CN108841840B - Application of protein TaNADH-GoGAT in regulation and control of plant yield - Google Patents

Abstract

The invention discloses application of a protein TaNADH-GoGAT in regulating and controlling plant yield. The invention provides an application of TaNADH-GoGAT protein or related biological materials thereof in regulating and controlling plant yield; the related biological material is a nucleic acid molecule capable of expressing the TaNADH-GoGAT protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule. Compared with wild plants, the yield of single-plant grains, the number of ears per plant and the biomass of single-plant of the TaNADH-GoGAT overexpression transgenic plant are obviously improved; the yield of single plant grains, the number of ears per plant and the biomass of single plant of the TaNADH-GoGAT decrement expression transgenic plant are all obviously reduced. Thus, the protein TaNADH-GoGAT can be used to regulate plant yield. The invention has important application value for breeding new high-yield plant materials.

Description

Application of protein TaNADH-GoGAT in regulation and control of plant yield

Technical Field

The invention relates to the technical field of biology, in particular to application of a protein TaNADH-GoGAT in regulation and control of plant yield.

Background

Wheat is one of the three major grains, is almost all eaten, and only about one sixth of the wheat is used as feed. The two river basin is the earliest region for cultivating wheat in the world, and China is one of the earliest countries for cultivating wheat in the world. In 2010, wheat is the second grain crop (6.51 hundred million tons) in the world with the total production, is second to corn (8.44 hundred million tons), about 40 percent of people use wheat as main grain, the wheat is rich in nutrition, and the wheat can be ground into flour to be made into foods such as steamed bread, biscuits and the like.

The wheat has high nutritive value, and the contained B vitamins and minerals are beneficial to human health. Along with the improvement of the living standard of people in recent years, the demand of society for high-quality special wheat is increasingly large, the planting area of the high-quality wheat is rapidly increased, along with the upgrading of special flour products of processing enterprises and the continuous modernization of processing technology, the modern flour milling industry puts forward higher and higher requirements on the quality and stability of large-batch commercial wheat, and in order to produce the wheat with high protein content, unreasonable application of chemical fertilizers not only causes soil hardening, low fertilizer utilization rate and serious loss pollution, but also greatly influences the yield and quality of the wheat, and has great harm to the environment. Therefore, it is of great significance to culture high-yield excellent wheat varieties by biological means such as genetic engineering.

Disclosure of Invention

The invention aims to provide application of a protein TaNADH-GoGAT in regulating and controlling plant yield.

In a first aspect, the present invention claims the use of a TaNADH-GoGAT protein or its related biological material for modulating plant yield.

The related biological material can be a nucleic acid molecule capable of expressing the TaNADH-GoGAT protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

Further, the expression level and/or activity of the TaNADH-GoGAT protein or the coding gene thereof in the plant is increased, and the yield of the plant is increased; the expression level and/or activity of the TaNADH-GoGAT protein or the coding gene thereof in the plant is reduced, and the yield of the plant is reduced.

Wherein said plant yield may be embodied as at least one of:

(a1) the yield of single plant seeds of the plant;

(a2) the number of plant ears per plant;

(a3) plant individual biomass (dry weight);

(a4) plant single ear biomass (dry weight).

In a second aspect, the invention claims a method of breeding a plant variety with increased yield.

The method for breeding a plant variety with improved yield provided by the invention can comprise the step of improving the expression level and/or activity of the TaNADH-GoGAT protein in a receptor plant. Wherein the yield improvement can be embodied as single plant kernel yield improvement and/or plant spike number increase and/or single plant biomass improvement and/or single spike biomass improvement.

Further, the present invention provides a method of breeding a transgenic plant with increased yield.

The method for cultivating the transgenic plant with improved yield, provided by the invention, can specifically comprise the following steps: introducing a nucleic acid molecule capable of expressing a TaNADH-GoGAT protein into a receptor plant to obtain a transgenic plant; the transgenic plant has increased yield as compared to the recipient plant. Wherein the yield improvement can be embodied as single plant kernel yield improvement and/or plant spike number increase and/or single plant biomass improvement and/or single spike biomass improvement.

In a third aspect, the invention claims a method of breeding a plant variety with reduced yield.

The method for breeding a plant variety with reduced yield provided by the invention can comprise the step of reducing the expression level and/or activity of the TaNADH-GoGAT protein in a receptor plant. Wherein the yield reduction can be embodied as single plant kernel yield reduction and/or single plant ear number reduction and/or single plant biomass reduction and/or single ear biomass reduction.

Further, the present invention provides a method for breeding a transgenic plant with reduced yield.

The method for cultivating the transgenic plant with reduced yield provided by the invention specifically comprises the following steps: inhibiting and expressing coding genes of TaNADH-GoGAT protein in a receptor plant to obtain a transgenic plant; the transgenic plant has reduced yield as compared to the recipient plant. Wherein the yield reduction can be embodied as single plant kernel yield reduction and/or single plant ear number reduction and/or single plant biomass reduction and/or single ear biomass reduction.

In the second aspect, the "introducing into a recipient plant a nucleic acid molecule capable of expressing the TaNADH-GoGAT protein" may be achieved by introducing into the recipient plant a recombinant expression vector containing a gene encoding the TaNADH-GoGAT protein.

The recombinant expression vector can be constructed by using the existing plant expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like, such as pCAMBIA-1300-221, pGreen0029, pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UBIN or other derivative plant expression vectors. The plant expression vector may also comprise the 3' untranslated region of the foreign gene, i.e., a region comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The poly A signal can direct the addition of poly A to the 3' end of the mRNA precursor. When the gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as a cauliflower mosaic virus (CAMV)35S promoter, a Ubiquitin gene Ubiquitin promoter (pUbi), a stress-inducible promoter rd29A and the like, 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 a recombinant expression vector, 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, the recombinant expression vectors used may be processed, for example, by adding genes encoding enzymes or luminescent compounds which produce a color change, antibiotic markers having resistance or chemical resistance marker genes, etc., which are expressed in plants. Or directly screening the transformed plants in a stress environment without adding any selective marker gene.

In the invention, the promoter for starting the transcription of the coding gene of the TaNADH-GoGAT protein in the recombinant vector is a Ubi promoter.

More specifically, the recombinant expression vector is a recombinant plasmid obtained by inserting the coding gene of the TaNADH-GoGAT protein into the multiple cloning site (Hind III and EcoR I) of pUbi-163 vector.

In a third aspect, the "suppression expression of a gene encoding a TaNADH-GoGAT protein in a recipient plant" can be achieved by introducing an interference vector containing a DNA fragment represented by the formula (I) into the recipient plant;

SEQ_{forward direction}-X-SEQ_{Reverse direction}(I)

Said SEQ_{Forward direction}Has the sequence of SEQ ID No.3 at the 1 st to 426 st positions;

said SEQ_{Reverse direction}And the sequence of SEQ_{Forward direction}Is reverse complementary (in particular at position 608-1033 of SEQ ID No. 3);

said X is said SEQ_{Forward direction}And said SEQ_{Reverse direction}In the sequence, the X and the SEQ_{Forward direction}And said SEQ_{Reverse direction}Are not complementary.

In the present invention, X in formula (I) is specifically shown as 433-601 of SEQ ID No. 3.

More specifically, the nucleotide sequence of the DNA fragment shown in the formula (I) is shown in SEQ ID No. 3.

In a specific embodiment of the invention, the interference vector is specifically a recombinant plasmid obtained by inserting the DNA fragment shown in formula (I) into the multiple cloning site (BamH I and KpnI) of pUbi-163 vector.

In the above method, the recombinant expression vector carrying the coding gene of the TaNADH-GoGAT protein or the interference vector carrying the DNA fragment represented by formula (I) is introduced into the recipient plant, which may specifically be: plant cells or tissues are transformed by conventional biological methods using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and the transformed plant tissues are grown into plants.

In the first, second and third aspects, the TaNADH-GoGAT protein may be any one of the following proteins:

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

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

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

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

Accordingly, in the first, second and third aspects, the "nucleic acid molecule capable of expressing the TaNADH-GoGAT protein" may be a gene encoding the TaNADH-GoGAT protein. Furthermore, the coding gene of the TaNADH-GoGAT protein can be specifically any one of the following DNA molecules:

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

(B2) a DNA molecule which hybridizes under stringent conditions to the DNA molecule defined in (B1) and which encodes said TaNADH-GoGAT protein;

(B3) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% homology with the DNA sequence defined in (B1) or (B2) and encodes the TaNADH-GoGAT protein.

The stringent conditions may be hybridization with a solution of 6 XSSC, 0.5% SDS at 65 ℃ followed by washing the membrane once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In the first, second and third aspects, the plant may be a monocotyledonous plant, and may be a dicotyledonous plant.

Further, the monocotyledon may be a gramineae.

Still further, the gramineous plant may be wheat.

In a particular embodiment of the invention, the plant is in particular the wheat variety KN199 (cronong 199).

Experiments prove that the protein TaNADH-GoGAT provided by the invention regulates and controls the plant yield: compared with wild plants, the yield of single-plant grains, the number of ears per plant and the biomass of single-plant of the TaNADH-GoGAT overexpression transgenic plant are obviously improved; the yield of single plant grains, the number of ears per plant and the biomass of single plant of the TaNADH-GoGAT decrement expression transgenic plant are all obviously reduced. Thus, the protein TaNADH-GoGAT can be used to regulate plant yield. The invention has important application value for breeding new high-yield plant materials.

Drawings

FIG. 1 shows the expression level identification of TaNADH-GoGAT transgenic line. Wherein A is TaNADH-GoGAT overexpression identification. And B is TaNADH-GoGAT decrement expression identification. NC stands for empty control. The expression level in the figure is relative to the expression level of TaActin. Data in the figure are mean ± s.e. of 3 replicates. Represents that the difference reached P <0.05 significance.

FIG. 2 is an assay of the phenotype and yield-related traits of the TaNADH-GoGAT overexpression line during the 2015-2016 growth season. Wherein A is the yield of single-plant grains. B is the grain number of the spike. C is the thousand grain weight. D is the number of spikes of the single plant. NC stands for empty control. Data in the figure are mean ± s.e. of 3 replicates. Represents that the difference reached P <0.05 significance.

FIG. 3 is a graph showing the determination of phenotype and yield-related traits of 2016-2017 growing season TaNADH-GoGAT transgenic line. Wherein A is plant biomass. And B is the yield of the seeds. GoOE1, GoOE17 and GoOE18 are overexpression lines, GoOE1-, GoOE 17-and GoOE 18-are corresponding negative lines, GoR1, GoR2 and GoR3 are decrement expression lines, and GoR1-, GoR 2-and GoR 3-are corresponding negative lines. Data in the figure are mean ± s.e. of 3 replicates. Represents that the difference reached P <0.05 significance.

Detailed Description

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

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

Wheat variety KN199 (cronong 199): the variety cultivated by the genetic institute Lijunming is publicly available or obtained from the institute of genetics and developmental biology of Chinese academy of sciences.

pUbi-163 vector: described in "Shao, a., Ma, w.y., Zhao, x.q., Hu, m.y., He, x., Teng, w., Li, h., and Tong, Y.P. (2017), The auxin biosynthetic TRYPTOPHAN amine organic residue RELATED tara 2.1-3A microorganisms grain of leather. plant physical.174, 2274-2288," publicly available from The applicant and usable only for duplicate invention experiments.

Example 1 construction of TaNADH-transgenic GoGAT wheat

Preparation of TaNADH-GoGAT transgenic plant

(ii) obtaining TaNADH-GoGAT Gene

1. Extracting total RNA of wheat variety KN199, and reverse transcribing to obtain its genome cDNA.

2. Taking the cDNA obtained in the step 1 as a template, and taking the following primers as primers to carry out PCR amplification to construct a sequence required by overexpression of TaNADH-GoGAT transgenic wheat:

TaNADH-GoGAT-OE-F：5’-AAGCTTATGCCGACGGCGCAGGGGATTGG-3' (the sequence underlined is the Hind III restriction recognition site);

TaNADH-GoGAT-OE-R：5’-GAATTCCTATGTAAGGCCTGCGACATTTTG-3' (the sequence shown underlined is the EcoR I cleavage recognition site).

The following primers are used as primers to carry out PCR amplification to construct a sequence required by wheat of a transgenic line of the reduced expression TaNADH-GoGAT:

TaNADH-GoGAT-RNAi-F1：5’-GGATCCGGATTGGTTTGAAGCACG-3' (the sequence shown underlined is the BamHI cleavage recognition site);

TaNADH-GoGAT-RNAi-R1: 5'-GAATTCAGGTGTGTGGTAGAGCAACG-3' (the sequence underlined is the EcoRI cleavage recognition site);

TaNADH-GoGAT-RNAi-F2：5’-GGTACC GGATTGGTTTGAAGCACG-3' (the sequence underlined is the KpnI restriction recognition site);

TaNADH-GoGAT-RNAi-R2：5’-AAGCTTAGGTGTGTGGTAGAGCAACG-3' (the sequence shown underlined is the HindIII restriction recognition site).

And (3) PCR system: mu.l template cDNA, 1. mu.l KOD plus DNA polymerase, 4. mu.l 10 XPCR buffer for KOD plus, 4. mu.l dNTPs (2mM each)l, 25mM MgSO₄Mu.l of each 20mM primer was added to the reaction mixture, and the reaction mixture was made up to 40. mu.l with double distilled water.

PCR reaction procedure: 2min at 98 ℃; 30sec at 98 ℃, 30sec at 58 ℃, 45sec at 68 ℃ and 38 cycles.

The nucleotide sequence of the PCR product (denoted as PCR product 1) for over-expressing the TaNADH-GoGAT gene is '5' -AAGCTT+SEQ ID No.2+GAATTC-3'". Wherein, SEQ ID No.2 is a cDNA sequence of a TaNADH-GoGAT gene (coding TaNADH-GoGAT protein shown in SEQ ID No. 1).

Two PCR products for reducing and expressing the TaNADH-GoGAT gene are provided, one is marked as a PCR product 2, and the other is marked as a PCR product 3; wherein the nucleotide sequence of the PCR product 2 is "5-GGATCCPosition 17-442 of + SEQ ID No.2GAATTC-3 '", the nucleotide sequence of the PCR product 3 being" 5' -GGTACCPosition 17-442 of + SEQ ID No.2AAGCTT-3’”。

(II) construction of TaNADH-GoGAT gene cloning vector

Carrying out double enzyme digestion on the PCR product 1 by using Hind III and EcoR I to obtain a gene fragment; hind III and EcoR I are used for double enzyme digestion of pUbi-163 vector to obtain a vector large fragment, the gene fragment is connected with the vector large fragment to obtain a recombinant plasmid, the recombinant plasmid is named as pUbi-163-TaNADH-GoGAT, the recombinant plasmid is sequenced, and the result is correct. The TaNADH-GoGAT gene in pUbi-163-TaNADH-GoGAT is driven by Ubi promoter. Structural description of pUbi-163-TaNADH-GoGAT vector: and (3) replacing a small fragment between enzyme cutting sites Hind III and EcoR I of the pUbi-163 vector by the DNA fragment shown in SEQ ID No.2 to obtain the recombinant plasmid.

The above PCR product 2 was digested with BamHI and EcoRI to give a gene fragment S1, the above PCR product 3 was digested with KpnI and HindIII to give a gene fragment S2, the pUbi-163 vector was digested with EcoRI and KpnI to recover the vector intron fragment S3, and the pUbi-163 vector was digested with BamHI and KpnI to recover the vector backbone V1. V1 and S3 were then ligated to S1 and S2, respectively, to construct pUbi-TaNADH-GOGAT-RNAi vectors. Sequencing and enzyme digestion verification.

Description of the structure of pUbi-TaNADH-GOGAT-RNAi vector: and (3) replacing a small fragment between enzyme cutting sites BamH I and KpnI of the pUbi-163 vector by the DNA fragment shown in SEQ ID No.3 to obtain the recombinant plasmid. The 1 st-426 nd site of SEQ ID No.3 is the forward sequence of the double-stranded part of the stem-loop structure, the 427 nd-432 nd site is the recognition sequence of EcoR I, the 433 nd-601 nd site is the intron sequence, the 602 nd-607 th site is the recognition sequence of HindIII, and the 608 th-1033 th site is the reverse sequence of the double-stranded part of the stem-loop structure.

(III) obtaining transgenic wheat

Respectively transferring the pUbi-163-TaNADH-GoGAT vector and the pUbi-TaNADH-GOGAT-RNAi vector into wild wheat KN199 by using a gene gun method to obtain T0 generation TaNADH-GoGAT overexpression and decrement expression transgenic wheat. Extracting genome DNA of T0 generation TaNADH-GoGAT overexpression and decrement expression transgenic wheat leaves, and performing PCR amplification by using the genome DNA as a template and using respective upstream primer and downstream primer to obtain fragments of about 1200bp and 800bp, namely positive T0 generation TaNADH-GoGAT overexpression and decrement expression transgenic wheat.

The primers used for identifying TaNADH-GoGAT overexpression transgenic wheat are as follows:

upstream primer T-OEpF: 5'-TCGGAGTAGAATACTGTTTCAAACTACC-3' (vector primers);

downstream primer T-OEpR: 5'-AGCATCTGAATGGCATCGAC-3' are provided.

Primers for identifying transgenic wheat with reduced expression of TaNADH-GoGAT:

upstream primer T-RNAi pF: 5'-TCGGAGTAGAATACTGTTTCAAACTACC-3' (vector primers);

downstream primer T-RNAi pR: 5'-AATGGTGATCATCCAGCTCTC-3' (vector primer).

And (3) culturing the transgenic wheat which is positively identified and is transformed from the T0 generation to TaNADH-GoGAT for overexpression and reduced expression until the T2 generation, identifying the T1-T2 generation according to the identification method of the T0 generation, and harvesting seeds.

The experiment is also provided with an empty-load control (hereinafter abbreviated as an empty-load control plant) for introducing pUbi-163 vector into a wild type wheat variety KN 199.

Second, detection of transgenic plants

(ii) detection of DNA level

DNA of leaves of T2 generations of TaNADH-GoGAT overexpression wheat, TaNADH-GoGAT decrement expression wheat and wild type wheat KN199 are respectively extracted, and the TaNADH-GoGAT overexpression lines are respectively identified by taking the DNA as a template and taking T-OEpF and T-OEpR (specific sequences are as above) as primers, the TaNADH-GoGAT decrement expression lines are identified by taking T-RNAi pF and T-RNAi pR as primers (specific sequences are as above), and simultaneously respective vectors are taken as Positive Controls (PC) and wild type wheat KN199 as negative controls (WT).

And (3) PCR reaction system: DNA template (about 20 ng/. mu.l) 2. mu.l; forward primer (10. mu.M) 0.5. mu.l; reverse primer (10. mu.M) 0.5. mu.l; 10 XPCR amplification buffer 2. mu.l; dNTP mix 1. mu.l; TaqDNA polymerase 0.2. mu.l; ddH₂Make up to 20. mu.l of O.

PCR reaction procedure: 94 ℃ for 3 min; 30s at 94 ℃, 30s at 60 ℃, 40s at 72 ℃ and 40 cycles; 5min at 72 ℃.

The target PCR amplification band of the TaNADH-GoGAT overexpression wheat is about 1200bp, and the target PCR amplification band of the TaNADH-GoGAT decrement expression wheat is about 800 bp. Wild-type wheat KN199 had no target band, and three T2-generation TaNADH-GoGAT overexpression wheat strains (GoOE1, GoOE17 and GoOE18) and three T2-generation TaNADH-GoGAT decrement expression wheat strains (GoR1, GoR2 and GoR3) were preliminarily identified as positive wheat.

(II) detection of RNA levels

1. Total RNA of stems and roots of T2 generations of TaNADH-GoGAT overexpression wheat, TaNADH-GoGAT decrement expression wheat and wild type wheat KN199 are respectively extracted and are reversely transcribed into cDNA.

2. Respectively taking the cDNA obtained in the step 1 as a template, taking TaNADH-GoGATRTpF and TaNADH-GoGAT RT pR as primers to carry out RT-PCR, amplifying the TaNADH-GoGAT gene, and simultaneously taking TaActin pF and TaActin pR as primers to carry out RT-PCR, amplifying the internal reference gene TaActin.

The primers are as follows:

the upstream primer TaNADH-GoGAT RT pF: 5'-GCGGAAGTGCCATACCAATAC-3', respectively;

the downstream primer TaNADH-GoGAT RT pR: 5'-AAAGTTAATGACATGCTCTGGTTCTC-3', respectively;

the upstream primer TaActin pF: 5'-ACCTTCAGTTGCCCAGCAAT-3', respectively;

the downstream primer TaActin pR: 5'-CAGAGTCGAGCACAATACCAGTTG-3' are provided.

And (3) PCR system: DNA template (about 20 ng/. mu.l) 2. mu.l; upstream primer (10. mu.M) 0.4. mu.l; 0.4. mu.l of downstream primer (10. mu.M); 2 × mixture (light Cycler SYBR Green I master, Roche)10 μ l; ddH₂Make up to 20. mu.l of O.

PCR procedure: 94 ℃ for 5 min; 94 ℃ 20s, 60 ℃ 20s, 72 ℃ 15s, 45 cycles.

Quantitative analysis: the CT value was analyzed by using Roche LightCycler 480 II real time PCR instrument. 2-gene for TaNADH-GoGAT in transgenic wheat with TaNADH-GoGAT of T2 generation and wild wheat KN199 by using TaActin gene as internal reference^-ΔctRelative quantification was performed.

The detection results of TaNADH-GoGAT genes in the wheat with overexpression of three T2 generations of TaNADH-GoGAT and three T2 generations of TaNADH-GoGAT of GoR1, GoR2 and GoR3 of GoOE1, GoOE17 and GoOE18 are shown in figure 1.

FIG. 1 shows that the expression level of TaNADH-GoGAT gene in T2 generations TaNADH-GoGAT over-expressed wheat of GoOE1, GoOE17 and GoOE18 is significantly increased compared with wild type wheat KN 199. And the expression quantity of the TaNADH-GoGAT gene in the wheat is obviously reduced and expressed by three T2 generations of TaNADH-GoGAT, namely GoR1, GoR2 and GoR 3.

And (3) determining that the construction of the T2-generation TaNADH-GoGAT overexpression wheat of GoOE1, GoOE17 and GoOE18 and the T2-generation TaNADH-GoGAT underexpression wheat of GoR1, GoR2 and GoR3 are successful through the DNA level detection of the step (one) and the RNA level detection of the step (two).

Example 2 identification of yield traits in TaNADH-GoGAT transgenic wheat

Wheat to be tested: TaNADH-GoGAT overexpresses wheat GoOE1, GoOE17 and GoOE18, TaNADH-GoGAT overexpresses wheat GoR1, GoR2 and GoR3, wild type wheat KN199, and negative control lines isolated from empty control plants (NC) and each transgenic line.

The determination of the wheat yield-related traits is carried out in a field test for two years (2015-2016 growing season and 2016-2017 growing season) under field conditions, and concretely comprises the following steps:

the homozygous T3 generation TaNADH-GoGAT transgenic line phenotype was identified during the 2015-2016 growth season. The field phenotype identification adopts 2m row length, 30cm row spacing and 10cm plant spacing. After maturation, the individual plants were harvested for phenotype determination. The following indices were determined for each wheat tested: the yield of each plant seed (g/plant), the number of ears per plant, the number of grains per ear and the thousand kernel weight (g). The results show that the individual grain yield (grams per plant) and ears per plant of the TaNADH-GoGAT overexpression lines (GoOE1, GoOE17, and GoOE18) were significantly increased compared to the receptor parent KN199, but there was no significant change in grain per ear, thousand kernel weight (grams). The yield (g/plant) of each seed and the number of ears per plant of the TaNADH-GoGAT decrement expression line (GoR1, GoR2 and GoR3) are obviously reduced, but the number of the seeds per ear and the thousand kernel weight (g) are not obviously changed. See fig. 2.

The phenotype of homozygous TaNADH-GoGAT over-and under-expressing the transgenic line T4 was identified in 2016-2017. Each transgenic line was seeded with 3 replicates, each replicate in1 line. The field phenotype identification adopts 2m row length, 30cm row spacing and 5cm plant spacing, and 10 representative single plants are selected for each row after the plant is mature to determine the agronomic characters. The following indexes of each wheat tested were determined in the field: kernel yield (g/10 plants), ear number (number/10 plants), plant biomass (g/10 plants) (dry weight). In addition, the number of grains per ear and the thousand grain weight (g) were measured. The results show that compared with the receptor parent KN199 and a negative control (a negative control line separated from each transgenic line), the grain yield, the plant biomass and the spike number of the TaNADH-GoGAT overexpression transgenic line are obviously increased, and other agronomic characters such as thousand kernel weight, spike kernel number and the like have no obvious difference. Compared with the receptor parent KN199 and a negative control (a negative control line separated from each transgenic line), the plant biomass, the grain yield and the spike number of the TaNADH-GoGAT decrement expression transgenic line are all obviously reduced, the spike grain number is also obviously reduced, and the thousand kernel weight has no obvious difference. See fig. 3.

In addition, the single spike test results randomly collected from the flowering stage to the mature stage show that the overground part dry weights of the single spikes of the transgenic line and the receptor parent KN199 have no significant difference in the flowering stage; the dry weight of the overground part of the single spike of the decrement expression system is obviously lower than KN199 in the mature period, and the dry weight of the overground part of the single spike of the decrement expression system is obviously higher than KN199 in the over-expression system, so that the accumulation of the dry matter of the single spike after the blossom is promoted by the over-expression of TaNADH-GoGAT, and the accumulation of the dry matter of the overground part is inhibited by the decrement expression of TaNADH-GoGAT. Combining the results of single plant and single ear test, the increase of ear number and single ear biomass is the main reason of the increase of yield of the overexpression system, and the reduction of ear number and single ear biomass is the main reason of the reduction of yield of the decrement expression system.

<110> institute of genetics and developmental biology of Chinese academy of sciences

Application of <120> protein TaNADH-GoGAT in regulation and control of plant yield

<130> GNCLN181551

<160> 3

<170> PatentIn version 3.5

<210> 1

<211> 2144

<212> PRT

<213> Triticum aestivum L.

<400> 1

Met Pro Thr Ala Gln Gly Ile Gly Leu Lys His Ala Ala Pro Pro Gly

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Gly Arg Arg Ala Arg Arg Ser Gln Ser Ala Ala Ala Pro Gly Arg Ser

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Ala Arg Gln Ala His Gly Ala Met Ser Leu Asp Gly Gly Phe Leu Gly

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Gly Ala Gln Arg Thr Glu Glu Arg Val Ala Pro Arg Pro Pro Arg Ala

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Ala Ala Arg Asp Ala Glu Ser Ile Arg Pro Met Ser Leu Leu Pro Glu

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Ser Ser Ile Gly Leu Tyr Asp Pro Ala Phe Glu Arg Asp Ser Cys Gly

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

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Val Asp Ala Ile Gln Met Leu Glu Arg Met Ala His Arg Gly Ala Cys

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Gly Cys Glu Lys Asn Thr Gly Asp Gly Ala Gly Ile Leu Val Ala Leu

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Pro His Thr Phe Phe Arg Glu Val Thr Lys Asp Ala Gly Phe Glu Leu

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Pro Pro Pro Gly Glu Tyr Ala Val Gly Met Val Phe Leu Pro Thr Asp

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Glu Lys Arg Arg Glu Arg Ser Lys Thr Glu Phe Thr Lys Val Ala Glu

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Ser Leu Gly His Ser Ile Leu Gly Trp Arg Gln Val Pro Thr Asp Asn

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Val Phe Leu Thr Lys Ser Ser Lys Ser Lys Ala Asp Phe Glu Gln Gln

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Leu Phe Ile Leu Arg Arg Leu Ser Ile Val Ser Ile Arg Ala Ala Leu

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Asn Leu Gln Arg Gly Gly Glu Arg Asp Phe Tyr Met Cys Ser Leu Ser

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Ser Arg Thr Ile Val Tyr Lys Gly Gln Leu Met Pro Ser Gln Leu Gln

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Gly Tyr Tyr Tyr Ala Asp Ile Gly His Glu Asn Phe Ser Ser Tyr Met

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Pro Ala Leu Ile Ser Phe Thr Asp Gly Arg Tyr Leu Gly Ala Thr Leu

435 440 445

Asp Arg Asn Gly Leu Arg Pro Gly Arg Phe Tyr Val Thr His Ser Gly

450 455 460

Arg Val Val Met Gly Ser Glu Val Gly Val Val Asp Ile Pro Ala Gln

465 470 475 480

Asp Val Leu Arg Lys Gly Arg Leu Asn Pro Gly Met Met Leu Leu Val

485 490 495

Asp Phe Asp Asn His Thr Val Val Asp Asp Glu Ala Leu Lys Ala Gln

500 505 510

Tyr Ser Lys Ala His Pro Tyr Gly Glu Trp Leu Lys Arg Gln Lys Met

515 520 525

Tyr Leu Lys Asp Ile Val Glu Ser Val Pro Glu Thr Asp Arg Val Ala

530 535 540

Pro Ser Ile Ser Gly Ser Ile Thr Gln Thr Asn Glu Asn Lys Glu Cys

545 550 555 560

Val Gly Ile Asn Ala Ile Val Thr Pro Leu Lys Ala Phe Gly Tyr Thr

565 570 575

Leu Glu Ala Leu Glu Met Leu Leu Leu Pro Met Ala Lys Asp Gly Val

580 585 590

Glu Ala Leu Gly Ser Met Gly Asn Asp Ala Pro Leu Ala Val Met Ser

595 600 605

Asn Arg Glu Lys Leu Thr Phe Glu Tyr Phe Lys Gln Met Phe Ala Gln

610 615 620

Val Thr Asn Pro Pro Ile Asp Pro Ile Arg Glu Lys Ile Val Thr Ser

625 630 635 640

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

645 650 655

Lys Gln Cys Asn Arg Leu Ala Leu Lys Gly Pro Leu Val Ser Met Asp

660 665 670

Glu Met Glu Ser Ile Lys Lys Met Asn Tyr Arg Gly Trp Arg Ser Lys

675 680 685

Val Leu Asp Ile Thr Tyr Pro Lys Asn Ser Gly Arg Lys Gly Leu Glu

690 695 700

Glu Thr Leu Asp Arg Ile Cys Ala Glu Ala Arg Glu Ala Ile Arg Glu

705 710 715 720

Gly Tyr Lys Ile Leu Val Leu Ser Asp Arg Gly Phe Ser Ser Asp Arg

725 730 735

Val Ala Val Ser Ser Leu Leu Ala Val Gly Ala Val His Gln His Leu

740 745 750

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

755 760 765

Glu Pro Arg Glu Val His His Phe Cys Thr Leu Val Gly Phe Gly Ala

770 775 780

Asp Ala Ile Cys Pro Tyr Leu Ala Ile Glu Ala Ile Trp Cys Leu Gln

785 790 795 800

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

805 810 815

Lys Tyr Phe Tyr Ala Ser Ile Tyr Gly Met Met Lys Val Leu Ala Lys

820 825 830

Met Gly Ile Ser Thr Leu Ala Ser Tyr Lys Gly Ala Gln Ile Phe Glu

835 840 845

Ala Leu Gly Leu Ser Ser Glu Val Ile His Lys Cys Phe Glu Gly Thr

850 855 860

Pro Ser Arg Ile Glu Gly Ala Thr Phe Glu Met Leu Ala Arg Asp Ala

865 870 875 880

Leu Arg Leu His Glu Leu Ala Phe Pro Ser Arg Thr Pro Pro Pro Gly

885 890 895

Ser Ala Asp Ala Lys Ala Leu Pro Asn Pro Gly Asp Tyr His Trp Arg

900 905 910

Lys Asn Gly Glu Val His Leu Asn Asp Pro Leu Ala Met Ala Lys Leu

915 920 925

Gln Glu Ala Ala Lys Val Asn Ser Arg Glu Ala Tyr Lys Glu Tyr Ser

930 935 940

Lys Arg Ile Gln Glu Leu Asn Lys Ala Cys Asn Leu Arg Gly Met Leu

945 950 955 960

Lys Phe Ile Asp Ser Thr Ser Lys Ile Ser Leu Asp Glu Val Glu Pro

965 970 975

Ala Ser Glu Ile Val Lys Arg Phe Cys Thr Gly Ala Met Ser Tyr Gly

980 985 990

Ser Ile Ser Leu Glu Ala His Thr Ala Leu Ala Val Ala Met Asn Lys

995 1000 1005

Leu Gly Gly Lys Ser Asn Thr Gly Glu Gly Gly Glu Gln Pro Ser

1010 1015 1020

Arg Met Glu Pro Leu Pro Asp Gly Ser Met Asn Pro Lys Arg Ser

1025 1030 1035

Ala Ile Lys Gln Val Ala Ser Gly Arg Phe Gly Val Ser Ser Tyr

1040 1045 1050

Tyr Leu Thr Asn Ala Asp Glu Leu Gln Ile Lys Met Ala Gln Gly

1055 1060 1065

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

1070 1075 1080

Gly Asp Ile Ala Val Thr Arg His Ser Thr Ala Gly Val Gly Leu

1085 1090 1095

Ile Ser Pro Pro Pro His His Asp Ile Tyr Ser Ile Glu Asp Leu

1100 1105 1110

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

1115 1120 1125

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

1130 1135 1140

Ser Gly Val Val Lys Gly His Ala Asp His Val Leu Ile Ser Gly

1145 1150 1155

His Asp Gly Gly Thr Gly Ala Ser Arg Trp Thr Gly Ile Lys Asn

1160 1165 1170

Ala Gly Leu Pro Trp Glu Leu Gly Leu Ala Glu Thr His Gln Thr

1175 1180 1185

Leu Val Ala Asn Gly Leu Arg Gly Arg Ala Val Leu Gln Thr Asp

1190 1195 1200

Gly Gln Leu Lys Thr Gly Arg Asp Val Ala Val Ala Cys Leu Leu

1205 1210 1215

Gly Ala Glu Glu Phe Gly Phe Ser Thr Ala Pro Leu Ile Thr Leu

1220 1225 1230

Gly Cys Ile Met Met Arg Lys Cys His Thr Asn Thr Cys Pro Val

1235 1240 1245

Gly Ile Ala Thr Gln Asp Pro Val Leu Arg Glu Lys Phe Ala Gly

1250 1255 1260

Glu Pro Glu His Val Ile Asn Phe Phe Phe Met Leu Ala Glu Glu

1265 1270 1275

Leu Arg Glu Ile Met Ala Gln Leu Gly Leu Arg Thr Ile Asn Glu

1280 1285 1290

Met Val Gly Arg Ser Asp Met Leu Glu Val Asp Pro Glu Val Val

1295 1300 1305

Lys Ser Asn Glu Lys Leu Glu Asn Ile Asp Leu Ser Leu Ile Leu

1310 1315 1320

Lys Pro Ala Ala Glu Ile Arg Pro Gly Ala Ala Gln Tyr Cys Val

1325 1330 1335

Glu Lys Gln Asp His Gly Leu Asp Met Ala Leu Asp Asn Lys Leu

1340 1345 1350

Ile Ala Leu Ser Arg Ala Ala Leu Glu Lys Glu Val Arg Val Phe

1355 1360 1365

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

1370 1375 1380

Leu Ser His Glu Val Thr Lys Arg Tyr His Met Lys Gly Leu Asp

1385 1390 1395

Pro Gly Thr Ile His Val Lys Leu Thr Gly Ser Ala Gly Gln Ser

1400 1405 1410

Phe Gly Ala Phe Leu Cys Pro Gly Ile Thr Leu Glu Leu Glu Gly

1415 1420 1425

Asp Ser Asn Asp Tyr Val Gly Lys Gly Leu Ser Gly Gly Lys Ile

1430 1435 1440

Val Val Tyr Pro Pro Arg Asn Ser Thr Phe Ser Ala Glu Asp Asn

1445 1450 1455

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

1460 1465 1470

Ala Tyr Phe Asn Gly Met Ala Ala Glu Arg Phe Cys Val Arg Asn

1475 1480 1485

Ser Gly Ala Arg Thr Val Val Glu Gly Ile Gly Asp His Gly Cys

1490 1495 1500

Glu Tyr Met Thr Gly Gly Thr Val Val Ile Leu Gly Lys Thr Gly

1505 1510 1515

Arg Asn Phe Ala Ala Gly Met Ser Gly Gly Ile Ala Tyr Val Tyr

1520 1525 1530

Asp Val Asp Gly Thr Phe Ser Val Arg Cys Asn Asn Glu Leu Val

1535 1540 1545

Asp Leu Tyr His Val Glu Glu Glu Asp Asp Val Thr Thr Leu Lys

1550 1555 1560

Met Met Ile Glu Gln His Arg Leu His Thr Glu Ser Val Leu Ala

1565 1570 1575

Lys Asp Ile Leu Ser Lys Phe Asp Thr Leu Leu Pro Lys Phe Val

1580 1585 1590

Lys Val Tyr Pro Arg Asp Tyr Lys Arg Val Leu Glu Glu Met Lys

1595 1600 1605

Ala Glu Lys Ala Ala Ala Arg Pro Thr Lys Glu Pro Lys Val Ala

1610 1615 1620

Asn Gly Val Ser Val Thr Thr Lys Lys Ile Gln Thr Glu Lys Ser

1625 1630 1635

Ser Ser Arg Pro Thr Arg Val Ala Asn Ala Lys Lys Tyr Arg Gly

1640 1645 1650

Phe Val Thr Tyr Glu Arg Glu Gly Val Ser Tyr Arg Asp Pro Asn

1655 1660 1665

Glu Arg Val Lys Asp Trp Asn Glu Val Ala Ile Glu Ser Val Pro

1670 1675 1680

Gly Pro Leu Leu Asn Thr Gln Ser Ala Arg Cys Met Asp Cys Gly

1685 1690 1695

Thr Pro Phe Cys His Gln Glu Ser Ser Gly Ala Gly Cys Pro Leu

1700 1705 1710

Gly Asn Lys Ile Pro Glu Phe Asn Glu Leu Val His Gln Asn Arg

1715 1720 1725

Trp Arg Glu Ala Leu Asp Arg Leu Leu Glu Thr Asn Asn Phe Pro

1730 1735 1740

Glu Phe Thr Gly Arg Val Cys Pro Ala Pro Cys Glu Gly Ser Cys

1745 1750 1755

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

1760 1765 1770

Cys Ser Ile Ile Asp Lys Gly Phe Glu Glu Gly Trp Met Val Pro

1775 1780 1785

Arg Pro Pro Leu Gln Arg Thr Gly Lys Lys Ile Ala Ile Val Gly

1790 1795 1800

Ser Gly Pro Ala Gly Leu Ala Ala Ala Asp Gln Leu Asn Lys Met

1805 1810 1815

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

1820 1825 1830

Leu Met Met Tyr Gly Val Pro Asn Met Lys Thr Asp Lys Ile Gly

1835 1840 1845

Val Val Gln Arg Arg Val Asn Leu Met Ala Glu Glu Gly Val Thr

1850 1855 1860

Phe Val Val Asn Ala Asn Val Gly Ser Asp Pro Leu Tyr Ser Ile

1865 1870 1875

Glu Arg Leu His Ser Glu Asn Asn Ala Val Ile Leu Ala Cys Gly

1880 1885 1890

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

1895 1900 1905

Gly Val His Phe Ala Met Glu Phe Leu His Ala Asn Thr Lys Ser

1910 1915 1920

Leu Leu Asp Ser Asn Leu Glu Asp Gly Arg Tyr Ile Ser Ala Gln

1925 1930 1935

Gly Lys Lys Val Val Val Ile Gly Gly Gly Asp Thr Gly Thr Asp

1940 1945 1950

Cys Ile Gly Thr Ser Val Arg His Gly Cys Ser Ser Ile Val Asn

1955 1960 1965

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

1970 1975 1980

Pro Trp Pro Gln Trp Pro Arg Val Phe Arg Val Asp Tyr Gly His

1985 1990 1995

Gln Glu Ala Ser Thr Lys Phe Gly Asn Asp Pro Arg Thr Tyr Glu

2000 2005 2010

Val Leu Thr Lys Arg Phe Ile Gly Asp Glu Asp Gly Lys Leu Lys

2015 2020 2025

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

2030 2035 2040

Phe Gln Phe Lys Glu Ile Glu Gly Ser Gln Glu Ile Ile Glu Ala

2045 2050 2055

Asp Leu Val Leu Leu Ala Met Gly Phe Leu Gly Pro Glu Glu Asn

2060 2065 2070

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

2075 2080 2085

Lys Ala Gln Phe Gly His Phe Gly Thr Ser Val Asp Gly Val Phe

2090 2095 2100

Ala Ala Gly Asp Cys Arg Arg Gly Gln Ser Leu Val Val Trp Ala

2105 2110 2115

Ile Thr Glu Gly Arg Glu Ala Ala Ala Ala Val Asp Lys Tyr Leu

2120 2125 2130

Ser Arg Asp Glu Gln Asn Val Ala Gly Leu Thr

2135 2140

<210> 2

<211> 6435

<212> DNA

<213> Triticum aestivum L.

<400> 2

atgccgacgg cgcaggggat tggtttgaag cacgcggcgc cgccgggcgg ccgcagggcc 60

cgccgcagcc agtccgcggc cgcgccgggc cgctccgcaa ggcaggcgca cggcgccatg 120

tccctggatg gcgggttcct cggcggcgcg cagcgcaccg aggaacgcgt cgcgccacgc 180

ccgcctcggg ccgcggcgcg cgacgccgag tccatcaggc ccatgtctct gctacccgag 240

agcagcattg ggctgtacga cccggcgttc gagcgtgact cgtgcggcgt tggcttcgtc 300

gccgagctgt cgggcgttga caaccgggcg accgtcgtcg atgccattca gatgcttgaa 360

agaatggcac accgaggtgc ctgcggctgt gagaaaaaca ctggtgatgg tgccggcatt 420

ctcgttgctc taccacacac cttcttccga gaggtgacaa aggatgccgg tttcgagtta 480

ccgccaccag gtgagtatgc tgttggaatg gtcttcctgc caaccgatga gaagcgccgc 540

gagaggagca aaactgagtt tacaaaggtc gctgagtctc taggacattc gatacttggg 600

tggcgccagg ttcccactga caattcagac ttgggccaag ctgcgctcga tactgaacca 660

gccattgaac aggttttcct caccaagagt tcaaaatcga aggccgactt cgaacagcag 720

ttgtttatcc tgaggaggct ctcaattgta tctatccggg ccgcgctgaa tcttcagcgt 780

ggaggagaga gagatttcta catgtgctct ctatcttcaa ggaccattgt ttacaagggc 840

caacttatgc catcccagct ccaagggtac tactatgcgg atataggtca tgaaaacttc 900

tccagttata tggctctggt tcactcaagg ttctccacca acaccttccc cagctgggac 960

cgtgcacagc caatgcgtgt cttaggccac aatggagaga tcaatactct caaagggaac 1020

aaaaactgga tgaaagctcg tgagggtctc ttggagtgtg agaagcttgg cctatcacag 1080

gatgaaatgt caaagattct tccaatagta gatgccactt cttcagattc aggtgcattt 1140

gatggtgttc tcgagctcct tatccgtggt ggaagaagcc tgccagaagc tgtgatgatg 1200

atgatccctg aggcgtggca gaatgatgta aacatggaac ctgataagaa agctctgtac 1260

gagttcttgt cagcccttat ggagccttgg gatggacctg ctctcatatc ttttaccgat 1320

ggccgctacc ttggagctac cctggatcgc aatggcctta ggcctggtcg attttatgta 1380

acccacagtg gacgtgtggt catgggtagt gaagttggtg ttgtggatat tcctgcccaa 1440

gatgtgttga gaaagggtcg gcttaaccct ggaatgatgt tactcgttga cttcgacaac 1500

catactgtag tagatgatga agcactcaag gcacagtact ctaaagctca cccgtatgga 1560

gaatggctca agcgacaaaa gatgtacctc aaagacattg tagaatctgt cccagaaact 1620

gacagagttg ctccaagcat ttctggttct attacgcaaa caaatgagaa caaggaatgt 1680

gtaggcatca atgcaattgt gactccacta aaggcgtttg ggtacacatt ggaagcccta 1740

gaaatgttgc tgctgccaat ggctaaagat ggagtggaag ctcttggatc aatgggaaat 1800

gatgcacccc tagcagtgat gtcaaacaga gagaagctga cttttgagta cttcaagcag 1860

atgtttgcac aagtaacaaa ccctccaatc gatccaatta gggagaagat tgttacatct 1920

atggaatgta tgattgggcc agaaggagat ttgctggaaa taaccgaaaa gcaatgcaac 1980

cgccttgcac ttaaaggtcc tttggtgtca atggatgaaa tggaatctat caagaagatg 2040

aactaccgtg gttggcgcag caaggtgctc gacataactt atccgaagaa ttctggaagg 2100

aagggcttgg aagaaacttt ggatagaatt tgtgctgaag cccgggaagc tatacgcgag 2160

ggatacaaaa ttttagttct ttcagacaga ggattttctt cagaccgtgt tgctgtcagt 2220

tccctcttag cagttggagc agtacatcaa caccttgttg caaatcttga gaggacacgc 2280

gtaggattat tggttgagtc tgctgaacct cgtgaagtgc accatttctg tacactcgtt 2340

ggatttggtg cagatgctat atgcccttat ttggccattg aagcaatttg gtgcttgcag 2400

actgatggga agattccccc taccgactca aaggaggaac ttgtcgaaaa atatttttat 2460

gcttccatct atggaatgat gaaggttctt gcaaagatgg gaatatccac ccttgcatct 2520

tacaaagggg cacagatttt tgaagctctt ggactttctt ctgaagtgat tcacaagtgt 2580

tttgaaggca ctcctagcag aattgagggt gcaacgttcg aaatgcttgc acgtgatgct 2640

ctccgtcttc acgagttggc attcccatca agaacacctc cgcctggcag tgcagatgca 2700

aaagcccttc ccaatccagg ggattaccac tggaggaaaa atggtgaagt ccatctaaat 2760

gatcctcttg caatggcaaa attacaagaa gcagctaaag taaacagccg ggaagcatac 2820

aaagaatatt ctaagcgaat tcaagagctt aacaaggcgt gcaatctgcg tggtatgctg 2880

aaatttatag atagcactag caagatttct ttggatgagg ttgaacctgc aagtgagata 2940

gtgaaacgct tttgtactgg ggcaatgagt tatggatcta tttcgttgga ggcacatact 3000

gcccttgctg tggccatgaa caaactggga ggcaaatcta acacagggga gggaggggag 3060

cagccttctc gtatggagcc tcttcctgat ggttcgatga atccaaaacg aagtgcaatc 3120

aagcaagttg ccagtggacg atttggagtt tccagctatt atctgactaa tgcagatgag 3180

ctgcagataa aaatggctca gggtgctaag cctggtgaag gtggtgagct accaggtcac 3240

aaggttatcg gtgacattgc agttaccaga cattctacag ctggtgttgg gcttattagt 3300

ccacctcctc accatgatat atattctatc gaggatcttg cacagcttat ccatgacctt 3360

aagaattcaa atcctcaagc tcgaatcagc gtgaagctag tgtctgaggc tggtgtygga 3420

gttgtagcta gcggtgttgt aaaaggacac gcagaccatg ttcttatttc tggccatgac 3480

ggtggcaccg gtgcatcaag atggacaggt atcaagaatg ctggacttcc atgggaacta 3540

ggattggcyg agacacatca aactttagtg gcaaatgggc ttcgtggtcg agctgtccta 3600

caaacagatg gccaattaaa aactggtaga gatgttgctg tggcgtgctt acttggtgca 3660

gaggaatttg gtttcagcac tgctccactg atcacacttg gctgcattat gatgcggaag 3720

tgccatacca atacttgccc tgttggtata gccactcaag atccagtgct ccgagaaaaa 3780

tttgctggag aaccagagca tgtcattaac tttttcttca tgcttgctga ggagctaaga 3840

gaaattatgg ctcagcttgg cctccgtaca attaatgaaa tggttggacg ctctgatatg 3900

cttgaagttg atccagaagt agttaagagc aatgagaaac ttgaaaatat tgatctctca 3960

ttgatcttaa aaccagctgc agagattcgt cctggggctg ctcagtactg tgttgaaaag 4020

caagaccatg gccttgacat ggctttggat aacaaactta tagctttatc aagggctgca 4080

cttgaaaagg aagttcgtgt tttcattgag actccaatca agaacactaa tcgggcagta 4140

ggcaccacac ttagccatga agtcacaaaa cgttatcaca tgaagggatt agatcctgga 4200

accattcatg tgaaactcac tggaagtgct ggtcagagtt ttggtgcttt tctctgtcct 4260

ggaataaccc ttgagcttga aggagacagc aatgattacg ttggcaaagg attatctggt 4320

ggaaagattg ttgtgtaccc acccaggaac agtacattta gtgcagaaga caatattgtc 4380

attggtaatg tggccctgta tggtgctacc aagggagaag catacttcaa tggaatggca 4440

gcagaaaggt tttgtgttcg taattctggt gctcgaacag tggttgaagg aattggtgat 4500

catggatgcg agtacatgac agggggtact gtagtcatcc ttggtaaaac aggaagaaat 4560

tttgctgctg ggatgagtgg aggcatcgct tatgtttatg atgttgatgg gacattcagt 4620

gtccgctgta acaatgagtt ggttgatcta tatcatgtgg aggaagagga tgatgtaacc 4680

actttgaaaa tgatgataga gcaacatcga cttcacacag agagtgtcct ggccaaagac 4740

atactctcta aattcgacac tcttcttcca aaatttgtaa aagtataccc aagggattat 4800

aagagggttc tagaagaaat gaaggcagaa aaagctgcag ctaggcctac aaaggaacct 4860

aaggtggcaa atggtgtttc tgtgacaact aagaaaatac aaacagagaa gtcatccagc 4920

cgaccaacac gtgttgccaa cgccaaaaag taccggggtt ttgtaacata tgagcgagag 4980

ggtgtttctt accgtgatcc aaatgagcgt gttaaagatt ggaacgaggt tgccattgaa 5040

tcagttccag ggccactgtt aaacacacag tctgctcgtt gtatggattg tggcactcct 5100

ttctgtcatc aggaaagctc aggtgctggc tgtcctcttg gaaataagat cccagagttc 5160

aatgaattag ttcaccagaa tagatggcgt gaagcattgg atcgcctact agagacaaac 5220

aacttccctg aatttactgg acgtgtgtgc cctgctcctt gtgaggggtc ttgtgttctt 5280

ggcattattg agaacccagt atcaatcaaa agcatagaat gctcgattat agacaaaggt 5340

tttgaagagg gatggatggt accacgacca ccacttcaaa gaacaggaaa gaaaatcgct 5400

atagttggca gtggtcctgc tggtttggct gccgctgatc aactaaataa aatgggccat 5460

tttgtaactg tatttgaacg ttcggatcgt ataggaggtc ttatgatgta tggagtacca 5520

aacatgaaga cagacaagat tggagttgtt cagcgtcgtg tcaatttaat ggccgaagag 5580

ggtgtaacat ttgtggtgaa tgctaatgta gggagtgatc ctttatactc aattgaacgt 5640

ctccattctg agaacaatgc agttattttg gcttgtggag ctacaaaacc aagggacctc 5700

agtattcctg gccgtgagct agctggagtt cattttgcca tggaatttct ccacgcaaat 5760

accaaaagtt tgcttgatag caacctggag gatggaagat acatatctgc ccagggtaag 5820

aaggtggtgg tcattggtgg tggagacaca ggcacagatt gcatcggtac gtctgttagg 5880

catggttgca gcagcattgt aaatctggag cttctcacca agccaccaag caagagagct 5940

tctgacaacc cctggcccca gtggcctaga gtcttccgag tggactatgg gcaccaggaa 6000

gcatctacca aatttggaaa tgatccaaga acttacgaag tcttaaccaa gcgtttcatt 6060

ggtgatgaag atggaaaatt gaaggccctt gaggtggtgc gcgtgaagtg ggagaaagta 6120

gatggaaaat tccagttcaa ggagattgaa ggatcacaag agatcatcga ggcagacctt 6180

gtcctccttg ccatgggatt cctgggccct gaagagaaca tcgctgacaa actgggttta 6240

gagaaagaca accgttccaa cttcaaagct caattcggac acttcgggac cagtgtggat 6300

ggcgtttttg ccgctgggga ttgcaggcgc gggcaatcgc tggttgtttg ggccatcacc 6360

gaagggcgtg aagctgctgc tgcagtagat aagtacttgt caagggatga acaaaatgtc 6420

gcaggcctta catag 6435

<210> 3

<211> 1033

<212> DNA

<213> Artificial sequence

<400> 3

ggattggttt gaagcacgcg gcgccgccgg gcggccgcag ggcccgccgc agccagtccg 60

cggccgcgcc gggccgctcc gcaaggcagg cgcacggcgc catgtccctg gatggcgggt 120

tcctcggcgg cgcgcagcgc accgaggaac gcgtcgcgcc acgcccgcct cgggccgcgg 180

cgcgcgacgc cgagtccatc aggcccatgt ctctgctacc cgagagcagc attgggctgt 240

acgacccggc gttcgagcgt gactcgtgcg gcgttggctt cgtcgccgag ctgtcgggcg 300

ttgacaaccg ggcgaccgtc gtcgatgcca ttcagatgct tgaaagaatg gcacaccgag 360

gtgcctgcgg ctgtgagaaa aacactggtg atggtgccgg cattctcgtt gctctaccac 420

acacctgaat tcaagcttac gtcctcccct gcgcggcgcg caacaaggga cgacgacggc 480

acccagatac aaaaaaaaat ggtgatcatc cagctctctc aagaaaatat caagttcttc 540

agagttcaga ttacacacac tctagcttga actagtaggc gtgcttgatc ttgatcttac 600

caagcttagg tgtgtggtag agcaacgaga atgccggcac catcaccagt gtttttctca 660

cagccgcagg cacctcggtg tgccattctt tcaagcatct gaatggcatc gacgacggtc 720

gcccggttgt caacgcccga cagctcggcg acgaagccaa cgccgcacga gtcacgctcg 780

aacgccgggt cgtacagccc aatgctgctc tcgggtagca gagacatggg cctgatggac 840

tcggcgtcgc gcgccgcggc ccgaggcggg cgtggcgcga cgcgttcctc ggtgcgctgc 900

gcgccgccga ggaacccgcc atccagggac atggcgccgt gcgcctgcct tgcggagcgg 960

cccggcgcgg ccgcggactg gctgcggcgg gccctgcggc cgcccggcgg cgccgcgtgc 1020

ttcaaaccaa tcc 1033

Claims (10)

1. The application of TaNADH-GoGAT protein or its related biological material in regulating and controlling plant yield;

   the TaNADH-GoGAT protein is a protein with an amino acid sequence of SEQ ID No. 1;

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

   the expression quantity and/or activity of the TaNADH-GoGAT protein or the coding gene thereof in the plant is improved, and the yield of the plant is improved; the expression level and/or activity of the TaNADH-GoGAT protein or the coding gene thereof in the plant is reduced, and the yield of the plant is reduced;

   said plant yield is embodied in at least one of:

   (a1) the yield of single plant seeds of the plant;

   (a2) the number of plant ears per plant;

   (a3) plant individual biomass;

   (a4) plant single ear biomass;

   the plant is wheat.
2. 2. Use according to claim 1, characterized in that: the nucleic acid molecule is a DNA molecule shown in SEQ ID No. 2.
3. 3. A method for producing a plant variety with increased yield, comprising the step of increasing the expression level and/or activity of a TaNADH-GoGAT protein in a recipient plant; the TaNADH-GoGAT protein is a protein with an amino acid sequence of SEQ ID No. 1; the yield improvement is embodied as single plant seed yield improvement and/or increased ear number per plant and/or single plant biomass improvement and/or single ear biomass improvement;

   the plant is wheat.
4. 4. A method of breeding a transgenic plant with increased yield comprising the steps of: introducing a nucleic acid molecule capable of expressing a TaNADH-GoGAT protein into a receptor plant to obtain a transgenic plant; the transgenic plant has increased yield as compared to the recipient plant; the TaNADH-GoGAT protein is a protein with an amino acid sequence of SEQ ID No. 1;

   the yield improvement is embodied as single plant seed yield improvement and/or increased ear number per plant and/or single plant biomass improvement and/or single ear biomass improvement;

   the plant is wheat.
5. 5. A method for breeding a plant variety with reduced yield, comprising the step of reducing the expression level and/or activity of a TaNADH-GoGAT protein in a recipient plant; the TaNADH-GoGAT protein is a protein with an amino acid sequence of SEQ ID No. 1;

   the yield reduction is embodied as the yield reduction of single plant grains and/or the reduction of the number of ears per plant and/or the reduction of single plant biomass and/or the reduction of single ear biomass;

   the plant is wheat.
6. 6. A method of breeding transgenic plants with reduced yield comprising the steps of: inhibiting and expressing coding genes of TaNADH-GoGAT protein in a receptor plant to obtain a transgenic plant; reduced yield of said transgenic plant compared to said recipient plant; the TaNADH-GoGAT protein is a protein with an amino acid sequence of SEQ ID No. 1;

   the yield reduction is embodied as the yield reduction of single plant grains and/or the reduction of the number of ears per plant and/or the reduction of single plant biomass and/or the reduction of single ear biomass;

   the plant is wheat.
7. 7. The method of claim 4, wherein: introducing into said recipient plant a nucleic acid molecule capable of expressing said TaNADH-GoGAT protein by introducing into said recipient plant a recombinant expression vector comprising a gene encoding said TaNADH-GoGAT protein.
8. 8. Use according to claim 4, characterized in that: the nucleic acid molecule is a DNA molecule shown in SEQ ID No. 2.
9. 9. The method of claim 6, wherein: the inhibition expression of the coding gene of the TaNADH-GoGAT protein in the receptor plant is realized by introducing an interference vector containing a DNA fragment shown in a formula (I) into the receptor plant;

   SEQ_{forward direction}-X-SEQ_{Reverse direction} (I)

   The above-mentionedSEQ_{Forward direction}Has the sequence of SEQ ID No.3 at the 1 st to 426 st positions;

   said SEQ_{Reverse direction}And the sequence of SEQ_{Forward direction}Is complementary in reverse direction;

   said X is said SEQ_{Forward direction}And said SEQ_{Reverse direction}In the sequence, the X and the SEQ_{Forward direction}And said SEQ_{Reverse direction}Are not complementary.
10. 10. The method of claim 6, wherein: the coding gene of the TaNADH-GoGAT protein is a DNA molecule shown in SEQ ID No. 2.

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