CN116947988A

CN116947988A - ZmECT2 protein and application of encoding gene thereof in regulation and control of corn kernel quality

Info

Publication number: CN116947988A
Application number: CN202211434185.8A
Authority: CN
Inventors: 宗娜; 赵军
Original assignee: Biotechnology Research Institute of CAAS
Current assignee: Biotechnology Research Institute of CAAS
Priority date: 2022-11-16
Filing date: 2022-11-16
Publication date: 2023-10-27

Abstract

The application discloses ZmECT2 protein and application of a coding gene thereof in regulating and controlling corn kernel quality. The present application provides the following uses of zmct 2 protein or its related biological material (nucleic acid molecule expressing zmct 2 protein or expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing said nucleic acid molecule); regulating and controlling the quality of plant plasmids, regulating and controlling the protein content and/or the starch content of plant seeds; the ZmECT2 protein is SEQ ID No.1 or a fusion protein obtained by substituting, deleting and/or adding one or more amino acids, or a protein which has more than 80% of identity compared with the protein and is derived from rice and has the same function, or a fusion protein obtained by connecting a label at the N end and/or the C end of the protein. The ZmECT2 provided by the application can regulate and control the quality of plant seeds, including regulating and controlling the protein content and the starch content of the seeds. The application has important application value for effectively regulating and controlling the quality of plant seeds by utilizing the gene resources through genetic breeding and genetic engineering methods.

Description

ZmECT2 protein and application of encoding gene thereof in regulation and control of corn kernel quality

Technical Field

The application relates to the technical field of biology, in particular to application of ZmECT2 protein and a coding gene thereof in regulation and control of corn kernel quality.

Background

Corn is the first large grain crop in China, is widely used as grain, feed and industrial raw materials, and plays an important role in guaranteeing grain safety in economic and social development in China.

Although researchers in China have made an important contribution to improving the corn yield in recent years, the total corn yield in China is greatly improved, but the research on improving the corn quality is relatively less. For example, corn is poor in quality, low in specific efficiency, low in essential amino acid content, low in nutritive value and the like, and the quality seriously hinders the requirements of the feed industry and the processing industry on domestic corn.

Therefore, improving the quality of corn is a great demand for guaranteeing the grain safety of China.

Disclosure of Invention

The application aims to provide an application of ZmECT2 protein and a coding gene thereof in regulating and controlling corn kernel quality.

In a first aspect, the application claims any of the following uses of zmct 2 protein or related biological material thereof;

p1, regulating and controlling the quality of plant seeds;

p2, regulating and controlling the protein content of the plant seeds;

p3, regulating and controlling the starch content of the plant seeds.

Wherein the relevant biological material may be a nucleic acid molecule capable of expressing the zmct 2 protein, or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule.

The expression cassette refers to a DNA capable of expressing zmct 2 in a host cell, which may include not only a promoter that initiates transcription of the zmct 2 gene, but also a terminator that terminates transcription of zmct 2. Further, the expression cassette may also include an enhancer sequence. Promoters useful in the present application 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: ubiquitin gene Ubiqutin promoter (pUbi); a constitutive promoter of cauliflower mosaic virus 35S; wound-inducible promoters 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 jasmonic acid ester); 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 2007 1 0099169.7)), seed storage protein-specific promoters (e.g., promoters of phaseolin, napin, oleosin and soybean beta-glycin (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: agrobacterium nopaline synthase terminator (NOS terminator), cauliflower mosaic virus CaMV 35S terminator, tml terminator, pea rbcS E9 terminator and nopaline and octopine synthase terminator (see, e.g., odell et al (I985) 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; joid et al (1987) Nucleic Acid Res. 15:9627).

Constructing a recombinant expression vector containing the ZmECT2 gene expression cassette. The plant expression vector used may be a binary Agrobacterium vector or a Gateway system vector, etc., such as pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pGWB411, pGWB412, pGWB405, pCAMBIA1391-Xa or pCAMBIA1391-Xb. When ZmECT2 is used to construct recombinant expression vectors, any one of enhanced, constitutive, tissue-specific or inducible promoters such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiqutin promoter (pUbi) and the like may be added before the transcription initiation nucleotide thereof, and they may be used alone or in combination with other plant promoters; in addition, when the gene of the present application is used to construct a plant expression vector, enhancers, including translational enhancers 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.

In order to facilitate the 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 markers with resistance (gentamicin markers, kanamycin markers, etc.), or anti-chemical marker genes (e.g., anti-herbicide genes), etc., which may be expressed in plants.

In the above applications, the vector may be a plasmid, cosmid, phage or viral vector.

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

The zmcct 2 protein may be any of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

(A2) The amino acid sequence shown in SEQ ID No.1 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues and is derived from rice protein with the same function;

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

(A4) A fusion protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in any one of (A1) to (A3) with a protein tag.

Among the above proteins, the protein tag (protein-tag) refers to a polypeptide or protein that is fusion expressed together with a target protein by using a DNA in vitro recombination technique, 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 above proteins, the identity refers to the identity of amino acid sequences. 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 above protein, the homology of 95% or more may be at least 96%, 97% or 98% identical. The 90% or more homology may be at least 91%, 92%, 93%, 94% identical. The 85% or more homology may be at least 86%, 87%, 88%, 89% identical. The 80% or more homology may be at least 81%, 82%, 83%, 84% identical.

In the plant, the expression amount and/or activity of the zmct 2 protein is reduced, the plant kernel protein content is increased and/or the plant kernel starch content is reduced. The expression amount and/or activity of the ZmECT2 protein is increased, the plant kernel protein content is reduced and/or the plant kernel starch content is increased.

In a second aspect, the present application claims the use of a substance capable of reducing the expression and/or activity of zmct 2 protein in a plant in (a 1) and/or (a 2):

(a1) Increasing the protein content of the plant seeds;

(a2) Reducing the starch content of the plant seeds.

The zmct 2 protein may be any one of the proteins set forth in any one of (A1) to (A4) above.

In a third aspect, the present application claims the use of a substance capable of increasing the expression and/or activity of zmct 2 protein in a plant in (b 1) and/or (b 2):

(b1) Reducing the plant kernel protein content;

(b2) And improving the starch content of the plant seeds.

In a fourth aspect, the application claims a plant cultivation method.

The plant cultivation method claimed by the application is the following method I or method II:

method I: a method of growing a plant having an increased grain protein content and/or a decreased grain starch content (alternatively referred to as a "method of increasing grain protein content and/or decreasing grain starch content of a plant") may comprise the step of decreasing the expression and/or activity of zmct 2 protein in a recipient plant.

Method II: a method of growing a plant having reduced grain protein content and/or increased grain starch content (alternatively referred to as a "method of reducing grain protein content and/or increasing grain starch content of a plant") may comprise the step of increasing the expression and/or activity of zmct 2 protein in a recipient plant.

The method can be realized by hybridization means or transgenic means.

In a fifth aspect, the application claims a method of growing a transgenic plant.

The method for cultivating the transgenic plant claimed by the application can be the following method III or method IV:

method III: a method of growing a transgenic plant having increased grain protein content and/or decreased grain starch content, comprising the steps of: inhibiting expression of a nucleic acid molecule capable of expressing a zmct 2 protein in a recipient plant to obtain a transgenic plant; the transgenic plant has an increased grain protein content and/or a decreased grain starch content as compared to the recipient plant.

Method IV: a method of growing a transgenic plant having reduced grain protein content and/or increased grain starch content, comprising the steps of: introducing a nucleic acid molecule capable of expressing a zmct 2 protein into a recipient plant to obtain a transgenic plant; the transgenic plant has a reduced grain protein content and/or an increased grain starch content compared to the recipient plant.

Further, in method III, the inhibition of expression of a nucleic acid molecule capable of expressing the ZmECT2 protein in the recipient plant may be achieved by any technical means capable of achieving this objective. Such as by gene editing techniques, such as CRISPR-cas9, and the like.

In a specific embodiment of the application, the method is realized by CRISPR-cas9 technology, and the specifically adopted sgRNA sequence is shown in SEQ ID No. 3.

Further, in method IV, the nucleic acid molecule capable of expressing the zmct 2 protein may be introduced into the recipient plant in the form of a recombinant vector.

In a specific embodiment of the application, the promoter in the recombinant vector that initiates transcription of the nucleic acid molecule is the pUbi promoter. The recombinant vector is specifically pCAMBIA3301-UBI-ZmECT2. The original skeleton carrier is pCAMBIA3301, contains ZmECT2 CDS sequence with the target gene length of 1914bp, the promoter is corn Ubi promoter, the terminator is nopaline synthase gene NOS terminator, the carrier also contains phosphinothricin acetyl transferase gene bar from streptomyces hygroscopicus, the phosphinothricin acetyl transferase gene bar can endow plants with glufosinate resistance, the promoter is 35S promoter, and the terminator is 35S terminator.

In the above method, the method of introducing the CRISPR-cas 9-based editing vector or the recombinant vector for overexpression into the recipient plant may specifically be: 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.

In the above methods, the transgenic plants are understood to include not only first to second generation transgenic plants but also their progeny. For transgenic plants, the gene may be propagated in that species, and may be transferred into other varieties of the same species, including particularly commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, calli, whole plants and cells.

In each of the above aspects, 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, and the like.

Further, the nucleic acid molecule capable of expressing the zmcct 2 protein may be any of the following:

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

(B2) A DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes the zmct 2 protein;

(B3) A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the DNA sequence defined in any one of (B1) to (B2) and encoding the zmct 2 protein.

In the above nucleic acid molecule, the stringent conditions may be as follows: 50℃in 7% Sodium Dodecyl Sulfate (SDS), 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5Mna ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; the method can also be as follows: 50℃in 7% SDS, 0.5M Na ₃ PO ₄ Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; the method can also be as follows: hybridization was performed in a solution of 6 XSSC, 0.5% SDS at 65℃and then washed once with 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

In the above nucleic acid molecules, homology refers to the identity of nucleotide sequences. The identity of nucleotide 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, by using blastp as a program, the Expect value is set to 10, all filters are set to OFF, BLOSUM62 is used as Matrix, gap existence cost, per residue gap cost and Lambda ratio are set to 11,1 and 0.85 (default values), respectively, and identity of a pair of nucleotide sequences is searched for and calculated, and then the value (%) of identity can be obtained.

In the nucleic acid molecule, the homology of 95% or more may be at least 96%, 97% or 98% identical. The 90% or more homology may be at least 91%, 92%, 93%, 94% identical. The 85% or more homology may be at least 86%, 87%, 88%, 89% identical. The 80% or more homology may be at least 81%, 82%, 83%, 84% identical.

In a fifth aspect, the application claims the use of a method as described in the third or fourth aspect hereinbefore in plant breeding.

In each of the above aspects, the plant is a monocot or dicot.

Further, the plant is a gramineous plant.

Still further, the plant is a maize plant, such as maize.

In a specific embodiment of the application, the plant is in particular maize inbred line C01 or maize inbred line KN5585.

Experiments prove that the plant type related protein ZmECT2 and the coding gene thereof can regulate and control the quality of plant seeds, including the protein content and the starch content of the seeds. Inhibiting the expression of the ZmECT2 coding gene can obviously improve the protein content of the plant seeds and reduce the starch content of the seeds; overexpression of zmcct 2 can significantly reduce plant grain protein content and increase grain starch content. The ZmECT2 protein and the coding gene thereof play an important role in regulating the quality of plant seeds. The application provides new gene resources and breeding resources for breeding in the aspect of improving the quality of plant seeds. The application has important application value for effectively regulating and controlling the quality of plant seeds by utilizing the gene resources through genetic breeding and genetic engineering methods.

Drawings

FIG. 1 shows ZmECT2 gene editing vector map and overexpression vector map. A is ZmECT2 gene editing vector map; b is ZmECT2 gene over-expression vector map.

FIG. 2 is an identification of ZmECT2 gene editing mutant maize. A is the alignment of nucleotide sequences before and after editing of ZmECT2. B is the alignment of the amino acid sequences before and after editing of ZmECT2.

FIG. 3 shows the results of a comparison of protein and starch content of ZmECT2 gene editing material and over-expressed material. Wherein ZmECT2-1 and ZmECT2-2 are two repeats of the ZmECT2 gene editing mutant; overexpression 1 represents zmct 2 overexpressing transgenic material 1; overexpression 2 means zmct 2 overexpressing transgenic material 2. In the figure, x represents significant differences (P < 0.01); * Represents significant differences (P < 0.001).

Detailed Description

The following detailed description of the application is provided in connection with the accompanying drawings that are presented to illustrate the application 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 application 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.

Example 1, application of ZmECT2 protein in regulating corn kernel quality

1. Cloning of ZmECT2 protein and its coding gene

The coding region sequence of ZmECT2 is amplified from cDNA of maize inbred line B73, as shown in SEQ ID No. 2. SEQ ID No.2 encodes a protein shown as SEQ ID No. 1.

2. Construction of ZmECT2 knockout mutant corn and grain quality identification

The application constructs a mutant corn material of ZmECT2 gene taking corn inbred line C01 as background by a CRISPR-cas9 method, and identifies the protein content and starch content of seeds.

1. Construction of ZmECT2 Gene knockout vector

Based on the zmct 2 gene sequence, the sgrnas were designed as follows: 5'-GGATGTATCTACTATTGGTG-3' (SEQ ID No. 3).

Then, the zmct 2 knockout vector was constructed as follows:

first, the U6 promoter was amplified from the maize genome, using maize inbred B73 genomic DNA as a template, to: MU61-3F:5'-TGCACTGCACAAGCTGCTGTTTTTGTTAGCCCCATCG-3' and MU61-1R:5'-AATTCGGTGCTTGCGGCTC-3' is a primer amplified U6 promoter. Next, DNA was used as a template in CPB vector (described in "Li C, liu C, qi X, wu Y, fei X, mao L, cheng B, li X, xie C. RNA-guided Cas9 as an in vivo desired-target mutator in mail; plant Biotechnol J.2017Dec;15 (12): 1566-1576.Doi:10.1111/pbi.12739.Epub 2017May 12.PMID:28379609;PMCID:PMC5698053." available to the public from the applicant, available only for repeat the experiments of the present application, not to be used for him "): 5'-GAGCCGCAAGCACCGAATTGGATGTATCTACTATTGGTGGTTTTAGAGCTAGAAATAGCAAGTT-3' and MUsgR-2R:5'-GGCCAGTGCCAAGCTTAAAAAAAGCACCGACTCG-3' primer amplification sgRNA. The amplified U6 promoter and sgRNA products were used as templates, amplified using MU61-3F and MUsgR-2R as primers, and fused together by overlap PCR, and the fused fragment was designated U6 promor-sgRNA. pCAMBIA3301 (https:// www.cambia.org) was digested and recovered with HindIII, then U6 promotor-sgRNA was ligated to the vector with an infusion enzyme, and sequencing verified, and the recombinant plasmid that was sequenced to verify correct was designated CPB-ZmECT2.

Finally, the map of the ZmECT2 gene knockout vector CPB-ZmECT2 obtained after the correct sequencing verification is shown as A in figure 1, and the vector size is 16.973Kb. Exogenous genes include Cas9 gene and sgrnas, with ZmU promoter and UBI promoter on the vector backbone, and terminators are Nos. The marker gene is bar, and no reporter gene exists.

2. Construction of ZmECT2 knockout mutant maize

And (3) transforming the ZmECT2 gene knockout vector constructed in the step (1) into a corn inbred line C01 by an agrobacterium-mediated transformation callus method. Obtaining a gene editing mutant, wherein the nucleotide sequence of the full-length coding region of the target gene ZmECT2 after editing is shown as SEQ ID No.4, translation is terminated in advance after ZmECT2 is edited, the generated amino acid sequence is shown as SEQ ID No.5, the nucleotide sequences of the target gene ZmECT2 before and after editing are aligned, and an A is inserted after editing (shown as A in figure 2). Alignment of amino acid sequences before and after editing of zmcct 2, after editing glycine, valine, aspartic acid and glutamic acid at positions 177, 178 and 179 were mutated into glutamic acid, cysteine, glycine and stop codon, respectively (as shown in B in fig. 2), zmcct 2 was terminated prematurely.

The experiment was also set up with an empty control introducing the CPB vector into maize inbred C01.

3. Determination of grain protein and starch content of ZmECT2 knockout mutant maize

The protein and starch content of the corn kernel was measured using a FUSS near infrared spectrometer (Infratec ^TM 1241 GrainAnalyzer) determination. Opening a corn measurement mode in a main menu of the instrument, placing 100-200 corn kernels in a sample pool, setting the corn kernels to be scanned ten times per measurement, and repeatedly measuring each sample for three times to eliminate the influence of factors such as granularity, uniformity inconsistency and the like of the samples on the spectrum, and taking an average value of the result. Protein content was verified by the Kjeldahl method.

The results are shown in fig. 3, where zmct 2 knockout mutant corn kernels have significantly increased protein content and significantly decreased starch content compared to non-transgenic control C01 (representing P <0.01, × representing P < 0.001). In addition, the results of the no-load control were substantially identical to those of C01, with no statistical differences.

3. Construction of ZmECT2 over-expressed corn and grain quality identification

1. Construction of the overexpression vector

Primer fragment. For from maize kernel cDNA: 5'-tgatgataaaggatcGATGGCGGCTGTAGG-3' and fragment. Rev:5'-caccgagctcaagctTTAACAGCCATTTAGACCCGC-3' the ZmECT2 coding region was amplified, amplified fragments were recovered, bamHI and HindIII were used to cleave pCAMBIA3301-UBI (this vector is a recombinant vector obtained by replacing the 35S promoter in pCAMBIA3301 vector with UBI promoter, pCAMBIA3301 vector was described in Li JT, yuG, sun XH, jia CG, du Q, li QY, pan HY. Modification of vectors for functional genomicanalysis in plants. Genet Mol Res.2014Sep 26;13 (3): 7815-25.Doi: 10.4238/4. September.26.20.201PMID: 25299096.), large fragments were recovered, and then the recovered PCR product and the cleaved product of pCAMBIA3301-UBI were ligated with an infusion enzyme. E.coli is transformed by the connection product, positive colonies are identified by PCR, sequencing is verified, and the correct recombinant plasmid is the ZmECT2 over-expression vector after sequencing verification, which is named pCAMBIA3301-UBI-ZmECT2 (plasmid map is shown in B in figure 1). The structure of pCAMBIA3301-UBI-ZmECT2 is described as: the DNA fragment shown in SEQ ID No.2 was cloned into pCAMBIA3301-ubi vector between the cleavage sites BamHI and HindIII to obtain a recombinant plasmid.

2. Construction of ZmECT2 overexpressing maize

The maize transformation method employs agrobacterium-mediated immature embryo transformation, see Ishida Y et al 1996Nat Biotechnol.14 (6): 745-750. The ZmECT2 over-expression vector pCAMBIA3301-UBI-ZmECT2 constructed in the step 1 is introduced into a maize inbred line KN5585.

The experiment was also set up with an empty control introducing pCAMBIA3301-ubi into maize inbred line KN 5588.

3. Determination of grain protein and starch content of ZmECT2 overexpressed corn

The protein and starch content of the corn kernel was measured using a FUSS near infrared spectrometer (Infratec ^TM 1241 Grain Analyzer). Opening a corn measurement mode in a main menu of the instrument, placing 100-200 corn kernels in a sample pool, setting the corn kernels to be scanned ten times per measurement, and repeatedly measuring each sample for three times to eliminate the influence of factors such as granularity, uniformity inconsistency and the like of the samples on the spectrum, and taking an average value of the result.

The results show that: in comparison to maize inbred line KN5585, zmct 2 overexpressed transgenic maize material had increased grain starch content and decreased protein content (×p <0.01, ×p <0.001, t-test). The results are shown in FIG. 3. In addition, the results of the no-load control were substantially identical to those of KN5585 with no statistical differences.

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

Claims

Any of the following uses of zmct 2 protein or related biological material;

p1, regulating and controlling the quality of plant seeds;

p2, regulating and controlling the protein content of the plant seeds;

p3, regulating and controlling the starch content of the plant seeds;

the ZmECT2 protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

(A2) The amino acid sequence shown in SEQ ID No.1 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues and is derived from rice protein with the same function;

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

(A4) A fusion protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in any one of (A1) to (A3) with a protein tag;

the relevant biological material is a nucleic acid molecule capable of expressing the zmct 2 protein, or an expression cassette, recombinant vector, recombinant microorganism or transgenic cell line containing the nucleic acid molecule.
2. The use according to claim 1, characterized in that: in the plant, the expression amount and/or activity of the zmct 2 protein is reduced, the plant kernel protein content is increased and/or the plant kernel starch content is reduced; and/or

In the plant, the expression amount and/or activity of the zmct 2 protein is increased, the plant kernel protein content is decreased and/or the plant kernel starch content is increased.
3. Use of a substance capable of reducing the expression level and/or activity of a zmct 2 protein in a plant in (a 1) and/or (a 2):

(a1) Increasing the protein content of the plant seeds;

(a2) Reducing the starch content of the plant kernel;

the ZmECT2 protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

(A2) The amino acid sequence shown in SEQ ID No.1 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues and is derived from rice protein with the same function;

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

(A4) A fusion protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in any one of (A1) to (A3) with a protein tag.
4. Use of a substance capable of increasing the expression level and/or activity of a zmct 2 protein in a plant in (b 1) and/or (b 2):

(b1) Reducing the plant kernel protein content;

(b2) Increasing the starch content of the plant kernel;

the ZmECT2 protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

(A2) The amino acid sequence shown in SEQ ID No.1 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues and is derived from rice protein with the same function;

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

(A4) A fusion protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in any one of (A1) to (A3) with a protein tag.
5. A plant cultivation method is as follows method I or method II:

method I: a method of growing a plant having an increased grain protein content and/or a decreased grain starch content comprising the step of decreasing the expression and/or activity of a zmct 2 protein in a recipient plant;

method II: a method of growing a plant having a reduced level of kernel protein and/or an increased level of kernel starch comprising the step of increasing the expression and/or activity of zmct 2 protein in a recipient plant;

the ZmECT2 protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

(A2) The amino acid sequence shown in SEQ ID No.1 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues and is derived from rice protein with the same function;

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

(A4) A fusion protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in any one of (A1) to (A3) with a protein tag.
6. A method of growing a transgenic plant, which is method III or method IV as follows:

method III: a method of growing a transgenic plant having increased grain protein content and/or decreased grain starch content comprising the steps of: inhibiting expression of a nucleic acid molecule capable of expressing a zmct 2 protein in a recipient plant to obtain a transgenic plant; the transgenic plant has an increased grain protein content and/or a decreased grain starch content compared to the recipient plant;

method IV: a method of growing a transgenic plant having reduced grain protein content and/or increased grain starch content, comprising the steps of: introducing a nucleic acid molecule capable of expressing a zmct 2 protein into a recipient plant to obtain a transgenic plant; the transgenic plant has a reduced grain protein content and/or an increased grain starch content compared to the recipient plant;

the ZmECT2 protein is any one of the following:

(A1) A protein with an amino acid sequence of SEQ ID No. 1;

(A2) The amino acid sequence shown in SEQ ID No.1 is subjected to substitution and/or deletion and/or addition of one or more amino acid residues and is derived from rice protein with the same function;

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

(A4) A fusion protein obtained by ligating the N-terminal and/or C-terminal of the protein defined in any one of (A1) to (A3) with a protein tag.
7. The method according to claim 6, wherein: in method III, inhibiting expression of a nucleic acid molecule capable of expressing the zmct 2 protein in the recipient plant is achieved by a gene editing technique;

further, the gene editing technology is CRISPR-Cas9, and the adopted sgRNA sequence is shown as SEQ ID No. 3;

and/or

In method IV, a nucleic acid molecule capable of expressing the zmcct 2 protein is introduced into the recipient plant in the form of a recombinant vector;

further, the promoter in the recombinant vector that initiates transcription of the nucleic acid molecule is the pUbi promoter.
8. The use or method according to any one of claims 1-7, wherein: the nucleic acid molecule capable of expressing the zmct 2 protein is any one of the following:

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

(B2) A DNA molecule that hybridizes under stringent conditions to the DNA molecule defined in (B1) and encodes the zmct 2 protein;

(B3) A DNA molecule having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the DNA sequence defined in any one of (B1) to (B2) and encoding the zmct 2 protein.
9. Use of the method of any one of claims 4-8 in plant breeding.
10. The use or method according to any one of claims 1-9, wherein: the plant is monocotyledonous plant or dicotyledonous plant;

further, the monocotyledonous plant is a plant of the Gramineae family;

still further, the gramineous plant is a maize plant;

more specifically, the maize plant is maize.