Maize ZmHPSC1 gene and its application in increasing plant nitrogen content and biomass

Abstract

The present invention discloses a corn ZmHPSC1 gene and its application in increasing the nitrogen content and biomass of plants, and relates to the field of biotechnology. The present invention has discovered a key gene, ZmHPSC1, which can both regulate plant biomass and green retention and increase plant protein content. Through in-depth analysis and identification of its function by transgenic methods, it was found that overexpression of the gene in plants can significantly increase plant biomass. Overexpressed plants show characteristics such as tall plant type, thick stems, and strong green retention. The nitrogen content of its tissue parts was measured, and the nitrogen content of the stem part was increased by 3 times, further clarifying the role of the ZmHPSC1 gene in increasing the biomass and protein content of corn plants. The present invention is of great significance to the cultivation of high-protein silage corn.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, in particular to a corn ZmHPSC1 gene and an application thereof in improving the nitrogen content and biomass of plants. Background Art

[0002] Silage corn, also known as feed corn, is a special type of corn that is harvested from the entire corn plant, including the cobs, and then chopped, processed, and fermented to make silage. This type of corn is mainly used to feed cattle, sheep, and other livestock in winter, especially in northern pastoral areas. The characteristics of silage corn include high biomass yield, good fiber quality, good green retention, and dry matter and moisture content suitable for closed silage by anaerobic fermentation. It has higher yield and better fiber than ordinary grain corn. After harvesting during the appropriate harvest period, the above-ground green plant part, including the cobs, will be chopped, processed, and ensiled to become the main feed for herbivorous livestock such as cattle and sheep. In addition, silage corn is rich in nutrients, including sugars, carotene, vitamin B1 and vitamin B2, making it an ideal feed for herbivores. Once the ensilage process is completed, it can be stored for a long time and can maintain the nutritional value and taste of the feed, regardless of the season, to ensure a balanced supply throughout the year. In summary, silage corn is a high-yield, nutritious and easy-to-store feed source, which is of great significance to the breeding industry.

[0003] Silage corn has low protein and mineral content. The protein content in livestock and poultry feed must reach 11% to 20% to meet nutritional needs, while the current protein content of ordinary corn is only 7% to 9%. Since silage corn alone as feed is difficult to meet the feed nutrition requirements for the best production performance of ruminants, soybean meal needs to be added to the feed to supplement protein. my country's corn production will reach 290 million tons in 2023. If the protein content increases by one percentage point, it will increase by 2.9 million tons of protein. The protein content of imported soybeans is about 35%, which is equivalent to the protein content of more than 8 million tons of soybeans. How to increase the protein content of silage corn while ensuring the characteristics of large biomass, strong green retention, and good fiber quality is an important research direction for cultivating high-protein silage corn. Summary of the invention

[0004] The purpose of the present invention is to provide a corn ZmHPSC1 gene and its application in increasing the nitrogen content and biomass of plants to solve the problems existing in the above-mentioned prior art. The present invention has found that the ZmHPSC1 gene has the function of increasing the nitrogen content and biomass of plants, thereby providing important technical support for the cultivation of high-protein silage corn.

[0005] The present study found that overexpression of the corn ZmHPSC1 gene in plants can significantly increase plant biomass. The overexpressing plants have the characteristics of tall plant shape, thick stems, and strong green retention. The nitrogen content of its tissue parts was measured, and the nitrogen content of the stem part was increased by 3 times.

[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) ZmHPSC1 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 ZmHPSC1 protein to obtain a protein having more than 90% identity with the ZmHPSC1 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.2.

[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)-(B5):

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

[0018] (B2) increasing plant biomass;

[0019] (B3) Improve plant stay-greenness;

[0020] (B4) preparing high-protein silage;

[0021] (B5) 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 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.

[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 (SEQ ID NO.3) 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 end product of the transformation process but also the transgenic progeny thereof.

[0033] The present invention discloses the following technical effects:

[0034] The present invention has discovered a key gene, ZmHPSC1, which can regulate plant biomass and green retention and increase plant protein content. Through in-depth analysis and identification of its function by transgenic methods, the role of the ZmHPSC1 gene in increasing corn plant biomass and protein content has been further clarified. The present invention is of great significance to the cultivation of high-protein silage corn. 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 The plant phenotypes of ZmHPSC1 protein overexpression material (HPSC1-OE) and control (WT);

[0037] Figure 2 It is the plant height statistics of ZmHPSC1 protein overexpression material (HPSC1-OE) and control (WT);

[0038] Figure 3 Statistical graph of plant fresh weight of ZmHPSC1 protein overexpression material (HPSC1-OE) and control (WT);

[0039] Figure 4 It is a statistical diagram of the stem protein content of ZmHPSC1 protein overexpression material (HPSC1-OE) and control (WT);

[0040] Figure 5 Statistical graph of grain protein content of ZmHPSC1 protein overexpression material (HPSC1-OE) and control (WT). DETAILED DESCRIPTION

[0041] 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.

[0042] 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.

[0043] 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.

[0044] 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.

[0045] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0046] Example 1 Obtaining ZmHPSC1 protein overexpressing corn material

[0047] By screening more than 800 maize transcription factor mutants with nitrogen concentration gradient, a nitrogen function defective mutant material was screened, which is a functional loss mutant of the transcription factor HPSC. The mutant showed reduced biomass, reduced plant height, and impaired root development, and it is speculated that it has a functional association with plant vegetative growth. Therefore, the present invention constructs a HPSC protein overexpression maize material.

[0048] 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) was replaced with the DNA molecule shown in SEQ ID NO.2 to obtain the overexpression vector UBI-HPSC-FLAG (which has been sequenced and verified).

[0049] 2. Introduce the overexpression vector UBI-HPSC-FLAG prepared in step 1 into Agrobacterium EHA105 to obtain recombinant bacteria.

[0050] 3. Use the recombinant bacteria obtained in step 2 to transform the recipient corn B73 inbred line to obtain T0 generation plants.

[0051] 4. Use the primer pair consisting of primer HPSC-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.

[0052] HPSC-F: 5'-CTGCAGCAGAACCTCGCG-3' (SEQ ID NO. 6);

[0053] FLAG-R: 5'-TGTCGTGATCCTTAGTCTCCATCATG-3' (SEQ ID NO. 7).

[0054] 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.

[0055] 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.

[0056] Amino acid sequence of ZmHPSC1 protein (SEQ ID NO.1):

[0057] MELYSGYLDDHFNPHKLSIAGSAVSPPEYMASASPAQFAAAPLRMGYGRPAPAPPPPVMGMWSSEPFRVDSGSAHATSASTVNTKLEETRLEDDAQVALEPARSTEQETSRPPERAQRRLAQNREAARKSRLRKKAYIQQLETSRMKLSQLELELQRARQQGAYANSGSMGDSALGYRCPIDPGVSVFEIDYSHWVDEQKRHTAELTSALQGQQTSELELRLLVETGLSNYEHLFKIKAAAANADVFYVMSGLWKTPAERFFLWIGGFRPSEVLKILSPQLEPLAEAQRMLVGGLQHTSAQAEDALSQGMEKLQQNLAETLTAEADPFGPPDPYMLQMATAVGILKELVNFVTQADHLRLTTLQQMHKILTPRQAARGLLALGDYFQRLRTLSSMWAARPREAAVS。

[0058] Nucleotide sequence of the ZmHPSC1 gene (SEQ ID NO.2):

[0059]

[0060] Nucleotide sequence of vector UBI-FLAG (SEQ ID NO.3+SEQ ID NO.4+SEQ ID NO.5):

[0061] SEQ ID NO.3:

[0062]

[0063] SEQ ID NO.4:

[0064]

[0065] SEQ ID NO.5:

[0066]

[0067] Example 2 Phenotypic Investigation of ZmHPSC1 Protein Overexpression in Maize

[0068] 1. Methods

[0069] In 2023, a control (corn B73 inbred line) and a ZmHPSC1 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 and plant height) and plant protein content (stem and grain protein content).

[0070] Total biomass determination: Dig out the entire plant, clean the roots, remove excess soil, and weigh the entire plant.

[0071] Plant nitrogen content determination: The nitrogen content of ZmHPSC1 protein overexpressing maize plants was determined by high-throughput determination using 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.

[0072] 2. Results

[0073] (1) Plant phenotype and physiological index test results

[0074] The phenotypes of the plants with ZmHPSC1 protein overexpression (HPSC1-OE) and control (WT) are shown in Figure 1 , plant height and fresh weight test results are shown in Figure 2 and Figure 3 The results showed that the total fresh weight of the control plant was 1003g, and the total fresh weight of HPSC1-OE reached 1493g, and the biomass increased by 48.9% compared with the control. The plant height of the control plant was 234cm, while the plant height of HPSC1-OE reached 300cm, which was 28.2% higher than that of the control.

[0075] (2) Plant protein content test results

[0076] The results of protein content detection of stems and grains of ZmHPSC1 protein overexpression material (HPSC1-OE) and control (WT) are shown in Figure 4 and Figure 5 The protein content of the stem of the control plant was 2.36%, while that of the material with overexpression of ZmHPSC1 protein was 6.17%, which was 2.6 times higher. The protein content of the grain of the control plant was 11.87%, while that of the material with overexpression of ZmHPSC1 protein was 17.24%, which was 45% higher.

[0077] 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. Use of a gene encoding a ZmHPSC1 protein in increasing plant height, fresh weight, stalk protein and / or grain protein content of corn plants, characterized in that: The amino acid sequence of the ZmHPSC1 protein is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that: The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

3. Use of a gene expression cassette for increasing plant height, fresh weight, stalk protein and / or grain protein content of corn plants, characterized in that: The gene expression cassette comprises the coding gene described in claim 1 or 2.

4. Use of a recombinant expression vector in increasing plant height, fresh weight, stalk protein and / or grain protein content of corn plants, characterized in that: The recombinant expression vector comprises the gene expression cassette described in claim 3.

5. Use of a recombinant host cell in increasing plant height, fresh weight, stalk protein and / or grain protein content of corn plants, characterized in that: The host cell comprises the recombinant expression vector described in claim 4.

6. A method for increasing plant height, fresh weight, stalk protein and / or grain protein content of corn plants, characterized in that: The method comprises the steps of introducing the coding gene described in claim 1 or 2 into a plant to construct a transgenic plant that overexpresses the coding gene.

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