived from the phloem of Dangshan pear branches, and the pear flowering transcription factor PbrSPL15 comprises 492bp of open reading frame.

The pear flowering-regulating transcription factor PbrSPL15 is obtained by the following method: extracting RNA of the Dangshan pear from the phloem of the Dangshan pear branch; reverse transcribing the RNA to obtain cDNA; and amplifying by using a specific primer F1 and a reverse specific primer R1 by using the cDNA as a template to obtain the pear flowering regulating transcription factor PbrSPL 15. In the invention, the sequence of the forward primer F1 is shown as SEQ ID No.3, specifically 5' -CACCGAATTCATGGAGGGGATGAGTAAATTG-3’；

The sequence of the reverse primer R1 is shown as SEQ ID No.4, in particular to 5-CTCGAGTTTGATTTGGAAGTGCTTGT-3’。

In the invention, the Dangshan pear branches are preferably grown in a robust annual branch, RNA of the Dangshan pear is extracted by a CTAB method, and no other special limitation exists.

According to the method, after the Dangshan pear RNA is obtained, the cDNA is obtained through reverse transcription; in the present invention, the reverse transcription is carried out by a method conventional in the art, preferably by using a Thermo reverse transcription kit (U.S.A.), and the specific steps are described in the specification of the kit.

After the cNDA is obtained, the cDNA obtained is used as a template, and a forward primer F1 and a reverse primer R1 are used for amplificationObtaining the gene sequence of the pear flowering regulating transcription factor PbrSPL 15. The sequence of the forward primer F1 is shown as SEQ ID No. 3; the sequence of the reverse primer R1 is shown as SEQ ID No. 4. In the practice of the present invention, the amplification system is preferably a 50. mu.L system comprising 100ng of template DNA, I-5^TM2 × High-Fidelity Master Mix, 10.0 μ M forward primer and 10.0 μ M reverse primer. I-5 in the present invention ^TM2 × High-Fidelity Master Mix is preferably purchased from Molecular Cloning laboratories. The procedure of the amplification reaction in the present invention is preferably: pre-denaturation at 98 ℃ for 2 min; 35 amplification cycles comprising denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 15s, and extension at 72 ℃ for 1 min; after completion of the cycle, the extension was carried out at 72 ℃ for 10min, followed by incubation at 20 ℃.

After the pear flowering-regulating transcription factor PbrSPL15 is obtained by amplification, the nucleotide sequence of the pear flowering-regulating transcription factor PbrSPL15 is obtained by preferably performing 1% agarose gel electrophoresis and recovery and sequencing verification on the product obtained by amplification (as shown in FIG. 1 and FIG. 2).

The invention also provides a protein coded by the pear flowering regulating transcription factor PbrSPL15, the amino acid sequence of the protein is shown as SEQ ID No.2, and the protein coded by PbrSPL15 comprises 163 amino acids. The protein is positioned in a cell nucleus and belongs to nucleoprotein; the protein can obviously reduce the number of rosette leaves before bolting of plants, and shorten the time from the date of receiving light to the first flower of the plants, thereby promoting the early blossoming of the plants.

The invention provides application of the pear flowering regulating transcription factor PbrSPL15 or the protein in the aspect of advancing flowering capability of plants.

In the present invention, the plant is preferably Arabidopsis thaliana. When the plant is arabidopsis, the method comprises the following steps: 1) providing the pear flowering regulating transcription factor PbrSPL 15; 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 transforming the recombinant agrobacterium tumefaciens into arabidopsis by a floral dip method to obtain the arabidopsis with over-expression pear flowering regulating transcription factor PbrSPL 15.

In the present invention, step 1) is preferably: PCR amplification is carried out by taking the Dangshan pear branch phloem cDNA as a template to obtain a pear flowering regulating transcription factor PbrSPL 15; the specific method and steps for obtaining the pear flowering-regulating transcription factor PbrSPL15 by PCR amplification in the invention are described in the method for obtaining the pear flowering-regulating transcription factor PbrSPL15, and are not described in detail herein.

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

the obtained pear flowering transcription factor PbrSPL15 gene containing EcoRI and XhoI enzyme cutting sites is connected with the entry vector TOPO to obtain a recombinant vector, the TOPO cloning kit produced by Thermo Fisher company is preferably adopted in the specific implementation process of the invention, and the specific method steps refer to the instruction of the kit.

After obtaining the recombinant vector, the recombinant vector is preferably transferred into an escherichia coli strain DH5 alpha, and colony PCR sequencing is carried out to verify whether the recombinant expression vector is successfully constructed; the colony PCR sequencing verification method in the invention adopts a colony PCR sequencing verification method which is conventional in the field; after the PCR verification in the invention is successful, the bacterial liquid is verified by Sanger sequencing.

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

After obtaining the recombinant vector, preferably transforming the recombinant vector into an escherichia coli strain DH5 alpha, and carrying out colony PCR sequencing to verify whether the recombinant expression vector is successfully constructed; the colony PCR sequencing verification method in the invention adopts a colony PCR sequencing verification method which is conventional in the field; after the PCR verification in the invention is successful, the bacterial liquid is verified by Sanger sequencing.

After obtaining a recombinant vector, 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 a freeze-thaw method, and the specific steps of the freeze-thaw method in the present invention are described in molecular cloning protocols (Samsburg, Huangpetang, molecular cloning protocols, third edition, scientific Press, 2002).

After the recombinant agrobacterium tumefaciens is obtained, the recombinant agrobacterium tumefaciens is infected into arabidopsis thaliana by the floral dip method to obtain homozygote arabidopsis thaliana which excessively expresses the transcription factor PbrSPL 15. In the invention, the method for infecting arabidopsis thaliana by the recombinant agrobacterium tumefaciens comprises the following steps: and dissolving the thallus of the recombinant agrobacterium tumefaciens in an inducing solution, soaking unopened inflorescence of arabidopsis into a bacterial solution, infecting the inflorescence by a vacuumizing method, and screening to obtain the arabidopsis with the transcription factor PbrSPL15 overexpressed. The recombinant agrobacterium tumefaciens is subjected to liquid amplification culture to obtain a bacterial liquid, and the OD of the bacterial liquid₆₀₀Preferably 0.6 to 0.8, and more preferably 0.7. In the invention, the time for vacuuming and infecting is preferably 5-10 min, and more preferably 7 min. The amount of the bacterial liquid during vacuum infection in the invention is preferably that the whole plant can be completely immersed.

In the invention, the inducing liquid preferably contains MS, sucrose, 6-BA and Silwet L-77; in the invention, the addition amount of MS in the inducing liquid is preferably 4.747g/L, the addition amount of sucrose is preferably 50g/L, the addition amount of 6-BA is preferably 10 μ g/L, the pH is adjusted to be most preferably 5.70 by using KOH, and the addition amount of Silwet L-77 is preferably 250-400 μ L/L, and more preferably 350 μ L/L. The infection is preferably followed by a dark culture for 24h, followed by a normal light culture for 16h/8 h. The culture temperature after infection is 24-26 ℃.

After the infection, the selection of Arabidopsis thaliana for over-expressing pear to regulate flowering transcription factor PbrSPL15 is preferably carried out. In the invention, the screening is preferably carried out by using a screening culture medium added with hygromycin, and the arabidopsis thaliana capable of growing in the screening culture medium added with hygromycin is the arabidopsis thaliana over-expressing pear flowering transcription factor PbrSPL 15.

After the Arabidopsis thaliana strain of the overexpression pear flowering regulating transcription factor PbrSPL15 is obtained, generation-by-generation screening is carried out to obtain T3 generation homozygote, and detection and verification of each generation of transgenic positive plants are preferably carried out. In the invention, the detection is preferably performed by PCR amplification detection by using a specific primer of a pear controlled flowering transcription factor PbrSPL 15. The reaction procedure of the PCR amplification detection in the invention is preferably pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, extension at 72 ℃ for 1min for 10s, and 35 cycles, extension at 72 ℃ for 10min after the completion of the cycles, and then heat preservation at 20 ℃. The 20 μ L system is preferably: 15.2 μ L ddH₂O, 1.6. mu.L of 2.5 XdNTP, 1. mu.L of primer F1, 1. mu.L of primer R2, 1. mu.L of template cDNA, 0.2. mu.L of Taq enzyme (200U). In the obtained Arabidopsis lines, fragments of expected sizes can be amplified, and the Arabidopsis lines are positive transgenic lines. The arabidopsis planting and transplanting management in the invention can be realized by adopting a conventional method in the field.

In the invention, the number of rosette leaves of each transgenic arabidopsis thaliana and the time from the date of illumination to the first flower of the plant are preferably recorded, and statistics are preferably carried out after the detection and verification of the obtained transgenic positive plants. The arabidopsis planting and transplanting management in the invention can be realized by adopting a conventional method in the field.

After obtaining the Arabidopsis T3 generation homozygote strain of the transgenic pear flowering regulating transcription factor PbrSPL15, the expression levels of a downstream flowering integration factor SOC1 regulated by the transcription factor PbrSPL15 and another flowering integration factor FT of a plant are preferably measured, and the expression levels are shown in a figure 6A and a figure 6B.

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

Example 1

Cloning of full-length cDNA of pear PbrSPL15 gene

A flowering transcription factor PbrSPL15 is screened by screening a pear full-length cNDA library, primers are designed by using a Primer premier 5.0 according to the recombination characteristics of a Gateway system and the sequence of a PbrSPL15 gene, and PCR amplification full length is carried out by using the cDNA of the bast part of a Dangshan pear branch as a template. The detailed steps are as follows:

research material Dangshan crisp pear is planted in the national pear engineering center of Nanjing agriculture university, and the seedling age of the Dangshan crisp pear is 3-5 years. And (3) selecting annual Dangshan pear branches with robust growth potential, randomly weighing 500mg of samples, and immediately quickly freezing by using liquid nitrogen. Extracting total RNA by adopting a CTAB method, and preparing a blue suction head, a yellow suction head, a white suction head and a 1.5ml centrifuge tube of RNA-free before an experiment; the mortar, pestle and small key need to be sterilized with alcohol at high temperature in advance, and then cooled and quickly frozen with liquid nitrogen. CTAB extraction buffer: 2% CTAB, 2% PVP K-30, 10mM Tris-HCl (pH 8.0), 25mM EDTA, 2M NaCl, 0.5g/L spermidine. After extraction was complete, the quality of the extracted RNA was checked by electrophoresis on a 1% agarose gel and the concentration and quality of the RNA was determined using a NanoDrop 2000 spectrophotometer.

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

Example 2

qRT-PCR analysis of pear flowering transcription factor PbrSPL15 gene in branches of Dangshan pear of different ages

In order to analyze the response pattern of PbrSPL15 gene to age in Dangshan pear, the expression pattern of PbrSPL15 gene was analyzed using Real-time PCR technique. Based on the coding region sequence of PbrSPL15 gene, the Primer 5.0 software was used to design the upstream and downstream PCR primers for amplifying the entire coding region of gene according to the general Primer design principle. RNA was extracted using the Kit and First Strand cDNA Synthesis was performed according to the operating manual of Thermo Scientific RevertAID First Strand cDNA Synthesis Kit. 20 μ L of the reaction system included: 10 μ L SYBR Green, 5 μ L sterilized ultrapure water, 1 μ L cDNA, 2 μ L forward primer, F2: 5'-GTCGTAGGCGTTTGTCAGGA-3' (SEQ ID No.5), 2. mu.L reverse primer, R2: 5'-AGCCTTCCACAGCATGAGAC-3' (SEQ ID No. 6). (UBQ is used as an internal reference, and the sequence is shown as UBQ-F: 5'-CCCTTCACTTGGTTCTCCGT-3' (SEQ ID No. 7);

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

the procedure for qRT-PCR was as follows: pre-denaturation at 94 ℃ for 5 min; denaturation at 94 ℃ for 3s, annealing at 60 ℃ for 10s, extension at 72 ℃ for 30s, and 55 cycles; extending for 3min at 72 ℃, and keeping the temperature for 30s at 40 ℃.

Taking the bast of the Dangshan pear trees of different ages as a material, the relative expression level of the PbrSPL15 gene gradually decreases with the increase of the age of the branches, as shown in FIG. 4. Indicating that the PbrSPL15 gene responds to age mechanism.

Example 3

Subcellular localization of pear regulated flowering transcription factor PbrSPL15 protein

According to the nucleotide sequence of the PbrSPL15 gene and the maps of a TOPO vector and a pMDC83-GFP vector, EcoRI enzyme cutting sites and XhoI enzyme cutting sites are respectively added before and after the gene sequence. The sequence of the cleavage site is shown below:

EcoRI：GAATTC

XhoI：CTCGAG

using cDNA of the phloem of the Dangshan pear branch as a template, and amplifying by using a primer added with a restriction enzyme site, wherein the PCR program is as follows: pre-denaturation at 98 ℃ for 2 min; denaturation at 98 ℃ for 10s, annealing at 58 ℃ for 15s, extension at 72 ℃ for 1min, and 35 cycles; extension at 72 ℃ for 10 min. The primer sequences of the cleavage sites are shown below:

F1：5’-CACCGAATTCATGGAGGGGATGAGTAAATTG-3’(SEQ ID No.3)

R1：5’-CTCGAGTTTGATTTGGAAGTGCTTGT-3’(SEQ ID No.4)

the stop codon TAG was removed 3' of the gene in order to allow the gene to be fused to GFP. After the PCR product was electrophoresed through 1% agarose gel, the objective band was recovered by using a gel kit. The recovered and purified amplified fragment was ligated with TOPO entry vector and transformed into E.coli competent DH5 alpha by heat shock. And detecting the transformed bacterial liquid by using PCR, and sequencing the bacterial liquid with positive PCR identification.

And extracting bacterial liquid with correct sequencing result and plasmid of pMDC83-GFP vector. The TOPO vector plasmid is digested by MluI as a linear fragment and is fused with a pMDC83-GFP vector plasmid to carry out LR reaction, the two are recombined and connected by LR, the LR is transferred into escherichia coli competence DH5 alpha by a heat shock method, the transformed bacterial liquid is detected by PCR, the bacterial liquid with positive PCR identification is sequenced, and the correct recombinant target vector is named as pMDC83-GFP-PbrSPL 15. The recombinant vector pMDC83-PbrSPL15 was introduced into Agrobacterium GV3101 by freeze-thaw method (see Sam Brooks, Huangpetang, third edition, molecular cloning, A laboratory Manual, science Press, 2002).

By adopting a tobacco leaf instantaneous transformation method, the inducing liquid is prepared as follows:

TABLE 1 method for preparing inducing liquid for tobacco leaf instantaneous transformation

The specific operation method comprises the following steps:

1) taking out the preserved Agrobacterium liquid from a-80 deg.C refrigerator, streaking on LB solid culture medium with corresponding resistance (generally kanamycin and rifampicin), placing in 30 deg.C incubator in reverse manner, and activating bacteria liquid;

2) after the monoclone grows out, carrying out expansion culture in an LB liquid culture medium of kanamycin and rifampicin antibiotics, shaking and culturing at the temperature of 28-30 ℃ by using a shaking table at 220-240rpm overnight by shaking;

3) collecting bacterial liquid, wherein the bacterial liquid is collected twice by using a 10mL centrifuge tube, and the quantity of bacteria at the bottom of the tube is determined and more bacteria can be collected;

4) pouring off the supernatant, draining off the liquid, and adding 2mL of induction liquid;

5) uniformly mixing by vortex, and inducing by shaking up and down on a shaking table for more than 3 h;

6) infecting the tobacco by injection, injecting from the back of the native tobacco; after 2-3 days, the fluorescence was observed by confocal laser scanning microscope, and the leaves were evacuated by vacuum pump or syringe before observation for clearer observation.

The localization of PbrSPL15 protein localization was determined by injecting PbrSPL15-GFP and control empty 35S-GFP plasmids into epidermal cells of tobacco, respectively, and detecting the position of GFP fluorescence in epidermal cells of leaf blades, and the results are shown in FIG. 5, in which FIG. 5A shows the localization of control empty vector, and green fluorescence is distributed over cell membrane and cell nucleus; FIG. 5B shows the transient expression of PbrSPL15-GFP in tobacco epidermal cells, with green fluorescence distributed in the nucleus and not found elsewhere. The result shows that the PbrSPL15 protein is localized in the nucleus and is a nuclear localization protein.

Example 4

Application of pear flowering regulating transcription factor PbrSPL15 in early flowering of arabidopsis thaliana

1. Construction of plant transformation vectors

As shown in FIG. 3, the termination codon TAG was removed 3' from the gene in order to allow the gene to be fused with GFP, based on the recombination characteristics of the Gateway system and the sequence of the PbrSPL15 gene. After the PCR product was electrophoresed through 1% agarose gel, the objective band was recovered by using a gel kit. The recovered and purified amplified fragment was ligated with TOPO entry vector and transformed into E.coli competent DH5 alpha by heat shock. And detecting the transformed bacterial liquid by using PCR, and sending the bacterial liquid which is positive in PCR identification to sequencing.

And extracting bacterial liquid with correct sequencing result and plasmid of pMDC83-GFP vector. And (3) digesting the constructed TOPO entry vector into a linear fragment, carrying out electrophoresis on the PCR product through 1% agarose gel, and recovering a target band by using a gel kit. And (2) carrying out LR reaction on the recombinant plasmid and plasmid of a pMDC83-GFP vector, carrying out LR recombination connection on the two, transforming escherichia coli competence DH5 alpha by adopting a heat shock method, detecting the transformed bacterial liquid by using PCR, and sequencing the bacterial liquid which is identified to be positive by the PCR to obtain a correct recombinant target vector named as pMDC83-GFP-PbrSPL 15. The recombinant vector pMDC83-PbrSPL15 was introduced into Agrobacterium GV3101 by freeze-thaw method (see Sam Brooks, Huangpetang, third edition, molecular cloning, A laboratory Manual, science Press, 2002).

2. The agrobacterium-mediated genetic transformation of arabidopsis thaliana was as follows:

(1) and (3) agrobacterium culture: taking the Agrobacterium tumefaciens liquid stored in an ultra-low temperature refrigerator at (-80 ℃), streaking on a plate culture medium added with LB of 50mg/L kanamycin and 50mg/L rifampicin, picking a single clone, carrying out shake culture in LB liquid culture medium of 50mg/L kanamycin and 50mg/L rifampicin, and carrying out shake culture at 28-30 ℃ and 240rpm/min 220-24 h.

(2) To be OD₆₀₀When the concentration reaches 0.6-0.8, collecting thallus, centrifuging at 5000rpm/min for 20 min.

(3) Preparing an induced transformation solution as follows:

TABLE 2 preparation method of inducing liquid for flower soaking of Arabidopsis thaliana

(4) The cells were resuspended in equal volumes of transformation medium.

(5) The Arabidopsis thaliana to be transformed is reduced for siliques and already opened flowers.

(6) And (3) reversely buckling the arabidopsis thaliana into a 100ml beaker, soaking the whole inflorescence in the infection solution, vacuumizing to 380 mmHg, and infecting for 5-10 min, preferably 7 min.

(7) The infection is preferably followed by a dark culture for 24h, followed by a normal light culture for 16h/8 h. And the culture temperature after infection is 22-25 ℃, normal management is carried out, and the final collection of the arabidopsis seeds is waited.

3. Screening for transgenic Positive seedlings

The PbrSPL 15-transferred gene T was obtained according to the above-mentioned method₀The seeds of Arabidopsis thaliana are marked with hygromycin resistance (20 mug/mL) as the phenotype characterWhen the strain is used, the MS culture medium containing the antibiotics of carbenicillin (100 mu g/mL) and timentin (200 mu g/mL) for inhibiting the contamination of the mixed bacteria is used for screening seeds, and the seeds are screened to T generation by generation₃The generation homozygote overexpresses the plant.

3.1 transgenic Arabidopsis thaliana

(1) The MS culture medium is prepared by the following steps:

TABLE 3 MS culture Medium preparation method

Sterilizing at 121 deg.C for 20min, cooling to 40 deg.C (not scalding hands), adding antibiotics at the following concentrations:

TABLE 4 MS culture Medium antibiotic preparation method

(2) Arabidopsis thaliana planting

Sterilizing the Arabidopsis seeds for 1min by using 70 percent alcohol, and washing the Arabidopsis seeds for 5 times by using sterile water; 8.33% sodium hypochlorite for 5min, washing with sterile water for 5 times, and uniformly sowing on the surface of MS solid culture medium containing hygromycin (20 mu g/ml), carbenicillin (100 mu g/ml) and timentin (200 mu g/ml) resistance. Vernalizing for 48h at 4 ℃ under the dark condition, culturing for 16h/8h under normal illumination, transplanting the screened resistant seedlings into a nutrition pot when two true leaves grow out, and culturing for 16h/8h under the temperature of 22-25 ℃ under illumination. The nutrient soil formula is as follows: 1, vermiculite: 1.5. and covering a plastic transparent cover after transplanting, preserving heat and moisture, removing the plastic transparent cover after the seedlings are revived for 2-3 days, and normally culturing.

3.2 extraction of Arabidopsis thaliana leaf RNA

The transgenic arabidopsis plant leaves are used as materials, and RNA is extracted by adopting a CTAB method. The above description is for a detailed description and will not be further described herein.

First Strand cDNA Synthesis was performed according to the operating manual of Thermo Scientific RevertAID First Strand cDNA Synthesis Kit. The above description is for a detailed description and will not be further described herein.

Example 5

Detection of early flowering function of PbrSPL15 transgenic positive plant

1. Transgenic positive plant detection

Gene specific forward primer F1 was used: 5'-CACCGAATTCATGGAGGGGATGAGTAAATTG-3' (SEQ ID No.3) and vector specific primers; r3: 5'-CGTCGTCCTTGAAGAAGATG-3' (SEQ ID No.9) were subjected to PCR amplification. The reaction system is 20 mu L of Taq mix 10 mu L of ddH₂O7. mu.L, F1 primer 1. mu.L, R2 primer 1. mu.L, cDNA 1. mu.L; the PCR procedure was: 94 ℃ for 3min, 94 ℃ for 30s, 58 ℃ for 30s, 72 ℃ for 2min, 72 ℃ for 10min and 20 ℃ for 5 min. The PCR product was detected by 1% agarose gel electrophoresis, and the selected transgenic line was amplified to a fragment of the expected size, indicating a positive transgenic line, as shown in FIG. 5A.

2. Statistics of transgene phenotype

In order to identify whether Arabidopsis infected with PbrSPL15 gene is related to flowering, a control line (wild type Arabidopsis WT) and Arabidopsis infected with PbrSPL15 gene are normally managed in a culture room at a temperature of preferably 22 to 25 ℃ and with light preferably for 16h/8h, the number of rosette leaves before bolting the plants and the time when the plants bloom first from the day of receiving light are recorded, and photographing is recorded when the plants bloom.

The phenotype of the growth of Arabidopsis plants (OE1 and OE2) transformed with PbrSPL15 gene and wild type plants (WT) under normal management conditions was statistically determined, and the results are shown in FIG. 6. Wherein: FIG. 6A is a PCR identification of transgenic Arabidopsis plants with PbrSPL15 gene, and FIG. 6B is a phenotype of transgenic plants with PbrSPL15 and wild-type WT plants under normal growth conditions. FIG. 6C is a statistical plot of the time required for the plant to bloom to the first flower under normal growth conditions. FIG. 6D is a statistical plot of the number of rosette leaves before bolting of plants under normal growth conditions. The results show that PbrSPL15 transgenic lines (OE1 and OE2) appeared to flower significantly earlier than Wild Type (WT) under normal growth conditions (fig. 6).

Example 6

Expression of flowering integration factor in PbrSPL15 transgenic positive homozygote plant

In the transgenic Arabidopsis lines, the low number of rosette leaves before bolting and the shortened time that the plants spend from the date of receiving light to the first flower indicates that they may have the ability to flower earlier than WT. Therefore, the identification of the expression level of the promoter integration factor gene in the plant is necessary. In T3 transgenic plants, homozygote was identified, RNA of transgenic homozygote positive seedlings was extracted and reverse-transcribed into cDNA, and qRT-PCR analysis was performed on expression levels of flowering integrators SOC1 and FT in plants using Actin of Arabidopsis thaliana as an internal reference, as shown in FIG. 7. The results show that under the same long day conditions, the expression level of SOC1 and FT in the plants of the transgenic line (OE1, OE2) is higher than that of the Wild Type (WT) (FIG. 7). Further shows that the over-expressed PbrSPL15 gene can effectively regulate and control downstream flowering integration factors and enhance the expression of the flowering integration factor gene in transgenic plants, thereby advancing the flowering of the plants.

The Actin primer nucleotide sequence is shown below:

the 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 SOC 1-F: 5'-AGCTACATGACGCCATTTCC-3' (SEQ ID No.12)

Reverse primer SOC 1-R: 5'-CCCTGTAAAGCAGCACCTTC-3' (SEQ ID No.13)

The FT primer nucleotide sequence is shown below:

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

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

According to the embodiments, the pear flowering regulating transcription factor PbrSPL15 has the function of advancing the plant flowering capability through biological function verification, and the over-expression of the flowering regulating transcription factor PbrSPL15 can effectively reduce the number of rosette leaves before bolting and flowering of the planted trees, so that the plant flowering performance is advanced. Compared with a control wild type, the flowering time of the transgenic over-expression strain of the arabidopsis is greatly advanced, the number of rosette leaves in the transgenic over-expression strain of the arabidopsis is less, the time from the date of receiving illumination to the first flower of the plant is shortened compared with that of the wild type, and the plant flowers earlier.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> Nanjing university of agriculture

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aaggtttgtg accttcattc caagtctcag gttgtgcttg tcgctggcct ccaccaaagg 300

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Claims

1. The gene shown as SEQ ID No.1, the protein shown as SEQ ID No.2, a recombinant expression vector, an expression kit or a recombinant strain containing the gene shown as SEQ ID No.1, or the application of primer pairs shown as SEQ ID No.3 and SEQ ID No.4 in promoting early flowering of plants; it is characterized in that the gene shown in SEQ ID No.1 or the protein shown in SEQ ID No.2 is over-expressed.

2. Use according to claim 1, wherein the plant is Arabidopsis thaliana or pear.

3. Use according to claim 2, characterized in that it comprises the following steps:

1) providing a pear flowering regulating transcription factor PbrSPL15 shown in SEQ ID No. 1;

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

4. Use according to claim 3, characterized in that 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 specific primer pair for amplification comprises a forward primer F1 and a reverse primer R1; the sequence of the forward primer F1 is shown as SEQ ID No. 3; the sequence of the reverse primer R1 is shown as SEQ ID No. 4.