Application of TaNAS1 protein or coding gene of TaNAS1 protein in regulation and control of plant nicotinamide content

Abstract

The invention belongs to the technical field of gene engineering, and particularly relates to application of TaNAS1 protein or a coding gene thereof in regulation and control of plant nicotinamide content. The invention provides application of a TaNAS1 protein or a coding gene thereof in regulating and controlling the content of nicotinamide in plants. The amino acid sequence of the TaNAS1 protein is as shown in SEQ ID NO: 3. The TaNAS1 protein or the coding gene thereof is overexpressed in a plant, so that the nicotinamide content of the plant can be increased, the agronomic traits of wheat can be improved, and the nutrient element content of wheat grains can be increased. Specifically, overexpression of the TaNAS1 protein or the coding gene of the TaNAS1 protein increases the grain length, grain width and hundred-grain weight of wheat, and increases the contents of zinc, iron and copper elements in wheat grains.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to the application of TaNAS1 protein or its encoding gene in regulating the nicotinamide content of plants. Background Art

[0002] The accumulation of trace elements in plants includes absorption from the soil, translocation from roots to the aboveground parts, and enrichment in grains. However, due to the limited solubility of zinc and iron ions in the alkaline environment of sieve tubes, the transport of trace elements in combination with chelating agents is an essential way for plants to absorb them.

[0003] Nicotinamide is a zinc-related chelator, primarily formed by the enzyme nicotinamide synthase from three S-adenosylmethionine molecules. Nicotinamide is present in all higher plants and is crucial for the long-distance transport of zinc and other metals to sinking organs, playing a crucial role in metal homeostasis in plants. Nicotinamide synthases have been reported in plants such as maize, rice, and Arabidopsis. Two types of nicotinamide synthases identified in the Poaceae family are classified as class I and class II. In rice and maize, expression of class I proteins is root-specific, primarily involved in nicotinamide biosynthesis, ion uptake, and long-distance transport regulation; expression of class II proteins is stem-specific, primarily involved in nicotinamide synthesis, ion loading, and maintaining cellular iron homeostasis. These reported nicotinamide synthase overexpression lines exhibit significantly elevated zinc content. Identifying genes associated with nicotinamide synthase in wheat and elucidating its regulatory pathways are crucial for improving zinc absorption and accumulation in wheat. Summary of the Invention

[0004] The purpose of the present invention is to provide the use of TaNAS1 protein or its encoding gene in regulating the nicotinamide content of plants, regulating the nicotinamide content of plants, especially wheat, improving the agronomic traits of wheat, and increasing the content of zinc, iron and copper in wheat grains.

[0005] In order to achieve the above objectives, the present invention provides the use of TaNAS1 protein or its encoding gene in regulating the nicotinamide content of plants;

[0006] The amino acid sequence of the TaNAS1 protein is shown in SEQ ID NO: 3.

[0007] Preferably, the regulating the nicotinamide content of the plant includes: overexpressing TaNAS1 protein or its encoding gene to increase the nicotinamide content of the plant.

[0008] Preferably, the plants include wheat and tobacco.

[0009] The present invention provides the use of TaNAS1 protein or its encoding gene in regulating wheat agronomic traits and / or grain nutrient element content;

[0010] The agronomic traits include one or more of grain length, grain width and 100-grain weight;

[0011] The nutrient element content includes one or more of zinc, iron and copper;

[0012] The amino acid sequence of the TaNAS1 protein is shown in SEQ ID NO: 3.

[0013] Preferably, the regulation of wheat agronomic traits includes: overexpressing TaNAS1 protein or its encoding gene to increase one or more of wheat grain length, grain width and 100-grain weight;

[0014] The method for regulating the nutrient content of wheat grains includes: overexpressing TaNAS1 protein or its encoding gene to increase the content of one or more of zinc, iron and copper in wheat grains.

[0015] Preferably, the nucleotide sequence of the coding region of the gene encoding the TaNAS1 protein is shown in SEQ ID NO: 2.

[0016] Preferably, the full-length genomic nucleotide sequence of the encoding gene is shown as SEQ ID NO: 1.

[0017] The present invention provides a biomaterial comprising a recombinant vector and / or a recombinant bacterium.

[0018] The recombinant vector includes a basic vector and a TaNAS1 gene inserted into the basic vector;

[0019] The recombinant bacteria include a basic bacteria and a TaNAS1 gene introduced into the recombinant bacteria;

[0020] The amino acid sequence of the TaNAS1 protein encoded by the TaNAS1 gene is shown in SEQ ID NO: 3.

[0021] Preferably, the basic vector comprises pLGY;

[0022] The basic bacteria include Escherichia coli and / or Agrobacterium.

[0023] The present invention provides a method for increasing the nicotinamide content in plants, comprising the following steps: overexpressing the TaNAS1 gene in the plant; the amino acid sequence of the TaNAS1 protein encoded by the TaNAS1 gene is shown in SEQ ID NO: 3.

[0024] Beneficial effects:

[0025] The present invention provides the use of a TaNAS1 protein or its encoding gene for regulating nicotinamide content in plants; the amino acid sequence of the TaNAS1 protein is shown in SEQ ID NO: 3. Overexpressing the TaNAS1 protein or its encoding gene in plants can increase the nicotinamide content of the plants. Nicotinamide chelates with trace elements (such as zinc and iron ions), increasing the solubility of trace elements in sieve tubes and improving plant agronomic traits.

[0026] Furthermore, the present invention overexpresses the TaNAS1 protein or its encoding gene in wheat, which can increase the nicotinamide content of the wheat plant, improve the agronomic traits of the wheat, and increase the nutrient content of the wheat grains. Specifically, overexpression of the TaNAS1 protein or its encoding gene increases wheat grain length, grain width, and 100-grain weight, and increases the zinc, iron, and copper content of the wheat grains. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] 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 are briefly introduced below.

[0028] Figure 1 The results of the genome-wide association analysis of nicotinamide content in wheat are shown in Figure 1.

[0029] Figure 2 The figure shows the sequence alignment results of TaNAS1 and homologous genes;

[0030] Figure 3 Phylogenetic tree (A) and expression pattern (B) of TaNAS1 protein homologous sequence analysis;

[0031] Figure 4 This figure shows the functional validation results of TaNAS1 transiently expressed in tobacco; ** indicates P < 0.01; *** indicates P < 0.001;

[0032] Figure 5 Schematic diagram of the genetic transformation vector structure for overexpressing TaNAS1 in wheat;

[0033] Figure 6 Differences in TaNAS1 gene expression, wheat grain agronomic traits, and zinc and related element contents between wild-type wheat and transgenic wheat overexpressing TaNAS1; * indicates P < 0.05; ** indicates P < 0.01; *** indicates P < 0.001. DETAILED DESCRIPTION

[0034] The present invention provides an application of a TaNAS1 protein or a gene encoding the same in regulating the nicotinamide content in plants; the amino acid sequence of the TaNAS1 protein is shown in SEQ ID NO: 3.

[0035] In one embodiment, regulating plant nicotinamide content in the present invention comprises: overexpressing the TaNAS1 protein or its encoding gene to increase the plant nicotinamide content. In one embodiment, increasing the plant nicotinamide content in the present invention comprises increasing the nicotinamide content in plant leaves. In one embodiment, the plants in the present invention include wheat and tobacco.

[0036] The present invention provides the use of a TaNAS1 protein or a gene encoding it in regulating agronomic traits and / or grain nutrient content of wheat; the agronomic traits include one or more of grain length, grain width and 100-grain weight; the nutrient content includes one or more of zinc, iron and copper; the amino acid sequence of the TaNAS1 protein is shown in SEQ ID NO: 3.

[0037] In one embodiment, the present invention comprises regulating agronomic traits of wheat by overexpressing the TaNAS1 protein or its encoding gene to increase one or more of wheat grain length, grain width, and 100-grain weight. In one embodiment, the present invention comprises regulating the nutrient content of wheat grains by overexpressing the TaNAS1 protein or its encoding gene to increase the content of one or more of zinc, iron, and copper in wheat grains.

[0038] In one embodiment, the nucleotide sequence of the coding region of the gene encoding the TaNAS1 protein of the present invention is shown in SEQ ID NO: 2. In one embodiment, the full-length genomic nucleotide sequence of the gene encoding the TaNAS1 protein of the present invention is shown in SEQ ID NO: 1.

[0039] The present invention provides a biomaterial, including a recombinant vector and / or a recombinant bacterium, wherein the recombinant vector includes a basic vector and a TaNAS1 gene inserted into the basic vector; the recombinant bacterium includes a basic bacterium and a TaNAS1 gene introduced into the recombinant bacterium; the amino acid sequence of the TaNAS1 protein encoded by the TaNAS1 gene is shown in SEQ ID NO: 3.

[0040] In one embodiment, the basic vector of the present invention comprises pLGY. In another embodiment, the basic bacteria of the present invention comprise Escherichia coli and / or Agrobacterium. The biomaterial provided by the present invention is capable of overexpressing the TaNAS1 gene in plants, thereby increasing the plant's nicotinamide content, positively improving wheat agronomic traits, and increasing the nutrient content of wheat grains.

[0041] The present invention provides a method for increasing the nicotinamide content in plants, comprising the following steps: overexpressing the TaNAS1 gene in the plant; the amino acid sequence of the TaNAS1 protein encoded by the TaNAS1 gene is shown in SEQ ID NO: 3.

[0042] As an embodiment, the plants of the present invention include wheat and tobacco. The present invention has no strict requirements on the method of overexpressing the TaNAS1 gene, and conventional methods in the art can be used.

[0043] To further illustrate the present invention, the application of the TaNAS1 protein or its encoding gene provided by the present invention in regulating the nicotinamide content in plants is described in detail below with reference to the accompanying drawings and examples, but they should not be construed as limiting the scope of protection of the present invention.

[0044] Example 1

[0045] Acquisition of the TaNAS1 gene

[0046] 1. TaNAS1 gene identification and structural analysis

[0047] The inventors used inductively coupled plasma mass spectrometry (ICP-MS) technology to detect the zinc content of natural wheat population materials and located a SNP site (AX02526944) on chromosome 2D. Within 1Mb (linkage disequilibrium distance) of the SNP site, there is a gene that is significantly associated with the zinc content in wheat grains ( Figure 1 ), the gene was annotated as nicotinamide synthase, the gene number was TraesCS2D01G049200, and it was named TaNAS1. The full-length genomic sequence of TaNAS1 is 1431bp (SEQ ID NO: 1), the coding sequence is 993bp (SEQ ID NO: 2), encoding 330 amino acids (SEQ ID NO: 3), and the gene has 1 exon. Sequence alignment analysis of NAS and TaNAS1 in rice and Arabidopsis showed that TaNAS1 and other NAS genes with proven functions all share the same conserved sequence motifs ( Figure 2 Phylogenetic analysis of TaNAS1 showed that TaNAS1 is highly homologous to OsNAS1 and OsNAS2 reported in rice ( Figure 3 ). The specific sequence information of SEQ ID NO: 1 to 3 is as follows:

[0048]

[0049]

[0050] SEQ ID NO:3:MAAQNKEVDALVQKITGLHAAIAKLPSLSPSPDVDALFT ELVTACVPPSPVDVTKLGPEAQKMREGLIRLCSEAEGKLEAHYSDMLAAFDNPLDHLGMFPYYNNYINLSKLEYELLARYVPGGIAPARVAFIGSGPLPFSSFVLAARHLPDTMFDNYDLCGAANDRASKLFRADKDVGARMSFHT ADVADLAGELAKYDVVFLAALVGMAAEDKAKVIAHLGAHMADGAALVVRSAHGARGFLYPIVDPQDIAGGGFEVLAVCHPDDDVVNSVIIAQKSKDVHAIGLRRGHGGQYAYGTVPVVSPPCRFGEMVPDMAQKREEFANAEVVF*.

[0051] 2. Amplification of the TaNAS1 gene

[0052] (1) DNA of Fielder wheat was extracted and PCR amplified using specific primers (TaNAS1-F: 5'-AGCTCCATCAACCACTCTCT-3', SEQ ID NO: 4; TaNAS1-R: 5'-GCGAGGCATGGATGATGATCA-3', SEQ ID NO: 5) to obtain PCR amplification products. The PCR amplification system consisted of 0.5 μL cDNA, 0.2 μL TaNAS1-F, 0.2 μL TaNAS1-R, 0.2 μL FastPfu Fly, 2 μL 5× Fly Buffer, 0.8 μL 2.5 mmol / L dNTP, and 6.1 μL H2O. The PCR amplification procedure was 95°C for 5 min, 95°C for 30 s, 58°C for 15 s, 72°C for extension, 72°C for 2 min for 35 cycles, and 12°C for 10 min. After the PCR reaction was completed, 3 μL of PCR product was taken for PCR electrophoresis on 0.8% agarose gel at 120V for 12 min to detect the presence of the target fragment.

[0053] (2) Connecting the PCR amplification product obtained in step (1) with the Blunt vector to obtain a ligation product; wherein the ligation system is: Blunt 0.5 μL, 10× Ehancer 0.25 μL, and amplification product 1.75 μL; the ligation conditions are 37°C metal bath for 25 minutes.

[0054] (3) The ligation product obtained in step (2) was transformed into Escherichia coli DH5α and revived in 1 mL of LB for 1 h. 150 μL of the revived bacterial solution was spread on a plate containing LA medium containing AMP resistance and incubated in a 37°C incubator for 12 h. A single clone was selected for PCR detection, and the plasmid was extracted and sequenced to obtain the cDNA sequence of the TaNAS1 gene. Sequencing analysis of the PCR product showed that a 993 bp TaNAS1 gene sequence was amplified from Fielder and the sequence alignment was correct.

[0055] Example 2

[0056] Transient expression of TaNAS1 in tobacco

[0057] 1. Using OsNAS1 and OsNAS2, previously reported in rice, as positive controls, the TaNAS1 gene obtained in Example 1 was ligated into the tobacco transient expression vector VB5 by homologous recombination. The recombinant plasmid was then transformed into competent Agrobacterium tumefaciens GV3101 (pSoup-p19) (Shanghai Weidi Biotechnology Co., Ltd.) using the instructions provided. After the bacteria reached an appropriate size, colonies were picked and plated in 12 mL of liquid YEP medium (containing 25 μg / mL rifampicin, 50 μg / mL kanamycin, and 15 μg / mL gentamicin) at 28°C, shaking at 200 rpm overnight.

[0058] 2. Detect TaNAS1 gene function according to the following steps

[0059] (1) Centrifuge the bacteria. Centrifuge the bacteria at room temperature at 3000g / min for 15 minutes. Discard the supernatant and aspirate the bacterial liquid near the bacterial mass with a pipette.

[0060] (2) Add resuspension buffer (final concentration: 2-(N-morpholino)ethanesulfonic acid (MES) 10 mM / L, pH = 5.6, MgCl2 10 mM / L, acetosyringone 150 mM / L) and mix by pipetting until OD 600 = about 0.5, incubate at room temperature for 1 to 2 hours.

[0061] (3) Inject tobacco into the tube using a disposable 1 mL syringe. After incubation overnight in the dark, incubate in the light for 3 to 5 days.

[0062] (4) The sample was placed in a 2 mL centrifuge tube and ground with liquid nitrogen (40 Hz, 60 s).

[0063] (5) Add 300 μL of methanol for every 0.1 g of fresh sample and vortex for 15 seconds until the powder is completely suspended.

[0064] (5) Ultrasound was performed at maximum power for 30 min, and the ultrasonic temperature was controlled below 20°C.

[0065] (7) Centrifuge at 4°C, 12,000 rpm for 10 min.

[0066] (8) The supernatant was drawn up by syringe, filtered through a 0.22 μM filter membrane into a sample injection bottle, and the sample was detected by LC-MS.

[0067] LC-MS test results are as follows Figure 4 As shown. Figure 4 It can be seen that the activity of TaNAS1 gene obtained in Example 1 in synthesizing nicotinamide (NA) is comparable to that of OsNAS1 and OsNAS2. This indicates that TaNAS1 has the function of synthesizing nicotinamide ( Figure 4 ).

[0068] Example 3

[0069] Regulation of nicotinamide content in wheat by TaNAS1 genetic material

[0070] 1. Construction of TaNAS1 genetic transformation vector

[0071] The 993 bp TaNAS1 gene fragment amplified from Fielder in Example 1 was subjected to homologous recombination reaction with the target vector pLGY to obtain the recombinant vector TaNAS1-pLGY. The mass spectrum is shown in FIG. Figure 5 The recombinant vector TaNAS1-pLGY is an Agrobacterium-mediated genetic transformation vector carrying a maize ubiquitin gene promoter with constitutive and overexpression characteristics.

[0072] 2. Referring to the prior art (Hayta S, Smedley MA, Demir SU, et al. An efficient and reproducible Agrobacterium-mediated transformation method for hexaploid wheat (Triticum aestivum L.) [J]. Plant Methods, 2019, 15: 1-15.), the plasmid correctly cloned in step 1 was introduced into the wheat variety Fielder through Agrobacterium EHA105-mediated and wheat genetic transformation system. After pre-culture, infection, co-cultivation, and screening of callus tissue with glufosinate resistance, differentiation, rooting, seedling hardening and transplanting were performed to obtain TaNAS1 overexpressing transgenic plants.

[0073] 3. Analysis of gene expression levels, agronomic traits, and zinc and related element contents in TaNAS1 overexpressing transgenic materials and wheat grains

[0074] The DNA level of the T0 transgenic wheat plants obtained in step 2 was tested to confirm the construction of TaNAS1 positive plants. The T1 generation positive transgenic wheat plants overexpressing TaNAS1 and wild-type wheat plants were grown to the rising stage, and RNA samples and metabolic samples of the leaves at the rising stage were taken to analyze the expression level of TaNAS1 in the plants and the content of metal elements in mature seeds (grains). The results are as follows: Figure 6 shown. Figure 6 A is a picture of mature seeds and the expression level of TaNAS1 gene; B is the nicotinamide content in the root; C is the length of the seed; D is the width of the seed; E is the weight of 100 seeds; F to J are the contents of zinc (Zn), iron (Fe), copper (Cu), manganese (Mn) and cadmium (Cd) in the seeds respectively. Figure 6 It can be seen that compared with the wild-type control Fielder, the expression level of the TaNAS1 gene in the positive transformed plants overexpressing TaNAS1 was greatly increased, the contents of zinc, iron and copper elements increased significantly, and the grain length, width and 100-grain weight increased significantly ( Figure 6 ).

[0075] Based on the above content, it can be seen that TaNAS1 provided by the present invention can increase the content of nicotinamide in wheat, improve the agronomic traits of wheat grains, and increase the content of zinc and related elements.

[0076] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Application of TaNAS1 protein or its encoding gene in regulating nicotinamide content in plants; The amino acid sequence of the TaNAS1 protein is shown in SEQ ID NO:

3.

2. The use according to claim 1, characterized in that The regulating plant nicotinamide content includes: overexpressing TaNAS1 protein or its encoding gene to increase the plant nicotinamide content.

3. The use according to claim 1, characterized in that Such plants include wheat and tobacco.

4. Application of TaNAS1 protein or its encoding gene in regulating agronomic traits and / or grain nutrient content in wheat; The agronomic traits include one or more of grain length, grain width and 100-grain weight; The nutrient element content includes one or more of zinc, iron and copper; The amino acid sequence of the TaNAS1 protein is shown in SEQ ID NO:

3.

5. The use according to claim 4, characterized in that The regulation of wheat agronomic traits includes: overexpressing TaNAS1 protein or its encoding gene to increase one or more of wheat grain length, grain width and 100-grain weight; The method of regulating the nutrient content of wheat grains includes overexpressing TaNAS1 protein or its encoding gene to increase the content of one or more of zinc, iron and copper in wheat grains.

6. The use according to any one of claims 1 to 5, characterized in that: The nucleotide sequence of the coding region of the gene encoding the TaNAS1 protein is shown in SEQ ID NO:

2.

7. The use according to claim 6, characterized in that The full-length genomic nucleotide sequence of the coding gene is shown in SEQ ID NO:

1.

8. A biomaterial comprising a recombinant vector and / or a recombinant bacterium, characterized in that: The recombinant vector includes a basic vector and a TaNAS1 gene inserted into the basic vector; The recombinant bacteria include a basic bacteria and a TaNAS1 gene introduced into the recombinant bacteria; The amino acid sequence of the TaNAS1 protein encoded by the TaNAS1 gene is shown in SEQ ID NO:

3.

9. The biomaterial according to claim 8, characterized in that The basic vector includes pLGY; The basic bacteria include Escherichia coli and / or Agrobacterium.

10. A method for increasing the nicotinamide content in plants, characterized in that: The method comprises the following steps: overexpressing the TaNAS1 gene in a plant; the amino acid sequence of the TaNAS1 protein encoded by the TaNAS1 gene is shown in SEQ ID NO: 3.

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