CN114230649A - Tn1 protein related to tillering force of rice and related biological material and application thereof - Google Patents

Abstract

The invention discloses Tn1 protein related to rice tillering force, a related biological material and application thereof. The Tn1 protein can be specifically the protein of A1), A2) or A3) as follows: A1) the amino acid sequence is protein of SEQ ID No.1 in a sequence table; A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues on the protein A1), has more than 90% of identity with the protein A1) and has the activity of regulating and controlling the tillering capacity of plants; A3) a fusion protein obtained by connecting a protein tag to the N-terminus or/and the C-terminus of A1) or A2). The Tn1 protein and related biomaterials can be used for regulating and controlling tillering force of plants.

Description

Tn1 protein related to tillering force of rice and related biological material and application thereof

Technical Field

The invention relates to a Tn1 protein related to rice tillering force in the field of biotechnology and an encoding gene and application thereof.

Background

Rice (Oryza Sativa L.) is one of the major food crops in the world, with rice being the staple food for more than half of the world's population. Under the severe situation that the available cultivated land area cannot be increased or even reduced year by year, the promotion of variety improvement and the exploration of the high-yield potential of rice are still important tasks of rice breeding in a period in the future. The ear number is one of three factors of the rice yield, and has important research significance in variety improvement. The number of spikes is usually based on the number of tillers. Therefore, the continuous development of a new gene for regulating and controlling rice tillering, the regulation and control mechanism of the gene is clear, and the gene can provide rich gene sources and theoretical guidance for the breeding of ideal plant types of rice.

The rice tillers essentially belong to stem branches formed by lateral meristems, each tillering starts from a tillering bud formed by the lateral meristems at a leaf axilla, the tillering bud which is initially in a dormant state extends out and grows into tillers after being activated under proper conditions, and the tillers can enter a reproductive growth stage under proper conditions and finally are converted into effective spikes. Improving the tillering number of the rice, promoting the early tillering and having important significance for establishing the spike number at the later stage. Meanwhile, the discovery of a new rice tillering gene has important application value and theoretical significance for further improving the molecular mechanism and the genetic control network of rice tillering morphogenesis.

Disclosure of Invention

The technical problem to be solved by the invention is how to regulate and control the tillering capacity of the plant.

The invention provides a protein, which is named as Tn1 and is A1), A2) or A3) as follows:

A1) the amino acid sequence is protein of SEQ ID No.1 in a sequence table;

A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues on the protein A1), has more than 90% of identity with the protein A1) and has the activity of regulating and controlling the tillering capacity of plants;

A3) a fusion protein obtained by connecting a protein tag to the N-terminus or/and the C-terminus of A1) or A2).

Wherein SEQ ID No.1 consists of 501 amino acid residues.

The protein can be derived from rice.

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

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 the 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, among others.

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 above protein, the 90% or more identity may be at least 91%, 92%, 95%, 96%, 98%, 99% or 100% identity.

Biomaterials associated with the protein Tn1 are also within the scope of the invention.

The protein Tn 1-related biomaterial provided by the invention is any one of the following B1) to B7):

is any one of the following B1) to B7):

B1) a DNA molecule encoding the protein;

B2) an expression cassette comprising the DNA molecule of B1);

B3) a recombinant vector containing the DNA molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the DNA 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, a transgenic plant tissue or a transgenic plant organ containing the DNA molecule of B1), or a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the expression cassette of B2);

B6) a nucleic acid molecule that reduces the expression of B1) the DNA molecule;

B7) an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ comprising the nucleic acid molecule according to B6).

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 biological material, the DNA molecule of B1) is the gene shown in the following B1) or B2):

b1) the coding sequence of the coding chain is a cDNA molecule or a DNA molecule of SEQ ID No. 2;

b2) the nucleotides of the coding strand are cDNA molecules or DNA molecules of SEQ ID No. 2.

Wherein, the sequence 2 in the sequence table consists of 1503 nucleotides and codes the protein shown as SEQ ID No.1 in the sequence table.

In the above biological material, the expression cassette containing the DNA molecule (Tn1 gene expression cassette) described in B2) refers to a DNA molecule capable of expressing Tn1 in a host cell, and the DNA molecule may include not only a promoter for initiating transcription of Tn1 gene, but also a terminator for terminating transcription of Tn 1. 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: the constitutive promoter of cauliflower mosaic virus 35S; 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) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato protease 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, oleocin, 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 by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (1985) 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 Gene Dev.,5: 141; Mogen et al (1990) Plant Cell,2: 1261; Munroe et al (1990) Gene,91: 151; Balla et al (1989) Nucleic Acids Res.17: 7891; Joshi et al (1987) Aclic, 9615).

The existing plant expression vector can be used for constructing a recombinant vector containing the protein Tn1 coding gene or the protein Tn1 coding gene expression cassette. The plant expression vector can be a Gateway system vector or a binary agrobacterium vector and the like, such as ProSuper1300, pGWB411, pGWB412, pGWB405, pBin438, pCAMBIA1302, pCAMBIA2300, pCAMBIA2301, pCAMBIA1301, pGWB18, pBI121, pCAMBIA1391-Xa or pCAMBIA 1391-Xb. When the Tn1 gene is used to construct a recombinant 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 the transcription initiation nucleotide, and 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 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 proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. 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.

In the above biological material, the recombinant microorganism can be yeast, bacteria, algae and fungi; the bacterium may be an Agrobacterium EHA105 strain, for example.

The protein or the biological material can be applied to any one of the following C1-C2:

C1) regulating and controlling tillering force;

C2) preparing the product for regulating and controlling tillering force.

The tillering force is regulated and controlled to improve the tillering force or reduce the tillering force. The tillering force is increased and/or tillering bud extension is promoted, and the tillering force is reduced and/or tillering bud extension is inhibited. The tillering force can be improved by inhibiting or reducing the expression of the coding gene of the protein Tn 1; the reduction of tillering force can be realized by promoting or increasing the expression of the gene coding for the protein Tn 1.

The invention also provides a method for improving the tillering capacity of rice, which comprises the following steps: inhibiting or reducing the expression of Tn1 gene in receptor rice to obtain target rice with tillering force higher than that of the receptor rice; the Tn1 gene is a gene for coding the Tn1 protein.

In the method, the inhibition or reduction of the expression of the Tn1 gene in the receptor rice is realized by performing gene editing on the Tn1 gene in the rice. The gene editing is realized by means of a CRISPR/Cas9 system.

In the method, the CRISPR/Cas9 system comprises a plasmid containing Cas9 and gRNA, and the target sequence of the gRNA is the 5' -N-matched DNA fragment shown in SEQ ID No.2_X-NGG-3 'or 5' -CCN-N_X-a fragment of the 3' sequence arrangement rule, wherein N represents any of A, G, C and T, 14. ltoreq. X.ltoreq.30, and X is an integer, N_XRepresents X consecutive deoxyribonucleotides. The target sequence of the gRNA can be specifically the 148 th and 167 th positions of SEQ ID No. 2.

In the method, the inhibition or reduction of the expression of the gene in the receptor rice is realized by deleting 6 nucleotides from 2075 th site to 2080 th site of a sequence 3 in a sequence table in a rice genome.

In order to solve the technical problems, the invention also provides a plant reagent which is used for regulating and controlling the tillering capacity of the plant.

The plant reagent provided by the invention contains the protein or/and biological materials related to the protein.

The active component of the plant agent can be the protein or/and the biological material related to the protein, the active component of the plant agent can also contain other biological components or/and non-biological components, and the other active components of the plant agent can be determined by a person skilled in the art according to the tillering effect of the plant.

The plant of interest may be a monocot or a dicot. The monocotyledon may be a gramineae plant, and specifically may be rice.

Experiments of knocking out Tn1 gene in rice prove that the tillering number of transgenic rice with the Tn1 gene is increased, and the extension of tillering bud is promoted. An overexpression experiment for introducing a Tn1 gene coding sequence into rice proves that compared with receptor rice, the transgenic rice overexpressing the Tn1 protein has the advantages that the tillering number is reduced, and the extension of tillering buds is inhibited. Both knockout experiments and overexpression experiments show that the Tn1 protein is a gene related to plant tillering force, and the Tn1 protein is inhibited from improving the tillering force of plants. The method of the invention has simple operation and low cost, greatly accelerates the breeding process and has wide application prospect.

Drawings

FIG. 1 is a Manhattan chart of genome-wide association analysis of 295 rice germplasm materials with respect to effective spike number in example 1 of the present invention.

FIG. 2 is a diagram showing the positions of the Tn1 gene targets in the Tn1 gene knockout test in example 2 of the present invention.

FIG. 3 is a drawing showing the DNA level identification of a Tn1 gene overexpression material in example 2 of the present invention.

FIG. 4 is a T0 sequencing peak diagram of a Tn1 gene knock-out material in example 2 of the present invention.

FIG. 5 is a statistical chart showing the tillering phenotype of Tn1 knock-out material Tn1-1 and wild type Nipponbare (NIP) in example 2 of the present invention. Data shown in the figure are mean ± sd, number of repeats is 15, represents significance analysis result P <0.01, Tiller number is number of tillers, unit is number per strain.

FIG. 6 shows the tillering phenotype and statistical plots of Tn1 gene overexpression materials (Tn1-OE1 and Tn1-OE2) and wild type in example 2 of the present invention. The data shown in the figure are mean ± standard deviation, number of replicates is 15, a significance analysis result is P <0.05, a significance analysis result is P <0.01, Tiller number is tillering number, units are per strain. In the left panel, three plants from left to right are Nipponbare (NIP), overexpression materials Tn1-OE1 and Tn1-OE2, respectively.

FIG. 7 is a photograph showing a tillering bud phenotype of Tn1 knock-out material, over-expressed material and wild type in example 2 of the present invention after three weeks of sprouting.

FIG. 8 shows the expression of Tn1 gene at the base of tillering bud at each stage in example 3 of the present invention. The internal reference is LOC _ Os03g13170(Ubiquitin1) gene. Root, sheath and leaf respectively represent roots, leaf sheaths and leaf blades in vegetative growth period, DT30_ Ab, DT45_ Ab and DT60_ Ab respectively represent tiller bud bases 30 days, 45 days and 60 days after seedling transplantation, and ST _1, ST _2 and ST _3 respectively represent inverted bases of one, two and three internodes 75 days after seedling transplantation. Data shown in the figure are mean ± sd, with 3 repeats, representing a significance analysis of P <0.01, and ns representing no difference.

FIG. 9 is a map of the subcellular localization of Tn1 in example 3 of the present invention. 35S:GFP (Prosuper1300 empty) was used as a 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.

In the quantitative tests in the following examples, three replicates were set up and the results averaged.

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

The SK-gRNA vector and the pC1300-Cas9 vector in the following examples are described in non-patent literature "Wang, Chun et al, A simple CRISPR/Cas9 system for multiplex genome editing in rice.journal of Genetics and Genomics", publicly available from the university of agriculture (i.e., the applicant) in China, to repeat the experiments of the present application, and are not applicable for other uses.

The vector ProSuper1300 in the following examples is described in non-patent literature "Li, Gangling et al, RGN1 control gain number and maps across agriculture architecture in rice plant biotechnology journel", publicly available from the university of agriculture in China (i.e., the applicant), to repeat the experiments of the present application, and is not useful for other applications.

Example 1 obtaining of Tn1 Gene

According to the invention, 295 parts of rice seed materials are used, effective spike numbers of the rice seed materials are planted and investigated in Beijing, MLM (multi-level molecular mass) model is used for carrying out whole genome correlation analysis, and the Tn1 gene is obtained by screening in combination with the gene expression condition of a RiceXPro website.

295 parts of rice seed material is utilized to carry out effective spike number correlation analysis, and P is 10^-4For the threshold, at least three consecutive significant SNP sites, and the interval not more than 170kb apart from each other, were defined as one QTL, defining 13 QTLs (qTn1-qTn13) (as shown in fig. 1). According to qTn2, 12 candidate genes are annotated, wherein 5 genes are not expressed in each stage of rice growth and development, only Tn1 gene in the remaining 7 genes is highly expressed in roots in vegetative growth phase, and significant non-synonymous mutation SNP sites exist, so that the expression mode of the tillering gene is met.

Tn1 gene is obtained by PCR amplification with cDNA of Nipponbare of rice variety as template, and the primers are as follows:

Tn1-F：5’-ATGGATGGTAGTAATGAGAATATC-3’；

Tn1-R：5’-TTACTTTCCCATCTTACTCGCAAAG-3’。

through sequencing, the nucleotide sequence of the coding sequence of the Tn1 gene is shown as SEQ ID No.2, and the protein Tn1 is coded, and the amino acid sequence of the protein is SEQ ID No. 1.

Example 2 functional verification of Tn1 protein

Construction of first, knockout vectors

(1) The nucleotide sequence of the genomic gene of Tn1 is SEQ ID No.3, 10 exons in total, 10 introns (wherein, SEQ ID No.3 is 5 'UTR at positions 1-139, first intron at positions 140-1872, 5' UTR at positions 1873-1916, first exon at positions 1917-2249, second intron at positions 2250-3035, second exon at positions 3036-3204, third intron at positions 3205-3317, third exon at positions 3318-3763, fourth intron at positions 3764-4558, fourth exon at positions 4559-4582, fifth intron at positions 4583-4754, fifth exon at positions 4755-4847, fifth exon at positions 4848-5824, sixth exon 5825, seventh exon at positions 605904, 595998, the 6083-. Html was logged into the website http:// www.genome.arizona.edu/criprpr/crispr search. html, and the target sites were screened. The off-target was then assessed to the http:// www.rgenome.net/cas-offder/website. The sequences with low off-target rates were selected as target sequences for this study, as follows:

target sequence: 5'-GGTGAGTCTGAACCTTACAT-3' (corresponding to positions 148 and 167 of SEQ ID No.2 and 2064 and 2083 of SEQ ID No. 3).

The Tn1 knockout target sequence is located in the first exon in the genome as shown in FIG. 2.

(2) Designing two complementary DNA sequences, adding GGCA before the forward target sequence and AAAC before the reverse complementary target sequence, and specifically, the following steps are carried out:

F：5’-GGCAGGTGAGTCTGAACCTTACAT-3’；

R：5’-AAACATGTAAGGTTCAGACTCACC-3' (the underlined sequence is reverse complementary to the underlined sequence in F).

(3) Construction of intermediate vectors:

a. carrying out AarI enzyme digestion (Ferent company) on the SK-gRNA vector to form a linear vector with a viscous tail end;

b.F strand and R strand are mixed and then denatured and annealed to form a fragment with sticky ends;

c. connecting the linear vector obtained in the step a with the fragment obtained in the step b (the molar concentration is 1:3-10), and transforming DH5 alpha to obtain a recombinant plasmid; the colony PCR positive detection can be carried out by matching a primer T3 with the R chain, and the primer sequence is as follows:

T3：5’-ATTAACCCTCACTAAAGGGA-3’；

R：5’-AAACATGTAAGGTTCAGACTCACC-3’。

d. the common primer T7 (5'-TAATACGACTCACTATAGGG-3') or T3 (5'-ATTAACCCTCACTAAAGGGA-3') is used for sequencing detection to verify whether the vector is correct or not, and the correct vector is named as SK-gRNA-Tn 1.

(4) Construction to final vector:

the vector pC1300-Cas9 was digested with KpnI and BamHI to give a linearized pC1300-Cas9 vector. And (4) carrying out enzyme digestion on the SK-gRNA-Tn1 constructed in the step (3) through KpnI and BglII, and then recovering a target fragment. Connecting the target fragment to a linearized pC1300-Cas9 vector to obtain a knockout vector, wherein the specific structure is as follows: the objective fragment cut by SK-gRNA-Tn1 is used to replace the fragment between the KpnI and BamHI recognition sites of the restriction endonuclease of the vector pC1300-Cas9 (small fragment including the recognition site of KpnI and the recognition site of BamHI), and the other sequences of the vector pC1300-Cas9 are kept unchanged to obtain the recombinant vector. After sequencing was correct, the resulting knockout vector was designated Tn 1-CR.

The vector Tn1-CR is knocked out to express gRNA, the target sequence of the gRNA is positioned on the first exon of the rice Tn1 gene, and the specific target sequence is shown as the 148-167 th position of SEQ ID No. 2.

Second, construction of overexpression vector

The over-expression vector used in the experiment is named as Tn1-OE, and is obtained by connecting CDS (1-1503 nucleotides in SEQ ID No. 2) with Tn1 removed stop codon in Nipponbare to a plant expression vector Prosuper1300 with a 35S strong promoter by a seamless connection method. The restriction sites are Hind III and KpnI, and the primers are shown as follows:

Tn1-OE-F：AATCTCGATACACCAAATCGACTCTAGAAAGCTTATGGATGGTTAATGAGAATATC

Tn1-OE-R：

CGGTGAACAGCTCCTCGCCCTTGCTCACCATGGTACCCTTTCCCATCTTACTCGCAAAGGTC

the primers are written in the 5 'to 3' direction.

The structure of the Tn1-OE vector is described as: and replacing a small fragment between enzyme cutting sites HindIII and KpnI of the ProSuper1300 vector with a positive plasmid obtained by keeping other sequences of the ProSuper1300 vector unchanged by using a Tn1 coding region sequence, namely the positive plasmid is a recombinant expression vector of the Tn1 protein. The Tn1 coding sequence is shown in1 st to 1503 st nucleotides of SEQ ID No. 2.

Third, obtaining transgenic rice

(1) Recombinant bacterium

And (3) transforming the knockout vector Tn1-CR prepared in the first step into the agrobacterium tumefaciens EHA105 by using a freeze-thaw method to obtain a recombinant strain EHA105-Tn 1-CR.

And (3) transforming the over-expression vector Tn1-OE prepared in the second step into the Agrobacterium tumefaciens EHA105 by a freeze-thawing method to obtain a recombinant strain EHA105-Tn 1-OE.

(2) Rice transplanting machine

The two recombinant bacteria are both subjected to a classical agrobacterium-mediated callus infection method, and the callus receptor varieties which are respectively used for infecting the receptor varieties are all Nipponbare. The method comprises the following specific steps:

a. obtaining embryogenic callus: removing hull of mature seed, sterilizing with 75% ethanol, sterilizing with 20% sodium hypochlorite solution, rinsing with sterile water, and air drying for 6 hr. Inoculating to NB medium, culturing at 28 deg.C in dark for 2 weeks, peeling off embryogenic callus, subculturing to new NB medium, and subculturing for 2 weeks.

b. Preparing a staining solution: sucking the preserved agrobacterium liquid, coating the liquid on a solid culture medium containing rifampicin and kanamycin, performing inversion dark culture at 28 ℃ for 2 days, scraping a small amount of agrobacterium into an AAM liquid culture medium, uniformly blowing and beating the liquid culture medium, and determining that the concentration OD600 of the bacterial liquid is about 0.3.

c. Co-culturing: selecting naturally dispersed granular callus with fresh yellow color and diameter of about 3-5 mm, adding the prepared infection solution into a triangular flask, infecting for 15min, sucking redundant infection solution by using sterile filter paper, placing on a co-culture medium paved with a layer of filter paper, and co-culturing for 2-3 d at 20 ℃.

d. Screening of resistant callus: and taking out the co-cultured callus, rapidly shaking and cleaning the callus for 5-6 times by using sterile water, cleaning the callus for 20min by using the sterile water containing the cefuroxime and the carbenicillin, and finally placing the callus on sterile filter paper and draining the callus for 3 h. Then transferred to a delayed screening medium. After one week, the cells were transferred to the first round of screening medium, and after two weeks, the cells were transferred to the second round of screening medium, and the cells were further cultured for two weeks.

e. Differentiation culture: and (3) inoculating the screened resistant callus into a pre-differentiation culture medium, performing dark culture at 28 ℃ for 2 weeks, then transferring to a differentiation culture medium, and performing illumination culture for 2-3 weeks to obtain a regenerated transgenic seedling plant.

f. And (3) transplanting the seedlings to a strong seedling culture medium, removing the culture bottle after the seedlings grow into roots, cleaning the culture medium on the roots, hardening the seedlings for 1-2 weeks, and transplanting the seedlings to a field for planting until the seedlings are mature.

The medium formulations used in the above transgenic procedures are shown in table 1.

TABLE 1 formulation of the respective media used in the transgenic procedure

Note: the basic components of the NB medium comprise macroelements N6, trace elements B5, organic components B5, inositol 150mg/L, casein hydrolysate 300mg/L, glutamine 500mg/L, proline 600mg/L, sucrose 30g/L and plant gel 3 g/L.

(3) PCR identification to obtain positive transgenic material

And (3) performing PCR identification and sequencing identification on the DNA level of the rice plants of the T0 generation Tn1-OE transgenic material and the T0 generation Tn1-CR transgenic material obtained in the step (2) respectively.

Tn1-OE transgenic material identification primers are Tn1-OE-check-F and Tn 1-OE-check-R:

Tn1-OE-check-F：5’-GACGCCATTTCGCCTTTTCAG-3’；

Tn1-OE-check-R：5’-CTTTCCAATGTAAGGTTCAG-3’。

the size of the target fragment is 310bp, the plant containing the target fragment in the amplification product is positive, the plant not containing the target fragment is negative, the identification result of a part of positive samples is shown in figure 3, and the positive plants are Tn1-OE transgenic materials (namely Tn1 gene overexpression materials) of the T0 generation.

Tn1-CR transgenic material identification sequencing primers are Tn1-CR-check-F and Tn 1-CR-check-R:

Tn1-CR-check-F：5’-GGTTTGTATGTTTGTTGACCACC-3’；

Tn1-CR-check-R：5’-TCTAGCTACCGATATGGCTTCTC-3’。

the sequencing peak pattern of Tn1-CR transgenic material (i.e., Tn1 knock-out material) T0 generation is shown in FIG. 4, and the homozygous material is named Tn 1-1.

Fourth, transgenic material tillering number correlation property identification

The homozygous positive strain of Tn1-CR is a T1 generation obtained after a T0 generation strain of Tn1-1 in the step III is harvested, and a homozygous positive strain of Tn1-CR (Tn1-1) is obtained by sequencing, and is also called Tn1 gene knockout material Tn1-1 below.

Compared with wild rice Nipponbare, the Tn1-CR homozygous positive strain (Tn1-1) has a mutation in the gene of Tn1 in the rice genome: in two homologous chromosomes, the genomic genes of Tn1 with nucleotide sequences of sequence 3 in the sequence table are changed as follows: 6 nucleotides from 2075 th to 2080 th in a sequence 3 in the sequence table are deleted, namely 6 nucleotides from 148 th to 167 th in a sequence 2 in the sequence table are deleted, so that three amino acids of 53 th glutamic acid (Glu), 54 th proline (Pro) and 55 th tyrosine (Tyr) in the sequence 1 are mutated into one aspartic acid (Asp), and the Tn1 gene is knocked out.

Homozygous positive strain of Tn1-CR (Tn1-1) and Nipponbare (NIP) were sown at Shanzhuang laboratory site at the university of agriculture in Beijing China. After seed soaking and germination accelerating, the seedlings grow for 30 days on a seedbed, and then the seedlings are transplanted to a field, 7 seedlings are planted in each row, the plant spacing is 20cm, the row spacing is 25cm, and 10 rows are planted respectively. The tillering phenotype at 50 days after transplantation was statistically investigated, and 15 plants (single plants in the same row were not statistically investigated) were investigated each for tn1-1 and NIP, and the results are shown in fig. 5, which indicates that the tillering number of the knockout material tn1-1 is significantly greater than that of the wild-type Nipponbare at 50 days after transplantation.

After the T0 generation positive strain of Tn1-OE is harvested, 20mg/L hygromycin solution is used for soaking seeds for sprouting before the T1 generation is sowed, seeds which can normally root are selected and transplanted to a field, after leaves are taken to extract DNA, the Tn1-OE-check-F and the Tn1-OE-check-R are used for DNA level identification to obtain a positive single strain. And (3) treating all positive single plant seeds of the T1 generation with 20mg/L hygromycin solution before sowing of the T2 generation, wherein all T1 single plants capable of normally rooting soaked seeds are homozygous lines, are marked as Tn1-OE1 and Tn1-OE2, transplanting the T1 single plants into a field, and counting the tillering phenotype 50 days after transplanting. And the expression quantity of Tn1 in Tn1-OE1 and Tn1-OE2 is quantitatively analyzed in real time. The results are shown in FIG. 6: on day 50 after transplantation, the overexpression materials Tn1-OE1 and Tn1-OE2 showed a reduced tillering number phenotype compared to wild-type Nipponbare.

To investigate the effect of Tn1 on rice tillering bud protrusion, seeds of wild type Nipponbare (NIP), Tn1 knock-out material Tn1-1, Tn1 overexpression material Tn1-OE1 and Tn1-OE2 were sterilized with sterile water. The plants were planted in 1/2MS medium, cultured in a light incubator for 3 weeks, and the phenotype of shoot-base tillering buds was investigated, and 10 plants were planted per line. FIG. 7 is a comparison graph of shoot buds of seedlings of NiP (NIP), knock-out material (Tn1-1) and overexpression material (Tn1-OE1 and Tn1-OE2) in three weeks, wherein the shoot buds of NIP are shorter than Tn1-1 but longer than Tn1-OE1 and Tn1-OE 2. Compared with a wild Nipponbare plant, the Tn1 knockout material Tn1-1 tillering bud extension is promoted, and the Tn1 overexpression materials Tn1-OE1 and Tn1-OE2 tillering bud extension is inhibited.

Example 3 Tn1 expression in various tissues of Rice and subcellular localization analysis

One, real time fluorescent quantitative PCR

Taking the tissue of the basal part of the tillering bud of each period of Nipponbare of a rice variety, extracting total RNA, utilizing reverse transcriptase M-MLV to carry out reverse transcription to synthesize a first cDNA chain, taking the first cDNA chain as a template, adopting a primer Tn1-RT-F and a primer Tn1-RT-R to amplify a specific fragment of a Tn1 gene, adopting a primer Ubiquitin-F and a primer Ubiquitin-R to amplify the specific fragment of the Ubiquitin gene of the rice to be used as an internal reference for real-time quantitative analysis. The sequences of the primers used are specifically as follows:

Tn1-RT-F：5’-GGCGTTGGCCTTGCTGAT-3’；

Tn1-RT-R：5’-GTTGACGATTCCTCTCATCCTTTG-3’。

Ubiquitin-F：5’-ACCAGCTGAGGCCCAAGA-3’；

Ubiquitin-R：5’-ACGATTGATTTAACCAGTCCATGA-3’。

Real-Time fluorescent quantitative PCR was performed on a Real-Time fluorescent quantitative PCR instrument Applied Biosystems 7500Real Time PCR system (ABI, USA) with 3 biological replicates per experiment, 3 mechanical replicates per biological replicate. The method reported by Livak KJ and Schmittgen TD (2001), 2^-ΔΔCTRelative table of calculationAnd (4) obtaining the amount.

ΔΔCT＝(CT.Target－CT.Ubiquitin)Time x－(CT.Target－CT.Ubiquitin)Time 0

Time x represents an arbitrary Time point, and Time 0 represents 1 Time of target gene expression after Ubiquitin correction.

As a result, as shown in FIG. 8, the Tn1 gene was detected in the tillering bud-based tissue at each stage of Nipponbare.

Two, subcellular localization of Tn1 in Rice protoplasts

In order to study the subcellular localization of Tn1, the Tn1-OE vector constructed in the second step of example 2 (the starting vector used is ProSuper1300) was used to transform rice protoplasts for observation of localization results.

(1) Sterilizing hulled Nipponbare seed of rice variety with 75% alcohol for 3-5min, and washing with sterilized water twice. Then the sodium hypochlorite solution is disinfected by 20 percent (160 revolutions of a shaking table at 28 ℃), and the sodium hypochlorite solution is changed once after 20min for two times. Finally rinsing with sterilized water for 5-8 times, and air drying in a clean bench for 4-5 hr

(2) The seeds after the disinfection treatment are inoculated in 1/2MS culture medium and grown for 12 days in a dark place.

(3) Cutting the rice seedlings in the step (2) by using a blade, adding the buffer Enzyme solution I, and mixing the mixture by gently shaking. After filtration through a 400 mesh nylon membrane, the minced tissue was added to Enzyme solution II.

(4) The Enzyme solution II containing the minced tissue was placed in a vacuum pump and evacuated at 50kpa for 0.5h, and then subjected to 40 rpm shaking for 3-4 h at room temperature to promote the dissociation and release of protoplasts.

(5) The Enzyme solution II containing protoplasts was filtered through a 400 mesh nylon membrane to remove minced rice tissue, and the protoplasts were collected in a 50ml centrifuge tube. The protoplasts were washed with 100ml of W5 solution and collected by centrifugation at 150g for 5min (3acel 9 brake).

(6) Resuspend protoplasts collected in (5) with MMG solution.

(7) Adding the extracted Tn1-OE plasmid into a 2ml tube, adding the mixed solution of the protoplast and the MMG obtained in the step (6), and gently and uniformly flicking by hand.

(8) Add 110. mu.l of 40% PEG, turn upside down and mix gently, leave in the dark for 15 minutes at 28 ℃.

(9) Adding W5, mixing, and centrifuging at 150g for 5min to remove supernatant.

(10) The cells were incubated overnight at 28 ℃ for 16 hours with 800. mu. l W5 added in the horizontal position.

(11) And (5) laser confocal observation.

The reagent formula used in the above protoplast extraction process is shown in Table 2.

TABLE 2 reagents for protoplast extraction

The results are shown in FIG. 9, which shows that Tn1 is a protein localized in the nucleus.

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 in 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 possible within the scope of the claims attached below.

Sequence listing

<110> university of agriculture in China

<120> Tn1 protein related to rice tillering force, related biological material and application thereof

<130> GNCSY213250

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 501

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 1

Met Asp Gly Ser Asn Glu Asn Ile Gln Phe Ser Trp Gly Lys Lys Arg

1 5 10 15

Ala Lys Gly Gly Ile Lys Met Asp Thr Gln Phe Tyr Asp Ser Phe Thr

20 25 30

Phe Asp Asn Val Lys Tyr Ser Leu Tyr Asp Asn Val Tyr Leu Phe Lys

35 40 45

Ser Gly Glu Ser Glu Pro Tyr Ile Gly Lys Ile Ile Lys Ile Trp Gln

50 55 60

Gln Asn Gln Ala Lys Lys Val Lys Ile Leu Trp Phe Phe Leu Pro Asp

65 70 75 80

Glu Ile Arg Lys His Leu Ser Gly Pro Val Met Glu Lys Glu Ile Phe

85 90 95

Leu Ala Cys Gly Glu Gly Val Gly Leu Ala Asp Ile Asn Pro Leu Glu

100 105 110

Ala Ile Gly Gly Lys Cys Thr Val Leu Cys Ile Ser Lys Asp Glu Arg

115 120 125

Asn Arg Gln Pro Ser Pro Arg Glu Leu Ala Met Ala Asp Tyr Ile Phe

130 135 140

Tyr Arg Phe Phe Asp Val Asn Ser Cys Thr Leu Ser Glu Gln Leu Pro

145 150 155 160

Glu Lys Ile Ala Gly Val Glu Gly Asn Leu Leu Leu Asn Ser Lys Val

165 170 175

Glu Gln Val Thr Ser Cys Ser Asp Gln Glu Val His Gly Val Asp Gln

180 185 190

Lys Met Leu Asn Val Pro Val Pro Leu Pro Gln Ser Thr Val Met Glu

195 200 205

Asp Glu Ser Pro Val Ala Ala Val Ser Leu Pro Pro Ser Val Phe Lys

210 215 220

Glu Glu Asn Val Ala Ser Ala Ile Pro Phe Pro Gln Pro Val Val Lys

225 230 235 240

Glu Glu Ser Ala Ala Ala Ala Ile Pro Pro Pro His Val Ala Leu Lys

245 250 255

Glu Glu Ser Val Ser Lys Ser Thr Glu Asn Ile Thr Lys Pro Ala Gln

260 265 270

Lys Val Leu Pro Gly Glu Arg Pro Pro Lys Arg Val Lys Phe Ser Glu

275 280 285

Asn Val Thr Val Gln Asn Val Pro Leu Asp Val Pro Glu Arg Pro Ser

290 295 300

Arg Thr Gly Pro Leu Glu Leu Ala Gly Arg Gln Ala Asp Arg Ser Lys

305 310 315 320

Trp Phe Lys Ile Pro Trp Asp Thr Arg Leu Arg Asn Ala Asp Glu Gln

325 330 335

Gly Thr Leu Val Tyr Ile Gln Asn Leu Asp Ile Gln Phe Ala Ala Ala

340 345 350

Asp Ile Glu Glu Leu Ile Arg Asp Ala Leu Gln Leu Asn Cys Ile Ala

355 360 365

Lys Pro Ile Asn His Pro Thr Tyr Asp Asp Pro Asn Asn Gly Lys Ala

370 375 380

Tyr Ala Ile Phe Lys Thr Lys Ser Ala Ala Asp Ser Ala Ile Ser Lys

385 390 395 400

Ile Asn Ser Gly Leu Val Val Gly Gly Arg Pro Leu Tyr Cys Ser Lys

405 410 415

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

420 425 430

Thr Ile Asn Asn Ile Arg Met Gly Ile Arg Gln Arg Glu Glu Gln Lys

435 440 445

Lys Ala Val Ser Thr Ser His Cys Ser Gln Pro Asn Thr Met Glu Tyr

450 455 460

Asp Leu Ala Leu Asp Trp Met Leu Val Arg Ala Lys Gln Glu Thr Lys

465 470 475 480

Phe Arg Thr Leu His Lys Lys His Lys Asp Glu Arg Lys Thr Phe Ala

485 490 495

Ser Lys Met Gly Lys

500

<210> 2

<211> 1506

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

atggatggta gtaatgagaa tatccaattc tcatggggga agaagagagc aaaaggtggt 60

attaagatgg atacacagtt ttatgactcc ttcacatttg acaatgtgaa gtactcactg 120

tatgacaatg tatatctttt taagagtggt gagtctgaac cttacattgg aaagataata 180

aagatatggc agcaaaatca ggctaagaaa gtaaagattc tttggttttt tctcccggat 240

gagattcgaa aacatttaag tggccctgta atggaaaagg agatatttct tgcttgtggt 300

gaaggcgttg gccttgctga tatcaaccca ctggaagcta ttggtgggaa atgcactgtg 360

ctttgcattt caaaggatga gaggaatcgt caaccttccc ccagggaact agcaatggct 420

gattatatct tctacaggtt ttttgatgtt aacagttgca cactttctga acaattacct 480

gagaaaattg caggggtgga aggaaatctt ttgcttaatt caaaagttga gcaagtgaca 540

tcctgttcag accaggaagt gcatggtgtt gatcagaaga tgcttaatgt cccagttccc 600

cttccccagt caacggttat ggaggatgaa agtccagttg ctgcagtttc ccttcccccg 660

tcagtattca aggaggaaaa tgtggcttca gccattccct ttccccagcc agtggtcaag 720

gaggaaagtg cggctgctgc cattccccct ccccatgtag cactgaaaga ggagagtgtg 780

tccaaatcta cagagaacat taccaaacct gcacagaaag ttctccctgg ggagaggcca 840

ccaaagaggg tcaaattttc tgaaaatgtt acagtgcaaa atgtgccatt agatgttcct 900

gaaagaccaa gtcgcactgg acctttggaa ctagcaggta gacaagctga cagaagcaaa 960

tggttcaaga ttccatggga taccagacta cgaaatgctg atgagcaggg gacacttgtg 1020

tacattcaaa atcttgacat acagtttgca gctgctgaca tagaggagct tatacgtgat 1080

gctttacaac taaattgtat cgctaagcct attaaccacc caacttatga tgatccaaac 1140

aatggaaaag catatgctat attcaaaaca aaaagtgccg cagactctgc tatttcaaaa 1200

attaattcag gcttggtggt cggtggaaga cccctttatt gcagcaaagg attgcttaag 1260

gttccaaaac cttcagaaac tcttctcggg cacttaacaa tcaacaatat tagaatgggt 1320

ataagacaac gagaagaaca gaagaaggca gtttcaacct cgcattgttc tcaacccaat 1380

acaatggagt atgatttggc cttggattgg atgcttgtcc gagcaaagca agaaacgaaa 1440

tttaggacac ttcacaagaa gcataaagat gagaggaaga cctttgcgag taagatggga 1500

aagtaa 1506

<210> 3

<211> 7468

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gcgtccgtac tgagacccaa ccacaaaaat ctcacctcct cctcctcctc cctctcccac 60

cgcggcctcc tcctgctacc tgggagccac gcgcccccgg cctccacctc gccgccctcc 120

tcgccggcgg ccgccacgcg tgagtcctct cccctcttcc tccccgccgt cccccgtcac 180

tgttccgcgc tggatttcgc tgcgcggccg cgcgtggttc cgcccccaac cccccaccgg 240

ggaccgcggg gctccggtcg ctggagagcg ctccgcgtcg ccccgtcggg ctccggggcc 300

gctggtcgtg gtcccccgcg cgcgcggcct ccggttgcgc ggcgcgggcg aggaatgccg 360

gcgcgcgtgc gggcgggcgg gccgaacgac cccggtcgaa cggctagggc gctaaatttt 420

ggtgctcgaa tggaggtgtc cttgcgaggt tttgggggat ttcgtgtggg ggcgtcgcac 480

gttcggtttg cgtttccccc gtctagggtt tggggtttgg tggattcgtc gtcggtgtga 540

tcgcatttag tggctctagc tgctgttggt ggtaatttta gttctcatat tatggattgg 600

tcatgtcaaa accctagatg agttccctgg aacccgttag ctcacctcat gggtttgttc 660

ttcgtcgtgc tggacaccct cctttgctct tgttctaaaa actaaggcat gcatgacgat 720

ccttgttgcg gtggtactgt tttatatatg cttatggtta atggtgagcc tattagtaag 780

gtgaaacagc ggatacgaac atgtcagttg aaaaagcatt catgttttta tttcctaccc 840

tatttaagaa cacacgtttg atcaaaggcg ctaactgttg ccttttaggt tgaaaacagg 900

gtactccaag ccattgttga gcctcaagtt ttatgcatgt actatgtagt tactcctcta 960

atggccttct tattcaccat gagcattcat gaaacttatt acattttctt tactcctgtt 1020

tagtgagcat gtagaattca tgccaaccgg aaaatgtagc agattgattt gtccaaaaga 1080

taaaaaatcg aacgacttcc tgtgggattt tattccagca taacctaaaa tgtaactaat 1140

cagcatgaca gatttattta tcaagcagat ggatctgtac ttccattatg tttgcttatt 1200

taccgcgaat attttggaac tttagaataa ccacagccta tccagaaaaa taagaattaa 1260

ttaggttggt ttaaaatgca tcaccatgca gcaatcggtt gcaatttcat atctttatat 1320

ttttttatgt gttatctttg atgttcccgc atgcattcag ctgtagataa aacattgcta 1380

cattcagttg caaaataggg ttcagctgtg aaataggact tttcaagaag tctgagagaa 1440

gctgtccctt tctttgttga gaaaataaca tctgctgcag ttaatatttc atctttctca 1500

attcattaaa ctaactaatg tatatacagc gtattagcta ggttaactaa tcttgcaaat 1560

gacatagatt tgctaggtta actaaaatgt taacaactca aattcaacat gtgtgaagaa 1620

actagattta cgattttcgt tatgaagcga atgtctaaat tcacacttga tacggtaata 1680

gtactattag tttgtaagca tggtttgtat gtttgttgac cacctagcat aaacagcaca 1740

attaacagac accacccttt cgagcttcag aaactacgta tgttctgtgc taacattatc 1800

aatagtcaag tctgatgtgt tgatattgcc taatttgatc taaagtttga cctgctgatt 1860

ttatcactct aggcttatgg tgagtgcaca gtgagctaca agttaccatt atcagaatgg 1920

atggtagtaa tgagaatatc caattctcat gggggaagaa gagagcaaaa ggtggtatta 1980

agatggatac acagttttat gactccttca catttgacaa tgtgaagtac tcactgtatg 2040

acaatgtata tctttttaag agtggtgagt ctgaacctta cattggaaag ataataaaga 2100

tatggcagca aaatcaggct aagaaagtaa agattctttg gttttttctc ccggatgaga 2160

ttcgaaaaca tttaagtggc cctgtaatgg aaaaggagat atttcttgct tgtggtgaag 2220

gcgttggcct tgctgatatc aacccactgg taagttcata tattttcccc ttcgtttttg 2280

aattggtttt cttgaatatc attttacctt atgctttctt gcaatatatt taaatatttt 2340

cattttgatc actcaatgga cagataatca ctattatgtg aacactatat caaatttgtt 2400

tctaattgtg aaaagtcatt ggatgggcaa tagtgtcgac attttgttaa ttccaaatat 2460

ctgttgttga gaagccatat cggtagctag aatcctagag ctacaagtta ccttatcaag 2520

ttgtatctag tactagttga tgtgaggaga tcatttccca cttttttttg ttgggaatcg 2580

tccaccttgt atttgtggta tatttagtta ttcacgcatg acatgaataa ttcgaagtta 2640

gagttttaat tctcatgtat aaaaagtgca tcacaatttt caaagagcaa tatttaaatt 2700

cacatttttg gaagtgaaaa tgcagtccaa ctgagttaga cagagtaggc attagcgtgt 2760

tacccatttg catgacctga gaagagccag cttggttgcg acaatcaact tgaaaattga 2820

gatcatttct agctggtaca gttaaacatc cttttgagct aattggcata ccattttagt 2880

agagcatggt taggtattaa cagtcatgat acaagattga aactttcctg ctattgtgta 2940

ttacctgtga atgttaagtc atttgccttt tccatccttt tactttttct gagaactaaa 3000

atattataat acccttttgt ttattggtca cccaggaagc tattggtggg aaatgcactg 3060

tgctttgcat ttcaaaggat gagaggaatc gtcaaccttc ccccagggaa ctagcaatgg 3120

ctgattatat cttctacagg ttttttgatg ttaacagttg cacactttct gaacaattac 3180

ctgagaaaat tgcaggggtg gaaggtcagc aatatatcag atttcatcag tgcattaaat 3240

tgcattttta atatggcaaa aaatttctcc accctcagta tccaaattgg tatttaatta 3300

tttttcttct ggcacaggaa atcttttgct taattcaaaa gttgagcaag tgacatcctg 3360

ttcagaccag gaagtgcatg gtgttgatca gaagatgctt aatgtcccag ttccccttcc 3420

ccagtcaacg gttatggagg atgaaagtcc agttgctgca gtttcccttc ccccgtcagt 3480

attcaaggag gaaaatgtgg cttcagccat tccctttccc cagccagtgg tcaaggagga 3540

aagtgcggct gctgccattc cccctcccca tgtagcactg aaagaggaga gtgtgtccaa 3600

atctacagag aacattacca aacctgcaca gaaagttctc cctggggaga ggccaccaaa 3660

gagggtcaaa ttttctgaaa atgttacagt gcaaaatgtg ccattagatg ttcctgaaag 3720

accaagtcgc actggacctt tggaactagc aggtagacaa gctgtaagta tctgaaccaa 3780

cactgaggat acatttccac cttatcaatg aatagcttta tgccattgat gttatgatat 3840

catgttaaac tttaaacatt cccccaggta actcagttaa tattatctcc acaactcatc 3900

tgcatctaat ctgttgtttg tatataagta aaataaaaca gttaacctgg actggccctg 3960

caatcttccc ataaaaaagg ccagaccgag ctcctgggct gatcccctgg acctgtccag 4020

cacaagggac tatatgagca gttgacctgg gaaaatctca gtgcaactgc tagttttttt 4080

tccccacctg ggcaagtcat tctatcctct atcgaccaac ggtgccccta tgtggcattt 4140

ggagttagca aaaattacat gaaaggctgg cctgggaacc caggaggcca ggatgtggta 4200

cttaaagctc tcatgtgaaa cctacttgta tgcattctta actaaggtat aaagtactat 4260

atgtgcctca tgcattctga ttgtcaattg agcagttttg ccgactgccc agttacccaa 4320

tgcattgaat gggttgattt acgatctggg tctaagagca atgagtaaaa gggccacatg 4380

ttccttcaat tatttctgtt caagaatttt attcggctga acatcatctg cttttactat 4440

taaggttcaa ccaatttcta agaaacaagc cataatcaaa atgttctggt tctacaattt 4500

tcctactgat agccgtgttt acttttgtgt gcttacaccc ttttctgcta tttaacagga 4560

cagaagcaaa tggttcaaga ttgtgagtaa tcatttttcc tacattgtct cttctctctt 4620

gcgtatatac tatgattgta cagaagattt acatctatgc ttcatgagtc aatgcttcaa 4680

aactattaga aaaacaaaaa tatttcagtt gaccttattt gtgggtgggt ttacctttta 4740

aatggaaatt tcagccatgg gataccagac tacgaaatgc tgatgagcag gggacacttg 4800

tgtacattca aaatcttgac atacagtttg cagctgctga catagaggta ttgtcttgtc 4860

tatctggttg acataaagcc caaactgctt tttttattat attcttggct tataatttat 4920

cacttcttta ctagctttgc tatgttctct gttgcacaaa ctttgcgatt tgttttcaat 4980

gtcttacacc tcttaatttc cacaaggcct tgtatatgag aatttgtaca gggaaggatt 5040

tcatatggct cggaaagaac tcccgtccga gtttccccag tgctgtagat ttccctcttc 5100

atgcgataaa catgacgcta actaataata ctaatatata tatatatata tatatatata 5160

tatatatatt gggcaccact tctgaacggg tgggcgaatg gatgagcaaa agatgcaaca 5220

aagtggggat tatcccgagt ctaaagtgtc atttttatgt aacctatttg tttttttgtt 5280

ttctctgctc tataaagtaa aaaaaaagat ccacttttac tatttttctc tcgatgagac 5340

aatggtacat gtttctctat tgagtgcgcc tttacgtacg cgtttccgct acccactaat 5400

tttgaaagat gtataaattg gaaagaatgt agggagcagt aaaaaagaga aatgttttgt 5460

ttacaggaac atatttgatt ttagaatctg ttttaatctt ttttgtgctt tctgtttcct 5520

aatttttgcc aattttcttg ggctggtgta gttaacactt ttgctataaa gagattaaaa 5580

atattggttc tacaacatca ctttctaccc ctaatttcag tacctaagag aggttgagta 5640

ggtgtaccat actatttgat gcttccttgt tctgccatac ttggacaata acttctaaaa 5700

tggatacgtg catgtcccac tctcttaggt gtccatgttt gctactttct gtgccttatc 5760

tgctcttttt tccagtatca tcaacatact taatgaatat ttatcttgat taccatgttt 5820

acaggagctt atacgtgatg ctttacaact aaattgtatc gctaagccta ttaaccaccc 5880

aacttatgat gatccaaaca atggtgagtg agttttgttt gtaatacctt gcaagtttgc 5940

cacattcagg caatgttctc gaatgcgatt taaaacaatc tgcttccttg tttgtaggaa 6000

aagcatatgc tatattcaaa acaaaaagtg ccgcagactc tgctatttca aaaattaatt 6060

caggcttggt ggtcggtgga aggtatctca ctctgttgct tgcagatttg ctggacgaaa 6120

tttactacta tatatgtgtt catgttgcat ttattgtgct ctgttttaag tggggtcact 6180

gtctgttata gttcacatga tggcatattg tcatctcatt acaaccccaa ctgcagaatt 6240

aagagatctt ttctttagga aagttaaggg aaacttctat tagcaattgg ttggttctat 6300

cttgatacca cctcagggac acattaccac actgcaattg cttattaggc aatttgtcaa 6360

caaaattgca tgtgatgcca tttgcactag ttacttactg ttattatttt tcaaatttct 6420

accgtggatt aaataacaat atgttgttca atatttttgg ttgacccttt ataattctct 6480

tattgaatta tttgcagacc cctttattgc agcaaaggat tgcttaaggt tccaaaacct 6540

tcagaaactc ttctcgggca cttaacaatc aacaatatta gaatgggtat aagacaacga 6600

gaagaacagg tgctgatgtt gtcctatgaa gtttgctcta tttcttttct ctgtcttaat 6660

atttttgcac aaccattatt ctttctccag aagaaggcag tttcaacctc gcattgttct 6720

caacccaata caatggagta tgatttggcc ttggattgga tgcttgtccg agcaaagcaa 6780

gaaacgaaat ttaggacact tcacaaggta tgtgcacttc cactggagat ctctgtgtat 6840

accagctttc agttttatcc atacagacct taaattttga atgctcaatt tatatctata 6900

gaagcataaa gatgagagga agacctttgc gagtaagatg ggaaagtaag tggcttccat 6960

ggatttggtt aagcgatggg aactgtcctg ttccgtgctt atcttggagt cctctactga 7020

gtactgacta tcattcctcg gagctttatg cactttttgc ctgaagcaac acttatctgc 7080

gagtttttct gctgcgcaag aatgaaatgc aacgaccatt tgagggggac aactaactgc 7140

accacactga ctctgctcat gttccgtaga gcattatttt tagaaaggaa agaattgtgc 7200

catagcttaa gaaaccaaag taacattggt agaagtagcc cttgcagcaa gttaggaaag 7260

ccagaggctg ttttaggtta ccccgcacat caatttgatc tcacatggac acatcagtta 7320

gatctgttgc tccatgttag atagcaaatc aacgcgtgcc gtcatgaaat gtgtatgtat 7380

atatttttaa attcatgacc tggctattag ttcattcatg tgactgctta tgtgtcttga 7440

gttacaagtt acaacttgac tccgttcc 7468

Claims (10)

1. A protein, wherein the protein is a protein of a1), a2), or A3) as follows:

A1) the amino acid sequence is protein of SEQ ID No.1 in a sequence table;

A2) a protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues on the protein A1), has more than 90% of identity with the protein A1) and has the activity of regulating and controlling the tillering capacity of plants;

A3) a fusion protein obtained by connecting a protein tag to the N-terminus or/and the C-terminus of A1) or A2).

2. The protein of claim 1, wherein: the protein is derived from rice.

3. The protein-related biomaterial according to claim 1 or 2, which is any one of the following B1) to B7):

B1) a DNA molecule encoding the protein of claim 1;

B2) an expression cassette comprising the DNA molecule of B1);

B3) a recombinant vector containing the DNA molecule of B1) or a recombinant vector containing the expression cassette of B2);

B4) a recombinant microorganism containing B1) the DNA 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, a transgenic plant tissue or a transgenic plant organ containing the DNA molecule of B1), or a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ containing the expression cassette of B2);

B6) a nucleic acid molecule that reduces the expression of B1) the DNA molecule;

B7) an expression cassette, a recombinant vector, a recombinant microorganism, a transgenic plant cell line, a transgenic plant tissue or a transgenic plant organ comprising the nucleic acid molecule according to B6).

4. The biomaterial of claim 3, wherein: B1) the DNA molecule is a gene shown as b1) or b 2):

b1) the coding sequence of the coding chain is a cDNA molecule or a DNA molecule of SEQ ID No. 2;

b2) the nucleotides of the coding strand are cDNA molecules or DNA molecules of SEQ ID No. 2.

5. The protein of claim 1 or 2, or the biomaterial of claim 3 or 4, for use in any of the following C1-C2, also falls within the scope of the present invention:

C1) regulating and controlling tillering force;

C2) preparing the product for regulating and controlling tillering force.

6. Use according to claim 5, characterized in that: the tillering force is regulated and controlled to be the tillering force; the tillering force is to increase tillering number and/or promote tillering bud extension.

7. Use according to claim 5, characterized in that: the tillering force is regulated and controlled to reduce the tillering force; the tillering force is reduced by reducing tillering number and/or inhibiting tillering bud from extending; the reduction of tillering ability is achieved by promoting or increasing the expression of a gene encoding the protein according to claim 1.

8. A method for improving tillering capacity of rice is characterized by comprising the following steps: the method comprises the steps of inhibiting or reducing the expression of genes in receptor rice to obtain target rice with tillering force higher than that of the receptor rice; the gene is a gene encoding the protein of claim 1.

9. The method of claim 8, wherein: the inhibition or reduction of the expression of the gene in the receptor rice is realized by knocking out the gene in the receptor rice through a CRISPR/Cas9 system; the CRISPR/Cas9 system includes expressing a plasmid containing Cas9 and a gRNA with the target sequence of SEQ ID No.2, position 148 and 167.

10. A plant reagent characterized by: the reagent contains the protein of claim 1 or 2 or/and the protein-related biological material of claim 3 or 4.

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