Corn ZmCNF1 gene and application thereof in increasing plant biomass, nitrogen utilization efficiency and plant protein content

Abstract

The invention discloses a corn ZmCNF1 gene and application thereof in improving plant biomass, nitrogen utilization efficiency and plant protein content, and relates to the technical field of biology. According to the invention, a key gene ZmCNF1 capable of regulating and controlling plant biomass and nitrogen utilization efficiency and increasing plant protein content is excavated. The functions of the ZmCNF1 gene are deeply analyzed and identified through a transgenosis method, it is found that the gene is over-expressed in a plant, the biomass of the plant can be remarkably improved, the nitrogen content of a tissue part of the plant can be measured, and the effect of the ZmCNF1 gene in improving the biomass of the corn plant, the nitrogen utilization efficiency and the protein content of the plant is further clarified. The method disclosed by the invention is of great significance to cultivation of excellent varieties with efficient utilization of nitrogen.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a corn ZmCNF1 gene and its application in improving plant biomass, nitrogen utilization efficiency and plant protein content. Background Art

[0002] As an important food crop and feed crop, corn has great production potential and high economic benefits. It can be used as food, feed and for a variety of industrial purposes. It plays an important strategic role in ensuring food security. Corn is a type of nitrogen-loving crop. In daily production, the principle of "applying sufficient basal fertilizer, applying seedling fertilizer early, and applying heavy ear fertilizer" is often followed. The application of nitrogen fertilizer to farmland will significantly increase yields. However, excessive application of nitrogen fertilizer will also lead to a decrease in crop utilization and cause environmental pollution problems. Improving the nitrogen utilization efficiency of corn crops, discovering high-efficiency nitrogen genes, cultivating new high-efficiency nitrogen varieties, and achieving green and sustainable yield increases are the core challenges of current corn genetic breeding.

[0003] Plants need to absorb a large number of elements and trace elements for their own growth and development during their growth. Nitrogen is the mineral element with the largest demand for plants and is also a key factor limiting plant growth. Nitrogen is an important component for protein synthesis in plants, and the average nitrogen content in protein can reach 16-18%. It is also a component element of genetic material nucleic acid, biocatalyst enzymes, chlorophyll and cytoskeleton phospholipids, and plays an important role in plant life activities. Most plants absorb and utilize nitrogen through their roots, and agricultural production generally increases yields by increasing nitrogen fertilizers. When plants are deficient in nitrogen, the protein synthesis ability of the plants is reduced, cell division and elongation are inhibited, and chlorophyll synthesis is reduced, which is specifically manifested as short and thin plants, yellow leaves, delayed growth cycle and low fruiting rate. When there is too much nitrogen, a large amount of carbohydrates will be used to synthesize substances such as protein and chlorophyll, which will greatly reduce the cellulose and pectin in the cell wall, resulting in large plant cells with thin walls, which are easily attacked by diseases and insects, and at the same time cause the mechanical tissue of the stem to be underdeveloped and prone to lodging. Therefore, studying the absorption and utilization of nitrogen by crops has important scientific and production significance. Summary of the invention

[0004] The purpose of the present invention is to provide a maize ZmCNF1 gene and its application in improving plant biomass, nitrogen utilization efficiency and plant protein content, so as to solve the problems existing in the above-mentioned prior art. The present invention has found that the ZmCNF1 gene has the function of improving plant biomass, nitrogen utilization efficiency and plant protein content, providing important gene resources and germplasm resources for cultivating new nitrogen efficient varieties.

[0005] The present invention found that overexpression of the corn ZmCNF1 gene in plants can significantly increase plant biomass, protein content and nitrogen utilization efficiency. Plant height is significantly increased and biomass is increased. Nitrogen content was measured and the average total protein content of the whole plant was increased by 11.97%.

[0006] Based on this, the present invention provides the following solutions:

[0007] 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):

[0008] (A1) ZmCNF1 protein, the amino acid sequence of which is shown in SEQ ID NO.1;

[0009] (A2) replacing, deleting and / or adding one or more amino acid residues in the amino acid sequence of the ZmCNF1 protein to obtain a protein having more than 90% identity with the ZmCNF1 protein and related to plant high protein;

[0010] (A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of (A1) or (A2).

[0011] The present invention also provides a gene encoding the above protein.

[0012] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.1.

[0013] The present invention also provides a gene expression cassette, comprising the above-mentioned coding gene.

[0014] The present invention also provides a recombinant expression vector, comprising the above gene expression cassette.

[0015] The present invention also provides a recombinant host cell, comprising the above-mentioned recombinant expression vector.

[0016] 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):

[0017] (B1) Increase the protein content of plants;

[0018] (B2) increasing plant biomass;

[0019] (B3) Improve plant nitrogen utilization efficiency;

[0020] (B4) Cultivate high-protein plant varieties.

[0021] Furthermore, the plant is corn.

[0022] 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.

[0023] The present invention also provides a method for increasing the nitrogen content of plants, comprising the steps of introducing the above encoding gene into plants to construct a transgenic plant that overexpresses the encoding gene.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] 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.

[0028] 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.

[0029] 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.1.

[0030] In the above method, the recombinant expression vector carrying the above-mentioned 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.

[0031] Transformed cells, tissues or plants are understood to include not only the end product of the transformation process but also the transgenic progeny thereof.

[0032] The present invention discloses the following technical effects:

[0033] The present invention has discovered a key gene, ZmCNF1, which can regulate plant biomass and nitrogen utilization efficiency and increase plant protein content. Through in-depth analysis and identification of its function by transgenic methods, the role of the ZmCNF1 gene in increasing corn plant biomass, nitrogen utilization efficiency and plant protein content has been further clarified. The present invention is of great significance to the cultivation of excellent varieties with efficient nitrogen utilization. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] 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.

[0035] Figure 1 Schematic diagram of the ZmCNF1 gene localization process; A is a schematic diagram of the F2 segregation population construction process; B is a gene localization peak diagram; C is a gene locus sequencing peak diagram;

[0036] Figure 2 The plant phenotypes of ZmCNF1 protein overexpression material (CNF1-OE) and control (WT);

[0037] Figure 3 It is the plant height statistics of ZmCNF1 protein overexpression material (CNF1-OE) and control (WT);

[0038] Figure 4 Statistical graph of plant fresh weight of ZmCNF1 protein overexpression material (CNF1-OE) and control (WT);

[0039] Figure 5 The statistical graph of the whole plant protein content of ZmCNF1 protein overexpression material (CNF1-OE) and control (WT); DETAILED DESCRIPTION

[0040] 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.

[0041] 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.

[0042] 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.

[0043] 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.

[0044] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0045] Example 1 Localization of ZmCNF1 gene

[0046] 1. The inventors previously used ethyl methanesulfonate (EMS) to induce mutagenesis in the maize B73 inbred line and created a batch of mutant materials with rich phenotypes. Through an efficient nitrogen screening system, a nitrogen-insensitive mutant material cnf1 was identified. An F2 segregation population was constructed using B73 and cnf1, and the ZmCNF1 gene was successfully cloned through BSA mixed pool sequencing. The results showed that the gene encodes a key enzyme in the nitrogen absorption and assimilation process of maize, and the results of the allelic mutant test further verified its function in maize nitrogen utilization.

[0047] A large number of M0 seeds were obtained by using EMS to induce mutagenesis of the B73 inbred line. The M0 seeds were planted and self-pollinated to obtain M1 seeds, which produced a rich variety of mutant phenotypes, including cnf1 mutant materials. Based on a large number of field phenotypic observations and biomass surveys, yellow and dwarf plants were hybridized with B73 to obtain F1.

[0048] 2. After self-pollination of the F1 obtained in step 1, the F2 was obtained for planting. The wild type and yellow dwarf plants were separated from the F2 offspring. The two high-quality data pools (wild type and mutant cnf1) obtained after filtering were aligned to the reference genome using the default parameters of the bwa program. The SNP information was obtained using the GATK software to calculate the SNP index, and the SNPs with SNP index <0.3 in both components were filtered. The SNP-index in each window was calculated with a window size of 4Mb and a step size of 200kb. The difference in SNP index of the two offspring pools was calculated to obtain △(SNP index) = SNP index (mutant) - SNP index (wild type). 10,000 permutation tests were performed, and the 95% confidence level was selected as the screening threshold, and the windows with a confidence level of more than 95% were selected as candidate intervals. △(SNP index) reflects the significant difference in genotype frequency between the two pools. The area above the threshold line was selected as the candidate area related to the trait to find the candidate gene. The candidate genes were further analyzed and located, and the ZmCNF1 gene was finally determined. The location process is shown in Figure 1 .

[0049] The nucleotide sequence of the ZmCNF1 gene is shown in SEQ ID NO.1, and the amino acid sequence of the ZmCNF1 protein encoded by it is shown in SEQ ID NO.2.

[0050] SEQ ID NO.1:

[0051]

[0052] SEQ ID NO.2:

[0053] .

[0054] Example 2 Obtaining ZmCNF1 protein overexpressing corn material

[0055] 1. The fragment between the restriction sites of SpeⅠ and BamhⅠ of the vector UBI-FLAG (the nucleotide sequence is obtained by connecting SEQ ID NO.3, SEQ ID NO.4 and SEQ ID NO.5 in sequence) is replaced with the DNA molecule shown in SEQ ID NO.1 to obtain the overexpression vector UBI-CNF1-FLAG (which has been sequenced and verified).

[0056] 2. Introduce the overexpression vector UBI-CNF1-FLAG prepared in step 1 into Agrobacterium EHA105 to obtain recombinant bacteria.

[0057] 3. Use the recombinant bacteria obtained in step 2 to transform the recipient corn B73 inbred line to obtain T0 generation plants.

[0058] 4. Use the primer pair consisting of primer CNF1-F and primer CNF1-R to perform PCR identification on the T0 generation plants obtained in step 3, and select the T0 generation plants identified as positive for self-pollination to obtain T1 generation plants. Among them, positive plants can amplify bands, while negative plants have no bands.

[0059] CNF1-F: 5'-GGCCGTCGATGCTGCTC-3' (SEQ ID NO. 6);

[0060] FLAG-R: 5'-TGTCGTGATCCTTATAGTCTCCATCATG-3' (SEQ ID NO. 7).

[0061] 5. Identify the T1 generation plants obtained in step 4 according to the method in step 4, select the T1 generation plants identified as positive for self-pollination to obtain T2 generation plants.

[0062] 6. Identify the T2 plants obtained in step 5 according to the method in step 4, select the T2 plants identified as positive for self-pollination, obtain T3 plants and identify them according to the method in step 4, and screen out positive transgenic T3 plants.

[0063] Example 3 Phenotypic Investigation of ZmCNF1 Protein Overexpression Maize

[0064] 1. Methods

[0065] In 2024, a control (corn B73 inbred line) and a ZmCNF1 protein overexpression material (T3 generation plants constructed in Example 1) were planted in the field in Wenjiang District, Chengdu City, and index detection was carried out 25 days after pollination, including plant phenotype, physiological indicators (total biomass, biomass of each tissue and plant height) and plant protein content.

[0066] Total biomass determination: Dig out the whole plant, clean the roots, remove excess soil, and weigh the whole plant. Take samples of leaves, stems, roots and seeds for biomass determination.

[0067] Plant nitrogen content determination: The nitrogen content of ZmCNF1 protein overexpressing maize plants was determined by high-throughput Dumas fast nitrogen analyzer. The samples were dried to constant weight at 65°C and then 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 fast nitrogen analyzer of Elementar, Germany. Before each round of measurement, 4 standards (asparagine 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 dosing time: 60s; O2 dosing 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 jar according to the corresponding serial number. 55 samples can be tested in each round. The data was exported to Excel format for analysis.

[0068] 2. Results

[0069] (1) Plant phenotype and physiological index test results

[0070] The phenotypes of ZmCNF1 protein overexpression materials (CNF1-OE) and control (WT) are shown in Figure 2 , plant height and fresh weight test results are shown in Figure 3 and Figure 4 The results showed that the total fresh weight of the control plant was 1000g, and the total fresh weight of CNF1-OE was 1100g, and the biomass increased by 10% compared with the control. The height of the control plant was 242cm, while the height of HPSC1-OE reached 260cm, which was 7.4% higher than the control.

[0071] (2) Plant protein content test results

[0072] The leaves, stems, roots and seeds of the plants overexpressing ZmCNF1 protein (CNF1-OE) and the control (WT) were sampled for biomass determination and protein content determination. The results are shown in Figure 5 The results showed that the average total protein content of the control plants was 9.226%, and the average total protein content of CNF1-OE was 10.330%, which was 11.97% higher than that of the control.

[0073] 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 plant biomass, nitrogen utilization efficiency and plant protein content, characterized in that: The protein is any one of (A1), (A2) and (A3): (A1) ZmCNF1 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 ZmCNF1 protein to obtain a protein having more than 90% identity with the ZmCNF1 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 as SEQ ID NO.

1.

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) increase 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 nitrogen content of plants, 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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