Maize Thn1 gene and application thereof in improving plant biomass and nitrogen utilization efficiency

Abstract

The invention discloses a corn Thn1 gene and application thereof in improving plant biomass and nitrogen utilization efficiency, and relates to the technical field of biology. According to the invention, a key gene Thn1 capable of regulating and controlling plant biomass and nitrogen utilization efficiency is excavated. The function of the gene is deeply analyzed and identified through a transgenosis method, and it is further clear that the biomass and nitrogen utilization efficiency of the corn plant can be effectively improved through overexpression of the Thn1 gene. The method is of great significance to cultivation of corn varieties with high nitrogen utilization efficiency.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a corn Thn1 gene and an application thereof in improving plant biomass and nitrogen utilization efficiency. Background Art

[0002] Nitrogen is one of the essential nutrients for plants and plays a vital role in their growth and development. Except for some leguminous plants that can fix nitrogen through rhizobia, most plants need to obtain nitrogen from the soil through their roots. 3- ) and ammonium nitrogen (NH4 + ) is the main inorganic nitrogen source. At present, poor basic soil fertility is one of the main factors affecting corn production, and the application of nitrogen fertilizer is an important measure to increase corn production. However, due to the low nitrogen utilization efficiency and excessive use of nitrogen fertilizers, the ecological environment has been severely damaged. Therefore, improving the utilization efficiency of nitrogen plays an important role. Such as: increasing nitrogen absorption, promoting the growth and development of corn, and increasing yields; reducing the application of nitrogen fertilizers, thereby reducing agricultural production costs; reducing the loss and waste of nitrogen fertilizers, thereby reducing pollution to the environment. In summary, cultivating new corn varieties that can efficiently utilize nitrogen is the key to solving the contradiction between environment and resources in my country's agricultural production. Summary of the invention

[0003] The purpose of the present invention is to provide a corn Thn1 gene and its application in improving plant biomass and nitrogen utilization efficiency to solve the problems existing in the above-mentioned prior art. The present invention has found that by overexpressing the Thn1 gene, the biomass and nitrogen utilization efficiency of corn plants can be effectively improved, which is of great significance for breeding corn varieties with high nitrogen utilization efficiency.

[0004] To achieve the above object, the present invention provides the following solutions:

[0005] The present invention provides a protein having the function of increasing plant biomass and nitrogen utilization efficiency, wherein the protein is any one of (A1), (A2) and (A3):

[0006] (A1) Thn1 protein, the amino acid sequence of which is shown in SEQ ID NO.1;

[0007] (A2) replacing, deleting and / or adding one or more amino acid residues in the amino acid sequence of the Thn1 protein to obtain a protein having more than 90% identity with the Thn1 protein and related to plant high protein;

[0008] (A3) A fusion protein obtained by connecting a protein tag to the N-terminus or / and C-terminus of (A1) or (A2).

[0009] The present invention also provides a gene encoding the above protein.

[0010] Furthermore, the nucleotide sequence of the encoding gene is shown in SEQ ID NO.2.

[0011] The present invention also provides a gene expression cassette, comprising the above-mentioned coding gene.

[0012] The present invention also provides a recombinant expression vector, comprising the above gene expression cassette.

[0013] The present invention also provides a recombinant host cell, comprising the above-mentioned recombinant expression vector.

[0014] 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)-(B2):

[0015] (B1) Increase plant biomass;

[0016] (B2) Improve the nitrogen utilization efficiency of plants.

[0017] Furthermore, the plant is corn.

[0018] The corns may specifically be varieties such as Zhongdan 808, Demeya 1, Xianyu 335, Jingnongke 728, Jingke 968, Suyu 29, Denghai 605, Zhengdan 958, Demeya 3, Heyu 187, and Zhengda 808.

[0019] The present invention also provides a method for improving nitrogen utilization efficiency of plants, comprising the steps of introducing the above encoding gene into plants to construct a transgenic plant that overexpresses the encoding gene.

[0020] 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.

[0021] 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.

[0022] 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 polyadenylic acid to be added 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 non-translated region transcribed at the 3' end of the plant gene (such as the soybean storage protein gene) all have similar functions.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] 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.

[0027] Transformed cells, tissues or plants are understood to include not only the end product of the transformation process but also the transgenic progeny thereof.

[0028] The present invention discloses the following technical effects:

[0029] The present invention has discovered a key gene, Thn1, that can regulate plant biomass and nitrogen utilization efficiency. Through the transgenic method, its function is deeply analyzed and identified, and it is further clarified that overexpression of the Thn1 gene can effectively improve the biomass and nitrogen utilization efficiency of corn plants. The present invention is of great significance for the cultivation of corn varieties with high nitrogen utilization efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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.

[0031] Figure 1 The phenotypes of the plants and seedlings of the THN1 protein overexpression material (Thn1-OE) and the control (WT) under LN and HN conditions;

[0032] Figure 2 It is a statistical chart of the total fresh weight of plants of THN1 protein overexpression material (Thn1-OE) and control (WT) under LN and HN conditions;

[0033] Figure 3 Statistical graph of fresh weight of aboveground part of plants under LN and HN conditions for THN1 protein overexpression material (Thn1-OE) and control (WT);

[0034] Figure 4 Statistical graph of fresh weight of underground part of plants under LN and HN conditions for THN1 protein overexpression material (Thn1-OE) and control (WT). DETAILED DESCRIPTION

[0035] 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.

[0036] 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.

[0037] 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.

[0038] 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 the skilled artisan. The present invention description and examples are exemplary only.

[0039] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0040] Example 1 Obtaining corn material with overexpression of Thn1 protein

[0041] By screening more than 100 corn materials with nitrogen concentration gradient, a corn material with significantly enhanced nitrogen utilization efficiency was obtained. The material was an overexpression of Thn1 protein, and the phenotype showed an increase in biomass and an increase in nitrogen utilization efficiency. It is speculated that it has a functional association with plant vegetative growth. Therefore, the present invention constructed a Thn1 protein overexpression corn material, and the method is as follows:

[0042] 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.2 to obtain the overexpression vector UBI-Thn1-FLAG (which has been sequenced and verified).

[0043] 2. Introduce the overexpression vector UBI-Thn1-FLAG prepared in step 1 into Agrobacterium EHA105 to obtain recombinant bacteria.

[0044] 3. Use the recombinant bacteria obtained in step 2 to transform the recipient corn B73 inbred line to obtain T0 generation plants.

[0045] 4. Use the primer pair consisting of primer Thn1-F and primer FLAG-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.

[0046] Thn1-F: 5'-CGTGGAGCCTGAACGCCA-3' (SEQ ID NO. 6);

[0047] FLAG-R: 5'-TGTCGTGATCCTTAGTCTCCATCATG-3' (SEQ ID NO. 7).

[0048] 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.

[0049] 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.

[0050] The amino acid sequence of Thn1 protein is shown in SEQ ID NO.1, and the nucleotide sequence of Thn1 gene is shown in SEQ ID NO.2.

[0051] SEQ ID NO.1:

[0052] MARQQSVQALCVLAALLFAASLPSPAAAGVHLSSLPKALDVTTSAKPGQVLHAGVDSLTVTWSLNATEPAGADAGYKGVKVKLCYAPASQKDRGWRKSEDDI SKDKACQFKVTEQAYAAAAPGSFQYAVARDVPSGSYYLRAFATDASGAEVAYGQTAPTAAFDVAGITGIHASLKIAAGVFSAFSVVALAFFFVIETRKKNK.

[0053] SEQ ID NO.2:

[0054]

[0055] Example 2 Phenotypic Investigation of Thn1 Protein Overexpression Corn

[0056] 1. Methods

[0057] In 2024, the control (corn B73 inbred line) and Thn1 protein overexpression material (T3 generation plants constructed in Example 1) were planted with vermiculite in the B118 light box of the Research Center of Sichuan Agricultural University in Wenjiang District, Chengdu. The seedlings were tested for indicators including plant phenotype and physiological indicators (total biomass) 11 days after nitrogen treatment (LN: 0.04 mM potassium nitrate / HN: 4 mM potassium nitrate).

[0058] Total biomass determination: Dig out the whole plant, clean the roots, remove excess vermiculite, and weigh the whole plant.

[0059] 2. Results

[0060] (1) Plant phenotype and physiological index test results

[0061] The phenotypes of the seedlings of Thn1 protein overexpression material (Thn1-OE) and control (CK) are shown in Figure 1 The results of total plant fresh weight, above-ground fresh weight and underground fresh weight under LN and HN treatments are shown in Figure 2 , Figure 3 and Figure 4 . The results showed that under LN conditions, the aboveground fresh weight of the control plant was 0.968g, and the aboveground fresh weight of Thn1-OE was 1.157g, which was 19.5% higher than that of the control plant; the underground fresh weight of the control plant was 0.923g, and the underground fresh weight of Thn1-OE was 0.913g, which was not much different from that of the control plant; the total fresh weight of the control plant was 1.891g, and the total fresh weight of Thn1-OE was 2.070g, and the total biomass increased by 9.5% compared with the control. Under HN conditions, the aboveground fresh weight of the control plant was 1.653g, and the aboveground fresh weight of Thn1-OE was 1.946g, which was 17.7% higher than that of the control. The fresh weight of the underground part of the control plant was 1.241g, and the fresh weight of the underground part of Thn1-OE was 1.338g. The fresh weight of the underground part of Thn1-OE increased by 7.8% compared with the control; the total fresh weight of the control plant was 2.894g, and the total fresh weight of Thn1-OE was 3.284g. The total biomass increased by 13.5% compared with the control.

[0062] 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 and nitrogen utilization efficiency, characterized in that: The protein is any one of (A1), (A2) and (A3): (A1) Thn1 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 Thn1 protein to obtain a protein having more than 90% identity with the Thn1 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)-(B2): (B1) Increase plant biomass; (B2) Improve the nitrogen utilization efficiency of plants.

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

9. A method for improving nitrogen utilization efficiency 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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