Application of TaPLT8A gene or TaPLT8A protein in regulating and controlling iron content of monocotyledon seeds

Abstract

The invention relates to the technical field of plant genetic improvement, in particular to application of a TaPLT8A gene or TaPLT8A protein to regulation and control of the iron content of monocotyledon seeds. The nucleotide sequence of the TaPLT8A gene disclosed by the invention is as shown in SEQ ID NO. 1; the amino acid sequence of the TaPLT8A protein is as shown in SEQ ID NO. 2. A result of a specific embodiment of the invention shows that the TaPLT8A gene is over-expressed in a wheat plant, so that the iron content of wheat grains can be remarkably increased; the gene is knocked out of wheat, so that the iron content of wheat grains is remarkably reduced. Therefore, the TaPLT8A gene or the TaPLT8A protein can regulate and control the iron content in the wheat grains, and the research provides an available gene resource for cultivating high-iron wheat varieties.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic improvement, and in particular to 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 JM. Iron homeostasis and plant immune responses: recent insights and translational implications [J]. Journal of Biological Chemistry, 2020, 295 (39): 13444-13457). Human iron deficiency can lead to poor development of young children, decreased resistance, adverse pregnancy, 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, about 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. In addition, due to the extremely low solubility of iron in the earth's crust, plants face many difficulties in absorbing iron, which in turn leads to insufficient iron intake in the population.

[0003] Wheat (Triticum aestivum L.) is one of the most important food crops in the world and is the staple food of many people. Unfortunately, the iron content in wheat grains is generally not high, which is difficult to meet the daily iron needs of the human body. According to relevant studies, the iron content of wheat grains is far lower than the dietary standard recommended by nutritionists. In this context, increasing the iron content of wheat grains has become one of the key ways to solve the problem of iron deficiency in the global population and an important research direction in the field of agricultural scientific 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 have been 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 breeding 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] Although genetic engineering has great potential in crop improvement and has been successful in increasing the iron content of crops such as rice and corn, research on genes that can effectively regulate the iron content of wheat grains is relatively scarce. At present, there is a lack of in-depth understanding of the regulation mechanism of wheat iron content and the discovery of key genes, which limits the research progress of increasing the iron content of wheat grains through genetic engineering. Summary of the invention

[0006] The purpose of the present invention is to provide an application of TaPLT8A gene or TaPLT8A protein in regulating the iron content of 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 TaPLT8A gene, TaPLT8A protein or biological material containing TaPLT8A gene in regulating the iron content of monocot seeds, the nucleotide sequence of the TaPLT8A gene is shown as SEQ ID NO.1; the amino acid sequence of the TaPLT8A protein is shown as 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 improving the iron content of monocotyledonous plant seeds, comprising the step of overexpressing the TaPLT8A gene in the monocotyledonous plant to improve 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 seed iron content and / or monocotyledonous plants with low seed iron content. The nucleotide sequence of the TaPLT8A gene is shown as SEQ ID NO.1; the amino acid sequence of the TaPLT8A protein is shown as 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 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 application 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, and this study provides an available 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 drawings required for use in the embodiments will be briefly introduced below. 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 creative work.

[0027] Figure 1The sequence cloning result of TaPLT8A gene; the left lane is the marker, and the right lane is the product;

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

[0029] Figure 3 The iron content in the grains of the wheat taplt8a knockout mutant; A is the result of Prussian blue staining, with the left side being the cross section and the right side being the longitudinal section; B is the result of the determination of iron in 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 result of Prussian blue staining, with the left side being the cross section and the right side being the longitudinal section; C is the result of iron determination 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 should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

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

[0033] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation 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 a conflict with any incorporated document, the content 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 of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present invention description and examples are exemplary 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, primers TaPLT8A-P1 and TaPLT8A-P2 were designed, and 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 recovery / DNA purification kit (Novozyme). The fragment was connected to the pEASY-Blunt vector using a Blunt gene cloning kit (Quanshijin Biotechnology) and transferred into E. coli DH5α. Then, by plating and overnight culture, a single spot was selected from the plate with kanamycin resistance for positive detection, and the positive clone plaque was 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 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 mutant is significantly reduced

[0047] According to the sequence information of wheat TaPLT8A gene, two specific targeting sites were designed on the second exon of TaPLT8A gene by 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 VK005-05 vector, and the insertion sites were Asc I and Avr II, and the knockout vector was successfully constructed. Among them, VK005-05 vector has been disclosed in the instructions of plant Cas9 / gRNA plasmid construction kit of Beijing Weishanglide Biotechnology Co., Ltd.

[0048] Wheat Fielder was used as the recipient material, and 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, recorded as taplt8a#11; the 45th line had a 16bp deletion compared to the WT, recorded as taplt8a#45 ( Figure 2 ). Both knockout events would 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 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 embodiment constructs 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, and then the vector is transformed into the wheat transgenic receptor 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 TaCCA1regulates 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 A. The results showed that the expression level of TaPLT8A gene in the two transgenic lines T2 plants (OE#5 and OE#13) was higher than that in WT. Therefore, these two transgenic lines T2 plants (OE#5 and OE#13) and WT were selected as research materials for further study.

[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 strains and WT materials were stained with Prussian blue. Figure 4 As shown in B and C. The 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 more iron is accumulated in the grains of the strain overexpressing the TaPLT8A gene. 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)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 As shown in C. The results showed that the iron content in the strains overexpressing the TaPLT8A gene was significantly higher than that in the WT, with OE#5 increasing by 14.99% and OE#13 increasing by 12.84%, proving 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 content in wheat grains.

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

[0059] The embodiments described above are only descriptions of the preferred modes of the present invention, and are not intended to limit the scope of the present invention. Without departing from the design spirit of the present invention, various modifications and improvements made to the technical solutions of the present invention by ordinary technicians in this field should all fall within the protection scope determined by the claims of the present invention.

Claims

1. Application of TaPLT8A gene, TaPLT8A protein or biological material containing TaPLT8A gene in regulating the iron content of monocotyledonous plant seeds, characterized in that: 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.

2. The use according to claim 1, characterized in that: By overexpressing the TaPLT8A gene in monocotyledonous plants, the iron content of the monocotyledonous plants' seeds is increased; by inhibiting the expression of the TaPLT8A gene in monocotyledonous plants, the iron content of the monocotyledonous plants' seeds is reduced.

3. The use according to claim 1 or 2, characterized in that: The monocotyledonous plants include wheat.

4. A method for increasing the iron content of monocotyledonous plant seeds, characterized in that: The method comprises the steps of overexpressing the TaPLT8A gene in a 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.

5. A method for reducing the iron content of monocotyledonous plant seeds, characterized in that: The method comprises the steps of inhibiting the expression of TaPLT8A gene in monocotyledonous plants to reduce the iron content of monocotyledonous plant seeds; the nucleotide sequence of the TaPLT8A gene is shown in SEQ ID NO.

1.

6. Use of the TaPLT8A gene, the TaPLT8A protein or a biological material containing the TaPLT8A gene in cultivating monocotyledonous plants with high iron content in seeds and / or monocotyledonous plants with low iron content in seeds, characterized in that: 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.

7. The use according to claim 6, characterized in that: By overexpressing the TaPLT8A gene in monocotyledonous plants, the iron content of the monocotyledonous plants' seeds is increased; by inhibiting the expression of the TaPLT8A gene in monocotyledonous plants, the iron content of the monocotyledonous plants' seeds is reduced.

8. The use according to claim 6 or 7, characterized in that: The monocotyledonous plants include wheat.

9. A method for cultivating monocotyledonous plants with high iron content in seeds, characterized in that: The method comprises the steps of overexpressing the TaPLT8A gene in a 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.

10. A method for cultivating monocotyledonous plants with low iron content in seeds, characterized in that: The method comprises the steps of inhibiting the expression of TaPLT8A gene in monocotyledonous plants to reduce the iron content of monocotyledonous plant seeds; the nucleotide sequence of the TaPLT8A gene is shown in SEQ ID NO.1.

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