Maize Thn1 Gene and Its Application in Improving Plant Biomass and Nitrogen Use Efficiency

Abstract

The present invention discloses a maize Thn1 gene and its application in improving plant biomass and nitrogen use efficiency, relating to the field of biotechnology. The present invention has discovered a key gene, Thn1, which can regulate plant biomass and nitrogen use efficiency. Through transgenic methods, its function has been deeply analyzed and identified, and it has been further clarified that overexpression of the Thn1 gene can effectively improve the biomass and nitrogen use efficiency of maize plants. The present invention is of great significance for the cultivation of maize varieties with high nitrogen use efficiency.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, in particular to the maize Thn1 gene and its application in improving plant biomass and nitrogen use efficiency. Background Art

[0002] Nitrogen is one of the essential nutrient elements for plants and plays a crucial role in plant growth and development. Except for some leguminous plants that can fix nitrogen through rhizobia, the vast majority of plants need to obtain nitrogen sources from the soil through their roots. In plants, nitrate nitrogen (NO 3- ) and ammonium nitrogen (NH4 + ) are the main inorganic nitrogen sources. At present, poor soil basal fertility is one of the main factors affecting maize yield increase, and applying nitrogen fertilizer is an important measure for maize yield increase. However, due to the excessively low nitrogen use efficiency and the overuse of nitrogen fertilizer, the ecological environment has been severely damaged. Therefore, improving nitrogen use efficiency plays an important role. For example, increasing the nitrogen absorption amount can promote maize growth and development and increase yield; reducing the application amount of nitrogen fertilizer can thus reduce agricultural production costs; reducing nitrogen loss and waste can thus reduce environmental pollution. In summary, cultivating new maize varieties that can efficiently utilize nitrogen is the key to solving the contradiction between environment and resources in China's agricultural production. Summary of the Invention

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

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

[0005] The present invention provides a protein having the function of improving plant biomass and nitrogen use efficiency, and 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) A protein obtained by substituting, deleting and / or adding one or several amino acid residues to the amino acid sequence of the Thn1 protein, having more than 90% identity with the Thn1 protein and being 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 coding gene for the above-mentioned protein.

[0010] Furthermore, the nucleotide sequence of the coding gene is as 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-mentioned 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 coding gene, gene expression cassette, recombinant expression vector or recombinant host cell in any one of the following (B1)-(B2):

[0015] (B1) increasing the biomass of plants;

[0016] (B2) increasing the nitrogen use efficiency of plants.

[0017] Furthermore, the plant is maize.

[0018] The maize can specifically be varieties such as Zhongdan 808, Demaiya 1, Xianyu 335, Jingnongke 728, Jingke 968, Suyu 29, Denghai 605, Zhengdan 958, Demaiya 3, Heyu 187, Zhengda 808, etc.

[0019] The present invention also provides a method for increasing the nitrogen use efficiency of plants, comprising the step of introducing the above-mentioned coding gene into a plant to construct a transgenic plant overexpressing the coding gene.

[0020] The present invention also provides a method for increasing the biomass of plants, comprising the step of introducing the above-mentioned coding gene into a plant to construct a transgenic plant overexpressing the coding gene.

[0021] The "introducing the above-mentioned coding gene into a plant" is achieved by introducing a recombinant expression vector containing the coding gene into a recipient plant.

[0022] An existing plant expression vector can be used to construct a recombinant expression vector containing the said coding gene. The plant expression vectors include binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment, etc. The plant expression vector may further contain the 3' untranslated region of a foreign gene, i.e., contain a polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylation signal can guide the addition of polyadenylic acid to the 3' end of the mRNA precursor. For example, the 3' untranslated regions transcribed from the genes of Agrobacterium tumefaciens Ti plasmid (such as the nopaline synthase gene Nos) and plant genes (such as the soybean storage protein gene) have similar functions.

[0023] When constructing a recombinant expression vector using the said 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 the 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 of 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 said enhancers are extensive and can be natural or synthetic. The translation initiation region can come from the transcription initiation region or the structural gene.

[0024] In order to facilitate the identification and screening of transgenic plant cells or plants, the used plant expression vector can be processed, such as adding a gene encoding an enzyme or a luminescent compound that can produce a color change and can be expressed in plants (GUS gene, luciferase gene, etc.), a resistance antibiotic marker (gentamicin marker, kanamycin marker, etc.) or an anti-chemical reagent marker gene (such as an anti-herbicide gene).

[0025] In the present invention, the said 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.

[0026] In the above method, introducing the recombinant expression vector carrying the said coding gene into the receptor 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.

[0027] The transformed cells, tissues or plants are understood to include not only the final products of the transformation process, but also their transgenic progeny.

[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 use efficiency. Through transgenic methods, its function was analyzed and identified in depth, and it was further clarified that overexpression of the Thn1 gene can effectively improve the biomass and nitrogen use efficiency of maize plants. The present invention is of great significance for the cultivation of maize varieties with high nitrogen use efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] 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 described below are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

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

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

[0033] Figure 3 Statistical charts of the fresh weight of the above-ground parts of plants of the THN1 protein overexpression material (Thn1-OE) and the control (WT) under LN and HN conditions;

[0034] Figure 4 Statistical charts of the fresh weight of the underground parts of plants of the THN1 protein overexpression material (Thn1-OE) and the control (WT) under LN and HN conditions. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0035] The various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0036] 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 can be independently included or excluded from the range.

[0037] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although this 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 this 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.

[0038] Without departing from the scope or spirit of this invention, various improvements and changes can be made to the specific embodiments of the specification of this invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of this invention are obvious to those skilled in the art. The specification and examples of this invention are merely exemplary.

[0039] Regarding the use of "comprising", "including", "having", "containing", etc. in this article, they are all open-ended terms, meaning including but not limited to.

[0040] Example 1 Obtaining Maize Materials with Overexpressed Thn1 Protein

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

[0042] 1. Replace the fragment between the SpeⅠ and BamhⅠ restriction enzyme sites 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) with the DNA molecule shown in SEQ ID NO.2 to obtain the overexpression vector UBI-Thn1-FLAG (which has been verified by sequencing).

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

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

[0045] 4. Using the primer pair consisting of primer Thn1-F and primer FLAG-R, perform PCR identification on the T0 generation plants obtained in step 3, and select the positive T0 generation plants for self-crossing to obtain T1 generation plants. Among them, positive plants can amplify a band, while negative plants have no band.

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

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

[0048] 5. Identify the T1 generation plants obtained in step 4 according to the method in step 4, and select the positive T1 generation plants for self-crossing to obtain T2 generation plants.

[0049] 6. Identify the T2 generation plants obtained in step 5 according to the method in step 4, select the positive T2 generation plants for self-crossing to obtain T3 generation plants and identify them according to the method in step 4, and screen out the positive transgenic T3 generation 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] MARQQSVQALCVLAALLFAASLPSPAAAGVHLSSLPKALDVTTSAKPGQVLHAGVDSLTVTWSLNATEPAGADAGYKGVKVKLCYAPASQKDRGWRKSEDDISKDKACQFKVTEQAYAAAAPGSFQYAVARDVPSGSYYLRAFATDASGAEVAYGQTAPTAAFDVAGITGIHASLKIAAGVFSAFSVVALAFFFVIETRKKNK.

[0053] SEQ ID NO.2:

[0054]

[0055] Example 2: Phenotypic Investigation of Maize with Overexpressed Thn1 Protein

[0056] 1. Method

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

[0058] Determination of total biomass: The whole plant was dug out, the roots were washed, the excess vermiculite was removed, and the whole plant was weighed.

[0059] 2. Results

[0060] (1) Results of Detection of Plant Phenotype and Physiological Index

[0061] The phenotypes of the plant seedlings of the material with overexpressed Thn1 protein (Thn1-OE) and the control (CK) are shown in Figure 1 , and the detection results of the total fresh weight, aboveground fresh weight and underground fresh weight of the plants under LN and HN treatments are shown in Figure 2 , Figure 3 and Figure 4 . The results show that under LN conditions, the aboveground fresh weight of the control plant was 0.968 g, and that of Thn1-OE was 1.157 g, with the aboveground fresh weight of Thn1-OE increasing by 19.5% compared to the control; the underground fresh weight of the control plant was 0.923 g, and that of Thn1-OE was 0.913 g, with little difference in the underground fresh weight compared to the control plant; the total fresh weight of the control plant was 1.891 g, and that of Thn1-OE was 2.070 g, with the total biomass increasing by 9.5% compared to the control. Under HN conditions, the aboveground fresh weight of the control plant was 1.653 g, and that of Thn1-OE was 1.946 g, with the aboveground fresh weight of Thn1-OE increasing by 17.7% compared to the control. The underground fresh weight of the control plant was 1.241 g, and that of Thn1-OE was 1.338 g, with the underground fresh weight of Thn1-OE increasing by 7.8% compared to the control; the total fresh weight of the control plant was 2.894 g, and that of Thn1-OE was 3.284 g, with the total biomass increasing by 13.5% compared to the control.

[0062] The embodiments described above 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 spirit of the present invention, various modifications 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 single coding gene, gene expression cassette, recombinant expression vector or recombinant host cell in improving nitrogen utilization efficiency of plants, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.2; The gene expression cassette includes the coding gene; The recombinant expression vector includes the gene expression cassette; The recombinant host cell includes the recombinant expression vector; the recombinant host cell is a non-plant cell; The plant is corn.

2. A method for improving nitrogen utilization efficiency of plants, characterized in that: The method comprises the steps of introducing only one coding gene into a plant to construct a transgenic plant that overexpresses the coding gene; The nucleotide sequence of the coding gene is shown in SEQ ID NO.2; The plant is corn.

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