Application of rice metal tolerance protein coding gene osmtp7

Abstract

The application discloses application of a rice metal tolerance protein coding gene OsMTP7 The rice metal tolerance protein coding gene OsMTP7 has the accession number of AK241225.1 in Genbank. The gene coding protein has zinc transport activity, can increase the zinc content of rice root system and above-ground part, and significantly increases the accumulation of zinc in rice grains.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology and relates to the application of the rice metal tolerance protein encoding gene OsMTP7. Background Technology

[0002] Zinc (Zn) is an essential micronutrient element for living organisms, often referred to as the "flower of life." It plays a vital role in human growth and development, intellectual development, immune function, reproductive function, digestive function, and metabolic function. It participates in the formation of over 200 enzymes and protein synthesis, exerting a wide range of physiological regulatory effects on human life and health (Yin Yueqin 2022). The human body cannot synthesize zinc and must obtain it from the diet. Zinc is mainly found in seafood and animal organs; other foods contain very little zinc, especially staple foods such as rice, noodles, and bread, as well as milk and eggs. Zinc deficiency is a risk factor for disease, affecting approximately one-third of the world's population. The zinc content in food is crucial for human health. Zinc deficiency is also a serious agricultural problem, as approximately half of the world's grain-growing soils have low zinc content. Rice is one of the most important food crops, feeding more than half of the world's population. Zinc deficiency can lead to a significant decline in rice yield and nutritional quality, and the zinc nutritional status of the population is also concerning. Reports indicate that approximately 31% of the global population suffers from zinc deficiency (Wessells et al., 2012). Incomplete statistics suggest that about four-fifths of my country's population has insufficient zinc intake, classifying it as zinc malnutrition. Statistics from the Chinese Academy of Preventive Medicine and other institutions found that approximately 60% of children are zinc deficient (Chen Xuecun et al., 1995). Recent reports indicate that zinc deficiency can lead to weakened immune function, lethargy, loss of appetite, delayed sexual development, disordered glucose metabolism, iron-deficiency anemia, and other diseases (Cao Jiqiong et al., 2014). Zinc deficiency in pregnant women can lead to premature birth or even miscarriage, and can also cause fetal malformations. Zinc deficiency in newborns can result in poor brain development and growth retardation. Therefore, pregnant women and newborns, as sensitive groups, especially need zinc supplementation. Since the human body cannot synthesize zinc, food is the only way to obtain it, and the absorption efficiency is only 20% (Kristensen et al., 2006). Therefore, for the sake of health, it is necessary to supplement zinc appropriately to avoid "hidden hunger." Therefore, rice-based biofortification is particularly important. By using appropriate biotechnology and genetic improvement, cultivating rice varieties that can tolerate zinc-deficient soil and increase the zinc content in rice can alleviate the widespread zinc deficiency problem in humans and is of great significance to human nutrition. Summary of the Invention

[0003] To address the shortcomings of existing technologies, the purpose of this invention is to provide the application of the rice metal tolerance protein encoding gene OsMTP7 in increasing the zinc content of rice.

[0004] The present invention provides the rice metal tolerance protein encoding gene OsMTP7, which has accession number AK241225.1 in GenBank and its sequence is shown in SEQ ID NO.1. Based on this, the technical solution protected by the present invention is as follows:

[0005] In a first aspect, the present invention protects the application of the rice metal tolerance protein encoding gene OsMTP7 in increasing the zinc content of rice, the sequence of which is shown in SEQ ID NO.1.

[0006] Secondly, the present invention protects the application of the rice metal tolerance protein encoding gene OsMTP7 in the preparation of products that increase the zinc content of rice, the sequence of which is shown in SEQ ID NO.1.

[0007] Thirdly, the present invention protects the application of the rice metal tolerance protein encoding gene OsMTP7 in the cultivation of rice with high zinc content, the sequence of which is shown in SEQ ID NO.1.

[0008] Fourthly, this invention protects the application of the rice metal tolerance protein encoding gene OsMTP7 in the preparation of rice products with high zinc content, the sequence of which is shown in SEQ ID NO.1.

[0009] In a specific implementation, the application is achieved by inhibiting / reducing / silencing the expression level of the rice metal tolerance protein encoding gene OsMTP7, or by inhibiting / reducing / silencing the activity and / or content of rice metal tolerance protein.

[0010] In a more specific implementation, the application is achieved by knocking out the rice metal tolerance protein encoding gene OsMTP7 using a CRISPR-CAS9 vector or a CRISPR-CAS9 vector composition.

[0011] This invention utilizes the CRISPR-CAS9 system to edit the metal tolerance protein encoding gene OsMTP7. Studies have shown that silencing the metal tolerance protein encoding gene OsMTP7 using the CRISPR-CAS9 system can significantly increase the absorption of zinc by rice roots and significantly increase the accumulation of zinc in the aboveground parts and grains of rice.

[0012] Fifthly, the present invention protects a method for increasing the zinc content of rice, said method being achieved by inhibiting / reducing / silencing the expression level of the rice metal tolerance protein encoding gene OsMTP7, or by inhibiting / reducing / silencing the activity and / or content of the rice metal tolerance protein.

[0013] In a specific implementation, the method is achieved by knocking out the rice metal tolerance protein encoding gene OsMTP7 using a CRISPR-CAS9 vector or a CRISPR-CAS9 vector composition.

[0014] Sixthly, the present invention protects the application of substances that knock out or silence the rice metal tolerance protein encoding gene OsMTP7 in increasing the zinc content of rice, wherein the substances that knock out or silence the rice metal tolerance protein encoding gene OsMTP7 are CRISPR-CAS9 vectors or CRISPR-CAS9 vector compositions targeting the rice metal tolerance protein encoding gene OsMTP7.

[0015] Beneficial effects of the present invention

[0016] This invention is the first to discover that knocking out the metal tolerance protein encoding gene OsMTP7 significantly increases the zinc content in rice roots and aboveground parts. Figure 3, Figure 4 ), significantly increasing the accumulation of zinc in rice grains ( Figure 5 ). Attached Figure Description

[0017] Figure 1 The ability of OsMTP7 to transport Zn was demonstrated after heterologous expression in the yeast mutant Zrc1. Vector represents yeast strain transformed with an empty vector as a negative control, and OsMTP7 represents a yeast strain heterologously expressing OsMTP7.

[0018] Figure 2 The gene editing types of the two mutant lines of the OsMTP7 gene knockout mutant are identified.

[0019] Figure 3 is Compared with the wild type, the Zn content in the roots and aboveground parts of the OsMTP7 knockout material was significantly increased under hydroponic conditions.

[0020] Figure 4 Compared with the wild type, the Zn content in the seeds of OsMTP7 knockout material was significantly increased under pot conditions.

[0021] Figure 5 Compared with the wild type, the Zn content in the grains of OsMTP7 knockout materials was significantly increased under field conditions in Hainan. Detailed Implementation

[0022] The following embodiments are used to illustrate the present invention, but are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0023] Example 1: Construction of the yeast expression vector for the OsMTP7 gene and verification of Zn transport activity

[0024] Using cDNA from wild-type rice variety Zhonghua 11 as a template, the full-length coding region sequence of OsMTP7 was amplified using GXL high-fidelity enzyme. The amplified fragment was inserted between the HindIII and EcoRI sites on pYES2.0 (Invitrogen) using a single-fragment recombinase (ClonExpress II One Step Cloning Kit, Vazyme, Nanjing). After sequencing verification, the constructed plasmid or empty vector (pYES2.0) was transformed into yeast (Saccharomyces cerevisiae) via LiAc-PEG transformation. The zinc-sensitive yeast strain ΔZrc1 was used to detect Zn transport activity. To assess Zn tolerance, transformed yeast cells were pre-cultured in synthetic SD-U medium containing 2% glucose, 0.67% amino acid-free and phosphate-free yeast nitrate (YNB), 2% agar, and 0.076% SD-U (Takara) at 30°C and 200 rpm for approximately 12 hours (to the logarithmic growth phase). Pre-cultured yeast cells were centrifuged, washed three times with sterile water, diluted, and titrated onto galactose SD-U plates containing 0, 0.3, 0.4, and 0.5 mM ZnSO4. 2.5 μl of the yeast cell suspension dilutions (OD600: 0.2, 0.02, 0.002, and 0.0002) were carefully titrated onto each SD-U plate and incubated at 30°C in the dark for 4 days. In both phosphorus-free and low-phosphorus media, the addition of Zn significantly inhibited the growth of yeast cells expressing OsMTP7. Figure 1 A). Yeast cells in the logarithmic growth phase were transferred to liquid SD-U medium (galactose as the carbon source) with an initial OD600 of 0.2 and cultured at 30°C and 200 rpm for 12 h. 200 μM ZnSO4 was added, and the cells were cultured for another 2 h. The cells were harvested by centrifugation at 4000 rpm and 4°C, washed three times with 10 μM EDTA (pH 5.0) at 4°C, then washed three times with ddH2O, lyophilized, and digested with 5 mL of analytical grade concentrated HNO3 using a microwave digester. Heavy metal content was determined by ICP-MS. Compared with the empty vector control, OsMTP7 expression reduced Zn accumulation in yeast cells by 90.5%. Figure 1 B).

[0025] The results of this embodiment indicate that heterologous expression of rice OsMTP7 in yeast has Zn transport activity. Figure 1 ).

[0026] Example 2 Construction of rice OsMTP7 gene knockout mutant

[0027] A knockout vector for the OsMTP7 gene was constructed using the CRISPR / Cas9 expression system in the getaway architecture. First, specific target sequences for OsMTP7 gene knockout were designed using TargetDesign (http: / / skl.scau.edu.cn / targetdesign / ). Considering both the lowest off-target probability and the highest specificity of the candidate sequences, the first exon of the OsMTP7 (LOC_Os04g23180) coding region was selected as the candidate region for mutation. The adapter primers for the target sequence were designed as follows:

[0028] OsU6a-MTP7-F:gccgAGCGGTTAACACAGCACTG;

[0029] OsU6a-MTP7-R:aaacCAGTGCTTGTGTTAACCGCT;

[0030] First, the artificially synthesized target adapter sequences were mixed at a 1:1 ratio and annealed at 95°C for 5 min to form double-stranded DNA. The sgRNA expression cassette vector pOsU3-sgRNA was digested with BsaI, and then the target adapter sequences and linearized pOsU3-sgRNA were ligated overnight at 4°C using NEB T4 ligase. The OsMTP7 knockout expression cassette vector, verified by transformation and sequencing, was named pOsU3-sgRNA-OsMTP7. The successfully constructed knockout expression cassette's introductory vector and the final vector pOs-Cas9 were used to construct a plant expression vector via an LR reaction. The constructed plant expression vector was transformed into callus tissue of Zhonghua 11 via Agrobacterium-mediated transformation. Positive transgenic seedlings were obtained through hygromycin selection, genomic DNA was extracted, and PCR amplification was performed using primers at both ends of the target site. Sequencing verification yielded two homozygous OsMTP7 knockout mutant lines. Figure 2 ).

[0031] Example 3: Comparison of total Zn accumulation in the aboveground parts and roots of osmtp7 mutant material and wild-type rice.

[0032] The specific implementation process is as follows;

[0033] 1) Soak wild-type rice seeds and osmtp7 mutant seeds at 37℃ for 48 hours, rinse them with tap water, and then sow them on a suspended plastic black net. Cultivate them in 0.5mM CaCl2 solution for 1 week, changing the solution every three days.

[0034] 2) Wild-type rice seedlings and osmtp7 mutant seedlings were transferred to 4L black plastic containers for culture, and cultured with 1 / 2 Kimura B (pH 5.5) for two weeks. Four wild-type and four osmtp7 mutant seedlings were planted in each 4L black container, with a total of three containers serving as three biological replicates.

[0035] 3) Collect samples of the aboveground parts and roots, wash them three times with deionized water to remove adsorbed ions and impurities from the surface, dry them in a 65℃ oven for 3 days, and then digest them.

[0036] 4) Weigh all the above-ground or root samples and place them in a graphite digestion tube. Add 5 mL of mixed acid solution (85%:15%, V / V, superior grade nitric acid:superior grade perchloric acid) and place the tube in a graphite digestion oven for digestion. The temperature control conditions are: 60℃ for 1 hour, 90℃ for 1 hour, 120℃ for 1 hour, and 150℃ for 1 hour. Then, completely remove the remaining acid from the tube at 190℃, and dilute to 25 mL with 2% nitric acid solution. Mix well and transfer to a 50 mL plastic centrifuge tube.

[0037] 5) After determining the Zn content in the sample solution using inductively coupled plasma mass spectrometry (ICP-MS), the total Zn content in the aboveground parts and roots of wild-type and mutant rice was finally calculated according to the sample amount and dilution factor. The results showed that loss of function of the rice OsMTP7 gene significantly increased Zn accumulation in both roots and aboveground parts under normal hydroponic conditions. Figure 3) .

[0038] The results of this embodiment show that, compared with the wild type, the osmtp7 mutant material accumulates more Zn in the roots and aboveground parts, by 19-24% and 29-32%, respectively. Figure 3) .

[0039] Example 4: Comparison of Zn accumulation in grains of osmtp7 mutant material and wild-type rice under pot cultivation conditions.

[0040] The specific implementation process is as follows:

[0041] 1) osmtp7 mutant material and background wild-type Nipponbare. Germinated for 2 weeks using 1 / 2 MS.

[0042] 2) Transfer the seedlings to a 5L plastic bucket and culture them in 1 / 2 Kimura nutrient solution for 1 week.

[0043] 3) Select osmtp7 mutant and wild-type (Zhonghua 11) seedlings of the same size and plant them in plastic pots filled with 10kg of paddy field soil for soil cultivation. Plant one wild-type and two mutant lines in each pot and set up four pot replicates.

[0044] 4) Harvest mature rice and dry it in a 70℃ oven for 2 days.

[0045] 5) Weigh about 0.2g of dehulled seeds into a digestion tube, add 5mL of high-purity nitric acid and microwave digest. After completion, place the tube at 160℃ to remove residual acid.

[0046] 6) Dilute the sample digest to 10 mL with 2% HNO3, shake well and filter (0.45 μm).

[0047] 7) The Zn content in each sample was determined using ICP-MS.

[0048] The results of this example show that, under pot cultivation conditions, the Zn content in the grains of the OsMTP7 mutant material was significantly increased by approximately 21-23% compared to the wild type. Figure 4 ).

[0049] Example 5: Comparison of Zn accumulation in grains of osmtp7 mutant material and wild-type rice under field conditions.

[0050] The comparison of total Zn accumulation in grains of wild-type rice and osmtp7 mutant materials in field experiments in Hainan Province was conducted as follows:

[0051] 1) Sow seeds of wild-type rice and the osmtp7 mutant in a seedbed and raise seedlings. When the seedlings have grown to three or four leaves, transplant them into paddy fields (experimental field in Lingshui, Hainan). Plant one seedling per hill, with a spacing of 20cm between seedlings and 25cm between rows. During this period, manage and control water and nutrients normally, and prevent and control pests and diseases.

[0052] 2) After the rice matures, select 10 rice plants in the middle of each row to harvest the rice grains, and take 4 individual plants from each line to determine the Zn content.

[0053] 3) Determination of total Zn content in rice grains: Weigh 0.25g of dried grains into a microwave digestion tube, add 5mL of analytical grade nitric acid for microwave digestion, and then place at 160℃ to remove residual acid. After acid removal, dilute to 10mL with 2% nitric acid and transfer to a 15mL centrifuge tube for storage. Determine the Zn content in the grain sample using ICP-MS.

[0054] The results of this example show that, under field conditions in Hainan, the Zn content in the grains of the OsMTP7 mutant material was significantly increased by approximately 25-28% compared to the wild type. Figure 5 ).

[0055] The scope of protection of this invention is not limited to the above embodiments. Variations and advantages that can be conceived by those skilled in the art without departing from the spirit and scope of the inventive concept are included in this invention and are protected by the appended claims.

Claims

1. Suppress / reduce / silence genes encoding rice metal tolerance proteins OsMTP7 The application of [expression] in increasing zinc content in rice is characterized by, The rice metal tolerance protein encoding gene OsMTP7 The sequence is shown in SEQ ID NO.1, and the rice variety is Zhonghua 11.

2. Suppress / reduce / silence genes encoding rice metal tolerance proteins OsMTP7 The application of the expression of [a specific ingredient] in the preparation of products that increase the zinc content of rice is characterized by [a specific feature]. The rice metal tolerance protein encoding gene OsMTP7 The sequence is shown in SEQ ID NO.1, and the rice variety is Zhonghua 11.

3. Suppress / reduce / silence genes encoding rice metal tolerance proteins OsMTP7 The application of the expression of [a specific ingredient] in the cultivation of high-zinc-content rice is characterized by [a specific feature]. The rice metal tolerance protein encoding gene OsMTP7 The sequence is shown in SEQ ID NO.1, and the rice variety is Zhonghua 11.

4. Suppress / reduce / silence genes encoding rice metal tolerance proteins OsMTP7 The application of the expression of [a specific ingredient] in the preparation of rice products with high zinc content is characterized by [a specific characteristic]. The rice metal tolerance protein encoding gene OsMTP7 The sequence is shown in SEQ ID NO.1, and the rice variety is Zhonghua 11.

5. The application according to any one of claims 1-4, characterized in that, The application is through CRISPR-CAS9 carrier or CRISPR-CAS9 The vector composition knocks out the rice metal tolerance protein encoding gene. OsMTP7 To achieve this.

6. A method for increasing the zinc content of rice grains, characterized in that, The method works by inhibiting / reducing / silencing the gene encoding rice metal tolerance protein. OsMTP7 The expression level is achieved by the rice metal tolerance protein encoding gene. OsMTP7 The sequence is shown in SEQ ID NO.1, and the rice variety is Zhonghua 11.

7. The method according to claim 6, characterized in that, The method is achieved by CRISPR-CAS9 carrier or CRISPR- CAS9 The vector composition knocks out the rice metal tolerance protein encoding gene. OsMTP7 To achieve this.

8. Knockout or silencing of genes encoding rice metal tolerance proteins OsMTP7 The application of a substance in increasing the zinc content of rice is characterized by, The knockout or silencing of the rice metal tolerance protein encoding gene OsMTP7 The substance is a gene encoding a metal tolerance protein in rice. OsMTP7 of CRISPR-CAS9 carrier or CRISPR-CAS9 Vector composition, wherein the rice metal tolerance protein encoding gene OsMTP7 The sequence is shown in SEQ ID NO.1, and the rice variety is Zhonghua 11.

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