Application of ZmPEPCK2 protein and coding gene thereof in regulation and control of corn yield and improvement of corn quality

Abstract

The invention discloses application of ZmPEPCK2 protein and a coding gene thereof in regulation and control of corn yield and improvement of corn quality. According to the invention, the ZmPEPCK2 gene is overexpressed in corn, so that the ZmPEPCK2 overexpressed transgenic corn is obtained. Experiments prove that compared with wild corn, the plant height, spike length, hundred-grain weight and grain width of ZmPEPCK2 overexpressed transgenic corn are increased, and the contents of partial amino acid, carotenoid and succinic acid in grains are increased. The result shows that the ZmPEPCK2 can increase the corn yield and improve the corn quality. The invention lays a foundation for research on cultivation of high-yield and high-quality transgenic plants.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of ZmPEPCK2 protein and its encoding gene in regulating maize yield and improving maize quality. Background Art

[0002] Maize is one of the three major important food crops in the world, and can also be widely used in animal feed, industrial and clean energy production. Maize is rich in starch and serves as an important food source in many regions, providing energy for people. With the increasing population and limited arable land area, it is particularly important to improve maize yield.

[0003] The increase in yield not only depends on the supply of carbohydrates, nitrogenous substances and other nutrients from the vegetative tissues - the "source" organs to the grain "sink" organs, but the utilization of nitrogen by the grain "sink" organs is more important. Grain size and 100-grain weight are important indicators for measuring yield. Amino acids are unloaded into the endosperm of grains through long-distance transportation in the phloem, thus providing an important amino acid source for the synthesis of stored proteins in maize endosperm. Maize endosperm can convert organic acids into sugars through gluconeogenesis. This conversion process can provide a carbon skeleton for the unloading of glutamine. By searching for key genes of gluconeogenesis and overexpressing them, the unloading of amino acids can be accelerated and the utilization of amino acids by grains can be promoted, thereby changing grain size and 100-grain weight - two important components of sink strength. Therefore, changing the utilization of assimilates by sink organs by altering the gluconeogenesis process to achieve yield increase provides new ideas for increasing crop yield. Summary of the Invention

[0004] An object of the present invention is to provide a protein.

[0005] The protein provided by the present invention is named ZmPEPCK2 and is derived from maize inbred line B73. The ZmPEPCK2 protein is any one of the following proteins (a1)-(a4):

[0006] (a1) The protein shown in Sequence 3;

[0007] (a2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in (a1);

[0008] (a3) A protein related to plant yield or quality obtained by substituting and / or deleting and / or adding one or several amino acid residues to (a1);

[0009] (a4) A protein having more than 98% identity with (a1) and related to plant yield or quality.

[0010] In the protein described in (a2) above, the tag refers to a polypeptide or protein that is fusion-expressed with the target protein using in vitro DNA recombination technology to facilitate the expression, detection, tracing, and / or purification of the target protein. The tag can be a Flag tag, His tag, MBP tag, HA tag, myc tag, GST tag, and / or SUMO tag, etc.

[0011] In the protein described in (a3) above, the substitution and / or deletion and / or addition of one or several amino acid residues are substitutions and / or deletions and / or additions of no more than 10 amino acid residues or substitutions and / or deletions and / or additions of no more than 9 amino acid residues or substitutions and / or deletions and / or additions of no more than 8 amino acid residues or substitutions and / or deletions and / or additions of no more than 7 amino acid residues or substitutions and / or deletions and / or additions of no more than 6 amino acid residues or substitutions and / or deletions and / or additions of no more than 5 amino acid residues or substitutions and / or deletions and / or additions of no more than 4 amino acid residues or substitutions and / or deletions and / or additions of no more than 3 amino acid residues or substitutions and / or deletions and / or additions of no more than 2 amino acid residues or substitutions and / or deletions and / or additions of no more than 1 amino acid residue.

[0012] In the protein described in (a4) above, the identity refers to the identity of the amino acid sequence. The identity of the amino acid sequence can be determined using homology search sites on the Internet, such as the BLAST web page on the NCBI home page website. For example, in Advanced BLAST 2.1, by using blastp as the program, setting the Expect value to 10, setting all Filters to OFF, using BLOSUM62 as the Matrix, setting the Gap existence cost, Per residue gap cost, and Lambda ratio to 11, 1, and 0.85 (default values) respectively and performing a search to calculate the identity of a pair of amino acid sequences, and then the identity value (%) can be obtained. The identity includes amino acid sequences having 90% or higher, or 91% or higher, or 92% or higher, or 93% or higher, or 94% or higher, or 95% or higher, or 96% or higher, or 97% or higher, or 98% or higher, or 99% or higher homology with the amino acid sequence shown in Sequence 3 of the present invention.

[0013] The protein described in (a1) or (a2) or (a3) or (a4) above can be artificially synthesized or can be obtained by first synthesizing its coding gene and then performing biological expression.

[0014] Another object of the present invention is to provide biological materials related to the ZmPEPCK2 protein.

[0015] The biomaterial related to the ZmPEPCK2 protein provided by the present invention is a nucleic acid molecule encoding the ZmPEPCK2 protein, or an expression cassette, recombinant vector, or recombinant microorganism containing the nucleic acid molecule.

[0016] Wherein, the nucleic acid molecule may be a DNA molecule as described in any one of the following (b1)-(b2):

[0017] (b1) The DNA molecule shown in Sequence 1 or Sequence 2;

[0018] (b2) A DNA molecule having more than 75% identity with (b1) and encoding the above-mentioned ZmPEPCK2 protein.

[0019] The nucleic acid molecule may be DNA, such as cDNA, genomic DNA, or recombinant DNA; the nucleic acid molecule may also be RNA, such as mRNA or hnRNA, etc.

[0020] Those of ordinary skill in the art can easily mutate the nucleotide sequence encoding the ZmPEPCK2 protein of the present invention by using known methods, such as directed evolution and point mutation methods. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence encoding the ZmPEPCK2 protein, as long as they encode the ZmPEPCK2 protein and have the same function, are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention.

[0021] As used herein, the term "identity" refers to the sequence similarity to the natural nucleic acid sequence. "Identity" includes a nucleotide sequence having 75% or higher, or 85% or higher, or 90% or higher, or 95% or higher identity with the nucleotide sequence of the protein composed of the amino acid sequence shown in Coding Sequence 3 of the present invention. Identity can be evaluated by the naked eye or by computer software. Using computer software, the identity between two or more sequences can be expressed as a percentage (%), which can be used to evaluate the identity between related sequences.

[0022] The above-mentioned identity of 75% or more than 75% may be 80%, 85%, 90%, or 95% or more identity.

[0023] The expression cassette may be DNA capable of expressing the ZmPEPCK2 protein in a host cell. This DNA may not only include a promoter that initiates the transcription of the ZmPEPCK2 gene, but also include a terminator that terminates the transcription of the ZmPEPCK2 gene. Further, the expression cassette may also include an enhancer sequence.

[0024] The recombinant vector may be a vector containing the DNA molecule encoding the ZmPEPCK2 protein shown in Sequence 1 or Sequence 2. For the convenience of identifying and screening transgenic plant cells or plants, the used plant expression vector can be processed, such as adding a gene encoding an enzyme or a luminescent compound that can produce a color change and can be expressed in plants (GUS gene, luciferase gene, etc.), an antibiotic marker with resistance (gentamicin marker, kanamycin marker, etc.) or an anti-chemical reagent marker gene (such as an anti-herbicide gene), etc.

[0025] The recombinant microorganism may be yeast, bacteria, algae and fungi containing the above nucleic acid molecule, the above expression cassette or the above recombinant vector. Specifically, the bacteria may be Agrobacterium (such as Agrobacterium EHA105).

[0026] Another object of the present invention is to provide a new use of the ZmPEPCK2 protein or its related biological material.

[0027] The present invention provides the application of the ZmPEPCK2 protein or its related biological material in any one of the following (c1)-(c10):

[0028] (c1) Regulating plant yield;

[0029] (c2) Regulating plant plant height;

[0030] (c3) Regulating plant ear length;

[0031] (c4) Regulating the grain width of plant grains;

[0032] (c5) Regulating the starch content of plant grains;

[0033] (c6) Regulating the content of amino acids (such as free amino acids) in plant grains;

[0034] (c7) Regulating the carotenoid content of plant grains;

[0035] (c8) Regulating the content of organic acids (such as organic acids in the tricarboxylic acid cycle) in plant grains;

[0036] (c9) Cultivating transgenic plants with improved yield and / or quality;

[0037] (c10) Plant breeding.

[0038] Furthermore, the carotenoids include β-cryptoxanthin, lutein and zeaxanthin.

[0039] The amino acids include L-alanine, glycine, valine, leucine, isoleucine, proline, phenylalanine, aspartic acid, glutamic acid and asparagine.

[0040] The organic acids include succinic acid, fumaric acid, L - malic acid, and citric acid.

[0041] Furthermore, the regulation of plant yield is manifested as an increase in the 100 - grain weight of the plant and / or an increase in the grain width of the plant grains.

[0042] The regulation of the carotenoid content in plant grains is manifested as an increase in the contents of β - cryptoxanthin, lutein, and zeaxanthin in the plant grains.

[0043] The regulation of the amino acid content in plant grains is manifested as an increase in the contents of L - alanine, glycine, valine, leucine, isoleucine, proline, and phenylalanine in the plant grains and / or a decrease in the contents of aspartic acid, glutamic acid, and asparagine in the plant grains.

[0044] The regulation of the organic acid content in plant grains is manifested as an increase in the content of succinic acid in the plant grains and / or a decrease in the contents of fumaric acid, L - malic acid, and citric acid in the plant grains.

[0045] The purpose of the plant breeding is to cultivate plant varieties with high yield and quality.

[0046] The last object of the present invention is to provide a method for cultivating transgenic plants with increased yield and / or quality.

[0047] The method for cultivating transgenic plants with increased yield and / or quality provided by the present invention includes the step of increasing the content and / or activity of ZmPEPCK2 protein in a recipient plant to obtain a transgenic plant; the yield and / or quality of the transgenic plant is higher than that of the recipient plant.

[0048] Furthermore, the fact that the yield of the transgenic plant is higher than that of the recipient plant is manifested as the 100 - grain weight of the transgenic plant being higher than that of the recipient plant and / or the grain width of the transgenic plant grains being longer than that of the recipient plant.

[0049] The fact that the quality of the transgenic plant is higher than that of the recipient plant is manifested as any one of the following 1) - 6):

[0050] 1) The plant height of the transgenic plant is higher than that of the recipient plant;

[0051] 2) The ear length of the transgenic plant is longer than that of the recipient plant;

[0052] 3) The starch content in the grains of the transgenic plant is higher than that of the recipient plant;

[0053] 4) The contents of amino acids (such as L - alanine, glycine, valine, leucine, isoleucine, proline, and phenylalanine) in the grains of the transgenic plant are higher than those of the recipient plant;

[0054] 5) The content of carotenoids (such as β-cryptoxanthin, lutein, and zeaxanthin) in the transgenic plant seeds is higher than that in the recipient plant;

[0055] 6) The content of organic acids (such as succinic acid) in the transgenic plant seeds is higher than that in the recipient plant.

[0056] Furthermore, the method for increasing the content and / or activity of ZmPEPCK2 protein in the recipient plant is to overexpress ZmPEPCK2 protein in the recipient plant.

[0057] The method of overexpression is to introduce the coding gene of ZmPEPCK2 protein into the recipient plant.

[0058] In any of the above applications or methods, the plant is a monocotyledon or a dicotyledon; the monocotyledon can be a gramineous plant; the gramineous plant can be maize; the specific maize variety can be B73-329.

[0059] In the above method, the transgenic plant is understood to include not only the first-generation transgenic plant obtained by transforming the recipient plant with the ZmPEPCK2 gene, but also its offspring. For transgenic plants, the gene can be propagated in this species, or transferred into other varieties of the same species by conventional breeding techniques, especially including commercial varieties. The transgenic plants include seeds, callus, whole plants, and cells.

[0060] In the present invention, the ZmPEPCK2 gene is overexpressed in maize to obtain ZmPEPCK2 overexpressing transgenic maize. Experiments have shown that compared with wild-type maize, the plant height, ear length, 100-grain weight, and grain width of ZmPEPCK2 overexpressing transgenic maize increase, and the contents of some amino acids, carotenoids, and succinic acid in the grains are increased. It shows that ZmPEPCK2 can increase the yield of maize and improve the quality of maize. The present invention lays a foundation for the research on cultivating transgenic plants with high yield and high quality. Description of the Drawings

[0061] Figure 1 It is the positive detection results of different transgenic maize lines. A is the detection at the DNA level. B is the qRT-PCR detection of the relative expression level of ZmPEPCK2 in transgenic positive seedlings, with the Actin gene in the wild type as a reference. C is the protein level detection of transgenic positive seedlings using total maize PEPCK antibody and GFP antibody, with Actin protein as an internal reference.

[0062] Figure 2Phenotypes, plant heights, and ear lengths of ZmPEPCK2 gene overexpression plants and wild-type plants. A shows the phenotypes of ZmPEPCK2 gene overexpression plants and wild-type plants. B shows the statistical results of the plant heights of ZmPEPCK2 gene overexpression plants and wild-type plants. C shows the statistical results of the ear lengths of ZmPEPCK2 gene overexpression plants and wild-type plants. **** indicates p < 0.0001; ** indicates p < 0.01.

[0063] Figure 3 Statistical analysis of grain size and phenotypic analysis of ZmPEPCK2 gene overexpression plants and wild-type plants. A shows the statistical results of the 100-grain weight of ZmPEPCK2 gene overexpression plants and wild-type plants. B shows the statistical results of the grain length of ZmPEPCK2 gene overexpression plants and wild-type plants. C shows the statistical results of the grain width of ZmPEPCK2 gene overexpression plants and wild-type plants. D shows the statistical results of the grain thickness of ZmPEPCK2 gene overexpression plants and wild-type plants. E and F are the phenotypic diagrams of the seed width and grain length of ZmPEPCK2 gene overexpression plants and wild-type plants, respectively. ** indicates p < 0.01; * indicates p < 0.05.

[0064] Figure 4 Analysis of the starch, soluble protein, and prolamin contents in the grains of ZmPEPCK2 gene overexpression plants and wild-type plants. A shows the statistical results of the starch content in the grains of ZmPEPCK2 gene overexpression plants and wild-type plants (n = 4). B shows the statistical results of the soluble protein content in the grains of ZmPEPCK2 gene overexpression plants and wild-type plants (n = 6). C shows the statistical results of the prolamin content in the grains of ZmPEPCK2 gene overexpression plants and wild-type plants (n = 6). *** indicates p < 0.001.

[0065] Figure 5 Analysis of the free amino acid, organic acid, and carotenoid contents in the grains of ZmPEPCK2 gene overexpression plants and wild-type plants. A shows the statistical results of the free amino acid contents that changed significantly in the grains of ZmPEPCK2 gene overexpression plants and wild-type plants. B shows the statistical results of the carotenoid content in the grains of ZmPEPCK2 gene overexpression plants and wild-type plants. C shows the statistical results of the organic acid contents that changed significantly in the TCA cycle in the grains of ZmPEPCK2 gene overexpression plants and wild-type plants (n = 3). *** indicates p < 0.001; ** indicates p < 0.01; * indicates p < 0.05. Detailed implementation methods

[0066] The present invention will be further described in detail below in conjunction with specific embodiments. The provided embodiments are only for clarifying the present invention and not for limiting the scope of the present invention. The following provided embodiments can be used as a guide for those of ordinary skill in the art to make further improvements and do not constitute any limitation to the present invention in any way.

[0067] In the following embodiments, the experimental methods, unless otherwise specified, are all conventional methods and are carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. The materials, reagents, etc. used in the following embodiments, unless otherwise specified, can be obtained from commercial channels.

[0068] The pCAMBIA3301 vector driven by the ubiquitin promoter in the following embodiments is described in the literature "Mingda Luan, Miaoyun Xu, Yunming Lu, Lan Zhang, Yunliu Fan, Lei Wang, Expression of zma-miR169 miRNAs and their target ZmNF-YA genes in response to abiotic stress in maize leaves, Gene, Volume 555, Issue 2, 2015, Pages 178 - 185, ISSN 0378 - 1119", and the public can obtain it from the Institute of Botany, Chinese Academy of Sciences. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0069] The Agrobacterium tumefaciens EHA105 strain in the following embodiments is described in the literature "Qu LQ, Xing YP, Liu WX, Xu XP, Song YR. (2008) Expression pattern and activity of six glutelin gene promoters in transgenic rice. Journal of Experimental Botany 59: 2417 - 2424.", and the public can obtain it from the Institute of Botany, Chinese Academy of Sciences. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0070] The maize transgenic transformation receptor material B73-329 in the following examples is described in the literature "Zhu, J., Song, N., Sun, S., Yang, W., Zhao, H., Song, W. and Lai, J. (2016) Efficiency and inheritance of targeted mutagenesis in maize using CRISPR-Cas9. J Genet Genomics, 43, 25-36." and can be obtained from the Institute of Botany, Chinese Academy of Sciences by the public. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0071] The maize inbred line experimental material B73 in the following examples is described in the literature "Jiao, Y., Peluso, P., Shi, J., Liang, T., Stitzer, M.C., Wang, B.,... & Ware, D. (2017). Improved maize reference genome with single-molecule technologies. Nature, 546(7659), 524-527." and can be obtained from the Institute of Botany, Chinese Academy of Sciences by the public. This biological material is only used for repeating the relevant experiments of the present invention and cannot be used for other purposes.

[0072] The starch content determination kit in the following examples is a product of Beijing Solarbio Science & Technology Co., Ltd., and the product number is BC0700.

[0073] Example 1. Obtaining of the ZmPEPCK2 protein-coding gene

[0074] 1. Place the seeds of maize inbred line B73 in the middle of a moist gauze (placed in a glass petri dish) and soak them in a 4°C refrigerator for 24 h to allow the seeds to absorb water without germinating. After removing the endosperm of the seeds using a surgical blade, quickly freeze the embryos in liquid nitrogen, grind them to extract total RNA, and reverse transcribe to obtain cDNA.

[0075] 2. Using the cDNA obtained in step 1 as a template, perform PCR amplification with primer F and primer R to obtain a PCR product.

[0076] F: 5'-CGGAAGCTTATGGCGACGCCGAACGGG-3';

[0077] R: 5'-CCGTCTAGAGAAGTTGGGGCCTGCGGCGAG-3'

[0078] 3. Perform agarose gel electrophoresis on the PCR products obtained in step 2, separate and purify the fragment approximately 2.0 kb in length, ligate this fragment into a T-vector and perform sequencing.

[0079] The sequencing results showed that the nucleotide sequence of this fragment was as shown in positions 1 - 1980 of Sequence 2 in the sequence listing.

[0080] Example 2. Obtaining of ZmPEPCK2 gene overexpression transgenic maize lines and analysis of their yield-related traits. I. Obtaining of ZmPEPCK2 gene overexpression transgenic maize lines

[0081] 1. Construction of ZmPEPCK2 gene overexpression vector

[0082] 1) Use the restriction endonuclease BamHI-HF (NEB) to perform single enzyme digestion on the 3301UBIGFP vector (the 3301UBIGFP vector is a vector obtained by inserting the GFP tag between the BamHI and SacI restriction sites of the pCAMBIA3301 vector driven by the ubiquitin promoter and keeping the other sequences of the pCAMBIA3301 vector unchanged), react at 37 °C for 3 h to obtain the BamHI-HF single enzyme digestion product of the 3301UBIGFP vector.

[0083] 2) Then use the CIP enzyme kit (NEB) to perform dephosphorylation treatment on the product of the 3301UBIGFP vector digested with BamHI-HF to avoid its self-ligation. After the dephosphorylation reaction is completed, immediately perform agarose gel electrophoresis and recover the enzyme digestion and dephosphorylation product. When recovering the DNA fragment, use the DNA gel recovery kit (TIANGEN), and the specific recovery process is operated according to the instructions provided by the kit.

[0084] 3) Use the cDNA of maize inbred line B73 as a template, perform PCR amplification with primers F / R to obtain the PCR product (the CDS sequence of the ZmPEPCK2 gene), and recover the cloned PCR product by agarose gel electrophoresis. When recovering, use the DNA gel recovery kit.

[0085] 4) Use the GBclonart seamless cloning kit to ligate the CDS sequence of the ZmPEPCK2 gene into the enzyme-digested and dephosphorylated 3301UBIGFP vector to obtain the homologous recombination product of the gene and the vector.

[0086] 5) Transform the homologous recombination product of the gene and the vector into DH5α competent cells by heat shock transformation method, and culture it using a resistant solid LB medium containing 50 mg / mL kanamycin.

[0087] 6) PCR detection of positive bacterial plaques in the bacterial solution. Using the 3301UBI-L primer designed according to the ubiquitin sequence and the antisense primer PCK924-R1 of the ZmPEPCK2 gene, PCR amplification was performed with the bacterial solution prepared from the grown bacterial plaques after transformation as the template. If a fragment with the correct size (fragment size is 2116bp) was detected after agarose gel electrophoresis, it indicated that the corresponding bacterial plaque was a positive bacterial plaque.

[0088] 3301UBI-L: 5'-GATGATGGCATATGCAGCAG-3';

[0089] PCK924-R1: 5'-CGGTAGACGTGATGAACGAC-3'.

[0090] 7) Pick positive bacterial plaques and culture them in large quantities using liquid LB medium containing 50 mg / mL kanamycin resistance. Extract the plasmid and name the positive plasmid 3301UBI-PEPCK2-GFP. The 3301UBI-PEPCK2-GFP plasmid can express the ZmPEPCK2 protein with a GFP tag at the C-terminus. Determine the plasmid concentration using NanoDrop2000. The plasmid extraction was performed using a plasmid rapid extraction kit (TIANGEN), and the experimental procedure was carried out according to the attached instructions in the kit.

[0091] 2. Construction of recombinant bacteria

[0092] Transform the 3301UBI-PEPCK2-GFP plasmid into Agrobacterium tumefaciens strain EHA105. After PCR identification, recombinant Agrobacterium containing the 3301UBI-PEPCK2-GFP plasmid was obtained, and this recombinant Agrobacterium was denoted as recombinant Agrobacterium OE.

[0093] 3. Obtaining and identification of ZmPEPCK2 gene overexpression transgenic maize lines

[0094] The recombinant Agrobacterium OE was transformed into the improved maize line B73-329. The specific transformation steps refer to the method in the literature "Zhu, J., Song, N., Sun, S., Yang, W., Zhao, H., Song, W. and Lai, J. (2016) Efficiency and inheritance of targeted mutagenesis in maize using CRISPR-Cas9. J Genet Genomics, 43, 25-36." The T0 generation of transgenic maize lines was harvested and the T1 generation of seeds was obtained. After the T1 generation of seeds germinated, the T1 generation of transgenic maize lines were screened by detecting at the DNA level, RNA level and protein level, and then the seeds of the T2 generation were obtained after harvesting; after the T2 generation of seeds germinated, the transgenic maize line with the highest expression level of the ZmPEPCK2 gene was selected for phenotypic analysis and named the ZmPEPCK2-OE line.

[0095] The T0 generation represents the plants in the transformation generation, the T1 generation represents the seeds produced by self-crossing of the T0 generation and the plants grown from them, and the T2 generation represents the seeds produced by self-crossing of the T1 generation and the plants grown from them.

[0096] The methods for detecting the above transgenic lines are as follows:

[0097] 1) Detection at the DNA level

[0098] Take the middle section of the first fully expanded leaf at the three-leaf and one-heart stage of the overexpression transgenic material and the wild type, extract the total DNA. First, use the specific primers for the resistance gene Bar-L / Bar-R to detect whether the resistance gene is inserted into the seedling genome; then use the combination of the primers on the vector and the gene-specific primers (3301UB1-L / PCK924-R1) to detect whether the vector is successfully inserted. The sequences of the above primers are as follows:

[0099] Bar-L: 5'-GAAGTCCAGCTGCCAGAAAC-3';

[0100] Bar-R: 5'-TCTGCACCATCGTCAACCAC-3';

[0101] 3301UBI-L: 5'-GATGATGGCATATGCAGCAG-3';

[0102] PCK924-R1: 5'-CGGTAGACGTGATGAACGAC-3';

[0103] 2) Detection at the RNA level

[0104] Take the middle section of the first fully expanded leaf at the three-leaf stage of transgenic materials and wild-type plants, extract the total RNA of the leaves using a plant RNA small-scale extraction kit from Megan Company, and synthesize cDNA using an Invitrogen reverse transcription kit with Oligo d(T) as the primer to obtain leaf cDNA. Use the specific quantitative detection primers PCK2-RT-1L / PCK2-RT-1R for ZmPEPCK2 to perform qRT-PCR analysis on ZmPEPCK2, with the Actin gene in maize as the internal reference. After analyzing the quantitative results, obtain the expression levels of the ZmPEPCK2 gene in different transgenic lines. The sequences of the above primers are as follows:

[0105] PCK2-RT-1L: 5'-GCGACCAAGCCCGACAAC-3';

[0106] PCK2-RT-1R: 5'-GTCCTTGATGGGCGTGGTTG-3';

[0107] Actin-L: 5'-CCTATCGTATGTGACAATGGCACT-3';

[0108] Actin-R: 5'-GCCTCATCACCTACGTAGGCAT-3'.

[0109] 3) Protein level detection

[0110] Extract the total proteins in the overexpressed transgenic materials and wild-type maize leaves using an extraction buffer with high pH value (250 mM bicine, pH = 9.0), and use a specific antibody against the PEPCK protein in maize to detect the expression level of the PEPCK2 protein by immunoblotting. When detecting, use the Actin protein as the internal reference.

[0111] The detection results of different transgenic lines are as Figure 1 shown, among which, Figure 1 A is the detection result at the DNA level of different lines, Figure 1 B is the detection result at the RNA level of different lines, Figure 1C shows the results of protein level detection for different strains. The results indicate that at the DNA level, compared with the negative control wild type, the vector sequences were successfully inserted into the genomes of the ZmPEPCK2 overexpression strains OE-L1, OE-L2, and OE-L3, and their PCR amplification results were consistent with the positive plasmid. At the RNA level, relative to the wild type, the RNA expression levels of ZmPEPCK2 in the ZmPEPCK2 overexpression strains OE-L1, OE-L2, and OE-L3 all increased significantly. Among them, the expression level of ZmPEPCK2 in OE-L2 was the highest. At the protein level, two bands were detected in the ZmPEPCK2 overexpression strains OE-L1, OE-L2, and OE-L3. The band at the lower position was approximately 90 kD in size, which was the size of the normal PEPCK protein, while the band at the upper position was approximately 30 kD larger than the PEPCK protein and was consistent with the size of the GFP protein (27 kD). When detected with the GFP antibody, bands were detected at approximately 120 kD in the materials of these events, indicating that the ZmPEPCK2 gene was successfully overexpressed in these events. From the perspectives of protein and RNA expression levels, the expression level of the ZmPEPCK2 overexpression strain OE-L2 was the highest. Therefore, the L2 strain was selected for the following phenotypic analysis.

[0112] II. Analysis of Yield and Quality-Related Traits of Transgenic Maize

[0113] 1. Overexpression of ZmPEPCK2 Increases Maize Plant Height and Ear Length

[0114] Seeds of the T2 generation ZmPEPCK2-OE homozygous strain and the wild type maize B73-329 strain (hereinafter referred to as WT) were taken respectively and planted according to the conventional method. The phenotypes of ZmPEPCK2-OE and WT were observed at the reproductive growth stage (grown for 56 days), and the plant height was statistically analyzed. The ears of mature and dehydrated WT and ZmPEPCK2-OE were taken for ear length statistics.

[0115] The results are as Figure 2 shown. Among them, A shows the phenotypes of ZmPEPCK2-OE and WT; B shows the plant heights of ZmPEPCK2-OE and WT; C shows the ear lengths of ZmPEPCK2-OE and WT. The results indicate that the average plant heights of ZmPEPCK2-OE and WT were 141.88 cm and 125.7 cm respectively, and the average ear lengths of ZmPEPCK2-OE and WT were 22 cm and 12.54 cm respectively. Compared with WT, the plant height and ear length of ZmPEPCK2-OE both increased significantly.

[0116] 2. Overexpression of ZmPEPCK2 Increases Kernel Width and 100-Kernel Weight of Maize

[0117] Yield-related traits of ZmPEPCK2-OE and WT grains were analyzed, including grain length, grain width, grain thickness, and 100-grain weight.

[0118] The results are shown as Figure 3 follows. Among them, A is the 100-grain weight of ZmPEPCK2-OE and WT; B is the grain length of ZmPEPCK2-OE and WT; C is the grain width of ZmPEPCK2-OE and WT; D is the grain thickness of ZmPEPCK2-OE and WT; E is the comparison diagram of the grain width of ZmPEPCK2-OE and WT at 30 days after pollination (30 DAP); Figure 3 F is the comparison diagram of the grain length of ZmPEPCK2-OE and WT at 30 days after pollination (30 DAP). The results show that after overexpression of ZmPEPCK2, its 100-grain weight increased significantly. Among them, the average 100-grain weights of ZmPEPCK2-OE and WT were 21.95 g and 17.924 g, respectively, and the 100-grain weight of ZmPEPCK2-OE increased by about 22.5% compared with WT. In addition, the grain width also increased significantly. The average grain width of ZmPEPCK2-OE grains was 7.72 ± 0.54 mm, and the average grain width of WT grains was 6.71 ± 0.39 mm. Grain length and grain thickness were not affected by the overexpression of ZmPEPCK2.

[0119] 3. Overexpression of ZmPEPCK2 increases the starch content in maize grains

[0120] Grains of ZmPEPCK2-OE and WT at 30 days after pollination were ground into powder under liquid nitrogen environment, and the starch content of the grains was measured.

[0121] The results are shown as Figure 4 follows. Among them, A is the starch content of ZmPEPCK2-OE and WT grains; B is the soluble protein content in ZmPEPCK2-OE and WT grains; C is the prolamin content in ZmPEPCK2-OE and WT grains. The results show that the average starch content in the grains of ZmPEPCK2-OE plants was 662.01 mg / g, which was significantly higher than that in WT grains (610.48 mg / g). Compared with WT, the starch content in ZmPEPCK2-OE grains increased by 8.4%. Overexpression of ZmPEPCK2 had no significant effect on the soluble protein content and prolamin content of the seeds.

[0122] 4. Overexpression of ZmPEPCK2 increases the contents of amino acids, organic acids, and carotenoids in maize grains

[0123] Grains of ZmPEPCK2-OE and WT at 30 days after pollination were taken, and the metabolite contents in the grains were detected by liquid chromatography-mass spectrometry technology.

[0124] The results are shown asFigure 5 As shown in the figure, where A is the difference map showing the amino acid content in ZmPEPCK2-OE and WT grains, B is the difference map showing the carotenoid content in ZmPEPCK2-OE and WT grains, and C is the difference map showing the organic acid content in ZmPEPCK2-OE and WT grains. The results show that compared with WT, a total of 17 metabolites changed significantly in ZmPEPCK2-OE grains, including amino acids, organic acids, and carotenoids.

[0125] The detection results of amino acid content show that: in WT grains, the contents of L-alanine, glycine, valine, leucine, isoleucine, proline, and phenylalanine are 84.82 μg / g, 9.43 μg / g, 15.17 μg / g, 9.05 μg / g, 9.51 μg / g, 395.63 μg / g, and 13.54 μg / g respectively. In ZmPEPCK2-OE grains, the contents of L-alanine, glycine, valine, leucine, isoleucine, proline, and phenylalanine are 106.43 μg / g, 11.41 μg / g, 17.45 μg / g, 11.64 μg / g, 10.83 μg / g, 469.36 μg / g, and 15.05 μg / g respectively, and the contents of these several amino acids increased significantly (p < 0.05); in WT grains, the contents of aspartic acid, glutamic acid, and asparagine are 59.01 μg / g, 92 μg / g, and 153.87 μg / g respectively. In ZmPEPCK2-OE grains, the contents of aspartic acid, glutamic acid, and asparagine are 47.15 μg / g, 71.66 μg / g, and 136.25 μg / g respectively, and the contents of these several amino acids decreased significantly; in addition, the contents of the other 8 amino acids (arginine, β-alanine, histidine, methionine, serine, threonine, cysteine, lysine) showed no significant changes in ZmPEPCK2-OE and WT grains.

[0126] The detection results of carotenoid content show that the contents of the three carotenoids, β-cryptoxanthin, lutein, and zeaxanthin, all increased significantly with the increase in the expression level of ZmPEPCK2. Among them, in WT grains, the contents of β-cryptoxanthin, lutein, and zeaxanthin are 3.28 μg / g, 25.78 μg / g, and 7.71 μg / g respectively. However, in ZmPEPCK2-OE grains, the contents of β-cryptoxanthin, lutein, and zeaxanthin are 4.28 μg / g, 34.47 μg / g, and 9.32 μg / g respectively. Compared with the wild type, the contents of these three carotenoids in ZmPEPCK2-OE grains are 1.3 times, 1.3 times, and 1.2 times that of the wild type respectively.

[0127] The detection results of various organic acids in the tricarboxylic acid cycle showed that the content of succinic acid in WT grains was 28.63 μg / g, while in ZmPEPCK2-OE grains, the content of succinic acid was 34.28 μg / g. The content of succinic acid in ZmPEPCK2-OE grains was 1.2 times that in WT grains, indicating that overexpression of ZmPEPCK2 could significantly increase the content of succinic acid. The content of fumaric acid in WT grains was 22.89 μg / g, and the content of fumaric acid in ZmPEPCK2-OE grains was 17.73 μg / g. In addition, the content of L-malate in WT grains was 101.75 μg / g, while the content of L-malate in ZmPEPCK2-OE grains was 57.1 μg / g. The content of L-malate was only 56% of that in WT grains. The content of citric acid in WT grains was 1020.21 μg / g, and the content of citric acid in ZmPEPCK2-OE grains was 807.39 μg / g. Generally speaking, the contents of fumaric acid, L-malate and citric acid in ZmPEPCK2-OE grains all decreased significantly.

[0128] The present invention has been described in detail above. For those skilled in the art, without departing from the gist and scope of the present invention and without unnecessary experiments, the present invention can be implemented within a relatively wide range under equivalent parameters, concentrations and conditions. Although specific embodiments of the present invention are given, it should be understood that the present invention can be further improved. In short, according to the principle of the present invention, this application intends to cover any modifications, uses or improvements of the present invention, including those that depart from the scope disclosed in this application but are made by conventional techniques known in the art. Some basic features can be applied according to the scope of the appended claims below.

Claims

1. A protein, which is any one of the following (a1)-(a4): (a1) The protein shown in Sequence 3; (a2) A fusion protein obtained by connecting a tag to the N-terminus and / or C-terminus of the protein described in (a1); (a3) A protein related to plant yield or quality obtained by substituting and / or deleting and / or adding one or several amino acid residues to the protein described in (a1); (a4) A protein having more than 98% identity with the protein described in (a1) and related to plant yield or quality.

2. A biological material related to the protein described in Claim 1, wherein the biological material is a nucleic acid molecule encoding the protein described in Claim 1, or an expression cassette, a recombinant vector, or a recombinant microorganism containing the nucleic acid molecule.

3. The biomaterial according to claim 2, characterized in that: The nucleic acid molecule is any one of the following (b1)-(b2) DNA molecules: (b1) The DNA molecule shown in Sequence 1 or Sequence 2; (b2) A DNA molecule having more than 75% identity with (b1) and encoding the protein described in Claim 1.

4. The application of the protein described in Claim 1 in any one of the following (c1)-(c10): (c1) Regulating plant yield; (c2) Regulating plant plant height; (c3) Regulating plant ear length; (c4) Regulating the grain width of plant grains; (c5) Regulating the starch content of plant grains; (c6) Regulating the amino acid content of plant grains; (c7) Regulating the carotenoid content of plant grains; (c8) Regulating the organic acid content of plant grains; (c9) Cultivating transgenic plants with improved yield and / or quality; (c10) Plant breeding.

5. The application according to claim 4, characterized in that: The carotenoids include β-cryptoxanthin, lutein, and zeaxanthin.

6. The application according to claim 4 or 5, characterized in that: The amino acids include L-alanine, glycine, valine, leucine, isoleucine, proline, phenylalanine, aspartic acid, glutamic acid, and asparagine.

7. The application according to any one of claims 4-6, characterized in that: The organic acids include succinic acid, fumaric acid, L-malic acid, and citric acid.

8. A method for cultivating transgenic plants with improved yield and / or quality, comprising the step of increasing the content and / or activity of the protein described in Claim 1 in a recipient plant to obtain a transgenic plant; the yield and / or quality of the transgenic plant is higher than that of the recipient plant.

9. The method according to claim 8, wherein: The method for increasing the content and / or activity of the protein described in Claim 1 in the recipient plant is to overexpress the protein described in Claim 1 in the recipient plant.

10. The application according to any one of claims 4 to 7 or the method according to claim 8 or 9, characterized in that: The plant is a monocotyledonous plant or a dicotyledonous plant; and / or, the monocotyledonous plant is a gramineous plant; and / or, the gramineous plant is maize.

Images