Maize ZmGLN3 gene and its application in increasing the protein content in plant stems

Abstract

The present invention discloses the maize ZmGLN3 gene and its application in increasing the protein content in plant stems. Based on the BC2S2 population of wild maize Ames21814 and modern cultivated maize B73, the present invention conducts QTL mapping, discovers the key gene ZmGLN3 controlling the protein content in maize stems, and through structural variation analysis, it is found that there is a tandem repeat variation in the ZmGLN3 gene in Ames21814, resulting in a significant increase in the expression level of this gene in Ames21814, thereby effectively increasing the total protein content in the stems and whole plants of Ames21814. The present invention also discovers that the protein content in the stems of the ZmGLN3 gene overexpression materials is also significantly increased. The discovery of the ZmGLN3 gene and its excellent haplotypes provides an efficient means for the genetic improvement and germplasm resource innovation of whole-plant silage maize, and has important application value and economic prospects.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to the maize ZmGLN3 gene and its application in increasing the protein content in plant stems. Background Art

[0002] Maize is one of the important food crops globally, and China has long led in maize planting area and output. In 2023, the national maize planting area exceeded 44 million hectares, of which approximately 70% was used for feed, mainly for livestock and poultry farming. Silage maize, due to its high yield, high energy, rich nutrition, and good palatability, has become one of the forage raw materials with the largest planting area and the widest distribution globally, and is also an important feed source for the development of China's animal husbandry.

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

[0004] Protein content is an important indicator for measuring the quality of silage maize, and its level determines the quality of silage maize. Currently, the protein content of the vast majority of silage maize is relatively low (only 7% - 9%), while the whole-plant protein content of the related wild maize teosinte is high (greater than 13%). Therefore, exploring and utilizing the high-protein genes of wild maize has important application potential for the genetic improvement of silage maize quality. The present invention aims to develop new genes with the function of increasing the protein content in plant stems and the whole-plant protein content, thereby providing technical support for the genetic improvement and germplasm resource innovation of whole-plant silage maize. Summary of the Invention

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

[0006] In the BC2S2 population constructed from Ames21814 and B73, through QTL analysis, the present invention discovered a QTL signal significantly associated with the stem protein content on chromosome 9. This signal is different from the grain protein QTL located by the previous ASN4 gene, indicating that there is also a QTL region related to stem protein independent of grain protein regulation on chromosome 9. Through the analysis of gene expression differences and nitrogen metabolism pathways in this QTL region, 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 identified as a candidate gene. Genome alignment found that there is a tandem repeat structural variation in ZmGLN3 in Ames21814, and RT-qPCR analysis showed that this variation significantly increased the expression of ZmGLN3. For this reason, the present invention designed molecular markers, 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 with the function of increasing the nitrogen content and biomass of plants, and 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) A protein obtained by substituting, deleting, and / or adding one or several amino acid residues to the amino acid sequence of the ZmGLN3 protein, which has more than 90% identity with the ZmGLN3 protein and is related to high plant protein;

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

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

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

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

[0015] The present invention also provides a recombinant expression vector, including 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 coding gene, gene expression cassette, recombinant expression vector or recombinant host cell in any one of the following (B1)-(B4):

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

[0019] (B2) increasing the biomass of plants;

[0020] (B3) increasing the nitrogen use efficiency of plants;

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

[0022] Further, the plant is maize.

[0023] The maize can specifically be varieties such as Zhengdan 958, Xianyu 335, Jingke 968, Denghai 605, Demeiya 1, Demeiya 3, Heyu 187, Suyu 29, Jingnongke 728, Zhongdan 808, Zhengda 808, etc.

[0024] The present invention also provides a method for increasing the protein content in the stem of a plant, comprising the step of introducing the above-mentioned coding gene into the plant to construct a transgenic plant overexpressing the coding gene.

[0025] The present invention also provides a method for increasing the biomass of a plant, comprising the step of introducing the above-mentioned coding gene into the plant to construct a transgenic plant overexpressing the coding gene.

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

[0027] An existing plant expression vector can be used to construct a recombinant expression vector containing the coding gene. The plant expression vector includes binary Agrobacterium vectors and vectors for plant microprojectile bombardment, etc. The plant expression vector may further contain the 3'-untranslated region of the foreign gene, i.e., containing a polyadenylation signal and any other DNA fragment involved in mRNA processing or gene expression. The polyadenylation signal can direct the addition of polyadenylic acid to the 3' end of the mRNA precursor. For example, the nopaline synthase gene (Nos) of the Agrobacterium tumefaciens Ti plasmid gene and the 3'-transcribed untranslated region of plant genes (such as soybean storage protein genes) have similar functions.

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

[0029] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used 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).

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

[0031] In the above method, introducing the recombinant expression vector carrying the above-mentioned coding gene into the recipient plant can specifically be: transforming plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated transformation, etc., and cultivating the transformed plant tissues into plants.

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

[0033] Based on the BC2S2 population of wild maize Ames21814 and modern cultivated maize B73, the present invention conducts QTL mapping, discovers the key gene ZmGLN3 that controls the protein content in maize stalks, and through structural variation analysis, it is found that there is a tandem repeat variation in the ZmGLN3 gene in Ames21814, resulting in a significant increase in the expression level of this gene in Ames21814, and thus effectively increasing the total protein content in the stalks and the whole plant of Ames21814. The present invention also finds that the protein content in the stalks of the ZmGLN3 gene overexpression materials is also significantly increased.

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

[0035] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

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

[0037] Figure 2 Graph of determination and phenotypic distribution of stem protein content;

[0038] Figure 3 QTL mapping graph of stem protein content;

[0039] Figure 4 Gene structure and molecular marker identification diagram of tandem repeat variation of the ZmGLN3 gene in B73 and Ames21814; where A is a schematic diagram of the gene structure of tandem repeat variation of the ZmGLN3 gene; B is the result of detecting the expression level of the ZmGLN3 gene using primers F1 and R1; C is the electrophoresis diagram of molecular marker identification;

[0040] Figure 5 Statistical graph of the 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 Statistical graph of stem protein content of NIL-Ames21814 and NIL-B73; where the ZmGLN3 of the NIL-Ames21814 material is in the background of Ames21814; the ZmGLN3 of the NIL-B73 material is in the background of B73;

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

[0044] The various exemplary embodiments of the present invention will be described in detail below. This detailed description should not be construed as a limitation on the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0045] It should be understood that the terms used in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0046] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can 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 related to the said documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0047] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific embodiments of the present invention specification, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0048] Regarding the use of "comprising", "including", "having", "containing", etc. herein, they are all open-ended terms, meaning including but not limited to.

[0049] Self-crossing, hybridization, field breeding, etc. 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 examples, including plasmid construction, enzyme digestion, ligation, preparation of competent cells, transformation, medium preparation, etc., mainly refer to "Molecular Cloning: A Laboratory Manual" (Third Edition), edited by J. Sambrook, D.W. Russell (USA), translated by Huang Peitang et al., Science Press, Beijing, 2002). Specific experimental conditions can be determined through simple experiments when necessary.

[0052] The PCR amplification experiment is carried out according to the reaction conditions provided by the reagent supplier or the kit instruction manual. It can be adjusted through simple experiments when necessary.

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

[0054] Table 1 Primer sequences

[0055]

[0056] The wild maize Ames21814 has been disclosed in the literature "THP9 enhances seed protein content and nitrogen - use efficiency in maize", and the present invention promises to make it available to the public for 20 years from the filing date of the present invention application.

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

[0058] Construct a BC2S2 population of wild maize Ames21814 and modern cultivated maize B73 ( Figure 1 ) which is planted and self - crossed in Sanya. Take the ear - height stems 25 days after pollination, then dry the samples at 65 °C to constant weight, and finally grind them into powder using a grinder (60 Hz, 60 s). Wrap 50 - 70 mg of the powder with tin foil as the test sample. The determination of total nitrogen is carried out using a Dumas rapid nitrogen analyzer from Elementar, Germany. Before each round of measurement, weigh 4 standard products (aspartic acid samples) as internal references. After the machine is debugged, enter the weight of each sample in the weight column of the rapid N super software (v1.1.25), and select (O2 injection time: 60 s; O2 injection flow rate: 120 mL / min; O2 cut - off threshold: 15%; automatic zero - delay: 30 s; select Peak anticip: 90 s) as the program settings. At the same time, put the packaged samples into the sample cans according to the corresponding serial numbers, and 55 samples can be tested in each round. Finally, the data of stalk protein content and phenotype distribution are obtained ( Figure 2 ) Perform high - throughput re - sequencing on the BC2S2 population and combine it with the stalk protein content for QTL mapping. Finally, the most significant QTL region appears on chromosome 9. Combining the nitrogen metabolism network and transcriptome data, the candidate gene ZmGLN3 is finally screened out in this QTL region ( 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 variations in ZmGLN3 - Ames21814 and analysis of gene expression levels

[0065] By comparing the differences in the ZmGLN3 gene between the Ames21814 and B73 genomes using the previously assembled high - precision Ames21814 genomic data, it was found that there might be tandem repeat variations in the ZmGLN3 gene in Ames21814. First, primers F1 and R1 were designed. Through RT - qPCR analysis with DNA as the template, it was preliminarily determined that there were tandem repeat variations in ZmGLN3 in Ames21814 ( Figure 4 A and B in the figure). Further, molecular marker primers F2, F3, and R2 for identifying tandem repeat variations in ZmGLN3 were designed. When PCR amplification was performed with F3 and R2, DNA from both Ames21814 and B73 could amplify a sequence with a length of 152 bp; when PCR amplification was performed with F2 and R2, only DNA from Ames21814 could amplify a sequence with a length of 712 bp ( Figure 4 C in the figure), and this result fully confirmed the existence of tandem repeat variations in ZmGLN3 in Ames21814.

[0066] In the present invention, leaf RNA of Ames21814 and B73 was further extracted, and quantitative primers F4 and R4 were designed. RT - qPCR analysis was performed for the ZmGLN3 gene, and it was found that the expression level of this gene in the leaves of Ames21814 was significantly higher than that of B73 ( Figure 5 ), and the difference in expression level was caused by the occurrence of tandem repeat structural variations.

[0067] Genomic DNA extraction and PCR identification method:

[0068] Extract corn DNA using the CTAB method for subsequent identification. Put corn leaves into a 2 mL tube, add steel beads, and grind them after liquid nitrogen treatment (60 Hz, 60 s); after grinding, add 0.6 mL of CTAB extraction buffer and mix well; place in a 65 °C oven for 60 min, and mix well every 10 - 15 min during this period; 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 13000 rpm at room temperature for 15 min, and transfer the supernatant to a new 1.5 mL centrifuge tube; add an equal volume of isopropanol, invert back and forth to mix well, and place at -20 °C for 20 min; centrifuge at 12000 rpm at room temperature for 1 min, and pour out the supernatant. Wash the DNA pellet twice with 1 mL of 75% ethanol, centrifuge at 12000 rpm for 1 min each time, and then pour out the ethanol; centrifuge briefly, aspirate the excess liquid, and air-dry the DNA pellet at room temperature; add 0.3 mL of ddH2O to dissolve the DNA pellet.

[0069] The PCR detection reaction was carried out using the Master Mix(With Dye) kit from Shanghai Yisheng Biotechnology Co., Ltd. Configure a 20 μL reaction system: 10 μL of Master Mix, 1 μL of each primer, 2 μL of DNA, 6 μL of ddH2O. The specific detection primers were designed using NCBI. The reaction conditions were: pre-denaturation at 94 °C for 5 min, denaturation at 94 °C for 30 s, annealing at 58 °C for 30 s, extension at 72 °C for 30 - 60 s / Kb, 35 cycles, and final extension at 72 °C for 10 min.

[0070] RT-qPCR analysis at the RNA level:

[0071] (1) RNA extraction: The plant RNA extraction kit (OminiPlant RNAKit) from CW Biotech Co., Ltd. was used for RNA extraction, following the standard method in the kit.

[0072] (2) Reverse transcription of RNA: The reverse transcription kit (HiFiScript gDNA Removal cDNA Synthesis Kit) from CW Biotech Co., Ltd. was used for reverse transcription of RNA, following the standard method in the kit.

[0073] (3) Specific primers were designed through NCBI and specific analysis was carried out, 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 CWBIO Co., Ltd. Each sample was subjected to three technical replicates. The above-mentioned reverse-transcribed cDNA samples were uniformly diluted 10-fold, and a 10 μL reaction system was used for quantitative analysis. 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 the diluted cDNA, and 1.6 μL of ddH2O were added.

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

[0076] (5) The ΔΔCT method was used to analyze the quantitative data.

[0077] DNA-level RT-qPCR analysis:

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

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

[0080] Starting from the F1 hybrid offspring of Ames21814 and B73, according to the sequence differences between Ames21814 and B73, based on the results of molecular markers and phenotypic measurements, the present invention was backcrossed generation by generation to the BC9F1 generation and self-crossed to obtain a homozygous near-isogenic line of the ZmGLN3-Ames21814 candidate interval ( Figure 6 ), which was planted and the ear-position stem was taken 25 days after pollination for the determination of the stem protein content ( Figure 7 ), and it was found that the stem protein content of the plants containing ZmGLN3-Ames21814 was significantly increased. It further verified the possibility that the tandem repeat variation of ZmGLN3 led to an increase in the total protein content of maize stems and whole plants.

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

[0082] The above-described embodiments are only descriptions of the preferred embodiments of the present invention and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. Use of a coding gene, gene expression cassette, recombinant expression vector or recombinant host cell in increasing the protein content in corn stalks, characterized in that, The nucleotide sequence of the coding gene is as shown in SEQ ID NO.2; The gene expression cassette comprises the coding gene; The recombinant expression vector comprises the gene expression cassette; The recombinant host cell comprises the recombinant expression vector.

2. A method for increasing the protein content in the stems of plant plants, characterized in that, It includes the step of introducing the coding gene described in claim 1 into a plant to construct a transgenic plant overexpressing the coding gene; The plant is maize.

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