CN115724927A - Application of tillering regulation gene from corn - Google Patents

Abstract

The invention discloses an application of a tillering regulation gene derived from corn. The invention provides an application of Tin8 protein or related biological materials thereof in regulating and controlling the tillering number and/or flowering period of plants; the related biological material is a nucleic acid molecule capable of expressing the Tin8 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule; the Tin8 protein is a protein with an amino acid sequence of SEQ ID No. 1. The Tin8 gene has great application potential in corn molecular design breeding.

Description

Application of tillering regulation gene from corn

Technical Field

The invention relates to the technical field of biology, in particular to application of a tillering regulation gene derived from corn.

Background

Corn was acclimatized to teosinte in the region of mexico approximately 1 million years ago. A key change in maize domestication is the conversion of multi-tillering of teosinte into a single stalk of modern maize cultivation. The change enhances the close planting resistance of the corn, increases the number of plants per mu and improves the yield per mu. However, when the modern cultivated corn is stressed by the environment (particularly when seedlings are in cold damage), redundant tillering can still be generated to cause yield reduction, so that the fine control of the tillering number of the corn is very important for corn breeding. The genetic diversity of parents in modern corn breeding is low, so that the genetic basis of a modern cultivated corn variety is narrow, the development of corn breeding in China is severely limited, and the utilization of tropical and subtropical corn germplasm resources is an effective way for widening the existing corn germplasm basis. Therefore, the significance of developing other tillering regulation factors capable of widening the corn germplasm genetic basis is great.

Disclosure of Invention

The invention aims to provide application of a tillering regulation gene derived from corn.

In a first aspect, the present invention claims the use of Tin8 protein or its related biomaterials in modulating the tillering number (number of branches) and/or flowering phase of plants.

The related biological material is a nucleic acid molecule capable of expressing the Tin8 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

The expression cassette refers to a DNA capable of expressing Tin8 in a host cell, and the DNA may include not only a promoter that initiates the transcription of the Tin8 gene but also a terminator that terminates the transcription of Tin8. 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; a wounding-inducible promoter from tomato; a chemically inducible promoter from tobacco; tomato proteinase inhibitor II promoter or LAP promoter; a heat shock promoter; a tetracycline-inducible promoter; seed-specific promoters, such as the millet seed-specific promoter pF128, seed storage protein-specific promoters. They can be used alone or in combination with other plant promoters. Suitable transcription terminators include, but are not limited to: an Agrobacterium nopaline synthase terminator (NOS terminator), a cauliflower mosaic virus CaMV 35S terminator, a tml terminator, a pea rbcS E9 terminator and a nopaline and octopine synthase terminator.

Constructing a recombinant expression vector containing the Tin8 gene expression cassette. The plant expression vector can be binary Agrobacterium tumefaciens vector, such as pCAMBIA1305, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb. When using Tin8 to construct recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene ubiqiutin promoter (pUbi), etc. can be added before its transcription initiation nucleotide, and they can be used alone or in combination with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational enhancers or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codons or initiation codons of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper 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 the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc., which can be expressed in plants.

The Tin8 protein is any one of the following proteins:

(A1) Protein with an amino acid sequence of SEQ ID No. 1;

(A2) Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

(A3) A protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) A fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of a protein defined in any one of (A1) to (A3).

In the above protein, the protein tag (protein-tag) refers to a polypeptide or protein that is expressed by fusion with a target protein using in vitro recombinant DNA technology, so as to facilitate expression, detection, tracking 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. The identity of the amino acid sequences can be determined using homology search sites on the Internet, such as the BLAST web pages of the NCBI home website. For example, in the advanced BLAST2.1, by using blastp as a program, setting the value of Expect 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 by searching for the identity of a pair of amino acid sequences, a value (%) of identity can be obtained.

In the application, the expression quantity and/or activity of the Tin8 protein in the plant is improved, the tillering number (branch number) of the plant is increased, and/or the flowering phase of the plant is advanced.

In a second aspect, the invention claims a method of breeding plant varieties with increased tiller number (number of branches) and/or advanced flowering stage.

The method for cultivating the plant variety with the increased tillering number (branch number) and/or the advanced flowering phase, which is claimed by the invention, can comprise the step of improving the expression amount and/or activity of Tin8 protein in a receptor plant. The Tin8 protein can be a protein shown in any one of (A1) to (A4) above.

The method for cultivating the plant variety with increased tiller number (branch number) and/or advanced flowering phase can be realized by a hybridization method or a transgenic method.

Further, the present invention claims a method for breeding transgenic plants with increased tiller number (branch number) and/or advanced flowering stage.

The method for cultivating transgenic plants with increased tillering number (branch number) and/or advanced flowering phase, which is claimed by the invention, can comprise the following steps: introducing a nucleic acid molecule capable of expressing the Tin8 protein into a receptor plant to obtain a transgenic plant; the transgenic plant has an increased number of tillers (number of branches) and/or an advanced flowering phase compared to the recipient plant.

In the method, the nucleic acid molecule capable of expressing Tin8 protein may be introduced into the recipient plant in the form of a recombinant expression vector.

In the present invention, the promoter for promoting transcription of the nucleic acid molecule in the recombinant expression vector is a 35S promoter.

More specifically, the recombinant vector is a recombinant plasmid obtained by inserting the nucleic acid molecule (SEQ ID No. 2) into the multiple cloning site of pCAMBIA1305 vector.

In the above method, the nucleic acid molecule (SEQ ID No. 2) may be modified as follows and then introduced into the recipient plant to achieve better expression:

1) Modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modifications are made 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 expression time and space requirements 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 shown 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.

In the above method, the introduction of the recombinant expression vector into the recipient plant may specifically be: plant cells or tissues are transformed by conventional biological methods using Ti plasmids, ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and the transformed plant tissues are grown into plants.

Further, the nucleic acid molecule capable of expressing Tin8 protein may be any one of the following DNA molecules:

(B1) DNA molecule shown in SEQ ID No. 2;

(B2) A DNA molecule which hybridizes with the DNA molecule defined in (B1) under stringent conditions and encodes the Tin8 protein;

(B3) A DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of identity with the DNA sequence defined in (B1) or (B2) and encodes the Tin8 protein.

In the above nucleic acid molecules, identity refers to the identity of nucleotide sequences. The identity of the nucleotide sequences 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, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as a Matrix, the Gap existence cost, the Per residual Gap cost, and the Lambda ratio are set to 11,1 and 0.85 (default values), respectively, the identity of a pair of nucleotide sequences is searched, calculation is performed, and then the value (%) of identity can be obtained.

In the above nucleic acid molecule, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na ₃ PO ₄ And 1mM EDTA, in 50 ℃,2 x SSC,0.1% SDS rinsing; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ And 1mM EDTA, and rinsing in 1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ And 1mM EDTA, and rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ And 1mM EDTA, and rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; it can also be: 50 ℃ in 7% SDS, 0.5M Na ₃ PO ₄ And 1mM EDTA, rinsed in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: in 6 XSSC, 0.5% SDS solution, hybridization was performed at 65 ℃ and then the SDS and 1 XSSC, 0.1% SDS were used to wash the membranes once each.

In each of the above aspects, the plant may be a monocot or a dicot.

Further, the monocotyledon may be a gramineae plant; the dicot may be a crucifer.

Further, the gramineous plant may be corn or rice; the cruciferous plant may be arabidopsis thaliana.

In a particular embodiment of the invention, the plant is in particular maize variety a661 or rice variety florae 11 or arabidopsis thaliana columbia ecotype.

The invention clones a gene Tin8 which can regulate and control the tillering and the flowering phase of the corn, the regulation and control of the flowering phase of the gene are not influenced by the light cycle, tropical and subtropical corn germplasm resources can be effectively utilized, and the corn breeding genetic basis is widened. Therefore, the clone of the Tin8 gene not only provides an important theoretical basis and a feasible new method for the fine improvement of the tillering number of corn in the future, but also has important significance for the utilization of tropical germplasm resources and the breeding of early-maturing varieties. Therefore, the Tin8 gene has great application potential in corn molecule design breeding.

Drawings

FIG. 1 shows the results of the identification of transgenic rice plants. OE represents overexpression; CK represents the transgenic receptor control. Different numbers represent different strains.

FIG. 2 shows the result of the identification of transgenic positive Arabidopsis thaliana. OE represents overexpression; CK represents the transgenic receptor control.

FIG. 3 shows the results of functional identification of transgenic rice. OE represents overexpression; CK represents the transgenic receptor control.

FIG. 4 shows the results of functional characterization of transgenic Arabidopsis thaliana. OE represents overexpression; CK represents the transgenic receptor control.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the 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, unless otherwise specified, were carried out in a conventional manner according to the techniques or conditions described in the literature in this field or according to the product instructions. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Maize inbred line A661: acquisition of an American germplasm resource library application (website: https:// www.ars-grin.gov/. ACCESSION: PI 607521).

Teosinte V.B.4: U.S. germplasm resources library application acquisition (website: https:// www.ars-grin. Gov/. ACCESSION: AMes 21861).

pCAMBIA1305 plasmid: youbao Bio Inc.

Example 1 cloning of maize Tin8 Gene and functional identification thereof

1. 35S construction of Tin8 overexpression vector

1. RNA of leaves of teosinte V.B.4 is extracted and inverted into cDNA

2. cDNA is specifically amplified by using specific primers 1305-Nco1-F/1305-Pml1-R, and high fidelity enzyme KOD FX is selected for amplification. The amplification system was as follows: 2 XKOD Buffer 25. Mu.L; dNTP 10 u L; KOD FX 1. Mu.L; is just for1. Mu.L of each of the forward primer and the reverse primer; 5 mu L of DNA; ddH ₂ O make up to 50. Mu.L.

1305-Nco1-F：5’-CATGCCATGGATGTCAGCAACCGATCATTTGG-3' (Nco 1 recognition sequence is underlined);

1305-Pml1-R：5’-GGCCACGTGCTACTTCCCTAAAACCTTCTCTCT-3' (the recognition sequence Pml1 is underlined).

3. And after the PCR reaction, tapping and recovering the PCR product.

4. The PCR product and pCAMBIA1305 plasmid were digested simultaneously. Double enzyme digestion is carried out on Nco1 and Pml1, and a product is purified and recovered after enzyme digestion. The digestion system and digestion program are shown in tables 1 and 2:

TABLE 1 enzyme digestion System

TABLE 2 digestion procedure

5. Ligation of the cleavage products

And (3) recovering the enzyme digestion product, and then carrying out concentration determination by using Nanodrop according to the PCR product: and the molar ratio of the vector to 1. The ligation system and procedure are shown in tables 3 and 4:

TABLE 3T 4 CONNECTION PROGRAM

TABLE 4T 4 connection System

6. Transformation of Escherichia coli

a, 50 mu L of Trans-T1 competent cells are thawed on ice (as if the competent activity is best when the cells are not dissolved), the ligation product is added into the competent cells, and the cells are flicked and mixed evenly and are ice-cooled for 30min.

b, performing heat shock on the water bath at 42 ℃ for 30s, and then quickly putting the mixture into an ice water bath for ice bath for 2min.

And c, adding 200 mu L of sterile LB culture medium (without antibiotics) with the temperature balanced to room temperature, placing the mixture in a shaker at 37 ℃, culturing for 1h at 200rpm, and recovering the bacteria.

d, sucking the recovered bacteria onto an LB solid culture medium containing kalin, uniformly coating the bacteria by using a bacteria coating rod, inverting the plate after the bacteria are absorbed by the plate, and culturing overnight at 37 ℃.

e, picking positive single clone, adding into 500 μ L of 1.5mL centrifuge tube containing Kaner's LB liquid medium, placing in shaker 37 deg.C and culturing at 220rpm for 1-2h.

And f, absorbing 1 mu L of bacterial liquid as a PCR template for monoclonal identification. Common Taq enzyme system is used for amplification, and the primer is only used for constructing an overexpression vector. And performing 1% concentration agarose electrophoresis after PCR amplification to determine whether the size of an amplified band is consistent with the expected size, and if so, taking 200 mu L of bacterial liquid for sequencing.

And g, comparing a sequencing result with a target sequence, if the sequencing result is completely correct, extracting the plasmid of the numbered bacterial liquid by a plasmid miniextraction method after amplification culture, and preserving the bacteria at-80 ℃ by using 20% glycerol.

The recombinant plasmid that was verified to be correct after sequencing was designated pCAMBIA1305-Tin8. The structure of the recombinant plasmid pCAMBIA1305-Tin8 is described as follows: a recombinant plasmid obtained by inserting a DNA fragment shown in SEQ ID No.2 between restriction enzyme sites Nco1 and Pml1 of pCAMBIA1305 vector. Wherein, SEQ ID No.2 is a cDNA sequence of the Tin8 gene and encodes Tin8 protein shown in SEQ ID No. 1.

2. Acquisition of transgenic Rice and transgenic Arabidopsis

1. Transformation of Agrobacterium

a, 50 μ L of EHA 105. Agrobacterium is thawed on ice, 1 μ g of plasmid (pCAMBIA 1305-Tin 8) is added to the Agrobacterium competence, flicked and mixed, ice-cooled for 30min.

And b, freezing in liquid nitrogen for 5min.

And c, transferring the mixture into a water bath kettle at 37 ℃ for water bath for 5min.

d, adding 1mL of LB culture medium without antibiotics, and restoring the culture at 28 ℃ and 200rpm for 4-6h.

e, centrifuging for 30s, removing the supernatant, and adding 200. Mu.L of LB liquid medium for heavy suspension.

f, paving the agrobacterium on an LB solid culture medium containing a kanamycin and rifampicin double-antibody, and culturing in the dark at 28 ℃ for 2-3 days to grow positive clones.

And g, selecting positive clones for PCR identification and sequencing, and selecting correct clones for bacteria preservation. The correctly verified recombinant Agrobacterium was named EHA105/Tin8.

An empty control, designated EHA105/1305, was also prepared for Agrobacterium EHA105 to which pCAMBIA1305 was introduced.

2. Agrobacterium infection of recipient plants

(1) Rice infected by agrobacterium

The test rice variety: middle flower 11.

And infecting the test rice middle flower 11 by using the recombinant agrobacterium EHA105/Tin8.

The experiment was also set up with an empty control of the test rice infected with EHA105/1305.

(2) Agrobacterium infection of Arabidopsis thaliana

Test arabidopsis thaliana varieties: col-0 type Arabidopsis thaliana

And infecting the Col-0 type of the arabidopsis to be tested by adopting the recombinant agrobacterium EHA105/Tin8.

The experiment was also set up with an empty control infected with EHA105/1305 in the Arabidopsis thaliana tested.

3. Identification of transgenic plants

(1) Identification of transgenic Rice

The transgenic rice is planted in a greenhouse, and the illumination is set to 14h illumination/10 h darkness. Taking rice leaves to extract RNA, and reversing the RNA into cDNA. And identifying the expression quantity of the Tin8 gene of the transgenic plant by using a semi-quantitative PCR (polymerase chain reaction) performed by using a specific primer Tin8-RT-F/Tin 8-RT-R.

Tin8-RT-F：5’-CTACACCCTGGTACTGATTG-3’；

Tin8-RT-R：5’-TAGTGATGGATGGCTTGGAC-3’。

The results are shown in FIG. 1, where it can be seen that: the expression of Tin8 in the transgenic positive rice is realized, and the expression of Tin8 in the receptor material is avoided.

(2) Identification of transgenic Arabidopsis thaliana

The transgenic arabidopsis thaliana was planted in a greenhouse with the light setting of 18h light/6 h dark. Arabidopsis leaves were taken to extract RNA and inverted to cDNA. And identifying the expression quantity of the Tin8 gene of the transgenic plant by performing semi-quantitative PCR by using a specific primer Tin8-RT-F/Tin8-RT-R (see the above).

The results are shown in FIG. 2, where it can be seen that: tin8 was expressed in transgenic positive arabidopsis, whereas no Tin8 was expressed in the receptor material.

3. Functional characterization of transgenic rice and transgenic Arabidopsis

1. Functional characterization of transgenic rice

The tillering number and flowering phase of the transgenic rice were investigated. The rice flowering period is counted from the beginning of the rice planting day to the end of the rice flowering day, and the total days between the beginning of the rice planting day and the end of the rice flowering day are the rice flowering period; the number of tillers of rice was counted as the number of tillers at the basal part of rice, and the total number of tillers of rice at the flowering stage was investigated.

The results are shown in FIG. 3, where it can be seen that: compared with receptor materials, the tillering number of the Tin8 over-expressed transgenic positive rice plants is increased, and the flowering phase is advanced (P is less than 0.05). And the tillering number and the flowering time of the plants of the no-load control group are basically consistent with those of the receptor material, and no statistical difference exists.

2. Functional characterization of transgenic Arabidopsis

The number of branches and flowering time of transgenic rice were investigated. The total leaf number (rosette leaves and cauline leaves) of arabidopsis when the first flower is produced is used as the time index of plant flowering; the branch number of the arabidopsis is the sum of the branch numbers of the main stem and the lateral branch of the arabidopsis, and the branch number of the arabidopsis is the branch number of the arabidopsis.

The results are shown in fig. 4, and it can be seen that: compared with receptor materials, the transgenic positive Arabidopsis plant with over-expressed Tin8 has more branches and less leaves (the number of leaves in the flowering period is earlier, the number of leaves is less, and P is less than 0.05). The plant branch number and leaf number of the unloaded control group are basically consistent with those of the receptor material, and no statistical difference exists.

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 examples, it will be appreciated that the invention may 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 possible within the scope of the claims attached below.

<110> university of agriculture in China

<120> application of tillering regulation gene from corn

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Claims (10)

1. The application of the Tin8 protein or the related biological materials thereof in regulating and controlling the tillering number and/or the flowering period of plants;

   the related biological material is a nucleic acid molecule capable of expressing the Tin8 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

   the Tin8 protein is any one of the following proteins:

   (A1) Protein with an amino acid sequence of SEQ ID No. 1;

   (A2) Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

   (A3) A protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

   (A4) A fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of a protein defined in any one of (A1) to (A3).
2. 2. Use according to claim 1, characterized in that: the expression quantity and/or activity of the Tin8 protein in the plant are improved, and the tillering number of the plant is increased and/or the flowering phase of the plant is advanced.
3. 3. A method for breeding a plant variety with increased tillering number and/or advanced flowering phase, comprising the step of increasing the expression level and/or activity of Tin8 protein in a recipient plant;

   the Tin8 protein is any one of the following proteins:

   (A1) Protein with an amino acid sequence of SEQ ID No. 1;

   (A2) Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

   (A3) A protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

   (A4) A fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of a protein defined in any one of (A1) to (A3).
4. 4. A method for breeding transgenic plants with increased tillering number and/or advanced flowering phase, comprising the steps of: introducing a nucleic acid molecule capable of expressing a Tin8 protein into a receptor plant to obtain a transgenic plant; the transgenic plant has an increased number of tillers and/or an advanced flowering stage as compared to the recipient plant;

   the Tin8 is protein shown in any one of the following formulas:

   (A1) Protein with an amino acid sequence of SEQ ID No. 1;

   (A2) Protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

   (A3) A protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identity to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

   (A4) A fusion protein obtained by attaching a protein tag to the N-terminus and/or C-terminus of a protein defined in any one of (A1) to (A3).
5. 5. The method of claim 4, wherein: the nucleic acid molecule capable of expressing the Tin8 protein is introduced into the recipient plant in the form of a recombinant expression vector.
6. 6. The method of claim 5, wherein: the promoter for starting the transcription of the nucleic acid molecule in the recombinant expression vector is a 35S promoter.
7. 7. Use or method according to any of claims 1-6, wherein: the nucleic acid molecule capable of expressing the Tin8 protein is any one of the following DNA molecules:

   (B1) DNA molecule shown in SEQ ID No. 2;

   (B2) A DNA molecule which hybridizes with the DNA molecule defined in (B1) under stringent conditions and encodes the Tin8 protein;

   (B3) A DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of identity with the DNA sequence defined in (B1) or (B2) and encodes the Tin8 protein.
8. 8. Use or method according to any of claims 1-7, wherein: the plant is a monocotyledon or a dicotyledon.
9. 9. The use or method according to claim 8, wherein: the monocotyledon is a gramineous plant; the dicotyledonous plant is a cruciferous plant.
10. 10. The use or method of claim 9, wherein: the gramineous plant is corn or rice; the cruciferae plant is arabidopsis thaliana.

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