Corn ZmGLN3 gene and application of corn ZmGLN3 gene in increasing plant stem protein content

Abstract

The invention discloses a corn ZmGLN3 gene and application of the corn ZmGLN3 gene in increasing the protein content of plant stems. QTL positioning is carried out based on a BC2S2 group of wild corn Ames21814 and modern cultivated corn B73, a key gene ZmGLN3 for controlling the corn stalk protein content is excavated, and structural variation analysis finds that the ZmGLN3 gene has tandem repeat variation in the Ames21814, so that the expression quantity of the gene in the Ames21814 is remarkably improved, and the total protein content of the stalk and the whole plant of the Ames21814 is further effectively improved. The invention also finds that the stem protein content of the ZmGLN3 gene overexpression material is also obviously improved. The discovery of the ZmGLN3 gene and the excellent haplotype thereof provides an efficient means for genetic improvement and germplasm resource innovation of whole-plant silage corn, and has important application value and economic prospect.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a corn ZmGLN3 gene and an application thereof in increasing the protein content of plant stems. Background Art

[0002] Corn is one of the most important food crops in the world. China has long been the leader in corn planting area and yield. In 2023, the national corn planting area will exceed 44 million hectares, of which about 70% will be used as feed, mainly for livestock and poultry breeding. Silage corn has become one of the largest and most widely distributed feed raw materials in the world due to its high yield, high energy, rich nutrition and good palatability. It is also an important feed source for the development of my country's animal husbandry.

[0003] Studies have shown that wild corn retains rich genetic diversity related to protein and biomass accumulation compared to modern cultivated corn. During the domestication of corn, some key genes were gradually lost, but these genes may play an important role in stress resistance, high protein accumulation and biomass increase. For example, the Teosinte High Protein9 (THP9) gene cloned from wild corn significantly improved the whole plant protein content and nitrogen utilization efficiency after being introduced into modern corn.

[0004] Protein content is an important indicator for measuring the quality of silage corn, and its content determines the quality of silage corn. At present, the protein content of most silage corn is low (only 7% to 9%), while the whole plant protein content of the closely related wild corn, Teosinte, is high (greater than 13%). Therefore, the exploration and utilization of high-protein genes in wild corn has important application potential for the genetic improvement of silage corn quality. The present invention intends to develop new genes that increase the protein content of plant stems and the protein content of the whole plant, thereby providing technical support for the genetic improvement of whole plant silage corn and the innovation of germplasm resources. Summary of the invention

[0005] The purpose of the present invention is to provide a maize ZmGLN3 gene and its application in increasing the protein content of plant stems to solve the problems existing in the above-mentioned prior art. The gene has the function of increasing the protein content of plant stems and the protein content of the whole plant, and provides technical support for the genetic improvement of high-protein plant varieties and the innovation of germplasm resources.

[0006] In the BC2S2 population constructed by Ames21814 and B73, the present invention found a QTL signal significantly correlated with the content of stem protein on chromosome 9 through QTL analysis. This signal is different from the grain protein QTL previously located by the ASN4 gene, indicating that there is also a stem protein-related QTL region on chromosome 9 that is independent of grain protein regulation. Through the expression difference of genes in the QTL region and the analysis of nitrogen metabolic pathways, it was found that ZmGLN3 (gene number: Zm00001eb399860) was highly expressed in Ames21814. The glutamine synthetase encoded by ZmGLN3 is responsible for the synthesis of glutamine and is a key enzyme in the nitrogen assimilation process. Therefore, ZmGLN3 was confirmed as a candidate gene. Genome comparison found that ZmGLN3 in Ames21814 had a tandem repeat structure variation, and RT-qPCR analysis showed that the variation significantly increased the expression of ZmGLN3. To this end, the present invention designs a molecular marker, and the haplotype variation of ZmGLN3 (ZmGLN3-Ames21814) can be identified by conventional PCR. Through the construction of molecular marker-assisted near-isogenic lines, NIL-Ames21814 significantly increased the protein content of the stem and the whole plant, indicating its regulatory role in nitrogen metabolism and accumulation.

[0007] Based on this, the present invention provides the following solutions:

[0008] The present invention provides a protein having the function of increasing the nitrogen content and biomass of plants, wherein the protein is any one of (A1), (A2) and (A3):

[0009] (A1) ZmGLN3 protein, the amino acid sequence of which is shown in SEQ ID NO.1;

[0010] (A2) replacing, deleting and / or adding one or more amino acid residues in the amino acid sequence of the ZmGLN3 protein to obtain a protein having more than 90% identity with the ZmGLN3 protein and related to plant high protein;

[0011] (A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of (A1) or (A2).

[0012] The present invention also provides a gene encoding the above protein.

[0013] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0014] The present invention also provides a gene expression cassette, comprising the above-mentioned coding gene.

[0015] The present invention also provides a recombinant expression vector, comprising the above gene expression cassette.

[0016] The present invention also provides a recombinant host cell, comprising the above-mentioned recombinant expression vector.

[0017] The present invention also provides the use of the above-mentioned encoding gene, gene expression cassette, recombinant expression vector or recombinant host cell in any one of the following (B1)-(B4):

[0018] (B1) Increase the protein content of plants;

[0019] (B2) increasing plant biomass;

[0020] (B3) Improve plant nitrogen utilization efficiency;

[0021] (B4) Cultivate high-protein plant varieties.

[0022] Furthermore, the plant is corn.

[0023] The corn may specifically be varieties such as Zhengdan 958, Xianyu 335, Jingke 968, Denghai 605, Demeya 1, Demeya 3, Heyu 187, Suyu 29, Jingnongke 728, Zhongdan 808, and Zhengda 808.

[0024] The present invention also provides a method for increasing the protein content of plant stems, comprising the steps of introducing the above encoding gene into plants to construct a transgenic plant that overexpresses the encoding gene.

[0025] The present invention also provides a method for increasing plant biomass, comprising the steps of introducing the above encoding gene into a plant to construct a transgenic plant that overexpresses the encoding gene.

[0026] The "introducing the above-mentioned encoding gene into plants" is achieved by introducing a recombinant expression vector containing the encoding gene into the recipient plant.

[0027] The existing plant expression vector can be used to construct a recombinant expression vector containing the coding gene. The plant expression vector includes a binary Agrobacterium vector and a vector that can be used for plant microprojectile bombardment. The plant expression vector can also contain 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 the addition of polyadenylic acid 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 plant gene (such as the soybean storage protein gene) 3' end transcribed non-translated region has similar functions.

[0028] When using the above-mentioned encoding gene to construct a recombinant expression vector, any enhanced promoter or constitutive promoter (such as cauliflower mosaic virus (CAMV) 35S promoter, corn ubiquitin promoter (Ubiquitin)) can be added before its transcription initiation nucleotide, or tissue-specific expression promoter (such as the promoter of seed-specific expression), which can be used alone or in combination with other plant promoters. In addition, when using this encoding gene to construct a recombinant expression vector, enhancers can also be used, including translation enhancers or transcription enhancers, and these enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be identical with the reading frame of the encoding sequence to ensure the correct translation of the entire sequence. The source of the enhancer is extensive, can be natural, and can also be synthetic. The translation initiation region can be from the transcription initiation region or a structural gene.

[0029] 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 resistance markers (gentamicin marker, kanamycin marker, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc.

[0030] In the present invention, the recombinant expression vector may specifically be a recombinant expression vector obtained by replacing the fragment between the SpeⅠ and BamhⅠ restriction sites of the UBI-cFLAG vector with the DNA molecule shown in SEQ ID NO.2.

[0031] In the above method, a recombinant expression vector carrying the above coding gene is introduced into the recipient plant, specifically by transforming plant cells or tissues using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, and the transformed plant tissue is cultivated into a plant.

[0032] Transformed cells, tissues or plants are understood to include not only the final product of the transformation process, but also its transgenic progeny. The present invention discloses the following technical effects:

[0033] The present invention conducts QTL positioning based on the BC2S2 population of wild corn Ames21814 and modern cultivated corn B73, and digs out the key gene ZmGLN3 that controls the corn stalk protein content, and finds through structural variation analysis that the ZmGLN3 gene has a tandem duplication variation in Ames21814, resulting in a significant increase in the expression of the gene in Ames21814, thereby effectively increasing the stalk and total protein content of the whole plant of Ames21814. The present invention also finds that the stalk protein content of the ZmGLN3 gene overexpression material is also significantly increased.

[0034] The discovery of the ZmGLN3 gene and its superior haplotypes provides an efficient means for the genetic improvement and germplasm resource innovation of whole-plant silage corn, and has important application value and economic prospects. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0036] Figure 1 Schematic diagram of the construction process of the BC2S2 population of B73×Ames21814;

[0037] Figure 2 This is the determination of stem protein content and phenotypic distribution diagram;

[0038] Figure 3 This is the QTL location map for stem protein content;

[0039] Figure 4 The figure is a diagram of the gene structure of the tandem duplication variation of the ZmGLN3 gene in B73 and Ames21814 and its molecular marker identification; wherein A is a schematic diagram of the gene structure of the tandem duplication variation of the ZmGLN3 gene; B is the result of detecting the expression of the ZmGLN3 gene using primers F1 and R1; C is an electrophoresis diagram of molecular marker identification;

[0040] Figure 5 Statistical graph of gene expression level of ZmGLN3 in B73 and Ames21814;

[0041] Figure 6 Schematic diagram of the construction process of the ZmGLN-Ames21814 near-isogenic line;

[0042] Figure 7 The statistical diagram of the stem protein content of NIL-Ames21814 and NIL-B73; among them, the ZmGLN3 of the NIL-Ames21814 material is the Ames21814 background; the ZmGLN3 of the NIL-B73 material is the B73 background;

[0043] Figure 8 Statistical graph of stem protein content in ZmGLN3 gene overexpression material (OE-ZmGLN3) and control receptor material (CK). DETAILED DESCRIPTION

[0044] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0045] It should be understood that the terms described in the present invention are only for describing a particular embodiment and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. The intermediate value in any stated value or stated range, and each smaller range between any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0046] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0047] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary only.

[0048] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0049] Self-pollination, hybridization, and field breeding of corn are carried out according to conventional breeding methods.

[0050] The primer synthesis and gene sequencing in the examples were all completed by Hangzhou Youkang Biotechnology Co., Ltd.

[0051] The molecular biology experiments in the embodiments include plasmid construction, enzyme digestion, ligation, competent cell preparation, transformation, culture medium preparation, etc., and are mainly carried out with reference to Molecular Cloning Experiment Guide (3rd edition), edited by J. Sambrook and DW Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). If necessary, the specific experimental conditions can be determined by simple experiments.

[0052] PCR amplification experiments were performed according to the reaction conditions provided by the reagent supplier or the kit instructions. If necessary, adjustments could be made through simple experiments.

[0053] The primer sequences involved in the following examples are shown in Table 1.

[0054] Table 1 Primer sequences

[0055]

[0056] Wild corn Ames21814 has been disclosed in the document “THP9 enhances seed protein content and nitrogen-use efficiency in maize”, and the present invention promises to release it to the public 20 years from the filing date of the present invention.

[0057] Example 1 QTL mapping of BC2S2 population revealed that ZmGLN3 is a key gene controlling the total protein content of maize stems and whole plants

[0058] Construction of the BC2S2 population of wild maize Ames21814 and modern cultivated maize B73 ( Figure 1 ) was planted and self-pollinated in Sanya, and the ear stems were taken 25 days after pollination. The samples were then dried at 65°C to constant weight and finally ground into powder using a grinder (60Hz, 60s). 50-70mg of powder was wrapped in tin foil as the test sample. The total nitrogen was determined using the Dumas rapid nitrogen analyzer from Elementar, Germany. Before each round of measurement, 4 standards (aspartic acid samples) were weighed as internal references. After the machine was debugged, the weight of each sample was entered in the weight column of the Fast N Ultra software (v1.1.25), and (O2 addition time: 60s; O2 addition flow: 120mL / min; O2 cut-off threshold: 15%; automatic zeroing delay: 30s; select Peak anticip: 90s) as the program setting. At the same time, the packaged samples were placed in the sample jars according to the corresponding serial numbers, and 55 samples could be tested in each round. Finally, the stem protein content and phenotypic distribution data were obtained ( Figure 2 ). High-throughput resequencing of the BC2S2 population and QTL localization combined with stem protein content revealed a QTL region with the most significant signal on chromosome 9. Combined with nitrogen metabolism network and transcriptome data, the candidate gene ZmGLN3 ( Figure 3 ).

[0059] The amino acid sequence of the ZmGLN3 protein is shown in SEQ ID NO.1; the CDS sequence of the ZmGLN3 gene is shown in SEQ ID NO.2.

[0060] SEQ ID NO.1:

[0061] MASLTDLVNLDLSDCTDRIIAEYIWVGGSGIDLRSKARTVKGPITDPSQLPKWNYDGSSTGQAPGEDSEVIFYPQAIFKDPFRKGNNILVMCDCYTPQGEPIPSNKRYKAATVFSHPDVAAEVPWYGIEQEYTLLQKDVSWPLGWPVGGYPGPQGPYYCAAGADKAFGRDVVDAHYKACLYAGINISGINGEVMPGQWEFQVGPSVGISAGDEIWVARYILERITEMAGIVLSLDPKPIKGDWNGAGAHTNYSTKSMREAGGYEVIKEAIEKLGKRHREHIAAYGEGNERRLTGRHETADINTFKWGVANRGASIRVGRDTEKEGKGYFEDRRPASNMDPYVVTGMIADTTILWKGN*.

[0062] SEQ ID NO.2:

[0063]

[0064] Example 2 Identification of tandem repeat variation and gene expression analysis of ZmGLN3-Ames21814

[0065] Using the high-precision Ames21814 genome data assembled earlier, we compared the differences between the ZmGLN3 gene in the Ames21814 and B73 genomes and found that the ZmGLN3 gene may have tandem duplication variation in Ames21814. First, we designed primers F1 and R1, and through RT-qPCR analysis using DNA as a template, we preliminarily determined that there was a tandem duplication variation in ZmGLN3 in Ames21814 ( Figure 4 A and B in Figure 1). Molecular marker primers F2, F3, and R2 were further designed to identify the tandem repeat variation of ZmGLN3. When PCR amplification was performed with F3 and R2, a 152 bp sequence could be amplified from both Ames21814 and B73 DNA; when PCR amplification was performed with F2 and R2, a 712 bp sequence could only be amplified from Ames21814 DNA ( Figure 4 This result fully confirmed the existence of tandem duplication variation in ZmGLN3 in Ames21814.

[0066] The present invention further extracted RNA from the leaves of Ames21814 and B73, designed quantitative primers F4 and R4, and performed RT-qPCR analysis on the ZmGLN3 gene. It was found that the expression level of this gene in the leaves of Ames21814 was significantly higher than that in B73 ( Figure 5 ), and the difference in expression levels is caused by the occurrence of tandem repeat structure variation.

[0067] Genomic DNA extraction and PCR identification method:

[0068] Extract maize DNA by CTAB method for subsequent identification. Place maize leaves in a 2 mL tube, add a steel column, treat with liquid nitrogen and grind (60 Hz, 60 s); after grinding, add 0.6 mL CTAB extraction buffer and mix; place in a 65 ° C oven for 60 min, mix every 10-15 min; take out and place at room temperature for 5-10 min, add an equal volume of chloroform: isoamyl alcohol (24:1) to the centrifuge tube, seal and shake for 5 min; centrifuge at room temperature at 13000 rpm for 15 min, and aspirate the supernatant into a new 1.5 mL centrifuge tube; add an equal volume of isopropanol, mix by inverting back and forth, and place at minus 20 degrees for 20 min; centrifuge at room temperature at 12000 rpm for 1 min, and pour out the supernatant. Wash the DNA precipitate twice with 1 mL of 75% ethanol, centrifuge at 12000 rpm for 1 min each time, pour out the ethanol; centrifuge briefly, aspirate excess liquid, and dry the DNA precipitate at room temperature; add 0.3 mL of ddH2O to dissolve the DNA precipitate.

[0069] PCR detection reaction was carried out using the Master Mix (With Dye) kit, prepare 20 μL reaction system: Master Mix 10μL, primers 1μL each, DNA 2μL, ddH2O 6μL, specific detection primers designed by NCBI, reaction conditions: pre-denaturation at 94℃ for 5min, denaturation at 94℃ for 30s, annealing at 58℃ for 30s, extension at 72℃ for 30-60s / Kb, 35 cycles, final extension at 72℃ for 10min.

[0070] RNA level RT-qPCR analysis:

[0071] (1) RNA extraction: RNA was extracted using the OminiPlant RNA Kit from OmniPlant Biotech Co., Ltd. according to the standard method in the kit.

[0072] (2) RNA reverse transcription: RNA reverse transcription was performed using HiFiScript gDNARemoval cDNA Synthesis Kit from HiFiScript Biotechnology Co., Ltd. according to the standard method in the kit.

[0073] (3) Specific primers were designed by NCBI and specific analysis was performed, using Actin as the internal reference gene.

[0074] (3) Quantitative analysis was performed according to the standard method of the 2×SuperFast Universal SYBR Master Mix kit of Kangwei Century Biotechnology Co., Ltd. Three technical replicates were performed for each sample, and the cDNA samples after reverse transcription were uniformly diluted 10 times and quantitative analysis was performed using a 10 μL reaction system. According to the 10 μL reaction system, 5 μL of 2×SuperFast Universal SYBR Master Mix, 0.2 μL of each primer, 3 μL of diluted cDNA, and 1.6 μL of ddH2O were added.

[0075] (4) The gene expression level was detected by two-step PCR amplification method using BIO-RAD fluorescence quantitative analyzer. The reaction conditions were: pre-denaturation at 95°C for 30 s; amplification at 95°C for 5 s, 60°C for 35 s, and 40 cycles; termination at 95°C for 15 s; 60°C for 60 s; and 95°C for 15 s.

[0076] (5) The quantitative data were analyzed using the ΔΔCT method.

[0077] DNA level RT-qPCR analysis:

[0078] The experimental method and its steps were the same as those for RNA level RT-qPCR analysis, but the quantitative template was 10 ng / μL DNA.

[0079] Example 3 Determination of stem protein content in ZmGLN3 near-isogenic lines and overexpression materials

[0080] Starting from the F1 hybrid offspring of Ames21814 and B73, the present invention backcrossed to the BC9F1 generation based on the sequence differences between Ames21814 and B73, and self-pollinated to obtain a near-isogenic line of the homozygous ZmGLN3-Ames21814 candidate interval ( Figure 6 ), planted and 25 days after pollination, the ear stems were taken and the stem protein content was determined ( Figure 7 ), and found that the stem protein content of plants containing ZmGLN3-Ames21814 was significantly increased. This further verified the possibility that the tandem repeat mutation of ZmGLN3 led to an increase in the total protein content of corn stems and the whole plant.

[0081] According to the expression characteristics of the ZmGLN3 gene, the present invention amplifies the full-length coding sequence of the candidate gene from the cDNA of the Ames21814 tissue, and clones it in front of the FLAG tag of the plant overexpression vector driven by the UBI promoter. Subsequently, the corn inbred line B73 was used as the background material, and the overexpression transgenic strain was constructed by the Agrobacterium-mediated method, and the most suitable positive transformation strain T0 generation was selected by screening and identification. The T0 generation plants were self-pollinated to obtain the T1 generation plants. The T1 generation plants were identified, and the T1 generation plants identified as positive were selected for self-pollination to obtain the T2 generation plants. The T2 generation was planted and tested in Chengdu, Sichuan. The results showed that the stem protein content of the ZmGLN3 gene overexpression material was significantly increased ( Figure 8 ).

[0082] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. A protein having the function of increasing the protein content in plant stems, characterized in that: The protein is any one of (A1), (A2) and (A3): (A1) ZmGLN3 protein, the amino acid sequence of which is shown in SEQ ID NO.1; (A2) replacing, deleting and / or adding one or more amino acid residues in the amino acid sequence of the ZmGLN3 protein to obtain a protein having more than 90% identity with the ZmGLN3 protein and related to plant high protein; (A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of (A1) or (A2).

2. A gene encoding the protein according to claim 1.

3. The coding gene according to claim 2, characterized in that The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

4. A gene expression cassette, characterized in that: Comprising the coding gene described in claim 2 or 3.

5. A recombinant expression vector, characterized in that: Comprising the gene expression cassette of claim 4.

6. A recombinant host cell, characterized in that Comprising the recombinant expression vector according to claim 5.

7. Use of the coding gene according to claim 2 or 3, the gene expression cassette according to claim 4, the recombinant expression vector according to claim 5 or the recombinant host cell according to claim 6 in any one of the following (B1) to (B4): (B1) Increase the protein content of plants; (B2) increasing plant biomass; (B3) Improve plant nitrogen utilization efficiency; (B4) Cultivate high-protein plant varieties.

8. The use according to claim 7, characterized in that: The plant is corn.

9. A method for increasing the protein content of plant stems, characterized in that: The method comprises the steps of introducing the coding gene according to claim 2 or 3 into a plant to construct a transgenic plant that overexpresses the coding gene.

10. A method for increasing plant biomass, characterized in that: The method comprises the steps of introducing the coding gene according to claim 2 or 3 into a plant to construct a transgenic plant that overexpresses the coding gene.

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