CN111850031B

CN111850031B - Application of protein GmFULc in regulation and control of plant yield

Info

Publication number: CN111850031B
Application number: CN201910289448.2A
Authority: CN
Inventors: 赵琳; 李文滨; 孙晶哲
Original assignee: Northeast Agricultural University
Current assignee: Northeast Agricultural University
Priority date: 2019-04-11
Filing date: 2019-04-11
Publication date: 2023-03-24
Anticipated expiration: 2039-04-11
Also published as: CN111850031A

Abstract

The invention discloses application of protein GmFULc in regulation and control of plant yield and/or photoperiod and/or plant height and/or flowering time, wherein an amino acid sequence of the protein GmFULc is shown as a sequence 2 in a sequence table. Experiments prove that the expression quantity and/or activity of protein GmFULc in soybean are improved, and transgenic soybean is obtained; compared with wild soybean, the transgenic soybean has the advantages of earlier flowering time, and obvious increase of plant height, pod number, four-grain pod number and single-plant grain number. The protein GmFULc can regulate and control the yield, the photoperiod, the plant height and the flowering time of plants. The invention has important application value.

Description

Application of protein GmFULc in regulation and control of plant yield

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of protein GmFULc in regulation and control of plant yield.

Background

Soybeans are Short Day Plants (SDP) and are sensitive to photoperiod reaction, and the characteristic seriously hinders the adaptability of soybean varieties, limits the soybean sowing range and influences the effective exertion of yield level.

Flowering time and maturity of soybeans are important agronomic traits that control yield, quality and adaptability of soybean varieties. Improvement of soybean growth time traits has been a hot issue of research. The earliness is a common requirement for soybean breeding in various regions, and is also a foundation for northward expanding planting of plants such as soybeans and the like in China and a foundation for land resource utilization in high-altitude regions. Modern bioengineering technology can break the boundary between organisms to realize recombination of genetic materials, and breeding is carried out according to human pre-design, so that the requirement of human is met. The bioengineering technology is combined with the traditional breeding technology, the new soybean variety with early maturity and high yield traits is cultivated by genetic breeding of excellent genes with early maturity and high yield, and necessary theoretical evidence is provided for soybean transgenic breeding, on one hand, the method can provide data for cultivating other new crop varieties by a gene transformation method, and simultaneously, huge social, economic and ecological benefits are generated.

Disclosure of Invention

The object of the present invention is to increase plant yield.

The invention firstly protects the application of the protein GmFULc, which can be at least one of the following S1) to S5):

s1) regulating and controlling the yield of plants;

s2) regulating and controlling the plant height of the plant;

s3) regulating and controlling the flowering time of the plants;

s4) regulating and controlling the plant photoperiod;

s5) cultivating transgenic plants with increased yield and/or increased plant height and/or earlier flowering time and/or altered photoperiod.

In the above application, the protein GmFULc may be a 1) or a 2) or a 3):

a1 ) the amino acid sequence is protein shown as a sequence 2 in a sequence table;

a2 A fusion protein obtained by connecting labels to the N terminal or/and the C terminal of the protein shown in the sequence 2 in the sequence table;

a3 Protein obtained by substituting and/or deleting and/or adding one or more amino acid residues to the protein shown in a 1) or a 2) and related to the yield and/or plant height and/or flowering time and/or photoperiod of the plant.

Wherein, the sequence 2 in the sequence table is composed of 239 amino acid residues.

In order to facilitate the purification of the protein in a 1), a tag as shown in Table 1 can be attached to the amino terminal or the carboxyl terminal of the protein shown in the sequence 2 in the sequence table.

TABLE 1 sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
FLAG	8	DYKDDDDK
			Strep-tag II	8	WSHPQFEK
c-myc	10	EQKLISEEDL

The protein according to a 3) above, wherein the substitution and/or deletion and/or addition of one or more amino acid residues is a substitution and/or deletion and/or addition of not more than 10 amino acid residues.

The protein in a 3) can be artificially synthesized, or can be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of a 3) above can be obtained by deleting one or several codons of amino acid residues from the DNA sequence shown in sequence 1 of the sequence table, and/or performing missense mutation of one or several base pairs, and/or connecting the coding sequence of the tag shown in Table 1 at the 5 'end and/or 3' end thereof.

The invention also protects the application of a nucleic acid molecule for coding any one of the proteins GmFULc, which can be at least one of the following S1) to S5):

s1) regulating and controlling the yield of plants;

s2) regulating and controlling the plant height of the plant;

s3) regulating and controlling the flowering time of the plants;

s4) regulating and controlling the plant photoperiod;

In the above application, the nucleic acid molecule encoding the protein GmFULc may be a DNA molecule represented by b 1) or b 2) or b 3) or b 4) as follows:

b1 The coding region is a DNA molecule shown as a sequence 1 in a sequence table;

b2 The nucleotide sequence is a DNA molecule shown as a sequence 1 in a sequence table;

b3 A DNA molecule which has 75 percent or more than 75 percent of identity with the nucleotide sequence defined by the b 1) or the b2 and codes the protein GmFULc;

b4 A DNA molecule which hybridizes with the nucleotide sequence defined in (b 1) or (b 2) under stringent conditions and codes for the protein GmFULc.

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

Wherein, the sequence 1 in the sequence table is composed of 720 nucleotides, and the nucleotide of the sequence 1 in the sequence table encodes an amino acid sequence shown as a sequence 2 in the sequence table.

The nucleotide sequence of the present invention encoding the protein GmFULc can be easily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which are artificially modified and have 75% or more identity with the nucleotide sequence of the protein GmFULc isolated in the invention are derived from the nucleotide sequence of the invention and are identical to the sequence of the invention as long as the nucleotide sequence encodes the protein GmFULc.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes a nucleotide sequence having 75% or more, or 80% or more, or 85% or more, or 90% or more, or 95% or more identity with the nucleotide sequence of protein GmFULc consisting of the amino acid sequence shown in sequence No. 2 of the coding sequence Listing of the present invention. Identity can be assessed visually or by 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 any of the above applications, the controlling plant yield may be increasing plant yield or decreasing plant yield.

In any of the above applications, the regulating plant height may be increasing plant height or decreasing plant height.

In any of the above applications, the regulating and controlling the flowering time of the plant may be early flowering time or late flowering time.

In the use of any of the above, the plant may be any of the following c 1) to c 8): c1 A dicotyledonous plant; c2 A monocot plant; c3 Leguminous plants; c4 Soybean); c5 Dongnong50 soybean variety; c6 Cruciferous plants; c7 Arabidopsis thaliana; c8 A wild type Arabidopsis thaliana Columbia-0 subtype.

The invention also provides a method for cultivating transgenic plants, which comprises the following steps: improving the expression quantity and/or activity of the protein GmFULc in the starting plant to obtain a transgenic plant; the transgenic plants have an increased yield and/or an increased plant height and/or an earlier flowering time and/or an altered photoperiod as compared to the starting plants.

In the above method, the "increasing the expression level and/or activity of the protein GmFULc in the starting plant" may be achieved by a method known in the art, such as transgene, multicopy, and change of a promoter and a regulatory factor, to increase the expression level and/or activity of any one of the above protein GmFULc in the starting plant.

In the above method, the "improvement of the expression level and/or activity of protein GmFULc in the starting plant" may be specifically achieved by introducing a nucleic acid molecule encoding protein GmFULc into the starting plant.

In the above method, the "introducing a nucleic acid molecule encoding a protein GmFULc into a starting plant" may be carried out by introducing a recombinant vector into a starting plant; the recombinant vector can be a recombinant plasmid obtained by inserting a nucleic acid molecule encoding the protein GmFULc into an expression vector. The recombinant vector can be specifically the recombinant plasmid pB7WG2-GmFULc mentioned in the examples. The recombinant plasmid pB7WG2-GmFULc contains a DNA molecule shown in a sequence 1 in a sequence table.

The transgenic plant can be specifically a GmFULc gene transferred Arabidopsis strain (such as OX-1, OX-5 and OX-23) or a GmFULc gene transferred soybean strain (such as GmFULcOX-1 and GmFULcOX-2) mentioned in the examples.

The invention also provides a plant breeding method, which comprises the following steps: increasing the content and/or activity of said protein GmFULc in plants, thereby increasing yield and/or increasing plant height and/or flowering time and/or altering photoperiod.

In any of the methods described above, the plant may be any of the following c 1) to c 8): c1 A dicotyledonous plant; c2 A monocot plant; c3 Leguminous plants; c4 Soybean); c5 Dongnong50 soybean variety; c6 Cruciferous plants; c7 Arabidopsis thaliana; c8 A wild type Arabidopsis thaliana Columbia-0 subtype.

Any of the above yields may be expressed as at least one of a weight of a hundred, a number of grains per plant and a number of pods.

Any of the above pod numbers may specifically be a four pod number.

In the above, the change in plant photoperiod may be manifested as a change in flowering time. The photoperiod refers to the alternation of light period and dark period in day and night. There are three types of responses of plant flowering to the length of sunlight, long-day plants, short-day plants, and neutral plants. A long-day plant is a plant that can flower after a certain number of days in a 24-hour day-night cycle. Short-day plants refer to plants that can flower in a 24h day-night cycle with less than a certain number of days of sunshine. The flower formation of the day neutral plant is not sensitive to the length of the day, and the plant can bloom in any length of the day as long as other conditions are met.

Experiments prove that under the condition of short sunshine, the overexpression of the GmFULc gene in wild arabidopsis thaliana can increase the plant height, advance the flowering time, reduce the number of rosette leaves and increase the pod bearing number; under the long-day condition, the GmFULc gene is over-expressed in wild arabidopsis thaliana, so that the plant height can be increased, the flowering time is advanced, the number of rosette leaves is reduced, the leaf surface area is increased, and the pod bearing number is increased; introducing recombinant plasmid pB7WG2-GmFULc into Dongnong50 of soybean variety, and screening to obtain T ₁ A positive plant of soybean with GmFULc gene. T is compared with Dongnong50 of soybean variety ₁ The positive plants of the soybeans with the GmFULc gene transferred are blossoming, and the plant height, the pod number, the four-pod number, the single-plant grain number and the hundred-grain weight are obviously increased. Therefore, the protein GmFULc can regulate and control the yield, the plant height, the flowering time and the photoperiod of the plant. The invention has important application value.

Drawings

FIG. 1 is a map of the construction process of recombinant plasmid pB7WG2-GmFULc.

FIG. 2 is T ₃ The molecular identification result of the transgenic Arabidopsis with GmFULc gene is obtained.

FIG. 3 shows the results of long-day 22-day long-day culture ₃ The generation is homozygous to transfer the phenotype of GmFULc gene Arabidopsis thaliana and wild Arabidopsis thaliana plants.

FIG. 4 shows T at 22 days of long-day culture ₃ Comparing the sizes of leaves of the generation homozygous GmFULc transgenic Arabidopsis plants with those of wild Arabidopsis plants.

FIG. 5 shows T at 52 days of long-day culture ₃ The generation is homozygous to transfer the phenotype of GmFULc gene Arabidopsis thaliana and wild Arabidopsis thaliana plants.

FIG. 6 shows T at 76 days of short-day cultivation ₃ The generation is homozygous to transfer the phenotype of GmFULc gene Arabidopsis thaliana and wild Arabidopsis thaliana plants.

FIG. 7 shows the detection of T by using an immune colloidal gold test strip ₀ And (3) a detection result of soybean with a GmFULc gene simulated.

FIG. 8 shows glufosinate-ammonium smearing identification T ₁ Transferring GmFULc gene soybean.

FIG. 9 is T ₁ And (4) carrying out molecular identification on soybean with GmFULc gene transfer.

FIG. 10 is a comparison of plant phenotypes during reproductive growth.

FIG. 11 shows inflorescence comparison for reproductive growth period.

FIG. 12 is T ₁ Transferring the pod shape of the soybean with the GmFULc gene.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.

The experimental procedures in the following examples are conventional unless otherwise specified.

The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified.

The quantitative tests in the following examples, all set up three replicates and the results averaged.

Soybean variety dongnong 42 is described in the following documents: research on dynamic change rules of soybean Dongnong 42 production quality traits at different sowing periods [ J ] soybean science, 2008. Hereinafter, the soybean variety dongnon 42 is simply referred to as dongnon 42. Dongnong50, a soybean variety, is described in the following documents: yu JIN, juanjuan QU, guangming REN, lei DONG. Effects of Transgenic DREB Soybean Dongnong50on the Diversity of Soil Ammonia-oxidizing Bacteria. Agricultural Science & technology.2013, 14 (7): 988-992. Hereinafter, soybean variety Donnong 50 is simply referred to as Donnong 50.

Wild type Arabidopsis thaliana (Arabidopsis thaliana) (Columbia-0 subtype) is described in the following references: kim H, hyun Y, park J, park M, kim M, kim H, lee M, moon J, lee I, kim J.A genetic link between colored responses and flowing time through FVE in Arabidopsis thaliana Nature genetics.2004, 36-167-171, publicly available from the northeast university of agriculture (i.e., at the Applicant) to repeat the experiments of the present application. Hereinafter, arabidopsis thaliana (Columbia-0 subtype) is simply referred to as wild type Arabidopsis thaliana.

Agrobacterium tumefaciens LBA4404 is described in: chenzhongjian, liu soldier, royal macro bin, royal golden hair, liulin. (2003) expression of rice repeat RRD3 deletion-mediated gusA in rice callus. 127-131.

The pB7WG2 vector is a product of Invitrogen corporation.

Example 1 cloning of a Gene encoding protein GmFULc (GmFULc Gene)

1. Extracting total RNA of leaves of Dongnong 42 seedlings growing for 30 days by using a Trizo1 method, and then performing reverse transcription by using reverse transcriptase to obtain first strand cDNA so as to obtain the Dongnon 42 cDNA.

2. PCR amplification was carried out using the cDNA of Dongnong 42 obtained in step 1 as a template and primer pairs consisting of 5'-ATGGGGAGGGGAAGAGTGGAG-3' and 5 '-CTATTCATTTTGTAGGAAGAGGCATCA-3' to obtain a double-stranded DNA molecule of about 720 bp.

The reaction conditions are as follows: 5min at 94 ℃; 30s at 94 ℃, 30s at 56 ℃, 45s at 72 ℃ and 35 cycles; 10min at 72 ℃.

And (3) sequencing the double-stranded DNA molecules obtained in the step (2). The sequencing result shows that the nucleotide sequence of the double-stranded DNA molecule (GmFULc gene) obtained in the step 2 is shown as a sequence 1 in a sequence table.

Example 2 obtaining and identification of GmFULc transgenic Arabidopsis thaliana

1. Construction of recombinant plasmid pB7WG2-GmFULc

The map of the construction process of the recombinant plasmid pB7WG2-GmFULc is shown in figure 1.

1. Total RNA of leaves of Dongnong 42 seedlings grown for 30 days was extracted by Trizo1 method, and then first strand cDNA was reverse-transcribed using reverse transcriptase to obtain Dongnon 42 cDNA.

2. PCR amplification was carried out using the cDNA of Dongnong 42 obtained in step 1 as a template and a primer pair consisting of 5 '-Bussner CACCATGGGAGGGAAGAGTGGGAG-3' and 5 '-Bussner CTATTCATTTTGTAGGAAGAGGCATCA-3', and a DNA fragment of about 720bp was recovered.

3. After completing step 2, the recovered DNA fragment and pENTRY D-TOPO vector (product of Invitrogen corporation) were subjected to TOPO reaction to obtain recombinant plasmid pENTRY D-TOPO-GmFULc.

4. After the step 3 is completed, the recombinant plasmid pENTRY D-TOPO-GmFULc and the pB7WG2 vector are subjected to LR reaction to obtain the recombinant plasmid pB7WG2-GmFULc.

The recombinant plasmid pB7WG2-GmFULc was sequenced. The sequencing result shows that the recombinant plasmid pB7WG2-GmFULc contains a DNA molecule shown in a sequence 1 in a sequence table.

The recombinant plasmid pB7WG2-GmFULc expresses protein GmFULc shown in a sequence 2 in a sequence table.

2. Obtaining of LBA4404/pB7WG2-GmFULc

The recombinant plasmid pB7WG2-GmFULc is introduced into Agrobacterium tumefaciens LBA4404 to obtain the recombinant Agrobacterium tumefaciens which is named as LBA4404/pB7WG2-GmFULc.

3. Obtaining of transgenic Arabidopsis with GmFULc gene

1. Planting of wild type Arabidopsis thaliana

(1) Taking a centrifuge tube, adding a small amount of dried wild type arabidopsis seeds, and sealing; then the mixture is placed at 4 ℃ for vernalization for 3-5d.

(2) After the step (1) is finished, taking the wild type arabidopsis seeds, washing the seeds for 10-30s by using 75% (v/v) ethanol water solution, and repeatedly blowing, sucking and washing the seeds for 2-3 times by using sterilized distilled water.

(3) And (3) after the step (2) is finished, taking the wild type arabidopsis seeds, carrying out vortex blowing on the seeds for 8min by using a 10% (m/v) sodium hypochlorite aqueous solution, and repeatedly carrying out blowing and sucking on the seeds for 3-5 times by using sterilized distilled water.

(4) After the step (3) is finished, taking the wild type arabidopsis seeds, adding sterilized water, and sucking the seeds by blowing to resuspend the seeds; then sucking the seeds evenly and dropping the seeds on an MS solid culture medium, sealing the MS solid culture medium by a sealing film, and culturing the MS solid culture medium in a tissue culture room. When 3-4 leaves are germinated from a wild type Arabidopsis seed (about 5-7 days), the wild type Arabidopsis seed is transferred to sterilized artificial soil (formed by mixing vermiculite, perlite and flower soil; the volume ratio of the vermiculite to the perlite to the flower soil is 3: 1), and then the wild type Arabidopsis seed is cultured in a light-dark alternating mode at 22 ℃. The light-dark alternate culture refers to that light culture (16 h) and dark culture (8 h) are alternately carried out; during light cultureIs white light with the illumination intensity of 350 mu mol m- ² s- ¹ 。

2. Transferring the LBA4404/pB7WG2-GmFULc obtained in the second step into wild type Arabidopsis thaliana to obtain T-shaped Arabidopsis thaliana by floral dip transformation (described in Clough, S.J., and Bent, A.F. Floraldip: amplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. (1998) 16, 735-743.) ₁ Transfer GmFULc gene Arabidopsis seeds. The method comprises the following specific steps:

(1) Taking a monoclonal of LBA4404/pB7WG2-GmFULc, adding 20mL YEP liquid culture medium containing 25mg/mL rifampicin, 25mg/mL streptomycin and 100mg/mL spectinomycin, and shake culturing at 28 ℃ and 200rpm to obtain OD _600nm And an agrobacterium liquid 1 with a value of about 0.5.

(2) Inoculating the agrobacterium liquid 1 obtained in the step (1) into 200mL YEP liquid culture medium containing 25mg/mL rifampicin, 25mg/mL streptomycin and 100mg/mL spectinomycin (the inoculation volume ratio is 1 _600nm And (3) an agrobacterium liquid 2 with a value of 1.6-2.0.

(3) And (3) centrifuging the agrobacterium liquid 2 obtained in the step (2) at room temperature and 5000rpm for 10min, and collecting the precipitate.

(4) Taking the precipitate collected in step (3), resuspending the precipitate in an osmotic medium (1/2 MS solid medium containing 5% (m/v) sucrose and 0.02% (v/v) silwet L-77) to obtain OD _600nm Resuspension with a value of 0.8.

(5) Pouring the heavy suspension obtained in the step (4) into a plate, and then transversely pouring wild type Arabidopsis plants on the edge of the plate to soak plant inflorescences in the heavy suspension for 30s; wild type Arabidopsis plants were then grown overnight in a incubator, horizontally, and then vertically. After infecting for 4-5d, continuing to soak for 1-2 times according to the growth condition of arabidopsis thaliana. Culturing for 3-4 weeks, collecting seeds after the seeds are mature, and storing in a dryer.

3. The T obtained in the step 2 ₁ The seeds of the GmFULc gene-transferred Arabidopsis are sown on an MS solid culture medium containing 5mg/LPPT, and the Arabidopsis (resistant seedlings) which can normally grow is T ₁ Transfer GmFULc gene positive seedling, T ₁ GmFULc baseThe seeds received by the positive seedlings are T ₂ Transfer GmFULc gene Arabidopsis seeds.

4. The T of different strains screened out in the step 3 ₂ Transgenic GmFULc gene Arabidopsis seeds are sown on MS solid medium containing 5mg/LPPT for screening, if the ratio of the number of Arabidopsis capable of normally growing (resistant seedlings) to the number of Arabidopsis incapable of normally growing (non-resistant seedlings) in a certain strain is 3:1, the strain is a strain with a copy of the GmFULc gene inserted therein, and the seeds received by the resistant seedlings in the strain are T ₃ Transfer GmFULc gene Arabidopsis seeds.

5. The T screened out in the step 4 ₃ The seeds of the GmFULc transgenic arabidopsis are sowed on an MS solid culture medium containing 5mg/LPPT again for screening, and the seeds which are all resistant seedlings are T ₃ The generation is homozygous and transformed into GmFULc gene Arabidopsis thaliana. 3 of them are T ₃ The generation homozygous GmFULc transgenic Arabidopsis strains are respectively named as GmFULcOX-1 (hereinafter, OX-1), gmFULcOX-5 (hereinafter, OX-5) and GmFULcOX-23 (hereinafter, OX-23), and subsequent experiments are carried out.

4. Molecular identification

The Arabidopsis seed to be tested is T of OX-1 ₃ T of seed generation, OX-5 ₃ Seed-producing, OX-23T ₃ Generation seed or wild type arabidopsis seed.

(1) Taking an arabidopsis seed to be detected, soaking the arabidopsis seed in 70% (v/v) ethanol water solution for 30s, and washing the arabidopsis seed with sterile water for 3 times; then spread on MS solid medium and vernalize for 2 days at 4 ℃.

(2) And (3) after the step (1) is finished, taking the arabidopsis thaliana seeds to be detected, and carrying out light-dark alternate culture (16 h illumination culture/8 h dark culture) at the temperature of 22 ℃ for 7 days to obtain the arabidopsis thaliana seedlings to be detected.

(3) Extracting the genome DNA of the arabidopsis seedling to be detected, taking the genome DNA as a template, and adopting a method comprising the following steps of GmFULc-F:5 'ATGGAGGGGAAGAGTGGAGTT-3' and GmFULc-R:5 'CTATTCATTTTGTAGGAAGAGGCATCA-3' to obtain a PCR amplification product; then, the following judgment is made: if some PCR amplification product contains DNA segment of about 720bp, the arabidopsis seedling to be detected corresponding to the PCR amplification product is a positive seedling.

Part of the identification results are shown in FIG. 2 (M is DNA Marker, WT is wild type Arabidopsis thaliana). The results showed that T of OX-1 ₃ Seed-substituted, OX-5T ₃ Seed and OX-23T ₃ The seedlings obtained by the generation of seeds are all positive seedlings.

5. Phenotypic analysis

1. Phenotypic analysis of Long-day treated Arabidopsis

The long-day culture refers to that light culture (16 h) and dark culture (8 h) are alternately carried out; the light culture is white light with illumination intensity of 350 μmol m ^-2 s ^-1 。

The Arabidopsis seed to be tested is T of OX-1 ₃ Seed-substituted, OX-5T ₃ Seed-producing, OX-23T ₃ Generation seed or wild type arabidopsis seed.

(1) Taking an arabidopsis seed to be detected, soaking the arabidopsis seed in 70% (v/v) ethanol water solution for 30s, and washing the arabidopsis seed with sterile water for 3 times; then vernalizing for 2-3 days at 4 ℃.

(2) And (3) after the step (1) is finished, spreading the arabidopsis thaliana seeds to be tested on an MS solid culture medium, and culturing for 7-10 days in long day to obtain an arabidopsis thaliana plant to be tested.

(3) And (3) after the step (2) is finished, transplanting the arabidopsis thaliana plant to be detected in nutrient soil, culturing for 22 days in long day, and observing the phenotype of the arabidopsis thaliana plant to be detected.

The results of the experiment are shown in FIG. 3 (WT is a wild type Arabidopsis thaliana) and FIG. 4 (WT is a wild type Arabidopsis thaliana). The results showed 3T's as compared to wild type Arabidopsis thaliana ₃ The number of rosette leaves of the generation homozygous GmFULc transgenic Arabidopsis strains (OX-1, OX-5 and OX-23) is obviously reduced, the flowering time is obviously advanced, and the leaf surface area is obviously increased.

(4) And (3) after the step (2) is finished, transplanting the arabidopsis thaliana plant to be detected in nutrient soil, culturing for 52 days in long day, and observing the phenotype of the arabidopsis thaliana plant to be detected.

The results of the experiment are shown in FIG. 5 (WT is wild type Arabidopsis thaliana) and Table 2 (WT is wild type Arabidopsis thaliana). The results showed 3T's as compared to wild type Arabidopsis thaliana ₃ The plant height of the generation homozygous GmFULc transgenic Arabidopsis strains (OX-1, OX-5 and OX-23) is obviously increased, the flowering time is obviously advanced, the number of rosette leaves is obviously reduced andthe number of pod bearing is obviously increased.

TABLE 2 phenotypic data under long day

	Flowering time (sky)	Number of rosette leaves (sheet)	Plant height (cm)	Number of pod
					WT	29.06±2.29	14.2±1.92	16.66±2.11	21.2±10.64
GmFULcox-1	24.65±1.93**	13.3±1.07*	29.94±1.46**	80.05±8.70**
					GmFULcox-5	25.82±1.74**	13.5±1.36*	27.02±0.99**	71.60±6.53**
GmFULcox-23	20.80±1.81**	11.9±0.93*	30.10±2.87**	86.85±10.35**

Note: * Denotes P <0.01, denotes P <0.05.

The results show that the GmFULc gene is overexpressed in wild arabidopsis thaliana under the long-day condition, so that the plant height can be increased, the flowering time is advanced, the number of rosette leaves is reduced, the leaf surface area is increased, and the pod bearing number is increased.

2. Phenotypic analysis of short-day treated Arabidopsis

Short-day culture refers to alternate light culture (8 h) and dark culture (16 h); the light culture is white light with illumination intensity of 350 μmol m ^-2 s ^-1 。

(2) And (3) after the step (1) is finished, spreading the arabidopsis thaliana seeds to be detected on an MS solid culture medium, and culturing for 7-10 days in short sunlight to obtain an arabidopsis thaliana plant to be detected.

(3) And (3) after the step (2) is finished, transplanting the arabidopsis thaliana plant to be detected in nutrient soil, culturing for 76 days in long day, and observing the phenotype of the arabidopsis thaliana plant to be detected.

The results of the experiment are shown in FIG. 6 (WT is Arabidopsis thaliana wild-type) and Table 3 (WT is Arabidopsis thaliana wild-type). The results showed 3T's as compared to wild type Arabidopsis thaliana ₃ The generation homozygous GmFULc transgenic Arabidopsis strains (OX-1, OX-5 and OX-23) have the advantages of obviously advanced flowering time, obviously reduced rosette leaf number, obviously increased plant height and obviously increased pod bearing number.

TABLE 3 short day tabular data

	Flowering time (sky)	Number of rosette leaves (sheet)	Plant height (cm)	Number of pod
					WT	77.93±3.32	39.33±4.56	19.11±2.98	32.86±9.24
GmFULcox-1	52.43±2.98**	25.40±1.38*	22.53±1.87*	50.77±5.36*
					GmFULcox-5	59.05±3.36**	26.05±2.03*	21.41±1.55	45.06±7.14*
GmFULcox-23	51.27±2.81**	23.22±1.45*	25.15±1.91*	54.35±6.73*

Note: * Denotes P <0.01, denotes P <0.05.

The results show that the overexpression of the GmFULc gene in wild Arabidopsis thaliana can increase the plant height, advance the flowering time, reduce the number of rosette leaves and increase the pod bearing number under the condition of short day.

Example 3 obtaining and identification of GmFULc transgenic Soybean

The light intensity is 350 μmol m and is white light during light culture ^-2 s ^-1 。

1. Construction of recombinant plasmid pB7WG2-GmFULc

The same procedure as in the first step of example 2.

2. Obtaining of LBA4404/pB7WG2-GmFULc

The same procedure as in step two of example 2.

3. T is ₁ Obtaining of positive plant of soybean with GmFULc gene

1、T ₀ Obtaining of soybean with pseudo-transferred GmFULc gene

Agrobacterium-mediated genetic transformation of soybean cotyledonary nodes (described in Olhoft P M, donovan C M, somers D A. Soybean (Glycine max) transformation using a method of using soybean cotyledonary node explants [ J ] as described in the following publications]Method Molecular Biology,2006, 343 (12): 385 Transferring LBA4404/pB7WG2-GmFULc obtained in the second step to the Dongnong50 soybean variety to obtain T ₀ Transferring GmFULc gene soybean. The method comprises the following specific steps:

(1) Taking the seeds of Dongnong50 of soybean variety, sterilizing with chlorine gas for 16h, and then soaking in sterile water for 12h to obtain imbibed soybean.

(2) After the step (1) is finished, the imbibed soybeans are taken, peeled, 2 cotyledons are separated, a wound is slightly scratched nearby a cotyledonary node by a blade, and then the cotyledons are placed into the agrobacterium tumefaciens heavy suspension for 30min for infection.

The preparation method of the agrobacterium tumefaciens resuspension comprises the following steps: taking the monoclonal of the recombinant agrobacterium obtained in the step two, adding 20mL YEP liquid culture medium, carrying out shake culture at 28 ℃ and 200rpm to obtain OD _600nm Bacterial liquid 1 with a value of about 0.5; inoculating the bacterial liquid 1 to 200mL YEP liquid culture medium, and performing shake culture at 28 ℃ and 200rpm to obtain OD _600nm A bacterial liquid 2 with a value of 1.6-2.0; centrifuging the bacterial liquid 2 at room temperature and 5000rpm for 10min, and collecting precipitate; taking the precipitate, and re-suspending with liquid staining culture medium to obtain OD _600nm Agrobacterium resuspension with a value of 0.8.

The liquid staining culture Medium has solute and concentration of 0.321g/LB5 culture Medium (GamborgB-5 basic Medium), 30g/L sucrose, 3.9g/LMES,1.67mg/L6-BA,0.25mg/LGA ₃ And 0.04g/LAS; the solvent is water; the pH was 5.4.

(3) After the step (2) is finished, taking soybean cotyledons, firstly sucking the dry weight suspension by using filter paper, then placing the suspension in a solid co-culture medium, and carrying out illumination culture at 25 ℃ for 5 days.

The solid co-culture medium has solute concentration of 0.321g/L B5 medium, sucrose concentration of 30g/L, LMES concentration of 3.9g/L, 6-BA concentration of 1.67mg/L, and LGA concentration of 0.25mg/L ₃ 0.04g/LAS,0.4g/LL-Cys,0.15g/LDTT,0.5% (w/v) agar powder; the solvent is water; the pH was 5.4.

(4) After the step (3) is completed, the soybean cotyledon is transferred to a cluster bud induction culture medium, and is cultured alternately in light and dark (16 h light culture/8 h dark culture) for 28 days at the temperature of 25 ℃.

The solute of the cluster bud induction culture medium and the concentration thereof are 0.321g/L B5 culture medium, 30g/L sucrose, 0.59g/LMES,1.67 mg/L6-BA, 200mg/LCef,200mg/LTim,5mg/L glufosinate-ammonium and 0.75% (w/v) agar powder; the solvent is water; the pH was 5.4.

(5) After the step (4) is completed, firstly cutting off the cluster buds from the soybean cotyledons, then transferring the cluster buds to a cluster bud elongation culture medium, and culturing alternately in light and dark at 25 ℃ (16 h illumination culture/8 h dark culture) until the regenerated plants in the cluster buds are elongated to 2cm.

The solute of the elongation culture medium of the cluster buds and the concentration thereof are 4.43g/L MS,30g/L sucrose, 0.59g/LMES and 0.5mg/LGA ₃ ，1mg/LZR，1mg/LAsp，0.1mg/LIAA,200mg/L Cef,200mg/L LTim,3mg/L glufosinate and 0.75% (w/v) agar; the solvent is water; the pH was 5.6.

(6) After the step (5) is completed, cutting the regenerated plants in the cluster buds from the base parts, transferring the cut plants to a rooting culture medium, and alternately culturing at 25 ℃ in light and dark (16 h of light culture/8 h of dark culture) to obtain T ₀ The soybean with the GmFULc gene is simulated.

The solute and the concentration of the rooting culture medium are 0.321g/LB5 culture medium, 20g/L sucrose, 3.9g/LMES,1mg/LIBA and 0.8% (w/v) agar powder; the solvent is water; the pH was 5.6.

2、T ₀ Identification of soybean with GmFULc gene transfer simulation

(1) Respectively taking the plant or T of Dongnong50 of soybean variety ₀ A plant with a GmFULc gene transferred soybean is replaced, and whether BAR protein is expressed or not is detected by adopting an immune colloidal gold test strip (a product of EnviroLosix corporation) (specific steps refer to a specification of the immune colloidal gold test strip). Expression of BAR protein is T ₀ Transferring GmFULc gene soybean.

Partial results of the assay are shown in FIG. 7 (WT is Dongnong50, 1 and 2 are T for soybean varieties) ₀ Soybean with GmFULc gene).

(2) Extraction of T ₀ Transferring genome DNA of leaves of a soybean plant with the GmFULc gene, taking the genome DNA as a template, and adopting a DNA sequence of 35S-GmFULc-F:5 'CGACAGTGGTTCCCAAAGATGGA-3' and GmFULc-R:5 'CTATTCATTTTGTAGGAAGAGGCATCA-3' to obtain a PCR amplification product; then, the following judgment is made: if some PCR amplification product contains about 1370bp DNA fragment, the corresponding T of the PCR amplification product ₀ The soybean plant transformed with the GmFULc gene is identified as T again ₀ Transferring GmFULc gene soybean plant.

3、T ₁ Acquisition and identification of soybean with GmFULc gene

(1) Wait for T ₀ After the soybean with the GmFULc gene is mature, harvesting and selfing to obtain T ₁ Transferring GmFULc gene soybean.

(2) Dongnong50 or T of soybean variety ₁ Sowing seeds of soybean with GmFULc gene in greenhouse, and introducing when three compound leaves are completely spreadThe resistance is identified by smearing glufosinate ammonium water solution with the concentration of 120mg/L on the leaves. The plant with glufosinate-ammonium resistance is T ₁ A positive plant of soybean with GmFULc gene.

Part of the results are shown in FIG. 8 (WT is Dongnong50,1 and 2 are T for soybean varieties) ₁ Positive plants of soybean with GmFULc gene transfer).

(3) Extraction of T ₁ Transferring genome DNA of leaves of a positive plant of the GmFULc gene soybean, taking the genome DNA as a template, and adopting a DNA sequence of 35S-GmFULc-F:5 'CGACAGTGGTTCCCAAAGATGGA-3' and GmFULc-R:5 'CTATTCATTTTGTAGGAAGAGGCATCA-3' to obtain a PCR amplification product; then, the following judgment is made: if some PCR amplification product contains DNA fragment of about 1370bp, the corresponding T of the PCR amplification product ₁ The positive plants of the soybean with the GmFULc gene are identified as T again ₁ Transferring GmFULc gene soybean plant.

Will be' T ₁ The positive plant of the soybean with the GmFULc gene is replaced by a plant of a soybean variety Dongnong50, and other steps are not changed and are used as negative control.

Will be' T ₁ The positive plant of soybean with GmFULc gene is replaced by water, and other steps are not changed and are used as a control.

The results are shown in FIG. 9 (M is DNA Marker, WT is Dongnong50, 1 and 2 are T of soybean variety) ₁ Soybean with GmFULc gene).

Through the steps, 2T of the three groups are treated ₁ Positive plants of soybean with the GmFULc gene transferred are named GmFULcOX-1 and GmFULcOX-2 respectively, and subsequent experiments are carried out.

4. Phenotypic analysis

Respectively sowing soybean (soybean variety Dongnong50, gmFULcOX-1 or GmFULcOX-2) to be tested, and performing long-day culture (light culture (13 h) and dark culture (11 h) alternately, wherein the light culture is white light and the illumination intensity is 350 μmol m ^-2 s ^-1 ) And observing the phenotype and counting the plant height, pod number, node number, branch number, four-pod number, single-plant grain number and soybean flowering time.

Partial results are shown in FIG. 10 (WT is Dongnong50 for soybean variety) and FIG. 11 (WT isSoybean variety dongnong 50), fig. 12 (WT is soybean variety dongnong 50), table 4 (R1 is the initial flowering phase, R2 is the full flowering phase, R3 is the initial pod phase, R4 is the full pod phase, R5 is the initial grain phase, R6 is the grain swelling phase, R7 is the initial ripening phase, R8 is the complete ripening phase), and table 5. In tables 4 and 5, gmFULc-OX was the average value of GmFULcOX-1 and GmFULcOX-2. The results show that T is compared with Dongnong50 of soybean variety ₁ The positive plants of the soybeans with the GmFULc gene transferred are blossoming, and the plant height, the pod number, the four-pod number, the single-plant grain number and the hundred-grain weight are obviously increased.

The results show that the flowering time of the overexpression GmFULc gene in the Dongnong50 soybean variety is advanced under the long-day condition, and the plant height, the pod number, the four-pod number, the single-plant grain number and the hundred-grain weight are obviously increased.

TABLE 4 flowering date

Note: * Representing P <0.05.

TABLE 5 phenotypic data

Note: * Denotes P <0.01, denotes P <0.05.

<110> northeast university of agriculture

Application of <120> protein GmFULc in regulation and control of plant yield

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 720

<212> DNA

<213> Glycine max

<400> 1

atggggaggg gaagagtgga gttgaagagg atcgagaaca agatcaatag gcaagtgacg 60

ttctcaaaga gaaggtctgg tttgctgaag aaagcgcgcg agatctctgt gctgtgcgat 120

gctgatgtgg ctctcatcgt cttctccacc aaaggcaagc ttttggacta ctccaaccaa 180

ccttgtacgg aaagaattct tgaacggtat gagaggtatt cgtatgcgga gaggcaactt 240

gttggagatg atcaaccacc aaatgaaaac tgggttatag aacatgaaaa gctcaaggct 300

agggtggagg tactacagag aaatcaaagg aattttatgg gagaagatct ggacagctta 360

aatcttagag gacttcagag tttggagcaa caacttgatt ccgctctcaa acacattaga 420

tcacgaaaga accaagccat gaatgaatct atttctgagc ttcagaaaaa ggataggaca 480

ctccgtgaac acaacaactt gctctccaag aagataaagg aaaaagagaa ggagctgacc 540

ccacaagagc aagaaggact gcaaaataac atggatgtga gctctgtcct tgtaactcaa 600

ccactggagt ccctgactat tggaggctcc ccagaagtca aatctaatga agaaactcca 660

acctcatgtc gacctaaaac cattcttccc cctttgatgc ctcttcctac aaatgaatag 720

<210> 2

<211> 239

<212> PRT

<213> Glycine max

<400> 2

Met Gly Arg Gly Arg Val Glu Leu Lys Arg Ile Glu Asn Lys Ile Asn

1 5 10 15

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

20 25 30

Arg Glu Ile Ser Val Leu Cys Asp Ala Asp Val Ala Leu Ile Val Phe

35 40 45

Ser Thr Lys Gly Lys Leu Leu Asp Tyr Ser Asn Gln Pro Cys Thr Glu

50 55 60

Arg Ile Leu Glu Arg Tyr Glu Arg Tyr Ser Tyr Ala Glu Arg Gln Leu

65 70 75 80

Val Gly Asp Asp Gln Pro Pro Asn Glu Asn Trp Val Ile Glu His Glu

85 90 95

Lys Leu Lys Ala Arg Val Glu Val Leu Gln Arg Asn Gln Arg Asn Phe

100 105 110

Met Gly Glu Asp Leu Asp Ser Leu Asn Leu Arg Gly Leu Gln Ser Leu

115 120 125

Glu Gln Gln Leu Asp Ser Ala Leu Lys His Ile Arg Ser Arg Lys Asn

130 135 140

Gln Ala Met Asn Glu Ser Ile Ser Glu Leu Gln Lys Lys Asp Arg Thr

145 150 155 160

Leu Arg Glu His Asn Asn Leu Leu Ser Lys Lys Ile Lys Glu Lys Glu

165 170 175

Lys Glu Leu Thr Pro Gln Glu Gln Glu Gly Leu Gln Asn Asn Met Asp

180 185 190

Val Ser Ser Val Leu Val Thr Gln Pro Leu Glu Ser Leu Thr Ile Gly

195 200 205

Gly Ser Pro Glu Val Lys Ser Asn Glu Glu Thr Pro Thr Ser Cys Arg

210 215 220

Pro Lys Thr Ile Leu Pro Pro Leu Met Pro Leu Pro Thr Asn Glu

225 230 235

Claims

1. The application of the protein GmFULc is S1) or S2) as follows:

s1) improving the yield of plants;

s2) cultivating transgenic plants with increased yield;

the protein GmFULc is a 1) or a 2):

a1 ) the amino acid sequence is a protein shown as a sequence 2 in a sequence table;

the plant is soybean or arabidopsis thaliana.

2. Use of a nucleic acid molecule coding for the protein GmFULc according to claim 1, as S1) or S2) as follows:

s1) improving the yield of plants;

s2) cultivating transgenic plants with increased yield;

the plant is soybean or arabidopsis thaliana.

3. Use according to claim 2, characterized in that: the nucleic acid molecule encoding the protein GmFULc according to claim 1 is a DNA molecule as shown in b 1) or b 2) below:

b2 The nucleotide sequence of the DNA molecule is shown as a sequence 1 in a sequence table.

4. Use according to any one of claims 1 to 3, characterized in that: the yield is expressed as at least one of a weight of a hundred, a number of grains per plant, and a number of pods.

5. Use according to claim 4, characterized in that: the number of the pods is four.

6. A method of breeding a transgenic plant comprising the steps of: increasing the expression level and/or activity of the protein GmFULc in the original plant according to claim 1 to obtain a transgenic plant; increased yield of transgenic plants compared to the starting plants;

the plant is soybean or arabidopsis thaliana.

7. The method of claim 6, wherein: the improvement of the expression level and/or activity of the protein GmFULc according to claim 1 in the starting plant is achieved by introducing a nucleic acid molecule encoding the protein GmFULc into the starting plant.

8. A method of plant breeding comprising the steps of: increasing the content and/or the activity of the protein GmFULc according to claim 1 in plants, thereby increasing yield;

the plant is soybean or arabidopsis thaliana.

9. The method according to any one of claims 6 to 8, wherein: the yield is expressed as at least one of a weight of a hundred, a number of grains per plant, and a number of pods.

10. The method of claim 9, wherein: the number of the pods is four.