Application of TaPIL1 protein in regulating and controlling trace element content of wheat grains
By overexpressing TaPIL1 protein in wheat, the problem of low iron and zinc content in wheat grains was solved, and the iron and zinc content in the grains was significantly increased, meeting human nutritional needs and promoting health.
Patent Information
- Application Number
- CN202510804191.5
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-06-16
- Publication Date
- 2025-09-16
AI Technical Summary
Wheat grains have low iron and zinc content, which leads to nutritional deficiencies in humans. Existing technologies make it difficult to effectively increase their content, which affects health.
Overexpression of TaPIL1 protein was achieved by constructing a recombinant expression vector and transferring it into wheat to increase the expression level of TaPIL1 protein, thereby increasing the iron and zinc content in wheat grains.
Significantly increase the iron and zinc content in wheat grains to meet human nutritional needs and promote health.
Smart Images
Figure CN120648735A_ABST
Abstract
Description
Technical Field
[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to the application of TaPIL1 protein in regulating the content of trace elements in wheat grains. Background Art
[0002] Wheat (Triticum aestivum L.), as an important staple food crop, not only provides 50% of daily energy needs but also a variety of essential trace elements. Trace element deficiencies can lead to "hidden hunger." Modern medicine has discovered that 70% of chronic diseases, including diabetes, cardiovascular disease, cancer, obesity, and sub-health, are related to an imbalance in nutrient intake. This "hidden hunger" is becoming a deadly threat to human health.
[0003] Wheat grains contain relatively low levels of essential trace elements. Conventional breeding and genetic modification methods can currently be used to increase the iron and zinc content of wheat grains, thereby preventing and alleviating human malnutrition. Studies have shown that the iron content in wheat grains reaches 59 mg / kg, which can meet people's daily iron intake needs, while the average iron content of most wheat grains is only 45 mg / kg; the zinc content is even lower, averaging between 28 and 30 mg / kg, which is lower than the threshold of 40 to 60 mg / kg required for human nutrition. Therefore, increasing the iron and zinc content in wheat grains is a key challenge in addressing iron and zinc deficiencies in human nutrition and is of great significance for ensuring people's health.
[0004] Studies have shown that the main proteins in plants are zinc-iron transporter (ZIP), heavy metal ATPase (HMA), cation diffusion assist protein (CDF), natural resistance macrophage protein (NRAMP) and Mg 2+ / H + Antiporters are involved in the absorption, transport and distribution of zinc in plants. At the same time, ferritin is a widely present iron storage protein that participates in iron homeostasis in plants, animals and microorganisms by continuously binding and releasing iron. The expression of ferritin genes in plants is regulated by multiple transcription factors, which in turn affects the iron content in seeds. However, due to the large and complex wheat genome and the difficulty of genetic transformation, research on iron storage in wheat grains and its regulatory genes has lagged behind. Therefore, how to increase the iron and zinc content in wheat grains remains a problem to be solved urgently in this field. Summary of the Invention
[0005] The purpose of the present invention is to provide an application of TaPIL1 protein in regulating the trace element content of wheat grains. Overexpression of the TaPIL1 protein in wheat plants can increase the trace element content of wheat grains.
[0006] The present invention provides the use of TaPIL1 protein in regulating the content of trace elements in wheat grains. The amino acid sequence of the TaPIL1 protein is shown in SEQ ID NO.1.
[0007] As a preferred solution, the regulation includes increasing the expression level of TaPIL1 protein to increase the trace element content in wheat grains.
[0008] As a preferred embodiment, the nucleotide sequence of the gene encoding the TaPIL1 protein is shown as SEQ ID NO.2.
[0009] As a preferred embodiment, the trace elements include iron and / or zinc.
[0010] The present invention also provides the use of TaPIL1 protein in cultivating wheat germplasm with high trace element content in grains. The amino acid sequence of the TaPIL1 protein is shown in SEQ ID NO.1.
[0011] As a preferred embodiment, the trace elements include iron and / or zinc.
[0012] The present invention also provides a recombinant expression vector comprising a gene encoding a TaPIL1 protein; the amino acid sequence of the TaPIL1 protein is shown in SEQ ID NO.1.
[0013] The present invention also provides a recombinant bacterium, which comprises the recombinant expression vector described in the above scheme.
[0014] The present invention also provides the use of the recombinant expression vector or the recombinant bacteria described in the above scheme in cultivating high-iron wheat and / or high-zinc wheat.
[0015] The present invention also provides a method for increasing the iron and / or zinc content of wheat grains, comprising: transferring the recombinant expression vector or the recombinant bacteria described in the above scheme into wheat.
[0016] The present invention provides the use of the TaPIL1 protein for regulating the trace element content of wheat grains. The amino acid sequence of the TaPIL1 protein is shown in SEQ ID NO. 1. Overexpression of the TaPIL1 protein in wheat plants can increase the trace element content of wheat grains. The results of the examples show that overexpression of TaPIL1 significantly increases the iron and zinc content in wheat grains, demonstrating the important application of TaPIL1 in increasing the iron and zinc content in wheat grains. This invention is of great significance for the cultivation of new high-quality wheat varieties and their widespread application in production. BRIEF DESCRIPTION OF THE DRAWINGS
[0017] 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.
[0018] Figure 1 Figure 1 is the result of PCR amplification of TaPIL1 gene;
[0019] Figure 2 This is a comparison chart of the sequencing results of the pUN1301-TaPIL1 recombinant vector;
[0020] Figure 3 This is a map of the overexpression vector pUN1301-TaPIL1;
[0021] Figure 4 The left picture shows the genetic transformation of the overexpression vector pUN1301-TaPIL1 and the growth of the T0 generation. The right picture shows the growth of the T0 generation plants. The scale in the figure is 5 cm.
[0022] Figure 5 Figure 1 is the result of PCR amplification of HptII gene in transgenic plants;
[0023] Figure 6 Figure 1 is the result of PCR amplification of TaPIL1 gene in transgenic plants;
[0024] Figure 7 The diagram shows the Fe and Zn contents in the grains of transgenic plants, where the left diagram shows the Fe content and the right diagram shows the Zn content. DETAILED DESCRIPTION
[0025] The present invention provides an application of TaPIL1 protein in regulating the content of trace elements in wheat grains. The amino acid sequence of the TaPIL1 protein is shown in SEQ ID NO.1: MSQFVPDWGNMGDISRPLG EDDDLMELLWCNGNVVMQSQGHRKLPPRPEKVPAPPVVQEDEAGLWFPFALADSLDKDIFTDLFCEEPPGVDAGKAGRDGAPVLGDADRRSSQSSAVSAASDLMPPPKSTHVSCSSRQQSMSLADCGDNAGGVLSDLVQARAGKAAMEEGASSTLSAMGASFCGSNQVQVQGAVSEQGRAGHTTAYGGSGAGSALPSAVGSGNANARGRGYEATVASSSGRSNYSFGVTATTAT TGTEPTSTSNRSSKRKRGLDTEDSESPSEDAESESSLALERKPPQKLTTARRSRAAEVHNLSERRRRDRINEKMRALQELIPHCNKTDKASMLDEAIEYLKTLQMQVQMMWMGSGMAP PAVMFPGMQMHQYLPQMGPPSMARMPFMAPPQQGHGVSLPEQYAHFLGVNPHHLQPPAHHHHHQHFAQGVGYYPLGAKALQQSPALHHVSNGNTGGGTPAATANATPGNAIHPNKR.
[0026] As an embodiment, the regulation includes increasing the expression of TaPIL1 protein to increase the content of trace elements in wheat grains. In a specific embodiment of the present invention, increasing the expression of TaPIL1 protein includes constructing a TaPIL1 overexpression vector and transferring it into wheat.
[0027]
[0028] As an embodiment, the trace elements include iron and / or zinc.
[0029] The present invention also provides the use of TaPIL1 protein in cultivating wheat germplasm with high trace element content in grains. The amino acid sequence of the TaPIL1 protein is shown in SEQ ID NO.1.
[0030] As an embodiment, the trace elements include iron and / or zinc.
[0031] The present invention also provides a recombinant expression vector comprising a gene encoding TaPIL1 protein, the amino acid sequence of which is shown in SEQ ID NO. 1. In a specific embodiment of the present invention, the base vector of the recombinant expression vector is the pUN1301 vector.
[0032] The present invention also provides a recombinant bacterium, wherein the recombinant bacterium comprises the recombinant expression vector described in the above embodiment. In a specific embodiment of the present invention, the base bacteria of the recombinant bacterium is Escherichia coli.
[0033] The present invention also provides the use of the recombinant expression vector or the recombinant bacteria described in the above scheme in cultivating high-iron wheat and / or high-zinc wheat.
[0034] The present invention also provides a method for increasing the iron and / or zinc content of wheat grains, comprising: transferring the recombinant expression vector or the recombinant bacteria described in the above scheme into wheat. In a specific embodiment of the present invention, the recombinant bacteria is transferred into wheat embryos.
[0035] In order to further illustrate the present invention, the technical solution provided by the present invention is described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.
[0036] Example 1 Construction of TaPIL1 overexpression vector
[0037] According to the map of the pUN1301 vector, the upstream primer TaPIL1-F and the downstream primer TaPIL1-R of TaPIL1 were designed. The primer sequences are shown below:
[0038] TaPIL1-F (SEQ ID NO.3): 5′-CGACGATAAG GGTACC ATGAGCCAATTC GTGCCA-3′; the underlined part is the KpnI recognition site;
[0039] TaPIL1-R (SEQ ID NO.4): 5′-TAGAGTCGAC GGATCCTCTTTTGTTTGG GTGTAT-3′; the underlined part is the BamHI recognition site.
[0040] The cDNA of Zhengmai 7698 was used as a template and the above primers were used for PCR amplification. The PCR system contained: 25 μL KOD OneTM PCRMasterMix (2×), 0.3 μL of upstream and downstream primers (10 μM), 200 ng cDNA template, and ddH2O was added to a total volume of 50 μL. The PCR reaction procedure was as follows: the first step was 94°C for 3 min; the second step was 98°C for 10 s, 59°C for 30 s, and 68°C for 3 min, for 32 cycles; the third step was 68°C for 5 min. After the amplification, agarose gel electrophoresis was performed to detect the size of the amplified band. A total of 8 groups of PCR amplifications were performed, and the results were as follows. Figure 1 .according to Figure 1 It can be seen that the size of the amplified fragment is consistent with the size of the target gene TaPIL1 (1461 bp), indicating that the PCR amplification was successful.
[0041] (2) A single 1461 bp target fragment of the amplified band was recovered using a gel recovery kit (purchased from Tiangen Company) to obtain the TaPIL1 fragment. At the same time, the vector pUN1301 was double-digested with KpnI and BamHI. The specific process was as follows: 10 μL (1 mg) of the vector plasmid, 5 μL of 10× enzyme digestion buffer, 1 μL of Kpn I, and 1.0 μL of BamHI were taken, and ddH2O was added to supplement the reaction system to 50 μL. The enzyme digestion was carried out at 37°C for 2 h. The enzyme digestion products were separated by agarose gel electrophoresis, and the large fragment obtained after the double enzyme digestion of the vector pUN1301 was recovered to obtain the linearized vector pUN1301.
[0042] The linearized vector pUN1301 and the PCR-amplified target fragment of TaPIL1 were then ligated using homologous recombination. The specific process was as follows: the linearized vector pUN1301 and TaPIL1 fragment were diluted to 10 ng / μL and 30 ng / μL, respectively, and then 2.0 μL of the vector and 6.0 μL of the TaPIL1 fragment were added. Then, 0.75 μL of homologous recombination ligase and 1.25 μL of 10× ligase buffer were added. The mixture was mixed, centrifuged, and ligated at 16°C for 1.5 hours. The ligation product was then transformed into E. coli DH5α competent cells for transformation. Three or more positive clones were randomly selected for expansion culture, and plasmids were extracted using the Tiangen plasmid extraction kit. The universal vector primer Ubi-F (SEQ ID NO. 7: 5′-TTTAGCCCTGCCTTCATAC-3′) was used for sequencing and additional testing. The sequencing results were spliced and sequenced using DNAMAN software (http: / / www.lynnon.com).
[0043] The sequence comparison results are as follows Figure 2 As shown, the TaPIL1-F sequence is the template reference sequence, and the TaPIL1-1 monoclonal splicing sequence is one of them. The results show that compared with the reference sequence, the monoclonal TaPIL1-1 nucleotide sequence has 99.9% similarity. This indicates that the overexpression vector pUN1301-TaPIL1 has been successfully constructed. The map of the overexpression vector pUN1301-TaPIL1 is shown in Figure 3 As shown, it contains the Ubquitin promoter, the pUN1301 gene, and Nos (Agrobacterium nopaline synthase terminator).
[0044] Example 2
[0045] Obtaining and identifying TaPIL1 transgenic wheat plants
[0046] (1) Obtaining TaPIL1 transgenic wheat plants
[0047] The gene gun method was used to transform wheat embryos. The specific process is as follows: First, young wheat ears were selected after anthesis and cultured in hydroponics at 4°C for about one week. The wheat seeds were separated and sterilized. The embryos were then removed using sterilized tweezers and a scalpel and placed in a culture dish for later use. At the same time, the overexpression vector pUN1301-TaPIL1 prepared in Example 1 was bombarded into the immature embryos of wheat using a gene gun; the culturing process of the immature embryos of wheat after bombardment was as follows: cultured in the dark at 25°C on N6 induction medium (purchased from Beijing Coolbo Technology Co., Ltd., article number: PM1311); then the immature embryos that produced callus tissue were transferred to differentiation medium (purchased from Beijing Coolbo Technology Co., Ltd., article number: PM10321) and cultured in a growth chamber at 25°C, 12h light / 12h dark; then, the recipient material that produced adventitious buds was transferred to screening medium (purchased from Beijing Coolbo Technology Co., Ltd., article number: PM1060) and cultured in a growth chamber at 25°C, 12h light / 12h dark, allowing the recipient material to take root and screen positive seedlings, and the screened positive seedlings were transferred to a cold storage, and after 14 days of vernalization culture, they were transferred to soil for culture (reference [Liu Huiyun, 2017, Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Master's thesis]). The growth process of the T0 generation is as follows Figure 4 shown.
[0048] (2) Identification of TaPIL1 transgenic wheat plants
[0049] DNA was extracted from leaves of each T0 transgenic seedling and wild-type plant using the CTAB method (Li et al. 2018). Hygromycin primers for the selection marker gene and primers specific to the overexpression vector were used for identification and detection. For the overexpression plants, the HptII gene was first detected using the selection marker gene hygromycin primers HptⅡ-F (SEQ ID NO. 5: 5′-CACGGCCTCCAGAAGAAGAT-3′) and HptⅡ-R (SEQ ID NO. 6: 5′-CCTGCCTGAAACCGAACTGC-3′). PCR was then performed on the transgenic plants to obtain positive plants screened for HptII. Then, PCR was performed using the vector primer Ubi-F (SEQ ID NO. 7: 5′-TTTAGCCCTGCCTTCATAC-3′) and the TaPIL1 gene's own R primer R (SEQ ID NO. 8: 5′-ATGTCCTTGTCGAGCGAGTC-3′). The specific procedures are as follows:
[0050] PCR detection uses the DNA of the overexpressed transgenic plant as a template and the wild type of Zhengmai 7698 as a control to amplify the marker gene. The amplified product is subjected to agarose gel electrophoresis to observe whether there is a marker gene band. The test results are as follows Figure 5 As shown, Figure 5 From left to right: Marker: DL2000; WT: Zhengmai 7698; OE1~OE7: TaPIL1 transgenic lines 1~7. Figure 5 It can be seen that, except for the wild type (WT), all other tested plants contain the hygromycin gene, indicating that these strains carry the selection marker gene of the overexpression vector.
[0051] On this basis, the target gene was amplified in the above plants using the forward primers Ubi-F (SEQ ID NO.7) and R (SEQ ID NO.8) upstream of the pUN1301 vector. Figure 6 As shown, Figure 6 From left to right: Marker: DL2000; WT: Zhengmai 7698; OE1~OE7: TaPIL1 transgenic lines 1~7. Figure 6 It can be seen that compared with the control plants, the transgenic lines 1 to 7 all detected bands representing the combination of the target gene and the vector, indicating that the TaPIL1 gene was overexpressed in these transgenic wheat lines.
[0052] Example 3
[0053] Effects of TaPIL1 gene on iron and zinc contents in wheat grains
[0054] Transgenic lines 1 to 3 constructed in Example 2 were planted individually, and the iron content in their T3 generation mature grains was determined at the harvest period. The method is as follows: When determining the iron content in wheat grains, wheat grains with full grain filling were selected for grinding and passed through a 100-mesh sieve. The flour obtained was the sample to be tested. Subsequently, 0.1 g of sample was weighed and placed at the bottom of a dry digestion tube and digested in 8 mL of a mixed acid solution (HNO3:H2O2=3:1, v / v) for 16 h. Subsequently, these sample mixtures were gradually heated to 180°C for 1 to 2 h until the digestion mixture became clear, and then cooled to 25°C. The digestion solution sample was diluted to 50 μM with nitric acid (1%, v / v) solution, and the Fe and Zn contents were determined after filtration. The Fe and Zn contents in the digestion solution were determined by inductively coupled plasma optical emission spectroscopy (ICP-OES, Hangzhou Xuhui Technology Co., Ltd., China). The results are shown in Tables 1 and Figure 7 As shown, Figure 7 WT Wei Zhengmai 7698; OE1 to OE7 are 7 lines transformed with TaPIL1 gene. Figure 7It can be seen that the iron content in mature grains increased by 181.97% to 525.00% compared with the wild type (WT), and the zinc content increased by 14.97% to 123.47% compared with the wild type (WT). After overexpression of TaPIL1, the iron and zinc content in the grains was significantly increased, indicating that TaPIL1 has important applications in increasing the iron and zinc content in wheat grains.
[0055] Table 1 Fe and Zn contents in grains of transgenic lines
[0056]
[0057]
[0058] In summary, after overexpression of TaPIL1 in wheat, the iron and zinc content in the grains was extremely significantly increased, indicating that TaPIL1 has important applications in increasing the iron and zinc content in wheat grains and can be used for the subsequent cultivation of new wheat varieties with high trace elements in the grains.
[0059] 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 TaPIL1 protein in regulating the content of trace elements in wheat grains, the amino acid sequence of the TaPIL1 protein is shown in SEQ ID NO.
1.
2. The use according to claim 1, characterized in that The regulation includes increasing the expression level of TaPIL1 protein and increasing the content of trace elements in wheat grains.
3. The use according to claim 1, characterized in that The nucleotide sequence of the gene encoding the TaPIL1 protein is shown in SEQ ID NO.
2.
4. The use according to any one of claims 1 to 3, characterized in that The trace elements include iron and / or zinc.
5. Application of TaPIL1 protein in cultivating wheat germplasm with high trace element content in grains, wherein the amino acid sequence of the TaPIL1 protein is shown in SEQ ID NO.
1.
6. The use according to claim 5, characterized in that The trace elements include iron and / or zinc.
7. A recombinant expression vector, characterized in that: The recombinant expression vector contains a gene encoding TaPIL1 protein; the amino acid sequence of the TaPIL1 protein is shown in SEQ ID NO.
1.
8. A recombinant bacterium, characterized in that The recombinant bacteria comprises the recombinant expression vector according to claim 7.
9. Use of the recombinant expression vector according to claim 7 or the recombinant bacteria according to claim 8 in cultivating high-iron wheat and / or high-zinc wheat.
10. A method for increasing the iron and / or zinc content of wheat grains, characterized in that: include: The recombinant expression vector according to claim 7 or the recombinant bacteria according to claim 8 is transformed into wheat.