CN117305353A

CN117305353A - Rice plant type related protein, coding gene and application thereof

Info

Publication number: CN117305353A
Application number: CN202210704514.XA
Authority: CN
Inventors: 万建民; 李帅; 程治军; 刘天珍; 张瑾辉
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: 2022-06-21
Filing date: 2022-06-21
Publication date: 2023-12-29

Abstract

The invention discloses a rice plant type related protein, and a coding gene and application thereof, and belongs to the technical field of genetic engineering breeding. The invention provides an application of an OsMYB86 protein or a substance for regulating and controlling the expression of an OsMYB86 protein coding gene or a substance for regulating and controlling the activity or content of the OsMYB86 protein in regulating and controlling the included angle of sword-leaf leaves of plants; the OsMYB86 protein is a protein with an amino acid sequence of SEQ ID No. 1. The invention also provides a method for regulating plant type and/or sword leaf angle, which comprises regulating plant type and/or sword leaf angle by regulating expression of the OsMYB86 protein coding gene or regulating activity or content of the OsMYB86 protein.

Description

Rice plant type related protein, coding gene and application thereof

Technical Field

The invention belongs to the technical field of genetic engineering breeding, and particularly relates to a rice plant type related protein, and a coding gene and application thereof.

Background

The included angle of the rice leaves refers to the included angle between the leaves and the stems, and the size of the included angle of the rice leaves can influence the photosynthetic efficiency of rice groups, so that the planting density of the rice is influenced, and the yield of the rice is further influenced. The yield problem is always a great problem affecting the grain safety of China, so that the improvement of the rice planting density and the grain yield per unit are significant. The transgenic technology is used for reducing the leaf angle of the rice, and has important significance for increasing the planting density of the rice and increasing the yield and the potential of the rice.

The current research shows that the included angle of rice leaves is mainly influenced by brassinolide, auxin and the mechanical strength of the joint of the leaves.

The reduction of the synthesis of brassinolide or the blockage of signal transmission can lead to the reduction of the leaf angle of rice, and auxin influences the mechanical strength of leaf pillow by crossing with other hormones and affecting the synthesis of cellulose so as to realize the regulation and control of the leaf angle of rice, so that the brassinolide has great scientific significance for the research of leaf angle phenotype.

The transcription factor encodes a DNA binding protein, which activates or inhibits the expression of the downstream gene through the promoter sequence of the direct binding gene so as to participate in various physiological and biochemical processes of rice, and besides, the transcription factor can form a protein complex with other proteins to realize precise and complex regulation of the downstream gene under specific conditions, so that the transcription factor plays an important role in the life process of eukaryotes.

Disclosure of Invention

The invention aims to solve the technical problems of how to regulate plant types, such as how to regulate the included angle of sword-leaf leaves of rice.

To solve the above technical problem, in a first aspect, the present invention provides an application, which may be any one of D1) to D4):

d1 An OsMYB86 protein or a substance regulating the expression of an OsMYB86 protein coding gene or the application of a substance regulating the activity or the content of the OsMYB86 protein in regulating plant types;

d2 An OsMYB86 protein or a substance regulating the expression of an OsMYB86 protein coding gene or the application of a substance regulating the activity or the content of the OsMYB86 protein in preparing a product regulating plant type;

d3 An OsMYB86 protein or a substance for regulating the expression of an OsMYB86 protein coding gene or a substance for regulating the activity or the content of the OsMYB86 protein is applied to regulating the leaf angle of a plant sword;

d4 An OsMYB86 protein or a substance for regulating the expression of an OsMYB86 protein coding gene or a substance for regulating the activity or content of the OsMYB86 protein in the preparation of a product for regulating the leaf angle of a plant sword-leaf;

the OsMYB86 protein can be A1), A2) or A3) as follows:

a1 A protein having an amino acid sequence of SEQ ID No. 1;

a2 A protein which is obtained by substituting and/or deleting and/or adding more than one amino acid residue in the amino acid sequence shown in the A1), has more than 80% of identity with the protein shown in the A1) and has the function of regulating plant type and/or sword leaf angle;

a3 A fusion protein obtained by linking protein tags at the N-terminal or/and C-terminal of A1) or A2).

Further, in the above application, the OsMYB86 protein is derived from rice.

In the invention, the breeding purpose can be to cultivate plants with small sword-leaf included angles.

In the invention, the substance for regulating and controlling the expression of the OsMYB86 protein coding gene can be a substance for inhibiting or reducing or down-regulating the expression of the gene, and the regulation and control of the leaf angle of the plant can be the reduction of the leaf angle of the leaf; the substance for regulating and controlling the expression of the OsMYB86 protein coding gene can be a substance for promoting or improving or up-regulating the expression of the gene, and the regulation and control of the leaf angle of the plant can be increasing the leaf angle of the leaf.

The protein can be synthesized artificially or obtained by synthesizing the coding gene and then biologically expressing.

Wherein SEQ ID No.1 consists of 301 amino acid residues.

In the present invention, the protein tag (protein-tag) refers to a polypeptide or protein which is fused and expressed together with a target protein by using a DNA in vitro recombination technology, so as to facilitate the expression, detection, tracing and/or purification of the target protein. The protein tag may be a Flag tag, his tag, MBP tag, HA tag, myc tag, GST tag, and/or SUMO tag, etc.

In the present invention, the identity refers to the identity of an amino acid sequence or a nucleotide sequence. The identity of amino acid sequences can be determined using homology search sites on the internet, such as BLAST web pages of the NCBI homepage website. For example, in advanced BLAST2.1, the identity of a pair of amino acid sequences can be searched for by using blastp as a program, setting the Expect value to 10, setting all filters to OFF, using BLOSUM62 as Matrix, setting Gap existence cost, per residue gap cost and Lambda ratio to 11,1 and 0.85 (default values), respectively, and calculating, and then obtaining the value (%) of the identity.

In the present invention, the 80% identity or more may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

Further, in the above application, the substance that regulates the expression of the gene encoding the OsMYB86 protein or the substance that regulates the activity or content of the OsMYB86 protein may be a biological material, and the biological material may be any one of the following B1) to B15):

b1 An RNA molecule that inhibits or reduces or down-regulates expression of a gene encoding the OsMYB86 protein or an RNA molecule that inhibits or reduces or down-regulates activity or content of the OsMYB86 protein;

b2 A gene encoding the RNA molecule of B1);

b3 An expression cassette containing the coding gene of B2);

b4 A recombinant vector comprising the coding gene of B2), or a recombinant vector comprising the expression cassette of B3);

b5 A recombinant microorganism comprising the coding gene of B2), or a recombinant microorganism comprising the expression cassette of B3), or a recombinant microorganism comprising the recombinant vector of B4);

b6 A transgenic plant cell line containing B2) the coding gene, or a transgenic plant cell line containing B3) the expression cassette, or a transgenic plant cell line containing B4) the recombinant vector;

b7 A transgenic plant tissue containing B2) the coding gene, or a transgenic plant tissue containing B3) the expression cassette, or a transgenic plant tissue containing B4) the recombinant vector;

b8 A transgenic plant organ containing B2) said coding gene, or a transgenic plant organ containing B3) said expression cassette, or a transgenic plant organ containing B4) said recombinant vector;

b9 A nucleic acid molecule encoding the OsMYB86 protein;

b10 An expression cassette comprising the nucleic acid molecule of B9);

b11 A recombinant vector comprising the nucleic acid molecule of B9), or a recombinant vector comprising the expression cassette of B10);

b12 A recombinant microorganism comprising the nucleic acid molecule of B9), or a recombinant microorganism comprising the expression cassette of B10), or a recombinant microorganism comprising the recombinant vector of B11);

b13 A transgenic plant cell line comprising the nucleic acid molecule of B9), or a transgenic plant cell line comprising the expression cassette of B10), or a transgenic plant cell line comprising the recombinant vector of B11);

b14 A transgenic plant tissue comprising the nucleic acid molecule of B9), or a transgenic plant tissue comprising the expression cassette of B10), or a transgenic plant tissue comprising the recombinant vector of B11);

b15 A transgenic plant organ comprising the nucleic acid molecule of B9), or a transgenic plant organ comprising the expression cassette of B10), or a transgenic plant organ comprising the recombinant vector of B11).

Further, in the above application, the RNA molecule B1) may be RNA transcribed from a DNA molecule represented by formula (I):

SEQ forward-X-SEQ reverse (I);

the SEQ forward direction is a partial fragment of sequence 2 or the full length thereof; the sequence of the SEQ reverse direction is reversely complementary to the sequence of the SEQ forward direction; and X is a spacer sequence between the SEQ forward direction and the SEQ reverse direction, and is not complementary with both the SEQ forward direction and the SEQ reverse direction.

Further, the forward nucleotide sequence of the SEQ is a sequence 4 in a sequence table; the nucleotide sequence of the SEQ reverse direction is a sequence 5 in a sequence table; the sequence 4 in the sequence table is the same as the 510 th to 764 th bit sequence shown in the sequence 2 in the sequence table.

Further, in the above application, the coding gene of B2) is shown in formula (I):

SEQ forward-X-SEQ reverse (I);

the SEQ forward direction is a partial fragment of sequence 2 or the full length thereof; the sequence of the SEQ reverse direction is reversely complementary to the sequence of the SEQ forward direction; the X is a spacer sequence between the SEQ forward direction and the SEQ reverse direction, and is not complementary to both the SEQ forward direction and the SEQ reverse direction;

b9 The nucleic acid molecule is a DNA molecule as shown in b 1) or b 2) below:

b1 A DNA molecule with the coding sequence of the coding strand shown in SEQ ID No. 2;

b2 The nucleotide sequence of the coding strand is a DNA molecule shown as SEQ ID No. 3.

Further, the forward nucleotide sequence of the SEQ is a sequence 4 in a sequence table; the nucleotide sequence of the SEQ reverse direction is a sequence 5 in a sequence table; the sequence 4 in the sequence table is the same as the sequence shown in the 510 th to 764 th bits of the sequence 2 in the sequence table.

Further, in the above application, the plant is any one of the following P1) -P4):

p1), monocot or dicot;

p2), gramineae;

p3), oryza plants;

p4), rice.

In order to solve the technical problem, in a second aspect, the invention provides a method for regulating plant type and/or leaf angle, which comprises regulating plant type and/or leaf angle by regulating expression of an OsMYB86 protein coding gene or regulating activity or content of the OsMYB86 protein.

Further, in the above method, the method comprises M1) and/or M2):

m1), introducing a coding gene shown in a formula (I) into a receptor plant, and inhibiting or reducing or downregulating the expression of the OsMYB86 protein coding gene or the activity or content of the OsMYB86 protein to obtain a target plant with a sword-leaf angle smaller than that of the receptor plant;

m2), introducing the encoding gene of the OsMYB86 protein into a receptor plant, and promoting or improving or up-regulating the expression of the encoding gene of the OsMYB86 protein or the activity or content of the OsMYB86 protein to obtain a target plant with a plant sword-leaf angle larger than that of the receptor plant.

In the invention, the inhibition or reduction or downregulation of the expression of the gene encoding the OsMYB86 protein can be realized through gene knockout or gene silencing.

The gene knockout (geneknockout) refers to a phenomenon in which a specific target gene is inactivated by homologous recombination. Gene knockout is the inactivation of a particular target gene by a change in DNA sequence.

The gene silencing refers to the phenomenon that the gene is not expressed or expressed under the condition of not damaging the original DNA. Gene silencing is premised on the fact that the DNA sequence is not altered, so that the gene is not expressed or is underexpressed. Gene silencing can occur at two levels, one is gene silencing at the transcriptional level due to DNA methylation, heterochromatin, and positional effects, and the other is post-transcriptional gene silencing, i.e., inactivation of a gene by specific inhibition of a target RNA at the post-transcriptional level of the gene, including antisense RNA, co-suppression (co-suppression), gene suppression (sequencing), RNA interference (RNAi), and microrna (miRNA) -mediated translational inhibition, among others.

Further, in the above method, the plant is any one of the following P1) -P4):

p1), monocot or dicot;

p2), gramineae;

p3), oryza plants;

p4), rice.

In order to solve the above technical problems, the third aspect of the present invention provides the above OsMYB86 protein or the above biological material.

In the present invention, the plant tissue of B7) or B14) may be derived from roots, stems, leaves, flowers, fruits, seeds, pollen, embryos and anthers.

In the present invention, the transgenic plant organ of B8) or B15) may be the root, stem, leaf, flower, fruit and seed of a transgenic plant.

In the present invention, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs may or may not include propagation material.

In the above method, the introduction of the coding gene into the recipient plant may be specifically: plant cells or tissues are transformed by conventional biological methods using Ti plasmids, ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium-mediated, etc., and the transformed plant tissues are grown into plants. Transformed cells, tissues or plants are understood to include not only the end product of the transformation process but also the material obtained by asexual propagation thereof and transgenic progeny.

The nucleotide sequence encoding the OsMYB86 protein of the present invention can be easily mutated by one of ordinary skill in the art using known methods such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or more identity to the nucleotide sequence of the OsMYB86 protein isolated by the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the OsMYB86 protein and function as the OsMYB86 protein.

In the above applications, the expression cassette (the expression cassette of the OsMYB86 gene) containing the nucleic acid molecule encoding the OsMYB86 protein described in B10) refers to DNA capable of expressing the OsMYB86 protein in a host cell, and the DNA may include not only a promoter for promoting the transcription of the OsMYB86 gene but also a terminator for terminating the transcription of the OsMYB86 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: constitutive promoter T7lac, constitutive promoter of cauliflower mosaic virus CaMV35S, tomato ribulose-1, 5-bisphosphate carboxylase small subunit (smallsubenitofacicular-1, 5-bisporcarboxidase, rbcs) gene promoter; wound-inducible promoter from tomato, leucine aminopeptidase ("LAP", chao et al (1999) plant Physiol 120:979-992); a chemically inducible promoter from tobacco, pathogenesis-related 1 (PR 1) (induced by salicylic acid and BTH (benzothiadiazole-7-carbothioic acid S-methyl ester); tomato protease inhibitor II promoter (PIN 2) or LAP promoter (both inducible with methyl jasmonate); heat shock promoters (U.S. Pat. No. 5,187,267); tetracycline-inducible promoters (U.S. Pat. No. 5, 057,422); seed-specific promoters, such as the millet seed-specific promoter pF128 (CN 101063139B (China patent 200710099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin, and soybean betacon (Beachy et al (1985) EMBO J. 4:3047-3053)). They may 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: t7 terminator, agrobacterium tumefaciens nopaline synthase terminator (NOS terminator), caMV35S terminator, tml terminator, pea rbcSE9 terminator and nopaline and octopine synthase terminator (see, e.g., odell et al (1985), nature,313:810; rosenberg et al (1987), gene,56:125; guerineau et al (1991), mol. Gen. Genet. 262:141; proudfot (1991), cell,64:671; sanfacon et al, genesDev.,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; shi et al (1987), nucleic acids Res., 15:9627).

The existing expression vector can be used for constructing a recombinant vector containing the OsMYB86 gene expression cassette. The plant expression vector comprises a binary agrobacterium vector, a vector which can be used for plant microprojectile bombardment and the like. Such as pET-28a, pCAMBIA2301, pSP72, pROKII, pBin438, pCAMBIA1302, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA). The plant expression vector may also comprise the 3' -untranslated region of a foreign gene, i.e., comprising a polyadenylation signal and any other DNA segments involved in mRNA processing or gene expression. The polyadenylation signal may direct the addition of polyadenylation to the 3 'end of the mRNA precursor and may function similarly to the 3' transcribed untranslated regions of Agrobacterium tumefaciens induction (Ti) plasmid genes (e.g., nopaline synthase gene Nos) and plant genes (e.g., soybean storage protein genes). 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 enhancers may be ATG initiation codon or adjacent region initiation codon, etc., but must be identical to the reading frame of the coding sequence to ensure proper translation of the entire sequence. The sources of the translational control signals and initiation codons are broad, and can be either natural or synthetic. The translation initiation region may be derived from a transcription initiation region or a structural gene. To facilitate identification and selection of transgenic plant cells or plants, the plant expression vectors used may be processed, for example by adding genes encoding enzymes or luminescent compounds which produce a color change (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene conferring resistance to kanamycin and related antibiotics, bar gene conferring resistance to the herbicide phosphinothricin, hph gene conferring resistance to antibiotic hygromycin, dhfr gene conferring resistance to methotrexate, EPSPS gene conferring resistance to glyphosate) or chemical marker genes, etc. (such as herbicide resistance genes), mannose-6-phosphate isomerase gene providing mannose metabolization ability, etc. From the safety of transgenic plants, transformed plants can be screened directly in stress without adding any selectable marker gene.

In one embodiment of the invention, the plant expression vector is a pcambia1305.1 vector.

In one embodiment of the invention, B11) the recombinant vector is pCAMBIA1305.1-OsMYB86. The pCAMBIA1305.1-OsMYB86 can express an OsMYB86 protein shown in a sequence 1 in a sequence table, and the expression of the protein is driven by a constitutive promoter CaMV35S of cauliflower mosaic virus. The pCAMBIA1305.1-OsMYB86 is a recombinant vector obtained by inserting a DNA fragment shown in a sequence 2 in a sequence table into a multiple cloning site of the pCAMBIA1305.1 vector. The pCAMBIA1305.1-OsMYB86 is a recombinant vector obtained by replacing a small fragment between EcoRI and Ncoll recognition sequences with a DNA fragment shown in a sequence 2 of a sequence table.

Recombinant vectors containing DNA molecules for reducing the expression level of OsMYB86, such as pCubi 1390-delta FAD2 vectors, can be constructed by using existing RNA interference vectors. B23 The recombinant vector may specifically be pCubi 1390-delta FAD2-OsMYB86. The pCUbi 1390-delta FAD2-OsMYB86 is a recombinant vector obtained by utilizing Sac1I restriction endonuclease to insert a DNA fragment shown in sequence 4 in a sequence table in the forward direction in a multiple cloning site of the pCUbi 1390-delta FAD2 vector and utilizing SnaBI restriction endonuclease to insert a DNA fragment shown in sequence 5 in the sequence table in the reverse direction in the multiple cloning site of the pCUbi 1390-delta FAD2 vector.

In the above application, the microorganism may be yeast, bacteria, algae or fungi. Wherein the bacteria may be derived from Escherichia (Escherichia), erwinia (Erwinia), agrobacterium (Agrobacterium) such as Agrobacterium tumefaciens EHA105, flavobacterium (Flavobacterium), alcaligenes (Alcaligenes), pseudomonas (Pseudomonas), bacillus (Bacillus), etc.

The beneficial technical effects obtained by the invention are as follows:

1. the application of the OsMYB86 protein in regulating plant type and/or sword leaf angle is provided; reducing the expression of the OsMYB86 protein through RNA interference can reduce the leaf angle of the sword-leaved tree, and increasing the leaf angle of the receptor plant through over-expression of the OsMYB86 protein in the receptor plant; provides a new target for the regulation and breeding of rice plant types.

2. The method for regulating and controlling the sword-leaf angle of the plants can obtain the rice with the sword-leaf angle increased or reduced, enriches the means of rice breeding and provides research materials for rice breeding.

Drawings

FIG. 1 is a vector map of recombinant expression vector pCAMBIA1305GFP-OsMYB86.

FIG. 2 shows the Kitaake phenotype assay results for wild type, osMYB86 overexpression and OsMYB 86-RNAi.

Detailed Description

The following detailed description of the invention is provided in connection with the accompanying drawings that are presented to illustrate the invention and not to limit the scope thereof. The examples provided below are intended as guidelines for further modifications by one of ordinary skill in the art and are not to be construed as limiting the invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, and are carried out according to techniques or conditions described in the literature in the field or according to the product specifications. Materials, reagents and the like used in the examples described below are commercially available unless otherwise specified.

The japonica rice variety kitaake is preserved in the laboratory, belongs to common rice varieties, and is disclosed in documents Wu Yan, tangning and Zhang Bianjiang, namely, the influence of nitrogen deficiency on photosynthetic characteristics of different japonica rice varieties [ J ]. Hubei agricultural science, 2014 (8): 1762-1764; the public can obtain the materials from the crop science research institute of national academy of agricultural science, and the obtained materials can only be used for verifying the experiment of the invention and cannot be used as other purposes. The pCAMBIA1305GFP vector was stored in the laboratory and is disclosed in the literature "Cai, m., zhu, s, wu, m., zheng, x, wang, j, zhou, l, zheng, t, cui, s, zhou, s, li, c, et al (2021),. DHD4, a CONstans-like family transcription factor, delays heading date by affecting the formation of the FAC complex in rice. Mol Plant 14:330-343", which is available to the public from the applicant, and the resulting material was only useful for validating the experiments of the present invention and was not useful for other applications.

The pCubi 1390-delta FAD2 vector was stored for this laboratory in literature: "Tan, j., tan, z., et al, anovelchloroplast-localized pentatricopeptide repeat protein involved in splicingaffects chloroplast development and abiotic stress response in gear.mol.plant, 2014,7:1329-1349," which is available to the public from applicant, the resulting material is only useful for validating the experiments of the present invention, and not for other uses.

The quantitative tests in the following examples were all set up in triplicate and the results averaged.

The following examples used Excel statistical software to process data, with experimental results expressed as mean ± standard deviation, and Two-way t-test, with significant differences (P < 0.05), with very significant differences (P < 0.01), and with very significant differences (P < 0.001).

Example 1 OsMYB86 protein and encoding gene thereof

The OsMYB86 protein is a protein with an amino acid sequence shown as a sequence 1 in a sequence table; the nucleotide sequence of the coding sequence is sequence 2 in the sequence table; the nucleotide sequence of the genome where the coding sequence is located is a sequence 3 in a sequence table. The encoding gene of the OsMYB86 protein is derived from rice.

Example 2, osMYB86 protein and application of encoding gene thereof

2.1 construction of the overexpression vector

The small fragment between SpeI and BamHI recognition sequences of pCAMBIA1305GFP vector was replaced with a double-stranded DNA molecule shown in sequence 2 of the sequence table to obtain a recombinant vector, and the recombinant vector with correct sequencing was designated pCAMBIA1305GFP-OsMYB86. The pCAMBIA1305GFP-OsMYB86 contains a DNA fragment shown in a sequence 2 in a sequence table and a constitutive promoter CaMV35S of the cauliflower mosaic virus, can express an OsMYB86 protein shown in a sequence 1 in the sequence table, and is driven by the constitutive promoter CaMV35S of the cauliflower mosaic virus. The map of the recombinant expression vector pCAMBIA1305GFP-OsMYB86 is shown in FIG. 1.

2.2 construction of RNAi interference vector

Double-stranded DNA molecules (sense fragments) shown in sequence 4 of a sequence table are inserted between SacI cleavage sites of the pCUbi 1390-delta FAD2 vector, and double-stranded DNA molecules (antisense fragments) shown in sequence 5 of the sequence table are inserted between SnaBI cleavage sites, so that an RNAi interference vector pCUbi 1390-delta FAD2-OsMYB86 (which is verified by sequencing) is obtained.

Vector pCUbi 1390-delta FAD2-OsMYB86 contains coding gene shown in formula (I)

SEQ forward-X-SEQ reverse (I);

the forward nucleotide sequence of the SEQ is a sequence 4 in a sequence table; the nucleotide sequence of the SEQ reverse direction is a sequence 5 in a sequence table; the sequence of the SEQ reverse direction is reversely complementary to the sequence of the SEQ forward direction. And X is a spacer sequence between the SEQ forward direction and the SEQ reverse direction, and is not complementary with both the SEQ forward direction and the SEQ reverse direction. The sequence 4 in the sequence table is the same as the 510 th to 764 th bit sequence shown in the sequence 2 in the sequence table.

2.3 construction and identification of transgenic OsMYB86 plants

2.3.1, respectively introducing the recombinant vector pCAMBIA1305.1-OsMYB86 and the recombinant vector pCubi 1390-delta FAD2-OsMYB86 obtained from 2.1 and 2.2 into an agrobacterium EHA105 strain to obtain recombinant agrobacterium EHA105/pCAMBIA1305.1-OsMYB86 and EHA105/pCubi 1390-delta FAD2-OsMYB86.

2.3.2 preparation of Agrobacterium transformed plants

The recombinant agrobacterium obtained by 2.3.1 is used for respectively transforming japonica rice variety kitaake (wild type), and the specific steps are as follows:

(1) Respectively taking 2.3.1 recombinant agrobacterium EHA105/pCAMBIA1305.1-OsMYB86 and EHA105/pCUbi 1390-delta FAD2-OsMYB86, re-suspending with N6 liquid medium (Sigma Co., C1416) and adjusting the OD600nm of the bacterial liquid to 0.5;

(2) Infecting the mature embryo embryogenic callus of japonica rice variety kitaake (wild type, WT) cultivated to one month in the bacterial liquid obtained in the step (1) for 30min, transferring the callus into a solid N6 culture medium (Sigma Co., C1416) containing 10g/L agar after the bacterial liquid is absorbed by filter paper, and co-culturing at 24 ℃ for 3 days;

(3) Inoculating the callus cultured in the step (2) on a solid screening N6 solid medium containing 10g/L agar and 100mg/L hygromycin for 16 days (first screening);

(4) Inoculating the healthy callus cultured in the step (3) on a solid screening N6 medium containing 10g/L agar and 100mg/L hygromycin for 15 days (second screening);

(5) Inoculating the healthy callus cultured in the step (4) on a solid screening N6 medium containing 10g/L agar and 100mg/L hygromycin for 15 days (third screening);

(6) And (3) inoculating the healthy callus cultured in the step (5) on a differentiation medium (PhytoTechnology laboratories, M524) for differentiation culture to obtain a plant transformed with the OsMYB86 gene of the T0 generation and a plant subjected to RNA interference of the T0 generation (OsMYB 86-RNAi) respectively. Selfing the T0 generation plant to obtain a T1 generation plant, wherein the T1 generation plants of the OsMYB86 gene transferred plants are respectively named as OE-N, and N is the plant number; t1 generation plants with RNA interference (OsMYB 86-RNAi) are respectively named as R-N, and N is the plant number.

2.3.3 phenotypic assay

(1) Detection of relative expression level of target Gene

And (3) detecting the expression level of the OsMYB86 gene of the T1 generation transferred OsMYB86 gene plant OE-N obtained in 2.3.2 and the RNA interference (OsMYB 86-RNAi) plant R-N and the wild japonica rice variety kitaake. Extracting RNA of the occipital part of the plant growing for about 60 days, reversely transcribing the RNA into cDNA, taking the cDNA as a template, carrying out quantitative PCR, detecting the expression level of the OsMYB86 gene by using a primer OsMYB86-F and a primer OsMYB86-R, and detecting the expression level of a Ubiquitin gene (internal reference) by using a primer Ubi-F and a primer Ubi-R.

Primer OsMYB86-F:5'-AGGCTTTGGCACTGGCTCTGA-3';

primer OsMYB86-R:5'-ACGGCAGCATCGTCCTTGAAAG-3';

primer Ubi-F:5'-AGGCAACAGGTGGTCGCAAATC-3';

primer Ubi-R:5'-GCTTCTTCTTGAGGCAGCTGTTCC-3'.

The plants with the expression level of the OsMYB86 gene in the recombinant vector pCAMBIA1305.1-OsMYB86 plants being obviously higher than that of the control are the positive plants of the OsMYB86 gene, and three T1 generation-transferred OsMYB86 gene positive plants OE-1, OE-2 and OE-3 are selected for leaf angle measurement; and (3) transferring pCUbi 1390-delta FAD2-OsMYB86 gene expression level which is obviously lower than that of a control plant to obtain an RNAi positive plant, and selecting three T1 generation RNAi positive plants R-1, R-2 and R-3 for sword leaf angle measurement.

(2) Sword leaf included angle size measurement

Selecting 16 plants of the wild type and the transgenic family main spike completely extracted, cutting the main spike and the sword leaf completely, photographing, measuring the angle between the main spike and the sword leaf by using software ImgeJ, and taking an average value of 16 measurement results of each family and obtaining the angle of the sword leaf included angle of the family.

The results are shown in FIG. 2, and FIG. 2 shows the Kitaake phenotypes of wild type, osMYB86 overexpression and OsMYB 86-RNAi. In FIG. 2, kit shows that Kitaake wild type, OE-1, OE-2 and OE-3 are respectively Kitaake for over-expressing OsMYB86 protein, RNAi-1, RNAi-2 and RNAi-3 are respectively Kitaake for reducing OsMYB86 protein expression by RNA interference; wherein a in fig. 2 is the Kitaake phenotype assay result of wild type and OsMYB86 overexpression, from top to bottom: photographs of sword-leaf angles of Kitaake over-expressed by wild type and OsMYB86, relative expression quantity of OsMYB86 coding genes and angle of sword-leaf angles; FIG. 2B shows the Kitaake phenotypes of wild type and OsMYB86-RNAi, from top to bottom: photographs of wild type and OsMYB86-RNAi Kitaake sword-leaf angle, angle of sword-leaf angle, and relative expression level of OsMYB86 coding gene.

The results show that: the expression level of OsMYB86 genes of the OsMYB86 gene-transferred positive plants OE-1, OE-2 and OE-3 is obviously higher than that of the Wild Type (WT), and the included angle of sword-leaved leaves of OE-1, OE-2 and OE-3 is obviously larger than that of the wild type (P < 0.01); RNAi positive plants RNAi-1, RNAi-2 and RNAi-3 have significantly lower OsMYB86 gene expression levels than (P < 0.01) Wild Type (WT), and RNAi-1, RNAi-2 and RNAi-3 have significantly lower sword-leaf angles than wild type (P < 0.01).

The present invention is described in detail above. It will be apparent to those skilled in the art that the present invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with respect to specific embodiments, it will be appreciated that the invention may be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The application of some of the basic features may be done in accordance with the scope of the claims that follow.

Sequence listing

<110> institute of crop science at national academy of agricultural sciences

<120> a rice plant type related protein, and coding gene and application thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 301

<212> PRT

<213> Rice (Oryza sativa L.)

<400> 1

Met Gly Arg His Ala Cys Ser Ala Ala Gly Val Gln Gln Lys Leu Arg

1 5 10 15

Lys Gly Leu Trp Ser Pro Glu Glu Asp Glu Lys Leu Tyr Asn His Ile

20 25 30

Tyr Arg Tyr Gly Val Gly Cys Trp Ser Ser Val Pro Lys Leu Ala Gly

35 40 45

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

50 55 60

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

65 70 75 80

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

85 90 95

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

100 105 110

Ser Cys Leu Lys Lys Lys Leu Arg Gln Arg Gly Ile Asp Pro Ser Thr

115 120 125

His Gln Pro Ile Ser Thr Ala Ala Ala Ala Ala Ala Ala Ala Leu Asp

130 135 140

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

145 150 155 160

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

165 170 175

Phe Gly Ser Gly Phe Asp Ala Thr Gly Met Pro Leu Tyr Gly His Leu

180 185 190

Gly Gly Lys Asp Ala Ala Gly Phe Val Asp Tyr Ser Ser Val Leu Asp

195 200 205

Val Ser Glu Asn Leu Gly Tyr Gly Glu Ser Ser Ser Asn Ser Ser Asn

210 215 220

Trp Asn Cys Gly Val Gly Ala Pro Glu Val Asn Asn Ala Leu Glu Ser

225 230 235 240

Glu Pro Leu His Trp Ala Thr Glu Ser Lys Val Glu Pro Phe Val Gly

245 250 255

Tyr Gly Glu Gly Asp Ala Met Glu His Lys Phe Gly Leu Pro Cys His

260 265 270

Gly Gln Gln Glu Gln Gly Met Thr His Phe Asp Phe Asp Val Ser Arg

275 280 285

Ser Met Val Val Gly Asp Phe Asn Phe Glu Tyr Phe Arg

290 295 300

<210> 2

<211> 906

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 2

atgggacggc acgcgtgctc tgccgccggt gtccagcaga agctgcgcaa ggggctctgg 60

tctcccgagg aagacgagaa gctctacaat cacatctacc gctacggcgt cggttgctgg 120

agctccgttc ccaagctcgc agggctccag aggtgcggca agagctgcag gctgcggtgg 180

atcaactacc tgcgccccga cctgaagcgg ggcagcttct cgcagcagga ggaggacgcc 240

atcgtcggcc tgcatgagat cctaggcaac aggtggtcgc aaatcgcgtc gcacttgccg 300

gggaggacgg acaacgagat caagaacttc tggaacagct gcctcaagaa gaagctgagg 360

cagcgcggca tcgaccccag cacccaccag cccatctcca cggctgctgc tgctgcggcg 420

gcggcattgg acacgtcgac gcaggaccag aagccgccgg ccaccgccga cggcttcgcc 480

ctgaagcagc agcagcaggt gttcgacccg ttcccggtga tcgacagctt cggcagcggg 540

ttcgacgcca cgggcatgcc attgtacggc cacctcggcg gcaaggacgc ggccgggttc 600

gtggactaca gcagcgtgct tgacgtgtcg gagaacctgg gctacggcga gagctccagc 660

aacagcagca actggaactg cggcgtgggc gcgccggagg tgaacaatgc gctcgagagc 720

gagccgctgc attgggccac cgagagcaag gtcgagcctt tcgtgggcta cggcgagggg 780

gacgccatgg agcacaagtt cgggctgccc tgccatgggc agcaagagca gggcatgaca 840

catttcgact tcgacgtcag ccggagcatg gtggtcggcg acttcaactt cgagtacttc 900

cgatga 906

<210> 3

<211> 1130

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 3

atgggacggc acgcgtgctc tgccgccggt gtccagcaga agctgcgcaa ggggctctgg 60

tctcccgagg aagacgagaa gctctacaat cacatctacc gctacggcgt cggttgctgg 120

agctccgttc ccaagctcgc aggtcgtaag ctgctcctgc tctgctcgat ccgctctttg 180

catgcatgtg tctcagtgtc tcatcgctga cccgtgccgt gcaacgatct tcttctgtgc 240

tccagggctc cagaggtgcg gcaagagctg caggctgcgg tggatcaact acctgcgccc 300

cgacctgaag cggggcagct tctcgcagca ggaggaggac gccatcgtcg gcctgcatga 360

gatcctaggc aacaggttcg tacgtactgc cgctccggca ataatgcgta ccagtactat 420

tttcgtcgca atgtggttgt gtggaggtga tttgttagcg ataagtggcc tgatcagcga 480

ggcgaatccg atgcaggtgg tcgcaaatcg cgtcgcactt gccggggagg acggacaacg 540

agatcaagaa cttctggaac agctgcctca agaagaagct gaggcagcgc ggcatcgacc 600

ccagcaccca ccagcccatc tccacggctg ctgctgctgc ggcggcggca ttggacacgt 660

cgacgcagga ccagaagccg ccggccaccg ccgacggctt cgccctgaag cagcagcagc 720

aggtgttcga cccgttcccg gtgatcgaca gcttcggcag cgggttcgac gccacgggca 780

tgccattgta cggccacctc ggcggcaagg acgcggccgg gttcgtggac tacagcagcg 840

tgcttgacgt gtcggagaac ctgggctacg gcgagagctc cagcaacagc agcaactgga 900

actgcggcgt gggcgcgccg gaggtgaaca atgcgctcga gagcgagccg ctgcattggg 960

ccaccgagag caaggtcgag cctttcgtgg gctacggcga gggggacgcc atggagcaca 1020

agttcgggct gccctgccat gggcagcaag agcagggcat gacacatttc gacttcgacg 1080

tcagccggag catggtggtc ggcgacttca acttcgagta cttccgatga 1130

<210> 4

<211> 295

<212> DNA

<213> Rice (Oryza sativa L.)

<400> 4

gttcccggtg atcgacagct tcggcagcgg gttcgacgcc acgggcatgc cattgtacgg 60

ccacctcggc ggcaaggacg cggccgggtt cgtggactac agcagcgtgc ttgacgtgtc 120

ggagaacctg ggctacggcg agagctccag caacagcagc aactggaact gcggcgtggg 180

cgcgccggag gtgaacaatg cgctcgagag cgagccgctg cattgggcca ccgagagcaa 240

ggtcgagcct ttcgtgggct acggcgaggg ggacgccatg gagcacaagt tcggg 295

<210> 5

<211> 295

<212> DNA

<213> Artificial sequence (Artificial Sequence)

<400> 5

cccgaacttg tgctccatgg cgtccccctc gccgtagccc acgaaaggct cgaccttgct 60

ctcggtggcc caatgcagcg gctcgctctc gagcgcattg ttcacctccg gcgcgcccac 120

gccgcagttc cagttgctgc tgttgctgga gctctcgccg tagcccaggt tctccgacac 180

gtcaagcacg ctgctgtagt ccacgaaccc ggccgcgtcc ttgccgccga ggtggccgta 240

caatggcatg cccgtggcgt cgaacccgct gccgaagctg tcgatcaccg ggaac 295

Claims

1. The application is characterized in that: the application is any one of D1) to D4),

the OsMYB86 protein is A1), A2) or A3) as follows:

a1 A protein having an amino acid sequence of SEQ ID No. 1;

2. The use according to claim 1, characterized in that: the OsMYB86 protein is derived from rice.

3. Use according to claim 1 or 2, characterized in that: the substance regulating the expression of the gene encoding the OsMYB86 protein or the substance regulating the activity or content of the OsMYB86 protein is a biological material, and the biological material is any one of the following B1) to B15):

b2 A gene encoding the RNA molecule of B1);

b3 An expression cassette containing the coding gene of B2);

b9 A nucleic acid molecule encoding the OsMYB86 protein;

b10 An expression cassette comprising the nucleic acid molecule of B9);

4. A use according to claim 3, characterized in that: b1 The RNA molecule is RNA transcribed from a DNA molecule shown as a formula (I):

SEQ forward-X-SEQ reverse (I);

the SEQ forward direction is a partial fragment of sequence 2; the sequence of the SEQ reverse direction is reversely complementary to the sequence of the SEQ forward direction; and X is a spacer sequence between the SEQ forward direction and the SEQ reverse direction, and is not complementary with both the SEQ forward direction and the SEQ reverse direction.

5. A use according to claim 3, characterized in that: b2 The coding gene is shown as a formula (I):

SEQ forward-X-SEQ reverse (I);

the SEQ forward direction is a partial fragment of sequence 2; the sequence of the SEQ reverse direction is reversely complementary to the sequence of the SEQ forward direction; the X is a spacer sequence between the SEQ forward direction and the SEQ reverse direction, and is not complementary to both the SEQ forward direction and the SEQ reverse direction;

b9 The nucleic acid molecule is a DNA molecule as shown in b 1) or b 2) below:

6. The use according to any one of claims 1-5, characterized in that: the plant is any one of the following P1) -P4):

p1), monocot or dicot;

p2), gramineae;

p3), oryza plants;

p4), rice.

7. A method for regulating plant type and/or leaf angle, which is characterized in that: the method comprises the step of regulating plant type and/or sword leaf angle by regulating expression of the OsMYB86 protein coding gene or regulating activity or content of the OsMYB86 protein.

8. The method according to claim 7, wherein: the method comprises M1) and/or M2):

9. The method according to claim 7 or 8, characterized in that: the plant is any one of the following P1) -P4):

p1), monocot or dicot;

p2), gramineae;

p3), oryza plants;

p4), rice.

10. The protein in the use of claim 1 or 2 or the biological material in the use of any one of claims 3-5.