CN110358774B - Gene, protein, gene expression cassette, expression vector, host cell, method and application for controlling rice flowering time - Google Patents

Abstract

The invention discloses a gene, a protein, a gene expression cassette, an expression vector, a host cell, a method and application for controlling the flowering time of rice. The invention provides a sequence of an OsFBO14 gene and a method for reducing the expression of the OsFBO14 gene by an RNAi technical means so as to improve the flowering time of rice. The growth period of the rice can be artificially adjusted by increasing or reducing the expression quantity of the OsFBO14 gene according to the climate characteristics of a planting place, so that the ecological adaptability of the rice is improved.

Description

Gene, protein, gene expression cassette, expression vector, host cell, method and application for controlling rice flowering time

Technical Field

The invention belongs to the field of molecular genetics, and particularly relates to a gene, a protein, a gene expression cassette, an expression vector, a host cell, a method and application for controlling rice flowering time.

Background

In higher plants, flowering represents a transition from vegetative to reproductive growth, which plays an important role throughout the growth and development stages of the plant. The biological character of when to bloom is subjected to the dual functions of the genetic factors of the plant body and the external environmental factors. Under the influence of the dual action, a series of flowering induction processes are common in higher plants, namely, the leaves of the plants generate flowering substances (or florigen) at proper time by sensing external growth conditions (light, temperature, humidity and the like), and the flowering substances are conveyed to the stem tip growing point from the leaves through the conduction tissues to stimulate the apical meristem to flower.

The flowering phase is an important character in the rice evolution and adaptation process, the understanding of the genetic basis of the character of the rice flowering phase and the cloning of candidate genes can improve the environmental adaptability and plasticity of the rice, which has important significance for cultivating excellent rice varieties adapting to different ecological regions, and simultaneously, the genetic improvement process of important production characters such as yield and the like closely related to the flowering phase can be promoted. In addition, the overall growth period of the rice is mainly determined by the length of the flowering period, and the overall growth period can be reduced by appropriately shortening the flowering period, so that the planting cost is reduced.

F-box proteins are members of a family of proteins containing 40-50 conserved amino acid F-box domains, widely found in eukaryotes. In the ubiquitin-proteasome pathway (UPP), F-box protein participates in life activities such as cell cycle regulation, transcription regulation, apoptosis, cell signal transduction, etc. due to specific recognition of substrate protein. The F-box protein family is one of the largest, and most rapidly evolving, families in plants. The genes encoding F-BOX proteins in rice are up to 687 (Jain M, Nijhawan A, Arora R, et al. F-BOX proteins in rice genome-wide analysis, classification, temporal and spatial gene expression and growth of rice, and regulation by light and antigenic stress [ J ]. Plant physical, 2007,143(4):1467-83.) they have regulatory effects on the development, seed germination and cytokinin signals of leaves, roots and floral organs of rice (Schopper, Zuille. F-BOX protein regulating inclination of leaves and its application: Chinese patent, CN 108479 [ P ], 2018-11-02.; Song S, Dai X, Zhang W H.A, Kivoid and growth [ J-78 ] J-gene, growth and growth H-352 ],59, kieber J, and Schaller G E.the rice F-box protein KISS ME DEADLY2functions as a negative regulator of cell signaling [ J ]. Plant Signal Behav,2013,8(12): e26434.; yan Y S, Chen X Y, Yang K, et al.overexpression of an F-box protein genes involved in biological stress tolerance and proteins root growth in rice [ J ]. Mol Plant,2011,4(1): 190-7.; li L, Li Y, Song S, et al. Anher reduction F-box (ADF) protein regulated by tape production reduction (TDR) control edge anti-reduction [ J ] plant, 2015,241(1): 157-66.). Since there are many F-box protein genes in rice, the functions of many members are still unknown.

The invention discovers the regulation and control effect of the OsFBO14 gene on rice flowering time by inhibiting the expression level of the rice F-box protein coding gene OsFBO14 by an RNAi method. By using the OsFBO14-RNAi vector and the RNAi method, the flowering time of rice can be advanced, so that the rice growth period can be shortened, the planting cost can be reduced, and the ecological adaptability of rice can be improved.

Disclosure of Invention

In order to achieve the above objects, according to one aspect of the present invention, there is provided a gene (OsFBO14) for controlling flowering-time in rice, comprising: comprises SEQ ID NO.1 or a complementary sequence thereof, and/or SEQ ID NO.3 or a complementary sequence thereof.

In another aspect, the present invention provides a protein, characterized in that: the amino acid sequence of the protein is shown as SEQ ID NO. 2; the amino acid sequence of the protein is a sequence which is shown in SEQ ID NO.2, is subjected to substitution and/or deletion and/or addition of one or more amino acids, and has the same function as the sequence shown in SEQ ID NO. 2.

In still another aspect, the present invention provides a gene expression cassette comprising the above nucleic acid sequence, characterized in that: the expression cassette comprising the nucleic acid sequence of claim 1 and its reverse complement; the nucleic acid sequence and the reverse complementary sequence are separated by a rice waxy-a intron 1 sequence; the nucleic acid sequence, the reverse complementary sequence and the rice waxy-a intron 1 sequence are operably linked with a ubiquitin promoter and a CaMV35S terminator.

In still another aspect, the present invention provides an expression vector comprising the above gene expression cassette, characterized in that: the vector also comprises a CaMV35S hygromycin CaMV35S polyA expression cassette and a CaMV35S GUS nos expression cassette.

In still another aspect, the present invention provides a host cell comprising the above expression vector, characterized in that: the host cell is a plant cell or a prokaryotic cell; optionally, the host cell is an escherichia coli or agrobacterium cell.

In yet another aspect, the present invention provides a method of producing a transgenic crop characterized by: the method is characterized in that the expression vector or the host cell is used for transforming crops to obtain transgenic crops.

In yet another aspect, the present invention provides a method of producing transgenic seed, characterized in that: transgenic plants produced using the above methods produce transgenic seed.

In yet another aspect, the present invention provides the use of a nucleic acid sequence, protein, gene expression cassette, expression vector, host cell, method of producing a transgenic crop, as described above, for altering flowering time in a crop.

Further, the crop is rice; the manner of changing the flowering time of the crop is early.

The amino acid sequence of the protein and the protein in the invention is shown in SEQ ID NO. 2; in some embodiments, the amino acid sequence of the protein is a sequence represented by SEQ ID No.2, which is substituted and/or deleted and/or added with one or more amino acids and has the same function as the sequence represented by SEQ ID No. 2.

The above-mentioned gene expression cassette of the present invention, which comprises the above-mentioned nucleic acid sequence and its reverse complement; in some embodiments, the nucleic acid sequence and its reverse complement are separated by a rice waxy-a intron 1 sequence; in some embodiments, the nucleic acid sequence, the reverse complement sequence, the rice wax-a intron 1 sequence are operably linked to a ubiquitin promoter and the CaMV35S terminator.

The expression vector of the present invention comprises the above expression cassette; in some embodiments, the vector further comprises a CaMV35S: hygromycin: CaMV35S polyA expression cassette and a CaMV35S: GUS: nos expression cassette.

The host cell of the present invention comprises the above expression vector; in some embodiments, the host cell is a plant cell or a prokaryotic cell; in some embodiments, the host cell is an escherichia coli or agrobacterium cell.

In the method for producing a transgenic crop of the present invention, a crop is transformed with the above expression vector or the above host cell to obtain a transgenic crop.

As described above, the method for producing transgenic seed according to the present invention produces transgenic seed from the transgenic crop produced by the above method.

The application of the nucleic acid, the protein, the gene expression cassette, the expression vector, the host cell and the method in the invention in changing the flowering time of crops; in some embodiments, the crop is rice; in some embodiments, the manner of altering the flowering time of the crop is early.

The invention has the following advantages and beneficial effects: the function of the OsFBO14 gene in regulating flowering-time has not been reported previously. The invention can artificially adjust the growth period of rice by increasing or reducing the expression quantity of OsFBO14 gene according to the climate characteristics of the planting site, thereby improving the ecological adaptability of rice. In addition, the overall growth period of the rice is mainly determined by the length of the flowering period, and the overall growth period can be reduced by appropriately shortening the flowering period, so that the planting cost is reduced.

Drawings

FIG. 1 is a schematic diagram showing the domain distribution of a protein encoded by OsFBO14 gene of the present invention and the position of RNAi interference fragment.

FIG. 2 is a diagram of an OsFBO14-pDS1301 vector of the invention.Wherein English and each abbreviation of each element are listed as follows:

FIG. 3 shows the detection of OsFBO14 gene expression in OsFBO14-RNAi material of the present invention.Wherein ZH 11-1: in the first part Flower 11 wild type rice; ZH 11-2: second medium 11 wild type rice; R1-R13: RNAi (ribonucleic acid interference) implantation of different OsFBO14 genes And (4) strain.

FIG. 4 shows the heading stage expression of OsFBO14-RNAi material of the present invention.Wherein, ZH 11: middle 11 wild type rice; r3, R11: different OsFBO14 gene RNAi plants. Photographs were taken 73 days after sowing.

Detailed Description

The following definitions and methods are provided to better define the present application and to guide those of ordinary skill in the art in the practice of the present application. Unless otherwise indicated, terms are to be understood in accordance with their ordinary usage by those of ordinary skill in the relevant art.

As used herein, the term "rice" refers to rice (Oryza sativa) and includes all plant species capable of reproduction with rice, including wild rice species as well as those plants belonging to the genus Oryza that allow for inter-species reproduction.

Unless otherwise indicated, nucleic acids are written from left to right in the 5 'to 3' direction; amino acid sequences are written from left to right in the amino to carboxy direction. Amino acids may be referred to herein by their commonly known three letter symbols or by the one letter symbols recommended by the IUPAC-IUB Biochemical nomenclature Commission. Similarly, nucleotides may be represented by commonly accepted single-letter codes. Numerical ranges include the numbers defining the range. As used herein, "nucleic acid" includes reference to deoxyribonucleotide or ribonucleotide polymers in either single-or double-stranded form, and unless otherwise limited, includes known analogs (e.g., peptide nucleic acids) having the basic properties of natural nucleotides that hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides. As used herein, the term "encode" or "encoded" when used in the context of a particular nucleic acid means that the nucleic acid contains the necessary information to direct translation of the nucleotide sequence into a particular protein. The information encoding the protein is represented using a codon. As used herein, "full-length sequence" in reference to a particular polynucleotide or protein encoded thereby refers to the entire nucleic acid sequence or the entire amino acid sequence having a native (non-synthetic) endogenous sequence. The full-length polynucleotide encodes the full-length, catalytically active form of the particular protein. The terms "polypeptide," "polypeptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The term is used for amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding naturally occurring amino acids. The term is also used for naturally occurring amino acid polymers. The terms "residue" or "amino acid" are used interchangeably herein to refer to an amino acid that is incorporated into a protein, polypeptide, or peptide (collectively, "protein"). The amino acid can be a naturally occurring amino acid, and unless otherwise limited, can include known analogs of natural amino acids that can function in a similar manner as naturally occurring amino acids.

The term "trait" refers to a physiological, morphological, biochemical or physical characteristic of a plant or a particular plant material or cell. In some cases, this property is visible to the human eye, such as seed or plant size, or can be measured by biochemical techniques, such as detecting the protein, starch or oil content of the seed or leaf, or by observing metabolic or physiological processes, for example by measuring tolerance to water deprivation or specific salt or sugar or nitrogen concentrations, or by observing the expression levels of one or more genes, or by agronomic observations such as osmotic stress tolerance or yield.

By "transgenic" is meant any cell, cell line, callus, tissue, plant part or plant whose genome has been altered by the presence of a heterologous nucleic acid (such as a recombinant DNA construct). The term "transgene" as used herein includes those initial transgenic events as well as those generated by sexual crosses or asexual propagation from the initial transgenic events and does not encompass genomic (chromosomal or extra-chromosomal) alteration by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non-recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition or spontaneous mutation.

"plant" includes reference to whole plants, plant organs, plant tissues, seeds, and plant cells, and progeny of same. Plant cells include, but are not limited to, cells from seeds, suspension cultures, embryos, meristematic regions, callus tissue, leaves, roots, shoots, gametophytes, sporophytes, pollen, and microspores. "progeny" comprises any subsequent generation of the plant.

In this application, the words "comprise", "comprising" or variations thereof are to be understood as embracing elements, numbers or steps in addition to those described. By "subject plant" or "subject plant cell" is meant a plant or plant cell in which the genetic modification has been effected, or a progeny cell of the plant or cell so modified, which progeny cell comprises the modification. The "control" or "control plant cell" provides a reference point for measuring the phenotypic change of the test plant or plant cell.

Negative or control plants may include, for example: (a) a wild-type plant or cell, i.e., a plant or cell having the same genotype as the starting material for the genetic alteration that produced the test plant or cell; (b) plants or plant cells having the same genotype as the starting material but which have been transformed with an empty construct (i.e., a construct that has no known effect on the trait of interest, such as a construct comprising a target gene); (c) a plant or plant cell that is a non-transformed isolate of a subject plant or plant cell; (d) a plant or plant cell that is genetically identical to the subject plant or plant cell but that has not been exposed to conditions or stimuli that induce expression of the gene of interest; or (e) the subject plant or plant cell itself, under conditions in which the gene of interest is not expressed.

Those skilled in the art will readily recognize that advances in the field of molecular biology, such as site-specific and random mutagenesis, polymerase chain reaction methods, and protein engineering techniques, provide a wide range of suitable tools and procedures for engineering or engineering amino acid sequences and potential gene sequences of proteins of agricultural interest.

In some embodiments, changes may be made to the nucleotide sequences of the present application to make conservative amino acid substitutions. The principles and examples of conservative amino acid substitutions are further described below. In certain embodiments, substitutions that do not alter the amino acid sequence of the nucleotide sequences of the present application can be made in accordance with the codon preferences disclosed for monocots, e.g., codons encoding the same amino acid sequence can be substituted with monocot preferred codons without altering the amino acid sequence encoded by the nucleotide sequence. In some embodiments, a portion of the nucleotide sequence in this application is replaced with a different codon that encodes the same amino acid sequence, such that the nucleotide sequence is not altered while the amino acid sequence encoded thereby is not altered. Conservative variants include those sequences that, due to the degeneracy of the genetic code, encode the amino acid sequence of one of the proteins of the embodiments. In some embodiments, a partial nucleotide sequence herein is replaced according to monocot preferred codons. One skilled in the art will recognize that amino acid additions and/or substitutions are generally based on the relative similarity of the amino acid side-chain substituents, e.g., hydrophobicity, charge, size, etc., of the substituents. Exemplary amino acid substituent groups having various of the foregoing properties are known to those skilled in the art and include arginine and lysine; glutamic acid and aspartic acid; serine and threonine; glutamine and asparagine; and valine, leucine and isoleucine. Guidance regarding suitable amino acid substitutions that do not affect the biological activity of the Protein of interest can be found in the model of the Atlas of Protein sequences and structures (Protein Sequence and Structure Atlas) (Natl. biomed. Res. Foundation, Washington, D.C.) (incorporated herein by reference). Conservative substitutions such as exchanging one amino acid for another with similar properties may be made. Identification of sequence identity includes hybridization techniques. For example, all or part of a known nucleotide sequence is used as a probe for selective hybridization to other corresponding nucleotide sequences present in a population of cloned genomic DNA fragments or cDNA fragments (i.e., a genomic library or cDNA library) from a selected organism. The hybridization probes may be genomic DNA fragments, cDNA fragments, RNA fragments, or other oligonucleotides, and may be labeled with a detectable group such as 32P or other detectable marker. Thus, for example, hybridization probes can be prepared by labeling synthetic oligonucleotides based on the sequence of the embodiment. Methods for preparing hybridization probes and constructing cDNA and genomic libraries are generally known in the art. Hybridization of the sequences may be performed under stringent conditions. As used herein, the term "stringent conditions" or "stringent hybridization conditions" refers to conditions under which a probe will hybridize to its target sequence to a detectably greater degree (e.g., at least 2-fold, 5-fold, or 10-fold over background) than to other sequences. Stringent conditions are sequence dependent and differ in different environments. By controlling the stringency of hybridization and/or the washing conditions, target sequences can be identified that are 100% complementary to the probes (homologous probe method). Alternatively, stringency conditions can be adjusted to allow some sequence mismatches in order to detect lower similarity (heterologous probe method). Typically, probes are less than about 1000 or 500 nucleotides in length. Typically, stringent conditions are conditions in which the salt concentration is less than about 1.5M Na ion, typically about 0.01M to 1.0M Na ion concentration (or other salt) at pH 7.0 to 8.3, and the temperature conditions are: when used with short probes (e.g., 10 to 50 nucleotides), at least about 30 ℃; when used with long probes (e.g., greater than 50 nucleotides), at least about 60 ℃. Stringent conditions may also be achieved by the addition of destabilizing agents such as formamide. Exemplary low stringency conditions include hybridization at 37 ℃ using 30% to 35% formamide buffer, 1M NaCl, 1% SDS (sodium dodecyl sulfate), washing at 50 ℃ to 55 ℃ in1 × to 2 × SSC (20 × SSC ═ 3.0M NaCl/0.3M trisodium citrate). Exemplary moderately stringent conditions include hybridization in 40% to 45% formamide, 1.0M NaCl, 1% SDS at 37 ℃ and washing in 0.5X to 1 XSSC at 55 ℃ to 60 ℃. Exemplary high stringency conditions include hybridization in 50% formamide, 1M NaCl, 1% SDS at 37 deg.C, and a final wash in 0.1 XSSC at 60 deg.C to 65 deg.C for at least about 20 minutes. Optionally, the wash buffer may comprise about 0.1% to about 1% SDS. The duration of hybridization is generally less than about 24 hours, and typically from about 4 hours to about 12 hours. Specificity usually depends on the post-hybridization wash, the critical factors being the ionic strength and temperature of the final wash solution. The Tm (thermal melting point) of a DNA-DNA hybrid can be approximated by the formula of Meinkoth and Wahl (1984) anal. biochem.138: 267-284: tm 81.5 ℃ +16.6(logM) +0.41 (% GC) -0.61 (% formamide) -500/L; where M is the molar concentration of monovalent cations,% GC is the percentage of guanosine and cytosine nucleotides in the DNA,% formamide is the percentage formamide of the hybridization solution, and L is the base pair length of the hybrid. The Tm is the temperature (under defined ionic strength and pH) at which 50% of a complementary target sequence hybridizes to a perfectly matched probe. Washing is typically performed at least until equilibrium is reached and a low background level of hybridization is achieved, such as for 2 hours, 1 hour, or 30 minutes. Decrease Tm by about 1 ℃ per 1% mismatch; thus, Tm, hybridization and/or wash conditions can be adjusted to hybridize to sequences of desired identity. For example, if a sequence with > 90% identity is desired, the Tm can be lowered by 10 ℃. Typically, stringent conditions are selected to be about 5 ℃ lower than the Tm for the specific sequence and its complement under defined ionic strength and pH. However, under very stringent conditions, hybridization and/or washing can be performed at 4 ℃ below the Tm; hybridization and/or washing may be performed at 6 ℃ below the Tm under moderately stringent conditions; under low stringency conditions, hybridization and/or washing can be performed at 11 ℃ below the Tm.

In some embodiments, fragments of the nucleotide sequences and the amino acid sequences encoded thereby are also included. As used herein, the term "fragment" refers to a portion of the nucleotide sequence of a polynucleotide or a portion of the amino acid sequence of a polypeptide of an embodiment. Fragments of the nucleotide sequences may encode protein fragments that retain the biological activity of the native or corresponding full-length protein, and thus have protein activity. Mutant proteins include biologically active fragments of the native protein that comprise contiguous amino acid residues that retain the biological activity of the native protein. Some embodiments also include a transformed plant cell or transgenic plant comprising the nucleotide sequence of at least one embodiment. In some embodiments, plants are transformed with an expression vector comprising at least one embodiment of the nucleotide sequence and operably linked thereto a promoter that drives expression in plant cells. Transformed plant cells and transgenic plants refer to plant cells or plants that comprise a heterologous polynucleotide within their genome. Generally, the heterologous polynucleotide is stably integrated within the genome of the transformed plant cell or transgenic plant such that the polynucleotide is transmitted to progeny. The heterologous polynucleotide may be integrated into the genome alone or as part of an expression vector. In some embodiments, the plants to which the present application relates include plant cells, plant protoplasts, plant cell tissue cultures from which plants can be regenerated, plant calli, plant clumps, and plant cells, which are whole plants or parts of plants, such as embryos, pollen, ovules, seeds, leaves, flowers, branches, fruits, nuts, ears, cobs, husks, stalks, roots, root tips, anthers, and the like. The present application also includes plant cells, protoplasts, tissues, calli, embryos, and flowers, stems, fruits, leaves, and roots derived from the transgenic plants of the present application or progeny thereof, and thus comprising at least in part the nucleotide sequences of the present application.

Unless otherwise indicated, the examples follow conventional experimental conditions, such as the Molecular cloning Manual of Sambrook et al (Sambrook J & Russell DW, Molecular cloning: a laboratory Manual,2001), or following the conditions suggested by the manufacturer's instructions. Unless otherwise specified, the chemical reagents used in the examples are all conventional commercially available reagents, and the technical means used in the examples are conventional means well known to those skilled in the art.

Example one rice OsFBO14 gene sequence and interference fragment sequence

The coding region sequence of the rice OsFBO14(LOC _ Os03g27250) gene is shown in SEQ ID NO.1, the coded protein comprises a F-box structural domain (figure 1), and the sequence is shown in SEQ ID NO. 2. No data is available to show the function of the gene in rice and the specific character controlled by the gene. In order to determine the function of the gene in rice, the invention aims to reduce the expression level of the OsFBO14 gene in rice by adopting an RNAi method. The invention selects the region between 1115-1529 nucleotides of the sequence shown in SEQ ID NO.1 as the interference fragment for constructing the RNAi carrier (the sequence is shown in SEQ ID NO. 3).

Example two construction of rice OsFBO14 Gene RNAi vector.

Primers (SEQ ID NO.4 and SEQ ID NO.5) are designed according to the full-length cDNA sequence of OsFBO14, and an OsFBO14 interference fragment with a restriction enzyme cutting site is obtained by PCR amplification. Constructing the interference fragment and the reverse complementary sequence of the fragment on an expression vector, and specifically comprising the following steps:

1. the OsFBO14 interference fragment is cut by KpnI and BamHI, a target band is recovered, and is connected with an expression vector plasmid pDS1301(Chu et al, Promoter activities of an essential gene for polar definition in a gene in place Dev,2006,20: 1250. 1255.) cut by KpnI and BamHI (all the endonucleases are purchased from TAKARA company, the usage and the dosage are according to the product instruction of the company; the ligases are products of invitrogen company, the usage and the dosage are according to the product instruction of the company);

2. the ligation product was introduced into DH10B (purchased from Promega, Inc.) by an electrotransformation method (an electrotransformation apparatus is manufactured by eppendorf corporation, the voltage used was 1800v, the operation method is described in the apparatus manual), and cultured on a resistant medium containing 250ppm of kanamycin (Roche, Inc.) in LA (LA formulation, see J. SammBruke, EF Frizi, T Mannich Abies, Huangpetang, Wangjia, et al, molecular cloning instructions (third edition), scientific Press, 2002 edition);

3. a single colony grown on LA resistance medium was inoculated on a clean bench into a sterilized 10mL centrifuge tube, 3mL of LB resistance medium containing 250ppm kanamycin was previously added to the tube, and then cultured on a shaker at 37 ℃ for 16 to 18 hours. Plasmids were extracted as described in (molecular cloning, A laboratory Manual, J. SammBruk and D.W. Lassel, Huangpetang et al, science publishers, 2002), cleaved with KpnI and BamHI and electrophoretically detected to obtain a positive plasmid vector pDS1301-FBO14-1, depending on the size of the insert;

4. cutting the OsFBO14 interference fragment by SpeI and SacI, recovering a target band, connecting the target band with a SpeI and SacI cut plasmid pDS1301-FBO14-1, and obtaining an expression inhibiting vector according to the steps of 2) and 3): pDS1301-HDT 1-2;

5. the newly constructed expression vector pDS1301-FBO14-2 was introduced into Agrobacterium EHA105 (available from CAMBIA) strain by electrotransformation (references and voltage parameters used as described above).

The vector map of OsFBO14-pDS1301 is shown in FIG. 2.

EXAMPLE III preparation of rice OsFBO14 Gene RNAi transgenic plant

The invention adopts an agrobacterium-mediated method to transfer an OsFBO14 gene expression vector into rice, and the agrobacterium strain is EHA 105. Rice seeds are sterilized, callus is induced, then co-cultured with Agrobacterium to infect the callus, the transformed callus is selected by selection culture, and then plant regeneration is performed on the selection medium. The specific process is as follows:

1. inducing the rice callus: taking mature seeds of the rice of the Zhonghua 11(ZH11) (provided and bred by the crop science research institute of Chinese academy of agricultural sciences), manually or mechanically shelling, selecting full, smooth and plaque-free seeds, putting the seeds into a 100ml sterile beaker, and pouring 70% alcohol (15ml) for disinfection for 2 min; pouring off alcohol, adding 100ml 30% sodium hypochlorite (NaClO) solution, and soaking for 30 min; the sodium hypochlorite solution was poured off, the seeds were washed 4-5 times with sterile distilled water and soaked for the last 30 min. Putting the seeds on sterile filter paper, sucking the seeds to be dry, and putting the seeds into a mature embryo induction culture medium, wherein 20-30 seeds are placed in each dish; after the completion of the operation, the culture dish was sealed with a sealing film (Micropore (TM) protective Tape) and cultured in an incubator at 28 ℃. Inducing the callus under the dark culture condition for 7-10 days; the culture dish was opened on an ultraclean bench, and the naturally-divided embryogenic callus (pale yellow, dense and spherical) was picked up with forceps and placed in a subculture medium and subcultured in the dark at 28 ℃ for 1 week.

2. And (3) agrobacterium culture: and selecting the agrobacterium tumefaciens monoclonals transformed with the target expression vector to be placed in 15ml of YEP culture solution (containing corresponding antibiotics), and carrying out shaking culture at the temperature of 28 ℃ and the rpm of 250 for 12-16 h until the OD600 of the bacterial solution is 0.8-1.0.

3. Co-culture and selection of resistant calli: the cultured bacterial liquid is centrifuged at 4000rpm for 10min at room temperature, and the supernatant is removed. Picking out the rice callus growing to a certain size, putting the rice callus into the agrobacterium tumefaciens suspension, and co-culturing for 30min at 80rpm on a horizontal shaker; taking out the callus, placing on sterile filter paper, and draining for 30-40 min; the callus was placed on a co-culture medium with a sterile filter paper and cultured in the dark at 25 ℃ for 3 days.

4. Selecting and culturing: taking out the callus, and washing with sterile water for 5-6 times without oscillating. Washing with 300mg/L carbenicillin sodium (Carb) in sterile water for 2 times, shaking on a horizontal shaker for 30min, and draining on sterile filter paper for 2 hr. The air-dried calli were transferred to selection medium containing 300mg/L carbenicillin sodium (Carb) and corresponding selection pressure, and cultured in dark at 28 ℃ for 14 days for two rounds of selection until granule-resistant calli grew out.

5. Induced differentiation and rooting of resistant callus: 3-5 yellow resistant calli from different calli are picked, transferred into a plastic wide-mouth bottle filled with a differentiation medium, sealed by a sealing film, placed into a constant-temperature (25 ℃) culture chamber (16h/8h), and waiting for differentiation into seedlings (about 40 days). After the seedling grows to about 3cm, old roots and callus are cut off from the base of the seedling by scissors and placed in a rooting medium to strengthen the seedling (about 1 week).

6. Hardening and transplanting seedlings: picking out test tubes with well-differentiated seedling roots and stems and leaves, opening a sealing film, adding a proper amount of distilled water or sterile water (for preventing the growth of bacteria in a culture medium), hardening seedlings for 2-3 days, then washing off agar, and transplanting the seedlings into a soil pot of a greenhouse.

Example four OsFBO14 gene expression level in RNAi rice material was examined.

And detecting the transcription level expression quantity of the OsFBO14 gene in the obtained 13 RNAi rice materials (R1-R13 respectively) by adopting a qRT-PCR method. The method comprises the following steps:

1. and (4) extracting RNA. 1) Placing about 25-50mg of tissue into a homogenizer, adding 1mL of Trizol reagent for homogenizing, and transferring into a 1.5mL centrifuge tube. 2) Centrifuging at 12000g for 15min at 2-8 deg.C, and transferring the supernatant into a new centrifuge tube. 3) After incubating the supernatant at room temperature for 5min, chloroform was added at a rate of 0.2mL per 1mL Trizol reagent, the tube was closed, the tube was shaken vigorously by a vortex apparatus for 15sec, and incubated at room temperature for 2-3 min. 3) Centrifuging at 12000g for 15min at 2-8 deg.C, and separating the mixture into red phenol-chloroform layer as the lower layer and colorless water phase as the upper layer (RNA in water phase). 4) The aqueous phase was transferred to a new centrifuge tube using a pipette tip and the RNA precipitated by adding isopropanol to 1mL Trizol plus 0.5mL isopropanol and incubated at room temperature for 10 min. 5) Centrifuging at 12000g for 10min at 2-8 deg.C. 6) Removing supernatant, adding 75% ethanol into 1mL Trizol solution at a ratio of at least 1mL, washing, vortexing, and centrifuging at 2-8 deg.C and 7500g for 5 min. 7) The supernatant is carefully discarded, and then air-or vacuum-dried for 5-10min, 30-40. mu.L RNase-free (RNase-free) water is added, and after sucking several times with a pipette tip, the mixture is incubated at 55-60 ℃ for 10min to dissolve RNA. 8) And (3) electrophoresis detection: 2-5 μ L of total RNA extracted was electrophoretically detected.

2. And (3) reverse transcription PCR. 1) The DNase treatment was performed according to the instruction of DNase1Amplification Grade Kit (Invitrogen). 2) First Strand cDNA Synthesis was performed according to Roche translator First Strand cDNA Synthesis kit instructions. 3) Realtime-PCR. The rice internal reference gene Actin1 is used as an internal reference, primers SEQ ID NO.6 and SEQ ID NO.7 are designed, expression detection primers SEQ ID NO.8 and SEQ ID NO.9 of OsFBO14 gene are designed, and real-PCR is respectively carried out, wherein the reaction system is as follows:

note: primer dilution 10. mu.L of the stock solution was diluted to 600. mu.L.

The PCR procedure was: at 95 ℃ for 10 min; (95 ℃, 10 sec; 60 ℃, 55 sec). times.40 cycles.

After the Real time-PCR reaction is finished, calculating the relative expression RQ value of the corresponding sample according to the average Ct (amplification cycle number) value of the internal reference gene and the target gene generated by the instrument and a formula RQ ^ 2 (-delta Ct), namely the multiple of the balanced internal reference gene relative to the control expression.

The results of the assay are shown in FIG. 3. With ZH11-1 as a control reference, except for R7 material, the OsFBO14 gene in the rest RNAi plants All the expression levels were reduced to different degrees. Seeds of the R1-R13 plants were harvested.

Example five OsFBO14 Gene RNAi Rice Material Performance.

When seeds of R3 and R11 were planted and the resulting transgenic plants were subjected to phenotypic observation, it was found that the flowering (heading) time of the R3 and R11 plants was significantly earlier than that of the control flower 11(ZH11) (growth environment: seeding in Wuhan 5 middle ten days in 2017 and 2018, seedling transplanting No.6 month 12, in the university of agriculture test field in Huazhong), and the results are shown in FIG. 4. The character survey result shows that the flowering time of ZH11 is 77.8 days, and the flowering time of R3 and R11 plants is 71.5 days, and the result is shown in Table 1. The rice flowering can be earlier caused by the reduction of the expression level of the OsFBO14 gene, which shows that the OsFBO14 gene has a regulation and control effect on rice flowering time. The rice flowering time is advanced, so that the rice growth period is shortened, and the planting cost is reduced; meanwhile, the OsFBO14 gene has a regulation and control effect on rice flowering time, so that the rice growth period can be artificially adjusted by increasing or reducing the expression level of the OsFBO14 gene according to the climate characteristics of a planting place, and the ecological adaptability of rice is improved.

TABLE 1 flowering time of OsFBO14-RNAi Material

The significance of the difference in flowering-time data (α ═ 0.05) was examined by the LSD method, indicating a significant difference in flowering-time compared to ZH11 (p < 0.05).

Sequence listing

<110> university of Jianghan

<120> genes, proteins, gene expression cassettes, expression vectors, host cells, methods and uses for controlling flowering time of rice

<160> 9

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2862

<212> DNA

<213> Oryza sativa

<400> 1

atggcggcgg ggaacggccg gatggaggcg gcgctgggct gcctcgcggc gctccccgac 60

gaggtgctct gcgccgtcgt cgacctcctc ccgcccaccg acgtcggccg cctcgcctgc 120

gtcagcagtg tcatgtacat actttgcaat gaggagcctc tctggatgag caagtgtctt 180

tcagttgggg gtcttcttgt gtatagaggt tcttggaaga aaacagcatt gtctagactt 240

aatctttgtt cagaaaatga tgagatttac cagaagcctc gccattttga tgggttcaat 300

tccatgcact tatacaggag atggtacaga tgttttacta atttgagtag cttttccttt 360

gataatgggc acgttgaaag gaaagatgac ctttctctag accaatttcg cgctcagtat 420

gatggaaaat gtccagtttt gcttactaaa ctggctgaaa cctggccagc aaggactaaa 480

tggacagcgc agcaactgac acatgattat ggtgaagttc cctttaggat atctcagaga 540

agccctcaaa agataaaaat gaaactaaaa gattatgttt tttacatgga actccagcat 600

gacgaagatc cactttacat atttgatgat aagtttggag aatcagcacc tacactattg 660

gaagattaca gtgtccctca tctatttcaa gaagatttct ttgaaatcat ggattacgac 720

caacgaccag ctttcagatg gcttattatt ggaccagaga gatcaggtgc ttcttggcat 780

gttgatccag ggttgaccag tgcctggaat actcttcttt gtggccgaaa aaggtgggca 840

atgtaccctc ctggaagagt accaggtggt gtcacagtac atgtcagtga tgaagatggt 900

gatgttgaca ttgaaactcc tacatctttg cagtggtggc tagatatcta cccaaatctt 960

gctgagcatg agaaaccact ggaatgcaca caattaccag gagagaccat atttgttcct 1020

agtgggtggt ggcattgtgt tttgaacctt gacatgacaa ttgctgtcac gcaaaatttt 1080

gtcaaccaat caaattttaa gcatgtatgt ttggacatgg cacctggtta ctgtcacaaa 1140

ggagtttgcc gtgctggctt acttgctgct ccagacaaat ctattagaga tattgaaaat 1200

cttcctagta taacgagtag attgaaccac tctgacatgg cctgtaagga aaaaagactg 1260

aaaagttcag agcctataag aacttcaaat aatgcaaatc agtgttctgc atttgagttc 1320

tcagatgttc atgaaaactt gggggaccaa gttttttcgt atgatataga tttcttatcc 1380

caattccttg agaaagaaaa ggatcactat tcttctgtct ggagccctac taattcaatt 1440

ggccagagag aagcaagaga atggctacgt aggctatggg ttcttaaacc tgaattgaga 1500

gaactaatat ggaagggtgc atgtctagca attaatgtag acaagtggta ttcatgctta 1560

gaggaaataa gtgcatgcca tagtttacca ccaccttctg aagatgagaa gcttcctgtt 1620

ggcacaggta gcaacccagt cttcattgtt tctggcaatg tgatcaaaat ttatgctgaa 1680

ggagggttgg gttattctat acatggtttg ggcacagagc ttgagttcta tgatcttctg 1740

caaaaacttg gctcgccatt gatcaaccat gtccctgaga tcattgcaag tggctttctt 1800

gtgtacctgg atggtgtcta caagacagtt ccatgggatg gaaacggaat accagatgtt 1860

ctagctaaat actactcttt ggaggtgtct tatgcaaacg gctcttttcc tcttggatta 1920

tggagcaagc aactgtttgg attgagtaat tcaactgatg ctccagacag accaatttgt 1980

ccttacatgg ttaccagaaa atgcaaaggg gatatttttg ctcgcatacg tgataaattg 2040

accaagactg atgttttgaa tcttgcatca tccttgggag ttcaaatgcg aaatattcat 2100

caattacccc ttccacatgt ggaacacata tccaaatctg ggaacgaaga tatcaaagca 2160

aaggaaaatt caatttctga tgtcactcat gttccgcctg aatggaaaca agtagtttct 2220

actctagaca ggagaaagaa aagtataaag aagcatctaa gtaactgggg tggttcaatt 2280

ccacaggttc taattgagaa ggctgaagaa tatctccctg acgacatccg ctttcttatc 2340

aagtttgtta aggacgatga tggtgattca gtctatgtgg taccttcttg gatacattca 2400

gatataatgg atgataacat tctcattgag gggaccacag aaccaggaac ttccactgat 2460

tgcattgccg ttgaagatct gaacaaaatg gatgcaattc atatcattga tttcagtgat 2520

ctgtccattg gggatcctct atgtgactta attccactgc acttggatgt attccgtggt 2580

gatattgatc ttctcaggca gtttttacga agctatcagc ttccttttct gagagcagaa 2640

tcaaataaag atatatacaa gtcaatacaa aattctaaat tcagcagggc atcgtatcgt 2700

gcgatgtgct actgcatact tcacgaggac aacgtcctgg gagccatatt tagcctgtgg 2760

aaggatctgg gcaccgcgac gtcatgggaa gatgttgaac acttggtttg gggagagctg 2820

aatcaatacc agcagtcatg cagcgtgggc gaaattaact ga 2862

<210> 2

<211> 953

<212> PRT

<213> Oryza sativa

<400> 2

Met Ala Ala Gly Asn Gly Arg Met Glu Ala Ala Leu Gly Cys Leu Ala

1 5 10 15

Ala Leu Pro Asp Glu Val Leu Cys Ala Val Val Asp Leu Leu Pro Pro

20 25 30

Thr Asp Val Gly Arg Leu Ala Cys Val Ser Ser Val Met Tyr Ile Leu

35 40 45

Cys Asn Glu Glu Pro Leu Trp Met Ser Lys Cys Leu Ser Val Gly Gly

50 55 60

Leu Leu Val Tyr Arg Gly Ser Trp Lys Lys Thr Ala Leu Ser Arg Leu

65 70 75 80

Asn Leu Cys Ser Glu Asn Asp Glu Ile Tyr Gln Lys Pro Arg His Phe

85 90 95

Asp Gly Phe Asn Ser Met His Leu Tyr Arg Arg Trp Tyr Arg Cys Phe

100 105 110

Thr Asn Leu Ser Ser Phe Ser Phe Asp Asn Gly His Val Glu Arg Lys

115 120 125

Asp Asp Leu Ser Leu Asp Gln Phe Arg Ala Gln Tyr Asp Gly Lys Cys

130 135 140

Pro Val Leu Leu Thr Lys Leu Ala Glu Thr Trp Pro Ala Arg Thr Lys

145 150 155 160

Trp Thr Ala Gln Gln Leu Thr His Asp Tyr Gly Glu Val Pro Phe Arg

165 170 175

Ile Ser Gln Arg Ser Pro Gln Lys Ile Lys Met Lys Leu Lys Asp Tyr

180 185 190

Val Phe Tyr Met Glu Leu Gln His Asp Glu Asp Pro Leu Tyr Ile Phe

195 200 205

Asp Asp Lys Phe Gly Glu Ser Ala Pro Thr Leu Leu Glu Asp Tyr Ser

210 215 220

Val Pro His Leu Phe Gln Glu Asp Phe Phe Glu Ile Met Asp Tyr Asp

225 230 235 240

Gln Arg Pro Ala Phe Arg Trp Leu Ile Ile Gly Pro Glu Arg Ser Gly

245 250 255

Ala Ser Trp His Val Asp Pro Gly Leu Thr Ser Ala Trp Asn Thr Leu

260 265 270

Leu Cys Gly Arg Lys Arg Trp Ala Met Tyr Pro Pro Gly Arg Val Pro

275 280 285

Gly Gly Val Thr Val His Val Ser Asp Glu Asp Gly Asp Val Asp Ile

290 295 300

Glu Thr Pro Thr Ser Leu Gln Trp Trp Leu Asp Ile Tyr Pro Asn Leu

305 310 315 320

Ala Glu His Glu Lys Pro Leu Glu Cys Thr Gln Leu Pro Gly Glu Thr

325 330 335

Ile Phe Val Pro Ser Gly Trp Trp His Cys Val Leu Asn Leu Asp Met

340 345 350

Thr Ile Ala Val Thr Gln Asn Phe Val Asn Gln Ser Asn Phe Lys His

355 360 365

Val Cys Leu Asp Met Ala Pro Gly Tyr Cys His Lys Gly Val Cys Arg

370 375 380

Ala Gly Leu Leu Ala Ala Pro Asp Lys Ser Ile Arg Asp Ile Glu Asn

385 390 395 400

Leu Pro Ser Ile Thr Ser Arg Leu Asn His Ser Asp Met Ala Cys Lys

405 410 415

Glu Lys Arg Leu Lys Ser Ser Glu Pro Ile Arg Thr Ser Asn Asn Ala

420 425 430

Asn Gln Cys Ser Ala Phe Glu Phe Ser Asp Val His Glu Asn Leu Gly

435 440 445

Asp Gln Val Phe Ser Tyr Asp Ile Asp Phe Leu Ser Gln Phe Leu Glu

450 455 460

Lys Glu Lys Asp His Tyr Ser Ser Val Trp Ser Pro Thr Asn Ser Ile

465 470 475 480

Gly Gln Arg Glu Ala Arg Glu Trp Leu Arg Arg Leu Trp Val Leu Lys

485 490 495

Pro Glu Leu Arg Glu Leu Ile Trp Lys Gly Ala Cys Leu Ala Ile Asn

500 505 510

Val Asp Lys Trp Tyr Ser Cys Leu Glu Glu Ile Ser Ala Cys His Ser

515 520 525

Leu Pro Pro Pro Ser Glu Asp Glu Lys Leu Pro Val Gly Thr Gly Ser

530 535 540

Asn Pro Val Phe Ile Val Ser Gly Asn Val Ile Lys Ile Tyr Ala Glu

545 550 555 560

Gly Gly Leu Gly Tyr Ser Ile His Gly Leu Gly Thr Glu Leu Glu Phe

565 570 575

Tyr Asp Leu Leu Gln Lys Leu Gly Ser Pro Leu Ile Asn His Val Pro

580 585 590

Glu Ile Ile Ala Ser Gly Phe Leu Val Tyr Leu Asp Gly Val Tyr Lys

595 600 605

Thr Val Pro Trp Asp Gly Asn Gly Ile Pro Asp Val Leu Ala Lys Tyr

610 615 620

Tyr Ser Leu Glu Val Ser Tyr Ala Asn Gly Ser Phe Pro Leu Gly Leu

625 630 635 640

Trp Ser Lys Gln Leu Phe Gly Leu Ser Asn Ser Thr Asp Ala Pro Asp

645 650 655

Arg Pro Ile Cys Pro Tyr Met Val Thr Arg Lys Cys Lys Gly Asp Ile

660 665 670

Phe Ala Arg Ile Arg Asp Lys Leu Thr Lys Thr Asp Val Leu Asn Leu

675 680 685

Ala Ser Ser Leu Gly Val Gln Met Arg Asn Ile His Gln Leu Pro Leu

690 695 700

Pro His Val Glu His Ile Ser Lys Ser Gly Asn Glu Asp Ile Lys Ala

705 710 715 720

Lys Glu Asn Ser Ile Ser Asp Val Thr His Val Pro Pro Glu Trp Lys

725 730 735

Gln Val Val Ser Thr Leu Asp Arg Arg Lys Lys Ser Ile Lys Lys His

740 745 750

Leu Ser Asn Trp Gly Gly Ser Ile Pro Gln Val Leu Ile Glu Lys Ala

755 760 765

Glu Glu Tyr Leu Pro Asp Asp Ile Arg Phe Leu Ile Lys Phe Val Lys

770 775 780

Asp Asp Asp Gly Asp Ser Val Tyr Val Val Pro Ser Trp Ile His Ser

785 790 795 800

Asp Ile Met Asp Asp Asn Ile Leu Ile Glu Gly Thr Thr Glu Pro Gly

805 810 815

Thr Ser Thr Asp Cys Ile Ala Val Glu Asp Leu Asn Lys Met Asp Ala

820 825 830

Ile His Ile Ile Asp Phe Ser Asp Leu Ser Ile Gly Asp Pro Leu Cys

835 840 845

Asp Leu Ile Pro Leu His Leu Asp Val Phe Arg Gly Asp Ile Asp Leu

850 855 860

Leu Arg Gln Phe Leu Arg Ser Tyr Gln Leu Pro Phe Leu Arg Ala Glu

865 870 875 880

Ser Asn Lys Asp Ile Tyr Lys Ser Ile Gln Asn Ser Lys Phe Ser Arg

885 890 895

Ala Ser Tyr Arg Ala Met Cys Tyr Cys Ile Leu His Glu Asp Asn Val

900 905 910

Leu Gly Ala Ile Phe Ser Leu Trp Lys Asp Leu Gly Thr Ala Thr Ser

915 920 925

Trp Glu Asp Val Glu His Leu Val Trp Gly Glu Leu Asn Gln Tyr Gln

930 935 940

Gln Ser Cys Ser Val Gly Glu Ile Asn

945 950

<210> 3

<211> 415

<212> DNA

<213> Artificial sequence (unknown)

<400> 3

acatggcacc tggttactgt cacaaaggag tttgccgtgc tggcttactt gctgctccag 60

acaaatctat tagagatatt gaaaatcttc ctagtataac gagtagattg aaccactctg 120

acatggcctg taaggaaaaa agactgaaaa gttcagagcc tataagaact tcaaataatg 180

caaatcagtg ttctgcattt gagttctcag atgttcatga aaacttgggg gaccaagttt 240

tttcgtatga tatagatttc ttatcccaat tccttgagaa agaaaaggat cactattctt 300

ctgtctggag ccctactaat tcaattggcc agagagaagc aagagaatgg ctacgtaggc 360

tatgggttct taaacctgaa ttgagagaac taatatggaa gggtgcatgt ctagc 415

<210> 4

<211> 34

<212> DNA

<213> Artificial sequence (unknown)

<400> 4

actagtggta ccacatggca cctggttact gtca 34

<210> 5

<211> 33

<212> DNA

<213> Artificial sequence (unknown)

<400> 5

gagctcggat ccgctagaca tgcacccttc cat 33

<210> 6

<211> 21

<212> DNA

<213> Artificial sequence (unknown)

<400> 6

tgtatgccag tggtcgtacc a 21

<210> 7

<211> 19

<212> DNA

<213> Artificial sequence (unknown)

<400> 7

ccagcaaggt cgagacgaa 19

<210> 8

<211> 21

<212> DNA

<213> Artificial sequence (unknown)

<400> 8

cgtgcgatgt gctactgcat a 21

<210> 9

<211> 20

<212> DNA

<213> Artificial sequence (unknown)

<400> 9

gcgtcagtta atttcgccca 20

Claims (1)

1. The application of a gene for controlling the flowering time of rice in advancing the flowering time of rice is characterized in that: the nucleic acid sequence of the gene is SEQ ID number 1; selecting a region between 1115-1529 nucleotides of a sequence shown by SEQ ID number 1 as an interference fragment for constructing an RNAi carrier, wherein the sequence of the interference fragment is shown as SEQ ID number 3.

Images