CN111534539A

CN111534539A - SiMYB4 protein related to plant stress resistance and related biological material and application thereof

Info

Publication number: CN111534539A
Application number: CN202010407074.2A
Authority: CN
Inventors: 陈明; 孙黛珍; 马有志; 黎毛毛; 张玥玮; 唐文思; 周永斌; 徐兆师; 陈隽
Original assignee: Shanxi Agricultural University; Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Shanxi Agricultural University; Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2020-05-14
Filing date: 2020-05-14
Publication date: 2020-08-14
Anticipated expiration: 2040-05-14
Also published as: CN111534539B

Abstract

The invention discloses SiMYB4 protein related to plant stress resistance and related biological material and application thereof. The invention provides an application of SiMYB4 protein and related biological materials thereof in improving stress tolerance of plants. 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

SiMYB4 protein related to plant stress resistance and related biological material and application thereof

Technical Field

The invention relates to SiMYB4 protein related to plant stress resistance and a related biological material and application thereof.

Background

China is one of countries with the lowest grain input and output of unit fertilizers in the world, the utilization rate of nitrogen fertilizers is only 30-35% (45% in developed countries), and the utilization rate of phosphate fertilizers is only 10-20%. According to the current consumption, for example, 10 percent of nitrogen fertilizer and 20 percent of phosphate fertilizer are saved, 241 million yuan of capital can be saved every year. The low-efficiency utilization of nitrogen and phosphorus fertilizers enables agricultural non-point source pollution to become the most important factor for water system eutrophication, soil acidification and heavy metal pollution, and threatens ecological safety and sustainable development. Therefore, our country urgently needs to cultivate new crop varieties with high nutrient utilization efficiency, thereby greatly improving the utilization efficiency of nitrogen and phosphorus fertilizers in our country.

The cultivation of new stress-resistant rice varieties improves the stress resistance of the rice varieties, and is an important measure for improving the yield level of the rice under the conditions of reduced application amount of chemical fertilizers and drought and water shortage.

Disclosure of Invention

The invention aims to provide SiMYB4 protein related to plant stress resistance, a related biological material and application thereof.

In a first aspect, the present invention first protects the use of a SiMYB4 protein or related biomaterial as follows (a1) and/or (a 2):

(a1) regulating and controlling plant yield-related traits;

(a2) regulating and controlling the stress resistance of the plants;

the related biological material is a nucleic acid molecule capable of expressing the SiMYB4 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the SiMYB4 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 4;

(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.4 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 identity 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 protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

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

In the above protein, the tag may be a Flag tag, a His tag, an MBP tag, an HA tag, a myc tag, a GST tag, and/or a SUMO tag, etc.

The nucleic acid molecule may specifically be a gene encoding a SiMYB4 protein. The encoding gene of the SiMYB4 protein is a DNA molecule described in any one of the following items:

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

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

(B3) a DNA molecule shown as SEQ ID No. 3;

(B4) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) or (B2) or (B3) and encodes the SiMYB4 protein;

(B3) a DNA molecule having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the DNA sequence defined in (B1) or (B2) or (B3) and encoding the SiMYB4 protein.

The stringent conditions may be as follows: at 50 ℃ in 7% dodecylSodium sulfate (SDS), 0.5M NaPO₄Hybridizing with 1mM EDTA, rinsing in 2 × SSC, 0.1% SDS at 50 deg.C, 7% SDS, 0.5M NaPO at 50 deg.C₄Hybridizing with 1mM EDTA, rinsing in 1 × SSC, 0.1% SDS at 50 deg.C, 7% SDS, 0.5M NaPO at 50 deg.C₄Hybridizing with 1mM EDTA, rinsing in 0.5 × SSC, 0.1% SDS at 50 deg.C, 7% SDS, 0.5M NaPO at 50 deg.C₄Hybridizing with 1mM EDTA, rinsing in 0.1 × SSC, 0.1% SDS at 50 deg.C, 7% SDS, 0.5M NaPO at 50 deg.C₄Hybridization with a mixed solution of 1mM EDTA, rinsing in 0.1 × SSC, 0.1% SDS at 65 ℃ or 6 × SSC, 0.5% SDS at 65 ℃ followed by washing once each with 2 × SSC, 0.1% SDS and 1 × SSC, 0.1% SDS.

The expression cassette can be specifically an expression cassette consisting of a ubiquitin constitutive promoter, the coding gene of the SiMYB4 protein and a terminator nos 3'. The expression cassette can be obtained by double enzyme digestion of a recombinant vector by Hind III and EcoRI. The recombinant vector can be specifically a recombinant vector obtained by cloning SEQ ID NO.3 into BamHI and SpeI sites of the vector LP 0471118-Bar-ubi-EDLL.

The recombinant strain can be obtained by introducing the expression cassette or the recombinant vector into an agrobacterium strain. The agrobacterium strain may specifically be agrobacterium strain EHA 105.

In said use, said plant yield-related traits comprise ear number, ear length, grain per ear and/or biomass; the biomass comprises the weight of rice straw and/or the weight of rice.

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

The modulation is a forward modulation.

In a second aspect, the invention features the use of a SiMYB4 protein or a related biomaterial thereof in plant breeding;

the SiMYB4 protein is as shown above.

In the application, the breeding aims to breed plants with high yield and/or high stress resistance. The high yield can be embodied in particular as a high number of ears and/or a high ear length and/or a high number of grains per ear and/or a high biomass. The biomass comprises the weight of rice straw and/or the weight of rice. The stress tolerance is in particular resistance to low nitrogen stress. The high resistance to low nitrogen stress is reflected by high yield and/or plant height under low nitrogen stress. The high resistance to low nitrogen stress is reflected by a high nitrogen content.

In a third aspect, the present invention provides a method for increasing plant yield and/or increasing plant stress tolerance, comprising the step of increasing the expression level and/or activity of a SiMYB4 protein in a recipient plant.

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

The biomass comprises the weight of rice straw and/or the weight of rice.

The stress tolerance is in particular resistance to low nitrogen stress.

The improved stress tolerance can be embodied as an increased yield and/or an increased plant height under low nitrogen stress.

The high resistance to low nitrogen stress is reflected by a high nitrogen content.

The SiMYB4 protein is as shown above.

In a fourth aspect, the present invention provides a method of growing a transgenic plant, comprising the steps of: introducing a nucleic acid molecule capable of expressing a SiMYB4 protein into a recipient plant to obtain a transgenic plant with increased SiMYB4 protein expression; the transgenic plants have increased yield and/or stress resistance as compared to the recipient plant.

The biomass comprises the weight of rice straw and/or the weight of rice.

The stress resistance is in particular resistance to low nitrogen stress.

The "introducing into a recipient plant a nucleic acid molecule capable of expressing a SiMYB4 protein" is effected by introducing into the recipient plant an expression cassette comprising a gene encoding the SiMYB4 protein.

The gene encoding the SiMYB4 protein is as indicated above.

The number of grains per spike is the number of solid grains per spike and/or the total grains per spike.

Any one of the plants is a dicotyledonous plant or a monocotyledonous plant;

further, the monocotyledon is a gramineous plant;

further, the gramineous plant is rice or millet.

The rice can be rice variety Kitaake.

The millet can be Yu Gu I.

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.

Drawings

FIG. 1 shows SSR detection results of stress-resistant rice transformed with SiMYB4 gene. Marker: DL1000 Marker; negative control: kitaake; positive control: the plant expression vector psSiMYB 4.

Fig. 2 shows the statistical result of 18-year field test data.

FIG. 3 is a comparison of transgenic plants with wild type phenotype.

FIG. 4 shows the statistical results of the test data under the condition of normal field treatment for 19 years.

FIG. 5 is a statistical result of detection data under 19-year field low nitrogen stress conditions.

FIG. 6 shows the statistical results of 19 years field straw and rice weight measurement data.

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.

LP0471118-Bar-ubi-EDLL vector: are described in the literature: ning bud, Wang Shuang, ju Peng Gao, Bai Xin Xuan, Ge Lin Hao, Qixin, Jiangqin, Sun Xijun, Chenming, Sun Dazhen, over-expression millet SiANT1 has influence on rice salt tolerance [ J ]. Chinese agricultural science, 2018,51(10): 1830) 1841.

Yugu I: are described in the literature: ning bud, Wang Shuang, ju Peng lifting, Bai Xin Xuan, Ge Lin Hao, Qixin, Jiangqin, Sun Shi Jun, Chenming, Sun Dazhen, overexpression of millet SiANT1 on rice salt tolerance [ J ]. Chinese agricultural science, 2018,51(10):1830 one 1841.; the public is available from the institute of crop science, academy of agricultural sciences, china.

bar gene expression vector pSBAR: are described in the literature: obtaining drought resistant transgenic wheat [ D ] using an improved minimum expression box technique, university of inner mongolia agriculture, 2012; the public is available from the institute of crop science, academy of agricultural sciences, china.

Agrobacterium strain EHA 105: beijing Ongke New Biotechnology Co.

Rice variety Kitaake: are described in the literature: ning bud, Wang Shuang, ju Peng lifting, Bai Xin Xuan, Ge Lin Hao, Qixin, Jiangqin, Sun Shi Jun, Chenming, Sun Dazhen, overexpression of millet SiANT1 on rice salt tolerance [ J ]. Chinese agricultural science, 2018,51(10):1830 one 1841.; the public is available from the institute of crop science, academy of agricultural sciences, china.

Example 1 acquisition of SimYB4 protein and Gene encoding the same

A stress resistance related gene is cloned from a first stress resistance millet and named as SiMYB4 gene, the genome sequence of the gene is shown as SEQ ID NO.1, the transcription sequence is shown as SEQ ID NO.2, and the CDS is shown as SEQ ID NO. 3. The protein (SiMYB4 protein) coded by the SiMYB4 gene is shown as SEQ ID NO. 4.

Example 2, SiMYB4 protein and application of encoding gene thereof in rice breeding

Preparation of linear minimal expression cassette

1. Obtaining of plant expression vector psSiMYB4

Extracting total RNA of Yugu No. one, and reverse transcribing into cDNA. The cDNA was used as a template, PCR amplification was carried out using primers F and R, and the CDS sequence of SiMYB4 (SEQ ID NO: 3) was cloned into the BamHI and SpeI sites of vector LP0471118-Bar-ubi-EDLL using the seamless cloning kit (cat # 639649) from Clotech according to the procedures described in the specification to obtain plant expression vector psSiMYB4 (which was sequence-verified).

And (3) primer F: 5'-AGACCGATCTGGATCATGGGGAAGGGCCGG-3', respectively;

and (3) primer R: 5'-CGATCGATCCACTAGTCAATACCCCATCTGGTTACTACTTGTAACAACA-3' are provided.

2. Preparation of Linear minimal expression cassette

Adopting HindIII and EcoRI to carry out double enzyme digestion on the plant expression vector psSiMYB4 obtained in the step 1, recovering a fragment of about 3200bp to obtain a SiMYB4 gene linear minimum expression frame, wherein the linear minimum expression frame consists of a ubiquitin constitutive promoter (1800bp), a SiMYB4 gene (1119bp) and a terminator nos 3' (300 bp). The minimal expression frame transformation method can remove the vector skeleton sequence, has no ampicillin resistance gene, has no obvious difference between the transgenic rice and the receptor, has no pathogenicity, and reduces the safety risk of inserting the plant genome exogenous fragment.

Preparing a marker gene expression frame by using a bar gene as a marker gene: performing enzyme digestion on the bar gene expression vector pSBAR by using Hind III, and recovering an enzyme digestion product to obtain a marker gene expression frame; the marker gene expression frame consists of a maize ubiquitin promoter, a marker gene bar and a nos terminator.

Second, transformation of Rice

1. And (3) transforming the SiMYB4 gene linear minimum expression frame and the marker gene expression frame prepared in the step one into an agrobacterium tumefaciens strain EHA105 to obtain the recombinant agrobacterium tumefaciens.

2. Soaking immature embryos of a rice variety Kitaake 12-14 days after pollination in 70% ethanol for 1 minute, then disinfecting the immature embryos with 10% sodium hypochlorite for 15 minutes, washing the immature embryos with sterile water for 3-5 times, taking out the immature embryos on an ultra-clean bench, and inoculating the immature embryos to SD (secure digital)₂The young embryo callus is induced on a culture medium (MS basic culture medium (containing no vitamin) +2 mg/L2, 4-D +1mg/LVB1+150mg/LAsn asparagine +30g/L sucrose +2.4g/L plant gel, pH is 5.8) for 7 days, and then the induced callus is transferred to a hypertonic culture medium (MS basic culture medium +5 mg/L2, 4-D +0.4mol/L mannitol +3g/L plant gel) to be subjected to hypertonic treatment for 4-6 hours.

3. After step 2, the callus was infected with Agrobacterium and soaked in OD₆₀₀And oscillating and infecting the recombinant agrobacterium tumefaciens liquid of 0.8 for about 30min at 180 r/min.

4. After the step 3 is finished, continuously culturing the callus on a hypertonic culture medium for 16-18 hours, and then transferring the infected callus to SD₂Dark culture was performed on the medium for two weeks.

5. After the step 4 is completed, transferring the callus onto a selection culture medium containing 2-3mg/L of herbicide Bialaphos (MS basic culture medium (containing no vitamin) +2 mg/L2, 4-D +1mg/L VB1+150mg/L Asn asparagine +30g/L sucrose +2.4g/L plant gel +2-3mg/L herbicide Bialaphos, pH is 5.8) to perform callus screening, differentiation and seedling strengthening, so as to obtain a T0 generation transgenic rice plant.

Identification of transgenic positive rice

Extracting DNA of T0 rice leaves to be detected by adopting an SDS method, designing a primer for amplifying a partial sequence according to a SiMYB4 gene sequence by taking the DNA as a template to carry out PCR amplification, carrying out PCR amplification by adopting a primer F and a primer R, detecting a PCR amplification product by 0.8% agarose gel, and carrying out ultraviolet photographing. Replacing T0 generation rice with Kitaake rice variety to carry out the operation as negative control; the plant expression vector psSiMYB4 was used as a positive control.

F：5’-AAGTACGGCCATGCCAACTG-3’；

R：5’-CTCCTCCGCGCCCGGCGACA-3’。

The PCR reaction system is shown in Table 1. And (3) PCR reaction conditions: denaturation at 94 deg.C for 5 min; 50sec at 94 ℃, 50sec at 62 ℃, 1min at 72 ℃ and 35 cycles; extension at 72 ℃ for 10 min.

TABLE 1

Composition (I)	Dosage (ul)
		(Takara)10x PCR buffer(Mg²⁺ Plus)	2.50
(Takara)25mM Mg²⁺	0.05
		(Takara)2.5mM dNTP Mixture	2.00
Primer F	0.80
		Primer R	0.80
(Takara)r-Taq DNA Ploymerase(5U/ul)	0.25
		Form panel	1.00
ddH₂O	17.6
		Total of	25

The results are shown in FIG. 1. 5 positive plants are obtained through PCR detection, and the positive rate is 2%. Through greenhouse generation adding, screening and identification, SiMYB 4-transgenic rice homozygous strains are selected and selected, and are sequentially named as OE02 and OE26 for the following experiments.

Fourth, phenotype detection of transgenic rice

1. 2018 field phenotype detection

And (3) the plant to be detected: rice variety kitaake (wt), transgenic line OE02, transgenic line OE 26.

Test site: transgenic test base of rice institute of agricultural science institute of high-safety Jiangxi province in Jiangxi province.

The seedling bed management is the same as the field production (no nitrogen fertilizer is added), and each test material can grow normally.

8 plants are harvested from each material, and the biological yield and the rice yield of each material are determined in three times.

The N content of the paddy and the N content of the straw of the reference material are respectively measured by a Kjeldahl method. The detection method is specifically described in the references "comparison of total nitrogen in plants by Yangyi, Chua-Shunlin. flow analysis and Kjeldahl method [ J ]. proceedings of the Seiki institute of America, 2016,32(08): 51-54" and "Dahonglin, Wu Xiaojun. determination of nitrogen content in dried samples of plants by Kjeldahl method [ J ]. proceedings of the Jiangsu institute of agriculture, 1995(03): 70").

3 plants of each material are respectively taken for indoor seed test, and the number of the single plant effective spikes (more than 5 single spikes are effective spikes), the solid grains per spike, the total grains per spike, the seed setting rate and the like are inspected.

The results are shown in FIG. 2. In FIG. 2, the nitrogen contents of rice and straw are expressed in g/8, and the nitrogen contents are expressed by the weight of 8 rice/straw. The unit of the total nitrogen content of the rice and the total nitrogen content of the rice is percent (%), which means the nitrogen content of the rice/the weight of the 8 rice straws/the rice.

The results show that the dry grain weight and the dry straw weight of the transgenic rice line are higher than those of the control rice variety Kitaake under the treatment without applying nitrogen fertilizer. The effective spike number, the solid grain number and the total grain number of each spike of the single plant are all higher than those of the control. The N content of the rice and the N content of the straw are higher than those of the control.

2. 2019 field data

And (3) the plant to be detected: rice variety kitaake (wt), transgenic line OE 02.

Seedling bed management is the same as field production. Each test material was able to grow normally.

The test sets two treatments of applying 12 kilograms of pure nitrogen and not applying nitrogen fertilizer per mu, and each treatment is set for two times.

Planting 6 rows of each material, 8 pockets of each row, 5 multiplied by 6 inches of row spacing, and planting a single material.

The test area is 4.0 mu. Other fertilizer and water management in the field is the same as local production.

20 plants of each material are harvested for each treatment, and the weight of the straw and the weight of the paddy are respectively measured for each material after three times of repetition.

Each material was harvested 8 plants per treatment, and the biological yield and rice yield of each material were determined in triplicate.

And 3 plants of each material are respectively taken for indoor seed test in each treatment, and the number of the effective ears of each plant, the solid grains of each ear, the total grains of each ear and the like are inspected.

The phenotypic observations are shown in figure 3.

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

The results show that under the condition of normal nitrogen fertilizer application, the biomass (straw weight and rice weight), the ear length and the grain number per ear of the transgenic plants are obviously higher than those of the receptor plants. Under the low nitrogen stress, the biomass (the weight of the rice straw and the weight of the paddy), the plant height, the spike number, the spike length and the grain number per spike of the transgenic plant are also higher than those of a receptor plant.

Sequence listing

<110> institute of crop science of Chinese academy of agricultural sciences

SHANXI AGRICULTURAL University

<120> SiMYB4 protein related to plant stress resistance, related biological material and application thereof

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>1832

<212>DNA

<213> millet (Setaria italica)

<400>1

cacaggcccc ggcacacgca cacacaagtg atcgaggcgc cgtcgtcgca gcaatagcat 60

cgaaccagag atcgagccgt cgaggcagcg atcgattcga tccgccgatc acgcggaggc 120

agcaggagct tggaggaacg atggggaagg gccgggcacc gtgctgcgcc aaggttgggc 180

tgaacaaggg ctcctggacg ccggaggagg acatgcgcct catcgcctac attcagaagt 240

acggccatgc caactggcgc gccctgccca agcaagcagg ttggtaccaa gcatagcaaa 300

tcctccctca tctgcagttg tccgcacatc catgcattca tggggggttc taatggtggt 360

tttgaattgc acggcacagg tctgctgcgg tgcgggaaga gctgccggct gcggtggatc 420

aactacctcc ggccggacct caagcgcggc aacttcaccg ccgaggagga ggagaccatc 480

atcaagctgc acggcctgct cggcaacaag tacggtgcag ctcgtcgttg cctggagaat 540

tcgcggttgc tgttctcgaa tccatggatg aagccgagac tgatagagct cgatcggatt 600

ttgtgtccag gtggtccaag atcgcggcgt gcctgccggg ccggacggac aacgagatca 660

agaacgtctg gaacacgcac ctgaagaagc gggtgtcgcc gggcgcggag gagcgcggcg 720

gcgccgccgg ttccaagaag aagaagaaag ccgccggcgc tggggtgccg gcggcggcgg 780

ccccgtcgcc gtcgccgtcc tcttccacga cgacggcgac gaccaactgc tccagcggcg 840

actccgggga gcagcagagc aaggcgagca aggacgagcc cggcgacgag ctgggcctgg 900

agaagctcga gataatcccc atgctcgacg accccgcctg cttcggcttc gacatgctgg 960

tggaccagat tccggatccg tactgcccgg acgtatccgt gcccacgtcg ccctgcgcct 1020

cgtccacgtc cccgccggcg cccgcccggc ccagcgtgga cgagctgctc gacctgccgg 1080

agatcgacat cgaccacgag ctgtggagca tcatcgacgg cgtcggcggc gacggcgctg 1140

gagctggcgc cgtcgcggcc gggactgcac cggcgccgtg ccagagcaat gcaacggagc 1200

cgaacgccgc cgccagcacg accagccacg ggacggagtg gtggttggag gacctggaga 1260

aggagctggg cctgtggggg cccatggagg actaccagta cccgatgggc ccacagtgtc 1320

cagtcgccca cccggaccca ctccctgcca tggtggacga ccctgtgtcg tgctacttcc 1380

aagcgggccc cgccccggcc atgctccacg aacccggtta ctcttctgtt gttacaagta 1440

gtaaccagat ggggtattga gatgttatcc tcgtcctttt ctgattttag atagaaaaaa 1500

tcctcagcct gatcgtacgg ccacgatcac taccagcatt atacttgtat aattgtacaa 1560

gtgcatggct caagacgagc acaaattcaa cacgaatacg tcgatggaat catcatcggc 1620

gtaatagtaa aaagggagat cagttaggct agtgtgcaca tacaaaaaga gccaaaggga 1680

tactgttttt gggcaggcag gcagtgttcg gcaatgcagt catagattca caaaaggagt 1740

caagattgcc ggtactcact gagttaacct gaaatgtaac ttaccttcat ttgccagttg 1800

gtatatacac actataaaac aatgactctt aa 1832

<210>2

<211>1631

<212>DNA

<213> millet (Setaria italica)

<400>2

cacaggcccc ggcacacgca cacacaagtg atcgaggcgc cgtcgtcgca gcaatagcat 60

cgaaccagag atcgagccgt cgaggcagcg atcgattcga tccgccgatc acgcggaggc 120

agcaggagct tggaggaacg atggggaagg gccgggcacc gtgctgcgcc aaggttgggc 180

tgaacaaggg ctcctggacg ccggaggagg acatgcgcct catcgcctac attcagaagt 240

acggccatgc caactggcgc gccctgccca agcaagcagg tctgctgcgg tgcgggaaga 300

gctgccggct gcggtggatc aactacctcc ggccggacct caagcgcggc aacttcaccg 360

ccgaggagga ggagaccatc atcaagctgc acggcctgct cggcaacaag tggtccaaga 420

tcgcggcgtg cctgccgggc cggacggaca acgagatcaa gaacgtctgg aacacgcacc 480

tgaagaagcg ggtgtcgccg ggcgcggagg agcgcggcgg cgccgccggt tccaagaaga 540

agaagaaagc cgccggcgct ggggtgccgg cggcggcggc cccgtcgccg tcgccgtcct 600

cttccacgac gacggcgacg accaactgct ccagcggcga ctccggggag cagcagagca 660

aggcgagcaa ggacgagccc ggcgacgagc tgggcctgga gaagctcgag ataatcccca 720

tgctcgacga ccccgcctgc ttcggcttcg acatgctggt ggaccagatt ccggatccgt 780

actgcccgga cgtatccgtg cccacgtcgc cctgcgcctc gtccacgtcc ccgccggcgc 840

ccgcccggcc cagcgtggac gagctgctcg acctgccgga gatcgacatc gaccacgagc 900

tgtggagcat catcgacggc gtcggcggcg acggcgctgg agctggcgcc gtcgcggccg 960

ggactgcacc ggcgccgtgc cagagcaatg caacggagcc gaacgccgcc gccagcacga 1020

ccagccacgg gacggagtgg tggttggagg acctggagaa ggagctgggc ctgtgggggc 1080

ccatggagga ctaccagtac ccgatgggcc cacagtgtcc agtcgcccac ccggacccac 1140

tccctgccat ggtggacgac cctgtgtcgt gctacttcca agcgggcccc gccccggcca 1200

tgctccacga acccggttac tcttctgttg ttacaagtag taaccagatg gggtattgag 1260

atgttatcct cgtccttttc tgattttaga tagaaaaaat cctcagcctg atcgtacggc 1320

cacgatcact accagcatta tacttgtata attgtacaag tgcatggctc aagacgagca 1380

caaattcaac acgaatacgt cgatggaatc atcatcggcg taatagtaaa aagggagatc 1440

agttaggcta gtgtgcacat acaaaaagag ccaaagggat actgtttttg ggcaggcagg 1500

cagtgttcgg caatgcagtc atagattcac aaaaggagtc aagattgccg gtactcactg 1560

agttaacctg aaatgtaact taccttcatt tgccagttgg tatatacaca ctataaaaca 1620

atgactctta a 1631

<210>3

<211>1119

<212>DNA

<213> millet (Setaria italica)

<400>3

atggggaagg gccgggcacc gtgctgcgcc aaggttgggc tgaacaaggg ctcctggacg 60

ccggaggagg acatgcgcct catcgcctac attcagaagt acggccatgc caactggcgc 120

gccctgccca agcaagcagg tctgctgcgg tgcgggaaga gctgccggct gcggtggatc 180

aactacctcc ggccggacct caagcgcggc aacttcaccg ccgaggagga ggagaccatc 240

atcaagctgc acggcctgct cggcaacaag tggtccaaga tcgcggcgtg cctgccgggc 300

cggacggaca acgagatcaa gaacgtctgg aacacgcacc tgaagaagcg ggtgtcgccg 360

ggcgcggagg agcgcggcgg cgccgccggt tccaagaaga agaagaaagc cgccggcgct 420

ggggtgccgg cggcggcggc cccgtcgccg tcgccgtcct cttccacgac gacggcgacg 480

accaactgct ccagcggcga ctccggggag cagcagagca aggcgagcaa ggacgagccc 540

ggcgacgagc tgggcctgga gaagctcgag ataatcccca tgctcgacga ccccgcctgc 600

ttcggcttcg acatgctggt ggaccagatt ccggatccgt actgcccgga cgtatccgtg 660

cccacgtcgc cctgcgcctc gtccacgtcc ccgccggcgc ccgcccggcc cagcgtggac 720

gagctgctcg acctgccgga gatcgacatc gaccacgagc tgtggagcat catcgacggc 780

gtcggcggcg acggcgctgg agctggcgcc gtcgcggccg ggactgcacc ggcgccgtgc 840

cagagcaatg caacggagcc gaacgccgcc gccagcacga ccagccacgg gacggagtgg 900

tggttggagg acctggagaa ggagctgggc ctgtgggggc ccatggagga ctaccagtac 960

ccgatgggcc cacagtgtcc agtcgcccac ccggacccac tccctgccat ggtggacgac 1020

cctgtgtcgt gctacttcca agcgggcccc gccccggcca tgctccacga acccggttac 1080

tcttctgttg ttacaagtag taaccagatg gggtattga 1119

<210>4

<211>372

<212>PRT

<213> millet (Setaria italica)

<400>4

Met Gly Lys Gly Arg Ala Pro Cys Cys Ala Lys Val Gly Leu Asn Lys

1 5 10 15

Gly Ser Trp Thr Pro Glu Glu Asp Met Arg Leu Ile Ala Tyr Ile Gln

20 25 30

Lys Tyr Gly His Ala Asn Trp Arg Ala Leu Pro Lys Gln Ala Gly Leu

35 40 45

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

50 55 60

Pro Asp Leu Lys Arg Gly Asn Phe Thr Ala Glu Glu Glu Glu Thr Ile

65 70 75 80

Ile Lys Leu His Gly Leu Leu Gly Asn Lys Trp Ser Lys Ile Ala Ala

85 90 95

Cys Leu Pro Gly Arg Thr Asp Asn Glu Ile Lys Asn Val Trp Asn Thr

100 105 110

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

115 120125

Ala Gly Ser Lys Lys Lys Lys Lys Ala Ala Gly Ala Gly Val Pro Ala

130 135 140

Ala Ala Ala Pro Ser Pro Ser Pro Ser Ser Ser Thr Thr Thr Ala Thr

145 150 155 160

Thr Asn Cys Ser Ser Gly Asp Ser Gly Glu Gln Gln Ser Lys Ala Ser

165 170 175

Lys Asp Glu Pro Gly Asp Glu Leu Gly Leu Glu Lys Leu Glu Ile Ile

180 185 190

Pro Met Leu Asp Asp Pro Ala Cys Phe Gly Phe Asp Met Leu Val Asp

195 200 205

Gln Ile Pro Asp Pro Tyr Cys Pro Asp Val Ser Val Pro Thr Ser Pro

210 215 220

Cys Ala Ser Ser Thr Ser Pro Pro Ala Pro Ala Arg Pro Ser Val Asp

225 230 235 240

Glu Leu Leu Asp Leu Pro Glu Ile Asp Ile Asp His Glu Leu Trp Ser

245 250 255

Ile Ile Asp Gly Val Gly Gly Asp Gly Ala Gly Ala Gly Ala Val Ala

260 265 270

Ala Gly Thr Ala Pro Ala Pro Cys Gln Ser Asn Ala Thr Glu Pro Asn

275 280 285

Ala Ala Ala Ser Thr Thr Ser His Gly Thr Glu Trp Trp Leu Glu Asp

290 295 300

Leu Glu Lys Glu Leu Gly Leu Trp Gly Pro Met Glu Asp Tyr Gln Tyr

305 310 315 320

Pro Met Gly Pro Gln Cys Pro Val Ala His Pro Asp Pro Leu Pro Ala

325 330 335

Met Val Asp Asp Pro Val Ser Cys Tyr Phe Gln Ala Gly Pro Ala Pro

340 345 350

Ala Met Leu His Glu Pro Gly Tyr Ser Ser Val Val Thr Ser Ser Asn

355 360 365

Gln Met Gly Tyr

370

Claims

Use of a SiMYB4 protein or related biomaterial thereof in (a1) and/or (a2) as follows:

(a1) regulating and controlling plant yield-related traits;

(a2) regulating and controlling the stress resistance of the plants;

the related biological material is a nucleic acid molecule capable of expressing the SiMYB4 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the SiMYB4 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 4;

(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.4 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 identity 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 protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
2. The use of claim 1, wherein:

said plant yield-related traits comprise ear number, ear length, grain per ear number and/or biomass;

the plant stress resistance is the resistance of a plant to low nitrogen stress.
Use of a SiMYB4 protein or a related biomaterial thereof in plant breeding;

the related biological material is a nucleic acid molecule capable of expressing the SiMYB4 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the SiMYB4 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 4;

(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.4 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 identity 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 protein tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).
4. Use according to claim 3, characterized in that: the breeding aims to breed plants with high yield and/or high stress resistance.
5. A method for increasing plant yield and/or increasing plant stress tolerance, comprising the step of increasing the expression level and/or activity of a SiMYB4 protein in a recipient plant.
6. A method of breeding a transgenic plant comprising the steps of: introducing a nucleic acid molecule capable of expressing a SiMYB4 protein into a recipient plant to obtain a transgenic plant with increased SiMYB4 protein expression; the transgenic plants have increased yield and/or stress resistance as compared to the recipient plant.
7. The method of claim 6, wherein: the "introducing into a recipient plant a nucleic acid molecule capable of expressing a SiMYB4 protein" is effected by introducing into the recipient plant an expression cassette comprising a gene encoding the SiMYB4 protein.
8. The method of claim 7, wherein:

the encoding gene of the SiMYB4 protein is a DNA molecule described in any one of the following items:

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

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

(B3) a DNA molecule shown as SEQ ID No. 3;

(B4) a DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) or (B2) or (B3) and encodes the SiMYB4 protein;

(B3) a DNA molecule having more than 99%, more than 95%, more than 90%, more than 85% or more than 80% identity with the DNA sequence defined in (B1) or (B2) or (B3) and encoding the SiMYB4 protein.
9. The method of any of claims 5-8, wherein: the stress resistance is the resistance of a plant to low nitrogen stress.
10. Use or method according to any of claims 1-9, wherein: the plant is a dicotyledonous plant or a monocotyledonous plant;

further, the monocotyledon is a gramineous plant;

further, the gramineous plant is rice or millet.