CN111518183B - Application of SiMYB61 protein and related biomaterial thereof in regulation and control of plant stress resistance - Google Patents

Abstract

The invention discloses an application of SiMYB61 protein and related biological materials thereof in regulation and control of plant stress resistance. The invention provides a new gene for rice stress genetic engineering, and simultaneously introduces japonica rice fine varieties through genetic transformation to culture new stress-resistant transgenic varieties, so that the stress resistance of rice can be effectively improved, the influence of stress on rice production is reduced, and important economic and social benefits are achieved.

Description

Application of SiMYB61 protein and its related biomaterials in regulating plant stress resistance

technical field

The present invention relates to the application of SiMYB61 protein and related biological materials in regulating plant stress resistance.

Background technique

my country is one of the countries with the lowest grain output per unit of fertilizer input in the world. The utilization rate of nitrogen fertilizer is only 30-35% (45% in developed countries), and the utilization rate of phosphate fertilizer is only 10-20%. According to the current consumption, such as saving 10% of nitrogen fertilizer and 20% of phosphate fertilizer, 24.1 billion yuan can be saved every year. The inefficient use of nitrogen and phosphorus fertilizers makes agricultural non-point source pollution the most important factor in water eutrophication, soil acidification and heavy metal pollution, threatening ecological security and sustainable development. Therefore, our country urgently needs to cultivate new crop varieties with efficient nutrient utilization, so as to greatly improve the utilization efficiency of nitrogen and phosphorus fertilizers in our country.

Cultivating new varieties of rice with resistance to stress and improving its own resistance to stress are important measures to improve the yield level of rice under conditions of reducing fertilizer application and drought and water shortages.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a SiMYB61 protein related to plant stress resistance and its related biological materials and applications.

In the first aspect, the present invention first protects the application of SiMYB61 protein or its related biological materials in the following (a1) and/or (a2):

(a1) regulating plant yield-related traits;

(a2) regulating plant stress resistance;

The relevant biological material is a nucleic acid molecule capable of expressing the SiMYB61 protein or an expression cassette, a recombinant vector, a recombinant bacteria or a transgenic cell line containing the nucleic acid molecule;

The SiMYB61 protein is any one of the following proteins:

(A1) protein whose amino acid sequence is SEQ ID No.4;

(A2) The amino acid sequence shown in SEQ ID No. 4 is subjected to substitution and/or deletion and/or addition of one or several amino acid residues and has the same function;

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

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

The above proteins can be artificially synthesized or obtained by first synthesizing their coding genes and then carrying out biological expression.

Among the above proteins, the tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag and/or SUMO tag, and the like.

The nucleic acid molecule can specifically be the gene encoding SiMYB61 protein. The gene encoding the SiMYB61 protein is any of the following DNA molecules:

(B1) the DNA molecule shown in SEQ ID No.1;

(B2) the DNA molecule shown in SEQ ID No.2;

(B3) the DNA molecule shown in SEQ ID No.3;

(B4) a DNA molecule that hybridizes to the DNA molecule defined in (B1) or (B2) or (B3) under stringent conditions and encodes the SiMYB61 protein;

(B3) A DNA molecule that is 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more identical to the DNA sequence defined in (B1) or (B2) or (B3) and encodes the SiMYB61 protein .

The stringent conditions may be as follows: 50°C, hybridization in a mixed solution of 7% sodium dodecyl sulfate (SDS), 0.5M NaPO ₄ and 1 mM EDTA, rinsing at 50°C, 2×SSC, 0.1% SDS; It can also be: 50°C, hybridize in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1mM EDTA, and rinse in 1×SSC, 0.1% SDS at 50°C; it can also be: 50°C, in 7% SDS, Hybridization in a mixed solution of 0.5M NaPO ₄ and 1 mM EDTA, rinsed at 50°C, 0.5×SSC, 0.1% SDS; also: 50°C, in a mixed solution of 7% SDS, 0.5M NaPO ₄ and 1 mM EDTA Hybridization, rinsed in 0.1×SSC, 0.1% SDS at 50°C; alternatively: 50°C, hybridization in a mixture of 7% SDS, 0.5M NaPO ₄ and 1 mM EDTA, at 65°C, 0.1×SSC, 0.1 Rinse in % SDS; alternatively: hybridize in 6×SSC, 0.5% SDS solution at 65°C, then wash the membrane once with 2×SSC, 0.1% SDS and 1×SSC, 0.1% SDS each.

Specifically, the expression cassette may be an expression cassette consisting of a ubiquitin constitutive promoter, the coding gene of the above-mentioned SiMYB61 protein, and the terminator nos 3'. Specifically, the expression cassette can be obtained by double digestion of the recombinant vector with HindIII and EcoRI. Specifically, the recombinant vector can be a recombinant vector obtained by cloning SEQ ID NO. 3 into the BamHI and SpeI sites of the vector LP0471118-Bar-ubi-EDLL.

The recombinant bacteria can be obtained by introducing the above-mentioned expression cassettes or recombinant vectors into Agrobacterium strains. The Agrobacterium strain can be specifically Agrobacterium strain EHA105.

In the application, the plant yield-related traits include ear number, ear length, grain number per ear and/or biomass; and the biomass includes straw weight and/or rice weight.

The plant stress resistance is the resistance of the plant to low nitrogen stress.

The regulation is positive regulation.

In the second aspect, the present invention protects the application of SiMYB61 protein or its related biological materials in plant breeding;

The relevant biological material is a nucleic acid molecule capable of expressing the SiMYB61 protein or an expression cassette, a recombinant vector, a recombinant bacteria or a transgenic cell line containing the nucleic acid molecule;

The SiMYB61 protein is as previously described.

In the application, the purpose of the breeding is to select plants with high yield and/or high stress resistance. The high yield can be embodied by the number of panicles and/or the height of the panicle and/or the number of grains per panicle and/or the high biomass. The biomass includes straw weight and/or rice weight. The stress tolerance is specifically resistance to low nitrogen stress. The high resistance to low nitrogen stress is reflected in high yield and/or high plant height under low nitrogen stress. The high resistance to the low nitrogen stress is reflected in the high nitrogen content.

In a third aspect, the present invention protects a method for increasing plant yield and/or increasing plant stress tolerance, comprising the step of increasing the expression and/or activity of SiMYB61 protein in recipient plants.

The yield is specifically ear number, ear length, grain number per ear, grain weight and/or biomass.

The biomass includes straw weight and/or rice weight.

The stress tolerance is specifically resistance to low nitrogen stress.

The improvement of stress tolerance can specifically be embodied as an increase in yield and/or an increase in plant height under low nitrogen stress.

The high resistance to the low nitrogen stress is reflected in the high nitrogen content.

The SiMYB61 protein is as previously described.

In a fourth aspect, the present invention protects a method for cultivating a transgenic plant, comprising the steps of: introducing a nucleic acid molecule capable of expressing SiMYB61 protein into a recipient plant to obtain a transgenic plant with increased expression of the SiMYB61 protein; The recipient plants have improved yield and/or stress tolerance compared to the recipient plants.

The yield is specifically ear number, ear length, grain number per ear, grain weight and/or biomass.

The biomass includes straw weight and/or rice weight.

The stress tolerance is specifically resistance to low nitrogen stress.

The improvement of stress tolerance can specifically be embodied as an increase in yield and/or an increase in plant height under low nitrogen stress.

The high resistance to the low nitrogen stress is reflected in the high nitrogen content.

The "introducing a nucleic acid molecule capable of expressing SiMYB61 protein into a recipient plant" is achieved by introducing an expression cassette containing the gene encoding the SiMYB61 protein into the recipient plant.

The gene encoding the SiMYB61 protein is shown above.

Specifically, the expression cassette may be an expression cassette consisting of a ubiquitin constitutive promoter, the coding gene of the above-mentioned SiMYB61 protein, and the terminator nos 3'. Specifically, the expression cassette can be obtained by double digestion of the recombinant vector with HindIII and EcoRI. Specifically, the recombinant vector can be a recombinant vector obtained by cloning SEQ ID NO. 3 into the BamHI and SpeI sites of the vector LP0471118-Bar-ubi-EDLL.

Any of the above mentioned number of grains per panicle refers to real grains per panicle and/or total grains per panicle.

Any of the above-mentioned plants is a dicotyledonous plant or a monocotyledonous plant;

Further, the monocotyledonous plant is a grass;

Further, the grasses are rice or millet.

The rice can be specifically the rice variety Kitaake.

The millet can be specifically Yugu No. 1.

The invention provides a new gene for rice stress genetic engineering, and at the same time, through genetic transformation to introduce an excellent variety of japonica rice, and cultivating a new anti-reversion gene variety, the stress resistance of rice can be effectively improved, and the impact of stress on rice production can be reduced. important economic and social benefits.

Description of drawings

Figure 1 shows the SSR detection results of the transgenic SiMYB61 gene resistant rice. Marker: DL1000 Marker; negative control: Kitaake; positive control: plant expression vector psSiMYB61.

Figure 2 shows the statistical results of field testing data in 18 years.

Figure 3 is a comparison of the phenotypes of transgenic plants and wild type.

Figure 4 shows the statistical results of the detection data under normal treatment conditions in the field for 19 years.

Figure 5 shows the statistical results of the detection data under the condition of low nitrogen stress in the field for 19 years.

Figure 6 shows the statistical results of the 19-year field straw and paddy weight detection data.

Detailed ways

The following examples facilitate a better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The test materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

LP0471118-Bar-ubi-EDLL vector: documented in the literature: Ning Lei, Wang Shuguang, Ju Pengju, Bai Xingxuan, Ge Linhao, Qi Xin, Jiang Qiyan, Sun Xianjun, Chen Ming, Sun Daizhen. Effects of overexpression of SiANT1 in millet on the salinity tolerance of rice[ J]. Chinese Agricultural Sciences, 2018, 51(10): 1830-1841. The public can obtain from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

Yugu No. 1: Recorded in the literature: Ning Lei, Wang Shuguang, Ju Pengju, Bai Xingxuan, Ge Linhao, Qi Xin, Jiang Qiyan, Sun Xianjun, Chen Ming, Sun Daizhen. Effects of overexpression of SiANT1 in millet on salt tolerance of rice[J]. Chinese Agriculture Science, 2018, 51(10): 1830-1841.; Publicly available from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

The bar gene expression vector pSBAR: recorded in the literature: Su Ruibo. The use of improved minimal expression cassette technology to obtain drought-resistant transgenic wheat [D]. Inner Mongolia Agricultural University, 2012.; Publicly available from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

Agrobacterium strain EHA105: Beijing Qingke Xinye Biotechnology Co., Ltd.

Rice variety Kitaake: documented in the literature: Ning Lei, Wang Shuguang, Ju Pengju, Bai Xingxuan, Ge Linhao, Qi Xin, Jiang Qiyan, Sun Xianjun, Chen Ming, Sun Daizhen. Effects of overexpression of SiANT1 in millet on salt tolerance of rice[J]. Chinese Agriculture Science, 2018, 51(10): 1830-1841.; Publicly available from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.

Example 1. Acquisition of SiMYB61 protein and its encoding gene

A resistance-related gene was cloned from the confirmation of retrograde foxtail millet Yugu No. 1 and named as SiMYB61 gene. Its genome sequence is shown in SEQ ID NO.1, its transcription sequence is shown in SEQ ID NO.2, and its CDS is shown in SEQ ID NO. .3 shown. The protein encoded by the SiMYB61 gene (SiMYB61 protein) is shown in SEQ ID NO.4.

Example 2. Application of SiMYB61 protein and its encoding gene in rice breeding

1. Preparation of linear minimal expression cassettes

1. Obtaining the plant expression vector psSiMYB61

Total RNA from Yugu No. 1 was extracted and reverse transcribed into cDNA. Using the cDNA as a template, primer F and primer R were used for PCR amplification, and the CDS sequence (SEQ ID NO. 3) of SiMYB61 was cloned into the vector using the seamless cloning kit of Clotech company (catalog number: 639649) according to the instructions. The BamHI and SpeI sites of LP0471118-Bar-ubi-EDLL were used to obtain the plant expression vector psSiMYB61 (which has been verified by sequencing).

Primer F: 5'-AGACCGATCTGGATCATGGGGAAGCACTCCTGC-3';

Primer R: 5'-CGATCGATCCACTAGCTAGATATTCTCGAAAGACAAGGACATTCTCTG-3'.

2. Preparation of linear minimal expression cassettes

The plant expression vector psSiMYB61 obtained in step 1 was digested with HindIII and EcoRI, and a fragment of about 3500 bp was recovered to obtain a linear minimal expression frame of the SiMYB61 gene. The terminator nos 3' (300bp) is composed. The minimal expression cassette transformation method can remove the vector backbone sequence, without ampicillin resistance gene, transgenic rice is not significantly different from the receptor, and has no pathogenicity, which reduces the safety risk of inserting exogenous fragments into the plant genome.

The bar gene was used as the marker gene to prepare the marker gene expression frame: the bar gene expression vector pSBAR was digested with Hind III enzyme, and the digested product was recovered, which was the marker gene expression frame; the marker gene expression frame was composed of the maize ubiquitin promoter and marker gene. bar and nos terminator.

The transformation of rice

1. The linear minimal expression cassette and marker gene expression cassette of the SiMYB61 gene prepared in step 1 were transformed into Agrobacterium strain EHA105 to obtain a recombinant Agrobacterium.

2. Take the immature embryos of the rice variety Kitaake 12 to 14 days after pollination, soak them in 70% ethanol for 1 minute, then disinfect with 10% sodium hypochlorite for 15 minutes, rinse with sterile water for 3 to 5 times, and remove the Embryos were taken out and inoculated in SD ₂ medium (MS minimal medium (without vitamins)+2mg/L 2,4-D+1mg/LVB1+150mg/LAsn asparagine+30g/L sucrose+2.4g/ L phytogel, pH=5.8) induced immature embryo callus for 7 days, then transferred the induced callus to hypertonic medium (MS minimal medium+5mg/L 2,4-D+0.4mol/ L mannitol + 3g/L vegetable gel) hypertonicity treatment for 4 to 6 hours.

3. After step 2 is completed, the callus is infected with Agrobacterium, and the callus is immersed in the recombinant Agrobacterium solution with OD ₆₀₀ = 0.8, and the callus is oscillated at 180 r/min for about 30 minutes.

4. After completing step 3, the callus was cultured on the hypertonic medium for 16-18 hours, and then the infected callus was transferred to SD ₂ medium for two weeks in the dark.

5. After completing step 4, transfer the callus to a selective medium containing 2-3 mg/L of the herbicide Bialaphos (MS minimal medium (without vitamins) + 2 mg/L 2,4-D+ 1mg/LVB1+150mg/LAsn asparagine+30g/L sucrose+2.4g/L phytogel+2-3mg/L herbicide Bialaphos, pH=5.8) to screen callus, differentiate and strengthen seedlings to obtain Transgenic rice plants of the T0 generation.

3. Identification of transgenic positive rice

The SDS method was used to extract the DNA of the rice leaves of the T0 generation to be tested. Using the DNA as a template, primers for amplifying part of the sequence were designed according to the SiMYB61 gene sequence for PCR amplification, and primer F and primer R were used for PCR amplification. The product was detected by 0.8% agarose gel and photographed by UV light. The rice variety Kitaake was used to replace the T0 generation rice for the above operation as a negative control; the plant expression vector psSiMYB61 was used as a positive control.

F: 5'-TGGCAACTTGAGCCATCTCC-3';

R: 5'-GTTGATGTTGAGAGAGTT-3'.

The PCR reaction system is shown in Table 1. PCR reaction conditions: denaturation at 94°C for 5 min; 35 cycles of 94°C for 50 sec, 62°C for 50 sec, 72°C for 1 min; extension at 72°C for 10 min.

Table 1

Element Dosage (ul) (Takara)10xPCRbuffer(Mg²⁺Plus) 2.50 (Takara) 25mMMg²⁺ 0.05 (Takara) 2.5mM dNTP Mixture 2.00 Primer F 0.80 Primer R 0.80 (Takara)r-TaqDNAPloymerase(5U/ul) 0.25 template 1.00 ddH₂O 17.6 total 25

The results are shown in Figure 1. After PCR detection, 5 positive plants were obtained, and the positive rate was 2%. After generation, screening and identification in the greenhouse, the homozygous lines of SiMYB61 transgenic rice were selected and named as OE21 and OE44 for the following experiments.

4. Phenotypic detection of transgenic rice

1. Field phenotype detection in 2018

Plants to be tested: rice variety Kitaake (WT), transgenic line OE21, and transgenic line OE44.

Test site: Gao'an, Jiangxi Province, Jiangxi Academy of Agricultural Sciences, Rice Research Institute, Transgenic Test Base.

Seedling management was the same as field production (no nitrogen fertilizer was added), and each test material could grow normally.

8 plants were harvested from each material, repeated three times, and the biological yield and rice yield of each material were determined respectively.

The Kjeldahl method was used to determine the N content of rice and straw of the test materials respectively. The specific reference for the detection method is "Yang Yali, Cai Shunlin. Comparison of flow analysis method and Kjeldahl method for determination of total nitrogen in plants [J]. Journal of Yuxi Normal University, 2016, 32(08): 51-54." and "Dai Honglin, Wu Xiaojun. Determination of nitrogen content in plant dry samples by Kjeldahl method [J]. Journal of Jiangsu Agricultural University, 1995(03):70.” Method in.

Three plants of each material were selected for indoor seed testing, and the number of effective panicles per plant (more than 5 per panicle was considered effective panicle), the number of grains per panicle, the total grains per panicle, and the seed setting rate were investigated.

The results are shown in Figure 2. In Fig. 2, the unit of nitrogen content of rice and nitrogen content of rice straw is g/8 plants, which is the nitrogen content of 8 rice plants/straw weight. The unit of the total nitrogen content of the straw is percentage (%), which means the nitrogen content of the straw/the weight of the 8 straws.

The results showed that the dry grain weight and straw dry weight of the transgenic rice lines were higher than those of the control rice variety Kitaake without nitrogen fertilizer. The number of effective panicles per plant, the number of real grains per panicle and the total number of grains per panicle were higher than those of the control. The N content of rice and the N content of rice straw were also higher than those of the control.

2. 2019 field data

Plants to be tested: rice variety Kitaake (WT), transgenic line OE44.

Test site: Gao'an, Jiangxi Province, Jiangxi Academy of Agricultural Sciences, Rice Research Institute, Transgenic Test Base.

Seedling management is the same as field production. Each test material can grow normally.

The test set up two treatments with 12 kg of pure nitrogen per mu and no nitrogen fertilizer, and each treatment set up two replicates.

Each material is planted in 6 rows, 8 pockets in each row, and the row spacing is 5 × 6 inches. Single planting.

The test area is 4.0 mu. The management of other fertilizers and water in the field is the same as that of local production.

20 plants were harvested for each material and each treatment, repeated three times, and the weights of straw and rice were determined for each material.

Eight plants were harvested from each treatment and each material was repeated three times, and the biological yield and rice yield of each material were determined respectively.

Three plants of each treatment were selected for indoor seed testing, and the number of effective panicles per plant, the number of grains per panicle, and the total grains per panicle were investigated.

The phenotypic observations are shown in Figure 3.

The statistical results are shown in Figure 4 (normal treatment), Figure 5 (low nitrogen treatment) and Figure 6.

The above results showed that the biomass (straw weight, rice weight), ear length and total grains per ear of the transgenic plants were significantly higher than those of the recipient plants under normal nitrogen fertilization conditions. Under low nitrogen stress, the biomass (straw weight, paddy weight), plant height, ear number, ear length, grains per ear, and total grains per ear of transgenic plants were also higher than those of recipient plants.

sequence listing

<110> Institute of Crop Science, Chinese Academy of Agricultural Sciences

Shanxi Agricultural University

<120> Application of SiMYB61 protein and related biomaterials in regulating plant stress resistance

<160> 4

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2271

<212> DNA

<213> Millet (Setaria italica)

<400> 1

tgataggaca taaggacaca cacgcttaca gagaagcaac aaagggaagg gctaagagcc 60

tgaggctccc acaaccacca gctttaaaag aaggggggca attggggagg gaggcctgca 120

aagaaaagga gaagagtgcc cttaagccta ccagtgcctt cctccaaatc ctgatattag 180

tagccactag ttgctgccgt tctcccacaa gctctactct atcagacacc actatatgag 240

cccgtataag cagttcatcc acaataatcc cacccttgaa ccacaaattg ttgagaggtg 300

ttcttagaac cagaggatta cctgtgaaga attctgtgct gctcctcaag gtgtgtgcag 360

attgcagagc tcagcttcag cttctgcatt gctttcaatg gggaagcact cctgctgtta 420

caagcagaag ctgaggaagg ggctctggtc tcctgaggaa gacgagaagc tcatgaacca 480

cataaccaag catgggcatg gctgctggag cactgttcca aagcttgcag gtacaatcaa 540

aatcctgcac actttattct tctcaattat ccattcccac tgtttcctat tcatctccaa 600

ggccacttct tctcccaatc ttctcatacc tgcccaatta atgattgcag ggcttcaaag 660

gtgtggcaag agctgcaggc tgaggtggat aaattacctg aggcctgacc ttaaaagagg 720

tgcattctct caggaggagg aagaccttat cattgaactt catgctgtct tgggcaacag 780

gtaaatttaa tcccaaatca cactgtcaga attttgcacc tattgcttca aagcaagctt 840

tccttaacta tgccttttttc acgtaaatga acaggtggtc tcagattgca acacggttgc 900

ctggaagaac tgataacgag atcaagaatc tctggaattc aagcatcaag aagaagctcc 960

ggcagaaagg catcgacccc aacacccaca agccccttgc tgaggttgat cgcaaaatag 1020

ctccaacaat cagtactgag agaacctccg agtccagcga tattgaccct tcaagtggtg 1080

gtgcacttgg caacttgagc catctcctca gtgagacagc acaatcacca gagctgctgc 1140

cagtgctcgg taagcatcgc aaagaaacta ctagtttggc acatctaagg gtgccatcga 1200

aggagctatt ccttgaccag cttgtttctg gtaatgataa cctccccagc tgccgctcaa 1260

caggcccaat tccaaatttc cctttccagc agttgatgtg ttacagcaat gaatttggcg 1320

gcaagcatgg aggcagcacg aatccactct ggtttaacca gaatgagtca agctgcagca 1380

ccatttccac tgtgatgcca ccagtttcgc catcaactct ctcaacatca acaggactca 1440

ataggtcacc ggacaatcca cactctggag gtactggcat tcagagtaac caattctact 1500

gggacaccac taatcctagc agcagtagca gtaaaggaag cagtggaagc aatagcttgg 1560

gatttgagct gcaaagcaca agctcaattc tggagaatag tatcttccca tggacagatt 1620

tatcgccaga taaaaatagc cacctagagg aagaactcaa gtggcctgac ctgctccatg 1680

gaaccttcac agatacacca gcaaccatgc agaatcttag ccaatcactg tatgaagatg 1740

tggtcaaagc cgagaaccaa ttcaacatgg agggcctctg tgcagcttgg tctcaaaatt 1800

tgcagccaca gcaacatctg caggtagcat cagatttgta tgacaaggat ttgcagagaa 1860

tgtccttgtc tttcgagaat atctaggcaa catgtttcag catgcatgga acagaacaat 1920

agcagaaagc tgagttaatg aagcctagaa aacacaacat ttcttcacag atttatttgc 1980

aggctatgtg gaggtgtttg cgtaggtaca ttcttatgat gatacagaga tcaacaatga 2040

taactgttca tcagttaaag atactgcagt acatgaaaaa aaggagacaa agtatgaatt 2100

tatattacag cataatgtat agcctagaga aaagtatgtc tcgaagttta ctttcctttc 2160

acttaaagtt gctgtcttta tctgtacatg catgcagctc gactttttgt atacgtgagt 2220

catggttgat ctttttctac caaataaatg aggcaaggat gtggaacact g 2271

<210> 2

<211> 2057

<212> DNA

<213> Millet (Setaria italica)

<400> 2

tgataggaca taaggacaca cacgcttaca gagaagcaac aaagggaagg gctaagagcc 60

tgaggctccc acaaccacca gctttaaaag aaggggggca attggggagg gaggcctgca 120

aagaaaagga gaagagtgcc cttaagccta ccagtgcctt cctccaaatc ctgatattag 180

tagccactag ttgctgccgt tctcccacaa gctctactct atcagacacc actatatgag 240

cccgtataag cagttcatcc acaataatcc cacccttgaa ccacaaattg ttgagaggtg 300

ttcttagaac cagaggatta cctgtgaaga attctgtgct gctcctcaag gtgtgtgcag 360

attgcagagc tcagcttcag cttctgcatt gctttcaatg gggaagcact cctgctgtta 420

caagcagaag ctgaggaagg ggctctggtc tcctgaggaa gacgagaagc tcatgaacca 480

cataaccaag catgggcatg gctgctggag cactgttcca aagcttgcag ggcttcaaag 540

gtgtggcaag agctgcaggc tgaggtggat aaattacctg aggcctgacc ttaaaagagg 600

tgcattctct caggaggagg aagaccttat cattgaactt catgctgtct tgggcaacag 660

gtggtctcag attgcaacac ggttgcctgg aagaactgat aacgagatca agaatctctg 720

gaattcaagc atcaagaaga agctccggca gaaaggcatc gaccccaaca cccacaagcc 780

ccttgctgag gttgatcgca aaatagctcc aacaatcagt actgagagaa cctccgagtc 840

cagcgatatt gacccttcaa gtggtggtgc acttggcaac ttgagccatc tcctcagtga 900

gacagcacaa tcaccagagc tgctgccagt gctcggtaag catcgcaaag aaactactag 960

tttggcacat ctaagggtgc catcgaagga gctattcctt gaccagcttg tttctggtaa 1020

tgataacctc cccagctgcc gctcaacagg cccaattcca aatttccctt tccagcagtt 1080

gatgtgttac agcaatgaat ttggcggcaa gcatggaggc agcacgaatc cactctggtt 1140

taaccagaat gagtcaagct gcagcaccat ttccactgtg atgccaccag tttcgccatc 1200

aactctctca acatcaacag gactcaatag gtcaccggac aatccacact ctggaggtac 1260

tggcattcag agtaaccaat tctactggga caccactaat cctagcagca gtagcagtaa 1320

aggaagcagt ggaagcaata gcttgggatt tgagctgcaa agcacaagct caattctgga 1380

gaatagtatc ttcccatgga cagatttatc gccagataaa aatagccacc tagaggaaga 1440

actcaagtgg cctgacctgc tccatggaac cttcacagat acaccagcaa ccatgcagaa 1500

tcttagccaa tcactgtatg aagatgtggt caaagccgag aaccaattca acatggaggg 1560

cctctgtgca gcttggtctc aaaatttgca gccacagcaa catctgcagg tagcatcaga 1620

tttgtatgac aaggatttgc agagaatgtc cttgtctttc gagaatatct aggcaacatg 1680

tttcagcatg catggaacag aacaatagca gaaagctgag ttaatgaagc ctagaaaaca 1740

caacatttct tcacagattt atttgcaggc tatgtggagg tgtttgcgta ggtacattct 1800

tatgatgata cagagatcaa caatgataac tgttcatcag ttaaagatac tgcagtacat 1860

gaaaaaaagg agacaaagta tgaatttata ttacagcata atgtatagcc tagagaaaag 1920

tatgtctcga agtttacttt cctttcactt aaagttgctg tctttatctg tacatgcatg 1980

cagctcgact ttttgtatac gtgagtcatg gttgatcttt ttctaccaaa taaatgaggc 2040

aaggatgtgg aacactg 2057

<210> 3

<211> 1275

<212> DNA

<213> Millet (Setaria italica)

<400> 3

atggggaagc actcctgctg ttacaagcag aagctgagga aggggctctg gtctcctgag 60

gaagacgaga agctcatgaa ccacataacc aagcatgggc atggctgctg gagcactgtt 120

ccaaagcttg cagggcttca aaggtgtggc aagagctgca ggctgaggtg gataaattac 180

ctgaggcctg accttaaaag aggtgcattc tctcaggagg aggaagacct tatcattgaa 240

cttcatgctg tcttgggcaa caggtggtct cagattgcaa cacggttgcc tggaagaact 300

gataacgaga tcaagaatct ctggaattca agcatcaaga agaagctccg gcagaaaggc 360

atcgacccca acacccacaa gccccttgct gaggttgatc gcaaaatagc tccaacaatc 420

agtactgaga gaacctccga gtccagcgat attgaccctt caagtggtgg tgcacttggc 480

aacttgagcc atctcctcag tgagacagca caatcaccag agctgctgcc agtgctcggt 540

aagcatcgca aagaaactac tagtttggca catctaaggg tgccatcgaa ggagctattc 600

cttgaccagc ttgtttctgg taatgataac ctccccagct gccgctcaac aggcccaatt 660

ccaaatttcc ctttccagca gttgatgtgt tacagcaatg aatttggcgg caagcatgga 720

ggcagcacga atccactctg gtttaaccag aatgagtcaa gctgcagcac catttccact 780

gtgatgccac cagtttcgcc atcaactctc tcaacatcaa caggactcaa taggtcaccg 840

gacaatccac actctggagg tactggcatt cagagtaacc aattctactg ggacaccact 900

aatcctagca gcagtagcag taaaggaagc agtggaagca atagcttggg atttgagctg 960

caaagcacaa gctcaattct ggagaatagt atcttcccat ggacagattt atcgccagat 1020

aaaaatagcc acctagagga agaactcaag tggcctgacc tgctccatgg aaccttcaca 1080

gatacaccag caaccatgca gaatcttagc caatcactgt atgaagatgt ggtcaaagcc 1140

gagaaccaat tcaacatgga gggcctctgt gcagcttggt ctcaaaattt gcagccacag 1200

caacatctgc aggtagcatc agatttgtat gacaaggatt tgcagagaat gtccttgtct 1260

ttcgagaata tctag 1275

<210> 4

<211> 424

<212> PRT

<213> Millet (Setaria italica)

<400> 4

Met Gly Lys His Ser Cys Cys Tyr Lys Gln Lys Leu Arg Lys Gly Leu

1 5 10 15

Trp Ser Pro Glu Glu Asp Glu Lys Leu Met Asn His Ile Thr Lys His

20 25 30

Gly His Gly Cys Trp Ser Thr Val Pro Lys Leu Ala Gly Leu Gln Arg

35 40 45

Cys Gly Lys Ser Cys Arg Leu Arg Trp Ile Asn Tyr Leu Arg Pro Asp

50 55 60

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

65 70 75 80

Leu His Ala Val Leu Gly Asn Arg Trp Ser Gln Ile Ala Thr Arg Leu

85 90 95

Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Leu Trp Asn Ser Ser Ile

100 105 110

Lys Lys Lys Leu Arg Gln Lys Gly Ile Asp Pro Asn Thr His Lys Pro

115 120 125

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

130 135 140

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

145 150 155 160

Asn Leu Ser His Leu Leu Ser Glu Thr Ala Gln Ser Pro Glu Leu Leu

165 170 175

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

180 185 190

Arg Val Pro Ser Lys Glu Leu Phe Leu Asp Gln Leu Val Ser Gly Asn

195 200 205

Asp Asn Leu Pro Ser Cys Arg Ser Thr Gly Pro Ile Pro Asn Phe Pro

210 215 220

Phe Gln Gln Leu Met Cys Tyr Ser Asn Glu Phe Gly Gly Lys His Gly

225 230 235 240

Gly Ser Thr Asn Pro Leu Trp Phe Asn Gln Asn Glu Ser Ser Cys Ser

245 250 255

Thr Ile Ser Thr Val Met Pro Pro Val Ser Pro Ser Thr Leu Ser Thr

260 265 270

Ser Thr Gly Leu Asn Arg Ser Pro Asp Asn Pro His Ser Gly Gly Thr

275 280 285

Gly Ile Gln Ser Asn Gln Phe Tyr Trp Asp Thr Thr Asn Pro Ser Ser

290 295 300

Ser Ser Ser Lys Gly Ser Ser Gly Ser Asn Ser Leu Gly Phe Glu Leu

305 310 315 320

Gln Ser Thr Ser Ser Ile Leu Glu Asn Ser Ile Phe Pro Trp Thr Asp

325 330 335

Leu Ser Pro Asp Lys Asn Ser His Leu Glu Glu Glu Glu Leu Lys Trp Pro

340 345 350

Asp Leu Leu His Gly Thr Phe Thr Asp Thr Pro Ala Thr Met Gln Asn

355 360 365

Leu Ser Gln Ser Leu Tyr Glu Asp Val Val Lys Ala Glu Asn Gln Phe

370 375 380

Asn Met Glu Gly Leu Cys Ala Ala Trp Ser Gln Asn Leu Gln Pro Gln

385 390 395 400

Gln His Leu Gln Val Ala Ser Asp Leu Tyr Asp Lys Asp Leu Gln Arg

405 410 415

Met Ser Leu Ser Phe Glu Asn Ile

420

Claims (6)

1. Use of a SiMYB61 protein or related biomaterial thereof in (a1) and/or (a2) as follows:

   (a1) improving plant yield-related traits;

   (a2) the stress resistance of the plants is improved;

   the amino acid sequence of the SiMYB61 protein is shown in SEQ ID No. 4;

   the related biological material is a nucleic acid molecule capable of expressing the SiMYB61 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

   the plant yield-related traits are ear number, ear length and/or grain per ear;

   the plant stress resistance is the resistance of the plant to low nitrogen stress;

   the plant is rice or millet.
2. The application of SiMYB61 protein or related biological materials thereof in breeding plants with high yield and/or high stress resistance;

   the amino acid sequence of the SiMYB61 protein is shown as SEQ ID No. 4;

   the related biological material is a nucleic acid molecule capable of expressing the SiMYB61 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

   the high yield is represented by a large number of spikes and/or a high spike length and/or a large number of grains per spike;

   the stress resistance is resistance to low nitrogen stress;

   the plant is rice or millet.
3. 3. A method for increasing yield-related traits in plants and/or increasing stress tolerance in plants, comprising the step of increasing expression level and/or activity of a SiMYB61 protein in a recipient plant;

   the amino acid sequence of the SiMYB61 protein is shown in SEQ ID No. 4;

   the yield-related traits are ear number, ear length and/or grain number per ear;

   the stress tolerance is resistance to low nitrogen stress;

   the plant is rice or millet.
4. 4. A method of growing a transgenic plant comprising the steps of: introducing a nucleic acid molecule capable of expressing a SiMYB61 protein into a recipient plant to obtain a transgenic plant with increased SiMYB61 protein expression; the transgenic plant has increased yield-related traits and/or stress resistance as compared to the recipient plant;

   the amino acid sequence of the SiMYB61 protein is shown as SEQ ID No. 4;

   the yield is the ear number, the ear length and/or the grain number per ear;

   the stress resistance is resistance to low nitrogen stress;

   the plant is rice or millet.
5. 5. The method of claim 4, wherein: the step of introducing into a recipient plant a nucleic acid molecule capable of expressing a SiMYB61 protein comprises introducing into the recipient plant an expression cassette comprising a gene encoding the SiMYB61 protein.
6. 6. The method of claim 5, wherein:

   the encoding gene of the SiMYB61 protein is a DNA molecule described in any one of the following items:

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

   (B2) a DNA molecule shown as SEQ ID No. 3.

Images