CN111187342B

CN111187342B - ZmG2 application in improving plant strong light stress resistance and yield

Info

Publication number: CN111187342B
Application number: CN201910756917.7A
Authority: CN
Inventors: 周文彬; 李霞; 王鹏; 李静; 魏少博
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2019-08-16
Filing date: 2019-08-16
Publication date: 2022-02-11
Anticipated expiration: 2039-08-16
Also published as: CN111187342A

Abstract

The invention discloses an application of ZmG2 in improving the strong light stress resistance and yield of plants. ZmG2 provided by the invention is A1), A2) or A3) as follows: A1) the amino acid sequence is the protein of sequence 4; A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 4 in the sequence table and has the same function; A3) a fusion protein obtained by connecting a label to the N-terminal or/and the C-terminal of A1) or A2). Experiments prove that the ZmG2 and the coding gene thereof can improve the capability of resisting highlight inhibition of plants and biomass and yield: the transgenic plant obtained by introducing the ZmG 2-encoding gene into the plant has higher capability of resisting high photoinhibition and higher biomass and yield compared with the wild type plant.

Description

ZmG2 application in improving plant strong light stress resistance and yield

Technical Field

The invention relates to the field of biotechnology, and discloses application of ZmG2 in improving strong light stress resistance and yield of plants.

Background

Light is an energy source for photosynthesis of plants on the earth, but when the amount of light energy absorbed by the photosynthetic mechanism of a plant exceeds the amount that can be utilized for photosynthesis, the excess light energy causes a decrease in the efficiency of conversion of light energy and even photo-oxidative damage to the photosynthetic mechanism, which is called a photo-inhibition phenomenon. In the absence of other environmental stresses, photoinhibition can reduce photosynthetic productivity of plants by more than 10%. Photoinhibition occurs primarily in photosystem ii (psii) of photosynthetic elements, and is a protective response of plants against external stresses, particularly in high temperature arid environments.

Photoinhibition includes both photoprotection and photodisruption, and is mainly manifested by a decrease in the maximum photochemical efficiency (expressed as the fluorescence parameter Fv/Fm) and photosynthetic efficiency of PSII, with the degree of photoinhibition depending on the equilibrium state between repair of the photosynthetic machinery protective machinery and disruption of the PSII reaction center. The PSII reaction center D1 protein is the core protein of PSII and is the main target for the damage of strong light to the photoperiodic mechanism. The photoinhibition leads to the oxidative inactivation and the final degradation of the D1 protein, and the D1 protein is the protein which is metabolized most rapidly in the leaf turnover, has a half-life period of only 20-30min under normal conditions, and maintains the dynamic balance through continuous turnover. Research shows that strong light directly inhibits the biosynthesis of D1 protein, so that the repair rate is lower than the degradation rate, photodamage is accumulated, and the photoinhibition degree is increased. When the light energy is excessive, heat dissipation taking non-photochemical quenching (NPQ) as a main form is an important way for effectively protecting plants from high light inhibition, and research of Demmig-Adams et al proves that lutein circulation has an important role in regulating the dissipation of the excessive light energy in the light inhibition.

Plants regulate protective photoinhibition during long-term evolution through structural evolution, alteration of photosynthetic pathways and improvement of photosynthetic operation, among others, C₄The plant photosynthetic cycle keeps higher electron transfer efficiency, lightens photoinhibition and has the most effective protection on photoinhibition. The effect of strong light on light inhibition is very obvious, the sunlight is strongest at noon, and the corresponding C₃The plant photosynthetic rate is obviously reduced. C₃The crop rice needs to consume a large amount of energy in the growth period, particularly in the late heading and grain filling period, and the photoinhibition and photooxidation damage caused by high illumination can cause the photosynthetic capacity of leaves to be reduced, poor grain filling and directly cause the reduction of yield. Therefore, it is considered to sufficiently and effectively utilize the resistance of rice to the inhibition of light due to high lightThe light energy resource is one of the key problems of further improving the plant yield and realizing stable and high yield.

Disclosure of Invention

The invention aims to provide application of a protein (named ZmG2) from corn (Zea mays L.) in improving the strong light stress resistance and yield of plants.

The invention firstly provides any one of the following applications of ZmG2 or a substance for regulating ZmG2 activity or content:

D1) regulating the yield of the plant or preparing a product for regulating the yield of the plant;

D2) regulating the light stress resistance of the plant or preparing a product for regulating the light stress resistance of the plant;

D3) regulating the protein transport capacity of a plant D1 or preparing a product regulating the protein transport capacity of a plant D1;

D4) regulating the content of plant D1 protein or preparing a product for regulating the content of plant D1 protein;

D5) cultivating a yield-enhancing plant or preparing a product for cultivating a yield-enhancing plant;

D6) cultivating a plant having increased resistance to light stress or preparing a product for cultivating a plant having increased resistance to light stress;

D7) cultivating the plant with the D1 protein movement-improving capability or preparing a product of cultivating the plant with the D1 protein movement-improving capability;

D8) cultivating the plant with the increased D1 protein content or preparing a product of cultivating the plant with the increased D1 protein content;

ZmG2 is A1), A2) or A3) as follows:

A1) the amino acid sequence is the protein of sequence 4;

A2) the protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in the sequence 4 in the sequence table and has the same function;

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

In order to facilitate the purification of the protein of A1), the amino terminal or the carboxyl terminal of the protein consisting of the amino acid sequence shown in sequence 4 of the sequence listing may be labeled as shown in the following table.

Table: sequence of tags

Label (R)	Residue of	Sequence of
			Poly-Arg	5-6 (typically 5)	RRRRR
Poly-His	2-10 (generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The protein in A2) above is a protein having 75% or more identity to the amino acid sequence of the protein shown in SEQ ID NO. 4 and having the same function. The identity of 75% or more than 75% is 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity.

The protein of A2) above may be artificially synthesized, or may be obtained by synthesizing the coding gene and then performing biological expression.

The gene encoding the protein of A2) above can be obtained by deleting one or several amino acid residues from the DNA sequence shown in SEQ ID No. 5, and/or by carrying out missense mutation of one or several base pairs, and/or by attaching the coding sequence of the tag shown in the above table to the 5 'end and/or 3' end thereof. Wherein, the DNA molecule shown in the sequence 5 encodes the protein shown in the sequence 4.

The invention also provides any one of the following uses of the biomaterial associated with ZmG 2:

the biomaterial is any one of the following B1) to B7):

B1) a nucleic acid molecule encoding ZmG 2;

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

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

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

B5) a transgenic plant cell line containing B1) the nucleic acid molecule, or a transgenic plant cell line containing B2) the expression cassette, or a transgenic plant cell line containing B3) the recombinant vector;

B6) a transgenic plant tissue containing B1) the nucleic acid molecule, or a transgenic plant tissue containing B2) the expression cassette, or a transgenic plant tissue containing B3) the recombinant vector;

B7) a transgenic plant organ containing B1) the nucleic acid molecule, or a transgenic plant organ containing B2) the expression cassette, or a transgenic plant organ containing B3) the recombinant vector.

In the above application, the nucleic acid molecule of B1) may be any one of the following B11) -B15):

b11) the coding sequence is cDNA molecule or DNA molecule of sequence 5 in the sequence table;

b12) a cDNA molecule or a DNA molecule shown in a sequence 5 in a sequence table;

b13) a DNA molecule shown as a sequence 6 in a sequence table;

b14) a cDNA or DNA molecule having 75% or more identity to the nucleotide sequence defined in b11) or b12) or b13) and encoding ZmG 2;

b15) hybridizes under stringent conditions with the nucleotide sequence defined in b11) or b12) or b13) or b14) and encodes a cDNA molecule or a DNA molecule of ZmG 2.

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

The nucleotide sequence encoding ZmG2 protein of the present invention can be readily mutated by one of ordinary skill in the art using known methods, such as directed evolution and point mutation. Those nucleotides which have been artificially modified to have 75% or more identity to the nucleotide sequence of the ZmG2 protein isolated in the present invention are derived from the nucleotide sequence of the present invention and are identical to the sequence of the present invention as long as they encode ZmG2 protein and have the function of ZmG2 protein.

The term "identity" as used herein refers to sequence similarity to a native nucleic acid sequence. "identity" includes nucleotide sequences that are 75% or more, or 85% or more, or 90% or more, or 95% or more identical to the nucleotide sequence of a protein consisting of the amino acid sequence shown in coding sequence 4 of the present invention. Identity can be assessed visually or by computer software. Using computer software, the identity between two or more sequences can be expressed in percent (%), which can be used to assess the identity between related sequences.

In the above application, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can also be: hybridization in 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; can also be: hybridization and washing of membranes 2 times, 5min each, at 68 ℃ in a solution of 2 XSSC, 0.1% SDS, and hybridization and washing of membranes 2 times, 15min each, at 68 ℃ in a solution of 0.5 XSSC, 0.1% SDS; can also be: 0.1 XSSPE (or 0.1 XSSC), 0.1% SDS at 65 ℃ and washing the membrane.

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

In the above application, the expression of the nucleic acid molecule containing the protein ZmG2 of B2)A cassette (ZmG2 gene expression cassette) refers to DNA capable of expressing ZmG2 protein in a host cell, which may include not only a promoter to initiate transcription of ZmG2 gene, but also a terminator to terminate transcription of ZmG2 gene. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present invention include, but are not limited to: constitutive promoters, tissue, organ and development specific promoters, and inducible promoters. Examples of promoters include, but are not limited to: the constitutive promoter of cauliflower mosaic virus 35S; the wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", Chao et al (1999) Plant Physiol 120: 979-992); chemically inducible promoter from tobacco, pathogenesis-related 1(PR1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (U.S. patent 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5,057,422); seed-specific promoters, such as the millet seed-specific promoter pF128(CN101063139B (Chinese patent 200710099169.7)), seed storage protein-specific promoters (e.g., the promoters of phaseolin, napin, oleosin, and soybean beta conglycin (Beachy et al (1985) EMBO J.4: 3047-3053)). They can be used alone or in combination with other plant promoters. All references cited herein are incorporated by reference in their entirety. Suitable transcription terminators include, but are not limited to: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminators (see, e.g., Odell et al (I)⁹⁸⁵) Nature 313: 810; rosenberg et al (1987) Gene,56: 125; guerineau et al (1991) mol.gen.genet,262: 141; proudfoot (1991) Cell,64: 671; sanfacon et al Genes Dev.,5: 141; mogen et al (1990) Plant Cell,2: 1261; munroe et al (1990) Gene,91: 151; ballad et al (1989) Nucleic Acids Res.17: 7891; joshi et al (1987) Nucleic Acid Res, 15: 9627).

The recombinant vector containing the ZmG2 gene expression cassette can be constructed using an existing expression vector. The plant expression vector comprises a binary agrobacterium vector, a vector for plant microprojectile bombardment and the like. Such as pAHC25, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, PSN1301, or pCAMBIA1391-Xb (CAMBIA Corp.), etc. 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 lead poly A to be added to the 3 'end of mRNA precursor, and the untranslated regions transcribed at the 3' end of Agrobacterium crown gall inducible (Ti) plasmid genes (such as nopaline synthase gene Nos) and plant genes (such as soybean storage protein gene) have similar functions. When the gene of the present invention is used to construct a plant 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 correct 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 plant expression vector to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound capable of producing a color change (GUS gene, luciferase gene, etc.), a marker gene for antibiotics (e.g., nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to phosphinothricin as an herbicide, hph gene conferring resistance to hygromycin as an antibiotic, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or a marker gene for chemical resistance (e.g., herbicide resistance), a mannose-6-phosphate isomerase gene providing the ability to metabolize mannose, which can be expressed in plants. From the safety of transgenic plants, the transgenic plants can be directly screened and transformed in a stress environment without adding any selective marker gene.

In the above application, the vector may be a plasmid, a cosmid, a phage, or a viral vector. The plasmid can be specifically a binary vector pVec 8-Gateway.

B3) The recombinant vector can be pVec8-ZmG 2. The pVec8-ZmG2 is a recombinant vector obtained by inserting a DNA fragment shown in a sequence 5 in a sequence table into the downstream of a ZmUBI promoter of pVec 8-Gateway. The pVec8-ZmG2 can express ZmG2, and the expression of the encoding gene of ZmG2 is driven by the promoter of maize ubiquitin (ZmUBI).

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacteria can be Agrobacterium, such as Agrobacterium rhizogenes EHA 105.

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

In the above application, the regulating and controlling of the plant yield can be improving of the plant yield;

the regulation and control of the plant light stress resistance can be the improvement of the plant light stress resistance;

the regulation of the protein transport capacity of the plant D1 can be the improvement of the protein transport capacity of the plant D1;

the regulation of the content of the plant D1 protein can be realized by increasing the content of the plant D1 protein.

The yield may be aboveground biomass or grain yield of the plant;

the light stress may be a strong light stress;

the D1 protein content can be D1 content in the leaf.

In the above application, the plant may be a C3 plant or a C4 plant.

Further, the C3 plant may be any one of M1) -M3):

m1) monocotyledonous or dicotyledonous plants;

m2) gramineous plants;

m3) rice.

The invention also provides any one of the following X1) -X8):

x1) method for increasing plant yield, comprising: ZmG2 is expressed in a receptor plant, or ZmG2 content in the receptor plant is increased, or ZmG2 activity in the receptor plant is increased, so that a target plant with yield increased compared with the receptor plant is obtained, and yield is increased;

x2) method for growing plants with increased yield, comprising: expressing ZmG2 in a recipient plant, or increasing the ZmG2 content in the recipient plant, or increasing the ZmG2 activity in the recipient plant, resulting in a plant of interest having increased yield as compared to the recipient plant;

x3) a method for increasing light stress resistance in a plant comprising: ZmG2 is expressed in a receptor plant, or the content of ZmG2 in the receptor plant is increased, or the activity of ZmG2 in the receptor plant is increased, so that a target plant with light stress resistance improved compared with the receptor plant is obtained, and the light stress resistance of the plant is improved;

x4) method for breeding a plant with increased light stress resistance, comprising: expressing ZmG2 in a recipient plant, or increasing the content of ZmG2 in the recipient plant, or increasing the activity of ZmG2 in the recipient plant, to obtain a target plant with increased light stress resistance compared with the recipient plant;

x5) a method for increasing the operational capacity of a plant D1 protein, comprising: ZmG2 is expressed in a receptor plant, or the content of ZmG2 in the receptor plant is increased, or the activity of ZmG2 in the receptor plant is increased, so that a target plant with improved D1 protein transport capacity compared with the receptor plant is obtained, and the improvement of the D1 protein transport capacity of the plant is realized;

x6) method for breeding plants with increased D1 protein trafficability comprising: ZmG2 is expressed in a receptor plant, or the content of ZmG2 in the receptor plant is increased, or the activity of ZmG2 in the receptor plant is increased, so that a target plant with improved D1 protein transport capacity compared with the receptor plant is obtained;

x7) a method for increasing the content of plant D1 protein, comprising: ZmG2 is expressed in a receptor plant, or the content of ZmG2 in the receptor plant is increased, or the activity of ZmG2 in the receptor plant is increased, so that a target plant with the content of D1 protein increased compared with the receptor plant is obtained, and the content of the D1 protein in the plant is increased;

x8) method for breeding plants with increased D1 protein content, comprising: ZmG2 is expressed in a receptor plant, or the content of ZmG2 in the receptor plant is increased, or the activity of ZmG2 in the receptor plant is increased, and a target plant with the content of D1 protein increased compared with the receptor plant is obtained.

Of the above methods, X1) -X8) can be performed by introducing a ZmG 2-encoding gene into the recipient plant and allowing the encoding gene to be expressed.

In the above method, the encoding gene may be B1).

In the above method, the ZmG2 encoding gene may be modified as follows and then introduced into the recipient plant to achieve better expression effect:

1) modifying and optimizing according to actual needs to enable the gene to be efficiently expressed; for example, the amino acid sequence of the gene encoding ZmG2 of the present invention may be changed to conform to plant preferences while maintaining its amino acid sequence according to the preferences of the recipient plant; during the optimization, it is desirable to maintain a GC content in the optimized coding sequence to best achieve high expression levels of the introduced gene in plants, wherein the GC content can be 35%, more than 45%, more than 50%, or more than about 60%;

2) modifying the sequence of the gene adjacent to the initiating methionine to allow efficient initiation of translation; for example, modifications are made using sequences known to be effective in plants;

3) linking with promoters expressed by various plants to facilitate the expression of the promoters in the plants; such promoters may include constitutive, inducible, time-regulated, developmentally regulated, chemically regulated, tissue-preferred, and tissue-specific promoters; the choice of promoter will vary with the time and space requirements of expression, and will also depend on the target species; for example, tissue or organ specific expression promoters, depending on the stage of development of the desired receptor; although many promoters derived from dicots have been demonstrated to be functional in monocots and vice versa, desirably, dicot promoters are selected for expression in dicots and monocot promoters for expression in monocots;

4) the expression efficiency of the gene of the present invention can also be improved by linking to a suitable transcription terminator; tml from CaMV, E9 from rbcS; any available terminator which is known to function in plants may be linked to the gene of the invention;

5) enhancer sequences, such as intron sequences (e.g., from Adhl and bronzel) and viral leader sequences (e.g., from TMV, MCMV, and AMV) were introduced.

The gene encoding ZmG2 can be introduced into a recipient plant using a recombinant expression vector containing the gene encoding ZmG 2. The recombinant expression vector can be specifically pVec8-ZmG 2.

The recombinant expression vector can be introduced into Plant cells by using conventional biotechnological methods such as Ti plasmid, Plant virus vector, direct DNA transformation, microinjection, electroporation, etc. (Weissbach,1998, Method for Plant Molecular Biology VIII, academic Press, New York, pp.411-463; Geiserson and Corey,1998, Plant Molecular Biology (2nd Edition)).

The plant of interest is understood to include not only the ZmG2 protein or the first generation plant in which the gene encoding it has been altered, but also its progeny. For the plant of interest, the gene may be propagated in the species, or transferred into other varieties of the same species, including commercial varieties in particular, using conventional breeding techniques. The plant of interest includes seeds, callus, whole plants and cells.

In the above method, the yield may be aboveground biomass or grain yield of the plant;

the light stress may be a strong light stress;

the D1 protein content can be D1 content in the leaf.

The recipient plant may be a C3 plant or a C4 plant.

Further, the C3 plant may be any one of M1) -M3):

m1) monocotyledonous or dicotyledonous plants;

m2) gramineous plants;

m3) rice.

The invention also provides a product with any one of the following functions D1) -D8), wherein the product contains ZmG2 or the biological material:

D1) regulating and controlling the yield of the plant;

D2) regulating and controlling the light stress resistance of the plant;

D3) regulating the protein transport capacity of the plant D1;

D4) regulating and controlling the content of plant D1 protein;

D5) the yield of the plants is improved;

D6) improving the light stress resistance of the plants;

D7) improving the protein transport capacity of the plant D1;

D8) increasing the content of the D1 protein in plants.

The product can use ZmG2 or the biological material as its active component, and can also combine substances with the same function with ZmG2 or the biological material as its active component.

In the above product, the yield may be aboveground biomass or grain yield of the plant;

the light stress may be a strong light stress;

the D1 protein content can be D1 content in the leaf.

The plant may be a C3 plant or a C4 plant.

Further, the C3 plant may be any one of M1) -M3):

m1) monocotyledonous or dicotyledonous plants;

m2) gramineous plants;

m3) rice.

In the invention, the plant light stress improving antibody can be embodied in the improvement of the running capability of the D1 protein in plant leaves, the increase of the content of the D1 protein in the plant leaves and/or the increase of the Fv/Fm value of the plant leaves.

The intense light may illuminate at a higher intensity than that normally required by the plant. In one embodiment of the present invention, the intense light is illumination intensity of 1200. mu. mol. m^-2·s^-1Of (2) is detected.

Experiments prove that the ZmG2 and the coding gene thereof can improve the capability of resisting highlight inhibition of plants and biomass and yield: compared with wild plants, transgenic plants obtained by introducing the coding gene of ZmG2 into plants have higher capability of resisting highlight inhibition and higher biomass and yield, and the ZmG2 and the coding gene thereof have wide application prospects.

Drawings

FIG. 1 shows the copy number and gene expression level detection results of ZmGLK1 and ZmG2 genes inserted into transgenic rice. A is the copy number detection result of ZmGLK1 and ZmG2 genes, and each strain detects three plants, which correspond to three lanes in the figure; b is the detection result of the expression levels of ZmGLK1 and ZmG2 genes, and CK is a negative control plant.

FIG. 2 is a graph showing the Fv/Fm change curves of each rice to be tested after 4 hours of the highlight treatment. A is a water treatment control group, and B is a lincomycin treatment group. Indicates that the difference reaches a significant level (p < 0.01) compared to wild-type rice.

FIG. 3 is the Fv/Fm change curve of each rice to be tested at 12-hour recovery. A is a water treatment control group, and B is a lincomycin treatment group. Indicates that the difference reaches a significant level (p < 0.05) compared with wild type rice.

FIG. 4 shows Western blot analysis results of D1 protein contents of wild rice and ZmGLK1 and ZmG2 transgenic rice after 0h and 4h of highlight treatment. The loading amount was 15. mu.g per lane, wherein 1/4WT indicates 1/4 in terms of loading amount WT, and 1/2WT indicates 1/2 in terms of loading amount WT. 0H HL-H₂O represents the highlight treatment for 0 hour in the water treatment control group, 0H HL-Lin represents the highlight treatment for 0 hour in the lincomycin treatment group, and 4H HL-H₂O represents the water treatment control group which is subjected to highlight treatment for 4 hours, 4h HL-Lin represents the lincomycin treatment group which is subjected to highlight treatment for 4 hours, and LSU represents a rubisCO large subunit band after Coomassie brilliant blue staining as an internal reference.

FIG. 5 shows the aboveground biomass and yield of wild-type rice and transgenic rice in Beijing and Hainan. The yield of the single plant is the yield of the single plant seeds, and the yield of the cell is the yield of the cell seeds.

Detailed Description

The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents, instruments and the like used in the following examples are commercially available unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged. In the following examples, unless otherwise specified, the 1 st position of each nucleotide sequence in the sequence listing is the 5 'terminal nucleotide of the corresponding DNA/RNA, and the last position is the 3' terminal nucleotide of the corresponding DNA/RNA.

donor vector pDONR^TM207:Invitrogen。

The binary vector pVec8-Gateway (destination vector pVec8-Gateway) (Kim, C.M. & Dolan, L.ROOT HAIR DEFECTIVE SIX-LIKE class I genes promoter extent in the grass Brachypodium distachyon. PLoS Gene.12, e1006211 (2016)) in the examples described below was publicly available from the applicant, and was only used for repeating the relevant experiments of the present invention, and was not used for other purposes.

The japonica rice variety Kitaake (Wang, P.et al.Re-creation of a key step in the evolution switch from C) in the examples described below₃ to C₄The biological material is available to the public from the applicant, and is only used for repeating the relevant experiments of the present invention, and is not used for other purposes.

Example 1, ZmGLK1 and ZmG2 improve the rice's ability to resist high light inhibition and biomass and yield

This example provides two proteins from maize variety B73, designated as ZmGLK1 and ZmG2, both of which can increase the resistance of rice against high light inhibition, thereby greatly increasing the biomass and yield of transgenic rice. The amino acid sequence of ZmGLK1 is sequence 1 in the sequence table, in corn variety B73, the genome sequence of ZmGLK1 is sequence 3 in the sequence table, and the CDS sequence is sequence 2; ZmG2 is shown as sequence 4 in the sequence table, in maize variety B73, the genome sequence of ZmG2 is shown as sequence 6 in the sequence table, and the CDS sequence is shown as sequence 5.

1. Construction of recombinant expression vectors

CDS of two genes is cloned to a vector pDONR through BP reaction^TM207, cloning the promoter and gene of maize ubiquitin (ZmUBI) to a binary vector pVec8-Gateway through LR reaction after correct sequencing to construct a recombinant expression vector, marking the obtained recombinant vector containing the CDS sequence of ZmGLK1 as pVec8-ZmGLK1, and marking the obtained recombinant vector containing the CDS sequence of ZmG2 as pVec8-ZmG 2. The specific construction method comprises the following steps:

vector construction: PCR products containing ZmG2(GenBank accession number AF318579) or ZmGLK1(GenBank accession number AF318580) full-length cDNA were amplified from maize variety B73 cDNA by PCR, and then ZmG2 and ZmGLK1 full-length cDNAs were cloned into BP reaction using the two PCR products, respectively

donor vector pDONR^TM207, the recombinant vectors with the correct sequence were named 207-ZmGLK1 and 207-ZmG2, respectively. The primers used (with

Border sequence) is ZmG2 gene:

ZmG2-cloningF：5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGCTTGAGGTGTCGACGCTG-3′；

ZmG 2-cloningR: 5'-GGGGACCACTTTGTACAAGAAAGCTGGGTTAGTATGTCATCCGGTGGCGC-3', respectively; ZmGLK1 gene:

ZmGLK1-cloningF：5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTATGCTTGCAGTGTCGCCGTC-3′；

ZmGLK1-cloningR：5′-GGGGACCACTTTGTACAAGAAAGCTGGGTTCATCCACAAGCTTGCGGCAC-3′。

207-ZmGLK1 and 207-ZmG2 were sequenced correctly and then the target cDNA was cloned downstream of the ZmUBI promoter in destination vector pVec8-Gateway by LR reaction, and the recombinant vectors with correct sequence were designated as pVec8-ZmGLK1 and pVec8-ZmG2, respectively.

pVec8-ZmGLK1 is a recombinant vector obtained by inserting a DNA fragment shown in a sequence 2 in a sequence table into the downstream of a ZmUBI promoter of pVec8-Gateway, the recombinant vector can express ZmGLK1 protein, and the expression of a ZmGLK1 coding gene is driven by a promoter of maize ubiquitin (ZmUBI). pVec8-ZmG2 is a recombinant vector obtained by inserting a DNA fragment shown in a sequence 5 in a sequence table into the downstream of a ZmUBI promoter of pVec8-Gateway, the recombinant vector can express ZmG2 protein, and the expression of ZmG2 coding genes is driven by a promoter of maize ubiquitin (ZmUBI).

2. Rice transformation and identification of transgenic plants

Respectively introducing pVec8-ZmGLK1 and pVec8-ZmG2 obtained in the step 1 into agrobacterium EHA105, transforming a japonica rice variety Kitaake (namely wild rice and WT) by using an agrobacterium-mediated rice genetic transformation method to respectively obtain ZmGLK1 gene-transformed rice and ZmG2 gene-transformed rice, and using a plant which is separated in a progeny identification process and does not contain ZmGLK1 gene or ZmG2 gene as a negative control plant.

Detection of two ZmGLK1 transgenic rice lines (ZmUBI) by Southern blot_proZmGLK1-2 and ZmUBI_proZmGLK1-3) and two ZmG2 transgenic rice lines (ZmUBI)_proZmG2-2 and ZmUBI_proZmG2-3) the copy number of the inserted foreign gene (A in FIG. 1), the probes used were all: F-CTTCTACACAGCCATCGGTC and R-CCGATGGTTTCTACAAAGATCG.

qRT-PCR detects the relative expression level of the genes transferred into ZmGLK1 and ZmG2, uses rice japonica rice Kitaake (WT) and a negative control plant (CK) as a control, and comprises the following detection steps:

the primers used for detecting the relative expression level of the ZmGLK1 gene in ZmGLK1 transgenic rice are as follows: 5'-GGACCTGGATTTCGACTTCA-3', 5'-CACTCCCCTTTCCCTTCTTC-3', respectively; primers used for detecting the relative expression level of ZmG2 gene in ZmG2 transgenic rice are as follows: 5'-CATGGTGGACGACAACCTC-3', 5'-CACATGTTTGCTCCAACGAC-3', respectively; the reference genes are rice Actin, and the reference gene primers are as follows: 5'-GGCACCACACCTTCTACAAT-3', 5'-CTCACACCATCACCAGAGT-3' are provided.

The results showed that two ZmGLK1 transgenic rice plants (ZmUBI)_proZmGLK1-2 and ZmUBI_proZmGLK1-3) has relative expression level of ZmGLK1 gene higher than that of WT and CK, and two transgenic ZmG2 rice (ZmUBI)_proZmG2-2 and ZmUBI_proZmG2-3) the relative expression level of ZmG2 gene was significantly higher than that of WT and CK, both of which had no expression of ZmGLK1 gene and ZmG2 gene, B in FIG. 1.

3. Photoinhibition experiments on transgenic Rice

And (3) testing the rice: wild type rice, negative control plant, ZmUBI_proZmGLK1-2 and ZmUBI_pro:ZmGLK1-3，ZmUBI_proZmG2-2 and ZmUBI_pro:ZmG2-3。

Cutting leaves of rice seedlings to be tested which grow 5 weeks old in a greenhouse in a hydroponic manner, randomly dividing the leaves into two groups, namely a water treatment control group and a lincomycin treatment group, soaking the leaves of the water treatment control group in water, soaking the leaves of the lincomycin treatment group in 1mM lincomycin (inhibitor for synthesizing D1 protein) aqueous solution, and adapting for 3 hours (the light intensity is 20-30 mu mol. m) at room temperature^-2·s^-1). Each set of leaves was then placed at 1200. mu. mol. m^-2·s^-1Irradiating for 4 hours under an LED lamp with illumination intensity to perform highlight treatment, taking the leaves every hour to determine the Fv/Fm value, and simultaneously sampling and quickly freezing in liquid nitrogen. After 4 hours of high light treatment, each group of leaves was then placed in room temperature and room light for 12 hours of recovery and Fv/Fm values were determined using a FluorPen FP100 hand-held chlorophyll fluorometer (Photon Systems Instruments).

The samples which are treated by high light for 0h and 4h are ground and extracted to carry out Western blot experiments, and SDS-PAGE gel is used for electrophoretic separation of proteins and membrane conversion, wherein the primary antibody is a D1 protein antibody (Agrisera, product number AS05084), and the secondary antibody is a goat anti-rabbit IgG-HRP antibody (Agrisera, product number AS 09602).

The Fv/Fm value changes are shown in Table 1.

TABLE 1 Fv/Fm values

In Table 1, GLK1-2, GLK1-3, G2-2 and G2-3 are ZmUBI, respectively_proZmGLK1-2 and ZmUBI_pro:ZmGLK1-3、ZmUBI_proZmG2-2 and ZmUBI_proZmG 2-3. Indicates that the difference reaches a significant level (p < 0.01) compared with the wild type rice, and indicates that the difference reaches a significant level (p < 0.05) compared with the wild type rice.

The results show that the most intuitive indicator of PSII photochemical efficiency (Fv/Fm value) decreases after plants have been subjected to high light irradiation. In the water treatment control group, the Fv/Fm values of both wild-type rice and transgenic rice decreased from 0.8 to about 0.6 at a relatively slow rate, and the values of each transgenic rice were higher than those of the wild-type rice (A in FIG. 2). In the recovery process of the water-treated control group, it was also observed that wild-type rice was similar to transgenic rice, and both of them had Fv/Fm values that eventually recovered to about 0.8 after 12 hours (A in FIG. 3). Lincomycin is an inhibitor of protein synthesis in chloroplasts, and can inhibit the turnover of D1 protein, thereby blocking the recovery process of PSII. Under the conditions of highlight treatment and the presence of lincomycin, the Fv/Fm value of the leaf blade of the wild rice is sharply reduced from 1h, and the Fv/Fm value is only 21% of that of the leaf blade of the wild rice in dark adaptation after 4h, which indicates that PSII of the leaf blade of the wild rice is seriously damaged. In the transgenic rice, particularly ZmG2 transgenic rice, the Fv/Fm value is always higher than that of wild rice in the highlight treatment process, and after 4 hours, the Fv/Fm value is nearly 2 times that of the wild rice (B in figure 2). During the recovery process, the transgenic rice also recovered faster than wild-type rice, and eventually to a higher level than wild-type rice (fig. 3B). In the whole process, wild rice and negative control plants have no obvious change.

Further detection of the D1 protein level of the highlight-treated leaves by Western blot shows that the D1 protein content of the transgenic rice is higher than that of wild rice before highlight treatment. After 4 hours of high light treatment, D1 protein in leaves of wild rice is greatly reduced, while D1 protein in transgenic rice, particularly ZmG2 transgenic rice, is still kept at a higher level, and the difference between the two is more obvious under the condition of lincomycin treatment (figure 4). The results show that after the ZmGLK1 and ZmG2 genes are transferred, the content of the transgenic rice D1 protein is increased, and the capability of resisting high light stress is greatly improved. In the whole process, the content of the D1 protein in the wild rice and the negative control plant is not obviously changed.

4. Yield enhancement of transgenic rice in field

Detecting the dry weight of the overground part and the yield of grains of the rice, wherein the rice to be detected: wild type rice (WT), negative control plant (CK), ZmUBI_proZmGLK1-2 and ZmUBI_pro:ZmGLK1-3，ZmUBI_proZmG2-2 and ZmUBI_pro:ZmG2-3。

The field experiments in Beijing and Hainan both show that the dry weight of the overground part and the yield of grains of the transgenic rice are obviously improved compared with wild type. The dry weight average of the overground part of the ZmG2 transgenic rice is obviously or extremely obviously higher than that of the wild type rice in Beijing or Hainan. From the yield results, the yield of each plant of the rice with the ZmGLK1 and ZmG2 genes is respectively increased by 16-25% and about 33% compared with the wild type, the yield of the rice in the cell is similar to the yield of the rice, the rice with the ZmGLK1 and ZmG2 genes can be increased by 13-15% and 28-32% in Beijing and 14-18% and 34-45% in Hainan. The results show that after the corn genes ZmGLK1 and ZmG2 are transferred into rice, the capability of resisting highlight inhibition can be improved, so that the biomass and the yield of the transgenic rice are greatly improved.

The method of measuring the dry weight of the above-ground part is as follows: in the field experiment, each rice material is provided with 3 cells which are repeated and randomly distributed. After the rice is mature, removing seeds from single straws, putting the straws into a nylon mesh bag, drying the straws at the temperature of 80 ℃ to constant weight, and weighing the sample. 20-30 individual replicates per rice material were taken for statistical analysis.

The measurement method of the grain yield is as follows: and (3) threshing the rice ears of the single rice plants, removing shrunken grains, weighing to obtain the yield of the single rice seeds, and repeatedly taking 20-30 single rice plants from each rice material for statistical analysis. And removing the border row when the cell yield is counted, taking the weight of the kernel measured by 30 rice plants in the middle as the cell yield, and repeatedly using 3 cells for counting analysis.

The results of the indices are shown in table 2 and fig. 5.

TABLE 2 measurement of the dry weight of the aerial parts and the grain yield of transgenic plants

In Table 2, GLK1-2, GLK1-3, G2-2 and G2-3 are ZmUBI, respectively_proZmGLK1-2 and ZmUBI_pro:ZmGLK1-3、ZmUBI_proZmG2-2 and ZmUBI_proZmG 2-3. Indicates that the difference reaches a significant level (p < 0.01) compared with the wild type rice, and indicates that the difference reaches a significant level (p < 0.05) compared with the wild type rice.

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

Application of <120> ZmG2 in improving strong light stress resistance and yield of plants

<160> 6

<170> PatentIn version 3.5

<210> 1

<211> 476

<212> PRT

<213> corn (Zea mays L.)

<400> 1

Met Leu Ala Val Ser Pro Ser Pro Val Arg Cys Ala Asp Ala Glu Glu

1 5 10 15

Cys Gly Gly Gly Gly Ala Ser Lys Glu Met Glu Glu Thr Ala Val Gly

20 25 30

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

35 40 45

Asp Phe Gly Asp Phe Phe Leu Arg Leu Asp Asp Gly Asp Asp Ala Leu

50 55 60

Pro Gly Leu Glu Val Asp Pro Ala Glu Ile Val Phe Ala Asp Phe Glu

65 70 75 80

Ala Ile Ala Thr Ala Gly Gly Asp Gly Gly Val Thr Asp Gln Glu Val

85 90 95

Pro Ser Val Leu Pro Phe Ala Asp Ala Ala His Ile Gly Ala Val Asp

100 105 110

Pro Cys Cys Gly Val Leu Gly Glu Asp Asn Asp Ala Ala Cys Ala Asp

115 120 125

Val Glu Glu Gly Lys Gly Glu Cys Asp His Ala Asp Glu Val Ala Ala

130 135 140

Ala Gly Asn Asn Asn Ser Asp Ser Gly Glu Ala Gly Cys Gly Gly Ala

145 150 155 160

Phe Ala Gly Glu Lys Ser Pro Ser Ser Thr Ala Ser Ser Ser Gln Glu

165 170 175

Ala Glu Ser Arg Arg Lys Val Ser Lys Lys His Ser Gln Gly Lys Lys

180 185 190

Lys Ala Lys Val Asp Trp Thr Pro Glu Leu His Arg Arg Phe Val Gln

195 200 205

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

210 215 220

Glu Ile Met Gly Ile Asp Ser Leu Thr Arg His Asn Ile Ala Ser His

225 230 235 240

Leu Gln Lys Tyr Arg Ser His Arg Lys His Met Leu Ala Arg Glu Val

245 250 255

Glu Ala Ala Thr Trp Thr Thr His Arg Arg Pro Met Tyr Ala Ala Pro

260 265 270

Ser Gly Ala Val Lys Arg Pro Asp Ser Asn Ala Trp Thr Val Pro Thr

275 280 285

Ile Gly Phe Pro Pro Pro Ala Gly Thr Pro Pro Arg Pro Val Gln His

290 295 300

Phe Gly Arg Pro Leu His Val Trp Gly His Pro Ser Pro Thr Pro Ala

305 310 315 320

Val Glu Ser Pro Arg Val Pro Met Trp Pro Arg His Leu Ala Pro Arg

325 330 335

Ala Pro Pro Pro Pro Pro Trp Ala Pro Pro Pro Pro Ala Asp Pro Ala

340 345 350

Ser Phe Trp His His Ala Tyr Met Arg Gln Gly Pro Ala Ala His Met

355 360 365

Pro Asp Gln Val Ala Val Thr Pro Cys Val Ala Val Pro Met Ala Ala

370 375 380

Ala Arg Phe Pro Ala Pro His Val Arg Gly Ser Leu Pro Trp Pro Pro

385 390 395 400

Pro Met Tyr Arg Pro Leu Val Pro Pro Ala Leu Ala Gly Lys Ser Gln

405 410 415

Gln Asp Ala Leu Phe Gln Leu Gln Ile Gln Pro Ser Ser Glu Ser Ile

420 425 430

Asp Ala Ala Ile Gly Asp Val Leu Thr Lys Pro Trp Leu Pro Leu Pro

435 440 445

Leu Gly Leu Lys Pro Pro Ser Val Asp Ser Val Met Gly Glu Leu Gln

450 455 460

Arg Gln Gly Val Ala Asn Val Pro Gln Ala Cys Gly

465 470 475

<210> 2

<211> 1431

<212> DNA

<213> corn (Zea mays L.)

<400> 2

atgcttgcag tgtcgccgtc gccggtgcgg tgtgccgatg cggaggagtg cggcggagga 60

ggcgccagca aggaaatgga ggagaccgcc gtcgggcctg tgtccgactc ggacctggat 120

ttcgacttca cggtcgacga catagacttc ggggacttct tcctcaggct agacgacggg 180

gatgacgcgc tgccgggcct cgaggtcgac cctgccgaga tcgtcttcgc tgacttcgag 240

gcaatcgcca ccgccggcgg cgatggcggc gtcacggacc aggaggtgcc cagtgtcctg 300

ccctttgcgg acgcggcgca cataggcgcc gtggatccgt gttgtggtgt ccttggcgag 360

gacaacgacg cagcgtgcgc agacgtggaa gaagggaaag gggagtgcga ccatgccgac 420

gaggtagcag ccgccggtaa taataatagc gactccggtg aggccggctg tggaggagcc 480

tttgccggcg aaaaatcacc gtcgtcgacg gcatcgtcgt cgcaggaggc tgagagccgg 540

cgcaaggtgt ccaagaagca ctcccaaggg aagaagaaag caaaggtgga ttggacgccg 600

gagcttcacc ggagattcgt tcaggcggtg gaggagctgg gcatcgacaa ggcggtgccg 660

tccaggatcc tcgagatcat ggggatcgac tccctcacgc ggcataacat agccagccat 720

ctgcagaagt accgttccca caggaagcac atgcttgcga gggaggtgga ggcagcgacg 780

tggacgacgc accggcggcc gatgtacgct gcccccagcg gcgccgtgaa gaggcccgac 840

tctaacgcgt ggaccgtgcc gaccatcggt ttccctccgc cggcggggac ccctcctcgt 900

ccggtgcagc acttcgggag gccactgcac gtctggggcc atccgagtcc gacgccagcg 960

gtggagtcac cccgggtgcc aatgtggcct cggcatctcg ccccccgcgc cccgccgccg 1020

ccgccgtggg ctccgccacc gccagctgac ccggcgtcgt tctggcacca tgcttacatg 1080

aggcaggggc ctgctgccca tatgccagac caggtggcgg tgactccatg cgtggcagtg 1140

ccaatggcag cagcgcgttt ccctgctcca cacgtgaggg gttctttgcc atggccacct 1200

ccgatgtaca gacctctcgt tcctccagca ctcgcaggca agagccagca agacgcgctg 1260

tttcagctac agatacagcc atcaagcgag agcatagatg cagcaatagg tgatgtctta 1320

acgaagccgt ggctgccgct gcccctcgga ctgaagcccc cttcggtaga cagtgtcatg 1380

ggcgagctgc agaggcaagg cgtagcgaat gtgccgcaag cttgtggatg a 1431

<210> 3

<211> 2347

<212> DNA

<213> corn (Zea mays L.)

<400> 3

gagctatagc tggctgcctg agctcgctcc aagaagtgta tctatagtat agacactagg 60

cttcgtgtgg cgtgctcgag atatgcttgc agtgtcgccg tcgccggtgc ggtgtgccga 120

tgcggaggag tgcggcggag gaggcgccag caaggaaatg gaggagaccg ccgtcgggcc 180

tgtgtccgac tcggacctgg atttcgactt cacggtcgac gacatagact tcggggactt 240

cttcctcagg ctagacgacg gggatgacgc gctgccgggc ctcgaggtcg accctgccga 300

gatcgtcttc gctgacttcg aggcaatcgc caccgccggc ggcgatggcg gcgtcacgga 360

ccaggaggtg cccagtgtcc tgccctttgc ggacgcggcg cacataggcg ccgtggatcc 420

gtgttgtggt gtccttggcg aggacaacga cgcagcgtgc gcagacgtgg aagaagggaa 480

aggggagtgc gaccatgccg acgaggtagc agccgccggt aataataata gcgactccgg 540

tgaggccggc tgtggaggag cctttgccgg cgaaaaatca ccgtcgtcga cggcatcgtc 600

gtcgcaggag gctgagagcc ggcgcaaggt gtccaagaag cactcccaag ggaagaagaa 660

agcaaaggta cgcagggcta cgcatgcagt ataggcgtat agcaataatt gcaaagccct 720

aaatatatag caaaacaaag cgatgatacc atgcatgtgt gctctgctga catgtcttaa 780

cttaacacag gtggattgga cgccggagct tcaccggaga ttcgttcagg cggtggagga 840

gctgggcatc gacaaggcgg tgccgtccag gatcctcgag atcatgggga tcgactccct 900

cacgcggcat aacatagcca gccatctgca ggtccgtgcc gcgacgtact ccagcagcta 960

ctaccaatgt ttcctttgtg ctaacccgcg accgattttg gctatctaaa ttggagcaga 1020

agtaccgttc ccacaggaag cacatgcttg cgagggaggt ggaggcagcg acgtggacga 1080

cgcaccggcg gccgatgtac gctgccccca gcggcgccgt gaagaggccc gactctaacg 1140

cgtggaccgt gccgaccatc ggtttccctc cgccggcggg gacccctcct cgtccggtgc 1200

agcacttcgg gaggccactg cacgtctggg gccatccgag tccgacgcca gcggtggagt 1260

caccccgggt gccaatgtgg cctcggcatc tcgccccccg cgccccgccg ccgccgccgt 1320

gggctccgcc accgccagct gacccggcgt cgttctggca ccatgcttac atgagggtat 1380

gtatgcattg tacgcacggc cggccggccg gcaagtctac gtgctagtta tttttggatg 1440

caccgtaagt taactagctt cctagctagg gagatttagc tagcgttctg acggccggat 1500

ttcgttttac cagcaggggc ctgctgccca tatgccagac caggtggcgg tgactccatg 1560

cgtggcagtg ccaatggcag cagcggtaat ggtcgttcgt caacaataat gctgtcaaac 1620

aaatggaaaa tggcgttctt ctttaataag aacttggtct tggtgtcctg attttcagcg 1680

tttccctgct ccacacgtga ggggttcttt gccatggcca cctccgatgt acagacctct 1740

cgttcctcca gcactcgcag gcaagagcca gcaagacgcg ctgtttcagc tacagataca 1800

gccagtaagt gtatagctag ctgtagtgac attcatgttt cttttcatgc aacaacaatc 1860

atgtattctg cagcactctc aactcagccc ttcttttttc ctttctgtat ttttcagtca 1920

agcgagagca tagatgcagc aataggtgat gtcttaacga agccgtggct gccgctgccc 1980

ctcggactga agcccccttc ggtagacagt gtcatgggcg agctgcagag gcaaggcgta 2040

gcgaatgtgc cgcaagcttg tggatgatcc ccggggctgc atagctgcta gctcgtccct 2100

gcacgcagtg caacaaaagg aatttgtcga catgcctttt atgttttgaa ttcccgtgaa 2160

ccgtttatgg agcactctca actcgttcag ggtcagatga acagaacatt ataggagtac 2220

attctttgag gtttcaggtt tgagggaaat caacctgagt gcatccaagt acagtgtact 2280

gccacagtgc cacgtcggaa aatgaagtct tgacccgaat aacaagtatt ccctcgggtt 2340

atttgtc 2347

<210> 4

<211> 461

<212> PRT

<213> corn (Zea mays L.)

<400> 4

Met Leu Glu Val Ser Thr Leu Arg Gly Pro Thr Ser Ser Gly Ser Lys

1 5 10 15

Ala Glu Gln His Cys Gly Gly Gly Gly Gly Phe Val Gly Asp His His

20 25 30

Val Val Phe Pro Thr Ser Gly Asp Cys Phe Ala Met Val Asp Asp Asn

35 40 45

Leu Leu Asp Tyr Ile Asp Phe Ser Cys Asp Val Pro Phe Phe Asp Ala

50 55 60

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

65 70 75 80

Ala Glu Phe Ser Ser Thr Pro Pro Ala Asp Asp Leu Leu Ala Val Ala

85 90 95

Val Phe Gly Ala Asp Asp Gln Pro Ala Ala Ala Val Ala Gln Glu Lys

100 105 110

Pro Ser Ser Ser Leu Glu Gln Thr Cys Gly Asp Asp Lys Gly Val Ala

115 120 125

Val Ala Ala Ala Arg Arg Lys Leu Gln Thr Thr Thr Thr Thr Thr Thr

130 135 140

Thr Glu Glu Glu Asp Ser Ser Pro Ala Gly Ser Gly Ala Asn Lys Ser

145 150 155 160

Ser Ala Ser Ala Glu Gly His Ser Ser Lys Lys Lys Ser Ala Gly Lys

165 170 175

Asn Ser Asn Gly Gly Lys Arg Lys Val Lys Val Asp Trp Thr Pro Glu

180 185 190

Leu His Arg Arg Phe Val Gln Ala Val Glu Gln Leu Gly Ile Asp Lys

195 200 205

Ala Val Pro Ser Arg Ile Leu Glu Ile Met Gly Thr Asp Cys Leu Thr

210 215 220

Arg His Asn Ile Ala Ser His Leu Gln Lys Tyr Arg Ser His Arg Lys

225 230 235 240

His Leu Met Ala Arg Glu Ala Glu Ala Ala Thr Trp Ala Gln Lys Arg

245 250 255

His Met Tyr Ala Pro Pro Ala Pro Arg Thr Thr Thr Thr Thr Asp Ala

260 265 270

Ala Arg Pro Pro Trp Val Val Pro Thr Thr Ile Gly Phe Pro Pro Pro

275 280 285

Arg Phe Cys Arg Pro Leu His Val Trp Gly His Pro Pro Pro His Ala

290 295 300

Ala Ala Ala Glu Ala Ala Ala Ala Thr Pro Met Leu Pro Val Trp Pro

305 310 315 320

Arg His Leu Ala Pro Pro Arg His Leu Ala Pro Trp Ala His Pro Thr

325 330 335

Pro Val Asp Pro Ala Phe Trp His Gln Gln Tyr Ser Ala Ala Arg Lys

340 345 350

Trp Gly Pro Gln Ala Ala Ala Val Thr Gln Gly Thr Pro Cys Val Pro

355 360 365

Leu Pro Arg Phe Pro Val Pro His Pro Ile Tyr Ser Arg Pro Ala Met

370 375 380

Val Pro Pro Pro Pro Ser Thr Thr Lys Leu Ala Gln Leu His Leu Glu

385 390 395 400

Leu Gln Ala His Pro Ser Lys Glu Ser Ile Asp Ala Ala Ile Gly Asp

405 410 415

Val Leu Val Lys Pro Trp Leu Pro Leu Pro Leu Gly Leu Lys Pro Pro

420 425 430

Ser Leu Asp Ser Val Met Ser Glu Leu His Lys Gln Gly Val Pro Lys

435 440 445

Ile Pro Pro Ala Ala Ala Thr Thr Thr Gly Ala Thr Gly

450 455 460

<210> 5

<211> 1386

<212> DNA

<213> corn (Zea mays L.)

<400> 5

atgcttgagg tgtcgacgct gcgcggccct actagcagcg gcagcaaggc ggagcagcac 60

tgcggcggcg gcggcggctt cgtcggcgac caccatgtgg tgttcccgac gtccggcgac 120

tgcttcgcca tggtggacga caacctcctg gactacatcg acttcagctg cgacgtgccc 180

ttcttcgacg ctgacgggga catcctcccc gacctggagg tagacaccac ggagctcctc 240

gccgagttct cgtccacccc tcctgcggac gacctgctgg cagtggcagt attcggcgcc 300

gacgaccagc cggcggcggc agtagcacaa gagaagccgt cgtcgtcgtt ggagcaaaca 360

tgtggtgacg acaaaggtgt agcagtagcc gccgccagaa gaaagctgca gacgacgacg 420

acgacgacga cgacggagga ggaggattct tctcctgccg ggtccggggc caacaagtcg 480

tcggcgtcgg cagagggcca cagcagcaag aagaagtcgg cgggcaagaa ctccaacggc 540

ggcaagcgca aggtgaaggt ggactggacg ccggagctgc accggcggtt cgtgcaggcg 600

gtggagcagc tgggcatcga caaggccgtg ccgtccagga tcctggagat catgggcacg 660

gactgcctca caaggcacaa cattgccagc cacctccaga agtaccggtc gcacagaaag 720

cacctgatgg cgcgggaggc ggaggccgcc acctgggcgc agaagcgcca catgtacgcg 780

ccgccagctc caaggacgac gacgacgacg gacgccgcca ggccgccgtg ggtggtgccg 840

acgaccatcg ggttcccgcc gccgcgcttc tgccgcccgc tgcacgtgtg gggccacccg 900

ccgccgcacg ccgccgcggc tgaagcagca gcggcgactc ccatgctgcc cgtgtggccg 960

cgtcacctgg cgccgccccg gcacctggcg ccgtgggcgc acccgacgcc ggtggacccg 1020

gcgttctggc accagcagta cagcgctgcc aggaaatggg gcccacaggc agccgccgtg 1080

acgcaaggga cgccatgcgt gccgctgccg aggtttccgg tgcctcaccc catctacagc 1140

agaccggcga tggtacctcc gccgccaagc accaccaagc tagctcaact gcatctggag 1200

ctccaagcgc acccgtccaa ggagagcatc gacgcagcca tcggagatgt tttagtgaag 1260

ccatggctgc cgcttccact ggggctcaag ccgccgtcgc tcgacagcgt catgtcggag 1320

ctgcacaagc aaggcgtacc aaaaatccca ccggcggctg ccaccaccac cggcgccacc 1380

ggatga 1386

<210> 6

<211> 4729

<212> DNA

<213> corn (Zea mays L.)

<400> 6

gcaataatgc ttgtgtgtat acagcgacac gacacgagac gagagggagg gtgaactgaa 60

agagagaccc ggctagagct ataaagcaag tgggggaaga gagagagaga ggagaaggag 120

aaggagaggt ttttatgtat gtgtggtgga catgcgtcct cctgtcgtct cgagagagcg 180

gaggccctaa tactccaatc accacgcacc aaactagcta gctagcagtc gtcttgcatt 240

gtagctagtc cattgctctc atcttctctt cttcttcccc cagtcctccc cccctttccc 300

ctcttcggcc tctctcgctc agctcactct tcattaagcg agctcacgtc gttcctccct 360

tcttcttctt cttatccgtt gttcaattcg ttcagctagc cggccagaga tcgagcatca 420

tctccatcat cgattcatcc atctcatctt tctcttcttc tattctatgt cgtcgtcgtc 480

ccagattaga tcgaagctgc tagcagtcta tccagtctct agctagctag ctagatcaag 540

cccgcagact atacaatata atacaagcta gctacgtgct tattattgct ttattagtct 600

agaataatct tgatatatac gatcgaacat atatctcgat ctccaccgat acacacccgg 660

ccctatccat gcttgaggtg tcgacgctgc gcggccctac tagcagcggc agcaaggcgg 720

agcagcactg cggcggcggc ggcggcttcg tcggcgacca ccatgtggtg ttcccgacgt 780

ccggcgactg cttcgccatg gtggacgaca acctcctgga ctacatcgac ttcagctgcg 840

acgtgccctt cttcgacgct gacggggaca tcctccccga cctggaggta gacaccacgg 900

agctcctcgc cgagttctcg tccacccctc ctgcggacga cctgctggca gtggcagtat 960

tcggcgccga cgaccagccg gcggcggcag tagcacaaga gaagccgtcg tcgtcgttgg 1020

agcaaacatg tggtgacgac aaaggtgtag cagtagccgc cgccagaaga aagctgcaga 1080

cgacgacgac gacgacgacg acggaggagg aggattcttc tcctgccggg tccggggcca 1140

acaagtcgtc ggcgtcggca gagggccaca gcagcaagaa gaagtcggcg ggcaagaact 1200

ccaacggcgg caagcgcaag gtgaaggtac taactgctgc tgctagcgct tatatattta 1260

attagctatc ttgctcttgc ttgccctagc tgttgagcta gcatgcacat catatcgcct 1320

gatggatgct acatctatat atctatctat atatgtggtt ttgctactag ttaatttccc 1380

tttcttgcat gctgaagaga aattaattaa gaaaaatcta tatgtgctag ctatttgaaa 1440

ctaattcgaa agattagaca aataaatcta tcagatctga cggccgggcc ggggggagga 1500

gccatcatcg tcgtcgtgca tggagggcaa ttgaagggca atgaatagag ccatgagaat 1560

ccccgtgtac tacatacata ggctcccttt ttccggttgg tcgggcacgg atgaagagga 1620

aaggatcata tcgtcgatag catcacatcc cccatccatc catccatcaa atgaaacacg 1680

cacaagcaga gagagagaga gagagagaga gagaaagaga gtgtcacggg atctcaagca 1740

ccagtgacag tgttctagct catcacccct ctcctctctt tatttcctgc tagtcagaca 1800

cggtacaagt cgcacagagt ccatcttgtc ctgcagctag ctagctagct gccacatgcg 1860

gcatgcatta tgcagccagg acgacaaaat ctacttttta tcgtttacat ttacacgccg 1920

gactgcatac ctgcctgctg ctcatctata tatatggttt tgttggtgtg agtagcactc 1980

ctcatgtcct tggttctact cctcgtgtga gtaaattttt aggcggtggg taaaaaaatc 2040

cactcgtctg tcccacgcca aagcacaggt ttaagacctg gtcgcggtca ttctcacata 2100

gactacggtg tcactatgta tgagtgaggc aggggtttgg aaattttgta gacctacgta 2160

ataaggtctt cttcttgtta acctccacag accaccttat ttgagtatag tactgtacac 2220

tgtggctagc aatctaacat gcatattgcc tgtattttag ttagttaaaa gttgcttatt 2280

tcagtaatat atagaattta catgcaatgc attggtcgat ctgctagctt aatttagctc 2340

catgcaagca tccatctata tatgtatctg atgatgacga cgacacatgt gcattaattt 2400

atgcattatt attgttaatt accaggtgga ctggacgccg gagctgcacc ggcggttcgt 2460

gcaggcggtg gagcagctgg gcatcgacaa ggccgtgccg tccaggatcc tggagatcat 2520

gggcacggac tgcctcacaa ggcacaacat tgccagccac ctccaggtac gtatgcatag 2580

catgtgaatc ttcctagctt gttaattagt tagttcttgc gaatgccaac atatatgtat 2640

atatatatat gtaactattg gacatgtgaa caacgtacaa cccaagcaag catgcatgca 2700

tatttacttt taccgcgcgc tgccaacgac aaagacaaat ctgtactact gtacaagcac 2760

cagcatgatc tgatcgattc tgaggtgtgg taaaagtgaa agcatgcata ttaactaatt 2820

tgaaaaacaa acgtacaact gtacaagtgg tggcatgcat gcatgcatta tgttagcagc 2880

acgcagacgc aggagcaaag aggcaggagg ccagcagcag gcaggcagct agagatcgat 2940

gcaagaaagc aaagcaaatt gtcaatgggg gccgggggca ggcaggcatg catcccgatg 3000

ttgcatgtag cttcttgttc ttggactgga ttggactgca gatattcctt ccgatccctt 3060

ttaatttgca aagagaggtg gtacatacat tacatactac ggccactact agctgcttgc 3120

ttgctttgct ttgcttgtgg ggcgcctctg cactgcactg cactgcatgc ctgtggatca 3180

ccagctgccg agactttctt tagtttgtga tgaatgaatt caaaggaaat tattaccaca 3240

agctagctag ttagctagaa acctcgtcat gtggcatgca ttgatgatgc acaacaataa 3300

taactgctaa atcatttgct tgcagaagta ccggtcgcac agaaagcacc tgatggcgcg 3360

ggaggcggag gccgccacct gggcgcagaa gcgccacatg tacgcgccgc cagctccaag 3420

gacgacgacg acgacggacg ccgccaggcc gccgtgggtg gtgccgacga ccatcgggtt 3480

cccgccgccg cgcttctgcc gcccgctgca cgtgtggggc cacccgccgc cgcacgccgc 3540

cgcggctgaa gcagcagcgg cgactcccat gctgcccgtg tggccgcgtc acctggcgcc 3600

gccccggcac ctggcgccgt gggcgcaccc gacgccggtg gacccggcgt tctggcacca 3660

gcagtacagc gtgagtgctc ctccgtcccc tgctggacct cgcaggcatg catgcatagt 3720

gcgtgcatgt gatccatcca ctctcgcttt tttctaattt ttttttggca gcttgcttcg 3780

tttggctcta ctagctagct agctacactg tgtcgtcgtt gtaattgctt actacctact 3840

gtgattcatt cattgacttg tcttccgcct cccgccccat gcaggctgcc aggaaatggg 3900

gcccacaggc agccgccgtg acgcaaggga cgccatgcgt gccgctgccg aggtttccgg 3960

tgcctcaccc catctacagc agaccggcga tggtacctcc gccgccaagc accaccaagc 4020

tagctcaact gcatctggag ctccaagcgc acccggtaat taataatcta gctatatata 4080

taaatgcacg cattattgtg tgtactgaat atatatatat atatataaat atatatataa 4140

tccaaatgtg caaaacaatg cagtccaagg agagcatcga cgcagccatc ggagatgttt 4200

tagtgaagcc atggctgccg cttccactgg ggctcaagcc gccgtcgctc gacagcgtca 4260

tgtcggagct gcacaagcaa ggcgtaccaa aaatcccacc ggcggctgcc accaccaccg 4320

gcgccaccgg atgacatact atctcgtcga caatacatgc atgtacaata gacggatgtc 4380

tagtagtata gtagattgct gctagctagc tagccgctag agtgtagtag catatgcatg 4440

cctttttttt cttcttcttt ttttgccctt attattaagc tggctaatag cgattgagat 4500

ggagcttgac acagatctgc tagctagcta tttgagggtt tctcttgtat gctacctatt 4560

gctgctgctt gctgctgaga caagtaatgt acgtacgcct gtgcaaacga cacatcggat 4620

tgtattgtat tactagttat atgaacaaca acaataataa taggcacatg catgcctgcc 4680

tagtatgtga gctgctagct agctacatgc atgttgcgca ttcctttcc 4729

Claims

1. Any of the following uses of the protein:

D1) regulating the rice yield or preparing a product for regulating the rice yield; the rice yield is regulated and controlled to improve the rice yield; the yield is the overground biomass or seed yield of the rice;

D2) regulating rice light stress resistance or preparing a product for regulating rice light stress resistance; the regulation and control of the rice light stress resistance is to improve the rice light stress resistance; the light stress is strong light stress;

D3) regulating the running ability of rice D1 protein or preparing a product for regulating the running ability of rice D1 protein; the rice D1 protein transport capacity is regulated and controlled to be improved, namely the rice D1 protein transport capacity is improved;

D4) regulating the content of rice D1 protein or preparing a product for regulating the content of rice D1 protein; the content of the rice D1 protein is regulated and controlled to be increased, namely the content of the rice D1 protein is increased; the content of the D1 protein is the content of D1 in the leaves;

D5) cultivating the rice with increased yield or preparing a product of cultivating the rice with increased yield;

D6) cultivating rice with improved light stress resistance or preparing a product for cultivating rice with improved light stress resistance;

D7) cultivating rice with improved D1 protein transport capacity or preparing products of cultivating rice with improved D1 protein transport capacity;

D8) cultivating rice with increased D1 protein content or preparing a product of cultivating rice with increased D1 protein content;

the protein is A1) or A2) as follows:

A1) the amino acid sequence is the protein of sequence 4;

A2) a1) at the N-terminus or/and the C-terminus.

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

the biomaterial is any one of the following B1) to B7):

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

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

3. Use according to claim 2, characterized in that: B1) the nucleic acid molecule is any one of the following b11) -b 12):

b11) the coding sequence is a DNA molecule of a sequence 5 in a sequence table;

b12) DNA molecule shown in sequence 6 in the sequence table.

4. Any one of the following X1) -X8):

x1) a method for increasing rice yield comprising: expressing the protein of claim 1 in a receptor rice, or increasing the content of the protein of claim 1 in the receptor rice to obtain a target rice with increased yield compared with the receptor rice, thereby realizing the increase of the yield; the yield is the overground biomass or seed yield of the rice;

x2) method for producing rice with increased yield, comprising: expressing the protein of claim 1 in a recipient rice plant, or increasing the content of the protein of claim 1 in the recipient rice plant to obtain a target rice plant with an increased yield as compared with the recipient rice plant; the yield is the overground biomass or seed yield of the rice;

x3) a method for increasing light stress resistance of rice, comprising: expressing the protein of claim 1 in receptor rice or increasing the content of the protein of claim 1 in receptor rice to obtain target rice with improved light stress resistance compared with the receptor rice, thereby realizing the improvement of the light stress resistance of rice; the light stress is strong light stress;

x4) method for breeding rice having increased light stress resistance, comprising: expressing the protein of claim 1 in a recipient rice plant, or increasing the content of the protein of claim 1 in the recipient rice plant to obtain a target rice plant having an increased light stress resistance as compared with the recipient rice plant; the light stress is strong light stress;

x5) a method for improving the protein transport ability of rice D1, comprising: expressing the protein of claim 1 in a recipient rice plant, or increasing the content of the protein of claim 1 in the recipient rice plant to obtain a target rice plant with improved D1 protein transport ability compared with the recipient rice plant, thereby realizing the improvement of the D1 protein transport ability of the rice plant;

x6) method for breeding rice with improved D1 protein transport capacity, comprising: expressing the protein of claim 1 in a recipient rice plant, or increasing the content of the protein of claim 1 in the recipient rice plant to obtain a target rice plant having an improved D1 protein transport ability as compared with the recipient rice plant;

x7) to increase the content of rice D1 protein, comprising: expressing the protein of claim 1 in receptor rice or increasing the content of the protein of claim 1 in receptor rice to obtain target rice with increased content of D1 protein compared with the receptor rice, thereby increasing the content of D1 protein in rice; the content of the D1 protein is the content of D1 in the leaves;

x8) method for cultivating rice with increased content of D1 protein, which comprises the following steps: expressing the protein of claim 1 in a recipient rice plant, or increasing the content of the protein of claim 1 in the recipient rice plant to obtain a target rice plant with an increased content of D1 protein compared to the recipient rice plant; the content of the D1 protein is the content of D1 in the leaves.

5. The method of claim 4, wherein: x1) -X8) by introducing a gene encoding the protein of claim 1 into the recipient rice and expressing the gene.