Application of the rice lead transporter gene OsNRAMP5 in reducing lead absorption in rice

Abstract

This invention discloses the application of the rice lead transporter gene OsNRAMP5 in reducing lead uptake in rice. Extensive experiments demonstrate that CRISPR / Cas9 editing to knock out the rice OsNRAMP5 gene significantly reduces Pb uptake by roots and accumulation in the aboveground parts. Under hydroponic conditions, increasing the Mn concentration in the nutrient solution significantly inhibits Pb uptake by rice roots. Furthermore, when rice is planted in Pb-contaminated paddy soil, the OsNRAMP5 knockout mutant shows a significant reduction in Pb accumulation in both grains and straw.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology and relates to the application of the rice cadmium-manganese transporter gene OsNRAMP5 in Pb control. Background Technology

[0002] Lead is one of the most toxic heavy metals and is widely distributed in the environment. It has been listed by the World Health Organization (WHO) as one of the top ten pollutants affecting public health (Nriagu, 1998; Kopittke et al., 2012). Excessive Pb exposure can cause a series of adverse effects on human health (EFSA, 2012). Furthermore, Pb is also a neurotoxin; excessive exposure can lead to cognitive developmental delays and intellectual decline in children. Food is one of the main sources of Pb exposure in humans, with rice contributing significantly to dietary Pb intake (Zhang et al., 2021). Rice grown in uncontaminated soil typically has low Pb content, but soil pollution caused by human activities (such as mining, metal smelting, and atmospheric deposition) increases the accumulation of Pb in rice grains. The mechanism of Pb absorption by plant roots is still unclear. Researching the molecular mechanism of Pb absorption by rice roots is crucial for cultivating superior rice varieties with low lead and cadmium accumulation and improving agricultural product safety. Previous studies have shown that OsNRAMP5 is the main transport protein for the absorption of manganese (Mn) and cadmium (Cd) in rice. However, it is unclear whether OsNRAMP5 also transports Pb. (Nriagu, JOTales told inlead. Science 1998, 281(5383), 1622-1623.)

[0003] Kopittke,PM; Blamey,FPC;Asher,CJ;Menzies,NWTrace metalphytotoxicity in solution culture:a review.J.Exp.Bot.2010,61(4),945-954.

[0004] EFSA. Scientific report of EFSA: Lead dietary exposure in the European population.

[0005] EFSA J. 2012, 10(7), 2831.

[0006] Zhang, X.; Wang, Z.; Liu, L.; Zhan, N.; Qin, J.; Lu, X.; Cheng, M. Assessment of the risks from dietary lead exposure in China. Summary of the Invention

[0007] The purpose of this invention is to provide the application of rice cadmium-manganese transporter gene in Pb blocking.

[0008] The objective of this invention can be achieved through the following technical solutions:

[0009] OsNRAMP5 was heterologously expressed in yeast, and its CDS sequence is shown in SEQ ID NO.1.

[0010] The application of the rice lead transporter gene OsNRAMP5 in reducing the Pb content of rice grains, as described in this invention, is characterized by inhibiting, silencing, or knocking out the rice lead transporter gene OsNRAMP5, thereby reducing the lead content in rice grains.

[0011] Application of substances that inhibit, silence, or knock out the rice lead transporter gene OsNRAMP5 in reducing Pb content in rice grains.

[0012] The knockout of the rice lead transporter gene OsNRAMP5 causes premature termination of its translation and complete loss of function.

[0013] As a preferred embodiment of the present invention, the substance used to inhibit, silence, or knock out the rice lead transporter gene OsNRAMP5 is selected from siRNA and gene editing systems.

[0014] As a further preferred embodiment of the present invention, the substance used to inhibit, silence, or knock out the rice lead transporter gene OsNRAMP5 according to claim 1 is selected from a CRISPR / Cas9 expression system for knocking out OsNRAMP5. The application of OsNRAMP5 knockout described in this invention in reducing Pb content in rice roots, aboveground parts, and grains. The application of the knockout expression vector described in this invention in reducing Pb content in rice grains.

[0015] Beneficial effects of the present invention

[0016] 1. Through systematic research, this invention has for the first time verified that OsNRAMP5 has Pb transport activity and plays an important role in Pb absorption in rice roots.

[0017] 2. Knocking out OsNRAMP5 in rice significantly reduced Pb accumulation in rice roots and aboveground parts under hydroponic conditions. Figure 2 , Figure 3 , Figure 5 ).

[0018] 3. Knocking out OsNRAMP5 in rice significantly reduced the rice root system's ability to absorb Pb under hydroponic conditions. Figure 4 ).

[0019] 4. Under hydroponic conditions with different Mn concentrations, the absorption of Pb by rice can be reduced. Figure 6 ).

[0020] 5. Knocking out OsNRAMP5 significantly reduced the accumulation of Pb in rice straw and grains under Pb-contaminated paddy soil conditions. Figure 7 ). Attached Figure Description

[0021] Figure 1 To investigate the Pb transport activity of heterologously expressed OsNRAMP5 in yeast, we examined its expression. OsNRAMP5 expression increased yeast sensitivity to Pb and increased Pb accumulation in yeast cells. OsNRAMP5 expression increased Pb uptake in yeast cells by 41% and 66% in low-phosphorus and phosphorus-free media, respectively.

[0022] Figure 2 A schematic diagram of the OsNRAMP5 gene structure and the DNA sequence of the OsNRAMP5 gene edited by CRISPR / Cas9.

[0023] Figure 3 Knocking out OsNRAMP5 significantly reduced Pb accumulation in both roots and shoots. In environments containing 0.5 or 1.0 μM lead... Treat with Kimura B nutrient solution (pH 5.0, phosphate-free) for 24 hours.

[0024] Figure 4 The graphs show the Pb uptake kinetics of the OsNRAMP5 knockout mutant and wild-type Pb, and the maximum Pb uptake rate (Vo) in the roots of the OsNRAMP5 knockout mutant. max The number of offspring has decreased by 70% compared to the wild type.

[0025] Figure 5 To investigate the elimination of Pb accumulation in roots and shoots after OsNRAMP5 knockout in nutrient solutions with different phosphorus conditions, 0, 10, 50, and 100 μM phosphate solutions were used, along with 0.5 μM Pb(NO3)2. Treat with Kimura B nutrient solution (pH 5.0) for 24 hours.

[0026] Figure 6 Knocking out OsNRAMP5 significantly reduced Pb accumulation in roots and shoots under different Mn concentrations. This was observed in samples containing 0, 0.5, 5, and 50 μM Mn along with 0.5 μM Pb(NO3)2. Treated in concentrated Kimura B nutrient solution (pH 5.0, phosphorus-free) for 3 days.

[0027] Figure 7 In a pot experiment on Pb-contaminated soil, the OsNRAMP5 knockout mutant showed a 50% and 70% reduction in Pb content in grains and straw, respectively, compared to the wild type. Detailed Implementation

[0028] 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.

[0029] Example 1

[0030] Using cDNA from wild-type rice variety Zhonghua 11 as a template, the full-length coding sequences of OsNRAMP5 and OsIRT1 were amplified using GXL high-fidelity enzyme. The amplified fragments were then inserted between the HindIII and BamHI 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. Wild-type yeast strain SEY6210 (MATa; leu2-3113; ura3-52; his3-Δ200; trp1-Δ901; suc2-Δ9; lys2-801) was used to detect Pb transport activity. To assess Pb 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) for approximately 12 hours (to the logarithmic growth phase) at 30°C and 200 rpm in a shaker. The pre-cultured yeast cells were centrifuged, washed three times with sterile water, diluted, and titrated onto SD-U plates containing 0, 10, 20, 30, and 50 μM Pb(NO3)2 galactose. 2.5 μL of the yeast cell suspension dilution (OD200) was carefully titrated onto each SD-U plate. 600Yeast cells were cultured at concentrations of 0.2, 0.02, 0.002, and 0.0002 μM Pb and incubated in a 30°C incubator in the dark for 4 days. In both phosphorus-free and low-phosphorus media, the addition of Pb significantly inhibited the growth of yeast cells expressing OsNRAMP5, with a more pronounced inhibitory effect in the phosphate-free medium. Yeast cells were exposed to 10 μM Pb for 4 hours or 12 hours in phosphate-free or low-phosphorus media, and the amount of Pb absorbed was measured. Compared with the empty vector control, OsNRAMP5 expression increased Pb absorption by 41% in phosphate-free medium, while in low-phosphorus medium, OsNRAMP5 expression increased Pb absorption by 66% (…). Figure 1 ).

[0031] Example 2

[0032] OsNRAMP5 knockout vectors were constructed using the CRISPR / Cas9 expression system in the getaway architecture. First, specific target sequences for OsNRAMP5 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 OsNRAMP5 (LOC_Os07g15370) coding region was selected as the candidate region for mutation. The adapter primers for the target sequence were designed as follows:

[0033] OsU3T1-NRAMP5-F:ggcaAGAGAGAGAGCAGTGAGAGA

[0034] OsU3T1-NRAMP5-R:aaacTCTCTCACTGCTCTCTCTCT

[0035] 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 OsNRAMP5 knockout expression cassette vector, verified by transformation and sequencing, was named pOsU3-sgRNA-NRAMP5T1. The successfully constructed knockout expression cassette's introductory vector and the final vector pOs-Cas9 were combined via an LR reaction to construct a plant expression vector. The constructed plant expression vector was then transformed into Zhonghua 11 callus tissue via Agrobacterium-mediated transformation. OsNRAMP5 knockout mutants were obtained through hygromycin selection.

[0036] Example 3

[0037] Wild-type (Zhonghua 11) and two OsNRAMP5 CRISPR knockout mutants (L1, L2) were grown in normal nutrient solution for 28 days and then used for Pb uptake experiments. Before treatment, seedlings were transferred to elution solution (0.5 mM CaCl2, 2 mM MMEs, pH 5.0) for 12 hours. The seedlings were then transferred to a solution containing 0.5 or 1.0 μM Pb(NO3)2. Treatment with Kimura B nutrient solution (pH 5.0, phosphate-free) for 24 hours resulted in Pb content of 74.1 mg / kg in wild-type roots and 12.8 mg / kg in aerial parts exposed to 0.5 μM Pb. -1 The root and shoot Pb contents of the knockout mutant L1 were 43.9 mg / kg and 3.7 mg / kg, respectively. -1 Compared to the wild type, the levels were reduced by 41% and 71%, respectively; the root and shoot Pb contents of the knockout mutant L2 were 54.8 and 3.4 mg / kg, respectively. -1 Compared with the wild type, the levels were reduced by 26% and 74%, respectively. Under conditions of exposure to 1.0 μM Pb, the Pb content in the roots and aboveground parts of the wild type was 80.2 and 18.1 mg / kg, respectively. -1 The root and shoot Pb contents of the L1 knockout mutant were 55.3 mg / kg and 4.3 mg / kg, respectively. -1 Compared with the wild type, the levels were reduced by 30% and 76%, respectively; the root and shoot Pb contents of the knockout mutant L2 were 56.0 and 6.1 mg / kg, respectively. -1 Compared to the wild type, the reduction was 30% and 66%, respectively.

[0038] Example 4

[0039] Absorption matrix solution formulation: 0.5mM CaCl2, 2mM MES, pH 5.0.

[0040] Rice seedlings (OsNRAMP5 CRISPR knockout mutants osnramp5-L1, L2 and wild-type Zhonghua 11) grown hydroponically for approximately 28 days were pretreated with an absorptive matrix solution for 12 hours. Pb treatment concentrations of 0, 0.2, 0.5, 1.0, 2.0, 5.0, and 7.0 μM Pb(NO3)2 were set at 25℃ and 4℃, with treatment times of 20 min. Immediately after treatment, the seedlings were transferred to a pre-cooled solution of 100 mM CaCl2 and 50 mM EDTA, washed three times with deionized water, and dried at 65℃ to constant weight. The maximum Pb uptake rate (VL) in the roots of the OsNRAMP5 knockout mutant was measured. max The number of offspring has decreased by 70% compared to the wild type.

[0041] Example 5

[0042] Wild-type (Zhonghua 11) and two OsNRAMP5 CRISPR knockout mutants (L1, L2) were grown in normal nutrient solution for 28 days. Prior to treatment, seedlings were transferred to an elution solution (0.5 mM CaCl2, 2 mM MES, pH 5.0) for 12 hours. The seedlings were then transferred to a solution containing 0, 10, 50, and 100 μM phosphate and 0.5 μM Pb(NO3)2. Treatment with concentrated Kimura B nutrient solution (pH 5.0) for 24 hours significantly reduced Pb accumulation in both wild-type and mutant individuals by increasing the phosphate concentration in the nutrient solution. Under low phosphorus conditions, knocking out OsNRAMP5 significantly reduced Pb concentrations in roots and shoots, while this difference was not significant under high phosphorus conditions.

[0043] Example 6

[0044] Wild-type (Zhonghua 11) and two OsNRAMP5 CRISPR knockout mutants (L1, L2) were grown in normal nutrient solution for 28 days. Before treatment, seedlings were transferred to an elution solution (0.5 mM CaCl2, 2 mM MES, pH 5.0) for 12 hours. The seedlings were then transferred to a solution containing 0, 0.5, 5, and 50 μM Mn and 0.5 μM Pb(NO3)2. Treatment with Kimura B nutrient solution (pH 5.0, phosphorus-free) for 3 days reduced Pb accumulation in the wild type. Increasing the Mn concentration in the nutrient solution reduced Pb accumulation in the wild type. Under all Mn concentration conditions, knocking out OsNRAMP5 significantly reduced Pb concentrations in roots and shoots.

[0045] Example 7

[0046] Two CRISPR / Cas9 knockout osnramp5 mutants and a wild-type (Zhonghua 11) were planted in plastic pots filled with 10 kg of Pb-contaminated paddy field soil. This Pb-contaminated soil was collected from a mining-contaminated paddy field in Guangdong Province, China, and contained 222 mg / kg Pb. -1 The total lead content, with available lead content of 1.62 mg / kg, was [data missing]. -1 Lead can be extracted; total manganese and phosphorus concentrations are 283 mg / kg. -1 and 920mg kg -1 The soil pH value was 5.03; the organic matter content was 22.1 g / kg. -1 After rice matured, the Pb content in grains and straw was measured, and it was found that the two OsNRAMP5 knockout mutants had 50% and 70% less Pb content in grains and straw compared with the wild type, respectively.

Claims

1. Rice lead transporter gene OsNRAMP5 Its application in reducing Pb content in rice grains is characterized by... Suppress, silence, or knock out the rice lead transporter gene. OsNRAMP5 This reduces the lead content in rice grains, and the rice lead transporter gene mentioned above... OsNRAMP5 The sequence is shown in SEQ ID NO.

1.

2. Suppress, silence, or knock out the rice lead transporter gene as described in claim 1. OsNRAMP5 The application of substances in reducing Pb content in rice grains, specifically the inhibition, silencing, or knockout of the rice lead transporter gene as described in claim 1. OsNRAMP5 The substance is selected from knockout OsNRAMP5 The CRISPR / Cas9 expression system.

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