CN111662366A - Preparation method of early-flowering high-yield tomato material - Google Patents

Abstract

The invention discloses a preparation method of early-flowering and high-yield tomato material. The preparation method of the early-flowering and high-yielding tomato material disclosed in the present invention comprises: reducing the activity of SlTOE1 in the recipient plant, reducing the content of SlTOE1 in the recipient plant, inhibiting the expression of the gene encoding SlTOE1 in the recipient plant, or knocking out the SlTOE1 in the recipient plant Compared with the recipient plant, the gene encoding SlTOE1 can obtain the target plant with shortened flowering time and increased yield. The present invention can obtain a new early-flowering and high-yielding plant variety within one year, while using traditional breeding methods requires continuous backcrossing and self-crossing, which takes at least 3-4 years. The method of the present invention is simple to operate, low in cost, and greatly accelerated. The breeding process has been improved, and it has overcome the insufficiency of both early flowering and high yield, which greatly meets the production needs and has broad application prospects.

Description

A kind of preparation method of early-flowering and high-yield tomato material

technical field

The invention relates to a preparation method of early-flowering and high-yield tomato material in the field of agricultural biotechnology.

Background technique

Flowering time is a key factor affecting yield. Generally, late flowering will prolong the growth period, increase biomass, and yield will also increase; early flowering will shorten the growth period, but biomass will decrease and yield will decrease. How to achieve early flowering and high yield has always been a difficult problem in breeding. At present, there are many varieties of late-flowering and high-yielding crops on the market, and few varieties with early-flowering and high-yielding. Compared with late-flowering and high-yielding varieties, early-flowering and high-yielding varieties have the advantages of shortening the growth period while maintaining high-yield, and they are more popular with farmers and are also the goal pursued by breeders.

The Arabidopsis TOE1 transcription factor can inhibit flowering. When the function of the Arabidopsis TOE1 gene is completely lost, the flowering is advanced, but the plants are thin and the seed yield is significantly reduced.

Genome editing is a genetic manipulation technique that can modify DNA sequences at the genome level. The CRISPR/Cas9 system (clustered regularly interspaced shortpalindromic repeats/Cas9endonuclease) is one of the most widely used genome editing tools. The working principle of this technology is that the endonuclease Cas9 protein cuts the target gene under the guidance of guide RNA (short guide RNA, sgRNA), resulting in DNA double-strand break, which will induce the DNA repair mechanism of the cell. During the process, variations such as insertion, deletion or substitution of bases will inevitably be introduced, so as to achieve site-directed mutagenesis of the target gene. The transgenic markers are then removed, and the resulting varieties are identical to those bred by traditional breeding methods.

SUMMARY OF THE INVENTION

The technical problem to be solved by the present invention is how to improve the yield of plants and shorten the flowering time of plants.

In order to solve the above-mentioned technical problems, the present invention firstly provides any one of the following applications of proteins or substances regulating the content and/or activity of the proteins:

D1) regulating plant flowering time;

D2) prepare a product for regulating plant flowering time;

D3) shorten the flowering time of plants;

D4) prepare a product that shortens the flowering time of plants;

D5) regulating plant yield;

D6) prepare the product of regulating and controlling plant yield;

D7) increase plant yield;

D8) preparing a product to improve plant yield;

D9) regulating plant flowering time and yield;

D10) prepare and control plant flowering time and yield products;

D11) shortening plant flowering time and increasing plant yield;

D12) preparation of shortening plant flowering time and improving plant yield products;

D13) Plant breeding;

Said protein is named SlTOE1 and is A1), A2) or A3) as follows:

A1) the amino acid sequence is the protein of sequence 3;

A2) The amino acid sequence shown in SEQ ID NO: 3 in the sequence listing has undergone the substitution and/or deletion and/or addition of one or several amino acid residues and has the same function;

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

In order to facilitate the purification of the protein in A1), the amino-terminal or carboxyl-terminal of the protein consisting of the amino acid sequence shown in SEQ ID NO: 3 in the sequence listing can be attached with the tags shown in the following table.

Table: Sequence of tags

Label Residues sequence Poly-Arg 5-6 (usually 5) RRRRR Poly-His 2-10 (usually 6) HHHHHH FLAG 8 DYKDDDDK Strep-tag II 8 WSHPQFEK c-myc 10 EQKLISEEDL

The SlTOE1 protein in the above A2) is a protein having 75% or more identity to the amino acid sequence of the protein shown in SEQ ID NO: 3 and having the same function. Having 75% or more identity is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical .

The SlTOE1 protein in the above A2) can be obtained by artificial synthesis, or by first synthesizing its encoding gene and then biologically expressing it.

The coding gene of the S1TOE1 protein in the above-mentioned A2) can be obtained by deleting the codons of one or several amino acid residues in the DNA sequence shown in SEQ ID NO: 2, and/or carrying out missense mutation of one or several base pairs, and / or the coding sequence of the tag shown in the above table is attached to its 5' end and/or 3' end. Wherein, the DNA molecule shown in sequence 2 encodes the SlTOE1 protein shown in sequence 3.

The present invention also provides any of the following applications of the biological material related to SlTOE1:

D1) regulating plant flowering time;

D2) prepare a product for regulating plant flowering time;

D3) shorten the flowering time of plants;

D4) prepare a product that shortens the flowering time of plants;

D5) regulating plant yield;

D6) prepare the product of regulating and controlling plant yield;

D7) increase plant yield;

D8) preparing a product to improve plant yield;

D9) regulating plant flowering time and yield;

D10) prepare and control plant flowering time and yield products;

D11) shortening plant flowering time and increasing plant yield;

D12) preparation of shortening plant flowering time and improving plant yield products;

D13) Plant breeding;

The biological material is any one of the following B1) to B9):

B1) a nucleic acid molecule encoding SlTOE1;

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

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

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

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

B6) a transgenic plant tissue containing the nucleic acid molecule of B1), or a transgenic plant tissue containing the expression cassette of B2);

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

B8) nucleic acid molecules that reduce the content and/or activity of SlTOE1;

B9) An expression cassette, recombinant vector, recombinant microorganism or transgenic plant cell line comprising the nucleic acid molecule of B8).

In the above-mentioned application, the nucleic acid molecule of B1) can be any one of the following b1)-b5):

b1) The coding sequence is the cDNA molecule or DNA molecule of sequence 2 in the sequence listing;

b2) the cDNA molecule or DNA molecule shown in sequence 2 in the sequence listing;

b3) the DNA molecule shown in sequence 1 in the sequence listing;

b4) has 75% or more identity with the nucleotide sequence defined in b1) or b2) or b3), and encodes a cDNA molecule or DNA molecule of SlTOE1;

b5) hybridizes to a nucleotide sequence defined in b1) or b2) or b3) or b4) under stringent conditions and encodes a cDNA molecule or DNA molecule of SlTOE1;

B8) The nucleic acid molecule is any one of the following c1)-c4):

c1) the RNA molecule shown in sequence 4 in the sequence listing;

c2) DNA molecule of RNA shown in sequence 4 in the transcription sequence table;

c3) RNA molecules or DNA molecules having 75% or more identity with the nucleotide sequence defined in c1) or c2) and having the same function;

c4) An RNA molecule or a DNA molecule that hybridizes under stringent conditions to the nucleotide sequence defined in c1) or c2) or c3) and has the same function.

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

Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the SLTOE1 protein of the present invention using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides with 75% or higher identity to the nucleotide sequence of the SLTOE1 protein isolated by the present invention, as long as they encode the SLTOE1 protein and have the function of the SLTOE1 protein, are all derived from the nucleus of the present invention. nucleotide sequences and are equivalent to the sequences of the present invention.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "Identity" includes 75% or higher, or 85% or higher, or 90% or higher, or 95% or Nucleotide sequences of higher identity. Identity can be assessed with the naked eye or with computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above application, the stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ and 1 mM EDTA, 50°C, 2×SSC, 0.1% Rinse in SDS; also: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA, rinse at 50°C, 1×SSC, 0.1% SDS; also: 50°C, Hybridize in a mixture of 7% SDS, 0.5M NaPO and ₁ mM EDTA, rinse at 50°C in 0.5×SSC, 0.1% SDS; also: 50°C in 7% SDS, 0.5M NaPO and ₁ mM Hybridize in a mixed solution of EDTA, rinse in 0.1×SSC, 0.1% SDS at 50°C; also: hybridize in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA at 50°C, at 65°C, Wash in 0.1×SSC, 0.1% SDS; also: in 6×SSC, 0.5% SDS solution, hybridize at 65°C, then use 2×SSC, 0.1%SDS and 1×SSC, 0.1%SDS each Wash the membrane once; it can also be: hybridize in a solution of 2×SSC, 0.1% SDS at 68°C and wash the membrane twice for 5 min each, and then in a solution of 0.5×SSC, 0.1% SDS at 68°C Hybridize and wash the membrane twice at 65°C, each time for 15 min; or in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS, hybridize and wash the membrane at 65°C.

The above-mentioned 75% or more identity may be 80%, 85%, 90% or more than 95% identity.

In above-mentioned application, B2) described expression cassette (SLTOE1 gene expression cassette) containing the nucleic acid molecule of coding SLTOE1 protein, refers to the DNA that can express SLTOE1 protein in host cell, this DNA can not only include the start that starts SLTOE1 gene transcription , and may also include a terminator that terminates transcription of the SLTOE1 gene. Further, the expression cassette may also include enhancer sequences.

The recombinant vector containing the SLTOE1 gene expression cassette can be constructed by using the existing expression vector.

In the above applications, the vector may be a plasmid, cosmid, phage or viral vector.

B9) The recombinant vector can be a recombinant vector prepared by using the crisper/cas9 system that can reduce the content of SLTOE1. The recombinant vector can express sgRNA targeting the nucleic acid molecule of B1). The target sequence of the sgRNA can be positions 317-336 of SEQ ID NO: 1 in the sequence listing.

B9) The recombinant vector can specifically be a recombinant vector obtained by inserting the double-stranded DNA of the target sequence into the pTX041 vector using the restriction endonuclease BsaI and capable of transcribing the sgRNA shown in SEQ ID NO: 4 in the sequence listing.

In the above applications, the microorganisms may be yeast, bacteria, algae or fungi. Wherein, the bacteria can be Agrobacterium, such as Agrobacterium rhizogenes LBA4404.

In the above application, the transgenic plant cell line, transgenic plant tissue and transgenic plant organ do not include propagation material.

The present invention also provides any of the following methods:

X1) A method for shortening the flowering time of plants, comprising: reducing the activity of SlTOE1 in recipient plants, reducing the content of SlTOE1 in recipient plants, inhibiting the expression of SlTOE1-encoding genes in recipient plants, or knocking out SlTOE1 encoding in recipient plants A gene to obtain a target plant with a shortened flowering time compared with the recipient plant;

X2) A method for cultivating plants with shortened flowering time, comprising: reducing the activity of SlTOE1 in the recipient plant, reducing the content of SlTOE1 in the recipient plant, inhibiting the expression of the gene encoding SlTOE1 in the recipient plant, or knocking out the expression of SlTOE1 in the recipient plant encoding a gene to obtain a target plant with a shortened flowering time compared with the recipient plant;

X3) A method for improving plant yield, comprising: reducing the activity of SlTOE1 in the recipient plant, reducing the content of SlTOE1 in the recipient plant, inhibiting the expression of the gene encoding SlTOE1 in the recipient plant, or knocking out the gene encoding SlTOE1 in the recipient plant , to obtain the target plant with increased yield compared with the recipient plant;

X4) A method for cultivating yield-improving plants, comprising: reducing the activity of SlTOE1 in recipient plants, reducing the content of SlTOE1 in recipient plants, inhibiting the expression of a gene encoding SlTOE1 in recipient plants, or knocking out the encoding of SlTOE1 in recipient plants A gene to obtain a target plant with increased yield compared with the recipient plant;

X5) A method for shortening plant flowering time and improving plant yield, comprising: reducing the activity of SlTOE1 in recipient plants, reducing the content of SlTOE1 in recipient plants, inhibiting the expression of genes encoding SlTOE1 in recipient plants, or knocking out recipient plants The encoding gene of SlTOE1 in the middle, obtains the target plant with shortened flowering time and improved yield compared with the recipient plant;

X6) A method for cultivating plants with shortened flowering time and increased yield, comprising: reducing the activity of SlTOE1 in recipient plants, reducing the content of SlTOE1 in recipient plants, inhibiting the expression of genes encoding SlTOE1 in recipient plants, or knocking out recipient plants Compared with the recipient plant, the encoding gene of SlTOE1 can be obtained to obtain the target plant with shortened flowering time and increased yield.

The recipient plant contains the gene encoding SlTOE1.

In the above method, knockout of the gene encoding SlTOE1 in recipient plants can be achieved by CRISPR/Cas9 method.

The target sequence used in the CRISPR/Cas9 method is positions 317-336 of Sequence 1 in the sequence listing.

The sgRNA used in the CRISPR/Cas9 method can specifically be the RNA shown in SEQ ID NO: 4 in the sequence listing.

In the above method, the knockout of the gene encoding SlTOE1 in the recipient plant can be specifically achieved by introducing the recombinant vector described in B9) into the plant.

The method may also include the step of screening for plants of interest that do not contain the exogenous DNA sequence.

Compared with the recipient plant, the gene encoding SlTOE1 in the target plant is mutated, resulting in decreased SlTOE1 content and/or loss of SlTOE1 function. The mutation may be a deletion, insertion and/or change of one to more nucleotides.

In an embodiment of the present invention, compared with the recipient plant, the nucleotides 320-323 of the gene encoding SlTOE1 in the target plant corresponding to sequence 2 in the sequence listing are deleted.

The present invention also provides a product having the function of shortening plant flowering time and/or increasing plant yield, said product containing said biological material.

The product may use the biological material as its active ingredient, or may combine the biological material with a substance having the same function as its active ingredient.

The application of the target plant prepared by the method in plant breeding also belongs to the protection scope of the present invention.

In the present invention, the plant can be any one of c1)-c4):

c1) Tubular flowers;

c2) Solanaceae;

c3) Tomato plants;

c4) Tomato.

Said specific yield is reflected in the fruit of said plant.

Experiments show that the present invention achieves shortening of flowering time and improvement of yield after knocking out the TOE1 gene in plants by using the CRISPR/Cas9 method, and successfully cultivates early-flowering and high-yielding plants. The present invention can obtain a new early-flowering and high-yielding plant variety within one year, while using traditional breeding methods requires continuous backcrossing and self-crossing, which takes at least 3-4 years. The method of the present invention is simple to operate, low in cost, and greatly accelerated. The breeding process has been improved, and it has overcome the insufficiency of both early flowering and high yield, which greatly meets the production needs and has broad application prospects.

Description of drawings

Figure 1 shows the sequence mutation of plant 2.

Figure 2 shows the flowering timetable of wild-type tomato and plant 2, and the flowering time is represented by the number of leaves before the first sequence of flowers. Wild type refers to wild type tomato.

Figure 3 shows the yield phenotypes of wild-type tomato and plant 2, A is a photo of the phenotype, B is the yield statistics, and wild-type refers to wild-type tomato.

Detailed ways

The present invention will be further described in detail below with reference to the specific embodiments, and the given examples are only for illustrating the present invention, rather than for limiting the scope of the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. Materials, reagents, instruments, etc. used in the following examples can be obtained from commercial sources unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged. In the following examples, unless otherwise specified, the first position of each nucleotide sequence in the sequence listing is the 5'-terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3'-terminal nucleus of the corresponding DNA/RNA. Glycosides.

Tomato was a conventional tomato variety M82 (also referred to as wild-type tomato hereinafter), which was obtained from the American Tomato Genetic Resources Center (TGRC, http://tgrc.ucdavis.edu/).

Example 1. Using CRISPR/Cas9 method to edit tomato SlTOE1 gene to obtain early-flowering and high-yielding tomato

In this example, the TOE1 gene (referred to as SlTOE1 gene) of tomato was gene-edited by CRISPR/Cas9 method to obtain tomato with early flowering time and high yield. In wild-type tomato, the sequence of the SlTOE1 gene is sequence 1 in the sequence table, Its CDS sequence is sequence 2, which encodes the protein shown in sequence 3 in the sequence listing (denoted as SlTOE1 protein). Specific steps are as follows:

1. Construction of recombinant vector

Positions 317-336 of sequence 1 in the sequence listing were used as the target sequence, and the sequence was 5'-ATCGGGTCATTCTCTCCTCC-3' to construct a recombinant vector capable of expressing the targeting target sequence sgRNA.

Synthesize the double-stranded DNA of the target sequence, and use the restriction endonuclease BsaI to insert the double-stranded DNA of the target sequence into the CRISPR/Cas9 carrier (that is, the document "Deng et al., Efficient generation of pink-fruited tomatoes using CRISPR/Cas9system, Journal of Genetics and Genomics 45 (2018) 51-54" in pTX041), the recombinant vector was obtained. The recombinant vector can express the sgRNA targeting the target sequence shown in Sequence 4 in the sequence listing.

2. Construction and identification of transgenic tomato

Construction of transgenic tomato: The recombinant vector obtained in step 1 was introduced into Agrobacterium LBA4404 strain, and the wild-type tomato was transformed by Agrobacterium-mediated transformation to construct TO transgenic tomato.

Identification of transgenic tomato: PCR amplification was performed on the genomic DNA of the above-mentioned transgenic plants by using primer pair 1 consisting of a forward primer (5'-ATGTTGGATCTCAATGTAAGCG-3') and a reverse primer (5'-GACCTTGGGCCTCTCCTACTCT-3') to obtain PCR Product, the obtained PCR product was sequenced, and a plant with a homozygous mutation of the SlTOE1 gene was identified and named as plant 2. Compared with the wild-type tomato, the 4th to 7th positions of the target sequence were missing in this plant (Fig. 1), this kind of deletion leads to a frameshift mutation in the CDS sequence of the SlTOE1 gene from position 320, resulting in a stop codon, and a truncated protein that loses the DNA binding domain is translated, so that the function of the SlTOE1 protein is lost, and the plant does not contain the SlTOE1 protein. . Plant 2 was grown in a greenhouse, and T1 generation transgenic seeds were obtained by selfing. PCR method was used to identify individuals in the seeds of T1 generation that did not contain exogenous sequences (that is, exogenous sequences other than tomato genome sequences used in the process of editing tomato SlTOE1 gene by CRISPR/Cas9 method) (the primer pair used was denoted as primer pair 2, The sequence of the primer pair is: forward primer: 5'-TTGACAAGCTGTTCATCCAG-3'; reverse primer: 5'-CCTTCGTAATCTCGGTGTTC-3'). 1/4 of the individuals identified from the T1 generation of plant 2 did not contain foreign sequences.

Whether the SlTOE1 gene of the T1 generation individuals identified without exogenous sequences was still homozygous mutation was detected by sequencing. The SlTOE1 gene fragment was amplified by PCR with primer pair 1, and then the PCR product was sequenced. The sequencing results were compared with the wild-type tomato SlTOE1 gene, and it was found that there was still a homozygous mutation at the position of the target sequence (that is, it was still missing in both chromosomes). 4-7 of the target sequence), indicating that the mutations generated by CRISPR/Cas9 can be stably inherited from the T0 generation to the T1 generation. These T1 generation plants that do not contain foreign sequences and have homozygous mutations in the SlTOE1 gene are the target SlTOE1 gene-edited plants.

3. Determination of flowering time and yield

The flowering time and yield of tomato plants were measured in Beijing, and the plants to be tested were the target SlTOE1 gene-edited plants obtained in step 2 and wild-type tomato (as control plants).

The plants to be tested were raised in a greenhouse, and cultivated in the light for 20 d at 25° C. under the conditions of 16 h light/8 h dark. Each selected 20 seedlings with the same growth and transplanted into the greenhouse, the plant spacing and row spacing were more than 60cm, and the two materials were randomly distributed. Normal water and fertilizer management ensures that all plants have basically the same water and fertilizer conditions. The plant is not interrupted and allowed to grow naturally. After flowering, the number of leaves before the first sequence of flowers is counted. The number of leaves before the first sequence of flowers can represent the flowering time. The more the number, the later the flowering, and vice versa. For all the fruits of the SlTOE1 gene-edited plants and the control plants, the fruit weights of each plant were weighed and recorded by taking pictures. The results are shown in Table 1.

Table 1. Phenotype of each material

Material Yield (kg/plant) Number of leaves before the first inflorescence (number) Objective SlTOE1 gene-edited plants 4.56 7.94 wild type tomato 3.01 6.50

The results showed that compared with wild-type tomato, the yield of the target SlTOE1 gene-edited plants was significantly increased, the number of leaves before the first inflorescence was reduced by about 1.5, and the flowering time was significantly shortened. It is shown that the SlTOE1 gene and its encoded protein can regulate the yield and flowering time of tomato, and SlTOE1 gene editing can produce early flowering and high-yielding materials of tomato.

<110> Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing Academy of Agriculture and Forestry

<120> A kind of preparation method of early-flowering and high-yield tomato material

<160> 4

<170> PatentIn version 3.5

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ggagctgaga ctagtaggag cagcaacaac gatgatgagg aaggatacgg taagaatcag 240

agagttactc attctcaatt cgtgactagg cagctgtttc ccgttgatga tggtgagttg 300

aaccggaaac aaaccgatcg ggtcattctc tcctccgctc gatccggtac ttctatcggt 360

tttggagatg tgcggataat acaacagcaa caaacggagc aaccgaaaca acaagtgaag 420

aagagtagga gaggcccaag gtcaagaagt tcacagtaca gaggtgtcac tttctaccgt 480

agaactggta gatgggaatc acatatatgg gactgtggga aacaagtata tttgggtggt 540

tttgatactg ctcacacagc agcaagagct tatgacagag ctgcaattaa atttaggggt 600

gttgatgctg atatcaactt tagcttaagt gattacgagg aggatatgca acagatgaaa 660

aaccttggta aagaagaatt tgtgcacttg ctgcgacgcc atagcactgg tttctcaaga 720

gggagctcca aattcagagg agtgacgcta cataaatgtg gcagatggga ggctcggatg 780

ggacagttcc tcgggaaaaa gtatatatat cttgggctgt tcgacagcga agtagaagct 840

gcaagggcct acgataaggc ggcaattaaa actagcggaa gggaagctgt taccaacttt 900

gagccaagta gctatgaagg ggaaacaatg tctttaccac agagtgaagg tagccaacat 960

gatcttgatc tgaacttggg gatatcgacc acttcttcaa aggaaaatga caggttggga 1020

ggttctcgct atcatcctta cgatatgcaa gacgcaacaa aacctaagat ggataaacct 1080

ggttcagtaa tagttggaag ttcacatctc aagggactac caatgtcgtc ccaacaagct 1140

caattgtgga ctggaatcta ttctaatttc tcttccagct atgagggaag agcatatgac 1200

aagagaaagg acacaggttc atcacaagga cctccaaatt gggcactgca aatgcctagt 1260

caggttgata caaacagccc attgacaatg ttctgcacgg cagcatcatc aggattcttc 1320

attccatcta ctacttctat cacttcatca acatctgcat tagcaacttc aacaaatgcc 1380

tcgcagtgct tttaccagat taatccccgc ctaccacttc cataa 1425

<210> 3

<211> 474

<212> PRT

<213> Tomato (Solanum lycopersicum)

<400> 3

Met Leu Asp Leu Asn Val Ser Val Ile Tyr Asn Asn Asp Leu Pro Gln

1 5 10 15

Val Ser Leu Leu Asp Glu Ser Ala Thr Ser Asn Ser Ser Leu Arg Asn

20 25 30

Ala Glu Ala Thr Thr Ser Ala Gly Asp Glu Asp Ser Cys Ala Gly Glu

35 40 45

Leu Phe Ala Phe Asn Phe Gly Ile Leu Lys Val Glu Gly Ala Glu Thr

50 55 60

Ser Arg Ser Ser Asn Asn Asp Asp Glu Glu Gly Tyr Gly Lys Asn Gln

65 70 75 80

Arg Val Thr His Ser Gln Phe Val Thr Arg Gln Leu Phe Pro Val Asp

85 90 95

Asp Gly Glu Leu Asn Arg Lys Gln Thr Asp Arg Val Ile Leu Ser Ser

100 105 110

Ala Arg Ser Gly Thr Ser Ile Gly Phe Gly Asp Val Arg Ile Ile Gln

115 120 125

Gln Gln Gln Thr Glu Gln Pro Lys Gln Gln Val Lys Lys Ser Arg Arg

130 135 140

Gly Pro Arg Ser Arg Ser Ser Gln Tyr Arg Gly Val Thr Phe Tyr Arg

145 150 155 160

Arg Thr Gly Arg Trp Glu Ser His Ile Trp Asp Cys Gly Lys Gln Val

165 170 175

Tyr Leu Gly Gly Phe Asp Thr Ala His Thr Ala Ala Arg Ala Tyr Asp

180 185 190

Arg Ala Ala Ile Lys Phe Arg Gly Val Asp Ala Asp Ile Asn Phe Ser

195 200 205

Leu Ser Asp Tyr Glu Glu Asp Met Gln Gln Met Lys Asn Leu Gly Lys

210 215 220

Glu Glu Phe Val His Leu Leu Arg Arg His Ser Thr Gly Phe Ser Arg

225 230 235 240

Gly Ser Ser Lys Phe Arg Gly Val Thr Leu His Lys Cys Gly Arg Trp

245 250 255

Glu Ala Arg Met Gly Gln Phe Leu Gly Lys Lys Tyr Ile Tyr Leu Gly

260 265 270

Leu Phe Asp Ser Glu Val Glu Ala Ala Arg Ala Tyr Asp Lys Ala Ala

275 280 285

Ile Lys Thr Ser Gly Arg Glu Ala Val Thr Asn Phe Glu Pro Ser Ser

290 295 300

Tyr Glu Gly Glu Thr Met Ser Leu Pro Gln Ser Glu Gly Ser Gln His

305 310 315 320

Asp Leu Asp Leu Asn Leu Gly Ile Ser Thr Thr Ser Ser Lys Glu Asn

325 330 335

Asp Arg Leu Gly Gly Ser Arg Tyr His Pro Tyr Asp Met Gln Asp Ala

340 345 350

Thr Lys Pro Lys Met Asp Lys Pro Gly Ser Val Ile Val Gly Ser Ser

355 360 365

His Leu Lys Gly Leu Pro Met Ser Ser Gln Gln Ala Gln Leu Trp Thr

370 375 380

Gly Ile Tyr Ser Asn Phe Ser Ser Ser Tyr Glu Gly Arg Ala Tyr Asp

385 390 395 400

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

405 410 415

Gln Met Pro Ser Gln Val Asp Thr Asn Ser Pro Leu Thr Met Phe Cys

420 425 430

Thr Ala Ala Ser Ser Gly Phe Phe Ile Pro Ser Thr Thr Ser Ile Thr

435 440 445

Ser Ser Thr Ser Ala Leu Ala Thr Ser Thr Asn Ala Ser Gln Cys Phe

450 455 460

Tyr Gln Ile Asn Pro Arg Leu Pro Leu Pro

465 470

<210> 4

<211> 97

<212> RNA

<213> Artificial sequences

<400> 4

gggaggagag aaugacccga uguuuuagag cuagaaauag caaguuaaaa uaaggcuagu 60

ccguuaucaa cuugaaaaag uggcaccgag ucggugc 97

Claims (10)

1. Use of a protein or a substance modulating the content and/or activity of said protein of any one of the following:

D1) regulating and controlling the flowering time of the plants;

D2) preparing products for regulating and controlling the flowering time of plants;

D3) shortening the flowering time of plants;

D4) preparing products for shortening the flowering time of plants;

D5) regulating and controlling the yield of the plant;

D6) preparing a product for regulating and controlling the yield of the plants;

D7) the yield of the plants is improved;

D8) preparing a product for increasing the plant yield;

D9) regulating and controlling the flowering time and yield of the plants;

D10) preparing products for regulating and controlling the flowering time and the yield of plants;

D11) shortening the flowering time of plants and increasing the yield of plants;

D12) preparing products for shortening the flowering time of plants and improving the yield of the plants;

D13) plant breeding;

the protein is A1), A2) or A3) as follows:

A1) the amino acid sequence is the protein of sequence 3;

A2) the 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 the sequence 3 in the sequence table and has the same function;

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

2. Use of a biological material related to a protein according to claim 1, wherein the biological material is selected from the group consisting of:

D1) regulating and controlling the flowering time of the plants;

D2) preparing products for regulating and controlling the flowering time of plants;

D3) shortening the flowering time of plants;

D4) preparing products for shortening the flowering time of plants;

D5) regulating and controlling the yield of the plant;

D6) preparing a product for regulating and controlling the yield of the plants;

D7) the yield of the plants is improved;

D8) preparing a product for increasing the plant yield;

D9) regulating and controlling the flowering time and yield of the plants;

D10) preparing products for regulating and controlling the flowering time and the yield of plants;

D11) shortening the flowering time of plants and increasing the yield of plants;

D12) preparing products for shortening the flowering time of plants and improving the yield of the plants;

D13) plant breeding;

the biomaterial is any one of the following B1) to B9):

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

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

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

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

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

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

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

B8) a nucleic acid molecule that reduces the content and/or activity of the protein of claim 1;

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

3. Use according to claim 2, characterized in that: B1) the nucleic acid molecule is any one of the following b1) -b 5):

b1) the coding sequence is cDNA molecule or DNA molecule of sequence 2 in the sequence table;

b2) a cDNA molecule or a DNA molecule shown in a sequence 2 in a sequence table;

b3) DNA molecule shown in sequence 1 in the sequence table;

b4) a cDNA or DNA molecule having 75% or more identity with the nucleotide sequence defined in b1) or b2) or b3) and encoding the protein of claim 1;

b5) a cDNA molecule or a DNA molecule which hybridizes under stringent conditions with the nucleotide sequence defined in b1) or b2) or b3) or b4) and encodes the protein of claim 1;

B8) the nucleic acid molecule is any one of c1) -c4) as follows:

c1) RNA molecule shown in sequence 4 in the sequence table;

c2) transcribing a DNA molecule of RNA shown in a sequence 4 in a sequence table;

c3) an RNA molecule or DNA molecule with 75 percent or more than 75 percent of identity with the nucleotide sequence defined by c1) or c2) and the same function;

c4) RNA molecule or DNA molecule which hybridizes with the nucleotide sequence defined by c1) or c2) or c3) under strict conditions and has the same function.

4. Any one of the following methods:

x1) a method for reducing flowering time in plants comprising: reducing the activity of the protein of claim 1 in a recipient plant, reducing the content of the protein of claim 1 in a recipient plant, inhibiting the expression of the gene encoding the protein of claim 1 in a recipient plant, or knocking out the gene encoding the protein of claim 1 in a recipient plant, to obtain a plant of interest with reduced flowering-time as compared to the recipient plant;

x2) method for cultivating plants with reduced flowering time comprising: reducing the activity of the protein of claim 1 in a recipient plant, reducing the content of the protein of claim 1 in a recipient plant, inhibiting the expression of the gene encoding the protein of claim 1 in a recipient plant, or knocking out the gene encoding the protein of claim 1 in a recipient plant, to obtain a plant of interest with reduced flowering-time as compared to the recipient plant;

x3) method for increasing plant yield, comprising: reducing the activity of a protein according to claim 1 in a recipient plant, reducing the content of a protein according to claim 1 in a recipient plant, inhibiting the expression of a gene encoding a protein according to claim 1 in a recipient plant, or knocking out a gene encoding a protein according to claim 1 in a recipient plant, resulting in a plant of interest having an increased yield as compared to the recipient plant;

x4) method for growing plants with increased yield, comprising: reducing the activity of a protein according to claim 1 in a recipient plant, reducing the content of a protein according to claim 1 in a recipient plant, inhibiting the expression of a gene encoding a protein according to claim 1 in a recipient plant, or knocking out a gene encoding a protein according to claim 1 in a recipient plant, resulting in a plant of interest having an increased yield as compared to the recipient plant;

x5) method for reducing flowering time and increasing plant yield in plants comprising: reducing the activity of the protein of claim 1 in a recipient plant, reducing the content of the protein of claim 1 in a recipient plant, inhibiting the expression of the gene encoding the protein of claim 1 in a recipient plant, or knocking out the gene encoding the protein of claim 1 in a recipient plant, to obtain a plant of interest with reduced flowering-time and increased yield as compared to said recipient plant;

x6) method for cultivating plants with reduced flowering-time and increased yield, comprising: reducing the activity of the protein of claim 1 in a recipient plant, reducing the content of the protein of claim 1 in a recipient plant, inhibiting the expression of the gene encoding the protein of claim 1 in a recipient plant, or knocking out the gene encoding the protein of claim 1 in a recipient plant, to obtain a plant of interest with reduced flowering-time and increased yield as compared to said recipient plant.

5. The method of claim 4, wherein: knocking out a gene encoding the protein of claim 1 in a recipient plant is achieved using the CRISPR/Cas9 method.

6. The method of claim 6, wherein: the target sequence used in the CRISPR/Cas9 method is 317-336 th site of sequence 1 in the sequence table.

7. A product having the function of shortening flowering-time and/or increasing plant yield, comprising the biomaterial as claimed in claim 2 or 3.

8. Use according to any one of claims 1 to 3, or a method according to any one of claims 4 to 6, or a product according to claim 7, wherein: the plant is any one of c1) -c 4):

c1) tubular plants of the order florida;

c2) a plant of the Solanaceae family;

c3) a plant of the genus Lycopersicon;

c4) tomato.

9. The use, method or product according to claim 8, wherein: said yield is represented on the fruit of said plant.

10. Use of a plant of interest prepared by the method of any one of claims 4-6 in plant breeding.

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