OsEME1 protein capable of changing leaf color and plant height of rice as well as encoding gene and application of OsEME1 protein

Abstract

The invention discloses an OsEME1 protein capable of changing leaf color and plant height of rice as well as a coding gene and application of the OsEME1 protein. The protein is A1) or A2) or A3) or A4) as follows: A1) a protein with an amino acid sequence as shown in SEQ ID NO: 2; a2) a protein which is obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence as shown in SEQ ID NO: 2 and has the same function; a3) a protein which has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more of identity with the amino acid sequence limited by any one of A1)-A2) and has the same function; and A4) a fusion protein obtained by connecting a label to the N end and / or C end of any protein defined in A1)-A3). Tests prove that the OsEME1 gene deleted rice plant shows obvious dwarfing and leaf color difference.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to an OsEME1 protein for changing rice leaf color and plant height, and a coding gene and application thereof. Background Art

[0002] Rice is the main food crop in my country. Changes in climate and environment have also brought great challenges to increasing crop yields. Therefore, high-yield crop breeding has always been one of the important goals of agricultural production. By improving rice plant type, such as plant height, number of stems and branches (tillers), number of panicles, etc., rice yield can be significantly increased. Using "dwarfing genes" to cultivate short-stem rice can not only ensure the high-yield characteristics of rice, but also improve the lodging resistance of the plant, thereby improving the theoretical basis for breeding more widely adaptable and excellent varieties, and providing the possibility of introducing more excellent varieties into these main rice producing areas. Summary of the invention

[0003] The technical problem to be solved by the present invention is how to regulate the leaf color and plant height of plants.

[0004] In order to solve the above technical problems, the present invention first provides an application of a protein related to plant leaf color / plant height in regulating plant leaf color / plant height.

[0005] The protein related to plant leaf color and plant height provided by the present invention is any one of the following proteins A1) or A2) or A3) or A4):

[0006] A1) the protein whose amino acid sequence is shown in SEQ ID NO: 2;

[0007] A2) a protein having the same function as that of the amino acid sequence shown in SEQ ID NO: 2 after one or more amino acid residues are substituted and / or deleted and / or added;

[0008] A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more identity with the amino acid sequence defined in any one of A1) to A2) and having the same function;

[0009] A4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of any of the proteins defined in A1) to A3).

[0010] Among them, SEQ ID NO: 2 consists of 650 amino acid residues.

[0011] In order to facilitate the purification of the protein in a), a tag as shown in Table 1 may be connected to the amino terminus or carboxyl terminus of the protein shown in SEQ ID NO: 2 in the sequence listing.

[0012] Table 1. Sequence of tags

[0013] Label Residue sequence Poly-Arg 5-6 (usually 5) RRRRR (SEQ ID NO: 15) Poly-His 2-10 (usually 6) HHHHHH (SEQ ID NO: 16) FLAG 8 DYKDDDDK (SEQ ID NO: 17) Strep-tag II 8 WSHPQFEK (SEQ ID NO: 18) c-myc 10 EQKLISEEDL (SEQ ID NO: 19)

[0014] The substitution and / or deletion and / or addition of one or several amino acid residues is a substitution and / or deletion and / or addition of no more than 10 amino acid residues.

[0015] The protein is named OsEME1, which can be synthesized artificially or its encoding gene can be synthesized first and then expressed biologically.

[0016] The coding gene of the above protein OsEME1 can be obtained by deleting one or several amino acid residue codons in the DNA sequence shown in SEQ ID NO: 1, and / or performing one or several base pair missense mutations, and / or connecting the coding sequence of the tag shown in Table 1 to its 5′ end and / or 3′ end.

[0017] The present invention also provides the use of a biological material related to the above protein in regulating plant leaf color and / or plant height, wherein the biological material is any one of the following B1)-B10):

[0018] B1) a nucleic acid molecule encoding the above protein;

[0019] B2) an expression cassette containing the nucleic acid molecule described in B1);

[0020] B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2);

[0021] B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3);

[0022] B5) a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3);

[0023] B6) transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3);

[0024] B7) a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3);

[0025] B8) a transgenic plant containing the nucleic acid molecule described in B1), or a transgenic plant containing the expression cassette described in B2), or a transgenic plant containing the recombinant vector described in B3);

[0026] B9) tissue culture produced from regenerable cells of the transgenic plant described in B8);

[0027] B10) Protoplasts produced from the tissue culture described in B9).

[0028] The above nucleic acid molecule is a cDNA molecule or a DNA molecule.

[0029] The nucleic acid molecule described in B1) above is any one of the following C1)-C3):

[0030] C1) cDNA molecule or DNA molecule shown in SEQ ID NO: 1;

[0031] C2) a cDNA molecule or a genomic DNA molecule that hybridizes with the DNA molecule defined in C1) under stringent conditions and encodes the above-mentioned protein;

[0032] C3) A cDNA molecule or genomic DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with the DNA sequence defined in C1) or C2) and encodes the above-mentioned protein.

[0033] 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.

[0034] Among them, SEQ ID NO:1 consists of 1953 nucleotides and encodes the amino acid sequence shown in SEQ ID NO:2.

[0035] The present invention also provides the use of a substance that inhibits the expression of the above-mentioned protein or the above-mentioned biological material in plants in any of the following ways: 1) causing plant leaves to become bleached; 2) reducing the content of chlorophyll and / or carotenoids in plants; 3) reducing the height of plants; 4) cultivating plants with bleached leaves; 5) cultivating plants with low chlorophyll and / or carotenoid content; 6) cultivating plants with low height.

[0036] Those skilled in the art can easily mutate the nucleotide sequence encoding OsEME1 of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of OsEME1 isolated from 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 OsEME1 and have the same function.

[0037] In the above biological materials, the stringent conditions are hybridization and washing at 68°C in a 2×SSC, 0.1% SDS solution for 5 min each time, and hybridization and washing at 68°C for 15 min each time in a 0.5×SSC, 0.1% SDS solution for 15 min each time; or hybridization and washing at 65°C in a 0.1×SSPE (or 0.1×SSC), 0.1% SDS solution.

[0038] In the above biological materials, the expression cassette (OsEME1 gene expression cassette) containing a nucleic acid molecule encoding OsEME1 described in B2) refers to a DNA capable of expressing OsEME1 in a host cell, and the DNA may include not only a promoter for initiating transcription of OsEME1, but also a terminator for terminating transcription of OsEME1. Furthermore, the expression cassette may also include an enhancer sequence. Promoters that can be used 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 35S of cauliflower mosaic virus; the wound-inducible promoter from tomato; the 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-thiocarboxylic acid S-methyl ester)); tomato proteinase inhibitor II promoter (PIN2) or LAP promoter (both can be induced by methyl jasmonate); heat shock promoter (U.S. Pat. No. 5,187,267); tetracycline-inducible promoter (U.S. Pat. No. 5,057,422); seed-specific promoters, such as millet seed-specific promoter pF128 (CN101063139B (Chinese Patent 200710099169.7)), seed storage protein-specific promoters (e.g., 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 cited in their entirety. Suitable transcription terminators include, but are not limited to, the Agrobacterium nopaline synthase terminator (NOS terminator), the cauliflower mosaic virus CaMV 35S terminator, the tml terminator, the pea rbcS E9 terminator, and the nopaline and octopine synthase terminators (see, for example, Odell et al. (1996). 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).

[0039] The recombinant vector containing the OsEME1 gene expression cassette can be constructed using an existing expression vector. The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment, etc. Such as pAHC25, pBin438, pRGEB32, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA Company), etc. The plant expression vector can also include the 3' non-translated region of the foreign gene, that is, a polyadenylic acid signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as the Agrobacterium crown gall induction (Ti) plasmid gene (such as the nopaline synthase gene Nos), the non-translated region transcribed at the 3' end of the plant gene (such as the soybean storage protein gene) all have similar functions. When using the gene construction plant expression vector of the present invention, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical to the reading frame of the coding sequence to ensure the correct translation of the entire sequence. The source of the translation control signal and the start codon is extensive and can be natural or synthetic. The translation initiation region can come 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 used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic marker genes (such as nptII gene that confers resistance to kanamycin and related antibiotics, bar gene that confers resistance to the herbicide phosphinothricin, hph gene that confers resistance to the antibiotic hygromycin, dhfr gene that confers resistance to methotrexate, EPSPS gene that confers resistance to glyphosate) or chemical resistance marker genes (such as herbicide resistance genes), mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose. Considering the safety of transgenic plants, no selective marker gene can be added, and transformed plants can be directly screened by adversity.

[0040] In the above biological materials, the vector may be a plasmid, a cosmid, a phage or a virus vector.

[0041] In the above biological materials, the microorganism may be yeast, bacteria, algae or fungi, such as Agrobacterium.

[0042] Among the above biological materials, the transgenic plant cell lines, transgenic plant tissues and transgenic plant organs do not include propagation materials.

[0043] The present invention also provides application of the above protein or biological material in plant breeding.

[0044] The present invention also provides a method for changing plant leaf color and / or plant height, comprising inhibiting the expression of the above protein encoding gene in the target plant to change the plant height and / or leaf color of the target plant.

[0045] The above-mentioned change in plant height is manifested as a plant height significantly lower than that of the recipient plant, and the above-mentioned change in leaf color is manifested as the leaf color of the plant turning white.

[0046] The present invention also provides a method for cultivating transgenic plants with albino and / or dwarfed leaves, comprising inhibiting the expression of the above-mentioned protein encoding gene in the target plant to obtain the transgenic plant; compared with the target plant, the transgenic plant has albino leaves and / or a plant height that is significantly lower than that of the recipient plant.

[0047] The method for inhibiting the expression of the protein encoding gene is to introduce a substance that inhibits the expression of the protein encoding gene into a recipient plant to obtain a transgenic plant, and at the same time use EMS to mutate the recipient plant to obtain a mutant plant in which the protein expression is inhibited due to a mutation in the gene sequence encoding the above protein. The leaves of the obtained mutant plant are white and the plant height is significantly reduced.

[0048] The transgenic plant has a lower plant height than the recipient plant, and the leaf color is significantly lighter than that of the recipient plant, which is specifically reflected in: under normal growth conditions, the leaf color of the transgenic plant in the seedling stage is albino, the chlorophyll content and carotenoid content are significantly reduced, and the plant height of the transgenic plant in the mature stage is significantly lower than that of the recipient plant.

[0049] In the above method, the substance that inhibits the expression of the above protein is the CRISPR / Ca9 genome editing system, which includes a DNA molecule as shown in SEQ ID NO:3, a DNA molecule as shown in SEQ ID NO:4, and a cas9 expression vector.

[0050] The plant is a monocotyledonous plant; preferably, the monocotyledonous plant is a Poaceae plant; further preferably, the Poaceae plant is preferably rice, and the rice is specifically Kongyu 131 (KY131).

[0051] In an embodiment of the present invention, the substance that inhibits the expression of the above-mentioned protein is introduced into the recipient plant through pRGEB32-OsEME1, and the pRGEB32-OsEME1 transformation vector is a vector obtained by connecting the DNA molecule shown in SEQ ID NO:3 with the DNA molecule shown in SEQ ID NO:4 to replace the DNA fragment between the BsaI restriction sites of the vector pRGEB32, while keeping the other sequences of the vector pRGEB32 unchanged.

[0052] The present invention cloned the gene OsEME1 related to DNA double-strand damage repair from rice, and constructed plants (k48 and Oseme1) with OsEME1 gene deletion by EMS mutagenesis and transgenic technology respectively. Experiments proved that under normal growth conditions, the plants with OsEME1 gene deletion showed dwarfing and reduced leaf pigment content phenotypes, indicating that the OsEME1 gene has the function of regulating plant chloroplast development and plant height, and by seeking rice with natural OsEME1 mutations and hybridizing with the main cultivated varieties, the plant height can be reduced, thereby reducing the yield reduction caused by crop lodging, providing a theoretical basis for cultivating better and higher-yield rice varieties; at the same time, the leaf color change caused by the deletion of the gene can be used as a trait marker for molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0053] Figure 1 The phenotypes of OsEME1-deficient plants and KY131 plants grown under long-day growth conditions for 12 days;

[0054] Figure 2 OsEME1-deficient plants and KY131 plants Figure 1 Determination of leaf pigment content in ;

[0055] Figure 3 The phenotypes of OsEME1-deficient plants and KY131 plants at maturity under normal growth conditions;

[0056] Figure 4 These are the plant height measurement results of OsEME1-deficient plants and KY131 plants at mature stage. DETAILED DESCRIPTION

[0057] The present invention is further described in detail below in conjunction with specific embodiments. The given examples are only for illustrating the present invention, but not for limiting the scope of the present invention.

[0058] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0059] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0060] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0061] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0062] Example 1. Acquisition of OsEME1 gene

[0063] 1. Extraction of plant cDNA

[0064] RNA was extracted from leaves of wild-type rice (Kongyu 131, KY131, Zhou Yaoqun et al., New Rice Variety Kongyu 131, Modern Agriculture, (11), 10-11) and reverse transcribed to obtain cDNA.

[0065] 2. PCR amplification

[0066] The cDNA obtained in step 1 was used as a template and PCR amplification was performed using primers F and R to obtain a PCR amplification product. The primer sequences are as follows: F: 5'-GGTACCATGGCGTCCCACGCCGC-3' (SEQ ID NO: 5) and R: 5'-CTCGAGCTAAGCTCCACGCCTATCAAC-3' (SEQ ID NO: 6).

[0067] 3. Acquisition of OsEME1 gene

[0068] The PCR amplification products were detected by 1% agarose gel electrophoresis, recovered and purified, and then sequenced.

[0069] The sequencing results showed that: PCR amplification obtained an amplification product of 1953 bp in size, whose nucleotide sequence is shown in SEQ ID NO: 1 in the sequence listing, the gene shown in SEQ ID NO: 1 is named OsEME1 gene, the amino acid sequence encoded by the OsEME1 gene is shown in SEQ ID NO: 2 in the sequence listing, and the protein shown in SEQ ID NO: 2 is named OsEME1 protein.

[0070] Example 2: Obtaining OsEME1 gene-deficient rice and phenotypic analysis

[0071] 1. Obtaining OsEME1 gene-deficient rice

[0072] Referring to the method in the literature “Ma X, Zhang Q, Zhu Q, Liu W, Chen Y, Qiu R, Wang B, Yang Z, Li H, LinY, Xie Y, Shen R, Chen S, Wang Z, Chen Y, Guo J, Chen L, Zhao X, Dong Z, Liu YG. (2015) A Robust CRISPR / Cas9 System for Convenient, High-Efficiency Multiplex Genome Editing in Monocot and Dicot Plants. Mol Plant. 8: 1274-1284.”, the OsEME1 gene CRISPR / Cas genome editing system was designed and constructed, and OsEME1 gene-deficient rice was obtained. Sequencing identification shows that, compared with wild-type rice, the OsEME1 gene-deficient rice is a rice plant obtained by deleting the nucleotide molecules at positions 1764-2230 (corresponding to positions 754-895 of the cDNA sequence of the wild-type rice OsEME1 gene SEQ ID NO: 1) of the wild-type rice OsEME1 genome sequence shown in SEQ ID NO: 20, while keeping other sequences of the rice genome unchanged. The specific construction steps are as follows:

[0073] 1. Construction of pRGEB32-OsEME1 transformation vector

[0074] (1) The following sequences were synthesized separately and dimers with sticky ends were formed by denaturation annealing:

[0075] Site1 F:5'-GCCGACAAGGAGAATTAGCACGAG-3' (SEQ ID NO:7);

[0076] Site1 R:5'-AAACCTCGTGCTAATTCTCCTTGT-3'(SEQ ID NO:8)

[0077] Site2 F:5'-GGCATGCCCTACCGATTGAAGAA-3'(SEQ ID NO:9)

[0078] Site2 R: 5'-AAACTTCTTCAATCGGTAGGGCA-3' (SEQ ID NO: 10);

[0079] (2) The two dimers with sticky ends obtained in step 1 were connected to the gRNA vectors digested with BsaI (Ma et al., 2015), where Site 1 was connected to the gRNA vector containing OsU6a, and Site 2 was connected to the gRNA vector containing OsU3;

[0080] (3) PCR amplification was performed using primers with BsaI restriction sites to obtain the gRNA expression frame containing the target site (both ends of the same expression frame contained BsaI restriction sites and different sticky ends were produced after BsaI restriction digestion). The amplification primer sequences were as follows:

[0081] F:5'-TTCAGAGGTCTCT-3'(SEQ ID NO:11)

[0082] R:5'-AGCGTGGGTCTCG-3'(SEQ ID NO:12)

[0083] (4) recovering the amplified target site fragment and digesting it with BsaI;

[0084] (5) The Cas9 vector digested with BsaI was ligated with the target site fragment digested in step (4) to obtain the pRGEB32-OsEME1 transformation vector, which was then sequenced.

[0085] The sequencing results showed that the pRGEB32-OsEME1 transformation vector is a DNA molecule shown in SEQ ID NO:3 directly connected to the DNA molecule shown in SEQ ID NO:4 [SEQ ID NO:3 is a CRISPR target prediction website http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR The predicted OsEME1 sequence knockout target (SEQ ID NO: 3, 481-500 from the 5' end is a part of the OsEME1 gene sequence) was linked to the OsU6a promoter; SEQ ID NO: 4 is a CRISPR target prediction website http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR The predicted OsEME1 sequence knockout target (SEQ ID NO: 4, 452-471 from the 5' end, which is a part of the OsEME1 gene sequence) was linked to the OsU3 promoter to replace the DNA fragment between the BsaI restriction site of the vector pRGEB32 (Addgene, catalog number: 63142), while keeping the other sequences of the vector pRGEB32 unchanged to obtain the vector.

[0086] 2. Obtaining recombinant bacteria

[0087] The pRGEB32-OsEME1 transformation vector obtained in step 1 was introduced into Agrobacterium EHA105 (EHA105, Beijing Huayueyang Biological, catalog number: GX0133-100) to obtain the recombinant bacterium EHA105-OsEME1sg.

[0088] 3. Obtaining OsEME1 gene-deficient rice

[0089] The recombinant bacteria EHA105-OsEME1sg was transferred into callus tissue induced by KY131 rice (hereinafter referred to as wild-type rice) seeds (Wuhan Tianwen Biotechnology Co., Ltd.) by conventional methods. The culture medium was conventional transformation medium. The detailed method is as follows:

[0090] The mature seeds were shelled, disinfected, and inoculated into the callus induction medium, and induced in the dark for 30 days. The callus was then transferred to the subculture medium and cultured in the dark for 20 days. The callus was infected with activated Agrobacterium on a medium containing 50 mg / L kanamycin, the bacterial solution was discarded, and the infected callus was transferred to the co-culture medium for 3 days. The callus was washed with sterile distilled water and an aqueous solution containing antibiotics, and after the surface water of the callus was dried, the callus was transferred to a screening medium containing 50 mg / L hygromycin for screening. The resistant callus was then transferred to the differentiation medium and cultured under light for 40 days. The differentiated seedlings were transferred to the rooting medium and cultured under light for 15 days to obtain rice transformants.

[0091] The obtained rice transformants were molecularly identified using the following primers: PF: 5'-TGTCCAATATCTCTGGATAGTG-3' (SEQ ID NO: 13); PR: 5'-CTCCAGCTTCTTCAATTCT-3' (SEQ ID NO: 14).

[0092] The sequencing analysis of the amplified products showed that, compared with the wild-type rice, the OsEME1 gene-deficient rice was a rice plant obtained by deleting the nucleotide molecules of positions 1764-2230 (corresponding to positions 754-895 of the cDNA sequence of the wild-type rice OsEME1 gene SEQ ID NO: 20) of the wild-type rice, while keeping the other sequences of the rice genome unchanged. The deletion of 467 bases of the OsEME1 gene resulted in the deletion of a large fragment of the reading frame, inhibiting the expression of the protein. This indicated that the OsEME1 gene in the OsEME1 gene-deficient rice was successfully deleted, and the OsEME1 gene-deficient rice was named Oseme1.

[0093] 2. Phenotypic analysis of rice plants lacking the OsEME1 gene

[0094] 1. Short-term cultivation of phenotypes

[0095] The seedlings of OsEME1 gene-deficient rice plants (k48 and Oseme1) and wild-type rice plants (KY131) were grown under long-day conditions for 12 days and their phenotypes were observed.

[0096] The results are as follows Figure 1 As shown: Under normal conditions, OsEME1 gene-deficient rice plants (k48 and Oseme1) have chloroplast development disorders and are whitened compared to wild-type rice plants (KY131). Collect the leaves of the above materials in the dark, weigh 0.1 g each, grind with liquid nitrogen and transfer to a 10 mL tube, add 10 mL of 80% acetone (Sinopharm Group, 10000418), shake and extract at 4°C for 1 hour until the powder turns from green to white, filter the supernatant into a 25 mL constant volume bottle using a magic filter cloth (Millipore, 475855), and dilute to 25 mL with 80% acetone. Take 80% acetone as the blank control, take 1 mL of the dilute extract to measure the OD 665 , O.D. 649 , O.D. 470 The pigment content of each plant was calculated according to the following formula: Chlorophyll a (Chl a) = 13.95 × D 665 -6.88×D 649 ; Chlorophyll b (Chl b) = 24.96 × D 649 -7.32×D 665 ; Carotenoids (Car) = (1000 × D 470 -2.05×Chl a-114×Chl b) / 245 (Porra et al., 1989). The results showed that the chlorophyll content and carotenoid content of OsEME1-deficient plants were significantly lower than those of wild-type plants ( Figure 2 ). This indicates that OsEME1 protein is involved in the chloroplast development process of rice.

[0097] 2. Long-term cultivation of phenotypes

[0098] Seedlings of OsEME1 gene-deficient rice plants (k48 and Oseme1) and wild-type rice plants (KY131) were grown under long-day conditions for more than four months and their phenotypes were observed. Figure 3 and 4 As shown: Under normal conditions, the plant height of rice plants lacking the OsEME1 gene (k48 and Oseme1) was significantly reduced compared with that of wild-type rice plants (KY131).

[0099] The present invention has been described in detail above. It will be apparent to those skilled in the art that the present invention may be implemented in a wide range under equivalent parameters, concentrations and conditions without departing from the spirit and scope of the present invention and without the need for unnecessary experimentation. Although the present invention provides specific embodiments, it should be understood that further improvements may be made to the present invention. In short, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by conventional techniques known in the art that depart from the scope disclosed in this application. Applications of some of the basic features may be made within the scope of the following appended claims.

Claims

1. Application of any of the proteins shown in A1) or A2) or A3) or A4) below in regulating plant leaf color and / or plant height: A1) the protein whose amino acid sequence is shown in SEQ ID NO: 2; A2) a protein having the same function as that of the amino acid sequence shown in SEQ ID NO: 2 after one or more amino acid residues are substituted and / or deleted and / or added; A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, 80% or more or 75% or more identity with the amino acid sequence defined in any one of A1) to A2) and having the same function; A4) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of any protein defined in A1) to A3).

2. Use of biological materials related to the protein described in claim 1 in regulating plant leaf color and / or plant height; The biological material is any one of the following B1)-B10): B1) a nucleic acid molecule encoding the protein according to claim 1; B2) an expression cassette containing the nucleic acid molecule described in B1); B3) a recombinant vector containing the nucleic acid molecule described in B1), or a recombinant vector containing the expression cassette described in B2); B4) a recombinant microorganism containing the nucleic acid molecule described in B1), or a recombinant microorganism containing the expression cassette described in B2), or a recombinant microorganism containing the recombinant vector described in B3); B5) a transgenic plant cell line containing the nucleic acid molecule described in B1), or a transgenic plant cell line containing the expression cassette described in B2), or a transgenic plant cell line containing the recombinant vector described in B3); B6) transgenic plant tissue containing the nucleic acid molecule described in B1), or transgenic plant tissue containing the expression cassette described in B2), or transgenic plant tissue containing the recombinant vector described in B3); B7) a transgenic plant organ containing the nucleic acid molecule described in B1), or a transgenic plant organ containing the expression cassette described in B2), or a transgenic plant organ containing the recombinant vector described in B3); B8) a transgenic plant containing the nucleic acid molecule described in B1), or a transgenic plant containing the expression cassette described in B2), or a transgenic plant containing the recombinant vector described in B3); B9) tissue culture produced from regenerable cells of the transgenic plant described in B8); B10) Protoplasts produced from the tissue culture described in B9).

3. The use according to claim 2, characterized in that: B1) The nucleic acid molecule is any one of the following C1)-C3): C1) cDNA molecule or DNA molecule shown in SEQ ID NO: 1; C2) a cDNA molecule or a genomic DNA molecule that hybridizes with the DNA molecule defined in C1) under stringent conditions and encodes the protein of claim 1; C3) A cDNA molecule or genomic DNA molecule that has 99% or more, 95% or more, 90% or more, 85% or more, 80% or more, or 75% or more identity with the DNA sequence defined in C1) or C2) and encodes the protein of claim 1.

4. Use of the protein according to claim 1 or the biological material according to claim 2 or 3 in plant breeding.

5. Use of a substance for inhibiting the expression of the protein according to claim 1 or the biological material according to claim 2 or 3 in a plant in any of the following: 1) Cause the leaves of plants to turn white; 2) Reduce the content of chlorophyll and / or carotenoids in plants; 3) Reduce plant height; 4) Cultivating plants with white leaves; 5) Cultivating plants with low chlorophyll and / or carotenoid content; 6) Cultivate plants with low plant height.

6. A method for changing plant leaf color and / or plant height, characterized in that: The method comprises inhibiting the expression of the gene encoding the protein according to claim 1 in the target plant, so that the plant height and / or leaf color of the target plant changes.

7. A method for cultivating transgenic plants with albino and / or dwarfed leaves, characterized in that: The method comprises inhibiting the expression of the protein encoding gene of claim 1 in the target plant to obtain a transgenic plant; compared with the target plant, the transgenic plant has albino leaves and / or a plant height significantly lower than that of the recipient plant.

8. The method according to claim 5 or 7, characterized in that: The method for inhibiting the expression of the gene encoding the protein of claim 1 is to obtain a transgenic plant by introducing a substance that inhibits the expression of the gene encoding the protein of claim 1 into a recipient plant.

9. The method according to claim 8, characterized in that: The substance that inhibits the expression of the protein described in claim 1 is the CRISPR / Ca9 genome editing system, which includes a DNA molecule as shown in SEQ ID NO: 3, a DNA molecule as shown in SEQ ID NO: 4, and a cas9 expression vector.

10. The method according to any one of claims 5 to 9, characterized in that: The plant is a monocotyledonous plant; preferably, the monocotyledonous plant is a grass plant.

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