ZmAAP14 gene, protein and application thereof for increasing protein content of corn kernels

Abstract

This invention discloses a ZmAAP14 protein and gene for increasing the protein content of corn kernels. The gene sequence is shown in SEQ ID NO.1, and the amino acid sequence is shown in SEQ ID NO.2. This invention constructs... ZmAAP14 Gene overexpression vectors, overexpression in maize ZmAAP14 Genes, determined ZmAAP14 Genes can increase the protein content of corn kernels and can be used to breed new high-protein corn varieties.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology, and more specifically, relates to a method for increasing the protein content of corn kernels. ZmAAP14 Genes, proteins and their applications. Background Technology

[0002] Corn is an important food and feed crop in my country, with about 70% used as livestock and poultry feed.

[0003] Discovering genes with high protein content in corn kernels will help breed new high-protein corn varieties, ensuring my country's food security and sustainable agricultural development. Summary of the Invention

[0004] To address at least one deficiency or improvement need in the prior art, this invention provides a superior gene for increasing the protein content of maize, which the inventors have cloned from wild maize through extensive and in-depth research. ZmAAP14 Introducing it into cultivated corn can effectively increase the protein content of corn kernels.

[0005] To achieve the above objectives, according to one aspect of the present invention, a ZmAAP14 protein for increasing the protein content of corn kernels is provided, wherein the ZmAAP14 protein is a protein having the amino acid sequence shown in SEQ ID NO.2.

[0006] Furthermore, the gene sequence encoding the protein is shown in SEQ ID NO.1.

[0007] According to another aspect of the invention, an isolated polynucleotide is also provided, said polynucleotide encoding the aforementioned protein.

[0008] According to another aspect of the invention, a gene expression cassette is also provided, the gene expression cassette comprising the aforementioned polynucleotide.

[0009] According to another aspect of the invention, a recombinant expression vector is also provided, the recombinant expression vector comprising the aforementioned polynucleotide.

[0010] 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.

[0011] According to another aspect of the invention, a method for increasing the protein content of corn kernels is also provided, comprising increasing the protein content of corn kernels... ZmAAP14 The steps for determining gene expression levels, as described ZmAAP14 The gene nucleotide sequence is shown in SEQ ID NO.1.

[0012] Furthermore, the method includes the following steps: (i) Transplanting the gene encoding the protein, or the gene thereof, into the plant cells, thereby causing the plant cells to express the protein of claim 1; and (ii) Regenerate plants from the plant cells in step (a); wherein the improvement is to increase the protein content of plant grains, and the plant is corn.

[0013] According to another aspect of the invention, a kit for increasing the protein content of plants or constructing high-protein plants is also provided, the kit comprising: (1) A vector or expression cassette for expressing the protein as described; and Optional (2) expression ZmAAP14 Vectors or expression cassettes for gene-encoded proteins; The plant in question is corn.

[0014] According to another aspect of the invention, the application of the aforementioned biological material, expression cassette, expression vector, host cell, method, and kit 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: This invention has discovered a novel gene and its protein that can positively regulate the protein content of maize kernels. ZmAAP14 By building ZmAAP14 The protein content of maize kernels was determined using a gene overexpression vector, and it was found that... ZmAAP14 The gene significantly increases the protein content of corn kernels and can be used to breed new corn varieties with high protein content. Attached Figure Description

[0016] Figure 1 This is provided by the embodiments of the present invention. ZmAAP14 Overexpression vector map; Figure 2 The construction provided by the embodiments of the present invention ZmAAP14 Image of sequencing results for overexpression vector; Figure 3 The construction provided by the embodiments of the present invention ZmAAP14 Image of sequencing results for overexpression vector; Figure 4 This is provided by the embodiments of the present invention. ZmAAP14 Electrophoresis detection image of positive clones of overexpression vector; Figure 5 This is provided by the embodiments of the present invention. ZmAAP14 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 Construction ZmAAP14 Gene overexpression recombinant vector 1. Constructing overexpression vectors The gene encoding the ZmAAP14 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] In one example ZmAAP14 The construction process of a gene overexpression vector is shown in the following steps: 1) The vector framework wmv00006 from Weimi Biotechnology Co., Ltd. was used for vector construction. The vector map is shown below. Figure 1 As shown, first use BamH I and PacI. 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. ZmAAP14 The CDS sequence and the required primer sequences are shown in Table 1: Table 1 Amplified Genes ZmAAP14 Primers for CDS sequences

[0024] Amplification of 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 was cut and recovered, ligated into the T-vector, and sent to a sequencing company for sequencing. The sequencing results are as follows: Figure 2 As shown, if the sequencing is successful, the gene is obtained. ZmAAP14 cDNA.

[0025] In genes 3) The genes obtained in step (2) ZmAAP14 Using cDNA as a template, amplification was performed using the primers listed in Table 2 below: Table 2 Amplified Genes ZmAAP14 Primers for CDS sequences

[0026] Amplify the 50µl system, then run the PCR reaction as described in 2), and after completion, cut the gel for recovery.

[0027] 4) Recombining the linker system (on-ice mixing) - the desired carrier can then be obtained as follows: 4 µl of PCR digestion product in step (3) 2 µl of the enzyme digestion vector product in step (1) 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, incubated 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 plaques, and bacterial suspensions with the correct band size were sent for sequencing. Sequencing results are shown below. Figure 3 As shown, after accurate sequencing, the vector plasmid was extracted by shaking the bacteria.

[0028] 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: 2.1 Callus preparation This type of embryogenic callus is characterized by rapid growth, soft texture, loose and brittle structure, and bright color. It can be subcultured for extended periods and retain its embryogenic capacity for a considerable time.

[0029] 2.2 Preparation of Agrobacterium 1) Two days before infection, streak Agrobacterium on LB medium and incubate in the dark at 28°C, or pick single clones and shake them in liquid LB medium. 2) Collect the bacterial cells and transfer them to a 50 mL centrifuge tube. Resuspend the bacterial cells in the infection medium to make the concentration of the infection solution between OD660 and 0.8 and 1.5. 3) Activate Agrobacterium by shaking the prepared infection solution on a shaker at 28°C and 200 rpm for 2 hours for infection.

[0030] 2.3 Infection 1) Select pretreated callus tissue and transfer it to an infection culture medium; 2) Soak the callus tissue in the invasion staining solution for 10-30 minutes, break up the callus tissue clumps, and shake well to ensure that the callus tissue is fully in contact with Agrobacterium. 3) Aspirate the remaining infection solution and transfer the infected callus tissue into a co-culture medium. Incubate in the dark at 19°C for 3 days. 2.4 Resumption of Culture Rinse the callus surface 3-5 times with sterile water containing antibiotics. Once the water is no longer cloudy, discard the liquid and transfer the callus to a Petri dish lined with filter paper. Dry the callus surface in a laminar flow hood. Transfer to recovery medium and incubate in the dark at 28°C for 7-10 days. Then transfer to selection medium.

[0031] 2.5 Screening 1) After recovery culture, the transformed callus tissue was transferred into a selection medium supplemented with antibiotics and cultured in the dark at 28°C for 20-30 days; 2) Transfer the callus tissue into a sterile petri dish, break it up, and then press it thin. 3) Pick out the brighter callus particles on the UV operating table, clump 3-5 particles together, and transfer them into a new selection medium; 4) Incubate in the dark at 28℃ for 20 days, then transfer to a new screening medium for subculture and propagation; 5) After two cycles (40 days), the callus tissue that emits fluorescence again is picked (the callus tissue is not broken up).

[0032] 2.6 Differentiation and Rooting 1) After heat shock, the callus tissue was transferred to predifferentiation medium and cultured in the dark at 28°C for 10 days, then transferred to light at 28°C for 10 days. 2) Transfer to differentiation medium, and when the regenerated shoots grow to 3-5 cm, transfer to rooting medium; 3) After the seedlings have developed a large number of strong roots, harden them off and transplant them. 2.7 Hardening off and transplanting 1) After rooting, remove the sealing film from the rooting bottle and harden the seedlings in the culture medium for 2-3 days; 2) Wash the roots and transplant the culture medium into sterilized nutrient soil, harden the seedlings indoors for 7 days; (4) After screening the positive seedlings, plant them in the field for harvesting.

[0033] For positive seedlings, a small number of leaves were collected to extract genomic DNA using the TPS method. Positive plants were identified by PCR and sequencing. The PCR primers are shown below: Table 3 PCR identification primers

[0034] PCR positive seedling electrophoresis results Figure 4 As shown, the rightmost well is the positive control.

[0035] To further verify ZmAAP14 To investigate the effect on maize kernel protein content, the inventors conducted a field study in 2025 at the Agricultural Science Research Institute base in Gasa Town, Jinghong City, Xishuangbanna Prefecture, Yunnan Province, planting the negative control maize material KN5585 and... ZmAAP14 For each independent transformation event, three plants were planted as negative controls and three as positive controls to serve as biological replicates. At 25 days post-pollination, indicators were measured, including plant phenotype, physiological indicators (total biomass and plant height), and plant protein content (stem and grain protein content).

[0036] The protein content determination method is as follows: (1) Firstly, regarding ZmAAP14 Eight positive and eight negative control kernels were selected from T2 seeds that overexpressed independent events, and the kernels were ground into corn powder using a ball mill.

[0037] (2) Protein content was determined using a Dumas nitrogen analyzer. First, a standard curve was established using aspartic acid as a standard. Then, 200 mg of each corn 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 corn kernels was then obtained. The average values ​​of three positive and three negative results were calculated, and the significance was determined. The results are as follows: Figure 5 As shown, in 3 independent ZmAAP14 The protein content in the overexpression transformation event was significantly higher than that in the control, with an average increase of approximately 0.9 percentage points, indicating that... ZmAAP14 Genes can positively regulate the protein content of corn kernels.

[0038] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

[0039] SEQ ID NO.1 SEQ ID NO.2 MEVAGNHVQSCRTELPEPQKPLVDDDGRPLRTGTLWTASAHIITAVIGSGVLSLAWGVAQLGWAGGPAAMVLFAAVIYYTSTLLAECYRCGDPTFGPRNRTYIDAVRATLGDSKERLCGAIQLSNLFGIGIGVSIAASVSMQAIRRAGCFHYRGHEDPCHASTSPYIAVFGVMQIVFSQIPDLDKVWWLSTVAAIMSFSYSTIGILLGVVQIVEHGGPRGSLAGVIGAGARVTMMQKVWRSLQAFGNIAFAYGFSIILLEIQDTIKSPPPSEAKVMKKATAVSVAVTTVIYLLCGCVGYAAFGGAAPDNLLTGFGFYEPFWLLDVANAFVVVHLVGTYQVMSQPVFAYVERRAAAAWPGSALVRDRHVRVGRAVAFSVSPARLAWRTAYVCVTTAVAMLLPFFGSVVGLIGAASFWPLTVYFPVEMYIAQHRVARGSMRWLLLQGLSAGCLVVSVAAAAGSIAGVVEDLKAHNPFCWSC

Claims

1. A ZmAAP14 protein for increasing the protein content of corn kernels, characterized in that, The ZmAAP14 protein is a protein having the amino acid sequence shown in SEQ ID NO.

2.

2. The protein as described in claim 1, characterized in that, The gene sequence encoding the protein is shown in SEQ ID NO.

1.

3. An isolated polynucleotide, characterized in that, The polynucleotide encodes the protein of claim 1.

4. A gene expression cassette, characterized in that, The gene expression cassette comprises the polynucleotide described in claim 3.

5. A recombinant expression vector, characterized in that, The recombinant expression vector comprises the polynucleotide described in claim 3.

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, Including increasing the corn ZmAAP14 The steps for determining gene expression levels, as described ZmAAP14 The gene nucleotide sequence is shown in SEQ ID NO.

1.

8. The method as described in claim 7, characterized in that, The method includes the following steps: (i) Transplanting the plant cell with the gene encoding the protein of claim 1 or the gene of claim 2, thereby causing the plant cell to express the protein of claim 1; and (ii) Regenerate plants from the plant cells in step (a); wherein the improvement is to increase the protein content of plant grains, and the plant is corn.

9. A kit for increasing the protein content of plants or constructing high-protein plants, characterized in that, The kit includes: (1) A vector or expression cassette for expressing the protein as described in claim 1; or Optional (2) expression ZmAAP14 Vectors or expression cassettes for gene-encoded proteins; The plant in question is corn.

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

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