ZmSAMS4 gene and application thereof in increasing zinc content of corn kernels

Abstract

The invention discloses a ZmSAMS4 gene and application of the ZmSAMS4 gene in increasing the zinc content of corn kernels. The polynucleotide sequence of the ZmSAMS4 gene is shown as SEQ ID No: 1 in a sequence table. Through overexpression of the corn ZmSAMS4 gene, the content of the zinc element in corn kernels is increased, and the recessive hunger problem caused by zinc element deficiency in resident diet is solved.

Description

Technical Field

[0001] This invention belongs to the field of plant biology technology, specifically involving ZmSAMS4 Genes and their application in increasing zinc content in maize kernels. Background Technology

[0002] Zinc (Zn) is an essential mineral element for normal human growth and development; long-term deficiency can cause irreversible damage to health. However, zinc deficiency is difficult to detect in the short term, hence it is often referred to as "hidden hunger." Zinc deficiency is difficult to detect in the diagnosis of mineral deficiencies in the human body. This is because zinc is an important component of hundreds of key enzymes in organisms (such as RNA polymerase, alkaline phosphatase, alcohol dehydrogenase, superoxide dismutase, and carbonic anhydrase), and also a structural cofactor for important proteins. Zinc deficiency is often misdiagnosed as other conditions, making the resolution of "hidden hunger" a challenge. Zinc deficiency can lead to impaired function in multiple organs and systems, including the skin, gastrointestinal tract, central nervous system, immune system, and reproductive system. Food is the primary source of zinc for the human body. It is generally believed that the most effective methods to address zinc deficiency are dietary diversification, the use of functional zinc supplements, or biofortification of staple crops (i.e., increasing the inherent zinc content of grains). However, due to low income levels and limited access to diverse foods, zinc-deficient individuals often struggle to obtain sufficient zinc through varied diets and nutritional supplements. Increasing the endogenous zinc content of food crops, known as zinc biofortification, is considered the most cost-effective and sustainable strategy for addressing zinc deficiency in humans.

[0003] Nicotinamide (NA) is a unique plant metabolite that can chelate with various metal ions such as iron, zinc, copper, and manganese within plants, participating in the transport and distribution of these metal ions and maintaining the balance of metal ions within the plant. The level of NA in plants directly affects the absorption of metal ions from the environment and the accumulation level of metal ions within the plant. The biosynthesis of NA in plants uses L-methionine as a substrate, which is catalyzed by S-adenosylmethionine synthase (SAMS) to produce S-adenosylmethionine (SAM). Three SAM molecules are then catalyzed by nicotinamide synthase (NAS) to produce one NA molecule. Currently, SAMS genes in various plants have been cloned and identified, and research indicates that NA in plants... SAMS Genes are induced to express and overexpressed by environmental stress. SAMS Genes can enhance a plant's resistance to adverse conditions. Corn contains four genes. ZmSAMS Whether these ZmSAMS genes can significantly increase grain zinc concentration and be truly used for zinc biofortification in maize has not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to provide ZmSAMS4 Genes and their application in increasing zinc content in maize kernels.

[0005] A sort of ZmSAMS4 Genes, the ones mentioned ZmSAMS4 The gene polynucleotides are shown in (a), (b), (c), or (d): (a) A polynucleotide as shown in SEQ ID No: 1; or (b) A polynucleotide whose complementary sequence to SEQ ID No: 1 can hybridize under strict hybridization conditions, and the protein encoded by the polynucleotide still has the function of increasing the zinc content of maize kernels; (c) A polynucleotide that has at least 90% or more homology with the polynucleotide shown in SEQ ID No: 1; or (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 1, wherein the protein encoded by the polynucleotide mutant still has the activity of increasing the zinc content of maize kernels.

[0006] A ZmSAMS4 protein, the amino acid sequence of which is shown in (a), (b), or (c): (a) The amino acid sequence as shown in SEQ ID No: 2 of the sequence listing; or (b) Amino acids that have at least 90% or more homology with the amino acid shown in SEQ ID No: 2; or (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids based on the protein shown in SEQ ID No: 2, and the protein still has the activity of increasing the zinc content of maize kernels.

[0007] Includes the ZmSAMS4 The carrier of genes.

[0008] Includes the ZmSAMS4 Engineered bacteria that act as vectors for genes.

[0009] The detection ZmSAMS4 Primers for any segment of a gene.

[0010] The ZmSAMS4 Application of genes in breeding maize varieties with high zinc content.

[0011] A method to increase the zinc content of maize kernels by increasing the expression level of ZmSAMS4 protein in maize.

[0012] The beneficial effects of this invention: This invention achieves the effect of overexpressing maize... ZmSAMS4Genes can increase the zinc content in corn kernels, helping to address the problem of hidden hunger caused by zinc deficiency in the diet of residents. Attached Figure Description

[0013] Figure 1 pBCXUN- ZmSAMS4 Schematic diagram of the carrier.

[0014] Figure 2 for ZmSAMS4 Overexpression of maize plants ZmSAMS4 Gene expression level detection.

[0015] Figure 3 for ZmSAMS4 Overexpression of total zinc content in corn kernels. Detailed Implementation

[0016] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0017] Example 1: Construction of the recombinant vector pBCXUN-ZmSAMS4 Total RNA was extracted from the aboveground parts of B73 seedlings and reverse transcribed into cDNA using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). Using this cDNA as a template, the upstream primer 5'-CG was used... GGATCC ATGGCGGCCGAGAGCTTCCTT-3' (underscore sequence is...) BamH I site) and downstream primer 5'-CG GGATCC TTAAGCCGATGCCTTGTCGAA-3' (underscore sequence is...) BamH PCR amplification was performed at site I, and a DNA fragment of approximately 1200 bp was purified and recovered to obtain... ZmSAMS4 The full-length protein-coding region of the gene was obtained. The pBCXUN plasmid vector was digested with BamH I restriction enzyme (NEB), and the digestion products were recovered. T4 DNA ligase was used to ligate... ZmSAMS4 The PCR product of the gene was ligated into a linearized pBCXUN plasmid vector to obtain the recombinant vector pBCXUN-ZmSAMS4. Figure 1 The recombinant vector was transformed into *E. coli* competent cells DH5α using the heat shock method, plated on LB agar plates containing kanamycin resistance, and positive clones were selected for sequencing. The correctly sequenced plasmid was then transformed into *Agrobacterium tumefaciens* competent cells GV3101 using electroporation.

[0018] Example 2 ZmSAMS4 Obtaining maize materials through overexpression The embryos of maize inbred line B73 were transformed using Agrobacterium-mediated transformation. Following infection, co-culture, callus induction, screening, differentiation, and seedling formation, T0 generation seeds were harvested in a greenhouse. After further propagation in Hainan and Beijing, two homozygous transgenic maize lines were obtained. OE #1 and OE #2 Total RNA was extracted from the aboveground parts of OE #1, OE #2, and wild-type B73 seedlings, respectively. cDNA was obtained by reverse transcription using the PrimeScript™ RT reagent Kit with gDNA Eraser (TaKaRa). This cDNA was then used as a template for quantitative real-time PCR detection. For quantitative real-time PCR, the primers for ZmSAMS4 detection were 5'-CAAGGAGCACGTCATCAAGCC-3' and 5'-TTGGTGGGGTCCTTGCCGGA-3'; ZmTub was used as an internal control for the maize gene, with primer sequences of 5'-ACACCACCATTGGGAGTCTA-3' and 5'-TTGTGGGGACCACTACTTTC-3'. Real-time quantitative PCR was performed using the Applied Biosystems 7500 Real-Time PCR system (Thermo Fisher Scientific), with each experiment set to three replicates. -ΔΔCT The relative expression level was calculated using this method. The results are as follows: Figure 2 As shown, OE#1 and OE#2 Transgenic pure lines ZmSAMS4 Gene expression levels were significantly increased compared to the wild type (B73).

[0019] Example 3: Zinc content analysis of ZmSAMS4 overexpressing maize grains Pick OE #1 and OE #2 Two g of mature seeds from the wild-type B73 strain were ground into powder using an agate mortar and pestle. The powder was then digested in 7 ml of nitric acid / perchloric acid (4:1 v / v) at 100°C for 2 h, followed by digestion at 150°C for 5 h. The volume was brought to 15 ml. The zinc content was determined by ICP-OES. The results are as follows: Figure 3 As shown, ZmSAMS4 The zinc content in maize kernels of the overexpression lines was significantly increased compared to the wild type, with lines #1 and #2 showing increases of approximately 12% and 8%, respectively, compared to the wild type.

[0020] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. A kind ZmSAMS4 Genes, characterized by, The ZmZIP5 The gene polynucleotides are shown in (a), (b), (c), or (d): (a) A polynucleotide as shown in SEQ ID No: 1; or (b) A polynucleotide whose complementary sequence to SEQ ID No: 1 can hybridize under strict hybridization conditions, and the protein encoded by the polynucleotide still has the function of increasing the zinc content of maize kernels; (c) A polynucleotide that has at least 90% or more homology with the polynucleotide shown in SEQ ID No: 1; or (d) A polynucleotide mutant obtained by deleting, substituting or inserting one or more bases based on the polynucleotide shown in SEQ ID No: 1, wherein the protein encoded by the polynucleotide mutant still has the activity of increasing the zinc content of maize kernels.

2. A ZmSAMS4 protein, characterized in that, The amino acid sequence of the ZmSAMS4 protein is shown in (a), (b), or (c): (a) The amino acid sequence as shown in SEQ ID No: 2 of the sequence listing; or (b) Amino acids that have at least 90% or more homology with the amino acid shown in SEQ ID No: 2; or (c) A protein mutant obtained by deleting, substituting or inserting one or more amino acids based on the protein shown in SEQ ID No: 2, and the protein still has the activity of increasing the zinc content of maize kernels.

3. Containing the contents of claim 1 ZmSAMS4 The carrier of genes.

4. Containing the contents of claim 3 ZmSAMS4 Engineered bacteria that act as vectors for genes.

5. The test according to claim 1 ZmSAMS4 Primers for any segment of a gene.

6. The claim 1 ZmSAMS4 Application of genes in breeding maize varieties with high zinc content.

7. A method for increasing the zinc content of corn kernels, characterized in that, Increase the expression level of ZmSAMS4 protein in maize.

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