CN111996211B - Application of OsFTIP7 gene and mutant thereof in relieving stress of metal oxide nanoparticles on rice - Google Patents

Abstract

The invention discloses application of an OsFTIP7 gene and a mutant thereof in relieving stress of metal oxide nanoparticles on rice, and relates to the technical field of crop physiology.

Description

Application of OsFTIP7 gene and mutant thereof in relieving stress of metal oxide nanoparticles on rice

Technical Field

The invention relates to the technical field of crop physiology, in particular to application of an OsFTIP7 gene and a mutant thereof in relieving stress of metal oxide nanoparticles on rice.

Background

With the rapid development of nanotechnology, it has become possible to produce nanomaterials of different types, sizes and shapes. Due to their unique physicochemical properties, nanoparticles have been widely used in various aspects of daily life, such as electronic communication, environmental protection, medical drug and agricultural production, and the like.

Copper oxide (CuO) nanoparticles, zinc oxide (ZnO) nanoparticles, and the like have been used in various fields as important metal oxide NPs. For example, ZnO nanoparticles are useful in cosmetics and sunscreens due to their strong ability to absorb ultraviolet rays; the ZnO nano-particles can enhance the toughness of the high polymer, so the ZnO nano-particles can be used for rubber industrial production. In addition, CuO nanoparticles have also been widely used in the production of semiconductors, gas sensors, and agricultural chemicals. However, excessive use of metal oxide nanoparticles, such as by agricultural activities such as pesticides and fertilizers into the soil environment, can have varying degrees of impact on the health of animals and plants. Solving the pollution caused by the excessive use of the metal oxide nano particles has important significance for guaranteeing the production and safety of food crops in China.

Oxidative stress caused by nanoparticles causes an increase in reactive oxygen species, thereby disturbing the redox system in the cell and causing oxidative damage to cellular macromolecules such as nucleic acids, proteins and lipids. The adverse effects of metal oxide nanoparticles on plant growth have been reported in several studies. For example: CuO nanoparticles can be transported from the root to the stem by xylem transport in maize plants; stress of 1.0mM CuO nanoparticles causes H₂O₂The sharp increase of the isocarboxide and the change of the activity of the antioxidant enzyme in the rice seedlings; 100mg/L of CuO nanoparticles can lead to O in Arabidopsis roots and leaves₂And H₂O₂(ii) accumulation of (d); this is achieved byIn addition, the ZnO nanoparticles can inhibit the growth of roots of different kinds of plants (such as rape, cucumber, corn and the like); ZnO nanoparticles cause cell death by increasing ROS accumulation; the ZnO nanoparticles of 800mg/kg in the soil can reduce the chlorophyll content of the corn seedlings; ZnO nanoparticles can also lead to changes in antioxidant activity and oxidative stress systems in rice plants; ZnO nanoparticles at concentrations of 100 and 200 μ M can increase H in wheat plants₂O₂Accumulation of (2).

Therefore, the alleviation of growth inhibition under stress of metal oxide nanoparticles is of great importance to the production of crops such as rice. At present, researches on how to solve the stress of the rice metal oxide nanoparticles are relatively few, and researches related to the rice specific genes responding to the stress of the metal oxide nanoparticles in the rice are not reported.

Disclosure of Invention

In order to solve and relieve the growth inhibition of the metal oxide nanoparticles on rice, the invention provides application of an OsFTIP7 gene and a mutant thereof in relieving the stress of the metal oxide nanoparticles on rice.

In order to realize the technical purpose of the invention, the invention provides the application of the OsFTIP7 gene and the mutant thereof in relieving the stress of the metal oxide nanoparticles on rice.

Wherein, the OsFTIP7 gene has the accession number of AK058522 in the Genbank of NCBI.

In order to achieve the technical purpose, the invention further provides application of the rice Osftip7 mutant obtained by editing the OsFTIP7 gene in relieving rice stress caused by metal oxide nanoparticles.

The rice Osftip7 mutant is obtained by editing an OsFTIP7 gene by using a CRISPR/Cas9 gene editing technology to mutate the OsFTIP7 gene, so that a rice plant of OsFTIP7 protein with a loss-function structural domain is obtained.

Specifically, the description of the functional domain of OsFTIP7 protein is described in the following documents: shiyong Song, Ying Chen, Lu Liu, Yen How Benjamin See, Chuanzao Mao, Yinbo Gan and Hao Yu, OsFTIP7 standards auxin-medium depth in rice, Nature Plants, VOL 4, JULY 2018: 495-504.

in one embodiment of the invention, the editing of the OsFTIP7 gene by using CRISPR/Cas9 gene editing technology is realized by inserting a single base A into the OsFTIP7 gene.

Of course, the rice Osftip7 mutant can be obtained by inserting other bases which can prevent the OsFTIP7 protein from being expressed into the OsFTIP7 gene by using CRISPR/Cas9 gene editing technology by a person skilled in the art.

In one embodiment of the invention, the rice Osftip7 mutant provided by the invention is obtained by constructing a pCAMBIA1300-CAS9-Os-OsFTIP7 vector, using BbsI to carry out enzyme digestion on psgR-CAS9-Os vector, connecting the psgR-CAS9-Os vector with synthesized sgRNA oligos, then connecting the obtained fragment into a rice pCAMBIA1300 binary vector (HindIII/EcoRI), transferring into agrobacterium, and carrying out genetic transformation by using rice variety Nipponbare to finally obtain the mutant with site mutation.

Preferably, the metal oxide nanoparticles are at least one of oxide nanoparticles of Cu and Zn.

Preferably, the metal oxide nanoparticles are CuO or ZnO.

Preferably, the rice is cultivated in a soil culture.

Preferably, the CuO concentration is in the range of 0.1 to 1g/kg, e.g., 0.1, 0.5 and 1 g/kg.

Preferably, the ZnO concentration is from 0.1 to 1g/kg, e.g., 0.1, 0.5 and 1g/kg

Preferably, the stress of the metal oxide nanoparticles on rice is relieved by at least one of the following means:

(1) the plant height and dry weight of the rice under the stress of the metal oxide nanoparticles are improved;

(2) the content of chlorophyll a, chlorophyll b and total chlorophyll in the rice leaves under the stress of the metal oxide nanoparticles is increased;

(3) hydrogen peroxide (H) in rice leaves under stress of metal oxide nanoparticles is reduced₂O₂) Malondialdehyde (MDA) and proline (Pro);

(4) increasing the content of Catalase (CAT), Peroxidase (POD) and superoxide dismutase (SOD) in rice leaves under the stress of the metal oxide nanoparticles;

(5) the content of auxin in rice leaves under the stress of metal oxide nanoparticles and the expression quantity of auxin synthesis and response related genes are improved.

The rice or rice plant can be rice in a seedling stage and can also be rice in a non-seedling stage.

Preferably, the auxin synthesis and response related gene is one of OsYUCCA1, OsYUCCA4, OsYUCCA6, OsYUCCA8, OsIAA10, OsIAA13, OsIAA14 and OsIAA 20.

Has the advantages that:

the Osftip7 mutant provided by the invention has the effects of promoting the growth of rice under the stress of metal oxide nanoparticles and relieving the toxic action of the metal oxide nanoparticles on the rice, such as improving the synthesis of rice chlorophyll under the stress of the metal oxide nanoparticles, increasing the plant height and dry weight of the rice, improving the oxidative stress induced by the metal oxide nanoparticles and the like.

Drawings

FIG. 1 is a schematic illustration of the mutation sites of the Osftip7 mutant used in the present application;

FIG. 2 shows that the rice material of the complementation line provided in test example 1 of the present invention, the wild type rice and the rice mutant material obtained in example 2 were planted under the stress of CuO NPs or ZnO NPs;

FIG. 3 is a graph of the effect of metal oxide nanoparticle stress on phenotype, plant height and dry weight of wild-type and Osftip7 mutants; wherein A is a growth schematic diagram; b and C are respectively plant height and dry weight under the stress of CuO and ZnO nanoparticles;

FIG. 4 is a graph of the effect of metal oxide nanoparticle stress on chlorophyll a, chlorophyll b and total chlorophyll content of wild-type and Osftip7 mutants; wherein, A and B are the chlorophyll a content, the chlorophyll B content and the total chlorophyll content under the stress of CuO and ZnO nanoparticles respectively;

FIG. 5 shows stress of metal oxide nanoparticles on wild plantsH of type and Osftip7 mutant₂O₂The effects of MDA and Pro content; wherein A and B are respectively the hydrogen peroxide content, MDA content and proline content under the stress of CuO and ZnO nanoparticles;

FIG. 6 is a graph of the effect of metal oxide nanoparticle stress on CAT, POD and SOD activity of wild-type and Osftip7 mutants; wherein A and B are respectively CAT activity, POD activity and SOD activity under the stress of CuO and ZnO nanoparticles;

FIG. 7 is a graph showing the effect of metal oxide nanoparticle stress on auxin content and auxin synthesis and response of related gene expression levels of wild-type and Osftip7 mutants; wherein A and B are the content of auxin under the stress of CuO and ZnO nanoparticles; c is the expression level of auxin synthetic genes (OsYUCCA1, OsYUCCA4, OsYUCCA6 and OsYUCCA 8); d is the expression level of auxin response related genes (OsIAA10, OsIAA13, OsIAA14 and OsIAA 20).

Detailed Description

The present invention is described below with reference to specific examples, which are intended to be illustrative only and are not to be construed as limiting the invention. Unless otherwise indicated, the techniques employed in the examples are conventional and well known to those skilled in the art, and the reagents and products employed are also commercially available. Various procedures and methods not described in detail are conventional methods well known in the art, and the sources, trade names, and components of the reagents used are indicated at the time of first appearance, and the same reagents used thereafter are the same as those indicated at the first appearance, unless otherwise specified.

The material of the mutant Osftip7 used in the present invention is known and described in the following documents: shiyong Song, Ying Chen, Lu Liu, Yen How Benjamin See, Chuanzao Mao, Yinbo Gan and Hao Yu, OsFTIP7 standards auxin-medium depth in rice, Nature Plants, VOL 4, JULY 2018: 495-504.

Example 1 origin of OsFTIP7 Gene

The description, source and function of the OsFTIP7 gene are described in the above-mentioned documents, and it is known from the description that the OsFTIP7 gene is obtained by: homology alignment of the FTIP1(AT5G0685) gene in Arabidopsis thaliana AT NCBI for the homologous gene in rice, a gene comprising high homology to the MCTP domain of FTIP1 (Os05G0370600) was found and designated OsFTIP7, which comprises three C2 domains and a phosphoribosyltransferase C-terminal domain (PRT _ C).

Of course, the gene can be obtained by artificial synthesis by those skilled in the art directly according to the accession number (the OsFTIP7 gene has the accession number AK058522 in NCBI's Genbank).

Example 2 obtaining of Osftip7 mutant

The description, source and function of the material of the mutant Osftip7 used in the invention are described in the above-mentioned documents, and according to the description of the documents, the mutant is obtained by applying CRISPR/Cas9 gene editing technology, specifically: firstly, constructing a pCAMBIA1300-CAS9-Os-OsFTIP7 vector according to the obtained OsFTIP7 gene, digesting the psgR-CAS9-Os vector by BbsI, connecting the psgR-CAS9-Os vector with synthesized sgRNA oligos, then connecting the obtained fragment into a rice pCAMBIA1300 binary vector (HindIII/EcoRI), transferring the plasmid into agrobacterium, and carrying out genetic transformation by using a rice variety Nipponbare to finally obtain a mutant with a mutation site as shown in figure 1.

Test example 1 complementation test

In order to verify the tolerance phenotype of the Osftip7 gene on metal oxide nanoparticles, due to the loss of function of the OsFTIP7 gene, a rice complementary line material Osftip73FLAG-gOsFTIP7 is created, and the rice complementary line material Osftip73FLAG-gOsFTIP7 is obtained by firstly constructing a pHGW-3FLAG-gOsFTIP7 vector, transferring the vector into agrobacterium and performing genetic transformation by using an Osftip7 mutant material.

The plant height and dry biomass data (i.e., the whole seedling weight) of the rice complementation line material, the wild type rice and the rice mutant material obtained in example 2 were collected after 35 days of treatment in soil with a concentration level of CuO NPs or ZnO NPs of 1g/kg, and the results of the data are shown in fig. 2, and it can be seen from the results shown in fig. 2 that the Osftip7 mutant material has higher plant height and dry biomass compared with the complementation line and wild type plants, and that the loss of function of the Osftip7 gene leads to the enhancement of the tolerance of Osftip7 under NPs stress treatment.

Test example 2 stress test of Metal oxide nanoparticles

1. Test materials and treatments

Rice seeds of uniform size and full grain (rice varieties used were Oryza sativa l. ssp. japonica cv. Nipponbare (Nipponbare, wild type) and Osftip7 mutant on the background of Nipponbare) were selected, sterilized with 2% sodium hypochlorite solution for 15min, followed by washing with distilled water 5 times, and then seeded with distilled water at 37 ℃ for 2 days. After germination in an artificial incubator at 30 ℃ for 4 days, the rice seeds were transplanted into different pots and cultured in an automatically controlled growth chamber. The growth chamber is set to be under light for 14 hours (30 +/-2 ℃), dark for 10 hours (26 +/-2 ℃) and relative humidity of 40-60%. Soil was sampled from a cowpea industry Co., Ltd, Zhejiang. After the soil was dried, it was screened through a 2mm screen, and selected according to our earlier concentration, different weights of metal oxide nanoparticles were mixed well with the soil to achieve final concentrations of 0.1, 0.5 and 1g/kg, and soil cultures without metal oxide nanoparticles were used as controls, with 6 replicates per group. After 35 days of treatment, rice seedlings were harvested for phenotypic, physiological, biochemical and molecular analysis.

2. Test method

2.1 measurement of phenotypic characteristics

The 35-day rice seedlings were sampled and thoroughly washed with sterile water, and phenotypic characteristics including plant height and dry weight of the rice seedlings were determined for six replicates. The seedlings were dried in an oven at 105 ℃ for 2 hours and then at 60 ℃ for about 48 hours until a constant weight was reached, at which time the total weight of the seedlings was determined to be dry biomass.

2.2 measurement of chlorophyll content

100mg of fresh leaves are ground with liquid nitrogen and then 5mL of 80% acetone is added. After incubation for 1 hour in the dark, the mixture was centrifuged at 12,000 Xg for 3 minutes. The supernatant was aspirated and the absorbance was measured spectrophotometrically at 645 and 663nm, using 80% acetone as a blank.

Chlorophyll a content (μ g/mL) of 12.7 (A)₆₆₃)-2.69(A₆₄₅)；

Chlorophyll b content (μ g/mL) ═ 22.9 (a)₆₄₅)-4.68(A₆₆₃)；

Total chlorophyll content (μ g/mL) 20.21 (A)₆₄₅)+8.02(A₆₆₃)。

2.3 measurement of Hydrogen peroxide content

According to the method of Zhang et al (2014), hydrogen peroxide (H) was performed using a hydrogen peroxide assay kit (Beijing Sorley technologies, Ltd.)₂O₂) And (6) measuring. Approximately 10mg of plant leaves were pulverized in liquid nitrogen and 500. mu.L of lysate was added and centrifuged at 8,000 Xg for 10 minutes at 4 ℃. Pipette 125 μ L of supernatant mixed with equal amounts of hydrogen peroxide detection reagent and incubate at room temperature for 5 minutes, then measure absorbance spectrophotometrically at 415nm and calculate hydrogen peroxide concentration from the standard curve.

2.4 measurement of MDA content

The Malondialdehyde (MDA) content was determined according to Tang et al (2013). Approximately 100mg of plant leaves were ground in liquid nitrogen and 10mL of 10% trichloroacetic acid was added. The homogenate was centrifuged at 10,000 Xg for 20 minutes. 2mL of the supernatant was added with the same amount of TBA, mixed, heated at 95 ℃ for 30 minutes, rapidly cooled on ice, and then centrifuged at 10,000 Xg for 20 minutes. The absorbance was then measured spectrophotometrically at 450,532 and 600 nm.

2.5 measurement of proline content

About 1g of dried plant leaves were ground in liquid nitrogen and 2% sulfosalicylic acid aqueous solution (about 10mL) was added. The mixture was incubated at room temperature for 1 hour at 200 Xg. The sample was then centrifuged at 12,000 Xg for 15 minutes, and 2mL of the supernatant was taken through a 0.22 μm Millex-LG membrane (Waters, USA), and Pro content was determined by a fully automatic amino acid analyzer L-8900(Hitachi, Japan).

2.6 measurement of antioxidant enzyme Activity

The activities of antioxidases were measured using CAT, POD and SOD assay kit (Beijing Solebao technologies, Ltd.) by the method of Aebi et al (1984), Hossain et al (2010) for POD and Zhang et al (2008) for SOD.

2.7 measurement of auxin content

Auxin (IAA) was extracted from leaves according to the method of Song et al (2018). The IAA content was measured using a commercial enzyme-linked immunosorbent assay kit (Brilliant Biotechnology Ltd.).

2.8 analysis of Gene expression

Total RNA was extracted from Plant leaves using the RNeasy Plant RNA Mini Kit (Qiagen, Hilden, Germany) and cDNA was reverse transcribed using 1. mu.g total RNA, oligo-dT18 primer and the GoScript reverse transcription system (Promega). Quantitative real-time polymerase chain reaction (qRT-PCR) was performed using SYBR Green GoTaq qPCRMaster Mix (Promega, Wis., USA). Rice ACTIN Gene as internal control and 2^-ΔΔCtRelative expression levels were calculated.

TABLE 1 quantitative real-time polymerase chain reaction (qRT-PCR) primer Table

2.9 data processing

All statistical analyses used one-way anova with significance differences between treatments of less than 5%.

3. Test results

3.1 physiological response of Osftip7 mutant to stress of metal oxide nanoparticles

As can be seen in fig. 3: under the stress of metal oxide nanoparticles at higher concentrations (0.5 and 1g/kg), the plant height and dry weight of wild-type rice plants are obviously reduced, and the stress of the metal oxide nanoparticles has no obvious influence on the plant height and dry weight of the Osftip7 mutant. The plant height and dry weight of 1g/kg CuO nanoparticle stressed wild-type rice plants were 72.1% and 80.4% of samples stressed without CuO nanoparticles (fig. 3B). Also, the plant height and dry weight of wild type were reduced by 36.6% and 19.2%, respectively, after stress with 1g/kg ZnO nanoparticles, compared to the samples stressed without ZnO metal oxide nanoparticles (fig. 3C). While the plant height and dry weight of the Osftip7 mutant did not change significantly after stress of metal oxide nanoparticles at different concentrations (FIGS. 3B and 3C).

Chlorophyll a, chlorophyll b and total chlorophyll content in wild type treated with 1g/kg CuO nanoparticles were 71.5%, 47.9% and 59.3% respectively, compared to samples without CuO nanoparticle stress (fig. 4A). Also, 1g/kg ZnO nanoparticle flank forced a reduction in wild type chlorophyll a, chlorophyll B and total chlorophyll content of 28.6%, 48.9% and 39.1% respectively in wild type compared to samples without ZnO nanoparticle stress (fig. 4B). However, after stress treatment of metal oxide nanoparticles with different concentrations, the chlorophyll content of the Osftip7 mutant did not change significantly (FIG. 4).

3.2, change of an Osftip7 mutant antioxidant system under stress of metal oxide nanoparticles

As can be seen in fig. 5 and 6: first, the metal oxide nanoparticle stress altered H in both groups of materials₂O₂MDA and Pro content (figure 5). These levels were found to be significantly lower in the Osftip7 mutant than in the wild-type rice plant under higher metal oxide nanoparticle stress (fig. 5). Under the stress of CuO nano-particles, except the concentration of 0.1g/kg, other concentrations enable H in wild type and mutant₂O₂MDA and Pro content increased (fig. 5A). While under the stress of high dose (0.5 and 1g/kg) CuO nanoparticles, H is in the wild type leaf₂O₂MDA and Pro contents were all significantly higher than the mutant (fig. 5A). No significant changes were detected in wild type and mutant leaves under the stress of 0.1 and 0.5g/kg ZnO nanoparticles; h of mutant under stress of 1g/kg ZnO nanoparticle₂O₂MDA and Pro accumulation were reduced by 12.9%, 26.2% and 16.8% respectively compared to wild type (fig. 5B).

Second, both CuO and ZnO nanoparticle stress altered the activities of CAT, POD and SOD in wild-type and mutant (figure 6). C at higher concentrations (0.5 and 1g/kg)uO and ZnO nanoparticles, the antioxidant enzyme activity of the Osftip7 mutant is obviously higher than that of wild rice. Under the stress of 1g/kg CuO nanoparticles, the enzyme activities of CAT, POD and SOD in the mutant leaves are respectively 1.28 times, 1.38 times and 1.63 times higher than those of wild type (figure 6A). And H₂O₂The contents of MDA and Pro are different, and compared with the Osftip7 mutant, the activities of CAT, POD and SOD in the leaf of the wild plant stressed by 0.5g/kg ZnO nanoparticles are respectively reduced by 35.1%, 35.0% and 16.5%. Whereas, after 1g/kg ZnO nanoparticle stress, wild-type CAT, POD and SOD activities were reduced by 49.0%, 32.8% and 37.7% respectively compared to the mutant (fig. 6B). These results indicate that the Osftip7 mutant relieves oxidative stress, reduces ROS levels, reduces stress-related accumulation of free amino acids, and induces antioxidant enzyme activity in rice under metal oxide stress.

3.3 biosynthesis and response change of auxin in Osftip7 mutant under stress of metal oxide nanoparticles

As can be seen in fig. 7: after stress treatment with CuO and ZnO nanoparticles, auxin content appeared to increase at all concentrations in leaves of both mutant and wild type plants (fig. 7A and 7B). After the stress treatment of the CuO and ZnO nanoparticles with different concentration levels, the auxin content in the leaves of the mutant plants is obviously greater than that of the wild type plants (FIGS. 7A and 7B). The auxin content of the Osftip7 mutant stressed with 1g/kg CuO and ZnO nanoparticles was 1.98 times and 1.81 times higher than that of the wild-type rice plant, respectively (FIGS. 7A and 7B).

Under the stress of metal oxide nanoparticles, the transcription expression levels of OsYUCCA1, OsYUCCA4, OsYUCCA6 and OsYUCCA8 in the mutant leaf are obviously up-regulated, and the transcription expression levels of the OsYUCCA1, the OsYUCCA4, the OsYUCCA6 and the OsYUCCA8 are increased by 1.50-2.38 times (CuO nanoparticles) and 1.51-2.54 times (ZnO nanoparticles) compared with the wild type (FIG. 7C). Similar to the expression of auxin biosynthesis genes, the expression levels of auxin response genes (OsIAA10, OsIAA13, OsIAA14 and OsIAA20) were also significantly higher in the Osftip7 mutant than in the wild type (FIG. 7D). Our results demonstrate that under the stress of metal oxide nanoparticles, the biosynthesis of auxin and the expression of response genes in rice leaves are inhibited, while in the Osftip7 mutant, the genes are significantly induced.

In summary, the present invention has found that by subjecting rice Oryza sativa l.ssp.japonica cv.nipponbare (nipponbare, wild type) and an Osftip7 mutant against nippon bare as a background to a soil culture treatment for 35 days in soil containing 0, 0.1, 0.5 and 1g/kg of metal oxide nanoparticles: the Osftip7 mutant not only obviously relieves the inhibition effect of the stress of the metal oxide nanoparticles on the growth of rice; the plant height, dry weight and chlorophyll biosynthesis of chlorophyll under the stress of the metal oxide nanoparticles are also improved; remarkably improves the oxidative stress induced by the metal oxide nano particles and reduces H₂O₂MDA and Pro, and increased CAT, POD and SOD activity. The invention further proves that the Osftip7 mutant can improve the content of auxin in rice leaves and the expression level of auxin synthesis (OsYUCCA1, OsYUCCA4, OsYUCCA6 and OsYUCCA8) and response related genes (OsIAA10, OsIAA13, OsIAA14 and OsIAA20) under the stress of metal oxide nanoparticles.

The invention is not limited to the specific embodiments illustrated, and any equivalent alterations to the technical solution of the invention, which are made by a person skilled in the art by reading the description, are covered by the claims of the present invention.

Claims (9)

1. A method of alleviating stress of metal oxide nanoparticles on rice comprising disabling the OsFTIP7 gene.

2. A method for alleviating stress of metal oxide nanoparticles on rice, comprising editing OsFTIP7 gene to obtain rice Osftip7 mutant.

3. The method of claim 1 or 2, wherein the metal oxide nanoparticles are at least one of Cu and Zn oxide nanoparticles.

4. The method of claim 3, wherein the metal oxide nanoparticles are CuO or ZnO.

5. The method according to claim 4, wherein the rice is cultivated in a soil culture.

6. The method of claim 5, wherein the CuO concentration is from 0.1 to 1 g/kg.

7. The method according to claim 5, wherein the ZnO concentration is 0.1 to 1 g/kg.

8. The method of claim 1 or 2, wherein the stress of the metal oxide nanoparticles on the rice is alleviated by at least one of:

(1) the plant height and dry weight of the rice under the stress of the metal oxide nanoparticles are improved;

(2) the content of chlorophyll a, chlorophyll b and total chlorophyll in the rice leaves under the stress of the metal oxide nanoparticles is increased;

(3) hydrogen peroxide (H) in rice leaves under stress of metal oxide nanoparticles is reduced₂O₂) Malondialdehyde (MDA) and proline (Pro);

(4) increasing the activity of Catalase (CAT), Peroxidase (POD) and superoxide dismutase (SOD) in rice leaves under the stress of the metal oxide nanoparticles;

(5) the content of auxin in rice leaves under the stress of metal oxide nanoparticles and the expression quantity of auxin synthesis and response related genes are improved.

9. The method of claim 8, wherein said auxin synthesis and response associated gene is one of OsYUCCA1, OsYUCCA4, OsYUCCA6, OsYUCCA8, OsIAA10, OsIAA13, OsIAA14 and OsIAA 20.

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