Application of TaPLT8A gene or TaPLT8A protein in regulating iron content in monocotyledonous plant grains

Abstract

The present invention relates to the technical field of plant genetic improvement, and in particular to the use of the TaPLT8A gene or TaPLT8A protein in regulating the iron content of monocotyledonous plant grains. The nucleotide sequence of the TaPLT8A gene of the present invention is shown in SEQ ID NO.1; the amino acid sequence of the TaPLT8A protein is shown in SEQ ID NO.2. The results of the specific embodiments of the present invention show that overexpressing the TaPLT8A gene in wheat plants can significantly increase the iron content of wheat grains; knocking out the gene in wheat significantly reduces the iron content of wheat grains. It can be seen that the TaPLT8A gene or TaPLT8A protein can regulate the iron content in wheat grains, and this study provides a usable gene resource for breeding high-iron wheat varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic improvement, and in particular to the application of TaPLT8A gene or TaPLT8A protein in regulating the iron content of monocotyledonous plant seeds. Background Art

[0002] Iron is an essential trace element for the survival and growth of various organisms (Herlihy JH, Long TA, McDowell J M. Iron homeostasis and plant immune responses: recent insights and translational implications[J]. Journal of Biological Chemistry, 2020, 295(39): 13444-13457). Iron deficiency in humans can lead to poor development in young children, decreased resistance, adverse pregnancy outcomes, and anemia (Pasricha SR, Tye-Din J, Muckenthaler MU, et al. Iron deficiency[J]. The Lancet, 2021, 397(10270): 233-248.). According to a survey by the World Health Organization, approximately 33% of non-pregnant women, 40% of pregnant women, and 42% of children worldwide suffer from iron deficiency anemia. Plants are one of the important sources of dietary iron for humans, but the iron content in most plants is low. Moreover, due to the extremely low solubility of iron in the earth's crust, plants face many difficulties in absorbing iron, which leads to insufficient iron nutrition intake in the population.

[0003] Wheat (Triticum aestivum L.) is one of the world's most important food crops, serving as a staple food for many people. Unfortunately, the iron content of wheat grains is generally low, failing to meet the body's daily iron needs. Studies have shown that the iron content of wheat grains is far below the recommended dietary allowance. In this context, increasing the iron content of wheat grains has become a key approach to addressing the global iron deficiency problem and a key research topic in agricultural research.

[0004] In recent years, with the rapid development of biotechnology, genetic engineering has shown great potential in crop improvement. Biofortification of crops through genetic engineering has become an effective strategy to improve the nutritional quality of crops. There are many successful cases in crops such as rice and corn. For example, in rice, overexpression of the OsIRT1 gene can significantly increase the iron content of shoots, roots and grains (Lee S, An G. Over-expression of OsIRT1 leads to increased iron and zinc accumulations in rice [J]. Plant, cell & environment, 2009, 32 (4): 408-416); in corn, overexpression of the ZmNAC78 gene effectively increases the iron content in corn grains, laying the foundation for the cultivation of high-iron corn varieties (Du Q, Li W X. Iron biofortification in maize by ZmNAC78 is a promising and sustainable way to fight iron-deficiency anaemia [J]. Clinical and Translational Medicine, 2024, 14 (1)).

[0005] While genetic engineering holds great potential for crop improvement, with successful examples of increasing iron content in crops like rice and corn, research into genes that can effectively regulate grain iron content in wheat remains relatively scarce. The current lack of a deep understanding of the mechanisms regulating iron content in wheat and the identification of key genes limits research progress in increasing wheat grain iron content through genetic engineering. Summary of the Invention

[0006] The purpose of the present invention is to provide an application of the TaPLT8A gene or TaPLT8A protein in regulating the iron content in monocotyledonous plant seeds, so as to solve the problems existing in the above-mentioned prior art.

[0007] To achieve the above object, the present invention provides the following solutions:

[0008] The present invention provides the use of the TaPLT8A gene, the TaPLT8A protein or a biological material containing the TaPLT8A gene in regulating the iron content of monocotyledonous plant seeds. The nucleotide sequence of the TaPLT8A gene is shown in SEQ ID NO.1; the amino acid sequence of the TaPLT8A protein is shown in SEQ ID NO.2.

[0009] Preferably, the iron content of the monocotyledonous plant seeds is increased by overexpressing the TaPLT8A gene in the monocotyledonous plant; and the iron content of the monocotyledonous plant seeds is reduced by inhibiting the expression of the TaPLT8A gene in the monocotyledonous plant.

[0010] Preferably, the monocotyledonous plant includes wheat.

[0011] Further preferably, the biological material includes a recombinant vector or a recombinant bacterium.

[0012] The present invention provides a method for increasing the iron content of monocotyledonous plant seeds, comprising the step of overexpressing the TaPLT8A gene in the monocotyledonous plant to increase the iron content of the monocotyledonous plant seeds; the nucleotide sequence of the TaPLT8A gene is shown in SEQ ID NO.1.

[0013] Further preferably, the iron content of the monocotyledonous plant seeds is increased by overexpressing the TaPLT8A gene in the monocotyledonous plant; and the iron content of the monocotyledonous plant seeds is reduced by inhibiting the expression of the TaPLT8A gene in the monocotyledonous plant.

[0014] The present invention provides a method for reducing the iron content of monocotyledonous plant seeds, comprising the steps of inhibiting the expression of TaPLT8A gene in the monocotyledonous plant to reduce the iron content of the monocotyledonous plant seeds; the nucleotide sequence of the TaPLT8A gene is shown in SEQ ID NO.1.

[0015] Further preferably, the iron content of the monocotyledonous plant seeds is increased by overexpressing the TaPLT8A gene in the monocotyledonous plant; and the iron content of the monocotyledonous plant seeds is reduced by inhibiting the expression of the TaPLT8A gene in the monocotyledonous plant.

[0016] The present invention provides the use of the TaPLT8A gene, the TaPLT8A protein or a biological material containing the TaPLT8A gene in cultivating monocotyledonous plants with high grain iron content and / or monocotyledonous plants with low grain iron content. The nucleotide sequence of the TaPLT8A gene is shown in SEQ ID NO.1; the amino acid sequence of the TaPLT8A protein is shown in SEQ ID NO.2.

[0017] Preferably, the iron content of the monocotyledonous plant seeds is increased by overexpressing the TaPLT8A gene in the monocotyledonous plant; and the iron content of the monocotyledonous plant seeds is reduced by inhibiting the expression of the TaPLT8A gene in the monocotyledonous plant.

[0018] Preferably, the monocotyledonous plant includes wheat.

[0019] Further preferably, the biological material includes a recombinant vector or a recombinant bacterium.

[0020] The present invention provides a method for cultivating monocotyledonous plants with high iron content in seeds, comprising the step of overexpressing the TaPLT8A gene in the monocotyledonous plants to increase the iron content in the seeds of the monocotyledonous plants; the nucleotide sequence of the TaPLT8A gene is shown in SEQ ID NO.1.

[0021] Further preferably, the iron content of the monocotyledonous plant seeds is increased by overexpressing the TaPLT8A gene in the monocotyledonous plant; and the iron content of the monocotyledonous plant seeds is reduced by inhibiting the expression of the TaPLT8A gene in the monocotyledonous plant.

[0022] The present invention provides a method for cultivating monocotyledonous plants with low iron content in seeds, comprising the steps of inhibiting the expression of TaPLT8A gene in the monocotyledonous plants to reduce the iron content in the seeds of the monocotyledonous plants; the nucleotide sequence of the TaPLT8A gene is shown in SEQ ID NO.1.

[0023] Further preferably, the iron content of the monocotyledonous plant seeds is increased by overexpressing the TaPLT8A gene in the monocotyledonous plant; and the iron content of the monocotyledonous plant seeds is reduced by inhibiting the expression of the TaPLT8A gene in the monocotyledonous plant.

[0024] The present invention discloses the following technical effects:

[0025] The present invention provides the use of the TaPLT8A gene in regulating the iron content of monocotyledonous plant grains. The nucleotide sequence of the TaPLT8A gene provided by the present invention is shown in SEQ ID NO.1; the amino acid sequence of the TaPLT8A protein is shown in SEQ ID NO.2. The results of the specific embodiments of the present invention show that overexpressing the TaPLT8A gene in wheat plants can significantly increase the iron content of wheat grains; knocking out the gene in wheat significantly reduces the iron content of wheat grains. It can be seen that the TaPLT8A gene or TaPLT8A protein can regulate the iron content in wheat grains. This study provides a usable gene resource for breeding high-iron wheat varieties. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments. 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 any creative work.

[0027] Figure 1The sequence cloning results of the TaPLT8A gene are shown in Figure 1. The left lane is the marker, and the right lane is the product.

[0028] Figure 2 Schematic diagram of gene knockout using CRISPR-Cas9 targeting the TaPLT8A gene;

[0029] Figure 3 The iron content in the grains of the wheat taplt8a knockout mutant is shown in Figure 1. A shows the Prussian blue staining result, with the left side showing the cross section and the right side showing the longitudinal section. B shows the iron content in the wheat grains.

[0030] Figure 4 is the iron content in transgenic wheat grains overexpressing TaPLT8A; A is the expression level of the TaPLT8A gene in different strains; B is the Prussian blue staining result, with the left side being the cross section and the right side being the longitudinal section; C is the iron determination result in wheat grains. DETAILED DESCRIPTION

[0031] 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 rather as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0032] It should be understood that the terms described herein are intended only to describe particular embodiments and are not intended to limit the present invention. In addition, for numerical ranges herein, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. The intermediate value within any stated value or stated range, and each smaller range between any other stated value or intermediate value within the stated range, is also encompassed within the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded within the scope.

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art. Although only preferred methods and materials are described herein, any methods and materials similar or equivalent to those described herein may also be used in the practice 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 any conflict with any incorporated document, the contents of this specification shall prevail.

[0034] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments described herein without departing from the scope or spirit of the invention. Other embodiments will be apparent to those skilled in the art from the description of the invention. The description and examples are intended to be illustrative only.

[0035] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0036] Example 1 Cloning of CDS sequence of wheat TaPLT8A gene

[0037] Total RNA was extracted from the roots of Fielder wheat (wild-type control material, hereinafter referred to as WT) seedlings grown for about 2 weeks using Trizol.

[0038] cDNA was obtained by reverse transcription using a reverse transcription kit, and primers TaPLT8A-P1 and TaPLT8A-P2 were designed. PCR amplification was performed using cDNA as a template to obtain the CDS sequence of the TaPLT8A gene (1398 bp).

[0039] TaPLT8A-P1: 5'-ATGTCTTCGCCTGCTCCGTC-3', SEQ ID NO.1;

[0040] TaPLT8A-P2: 5'-TCATCCCTTCCGCACCAGG-3', SEQ ID NO. 2.

[0041] The PCR products were detected by electrophoresis using 1.2% agarose gel. Figure 1 As shown, a band with a molecular weight of about 1400 bp was obtained, which was consistent with the expected result.

[0042] The fragment was recovered using a gel extraction / DNA purification kit (Novozymes). Using the Blunt Gene Cloning Kit (Golden Biotech), the fragment was ligated into the pEASY-Blunt vector and transformed into E. coli DH5α. The cells were then plated and cultured overnight. Single plaques from the kanamycin-resistant plates were selected for positive detection. Positive clones were then expanded and sent to the company for sequencing.

[0043] The sequencing results showed that the CDS sequence of the amplified TaPLT8A gene was shown in SEQ ID No. 3. Through codon translation, the TaPLT8A protein sequence encoded by the TaPLT8A gene was shown in SEQ ID NO. 4.

[0044]

[0045] The specific sequence of the TaPLT8A protein encoded by the TaPLT8A gene is: MSSPAPSSPPSTPCSSATRDLHITEVQQEVLVGCLSFVSLLGSLAGGRTADALGRKRTIGLAAAVFQAGALVMTLAPSFRVLMAGRLLAGIGIGFGVMIAPVYIAEISPAASRGSFTSFPEIFINLGILLGYISNYAFSGLPDHLGWRVMLAVGIVPSVSIVFALLVIPESPRWLVVQGRAAEAREVLLRVTDGEEEADERLAEIEAAAASAANSGETVWR ELSRPSPTIRRMLVTGLGIQCFQQITGIDALVYYSPTIFRDAGVTTESQLLLATVAVGFFKTAFIALAIVLIDRVGRKPLLYVSTVGMTVCLVILASTLWLLAHGAVPKALGVAVAVVAVCGDV AFFSVGIGPVCWVTSSEIFPLRLRSQAAALGAAVNRVTSGTVAMSFLSVSRAMTVAGAFSAFAAISALSVVFVHRFVPETKGKTLEEIELLFGGGEGGEGVTVSPGEVELGDAEHLVRKG(SEQ ID NO.4).

[0046] Example 2: The iron content of wheat taplt8a knockout mutants is significantly reduced.

[0047] Based on the sequence information of the wheat TaPLT8A gene, two specific targeting sites were designed in the second exon of the TaPLT8A gene using the CRISPR-Cas9 method. The target site sequences were gRNA1: 5'-TCTACATCGCCGAGATCTCC-3' (SEQ ID NO. 5) and gRNA2: 5'-GGGCTCCTTCACCTCCTTCC-3' (SEQ ID NO. 6). The designed gRNA1 and gRNA2 were inserted into the VK005-05 vector at the Asc I and Avr II sites, respectively, to successfully construct a knockout vector. The VK005-05 vector has been disclosed in the instructions for the plant Cas9 / gRNA plasmid construction kit of Beijing Weishanglide Biotechnology Co., Ltd.

[0048] Using wheat Fielder as the recipient material, the constructed knockout vector was transferred into the wheat recipient material by genetic transformation. Primers TaPLT8A-P3 and TaPLT8A-P4 were designed, and the gRNA target site sequence information was obtained by PCR amplification and sequencing in the T2 generation plants harvested from the taplt8a knockout mutant. The results showed that two homozygous knockout lines were found. The 11th line had a 34bp deletion compared to the WT and was recorded as taplt8a#11; the 45th line had a 16bp deletion compared to the WT and was recorded as taplt8a#45. Figure 2 ). Both knockout events lead to premature termination of the CDS of the TaPLT8A gene.

[0049] TaPLT8A-P3: 5'-AATCGCGTTGACTTGTTGCG-3', SEQ ID NO.7;

[0050] TaPLT8A-P4: 5'-GGCGTAGTTTGAGATGTAACCG-3', SEQ ID NO. 8.

[0051] The harvested grains of the taplt8a knockout mutant and WT materials were stained with Prussian blue. Figure 3 As shown. Staining results ( Figure 3 A) in the figure shows that the taplt8a knockout mutant grains are lighter in staining than the WT, indicating that the taplt8a knockout mutant grains have less iron accumulation. The iron content in wheat grains was further determined by referring to the method of Yao et al. (Yao Q, Li W, Liu Y, et al. FeCl3 and Fe2 (SO4) 3 differentially reduce Cd uptake and accumulation in Polish wheat (Triticumpolonicum L.) seedlings by exporting Cd from roots and limiting Cd binding in the root cell walls [J]. Environmental Pollution, 2023, 317: 120762). The results are as follows: Figure 3 As shown in Figure 2 B. The results showed that the iron content in taplt8a knockout mutants was significantly lower than that in WT, with taplt8a#11 reduced by 24.97% and taplt8a#11 reduced by 23.29%.

[0052] Example 3 Overexpression of wheat TaPLT8A significantly increased the iron content of wheat grains.

[0053] To further study the effect of the TaPLT8A gene in regulating the iron content of wheat grains, this example constructed an overexpression vector UBI-TaPLT8A of the UBI promoter, wherein the basic vector is pLGY02 and the insertion sites are Sma I and Spe I. Subsequently, the vector is transformed into the wheat transgenic recipient material Fielder. Among them, pLGY02 is disclosed in the document "The central circadian clock protein TaCCA1 regulates seedling growth and spike development in wheat (Triticum aestivum L.)" (Gong J, Tang Y, Liu Y, et al. The central circadian clock protein TaCCA1 regulates seedling growth and spike development in wheat (Triticum aestivum L.) [J]. Frontiers in plant science, 2022, 13: 946213).

[0054] Primers TaPLT8A-P5 and TaPLT8A-P6 were designed to detect the expression level of TaPLT8A gene in the expression strains. The results are as follows: Figure 4 As shown in Figure A. The results showed that the TaPLT8A gene expression levels in the T2 generation plants of two transgenic lines (OE#5 and OE#13) were higher than those in the WT. Therefore, these two T2 generation plants of the transgenic lines (OE#5 and OE#13) and the WT were selected for further research.

[0055] TaPLT8A-P5: 5'-TGTTCGTGCACAGGTTCGTC-3', SEQ ID NO.9;

[0056] TaPLT8A-P6: 5'-CTCACTGTCACTCCCTCGC-3', SEQ ID NO. 10.

[0057] The harvested grains of the TaPLT8A overexpressing strain and WT material were stained with Prussian blue. Figure 4 As shown in B and C. Staining results ( Figure 4B) in the figure shows that the grains of the strain overexpressing the TaPLT8A gene are stained darker than those of the WT, indicating that the grains of the strain overexpressing the TaPLT8A gene have accumulated more iron. The iron content in wheat grains was further determined by referring to the method of Yao et al. (Yao Q, LiW, Liu Y, et al. FeCl3 and Fe2(SO4)3differentially reduce Cd uptake and accumulation in Polish wheat(Triticum polonicum L.)seedlings by exporting Cdfrom roots and limiting Cd binding in the root cell walls[J]. Environmental Pollution, 2023, 317: 120762). The results are as follows: Figure 4 The results showed that the iron content in the lines overexpressing the TaPLT8A gene was significantly higher than that in the WT, with OE#5 and OE#13 increasing by 14.99% and 12.84%, respectively. This demonstrates that overexpressing the TaPLT8A gene can increase the iron content in transgenic wheat grains and play an important regulatory role in the accumulation of iron in wheat grains.

[0058] In summary, overexpressing the TaPLT8A gene in wheat plants significantly increases the iron content of wheat grains, while knocking out the gene significantly reduces the iron content of wheat grains. Therefore, the TaPLT8A gene or TaPLT8A protein can regulate the iron content of wheat grains and could be used to breed new wheat varieties.

[0059] The embodiments described above are merely descriptions of preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Without departing from the spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by persons skilled in the art should fall within the scope of protection defined by the claims of the present invention.

Claims

1. Use of the TaPLT8A gene, TaPLT8A protein or a biomaterial containing the TaPLT8A gene in increasing the iron content of wheat grains, characterized in that: The nucleotide sequence of the TaPLT8A gene is shown in SEQ ID NO.3; the amino acid sequence of the TaPLT8A protein is shown in SEQ ID NO.

4.

2. The use according to claim 1, characterized in that By overexpressing the TaPLT8A gene in wheat, the iron content of the wheat grains is increased.

3. A method for increasing the iron content of wheat grains, characterized in that: The method comprises the steps of overexpressing the TaPLT8A gene in wheat to increase the iron content of the wheat grains; the nucleotide sequence of the TaPLT8A gene is shown in SEQ ID NO.

3.

4. Use of the TaPLT8A gene, TaPLT8A protein or a biological material containing the TaPLT8A gene in cultivating wheat with a high iron content in grain, characterized in that: The nucleotide sequence of the TaPLT8A gene is shown in SEQ ID NO.3; the amino acid sequence of the TaPLT8A protein is shown in SEQ ID NO.

4.

5. The use according to claim 4, characterized in that By overexpressing the TaPLT8A gene in wheat, the iron content of the wheat grains is increased.

6. A method for cultivating wheat with high iron content in grain, characterized in that: The method comprises the steps of overexpressing the TaPLT8A gene in wheat to increase the iron content of the wheat grains; the nucleotide sequence of the TaPLT8A gene is shown in SEQ ID NO.3.

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