Use of a maize gene to increase protein content in maize kernels

Abstract

The application discloses application of a corn gene in improving corn kernel protein content, and relates to the technical field of biology. ZmAAP16 A new gene capable of improving corn kernel content is mined by the application. ZmAAP16 Through transgenic method, function of the gene is analyzed and identified in depth, it is found that the gene can significantly improve the corn kernel protein content by overexpression in plants, and the role of the gene in improving the corn kernel protein content is determined. The application has important significance for cultivation of high-protein ensiled corn.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, relates to the application of a maize gene in increasing the protein content of maize kernels. Background Technology

[0002] Corn is my country's largest grain crop, with an annual output of nearly 300 million tons, 70% of which is used for animal feed. Ordinary corn typically has a protein content of around 8%, which is insufficient to meet the protein requirements for animal feed. In recent years, my country has imported over 100 million tons of grain annually, more than 80% of which is soybeans, primarily to address the issue of feed protein sources. Domestic demand for soybeans is constantly increasing, but relevant departments estimate that to fully meet soybean self-sufficiency needs, an additional 600 million mu (approximately 40 million hectares) of arable land would be required, which is virtually impossible given the current arable land availability. The constantly changing international environment has exacerbated the uncertainty surrounding my country's soybean imports, making the exploration of new pathways for soybean protein substitution an urgent need to ensure my country's food security. Chinese scientists are continuously exploring various "protein substitution" methods, with high-protein corn being seen as a new avenue for "soybean protein substitution."

[0003] According to expert calculations, if the protein content of corn in my country's existing planting area increases by 4 percentage points, it is estimated that soybean imports can be reduced by nearly 30 million tons annually. This would not only alleviate my country's heavy dependence on imported soybeans, but also be of great significance to the development of modern large-scale agriculture, increasing the income of grain farmers, and consolidating the foundation of food security.

[0004] Therefore, identifying genes with high protein content in maize kernels will help breed new high-protein maize varieties, ensuring my country's food security and sustainable agricultural development. Summary of the Invention

[0005] In response to at least one deficiency or improvement need of the prior art, the present invention provides an application of a maize gene in increasing the protein content of maize kernels. This gene can significantly increase the protein content in maize kernels and can be used to breed new high-protein maize varieties.

[0006] To achieve the above objectives, according to one aspect of the present invention, an application of a maize gene in increasing the protein content of maize kernels is provided, wherein the nucleotide sequence of the maize gene is as follows: (1) As shown in SEQ ID NO.1; or (2) The nucleotide sequence of (1) encodes the same protein, but is different from the nucleotide sequence of (1) due to the degeneracy of genetic coding.

[0007] Furthermore, the amino acid sequence of the protein is shown in SEQ ID NO.2.

[0008] According to another aspect of the invention, a biological material is also provided, comprising the nucleotide sequence described above, wherein the biological material is one or more of a recombinant expression vector, plasmid, expression cassette, or recombinant bacteria.

[0009] According to another aspect of the invention, a gene expression cassette is also provided, the gene expression cassette comprising the aforementioned coding gene.

[0010] According to another aspect of the present invention, a recombinant expression vector is also provided, the recombinant expression vector comprising the gene expression cassette described above.

[0011] According to another aspect of the invention, a host cell is also provided, the host cell containing the recombinant expression vector, the host comprising any one of Escherichia coli, Agrobacterium tumefaciens, or non-renewable plant parts.

[0012] According to another aspect of the invention, a method for increasing the protein content of maize kernels is also provided, comprising the step of increasing the expression level of said gene in maize.

[0013] Furthermore, the method includes the following steps: (a) Introducing the gene of claim 1 into plant cells, thereby causing the plant cells to express the protein of claim 1; (b) Regenerate a plant from the plant cells in step (a); wherein the plant is corn.

[0014] According to another aspect of the invention, the application of the aforementioned biomaterial, expression cassette, expression vector, host cell, and method in maize breeding is also provided.

[0015] In summary, compared with the prior art, the above-described technical solutions conceived by this invention can achieve the following beneficial effects: The innovation of this invention lies in the fact that it reveals for the first time that corn ZmAAP16 Overexpression of the gene can significantly improve grain yield. The protein content of maize kernels was significantly increased. These findings not only provide a solid scientific foundation for a deeper understanding of the molecular mechanisms of maize kernel development, but also offer valuable theoretical guidance and practical direction for maize genetic improvement and breeding. Through these research results, we hope to cultivate new maize varieties with higher nutritional value and better yield performance, thereby making a significant contribution to agricultural production and food security. Attached Figure Description

[0016] Figure 1 This is a carrier skeleton map provided in an embodiment of the present invention; Figure 2 This is provided by the embodiments of the present invention. ZmAAP16Overexpression vector map; Figure 3 The construction provided by the embodiments of the present invention ZmAAP16 Image of sequencing results for overexpression vector; Figure 4 The construction provided by the embodiments of the present invention ZmAAP16 Image of sequencing results for overexpression vector; Figure 5 This is provided by the embodiments of the present invention. ZmAAP16 Statistical data on the protein content of maize kernels in overexpression maize materials and control maize materials; Detailed Implementation

[0017] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of the invention described below can be combined with each other as long as they do not conflict with each other. Where specific conditions are not specified in the embodiments, conventional conditions or conditions recommended by the manufacturer shall apply. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0018] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. While only preferred methods and materials have been described herein, any methods and materials similar or equivalent to those described herein may be used in the implementation or testing of this invention. All references to this specification are incorporated by way of citation to disclose and describe methods and / or materials associated with those references. In the event of any conflict with any incorporated reference, the content of this specification shall prevail.

[0019] Various modifications and variations can be made to the specific embodiments described in this specification without departing from the scope or spirit of the invention, as will be apparent to those skilled in the art. Other embodiments derived from this specification will also be readily apparent to those skilled in the art. This specification and embodiments are merely exemplary.

[0020] The terms “include,” “including,” “have,” “contain,” etc., used in this article are all open-ended terms, meaning that they include but are not limited to.

[0021] Example 1 ZmAAP16 Cloning of genes The gene encoding the ZmAAP16 protein has the gene number Zm00001d043382. Its nucleotide sequence is shown in SEQ ID NO.1, and its amino acid sequence is shown in SEQ ID NO.2. This nucleotide sequence was synthesized artificially.

[0022] SEQ ID NO.1: SEQ ID NO.2: MAQHHGSHSLEVGGVGAGGVELDDDGHAARTGNLWTCFAHIITAVIGCGVLALSWSVAQLGWVGGPVAMLCFAFVTYLSAFLLSHCYRSPASDDGSLKRQRNYTYMDAVRTHLGEKRTWLCGLFQYLN MYGTAIAYTITTATCLRAIVRANCYHSQGHSAPCGAGGDHLYMLLFGAAQAVLSLIPNFHSMAWLSAVAAVMSFTYATIGLGLGLAKTIENGAIKGSVAGVPMSTAPQKVWRVAQAIGDIAFAYPYTI VLLEIQVYYYYYTASIFTFFFLKVSFRLIYFIKKNSCNSLNCMQDTLKSPPPESETMQKGNVLAVLATTFFYLAVGCFGYAAFGNAAPGNLLTGFGFYEPYWLIDFANACIVLHLLGGYQMFSQQIFT FADRSLAARFPNSAFVNKSYAVKVPGAPASWSYSLNLQRLCFRTAYVASTTGLALLFPYFNEVLGVLGAVVFWPLAIYLPVEMYCVQRGVLPWTRTWVALQAFSVVCFVVGTFAFVGSVEGVIRKRLG Example 2, Gene ZmAAP16 Construction of overexpression lines 1. Construction ZmAAP16 Overexpression vector 1) Select carrier skeleton wmv00006 for... ZmAAP16 The overexpression vector was constructed using a vector backbone derived from Weimi Biotechnology Co., Ltd., and its vector map is shown below. Figure 1 As shown, the constructed overexpression vector map is as follows: Figure 2 As shown. First use BamH I and Pac I. The vector backbone was double-digested, and the digested vector was then recovered as a stock solution.

[0023] RNA was extracted from maize B73 leaves and reverse transcribed into cDNA. The gene was then amplified using primers. ZmAAP16 The CDS sequence and the required primer sequences are shown in Table 1: Table 1 Amplified Genes ZmAAP16 primer sequences sHP54-cds-F ATGGCGCAGCACCACGGCAG sHP54-cds-R CTAGCCGAGCCTCTTGCGGATGAC PCR amplification 50µl system: 1.5 μL each of F / R primers 2µl cDNA Pfu enzyme (high-fidelity enzyme) 20µl H2O 25µl 50µl The PCR instrument runs the following series of reactions: 95°C for 5 minutes 95°C for 30 seconds 58°C 45 sec 30 cycles 72°C for 1 minute 72°C for 5 minutes Storage temperature: 4°C After amplification, the gel is cut and recovered, ligated into the T-vector, and sent for sequencing. If the sequencing is successful, the gene is obtained. ZmAAP16 cDNA.

[0024] 3) Based on genes ZmAAP16 Using the cDNA as a template, amplification was performed using the primer sequences in Table 2: Table 2 Amplified Genes ZmAAP16 primer sequences sHP54-czFMB tttggtgttatacttctgcagATGGCGCAGCACCACGGCA sHP54-czRMB atcggggaaatTTAATTAACTAGCCGAGCCTCTTGCGG Amplify the 50µl system, then run the PCR reaction as described in 2), and after completion, cut the gel for recovery.

[0025] 4) Recombinant ligation system (mixing on ice) - This yields the desired overexpression vector. The ligation system is as follows: 3) 4 µl of PCR digestion product 1) 2µl of the enzyme digestion vector product 1 µl of recombinase Recombinant buffer 2µl H2O 1µl 10µl The PCR instrument was run with the recombination program at 37°C for 30 min, then placed on ice or at 4°C. The recombination products were then transformed into DH5α competent cells, shaken at 37°C and 200 rpm for 1 h, plated with LB+Kana, and incubated at 37°C for one day. Colony PCR was performed on selected colonies, and bacterial suspensions with the correct band size were sent for sequencing. A schematic diagram of the sequencing results is shown below. Figure 3 As shown, after accurate sequencing, the vector plasmid was extracted by shaking the bacteria.

[0026] 2. The overexpression vector was transformed into maize KN5585. This invention employs an Agrobacterium-mediated genetic transformation method, the specific method and process of which are as follows: (1) Plasmid vector transformation of Agrobacterium The overexpression vector was transferred into Agrobacterium EHA105 by electroporation. Single clones were picked, cultured, and identified by PCR, and then stored at -20°C for later use.

[0027] (2) Activation of microbial strains Remove Agrobacterium from the refrigerator and streak it onto YEP solid medium.

[0028] (3) Prepare Agrobacterium infection solution Fresh spores were scraped from the newly activated bacterial plate and resuspended in the infection solution.

[0029] (4) Take corn embryos Peel off the immature embryos of maize inbred line KN5585, which are about 1 mm thick. Take about 150 peeled immature maize embryos and put them into 2 mL plastic centrifuge tubes containing 1.8 mL of suspension (infection medium containing AS, but without Agrobacterium) and process for 30 min.

[0030] (5) Infection Remove the suspension from (4), leaving the corn embryo in the tube. Then add 1.0 mL of Agrobacterium infection solution, gently invert 10-15 times, and let stand for 5-10 minutes. Take a clean petri dish, place 3 sterile filter papers on it, and after infection, invert it a few times to quickly pour the bacterial solution onto the filter paper. Hold the petri dish and change its direction to ensure that the bacterial solution carrying the embryo is evenly distributed on the filter paper.

[0031] (6) Co-cultivation When the bacterial culture is no longer visible on the top layer of filter paper, use tweezers to pick up the top layer of filter paper and place the side with the embryo on the co-culture medium. Use tweezers to remove the air bubbles between the filter paper and the culture medium, then use tweezers to hold a corner of the filter paper and quickly peel it off. Transfer the embryo left on the filter paper to the culture medium with the embryo shield side facing up, and incubate in the dark at 23°C for 3 days.

[0032] (7) Restore culture After co-culturing for 3 days, the embryos were transferred to resting medium and cultured in the dark at 28°C for 6 days. Then, they were placed on selection medium containing 5 mg / L Bialaphos for 2 weeks of selection culture, and then transferred to selection medium containing 8 mg / L Bialaphos for 2 weeks of selection culture.

[0033] (8) Differentiation culture The resistant callus was transferred to differentiation medium 1 and cultured at 25°C, 5000 lx, under light for 1 week. The callus was then transferred to differentiation medium 2 and cultured under light for 2 weeks. The differentiated seedlings were transferred to rooting medium and cultured at 25°C, 5000 lx, under light until rooting occurred. The seedlings were then transferred to small pots for further growth. After a certain growth stage, they were transplanted into a greenhouse, and the offspring seeds were harvested after 3-4 months.

[0034] 3. After screening for positive seedlings, plant them in the field for harvesting. Transgenic positive seedlings obtained from genetic transformation of KN5585 were carefully cared for and harvested in the field.

[0035] For positive seedlings, a small number of leaves were taken to extract genomic DNA. PCR amplification was performed using the primer sequences in Table 3, and the PCR products were detected by agarose gel electrophoresis. The PCR product size of positive plants was 1536 bp. At the same time, three PCR positive samples were sequenced, and the results were analyzed using SnapGene software.

[0036] Genomic DNA extraction: (1) Place corn leaves in a 2mL tube, add steel balls, treat with liquid nitrogen and then grind (60Hz, 60s); (2) After grinding, add 0.6 mL of CTAB extraction buffer, mix well, and place in a 65℃ oven for 60 min, mixing every 10-15 min during the process; (3) Remove the tube and place it at room temperature for 5-10 minutes. Add an equal volume of chloroform:isoamyl alcohol (24:1) to the centrifuge tube, seal it, and shake for 5 minutes. (4) Centrifuge at 13000 rpm for 15 min at room temperature, and transfer the supernatant to a new 1.5 mL centrifuge tube; add an equal volume of isopropanol, mix by inverting back and forth, and place at -20 degrees for 20 min; centrifuge at 12000 rpm for 1 min at room temperature, and discard the supernatant. (5) Wash the DNA precipitate with 1 mL of 75% ethanol 1 to 2 times, centrifuge at 12000 rpm for 1 min each time, pour out the ethanol, and air dry the DNA precipitate at room temperature. (6) Add 0.3 mL ddH2O to dissolve the DNA precipitate.

[0037] Table 3 Primer sequences for PCR positive identification Bar-F1 CCATCGTCAACCACTACATCGAGACA Bar-R1 CTTCAGCAGGTGGGTGTAGAGCGT PCR-positive seedling electrophoresis results are as follows Figure 4 As shown, the rightmost well is the positive control.

[0038] Example 3 ZmAAP16 Phenotypic investigation of maize overexpression 1. Method In 2025, field planting control (negative control of transgenic maize KN5585 segregation) was conducted at the Agricultural Science Research Institute base in Gasa Town, Jinghong City, Xishuangbanna Prefecture, Yunnan Province. ZmAAP16 For each independent transformation event, three plants were planted for both negative and positive control to ensure biological replication. The protein content of the seeds was determined 25 days after pollination.

[0039] The protein content determination method is as follows: (1) Firstly, regarding ZmAAP16 Eight positive and eight negative control seeds were selected from T2 seeds that overexpressed independent events. The samples were dried to constant weight at 65°C and then ground into powder using a grinder (60Hz, 60s).

[0040] (2) The nitrogen content of maize plants overexpressing ZmAAP16 protein was determined using a Dumas rapid nitrogen analyzer for high-throughput analysis. First, aspartic acid was used as an internal control to create a standard curve. Then, 200 mg of each maize sample was weighed, wrapped in aluminum foil, and placed in the Dumas nitrogen analyzer to determine the nitrogen content via combustion. The protein content of the maize kernels was then obtained. The average values ​​of three positive and three negative samples were calculated, and the significance was determined.

[0041] 2. Results The results are as follows Figure 5 As shown, ZmAAP16 The protein content of overexpressing maize was significantly increased at three independent transformation events compared to the wild-type control. The average kernel protein content of control maize plants was 10.24%. ZmAAP16 The average grain protein content of the overexpressing maize plants was 11.26%, which was 10% higher than that of the control.

[0042] Therefore, we can conclude that: ZmAAP16 Genes play a role in regulating the protein content of corn kernels and have a positive effect on increasing the protein content of corn kernels.

[0043] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0044] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. The application of a maize gene in increasing the protein content of maize kernels, characterized in that, The nucleotide sequence of the maize gene is as follows: (1) As shown in SEQ ID NO.1; or (2) The nucleotide sequence of (1) encodes the same protein, but is different from the nucleotide sequence of (1) due to the degeneracy of genetic coding.

2. The application of the maize gene as described in claim 1 in increasing the protein content of maize kernels, characterized in that, The amino acid sequence of the protein is shown in SEQ ID NO.

2.

3. A biomaterial, characterized in that, Includes the nucleotide sequence as described in claim 1 or 2, wherein the biological material is one or more of a recombinant expression vector, plasmid, expression cassette, or recombinant bacteria.

4. A gene expression cassette, characterized in that, The gene expression cassette includes the coding gene described in 1 or 2.

5. A recombinant expression vector, characterized in that, The recombinant expression vector includes the gene expression cassette as described in claim 4.

6. A host cell containing the recombinant expression vector of claim 5, wherein the host includes any one of Escherichia coli, Agrobacterium tumefaciens, or a non-renewable plant part.

7. A method for increasing the protein content of corn kernels, characterized in that, The step includes increasing the gene expression level as described in claim 1 in maize.

8. The method as described in claim 7, characterized in that, The method includes the following steps: (a) Introducing the gene of claim 1 into plant cells, thereby causing the plant cells to express the protein of claim 1; (b) Regenerate a plant from the plant cells in step (a); wherein the plant is corn.

9. The application of the biomaterial as described in claim 3, the expression cassette as described in claim 4, the expression vector as described in claim 5, the host cell as described in claim 6, and the method as described in claims 7-8 in maize breeding.

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