CN111620936B - Protein related to plant flowering time and application thereof - Google Patents

Abstract

The invention relates to a protein related to plant flowering time and application thereof, wherein the protein is the protein of A1), A2) or A3): A1) the amino acid sequence is protein of a sequence 2 in a sequence table; A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in an amino acid sequence shown in a sequence 2 in a sequence table, has more than 90% of identity with the protein shown in A1), and is related to plant flowering time; A3) a fusion protein obtained by connecting protein tags at the N terminal or/and the C terminal of A1) or A2). The invention obtains an expansin gene PtoEXPB3 from populus tomentosa. When the gene is introduced into tobacco, compared with wild type, the flowering time of transgenic tobacco flowers is advanced by 21 days, and the PtoEXPB3 has the function of early flowering and can be used as an important gene resource for the molecular breeding of ornamental plants.

Description

Protein related to plant flowering time and application thereof

Technical Field

The invention relates to the technical field of biology, in particular to a protein related to plant flowering time and application thereof.

Background

Early flowering (i.e., early bud flowering) in plants is associated with genes, and some early flowering genes have been identified in different plants and have positive effects in production practice. For example, Smykal et al (2007) clones genes NtSOC1 and NtFUL related to flowering time from tobacco, and the genes are related to photoperiod characteristics, and the overexpression can cause early flowering of the tobacco. Plum fruit and the like (2014) are transferred into the populus tremuloides PsnAP1 gene in tobacco, and the transgenic plants bloom early. Wang (2012) found that the PtGT1 gene is overexpressed in tobacco, and the plants show early blossoming. Studies of Anxinmin et al (An et al, 2011) show that PtAP3 gene of Populus tomentosa plays An important role in inducing plant early flowering. The NTAG1 gene in narcissus is cloned by the method of RT-PCR by Dendrogen et al (2011), the NTAG1 gene is over-expressed in Arabidopsis thaliana, the leaves of transgenic plants are curled, the plants are short and the flowering is advanced. Guolijie (2015) utilizes RT-PCR technology to separate FT gene from Arabidopsis thaliana, tobacco varieties FC8 and Coker371 gold are transferred through Agrobacterium mediation method, tobacco plants transferred with single copy FT gene bud and flower in 3-4 leaves, and bud time is about 80 days earlier than that of control plants. In other studies FPA can inhibit the expression of the gene FLC and thus promote flowering (Sonmez et al, 2011; Willmann et al, 2011), CBF family genes have the effect of promoting FLC expression, resulting in late flowering of the plants (Tao et al, 2012).

The expansin is an important component of the cell wall, loosens the cell wall by breaking the connection between the cell wall cellulose and the hemicellulose, enhances the ductility of the cell, and widely participates in the processes of plant growth and development, stress resistance and the like. Castillo et al (Castillo et al, 2018) found that expansin genes EXPN4, EXPN10, EXPN7, EXPN1 and EXPN15 were involved in the extension of sunflower grain tissue, the expression of which was correlated with grain size. Low temperature scanning electron microscopy analysis shows that after the expression of OsEXPB2 is inhibited, the development of root hair is obviously inhibited, the root cortical cells are obviously reduced, and the average length and width of the cortical cells are reduced by about 70-80% (Zou et al, 2015). Lin et al (Lin et al, 2011) found that the root hair length was shortened by 25-48% after the expression of the Arabidopsis expansin gene AtEXPA7 was inhibited, but the root hair distribution range was increased. Plant height of plants overexpressing the populus nigra expansin gene PnEXPA3 increased by 20% -26% compared to the control line, but no significant difference was observed for the other phenotypes (Kuluev et al, 2013). In addition, increased expression of the durian expansin genes DzEXP1 and DzEXP2 promotes fruit dehiscence, and decreased expression delays fruit dehiscence (Palapol et al, 2015); the growth rate between tobacco nodes overexpressing the TaEXPB23 gene was significantly increased (Xing et al, 2009); the plant height of tobacco over-expressing the NtEXPA5 gene is increased by 24-46% compared with the wild type (Kuluev et al, 2013). Recent studies have found that a range of stress-related expansin genes have been identified in different species. Such as turf grass heat-resistance related gene AsEXP1(Xu et al, 2007), sweet potato cold-resistance related expansin genes IbEXPL1, IbEXP1 and IbEXP2(Noh et al, 2009), maize drought-resistance related expansin genes ZmEXPA1, ZmEXPA2, ZmEXPA6 and ZmEXPA8(Wu et al, 2001), wheat salt-resistance related expansin gene TaEXPB23(Han et al, 2015), Arabidopsis thaliana and rice disease-resistance related expansin genes AtEXLA2, OsEXPA1, OsEXPA5 and OsEXPA10(Abuqamar et al, 2013; Kong et al, 2010). However, no extender protein related to flowering time of plants has been reported at present.

Disclosure of Invention

The invention aims to solve the technical problem of how to regulate and control the flowering time of plants. In order to solve the above technical problems, the present invention provides a protein, named PtoEXPB3, derived from populus tomentosa TC1521, which is a protein of a1), a2) or A3) as follows, related to flowering time of plants:

A1) the amino acid sequence is protein of sequence 2 in the sequence table;

A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues in the amino acid sequence shown in the sequence 2 in the sequence table, has more than 90 percent of identity with the protein shown in A1) and is related to the flowering time of plants;

A3) a fusion protein obtained by connecting protein tags at the N terminal or/and the C terminal of A1) or A2).

In the protein, the sequence 2 in the sequence table consists of 262 amino acid residues.

The protein can be artificially synthesized, or can be obtained by synthesizing the coding gene and then carrying out biological expression.

In the above proteins, the protein-tag refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro DNA recombination technology, so as to facilitate expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, etc.

In the above proteins, identity refers to the identity of amino acid sequences. Amino acid sequence identity can be determined using homology search sites on the Internet, such as the BLAST web page of the NCBI home web site. For example, in the advanced BLAST2.1, by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as a Matrix, setting Gap existence cost, Per residual Gap cost, and Lambda ratio to 11, 1, and 0.85 (default values), respectively, and performing a calculation to search for identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the above proteins, the 90% or greater identity may be at least 91%, 92%, 95%, 96%, 98%, 99%, or 100% identity.

In the above protein, PtoEXPB3 can be derived from Populus tomentosa.

Biomaterials related to PtoEXPB3 are also within the scope of the invention.

The biological material related to the protein PtoEXPB3 provided by the invention is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding the protein of claim 1;

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector;

B5) a transgenic plant cell line containing the nucleic acid molecule according to B1) or a transgenic plant cell line containing the expression cassette according to B2);

B6) transgenic plant tissue comprising the nucleic acid molecule according to B1) or transgenic plant tissue comprising the expression cassette according to B2);

B7) a transgenic plant organ containing the nucleic acid molecule according to B1) or a transgenic plant organ containing the expression cassette according to B2);

B8) a nucleic acid molecule that reduces the expression of the protein of claim 1;

B9) an expression cassette, a recombinant vector, a recombinant microorganism or a transgenic plant cell line comprising the nucleic acid molecule according to B8).

Wherein the nucleic acid molecule may be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

In the above biological material, the nucleic acid molecule of B1) is a gene encoding the protein as shown in B1) or B2):

b1) the coding sequence is cDNA molecule or DNA molecule of 1 st-789 th nucleotide of sequence 1 in the sequence table;

b2) the nucleotide is a cDNA molecule or a DNA molecule of a sequence 1 in a sequence table.

In the above biological material, the nucleic acid molecule of B8) may specifically be a DNA molecule reverse-complementary to any fragment of the DNA molecule represented by nucleotides 1 to 789 of sequence 1 in the sequence table.

Wherein, the sequence 1 in the sequence table is composed of 789 nucleotides, the coding sequence is the sequence 1 in the sequence table, and the coding sequence is the protein shown in the sequence 2 in the sequence table.

In the above-mentioned biological materials, the expression cassette containing a nucleic acid molecule encoding PtoEXPB3 (PtoEXPB3 gene expression cassette) described in B2) refers to a DNA capable of expressing PtoEXPB3 in a host cell, and the DNA may include not only a promoter which initiates transcription of PtoEXPB3 gene but also a terminator which terminates transcription of PtoEXPB 3. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: constitutive promoter 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiology 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (from salicylic acid and BTH (benzothiadiazole-7-thiol)Acid S-methyl ester); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with jasmonic acid ester); heat shock promoters (us patent 5,187,267); tetracycline inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beach et al (1985) EMBO J.4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated herein in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV35S terminator, tml terminator, pea rbcSE9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (I)⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al GenesDev.,5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

The recombinant expression vector containing the PtoEXPB3 gene expression cassette can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pWMB123, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Corp.) etc. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant expression vector, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure correct translation of the entire sequence. The sources of the translational control signals and initiation codons are wide ranging from natural to synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), marker genes for antibiotics which are expressible in plants (e.g., nptII gene which confers resistance to kanamycin and related antibiotics, bar gene which confers resistance to phosphinothricin which is a herbicide, hph gene which confers resistance to hygromycin which is an antibiotic, dhS gene which confers resistance to methatrexate, EPSPS gene which confers resistance to glyphosate), or marker genes for chemical resistance (e.g., herbicide resistance), mannose-6-phosphate isomerase gene which provides the ability to metabolize mannose, etc. From the safety of transgenic plants, the transformed plants can be screened directly in stress without adding any selective marker gene.

In the above biological material, the recombinant microorganism may be specifically yeast, bacteria, algae and fungi.

In order to solve the above-mentioned problems, the present invention also provides a plant flowering-time regulating agent comprising the protein, or/and a biological material related to the protein. The plant flowering time regulator is an agent capable of advancing or delaying the flowering time of plants.

The active ingredient of the flowering promoter may be the protein or a biological material related to the protein, and the active ingredient of the flowering promoter may further contain other biological components or/and non-biological components, and the other active ingredients of the pharmaceutical agent may be determined by those skilled in the art according to the flowering promoting effect.

The protein or the biological material can be applied to any one of the following P1-P5:

use of P1, said protein, or said biological material for regulating flowering time in a plant;

use of P2, said protein, or said biomaterial in the preparation of a product for advancing the flowering time of a plant;

use of P3, said protein, or said biological material for growing early flowering plants;

the use of P4, said protein, or said biomaterial in the manufacture of a plant early flowering product;

use of P5, said protein, or said biological material in plant breeding.

In order to solve the technical problems, the invention also provides a method for cultivating early flowering plants, which comprises the steps of increasing the expression level of the protein or the coding gene thereof in a target plant to obtain the early flowering plants; the flowering time of the early flowering plant is earlier than that of the target plant.

In the above method, the improvement of the expression level of the protein according to claim 1 or a gene encoding the protein in a target plant is achieved by introducing a gene encoding the protein according to claim 1 into the target plant.

In the method, the coding gene of the protein can be modified as follows and then introduced into a target plant to achieve better expression effect:

1) modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modification is performed using sequences known to be effective in plants;

2) linking with various plant expression promoters to facilitate the expression of the plant expression promoters; the promoters may include constitutive, inducible, temporal regulated, developmental regulated, chemical regulated, tissue preferred and tissue specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

3) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; for example tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

4) enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

The gene encoding the protein can be introduced into Plant cells by conventional biotechnological methods using Ti plasmids, Plant virus vectors, direct DNA transformation, microinjection, electroporation, etc. (Weissbach,1998, Method for Plant Molecular Biology VIII, academic Press, Ne wYork, pp.411-463; Geiserson and Corey,1998, Plant Molecular Biology (2nd Edition).

The gene encoding the protein can be introduced into Plant cells by conventional biotechnological methods using Ti plasmids, Plant virus vectors, direct DNA transformation, microinjection, electroporation and the like (Weissbach,1998, Method for Plant Molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey,1998, Plant Molecular Biology (2nd Edition).

In the method, the early flowering plant can be a transgenic plant, and can also be a plant obtained by conventional breeding technology such as hybridization.

As described above, the plant and the target plant are both monocotyledons or dicotyledons.

As described above, the plant and the target plant are Nicotiana benthamiana.

The invention has the beneficial effects that: the invention utilizes molecular biology technology to obtain an expansin gene PtoEXPB3 from populus tomentosa. When the gene is introduced into tobacco, compared with wild type, the flowering time of transgenic tobacco flowers is advanced by 21 days, which shows that PtoEXPB3 has the function of early flowering and can be used as an important gene resource for the molecular breeding of ornamental plants. The flowering time is an important character of the plant, and for crops, the flowering is early, so that the growth period can be shortened, manpower and material resources are saved, and the yield per unit time is improved; for flowering plants, early flowering leads the flowering period to be advanced, the whole ornamental flowering period is prolonged by combining plants in normal flowering periods, and the value of the flowering plants can be effectively improved, so that the early flowering plants have important significance in cultivation.

Drawings

FIG. 1 shows the cloning, M, molecular labeling of the full-length gene PtoEXPB3 of the TC521 expansin of Populus tomentosa; b3, PtoEXPB 3.

Figure 2 is the nucleotide sequence and predicted amino acid sequence of PtoEXPB3 gene, "' is a stop codon.

FIG. 3 construction of PtoEXPB3 gene expression vector.

FIG. 4 is a diagram showing the transformation process of tobacco Bentoni with PtoEXPB3 gene.

FIG. 5 is a verification diagram of PtoEXPB3 transgenic tobacco, wherein FIG. 5a is an electrophoresis diagram of the DNA verification result of PtoEXPB3 transgenic tobacco, FIG. 5b is an electrophoresis diagram of the RNA analysis result of PtoEXPB3 transgenic tobacco, in the diagrams, M represents a molecular marker, and L1-L12 represents a PtoEXPB3 transgenic Nicotiana benthamiana strain; WT means a wild type Nicotiana benthamiana.

FIG. 6 is a photograph of plants grown for 100 days in wild type N.benthamiana (WT) and transgenic N.benthamiana (L1, L2, L3, L11).

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, and the examples are given only for illustrating the present invention and not for limiting the scope of the present invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.

The experimental procedures in the following examples are all conventional ones unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

The Populus tomentosa TC1521 in the following examples is a Populus tomentosa TC1521 genotype of a nursery national Populus tomentosa (Populus tomentosa Carr.) gene bank (Hao Zhuang, Yana Ding, Junkai Zhi, Xiaoyu Li, Huabo Liu, Jiche xu, over-expression of the polyplant expansin gene PtoEXPA12 in bacto plants enhanced calcium accummulation, International Journal of Biological macromolecules 2018,116:676 and Jing 682) publicly available from the university of forestry of North China to repeat the experiments of the present application and is not usable as other applications.

The cloning vectors pEASY-Blunt and E.coli competent top10 in the following examples were purchased from Kyoto Kogyo Biotech, Inc.

The plant expression vectors PEZR (K) -LC (Liu Jie, Xu Xiao, Xu Qian, Wang Shuhui, Xu Jiche. Transgenic Tobacco plants expressing PicW gene from Picea wilsonii exhibit enhanced from great storing tall plant Cell, Tissue and organic Culture,2014,118(3):391-400) in the following examples are publicly available from Beijing forestry university to repeat the experiments of the present application and are not useful for other purposes.

Agrobacterium tumefaciens strain LBA4404(Xu Qian, Xu Xiao, Shi Yang, xujiche, Huang Bingru. 2014, Transgenic tobaco plants overrepresentation a grass PpEXP1 gene expression enhanced strain to strain. plos One9(7): e100792) in the examples below was publicly available from Beijing university of forestry to repeat the experiments of this application and was not available for other uses.

Nicotiana tabacum cv.Xanthium (Liu Jie, Xu Xiao, Xu Qian, Wang Shuhui, Xu Jiche. Transgenic Tobacco plants expressing PicW gene from Picea wilsonii ex vivo modified tobacco free plant Cell, Tissue and organic Culture,2014,118(3): 391-.

In the present study, Chinese white poplar (Populus tomentosa Carr) with the TC1521 genotype in the gene library of Chinese poplar national nursery (Populus tomentosa Carr.) in Shandong province, China was used as a material (reference: Hao Zhuang, Yana Ding, Junkai Zhi, Xiaoyu Li, Huabo Liu, Jiche xu. over-expression of the potplan expansin gene PtoEXPA12 in tobaco plants expressed in nucleic acid amplification, International Journal of nal Biological macromolecules, 2018,116:676-682), leaf-extracted RNA was collected, cDNA was reverse-transcribed, and PtoEXPB3 was cloned. Cloning vectors pEASY-Blunt and E.coli competent top10 used in the gene cloning procedure were purchased from Beijing Quanyujin Biotechnology Ltd, plant expression vectors PEZR (K) -LC (references: Liu Jie, Xu Xiao, Xu Qian, Wang Shuhui, Xu Jiche. Transgenic Tobacco plants expressing PicW gene from Picea wilsonii expressed free from transformed tobacco strain plant, Tissue and Organ Culture,2014,118(3): LBA 391. 400), Agrobacterium (Agrobacterium tumefaciens) strain 4404 and the gene transformation receptor Nicotiana benthamiana were owned and stored in this laboratory.

Example 1 cloning of early flowering Gene PtoEXPB3

The inventor of the invention separates and clones a morning-blooming related gene PtoEXPB3 from Chinese white poplar TC1521, as shown in a sequence 1 of a sequence table, and names a protein coded by the gene PtoEXPB3 protein as shown in a sequence 2 of the sequence table.

The specific cloning method is as follows:

total RNA of TC1521 leaf of Populus tomentosa was extracted by Trizol method (Invitrogen, USA), and reverse-transcribed into cDNA, using cDNA as a template and a primer pair (PtoEXPB3F: 5'-CCAGAAAACTGTCGTCAAAGG-3';

PtoEXPB3R: 5'-GCTTGTTTCTCTCACGTGGA-3') was used as a primer, and amplification of gene PtoEXPB3 was performed using KODplusDNA polymerase. The amplification procedure was: 5min at 94 ℃; 30s at 94 ℃, 40s at 55 ℃ and 1min at 72 ℃ for 35 cycles. The amplification products were separated by electrophoresis on a 1.5% agarose gel, the electrophoresis results are shown in FIG. 1, where M in FIG. 1 represents a molecular marker; b3 shows PtoEXPB3, and FIG. 1 shows that the size of the gene PtoEXPB3 is about 750 bp.

After gel cutting and recovery, PtoEXPB3 is connected to the LacZ position of pEASY-Blunt cloning vector, Escherichia coli competent TOP10 is transformed, M13 primer (M13F: 5'-CAGGAAACAGCTATGAC-3'; M13R: 5'-GTAAAACGACGGCCAGT-3') is utilized to screen positive bacterial plaque, and sequencing is carried out after culture. The sequencing result shows that the amplified fragment comprises a complete PtoEXPB3 gene Open Reading Frame (ORF), the nucleotide length of a coding region is 789bp (shown as a sequence 1), the coding region is predicted to encode 262 amino acids (shown as a sequence 2), the sequence 2 comprises conserved 6 cysteine (C) residues (55 th, 84 th, 87 th, 92 th, 111 th and 157 th positions of the sequence 2), an HFD module sequence (122 nd and 124 th positions of the sequence 2) and 4 tryptophan (W) residues (203 th, 210 th, 214 th and 248 th positions of the sequence 2), and a signal peptide sequence is 1 st to 21 st positions of the sequence 2. The DNA shown in sequence 1 in the sequence table is named as PtoEXPB3 gene. The encoded protein is shown as a sequence 2 and is named as protein PtoEXPB 3. The recombinant vector containing PtoEXPB3 gene was named P-Blunt-PtoEXPB 3.

Example 2 obtaining and identification of PtoEXPB3 Gene-transfected Nicotiana benthamiana

Construction of recombinant expression vector

The recombinant vector P-Blunt-PtoEXPB3 in example 1 was used as a template, and PtoEXPB3F (5' -CCC)AAGCTTATGCAGCTCTTGGGGTTAC-3 ', HindIII restriction sites underlined) and PtoEXPB3R (5' -TGC)TCTAGATTAATGGAAGAAATTGAGCCTAGAGG-3', XbaI restriction sites underlined) as a primer pair, was amplified using a high fidelity enzyme KOD and the PCR product was recovered. The PCR product and the plasmid of the PEZR (K) -LC expression vector are respectively cut by Hind III and Xba I restriction enzymes, the cut PEZR (K) -LC vector and the target gene fragment are recovered, and a recombinant vector PEZR (K) -LC-PtoEXPB3 (the structural schematic diagram is shown in figure 3) is obtained, wherein the recombinant vector PEZR (K) -LC-PtoEXPB3 is a recombinant vector which replaces the sequence between Hind III and Xba I recognition sites of the PEZR (K) -LC expression vector with a PtoEXPB3 sequence and keeps other sequences unchanged, and the recombinant vector is transformed into escherichia coli competent TOP10 by a heat shock method after connection, so that escherichia coli-PEZR (K) -LC-PtoEXPB3 is obtained. The bacterial liquid of the Escherichia coli-PEZR (K) -LC-PtoEXPB3 is coated on an LB solid medium containing 50mg/Lkana, the mixture is cultured overnight at 37 ℃, single colonies are selected for PCR detection, positive colonies are screened and sent to companies for sequencing verification, and sequencing results show that the Escherichia coli-PEZR (K) -LC-PtoEXPB3 contains 1-789 nucleotides (protein of a coding sequence 2) in a sequence table 1. Extracting recombinant plasmid of positive colony, transforming Agrobacterium tumefaciens sensitive LBA4404 by electric shock transformation, selecting single colony for PCR detection, and screening positive colony (containingColonies with PtoEXPB3 gene) to obtain agrobacterium-PtoEXPB 3 containing PtoEXPB3 gene.

II, obtaining of PtoEXPB3 transgenic plant

The screened agrobacterium-ptoEXPB 3 bacterial colonies are cultured in 10mLYEB (containing 50mg/LCef and 50mg/Lkana) liquid culture medium overnight, 1mL of agrobacterium-ptoEXPB 3 bacterial liquid is transferred to 100mL of YEP liquid culture medium without antibiotics, and the culture is continued for 8h, so that the concentration of the agrobacterium-ptoEXPB 3 bacterial liquid reaches OD 600-0.6.

Selecting dark green tobacco leaf, cutting several wounds, and dip-dyeing in Agrobacterium-PtoEXPB 3 bacterial solution for 10 min. Taking out the bacterial liquid on the surface of the sucked dry leaf, and transferring to an MS solid culture medium for dark culture for 3 days. The leaves were taken out and washed once in water containing 200mg/L timentin and again 1 time with sterile water. The surface water of the leaf was blotted, transferred to a differentiation medium (MS +2mg/L6-BA +0.1mg/LNAA +50mg/Lkan +200mg/LCef + 3% sucrose + 0.8% agar) and the medium was changed once at 15 days. After 25 days, the regenerated adventitious buds were transferred to rooting medium (MS +0.1mg/LNAA +50mg/Lkan. +200mg/LCef + 3% sucrose + 0.8% agar) and cultured for 2 weeks to obtain rooted plant leaves (as shown in FIG. 4).

Taking the rooted plant leaves, extracting DNA by a CTAB method, and performing molecular detection on the DNA level by using a specific primer (PtoEXPB3F/PtoEXPB3R) of the PtoEXPB3 gene, wherein the result is shown in figure 5a, and determining a positive transgenic line (namely, a plant line containing the PtoEXPB3 gene).

Further, RNA of these lines was extracted by Trizol method, expression verification of positive transgenic seedlings was performed, and finally 4 positive lines of nicotiana benthamiana (shown in fig. 5b, L1, L2, L3, and L11) transformed with PtoEXPB3 gene were obtained. (primer ActinF: 5'-TACATTGCTCTTGACTTTG-3', ActinR: 5'-GGTCCAGATTCATCATATT-3' for the Actin gene in FIG. 5 b).

Third, analysis of flowering habit of wild type and transgenic plant

1. Cultivation of PtoEXPB3 Gene-transferred Nicotiana benthamiana

And (3) carrying out asexual propagation on the PtoEXPB3 gene-transferred Nicotiana benthamiana strains L1, L2, L3 and L11 prepared in the step two. After the same batch of the propagated strains grow for 20 days, each strain selects 6 plants (6 times of repetition) with the same growth vigor, the plants are respectively planted in pots, the substrate is vermiculite, the size of the pots is 10 multiplied by 10cm, one plant is planted in each pot, and the plants grow in an incubator (the illumination intensity is 2000lux, and the day-night ratio is 16/8 h). The water was applied every 3 days, and Hogland nutrient solution was applied weekly. Flowering time of mature plants was observed and recorded as shown in table 1.

2. Cultivation of wild type Nicotiana benthamiana

Carrying out asexual propagation on wild type Nicotiana benthamiana. After the same batch of propagated strains grow for 20 days, 6 plants with consistent growth vigor are selected from each strain (namely 6 times of repetition of each strain), and are respectively planted in pots, the substrate is vermiculite, the size of the pot is 10 multiplied by 10cm, and each pot is one plant and grows in a warm box (the illumination intensity is 2000lux, and the day-night ratio is 16/8 h). The water was applied every 3 days, and Hogland nutrient solution was applied weekly. Flowering time of mature plants was observed and recorded, and as shown in table 1, plant status at 100 days of cultivation is shown in fig. 6, and it can be seen that on day 100, plants L1, L2, L3 and L11 all had entered flowering status, but none of the wild type (WT shown in the figure) had flowering.

TABLE 1 flowering time of wild type and PtoEXPB3 gene-transgenic Nicotiana benthamiana

Statistics of the results in Table 1 show that the flowering time of wild-type tobacco is 110-115 days, and about 113 days on average, the flowering time of PtoEXPB3 transgenic line is 88-96 days, and the average time is 92 days, which is 21 days earlier than that of wild-type tobacco. Shown by the florescence statistics, the transgenic tobacco and the wild tobacco have no obvious change and are both about 15 days. The PtoEXPB3 is shown to have the effect of early flowering and can be used as an important gene resource for the molecular breeding of ornamental flowers.

The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced within a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is made possible within the scope of the claims attached below.

Sequence listing

<110> university of Beijing forestry

<120> protein related to plant flowering time and application thereof

<160> 2

<170> SIPOSequenceListing 1.0

<210> 1

<211> 789

<212> DNA

<213> Populus tomentosa Carr

<400> 1

atgcagctct tggggttata tgttgcggtg ttcttaaagt gctttttggg tgtctttggg 60

cagcaagttc atcctcaaca taatgtggct gacttacact ggaaaccggc tactgccacc 120

tggtatggca gtcctgacgg tgatggtagt gacggagggg catgtgggta cgggtcatta 180

gtggatgtga agccattcag ggccagagtt ggtgcagtga gcccagtact cttcaagaat 240

ggtgaaggtt gtggggcatg ttacaaagtt aggtgcctag acaagagcat atgctcaaga 300

agggcagtga ccataattgt tacagatgag tgtccaggtg ggtattgttc caatggcaac 360

actcactttg acctcagtgg tgcagccttt ggtcgcatgg ctatttccgg tgagaatggt 420

cagctcagga accgaggtga aattccagtc atttatcgca ggaccccatg caagtaccca 480

gggaagaaca ttgccttcca tgtcaatgaa ggctcaacag attattggct atcccttctg 540

gtagagtttg aagatggtga cggtgatgtt ggatcgatgc atataagaga agcaggaggc 600

actgagtggc tagagatgaa tcacgtatgg ggtgcaactt ggtgtattgt taggggaccc 660

ttgaaaggac cgttctccgt gaaattaaca acactgtcaa caggaagaac actgtctgca 720

agagaagtga ttccaagaaa ttgggctcca aaagctactt acacctctag gctcaatttc 780

ttccattaa 789

<210> 2

<211> 262

<212> PRT

<213> Populus tomentosa Carr

<400> 2

Met Gln Leu Leu Gly Leu Tyr Val Ala Val Phe Leu Lys Cys Phe Leu

1 5 10 15

Gly Val Phe Gly Gln Gln Val His Pro Gln His Asn Val Ala Asp Leu

20 25 30

His Trp Lys Pro Ala Thr Ala Thr Trp Tyr Gly Ser Pro Asp Gly Asp

35 40 45

Gly Ser Asp Gly Gly Ala Cys Gly Tyr Gly Ser Leu Val Asp Val Lys

50 55 60

Pro Phe Arg Ala Arg Val Gly Ala Val Ser Pro Val Leu Phe Lys Asn

65 70 75 80

Gly Glu Gly Cys Gly Ala Cys Tyr Lys Val Arg Cys Leu Asp Lys Ser

85 90 95

Ile Cys Ser Arg Arg Ala Val Thr Ile Ile Val Thr Asp Glu Cys Pro

100 105 110

Gly Gly Tyr Cys Ser Asn Gly Asn Thr His Phe Asp Leu Ser Gly Ala

115 120 125

Ala Phe Gly Arg Met Ala Ile Ser Gly Glu Asn Gly Gln Leu Arg Asn

130 135 140

Arg Gly Glu Ile Pro Val Ile Tyr Arg Arg Thr Pro Cys Lys Tyr Pro

145 150 155 160

Gly Lys Asn Ile Ala Phe His Val Asn Glu Gly Ser Thr Asp Tyr Trp

165 170 175

Leu Ser Leu Leu Val Glu Phe Glu Asp Gly Asp Gly Asp Val Gly Ser

180 185 190

Met His Ile Arg Glu Ala Gly Gly Thr Glu Trp Leu Glu Met Asn His

195 200 205

Val Trp Gly Ala Thr Trp Cys Ile Val Arg Gly Pro Leu Lys Gly Pro

210 215 220

Phe Ser Val Lys Leu Thr Thr Leu Ser Thr Gly Arg Thr Leu Ser Ala

225 230 235 240

Arg Glu Val Ile Pro Arg Asn Trp Ala Pro Lys Ala Thr Tyr Thr Ser

245 250 255

Arg Leu Asn Phe Phe His

260

Claims (5)

1. Use of any one of the following P1-P5 of a protein or biomaterial related to said protein:

use of P1, a protein or a biological material related to said protein for positively regulating flowering time in plants;

use of P2, a protein or a biomaterial related to said protein for the preparation of a product for advancing the flowering time of a plant;

use of P3, a protein or a biological material related to said protein for growing early flowering plants;

the use of P4, a protein or a biological material related to said protein for the preparation of a plant early flowering product;

use of P5, a protein or a biological material related to said protein in early flowering plant breeding;

the protein is the protein of A1) or A2):

A1) the amino acid sequence is protein of sequence 2 in the sequence table;

A2) a fusion protein obtained by attaching a protein tag to the N-terminus or/and the C-terminus of A1);

the plant is a dicotyledonous plant; the protein-related biomaterial is any one of the following B1) to B4):

B1) a nucleic acid molecule encoding the protein of A1) or A2);

B2) an expression cassette comprising the nucleic acid molecule of B1);

B3) a recombinant vector containing the nucleic acid molecule according to B1) or a recombinant vector containing the expression cassette according to B2);

B4) a recombinant microorganism containing B1) the nucleic acid molecule, or a recombinant microorganism containing B2) the expression cassette, or a recombinant microorganism containing B3) the recombinant vector.

2. Use according to claim 1, characterized in that: B1) the nucleic acid molecule is a cDNA molecule or DNA molecule shown as a sequence 1 in a sequence table.

3. A method for producing an early flowering plant, comprising increasing the expression level of the protein of claim 1 or a gene encoding the protein in a target plant to obtain an early flowering plant; the flowering time of the early flowering plant is earlier than that of the target plant, and the target plant is a dicotyledonous plant.

4. The method of claim 3, wherein: the improvement of the expression level of the protein of claim 1 or the gene encoding the protein in the target plant is achieved by introducing the gene encoding the protein of claim 1 into the target plant, which is a dicotyledonous plant.

5. The use according to any one of claims 1-2, or the method according to claim 3 or 4, wherein: the plant according to any one of claims 1 to 2, the plant of interest according to claim 3 or claim 4 is Nicotiana benthamiana.

Images