Production of low arsenic and low cadmium rice by overexpression of OSPCS1, OSABCC1 and OSHMA3 genes under regulation of rice ACTIN1 promoter

Abstract

The invention relates to a genetically modified rice plant or plant cell, which comprises a heterologous heavy metal ATP enzyme gene operably linked to an OsActin1 promoter, a heterologous ATP binding cassette (ABC) transporter gene operably linked to the OsActin1 promoter, and a heterologous plant chelating peptide synthase gene operably linked to the OsActin1 promoter, wherein the OsActin1 promoter has a lower activity in the seed endosperm of the modified rice plant compared to the activity in other nutritional tissues of the modified rice plant; wherein arsenic (As) and cadmium (Cd) in rice grains of the genetically modified rice plant are reduced as compared to a control rice plant in which the genetic modification is not performed. The invention also relates to a method for constructing such genetically modified rice plants or plant cells, and a kit for carrying out the method.

Description

Technical Field

[0001] The present invention relates to the production of transgenic rice plants that are genetically modified to contain reduced levels of arsenic and cadmium in their grains compared to the arsenic and cadmium levels in control non-transgenic rice grains, methods for making such plants, and bacteria for transforming rice plants into such plants. More specifically, the genetically modified plants of the present invention overexpress a heterologous P operably linked to an OsActin1 promoter. 1B The invention relates to a modified rice plant comprising: a plant-derived heavy metal ATPase gene, a heterologous ATP-binding cassette (ABC) transporter gene operably linked to an OsActin1 promoter, and a heterologous phytochelatin synthase gene operably linked to an OsActin1 promoter, wherein the OsActin1 promoter has low activity in the seed endosperm of the modified rice plant compared to its activity in other vegetative tissues of the modified rice plant. Background Art

[0002] Arsenic (As) is a highly toxic metalloid classified as a non-threshold Class I carcinogen, causing both acute and chronic health risks in humans (see Hughes, 2002). Cadmium (Cd) is a toxic heavy metal that preferentially accumulates in the liver and kidneys of humans, leading to renal tubular dysfunction, osteoporosis, cardiovascular disease, and cancer (see Fowler, 2009). Humans are primarily exposed to As and Cd through water and food contaminated by these two toxic elements. Rice (Oryza sativa), which feeds more than half of the global population, is the primary dietary source of Cd and As. Compared to other cereal crops, rice may be more efficient at taking up As and Cd from soil, attributed to its efficient uptake and transport systems for Cd and As, as well as the greater bioaccessibility of arsenite (As(III)), the predominant form in flooded rice paddies (see Ma et al., 2008; Sui et al., 2018; Xu et al., 2008). In Bangladesh and India, groundwater used for crop irrigation is contaminated with arsenic, making rice a major source of arsenic exposure, accounting for approximately 50% of total arsenic intake (Panda et al., 2010). In Japan and China, rice is the most important source of dietary cadmium intake for the general population (Song et al., 2017; Tsukahara et al., 2003). Therefore, the development and production of low-As and low-Cd rice is crucial for protecting public health.

[0003] Upregulating genes involved in As or Cd sequestration is an important and effective method to produce low As or low Cd rice grains. 1BOsHMA3 is a tonoplast transporter of the heavy metal ATPase family (Miyadate et al., 2011; Ueno et al., 2010). OsHMA3 is involved in transporting Cd from the cytosol to the vacuoles of root cells for storage, thereby limiting the transport of Cd from roots to shoots, alleviating Cd toxicity to the aerial parts of rice plants and reducing Cd accumulation in rice grains (Miyadate et al., 2011; Ueno et al., 2010). OsHMA2 is another P 1BOsHMA2 is a type of heavy metal ATPase, a plasma membrane-localized influx transporter expressed primarily in the vascular bundles of roots and stem nodes (Takahashi et al., 2012; Yamaji et al., 2013). OsHMA2 is involved in the transport of zinc (Zn) and cadmium (Cd) into developing tissues through the phloem (Takahashi et al., 2012; Yamaji et al., 2013). Overexpression of OsHMA3 under the control of the maize ubiquitin gene promoter or the rice OsHMA2 promoter selectively increased Cd sequestration in the root vacuole and reduced Cd translocation to rice shoots and grains (Shao et al., 2018; Ueno et al., 2010). In rice, OsABCC1 is a tonoplast transporter that plays a key role in arsenic detoxification. OsABCC1 knockout mutants exhibit hypersensitivity to As treatment, and As concentrations in rice grains are increased (Hayashi et al., 2017; Song et al., 2014). In the cytosol, phytochelatin (PC) chelates As(III) to form a PC-As(III) complex, which is then transported to the vacuole for sequestration by OsABCC1 (Hayashi et al., 2017; Song et al., 2014). PC is a non-encoded heavy metal-binding peptide with the general structure (γ-Glu-Cys)n(2-11)-Gly (Grill et al., 1985). PC is synthesized from glutathione (GSH) by phytochelatin synthase (PCS) (Ha et al., 1999). Two PCSs, OsPCS1 and OsPCS2, have been identified in rice (Das et al., 2017; Hayashi et al., 2017; Li et al., 2007). Overexpression of OsPCS1 was found to enhance PC-dependent arsenic sequestration and significantly reduce arsenic accumulation in rice grains (Hayashi et al., 2017). In a more complex study, rice with lower grain arsenic content was obtained by coexpressing two different vascular arsenic sequestration genes, ScYCF1 (Saccharomyces cerevisiae yeast cadmium factor) and OsABCC1, under the control of the RCc3 (rice root-specific cDNA clone 3) promoter, and c-ECS (a bacterial c-glutamylcysteine ​​synthetase gene) driven by the maize ubiquitin gene promoter (Deng et al., 2018). However, another report showed that heterologous expression of the wheat phytochelatin synthase gene (TaPCS1) in rice enhanced Cd sensitivity (Wang et al., 2012). Therefore, it is necessary to resolve the contradiction in using PCS to regulate As and Cd accumulation in rice grains.More importantly, it is noted that to date, no research reports have focused on the simultaneous reduction of As and Cd in rice grains via genetic engineering approaches.

[0004] The rice actin 1 gene (OsActin1) promoter was found to be highly active in both vegetative and reproductive tissues (see Park et al., 2010). However, notably, during seed development, OsActin1 promoter activity was primarily detected in the aleurone layer and embryo, rather than in the starchy endosperm (see Park et al., 2010), suggesting the potential to drive gene overexpression primarily in vegetative tissues. In the present invention, we report the generation and characterization of transgenic rice lines that overexpress or co-overexpress the OsPCS1, OsABCC1, and OsHMA3 genes under the control of the OsActin1 promoter. Our goal was to generate low-As and low-Cd rice grains by enhancing As and Cd sequestration in the vacuoles of vegetative tissues and organs without causing any pleiotropic phenotypes or yield losses in the transgenic plants. Summary of the Invention

[0005] In the present disclosure, a strategy for regulating As and Cd partitioning in rice is provided, resulting in a simultaneous and substantial reduction in the accumulation of As and Cd in the grain without compromising agronomic traits. Phytochelatins (PCs), which chelate heavy metal(s), play an important role in the detoxification of Cd and As in plants. Co-overexpression of the vacuolar PC-As transporter gene OsABCC1 and the PC synthase gene OsPCS1 exhibited a synergistic effect in reducing As concentrations in rice grains and improving As tolerance, thereby maximizing As sequestration. Overexpression of OsPCS1 rendered rice plants As-tolerant but resulted in Cd hypersensitivity. When exposed to Cd, the Cd concentration in vacuoles isolated from OsPCS1 transgenic plants was much lower than that in the vacuoles of T5105 control plants. Analysis of Cd and PC2-Cd transport activities in mesophyll protoplasts and vacuoles of T5105 and OsHMA3 transgenic plants revealed that PC2-Cd prevented Cd sequestration in the vacuole via OsHMA3. Simultaneous overexpression of OsHMA3 and OsPCS1 completely rescued the growth defect caused by Cd hypersensitivity induced by OsPCS1 overexpression, suggesting that OsHMA3 competes with PC for Cd binding. Finally, triple overexpression of OsABCC1, OsPCS1, and OsHMA3 under the control of the OsActin1 promoter in rice plants reduced As concentrations by 92% and Cd concentrations by 98% in brown rice compared with T5105 plants, without affecting plant growth or essential element homeostasis. This strategy could be applied to reduce dietary As and Cd intake through rice consumption.

[0006] According to a first aspect, the present invention provides a genetically modified rice plant or plant cell comprising a heterologous P operably linked to an OsActin1 promoter. 1B The invention relates to a rice plant comprising: a modified rice plant comprising: a heavy metal ATPase gene; a heterologous ATP-binding cassette (ABC) transporter gene operably linked to an OsActin1 promoter; and a heterologous phytochelatin synthase gene operably linked to the OsActin1 promoter, wherein the OsActin1 promoter has low activity in the endosperm of seeds of the modified rice plant compared to the activity in other vegetative tissues of the modified rice plant; and wherein arsenic (As) and cadmium (Cd) are reduced in rice grains of the genetically modified rice plant compared to control rice plants that have not undergone the genetic modification.

[0007] In some embodiments, the OsActin1 promoter comprises the nucleic acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 31 or a functional sequence variant thereof.

[0008] In some embodiments, the heterologous P 1B The type heavy metal ATPase gene encodes the amino acid sequence shown in SEQ ID NO: 39; the heterologous ABC transporter gene encodes the amino acid sequence shown in SEQ ID NO: 37, and the phytochelatin synthase gene encodes the amino acid sequence shown in SEQ ID NO: 38.

[0009] In some embodiments, the heterologous P 1B The type heavy metal ATPase gene comprises a nucleic acid sequence that, due to the degeneracy of the genetic code, has at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity with the polynucleotide sequence of SEQ ID NO: 22; the heterologous ABC transporter gene comprises a nucleic acid sequence that, due to the degeneracy of the genetic code, has at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity with the polynucleotide sequence of SEQ ID NO: 36; and the heterologous phytochelatin synthase gene comprises a nucleic acid sequence that, due to the degeneracy of the genetic code, has at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity with the polynucleotide sequence of SEQ ID NO: 32.

[0010] In some embodiments, the exogenous P 1B The heavy metal ATPase gene, the exogenous ABC transporter gene and / or the exogenous phytochelatin synthase gene are from cereal crops.

[0011] In some embodiments, the heterologous P 1B The type heavy metal ATPase gene is OsHMA3 and comprises the nucleic acid sequence shown in SEQ ID NO: 22, the heterologous ABC transporter gene is OsABCC1 and comprises the nucleic acid sequence shown in SEQ ID NO: 36, and the heterologous phytochelatin synthase gene is OsPCS1 and comprises the nucleic acid sequence shown in SEQ ID NO: 32.

[0012] In some embodiments, the genetically modified rice plant or plant cell comprises a heterologous OsHMA3 gene operably linked to an OsActin1 promoter, a heterologous OsABCC1 gene operably linked to an OsActin1 promoter, and a heterologous OsPCS1 gene operably linked to an OsActin1 promoter.

[0013] In some embodiments, the genetically modified rice plant or plant cell is of the species Oryza sativa L.

[0014] According to a second aspect, the present invention provides a method for constructing a genetically modified rice plant having reduced arsenic (As) and cadmium (Cd) in rice grains compared to rice grains of a control rice plant, the method comprising the steps of:

[0015] a) Producing a heterologous P gene operably linked to an OsActin1 promoter 1B Genetically modified rice plants expressing a heavy metal ATPase gene;

[0016] b) producing a genetically modified rice plant comprising a heterologous ATP-binding cassette (ABC) transporter gene operably linked to an OsActin1 promoter;

[0017] c) producing a genetically modified rice plant comprising a heterologous phytochelatin synthase gene operably linked to an OsActin1 promoter;

[0018] d) Select and overexpress the exogenous P 1B a genetically modified rice plant containing a type 1 heavy metal ATPase gene, the exogenous ATP-binding cassette (ABC) transporter gene, and the exogenous phytochelatin synthase gene;

[0019] e) crossing two of the three genetically modified rice plants to produce a plant that is doubly homozygous for the exogenous gene; and

[0020] f) crossing the double homozygous plant of step (e) with a third genetically modified rice plant to produce a triple homozygous plant overexpressing the exogenous gene.

[0021] In some embodiments, the OsActin1 promoter is as defined in the first aspect.

[0022] In some embodiments of the method according to the second aspect, the heterologous gene is as defined in the first aspect.

[0023] According to a second aspect, the present invention provides a kit for constructing a genetically modified rice plant having reduced arsenic (As) and cadmium (Cd) in rice grains compared to rice grains from a control rice plant, wherein the kit comprises: bacteria comprising a vector comprising a heterologous heavy metal ATPase gene operably linked to an OsActin1 promoter, and / or bacteria comprising a vector comprising a heterologous ATP-binding cassette (ABC) transporter gene operably linked to an OsActin1 promoter, and / or bacteria comprising a vector comprising a heterologous phytochelatin synthase gene operably linked to an OsActin1 promoter. In a preferred embodiment, the bacteria is Agrobacterium tumefaciens.

[0024] In some embodiments, the OsActin1 promoter comprises the nucleic acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 31 or a functional sequence variant thereof.

[0025] In some embodiments, the heterologous P 1B The type heavy metal ATPase gene encodes the amino acid sequence shown in SEQ ID NO: 39; the heterologous ABC transporter gene encodes the amino acid sequence shown in SEQ ID NO: 37, and the phytochelatin synthase gene encodes the amino acid sequence shown in SEQ ID NO: 38.

[0026] In some embodiments, the heterologous P 1BThe type heavy metal ATPase gene comprises a nucleic acid sequence that, due to the degeneracy of the genetic code, has at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity with the polynucleotide sequence of SEQ ID NO: 22; the heterologous ABC transporter gene comprises a nucleic acid sequence that, due to the degeneracy of the genetic code, has at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity with the polynucleotide sequence of SEQ ID NO: 36; and the heterologous phytochelatin synthase gene comprises a nucleic acid sequence that, due to the degeneracy of the genetic code, has at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity with the polynucleotide sequence of SEQ ID NO: 32.

[0027] In some embodiments, the exogenous P 1B The heavy metal ATPase gene, the exogenous ABC transporter gene and / or the exogenous phytochelatin synthase gene are from cereal crops.

[0028] In some embodiments, the heterologous P 1B The type heavy metal ATPase gene is OsHMA3 and comprises the nucleic acid sequence shown in SEQ ID NO: 22, the heterologous ABC transporter gene is OsABCC1 and comprises the nucleic acid sequence shown in SEQ ID NO: 36, and the heterologous phytochelatin synthase gene is OsPCS1 and comprises the nucleic acid sequence shown in SEQ ID NO: 32. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Fig. 1 shows the diagram of the binary construct and gene used in this study. (A) Schematic diagram of the binary construct. The lower sub-diagram of the figure shows the gene located in the T-DNA region of the binary construct, which has a backbone derived from pCAMBIA1305.1. The upper sub-diagram of the figure shows the target gene for replacing the GUSPlus in pCAMBIA1305.1. The cDNA clones of the OsHMA3 and OsPCS1 genes were used to construct constructs pCActin1-cHMA3 and pCActin1-cPCS1, respectively, while the genomic clone of the OsABCC1 gene was used to construct construct pCActin1-gABCC1. In all constructs, the CaMV35S promoter in pCAMBIA1305.1 was replaced with the OsActin1 promoter. The figure is not drawn to scale. (B) Contig map of pCActin1-gABCC1. The contig map was generated by Sequencher 5.1 (Gene Code, Inc., MI 48108, USA). It starts from the left border of the T-DNA to the right border, followed by the pC1305.1 backbone region. pCActin1-gABCC1 carries the hygromycin phosphotransferase gene (P 35S :Hpt:T 35S ), and the genomic clone of the OsABCC1 gene under the control of the OsActin1 gene promoter in the T-DNA region as shown in (A) (P Actin1 :gABCC1:T Nos ). P 35S :Hpt:T 35S The transcription direction of the gene is toward LB, while P Actin1 :gABCC1:T Nos The transcription direction of the gene is toward RB. (C) Contig map of pCActin1-cPCS1. The contig map was generated by Sequencher 5.1 (Gene Code, M148108, USA). It starts from the left border of the T-DNA to the right border, followed by the pC1305.1 backbone region. pCActin1-cPCS1 carries the hygromycin phosphotransferase gene (P 35S :Hpt:T 35S ), and the cDNA clone of the OsPCS1 gene under the control of the OsActin1 gene promoter in the T-DNA region as shown in (A) (P Actin1 :cPCS1:T Nos ). P 35S :Hpt:T 35S The transcription direction of the gene is toward LB, while P Actin1 :cPCS1:T NosThe transcription direction of the gene is toward RB. (D) Contig map of pCActin1-cHMA3. The contig map was generated by Sequencher 5.1 (Gene Code, MI 48108, USA). It starts from the left border of the T-DNA to the right border, followed by the pC1305.1 backbone region. pCActin1-cHMA3 carries the hygromycin phosphotransferase gene (P 35S :Hpt:T 35S ), and the cDNA clone of the OsHMA3 gene under the control of the OsActin1 gene promoter in the T-DNA region as shown in (A) (P Actin1 :cHMA3:T Nos ). P 35S :Hpt:T 35S The transcription direction of the gene is toward LB, while P Actin1 :cHMA3:T Nos The transcription direction of the gene is toward RB. Abbreviations in (A) to (D): LB, left boundary; T 35S , CaMV35S terminator; Hpt CDS, hygromycin phosphotransferase gene coding region P 35S , CaMV35S promoter; P Actin1 , OsActin1 promoter; GOI, target gene; T Nos , nopaline synthase gene terminator; RB, right border ORF, open reading frame

[0030] Figure 2 Results of generating OsHMA3 overexpression lines are shown. (A) Detection of transgenic P by Southern blot hybridization analysis Actin1 :cHAM3:T Nos The copy number of T-DNA in plants (T0). M, molecular marker. (B) Detection of T5105 and transgenic P by qRT-PCR Actin1 :cHAM3:T Nos The expression level of OsHMA3 gene in plants (T0). Only the plants carrying a single copy of P Actin1 :cHAM3:T Nos T0 plants of the gene. (C) Morphological phenotypes of T5105 and the OsHMA3-overexpressing line HMA3-L3 (T2). Plants were imaged at 120 days after sowing. (D) and (E) Plant height (D) and seed set rate (E) of T5105 and the OsHMA3-overexpressing line (T2). Values ​​are mean ± SD, based on three biological replicates. No significant differences were observed between T5105 and the OsHMA3-overexpressing lines (P > 0.05 by Student's t-test). L1, HMA3-L1; L3, HMA3-L3; L12, HMA3-L12.

[0031] Figure 3 Results of generating OsABCC1 overexpression lines are shown. (A) Detection of transgenic P by Southern blot hybridization analysis Actin1 :gABCC1:T Nos The copy number of T-DNA in plants (T0). M, molecular marker. (B) Detection of T5105 (NT) and transgenic P by qRT-PCR. Actin1 :gABCC1:T Nos The expression level of OsABCC1 gene in plants (T0). Only the plants carrying a single copy of P Actin1 :gABCC1:T Nos T0 plants of the gene. (C) Morphological phenotypes of T5105 and the OsABCC1 overexpression line ABCC1-L27 (T2). Plants were imaged at 120 days after sowing. (D) and (E) Plant height (D) and seed set rate (E) of T5105 and the OsABCC1 overexpression line (T2). Values ​​are mean ± SD, based on three biological replicates. No significant differences were observed between T5105 and the OsABCC1 overexpression lines (P > 0.05 by Student's t-test). L3, ABCC1-L3; L27, ABCC 1-L27; L31, ABCC1-L31.

[0032] Figure 4 Results of generating OsPCS1 overexpression lines are shown. (A) Detection of transgenic P by Southern blot hybridization analysis Actin1 :cPCS1:T Nos The copy number of T-DNA in plants (T0). M, molecular marker. (B) Detection of T5105 (NT) and transgenic P by qRT-PCR. Actin1 :cPCS1:T Nos The expression level of OsPCS1 gene in plants (T0). Only the plants carrying a single copy of P Actin1 :cPCS1:T Nos T0 plants of the gene. (C) Morphological phenotypes of T5105 and the OsPCS1 overexpression line PCS1-L1 (T2). Plants were photographed at 120 days after sowing. (D) and (E) Plant height (D) and seed set rate (E) of T5105 and the OsPCS1 overexpression line (T2). Values ​​are mean ± SD, based on three biological replicates. No significant differences were observed between T5105 and the OsPCS1 overexpression line (P > 0.05 by Student's t-test). L1, PCS1-L1; L3, PCS1-L3; L4, PCS1-L4.

[0033] Figure 5 Characterization results of OsHMA3 overexpression lines are shown. (A) Morphology of panicles of T5105 and three independent OsHMA3 overexpression lines. (B) Expression levels of OsHMA3 in T5105 and OsHMA3 overexpression lines. (C) Cd concentrations in grains of T5105 and OsHMA3 overexpression lines grown in soil with or without Cd treatment. (D) Cd concentrations in different straw tissues of T5105 and the OsHMA3 overexpression line HMA3-L3 grown in soil treated with Cd. (E) Seedlings of T5105 (NT) and OsHMA3 overexpression lines 14 days after Cd treatment. (F) and (G) Shoot length (F) and dry weight (DW) (G) of T5105 and OsHMA3 overexpression lines 14 days after Cd treatment. Data are mean ± SD, based on three biological replicates. Significant differences between T5105 and OsHMA3 overexpressing strains were calculated using Student's t-test (*P < 0.05; **P < 0.01). Control, soil without Cd treatment; Control+Cd, soil containing 3 mg / kg Cd in the form of CdSO4.

[0034] Figure 6 Characterization results of OsABCC1 overexpression lines are shown. (A) Morphology of panicles of T5105 and OsABCC1 overexpression lines. (B) Expression levels of OsABCC1 in T5105 and OsABCC1 overexpression lines. (C) As concentrations in grains of T5105 and OsABCC1 overexpression lines grown in soil with or without As treatment. (D) As concentrations in different straw tissues of T5105 and OsABCC1 overexpression line ABCC1-L27 grown in soil treated with As. (E) Seedlings of T5105 (NT) and OsABCC1 overexpression lines 14 days after As treatment. (F) and (G) Shoot length (F) and dry weight (DW) (G) of T5105 and OsABCC1 overexpression lines 14 days after As treatment. All data are mean ± SD and are based on at least three biological replicates. Significant differences between T5105 and OsABCC1 overexpression lines were calculated using Student's t-test (*P < 0.05; **P < 0.01). Control, soil without As treatment; Control+As, soil containing 10 mg / kg As in the form of NaAsO2.

[0035] Figure 7Figures show the results of a test of the tolerance of T5105 and OsABCC1 overexpression lines to Cd treatment. (A) Seedlings of T5105 (NT) and OsABCC1 overexpression lines 14 days after treatment in Cd-containing medium. NT, T5105; L3, ABCC1-L3; L27, ABCC1-L27; L31, ABCC11-L31. (B) and (C) Shoot length (B) and dry weight (DW) (C) of T5105 and OsABCC1 overexpression lines 14 days after treatment in Cd-containing medium. (D) Cd concentration in grains of T5105 and ABCC1-L27 grown in soil. Control, soil without Cd treatment; Control+Cd, soil containing 3 mg / kg Cd in the form of CdSO4. Values ​​are mean ± SD based on three biological replicates. No significant differences were observed between T5105 and the OsABCC1 overexpressing lines (P>0.05 by Student's t-test).

[0036] Figure 8 Characterization results of OsPCS1 overexpression lines are shown. (A) Morphology of panicles of T5105 and OsPCS1 overexpression lines. (B) Expression levels of OsPCS1 in T5105 and OsAPCS1 overexpression lines. (C) As concentrations in grains of T5105 and OsPCS1 overexpression lines grown in soils with or without As treatment. (D) Cd concentrations in grains of T5105 and OsPCS1 overexpression lines grown in soils with or without Cd treatment. (E) and (F) As (E) or Cd (F) concentrations in different straw tissues of T5105 and OsPCS1 overexpression line PCS1-L1 grown in soils treated with As or Cd. All data are mean ± SD, based on at least three biological replicates. Significant differences between T5105 and the OsPCS1 overexpression line were calculated using the Student's t-test (*P < 0.05; **P < 0.01). Control, soil without As or Cd treatment; Control+As, soil containing 10 mg / kg As in the form of NaAsO2. Control+Cd, soil containing 3 mg / kg Cd in the form of CdSO4.

[0037] Figure 9The results of the As and Cd tolerance test of the OsPCS1 overexpression strain are shown. (A) Seedlings of T5105 (NT) and the OsPCS1 overexpression strain 14 days after As treatment. (B) and (C) Shoot length (B) and dry weight (DW) (C) of T5105 and the OsPCS1 overexpression strain 14 days after As treatment. (D) Seedlings of T5105 (NT) and the OsPCS1 overexpression strain 14 days after Cd treatment. (E) and (F) Shoot length (E) and dry weight (DW) (F) of T5105 and the OsPCS1 overexpression strain 14 days after Cd treatment. All data are mean ± SD, based on three biological replicates. Significant differences between T5105 and the OsPCS1 overexpression strain were calculated using the Student's t-test (*P < 0.05; **P < 0.01). NT, T5105; L1, PCS1-L1; L3, PCS1-L3; L4, PCS1-L4.

[0038] Figure 10 As tolerance test results for OsABCC1 and OsPCS1 co-overexpressing lines are shown. (A) Seedlings of T5105 (NT), ABCC1-L27 (A27), PCS1-L1 (P1), and an OsABCC1 and OsPCS1 co-overexpressing line (AP) 14 days after As treatment. (B) and (C) Shoot length (B) and dry weight (DW) (C) of rice plants shown in (A). (D) and (E) As concentrations in roots (D) and shoots (E) of rice plants shown in (A). (F) As concentrations in grains of T5105, ABCC1-L27, PCS1-L1, and AP plants grown in soil with or without As treatment. Data are mean ± SD, based on three biological replicates. Different letters indicate significant differences calculated by one-way analysis of variance (ANOVA) followed by LSD test, P < 0.05. Control, soil without As treatment; Control+As, soil containing 10 mg / kg As in the form of NaAsO2.

[0039] Figure 11Results of a Cd tolerance test of OsPCS1 and OsHMA3 co-overexpressing lines are shown. (A) Seedlings of T5105 (NT), PCS1-L1 (P1), HMA3-L3 (H3), and an OsHMA3 and OsPCS1 co-overexpressing line (HP) 14 days after Cd treatment. (B) and (C) Shoot length (B) and dry weight (DW) (C) of rice plants shown in (A). (D) and (E) As concentrations in roots (D) and shoots (E) of rice plants shown in (A). (F) Cd concentrations in grains of T5105, HMA3-L3, PCS1-L1, and HP plants grown in soil with or without Cd treatment. Data are mean ± SD, based on three biological replicates. Different letters indicate significant differences calculated by one-way ANOVA followed by LSD test, P < 0.05. Control, soil without Cd treatment; Control+Cd, soil containing 3 mg / kg Cd in the form of CdSO4.

[0040] Figure 12 Results of tests evaluating the effects of OsPCS1 overexpression and phytochelatin 2-Cd complex (PC2-Cd) on OsHMA3-mediated Cd sequestration in vacuoles are shown. (A) Cd concentrations in vacuoles and protoplasts isolated from T5105 and PCS1-L1 seedlings grown in Cd-containing medium. Significant differences between T5105 and OsPCS1-L1 were calculated using a Student's t-test (**P < 0.01). Data are mean ± SD, based on three biological replicates. (B) Cd concentrations in vacuoles and protoplasts of T5105 and HMA3-L3 after incubation of protoplasts with Cd or PC2-Cd complex. Significant differences between Cd treatment and PC2-Cd treatment were calculated using a Student's t-test (**P < 0.01). Data are mean ± SD, based on three biological replicates.

[0041] Figure 13As and Cd concentrations in grains of lines co-overexpressing OsABCC1, OsPCS1, and OsHMA3 are shown. (A) Morphological phenotypes of T5105 and a line co-expressing OsABCC1, OsPCS1, and OsHMA3 (PAH). Plants were photographed 120 days after sowing. (B) Plant height of T5105 and PAH plants. (C) Panicles and unpolished rice grains of T5105 and PAH plants. (D) Seed set rate of T5105 and PAH plants. (E) Weight of 100 grains of T5105 and PAH plants. Data are mean ± SD; (B), (D), and (E) are based on at least three biological replicates. No significant differences were detected between T5105 and PAH plants using the Student's t-test (P > 0.05). (F) and (G) As (F) and Cd (G) concentrations in rice grains of T5105, PCS1-L1, ABCC1-L27, HMA3-L3, and PAH plants grown in soils with or without dual As and Cd treatment. Data are mean ± SD, based on three biological replicates. Different letters in (F) and (G) indicate significant differences calculated by one-way ANOVA followed by LSD test, P < 0.05. Control: soil without As or Cd treatment; Control+As+Cd: soil containing 10 mg / kg As in the form of NaAsO2 and 3 mg / kg Cd in the form of CdSO4.

[0042] Figure 14 Concentrations of other elements in the grains of T5105 and transgenic plants are shown. T5105, PCS1-L1, ABCC1-L27, HMA3-L3, and PAH plants were grown in soils with or without dual treatments of As and Cd. Concentrations of Co, Cu, Fe, Mn, Se, and Zn in the grains were determined by ICP-MS. Data are mean ± SD, based on three biological replicates. No significant differences were detected between T5105 and transgenic plants at P > 0.05 by the LSD test. Control, soil without As or Cd treatment; Control + As + Cd, soil containing 10 mg / kg As in the form of NaAsO2 and 3 mg / kg Cd in the form of CdSO4.

[0043] Figure 15Results show the tolerance of T5105, PCS1-L1, ABCC1-L27, HMA3-L3, and PAH plants to As and Cd. (A) Seedlings of T5105, PCS1-L1 (P1), ABCC1-L27 (A27), HMA3-L3 (H3), and PAH plants 14 days after As and Cd treatment. (B) and (C) Shoot length (B) and dry weight (DW) (C) of the rice plants shown in (A). (D) and (E) As concentrations in the roots (D) and shoots (E) of the rice plants shown in (A). (F) and (G) Cd concentrations in the roots (F) and shoots (G) of the rice plants shown in (A). Data are mean ± SD, based on three biological replicates. Different letters indicate significant differences calculated by one-way ANOVA followed by LSD test, P < 0.05. As+Cd treatment 1 (As+CD T1), half-strength MS medium containing 50 μM NaAsO2 + 10 μM CdSO4; As+Cd treatment 2 (As+Cd T2), half-strength MS medium containing 75 μM NaAsO2 and 20 μM CdSO4. DETAILED DESCRIPTION

[0044] For convenience, the references mentioned in this specification are listed in the form of a reference list and attached at the end of the examples. The entire contents of these references are incorporated herein by reference.

[0045] definition

[0046] For convenience, some terms used in the specification, examples, and appended claims are collected here.

[0047] As used herein, the term "comprising" is defined as the various components, ingredients, or steps that may be used in combination when practicing the invention. Thus, the term "comprising" encompasses the more restrictive terms "consisting essentially of" and "consisting of."

[0048] The term "Agrobacterium" refers to soil-borne, Gram-negative, rod-shaped plant pathogenic bacteria that cause crown root nodules. The term "Agrobacterium" includes, but is not limited to, strains of Agrobacterium tumefaciens (which typically cause crown root nodules in infected plants) and Agrobacterium rhizogens (which cause hairy root disease in infected host plants). Infection of plant cells with Agrobacterium typically results in the production of opines (e.g., nopaline, agropine, octopine, etc.) by the infected cells.

[0049] The term "expression" as used in reference to nucleic acid sequences such as genes refers to the process by which the genetic information encoded in a gene is converted into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) by the "transcription" of the gene (i.e., by the enzymatic action of an RNA polymerase), and, where applicable (when the gene encodes a protein), by the "translation" of the mRNA into a protein. Gene expression can be regulated at multiple stages of this process. "Upregulation" or "activation" refers to regulation that increases the production of a gene expression product (i.e., RNA or protein), while "downregulation" or "inhibition" refers to regulation that decreases production. The molecules involved in upregulation or downregulation (e.g., transcription factors) are typically referred to as "activators" and "inhibitors," respectively. "Overexpression" refers to the expression level of a particular gene being higher than that observed under normal circumstances.

[0050] The terms "nucleic acid sequence," "nucleotide sequence of interest," or "nucleic acid sequence of interest" refer to any nucleotide sequence (e.g., RNA or DNA) that one skilled in the art may deem desirable to manipulate for any reason (e.g., to treat a disease, to confer improved quality, etc.). Such nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selectable marker genes, oncogenes, drug resistance genes, growth factors, etc.), as well as non-coding regulatory sequences (e.g., promoter sequences, polyadenylation sequences, termination sequences, enhancer sequences, etc.) that do not encode mRNA or protein products.

[0051] As used herein, the term "amino acid" or "amino acid sequence" refers to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, as well as a naturally occurring or synthetic molecule. When an "amino acid sequence" as described herein refers to the amino acid sequence of a naturally occurring protein molecule, "amino acid sequence" and similar terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the protein molecule.

[0052] As used herein, the term "functional sequence variant" refers to a polynucleotide sequence that has one or more nucleic acid alterations relative to a reference or wild-type sequence, but the alterations do not eliminate or substantially alter the activity of the non-variant reference polynucleotide. For example, the OsActin1 promoter defined by SEQ ID NO: 21 or SEQ ID NO: 31 can be truncated or have one or more nucleic acid(s) removed from it and still retain activity.

[0053] The term "gene" encompasses the coding region of a structural gene and includes sequences adjacent to the 5' and 3' ends of the coding region and about 1 kb from each end, such that the gene corresponds to the length of the full-length mRNA. The sequences located at the 5' end of the coding region and present on the mRNA are referred to as 5' non-translated sequences. The sequences located at the 3' end or downstream of the coding region and present on the mRNA are referred to as 3' non-translated sequences. The term "gene" encompasses both cDNA and genomic forms of a gene. The genomic form or clone of a gene contains coding regions known as "exons" or "expressed regions" or "expressed sequences" that are interrupted by non-coding sequences known as "introns" or "intervening regions" or "intervening sequences."

[0054] As used herein, the term "seed" includes all tissues that develop from the fertilized plant egg; thus, it includes the mature ovule containing the embryo and stored nutrients, as well as one or more layers of integuments that differentiate into a protective seed coat or exocarp. Nutrients in seed tissues may be stored in the endosperm or in the embryo, particularly the cotyledons, or in both.

[0055] As used herein, the term "seed" may also refer to the mature and fertilized ovule of a seed plant, i.e., the mature ovule, which contains the plant embryo (i.e., a microplant) and also contains the endosperm (i.e., the nutrient supply source for the plant embryo), and may be surrounded by a seed coat.

[0056] The term "rice" as used herein when referring to a "rice plant" refers to Oryza spp., ie, cultivars, uncultivated rice plants, and progenitor rice plants. Preferably, the rice plant of the present invention is an Oryza sativa variety.

[0057] The term "heterologous" when referring to a gene or nucleic acid refers to a gene that has been manipulated in some way. For example, a heterologous gene includes a gene from one species introduced into another species. A heterologous gene also includes a native gene of an organism that has been altered in some way (e.g., mutated, with multiple copies added, linked to a non-native promoter or enhancer sequence, etc.). A heterologous gene can include a plant gene sequence that includes a cDNA form of a plant gene; the cDNA sequence can be expressed in sense (to produce mRNA) or in antisense (to produce an antisense RNA transcript that is complementary to the mRNA transcript). A heterologous gene is distinguished from an endogenous plant gene in that the heterologous gene sequence is typically linked to a nucleotide sequence containing regulatory elements such as a promoter, and the nucleotide sequence is not found in nature associated with a plant gene sequence in a gene or chromosome encoding the protein encoded by the heterologous gene, or is associated with a portion of a chromosome that does not exist in nature (e.g., a gene expressed in a locus where the gene is not normally expressed).

[0058] The term "transgenic" refers to the placement of foreign genes into an organism through a transfection process. The term "foreign gene" refers to any nucleic acid (e.g., a gene sequence) introduced into the genome of an organism through experimental manipulation, and may include gene sequences found in the organism, as long as the introduced gene is not co-located with a naturally occurring gene. Transgenic may also refer to "exogenous genes," which include, but are not limited to, reporter genes, marker genes, selection genes, and functional genes. The term "endogenous gene" refers to a naturally encoded and expressed gene.

[0059] The terms "transformants" and "transformed cells" include the primary transformed cell and cultures derived therefrom, regardless of the number of passages. Progeny produced may not be identical in DNA content due to intentional or accidental mutations. The definition of transformants includes functionally identical mutant progeny selected from the originally transformed cell.

[0060] The terms "in operable combination," "in an operable order," and "operably linked" refer to nucleic acid sequences being linked in such a manner as to produce a nucleic acid molecule capable of directing the transcription of a given gene and / or directing the synthesis of a desired protein molecule. The terms also refer to amino acid sequences being linked in such a manner as to produce a functional protein.

[0061] The terms "overexpressed," "overexpression," and "overexpressing," and their grammatical equivalents, when used with reference to mRNA levels, refer to expression levels that are about 3-fold higher than the expression levels typically observed in a given tissue of a control or non-transgenic animal. mRNA levels are determined using any of a variety of techniques known to those skilled in the art, including but not limited to qRT-PCR.

[0062] The term "promoter element," "promoter," or "promoter sequence" refers to a DNA sequence located 5' to (i.e., preceding) the coding region of a DNA polymer. Most promoters known in nature are located before the transcription region. The promoter acts as a switch, thereby activating the expression of a gene. If a gene is activated, it is said to be transcribed, or to participate in transcription. Transcription involves the synthesis of mRNA from a gene. Thus, a promoter acts as a transcriptional regulatory element and also provides a starting site for transcription of a gene into mRNA.

[0063] The term "vector" refers to a nucleic acid molecule that transfers DNA segments. Transfer can occur within a cell, between cells, or elsewhere. The term "vehicle" is sometimes used interchangeably with "vector."

[0064] The following examples are provided to demonstrate and further illustrate some preferred embodiments and aspects of the present invention, but should not be construed as limiting the scope thereof.

[0065] Example 1

[0066] Materials and methods

[0067] Plant materials and growth conditions

[0068] The rice variety used in this study was T5105, an improved aromatic rice based on the genetic background of the Thai aromatic KTML 105 (see Luo and Yin, 2013). Treated or untreated T5105 and transgenic plants were grown in potting soil in a greenhouse at 24°C to 33°C, with a photoperiod of 12 hours of daylight and 12 hours of darkness, and a relative humidity of 80% to 85%.

[0069] Genes, constructs, and rice transformation

[0070] As shown schematically in Figure 1, a construct for gene overexpression in rice was constructed based on the binary vector pCAMBIA1305.1 (Accession No. AF304545). Briefly, the GUSPlus promoter located in the T-DNA region of pCAMBIA1305.1 was replaced by a 1,414-bp promoter derived from the rice OsActin1 gene (Os03g0718100) (SEQ ID NO: 31 for pCActin1-cPCS1 and pCActin1-gABCC1; SEQ ID NO: 21 for pCActin1-cHMA3). TM The CaMV35S promoter (see Reece et al., 1990) was located upstream of the gene. The cDNA sequences of OsHMA3 (Os07g0232900) (SEQ ID NO: 22) and OsPCS1 (Os05g0415200) (SEQ ID NO: 32) were synthesized by GenScript (www.genscript.com.cn). The full-length genomic DNA fragment (from the start codon to the stop codon) of OsABCC1 (Os04g0620000) (SEQ ID NO: 35) was amplified from T5105 by PCR. GUSPlus TM The coding region of the gene was replaced by the coding region of the cDNA clone derived from OsHMA3 (Os07g0232900) (SEQ ID NO: 22), OsPCS1 (Os05g0415200) (SEQ ID NO: 32), or the coding region of the genomic clone derived from OsABCC1 (Os04g0620000) (SEQ ID NO: 35) to generate promoter fusion genes P, P, and OsHMA3, P, and OsPCS1, respectively. Actin1 :cHMA3:T Nos 、P Actin1 :cPCS1:TNos and P Actin1 :gABCC1:T Nos As well as the binary constructs pCActin1-cHMA3, pCActin1-cPCS1, and pCActin1-gABCC1 containing the NOS terminator (SEQ ID NO: 23), LB (SEQ ID NO: 27), and RB (SEQ ID NO: 28). All constructs were introduced into Agrobacterium tumefaciens strain AGL1 and used for rice transformation. Agrobacterium-mediated T5105 transformation was performed according to the previously described procedure with slight modifications (see Hiei et al., 1994). 1 mg / L KT and 0.2 mg / L NAA in the rice regeneration medium N6S3-CH were replaced with 1 mg / L BA and 1 mg / L NAA. Gene constructs such as Figures 1A to 1D And shown in SEQ ID NO: 19-20, 29-30, 33-34.

[0071] Southern blot hybridization analysis

[0072] use The genomic DNA of transgenic rice was extracted using the HP Plant DNA Mini Kit (Omega BIO-TEK). Approximately 2 μg of DNA was digested with restriction endonucleases Hind III and BamH I (NEB). The DNA fragments were separated by gel electrophoresis on a 0.8% (w / v) agarose gel. The fragments were then transferred from the agarose gel to a Hybond-N+ membrane (GE Healthcare). Digoxigenin-labeled hpt gene-specific nucleic acid probes were amplified by PCR using the DIG DNA labeling mixture (Roche) and the primer pairs listed in Table 1. Southern blot hybridization and DIG-labeled probe detection were performed using the DIG-High Prime DNA labeling and Detection Starter Kit II (Roche) according to the manufacturer's instructions. ChemiDoc TM The chemiluminescent signal was detected using the Touch Imaging System (Bio-Rad).

[0073] Table 1. Oligonucleotide primers used in this study

[0074]

[0075]

[0076] Gene expression analysis

[0077] Using Favorprep TMTotal RNA was extracted using the Plant Total RNA Purification Mini Kit (FAVORGEN), and then DNA digestion was performed using DNase I (Roche). First-strand cDNA was synthesized from 1 μg of total RNA using a cDNA Synthesis Kit (Bio-Rad). FAST qPCR Master Mix (KAPA Biosystems) was used in CFX96 TM Quantitative real-time PCR (qRT-PCR) was performed using a real-time system (Bio-Rad). The expression level of the rice elongation factor (EF) gene OsEF-1α (Os03g0178000) was used as an internal control. qRT-PCR primers for different genes are listed in Table 1.

[0078] Testing the tolerance of rice seedlings to As and Cd

[0079] Rice seeds were surface sterilized and grown in a tissue culture room at 25°C in a Phytatray. TM The seeds were germinated in half-strength MS medium (Sigma-Aldrich) in a 1.2-well container with a photoperiod of 16 hours of light and 8 hours of darkness. Two-week-old rice seedlings were transferred to half-strength MS medium containing varying concentrations of NaAsO2 (0 μM to 100 μM) and / or CdSO4 (0 μM to 40 μM) and cultured for 14 days. The roots of the treated seedlings were washed three times with 5 mM CaCl2 and deionized water, respectively. They were photographed before measuring the shoot length. The seedling samples were dried in a 70°C oven for 7 days, and then the dry weight of the seedlings was determined. The experiment was repeated three times.

[0080] Rice cultivation in soils treated with As and / or Cd

[0081] The control soil used in this study contained background levels of 2.09 mg / kg As and 0.44 mg / kg Cd. The control soil was supplemented with 10 mg / kg As in the form of NaAsO2 and / or 3 mg / kg Cd in the form of CdSO4. Rice seedlings were grown in the control soil in a nursery for 28 days. They were then transplanted into soil treated with or without As and / or Cd and grown to maturity in a greenhouse. Rice seeds and straw were harvested and dried for further analysis. The experiment was performed with three biological replicates.

[0082] Intact protoplasts and vacuoles were isolated from rice mesophyll cells and treated with CdSO4 or PC2-Cd complexes.

[0083] Protoplasts were isolated from rice mesophyll cells as previously described (see Trinidad et al., 2021). Briefly, buds of 10-day-old rice seedlings germinated and grown in half-strength MS medium were cut into 0.5 cm strips. Protoplasts were released from the strips by adding protoplast isolation buffer [0.6 mannitol, 10 mM methylethane sulfonate (MES), 10 mM CaCl2, 0.1% BSA (w / v), 1.5% (w / v) cellulase RS (C0615, Sigma, USA) and 0.75% (w / v) pectinase RS (P2401, Sigma, USA)] and then incubated with gentle shaking at 28 ° C in the dark for 4 hours. Protoplasts were collected by centrifugation at 150 g for 5 min at 20 ° C using a swinging bucket rotor with slow acceleration and slow deceleration. The pellet was washed twice with 20 ml of W5 buffer (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, and 2 mM MES) and recollected by spinning at 100 g for 3 min. To isolate intact vacuoles, 3 ml of lysis buffer [0.2 M mannitol, 10% Ficoll-400, 15 mM EDTA (pH 8.0), 5 mM sodium phosphate (pH 8.0)] preheated to 37°C was added to the protoplasts. The protoplasts were gently resuspended by pipetting up and down 5 to 8 times and incubated in a warm water bath at 37°C for 5 to 10 min to lyse. The protoplasts were lysed by lysis in three steps. The vacuoles released from the protoplasts were purified by centrifugation on a gradient. Two volumes of The solution (5% w / v) covered a volume of lysed protoplast suspension. The solution consisted of one volume of lysis buffer and one volume of vacuolar buffer (30 mM KCl, 20 mM HEPES-KOH, pH 7.5, 0.4 M betaine, 15 mg mL -1 A volume of vacuolar buffer was then carefully but quickly layered on top of the gradient. After centrifugation at 1,500 g for 20 min, the gradient was stirred at 5% Vacuoles were collected at the interface between the solution and the vacuolar buffer. CdSO4, PC2, and DDT were mixed at a molar ratio of 1:1:1 and incubated at 25°C for 1 hour to form a PC2-Cd complex. Protoplasts were resuspended in MMG buffer (0.4 M mannitol, 15 mM MgCl2, and 4 mM MES, pH adjusted to 7.5 with KOH). 10 μM CdSO4 or the PC2-Cd complex prepared as above was added to the protoplasts in MMG buffer and incubated at room temperature in the dark for 1 hour. After incubation, the protoplasts were collected by centrifugation at 150 g for 5 minutes at 20°C. The protoplasts were washed three times with W5 buffer to remove residual Cd in the buffer. The protoplasts and vacuoles isolated from the protoplasts were used for Cd determination by ICP-MS.

[0084] Elemental analysis by inductively coupled plasma mass spectrometry (ICP-MS)

[0085] The element concentrations in brown rice (hulled but not polished rice seeds) and straw were determined by ICP-MS. Approximately 0.1 g of dry rice seeds or straw tissue was pre-digested overnight with 3 ml of a concentrated HNO3 / H2O2 mixture (5:1, v:v) at room temperature and then digested in a microwave oven (Ethos One, Milestone Technologies). After dilution, the element concentrations in the digestate were determined by ICP-MS (7700S, Agilent Technologies, USA). 1568b was used as a certified reference material (CRM) to evaluate the precision and accuracy of the analytical procedures.

[0086] Statistical analysis

[0087] Data were analyzed using two-tailed Student's t-test (*P<0.05 or **P<0.01) or one-way ANOVA followed by LSD test (significance level P<0.05). All analyses were performed using IBM SPSS Statistics 19 software.

[0088] Example 2

[0089] Generation of OsHMA3, OsABCC1, and OsPCS1 overexpression lines

[0090] Three binary constructs containing the cDNA coding regions or genomic clone coding regions of the OsHMA3, OsPCS1, and OsABCC1 genes under the control of the OsActin1 promoter were constructed and used to generate transgenic rice plants by Agrobacterium-mediated rice transformation in the T5105 genetic background ( Figure 1ATo Figure ID). Transgenic T0 plants were characterized to screen for transgenic lines carrying a single copy of T-DNA and showing transgene overexpression for further study ( Figure 2 A and Figure 2 B. Figure 3 A and Figure 3 B. Figure 4 A and Figure 4 B. Figure 5 A and Figure 5 B. Figure 6 A and Figure 6 B. Figure 8 A and Figure 8 B, Table 2). Representative strains were screened using qPCR and Southern blot hybridization. The primers and probes used are listed in Table 1. Three independent transgenic lines were selected for each gene and characterized in detail for growth, development, yield, and gene expression. All of these transgenic lines exhibited morphological phenotypes similar to the non-transgenic control T5105 in terms of plant height, panicle, and seed set rate ( Figure 2 C to Figure 2 E. Figure 3 C to Figure 3 E. Figure 4 C to Figure 4 E).

[0091] Table 2. Summary of transgenic plants generated in this study

[0092]

[0093] Example 3

[0094] The Cd concentration in the grains of the OsHMA3 overexpressing strain was significantly reduced

[0095] T5105 and OsHMA3-overexpressing strains (HMA3-L1, HMA3-L3, and HMA3-L12) were grown in control soil and soil containing 3 mg / kg of Cd in the form of CdSO4. After harvest, seeds and straw were analyzed by ICP-MS. The Cd concentrations in the grains of the OsHMA3-overexpressing strains in the control and Cd-treated soils (0.003 ± 0.001 mg / kg and 0.041 ± 0.012 mg / kg) were only 2.0% and 2.0% of the Cd concentrations in the grains of T5105 in the control experiment (0.135 ± 0.064 mg / kg and 2.010 ± 0.813 mg / kg, respectively). Figure 5C). In the straw of T5105 grown in Cd-treated soil, the Cd concentration in the nodes (nodes I and II) was relatively higher than that in the roots, internodes, leaves (sheaths and blades), and panicles (pedicels, rachises, and bracts) (Figure SD). In the straw of HMA3-L3 grown in Cd-treated soil, the roots had the highest Cd concentration compared with other straw tissues ( Figure 5 D). The Cd concentration in the roots of HMA3-L3 (78.001±7.056 mg / kg) was 6.7 times that in the roots of T5105 (11.697±2.408 mg / kg), while the Cd concentrations in stem segments I (11.704±1.447 mg / kg) and II (9.002±0.590 mg / kg) of HMA3-L3 were 9.3% and 25.0% of those in stem segments I (125.837±24.001 mg / kg) and II (36.006±7.557 mg / kg), respectively. Figure 5 D) These results indicate that P in transgenic plants Actin1 :cHAM3:T Nos Expression of the gene significantly enhanced Cd accumulation in roots and drastically reduced Cd distribution to aerial parts of plants, including seeds.

[0096] The Cd tolerance of seedlings of T5105 and OsHMA3 overexpressing lines was tested in half-strength MS medium. T5105 plants showed increasing growth retardation in response to Cd treatment at CdSO4 concentrations ranging from 10 μM to 40 μM ( Figure 5 E). Compared with untreated T5105 plants, the shoot length and dry weight of treated T5105 plants were reduced on day 14 after treatment ( Figure 5 F and Figure 5 G). Compared with untreated T5105 and untreated transgenic plants, OsHMA3 overexpressing lines grown in Cd-containing medium showed no obvious growth retardation on day 14 after Cd treatment ( Figure 5 E and Figure 5 F). The dry weight of OsHMA3 overexpressing lines was slightly lower than that of untreated rice seedlings ( Figure 5 G). However, this reduction was much lower than that of T5105 after Cd treatment ( Figure 5 G) These results indicate that overexpression of OsHMA3 confers profound tolerance to Cd.

[0097] Example 4

[0098] As concentration in rice grains of OsABCC1-overexpressing lines is partially reduced

[0099] As concentrations in seeds and straw of T5105 and independent OsABCC1 overexpressing lines grown in control soil and soil containing 10 mg / kg As in the form of NaAsO2 were determined by ICP-MS. Figure 6 C and Figure 6 D). The average grain As concentrations of the OsABCC1 overexpressing lines grown in the control soil and As-treated soil (0.013±0.005 mg / kg and 0.197±0.024 mg / kg) were 72.2% and 53.7% of the average grain As concentration of T5105 in the control experiment (0.018±0.008 mg / kg and 0.367±0.068 mg / kg), respectively. Figure 6 C). For plants grown in As-treated soil, As concentrations in roots, stems (nodes I, II, and internode II), and lower leaves of ABCC1-L27 were higher than those of T5105, whereas As concentrations in flag leaves and panicles (pedicels, rachises, and bracts) of ABCC1-L27 were similar to or lower than those of T5105 ( Figure 6 D). The As tolerance of seedlings of T5105 and OsABCC1 overexpressing lines was tested in half-strength MS medium. When T5105 and OsABCC1 overexpressing lines were treated with 25 μM As(III) in the form of NaAsO2, no significant differences in shoot length and dry weight per plant were observed between T5105 and OsABCC1 overexpressing lines ( Figure 6 E to Figure 6 G). However, the OsABCC1 overexpressing lines showed enhanced tolerance to 50 μM As(III) to 100 μM As(III), with longer shoot lengths and higher dry weights than T5105 ( Figure 6 E to Figure 6 G). The OsABCC1 overexpression lines were also tested for Cd tolerance at the seedling stage. Compared with T5105, the OsABCC1 overexpression lines did not show any enhanced tolerance to Cd treatment ( Figure 7 A to Figure 7 C). Further scientific studies also demonstrated that grains harvested from ABCC1-L27 grown in control or Cd-treated soils had similar levels of Cd concentration as grains harvested from T5105 ( Figure 7 D). These results indicate that P Actin1 :gABCC1:T Nos Expression of the gene in transgenic plants increased As accumulation in roots, stems (nodes I, II, and internodes II), and lower leaves, and decreased As concentrations in grains.

[0100] Example 5

[0101] As and Cd concentrations in grains of OsPCS1 overexpressing lines were partially reduced

[0102] Overexpression of the OsPCS1 gene in T5105 resulted in a significant decrease in As and Cd concentrations in rice grains ( Figure 8 C and Figure 8 D). For rice plants grown in control soil, the average grain As concentration of the OsPCS1 overexpressing line (0.003 ± 0.001 mg / kg) was 37.5% of that of T5105 (0.008 ± 0.003 mg / kg). Figure 8 C). For rice plants grown in As-treated soil, the average grain As concentration of the OsPCS1 overexpressing line (0.064 ± 0.008 mg / kg) was 23.9% of that of T5105 (0.268 ± 0.023 mg / kg). Figure 8 In the Cd-treated planting experiment, the average grain Cd concentrations of the OsPCS1 overexpressing lines grown in the control soil and Cd-treated soil (0.053±0.011 mg / kg and 1.042±0.122 mg / kg) were 46.1% and 60.0% of the average grain Cd concentration of T5105 in the control experiment (0.115±0.018 mg / kg and 1.736±0.070 mg / kg), respectively. Figure 8 D). It was observed that in the OsPCS1 overexpression lines, the reduction in grain As concentration was more significant than that in grain Cd concentration ( Figure 8 C). As and Cd concentrations in different parts of rice straw were also measured. When grown in As-treated soil, the OsPCS1-overexpressing line PCS1-L1 had higher As concentrations in roots, stem nodes (nodes I and II), internode II, and leaves II, but lower As concentrations in peduncles, flag leaves, rachises, and bracts compared to T5105 ( Figure 8 E). When grown in Cd-treated soil, PCS1-L1 had higher Cd concentrations in the nodes (nodes I and II), internode II, and rachis compared to T5105, but similar or even lower Cd concentrations in the roots, flag leaves, and peduncles of the culms ( Figure 8 F). Unlike the highest As concentrations detected in the roots of T5105 and PCS1-L1, the Cd concentrations in the roots of these two lines were much lower than those in the stem nodes ( Figure 8 E and Figure 8F). In the seedling test of As or Cd tolerance, the OsPCS1 overexpressing line showed slightly enhanced tolerance to 75 μM As(III) to 100 μM As(III) in the form of NaAsO2, with longer shoot length and higher dry weight than T5105 ( Figure 9 A to Figure 9 C) However, the OsPCS1 overexpression line was resistant to 10 μM Cd 2+ to 40 μM Cd 2+ showed significant hypersensitivity, with shorter shoot length and lower dry weight than T5105 ( Figure 9 D to Figure 9 F) These results indicate that in the OsPCS1 overexpression line, P Actin1 :cPCS1:T Nos The expression of the gene increased As accumulation in roots, stems, and lower leaves, and reduced As partitioning in flag leaves, panicles, and seeds. Actin1 :cPCS1:T Nos Gene expression enhanced Cd accumulation mainly in the stems of transgenic plants.

[0103] Example 6

[0104] P Actin1 :gABCC1:T Nos Gene and P Actin1 :cPCS1:T Nos Co-expression of genes showed a synergistic effect in reducing As concentration in rice grains

[0105] In rice plants, As(lll) is sequestered in the vacuole as a phytochelatin-arsenic (PC-As) complex by ABCC1 (see Hayashi et al., 2017; Song et al., 2014). To further investigate whether co-overexpression of OsABCC1 and OsPCS1 has a synergistic effect in reducing As concentrations in rice grains, AP lines were generated by crossing ABCC1-L27 with PCS1-L1, followed by self-pollination and selection of double homozygotes to sequester P. Actin1 :gABCC1:T Nos Gene and P Actin1 :cPCS1:T Nos Compared with ABCC1-L27 or PCS1-L1, AP did show synergistically enhanced tolerance to 25 μM As(III) to 100 μM As(III) at the seedling stage ( Figure 10 A to Figure 10C). In addition, AP had higher As concentrations in roots and lower As concentrations in shoots compared with ABCC1-L27 or PCS1-L1, especially when treated with 75 μM As(III) at the seedling stage ( Figure 10 D and Figure 10 E). The As concentration in the grains of AP grown in As-treated soil (0.025 ± 0.009 mg / kg) was much lower than that in the grains of ABCC1-L27 (0.149 ± 0.000 mg / kg) or PCS1-L1 (0.067 ± 0.002 mg / kg), and was only 10.0% of that in the grains of T5105 (0.250 ± 0.008 mg / kg). Figure 10 F). These results indicate that P Actin1 :gABCCI:T Nos Gene and P Actin1 :cPCS1:T Nos Co-expression of the genes in transgenic rice plants showed synergistic effects in reducing As concentrations in rice grains and providing enhanced tolerance to As treatment at the seedling stage.

[0106] Example 7

[0107] Co-overexpression of the OsHMA3 gene alleviated the hypersensitivity of the OsPCS1-overexpressing strain to Cd treatment

[0108] To investigate whether co-overexpression of the OsHMA3 gene could alleviate the hypersensitivity of the OsPCS1 overexpressing line to Cd treatment, a double homozygous hybrid carrying P was generated by crossing PCS1-L1 and HMA3-L3, followed by self-pollination and selection. Actin1 :cPCS1:T Nos Gene and P Actin1 :cHMA3:T Nos The transgenic line (HP) of the gene. 2+ Seedlings of HP, PCS1-L1, HMA3-L3, and T5105 were tested on half-strength MS medium. Compared with HMA3-L3, the HP line provided enhanced tolerance to similar levels of Cd treatment and had longer shoot length and higher dry weight than PCS1-L1 or T5105 ( Figure 11 A to Figure 11 C). ICP-MS analysis of tissues from treated seedlings showed that HP plants accumulated more Cd in roots and distributed less Cd in shoots compared with PCS1-L1 or T5105 plants, a pattern similar to that observed in HMA3-L3 plants ( Figure 11 D and Figure 11E). The average Cd concentrations in the grains of HP plants grown in the control and Cd-treated soils (0.004 ± 0.001 mg / kg and 0.032 ± 0.011 mg / kg) were similar to those in the grains of HMA3-L3 plants (0.004 ± 0.001 mg / kg and 0.036 ± 0.006 mg / kg), but were significantly lower than those in the grains of PCS1-L1 (0.085 ± 0.008 mg / kg and 0.889 ± 0.093 mg / kg) and T5105 (0.115 ± 0.019 mg / kg and 1.913 ± 0.630 mg / kg) in the control experiment ( Figure 11 F).

[0109] To further investigate the effect of OsPCS1 overexpression on Cd sequestration in rice vacuoles, protoplasts and vacuoles were isolated from shoots of 10-day-old T5105 and PCS1-L1 seedlings grown in Cd-containing medium, and Cd concentrations were determined by ICP-MS analysis. 6 cells) and PCS1-L1 (49.839±3.694ng / 10 6 There was no significant difference in Cd concentration in the protoplasts of Figure 12 A). However, the Cd concentration in the vacuole of PCS1-L1 (24.429±1.498ng / 10 6 The Cd concentration in the vacuole of T5105 cells was only 46.809±1.933ng / 10 6 cells) of 52.2% ( Figure 12 A). These results indicate that P in PCS1-L1 Actin1 :cPCS1:T Nos The expression of the gene inhibits the sequestration of Cd in the vacuole. Overexpression of the OsPCS1 gene in rice will increase PC synthesis and form more PC-Cd complexes in the cytosol. The next step of the study is to investigate whether the PC2-Cd complex affects the sequestration of Cd in the vacuole by HMA3. Protoplasts isolated from T5105 and HMA3-L3 were respectively exposed to Cd 2+ The protoplasts were incubated with the PC2-Cd complex. The Cd concentrations in the incubated protoplasts and their vacuoles were then determined. Similar levels of Cd concentrations were detected in all protoplast samples, indicating that both Cd and PC2-Cd can be transported into the protoplasts of T5105 or HMA3-L3 with similar efficiency ( Figure 12 B) For Cd 2+ The Cd concentration in the vacuoles of HMA3-L3 protoplasts incubated together was 1.318±0.073ng / 106 The Cd concentration in the vacuole of T5105 cells was 0.366±0.081 ng / 10 6 This indicates that overexpression of OsHMA3 enhanced the 2+ Sequestration in vacuoles ( Figure 12 B). For protoplasts incubated with PC2-Cd, T5105 (0.141±0.029ng / 10 6 cells) and HMA3-L3 (0.423±0.058ng / 10 6 The Cd concentrations in the vacuoles of protoplasts and Cd 2+ The Cd concentrations in the vacuoles of T5105 and HMA3-L3 incubated together were 38.5% and 32.1% ( Figure 12 B). Meanwhile, the Cd concentration in the vacuoles of HMA3-L3 cells incubated with PC2-Cd (0.423±0.058ng / 10 6 cells) is the Cd concentration in the vacuoles of T5105 in the control experiment (0.141±0.029 ng / 10 6 cells) Figure 12 B) These results indicate that the PC2-Cd complex inhibits HMA3-mediated Cd 2+ OsHMA3 overexpression can alleviate the inhibition caused by the overproduction of PC2 or other PCs due to overexpression of OsPCS1 in rice cells.

[0110] Example 8

[0111] By stacking P in a single rice line Actin1 :cHMA3:T Nos 、P Actin1 :cPCS1:T Nos and P Actin1 :gABCC1:T Nos Producing rice grains with low As and Cd

[0112] To produce rice grains with low concentrations of both As and Cd, the P from PCS1-L1 was Actin1 :cPCS1:T Nos gene, P from ABCC1-L27 Actin1 :gABCC1:T Nos gene and P from HMA3-L3 Actin1 :cHMA3:T NosGenetic hybridization and marker-assisted selection were used to develop a rice line (PAH) that co-overexpresses the OsPCS1, OsABCC1, and OsHMA3 genes. Like its parental transgenic line, the PAH line grew and developed normally, and its plant architecture, growth period, panicle and seed size, seed set rate, and 100-grain weight were similar to those of T5105 ( Figure 13 A to Figure 13 E). PAH, PCS1-L1, ABCC1-L27, HMA3-L3, and T5105 plants were grown in control soil and soil treated with both As and Cd, and grain As and Cd concentrations were determined by ICP-MS analysis. For plants grown in control soil, the grain As concentration of PAH (0.002 ± 0.000 mg / kg) was 18.2% of the grain As concentration of T5105 (0.011 ± 0.001 mg / kg), while the grain Cd concentration of PAH (0.004 ± 0.001 mg / kg) was 3.5% of the grain Cd concentration of T5105 (0.115 ± 0.019 mg / kg). Figure 13 F and Figure 13 For plants grown in soils treated with As and Cd, the grain As concentration of PAH (0.027 ± 0.002 mg / kg) was 7.9% of the grain As concentration of T5105 (0.343 ± 0.017 mg / kg), while the grain Cd concentration of PAH (0.036 ± 0.006 mg / kg) was 2.0% of the grain Cd concentration of T5105 (1.809 ± 0.136 mg / kg). Figure 13 F and Figure 13 G). In both soil experiments, As or Cd concentrations in the grains of PAH were lower than or comparable to those in the grains of the parental line carrying a single transgene ( Figure 13 F and Figure 13 G). Notably, overexpression of the OsHMA3 gene in HMA3-L3 did not produce any significant changes in grain As concentration compared with T5105, and overexpression of the OsABCC1 gene in ABCC1-L27 did not affect grain Cd concentration ( Figure 13 F and Figure 13 G). These results indicate that PC-dependent and OsABCC1-mediated vacuolar sequestration of As and OsHMA3-mediated vacuolar sequestration of Cd are independent of each other. ICP-MS analysis also showed that the concentrations of micronutrients including cobalt (Co), copper (Cu), iron (Fe), manganese (Mn), selenium (Se), and zinc (Zn) in the grains of the four strains grown in control soil or soils treated with As and Cd did not show any significant differences ( Figure 14 ). In the presence of 50 μM As(III) and 10 μM Cd2+ (Treatment 1) or containing 75 μM As(III) + 20 μM Cd 2+ Seedlings of the transgenic lines and T5105 were tested in half-strength MS medium (treatment 2). In both treatments, PAH seedlings showed the highest enhanced tolerance to dual As and Cd treatments among all the lines tested and had the longest shoot length and the largest dry weight ( Figure 15 A to Figure 15 C). Compared with T5105, PAH seedlings accumulated much higher concentrations of As or Cd in roots and lower concentrations of As or Cd in shoots ( Figure 15 D to Figure 15 G) These results together indicate that the co-expressed P Actin1 :cPCS1:T Nos Gene, P Actin1 :gABCC1:T Nos Gene and P Actin1 :cHMA3:T Nos The gene significantly reduced the As and Cd concentrations in rice grains by increasing the accumulation and sequestration of two toxic elements, As and Cd, in the roots and reducing the transport of As and Cd to the aboveground tissues and organs, especially seeds. It also provided enhanced tolerance to heavy As and Cd treatments at the seedling stage without any impairment of plant growth, development, yield and micronutrients.

[0113] In summary, genetic engineering methods were used to generate transgenic plants overexpressing OsABCC1, OsPCS1, and OsHMA3 under the OsActin1 promoter in the T5105 genetic background. Actin1 :cPCS1:T Nos Gene or P Actin1 :gABCC1:T Nos The gene partially reduced the As concentration in the grains of transgenic rice plants. Actin1 :cPCS1:T Nos Gene and P Actin1 :gABCC1:T Nos Co-expression of the genes significantly reduced the As concentration in the grains of the double-gene overexpressing transgenic plants. Actin1 ;cHMA3:T Nos The expression of the gene significantly reduced the Cd concentration in the grains of the transgenic rice lines. Actin1 :cPCS1:T Nos Gene, P Actin1 :gABCC1:T Nos Gene and P Actin1 :cHMA3:T NosCoexpression of the genes significantly reduced As and Cd concentrations in the grains of the triple-gene-overexpressing transgenic plants. All transgenic plants displayed normal growth and development similar to the non-transgenic T5105 strain, without any pleiotropic phenotypes or yield loss. Low-As and low-Cd rice will reduce human exposure to both toxic elements through rice consumption and benefit human health.

[0114] References

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Claims

1. A genetically modified rice plant or plant cell comprising a heterologous P operably linked to an OsActin1 promoter. 1B a heavy metal ATPase gene, a heterologous ATP-binding cassette (ABC) transporter gene operably linked to an OsActin1 promoter, and a heterologous phytochelatin synthase gene operably linked to an OsActin1 promoter, wherein the OsActin1 promoter has low activity in the seed endosperm of the modified rice plant compared to its activity in other vegetative tissues of the modified rice plant; The genetically modified rice plants have reduced arsenic (As) and cadmium (Cd) in rice grains compared to control rice plants that have not undergone the genetic modification. 2 . The genetically modified rice plant or plant cell according to claim 1 , wherein the OsActin1 promoter comprises the nucleic acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 31 or a functional sequence variant thereof.

3. The genetically modified rice plant or plant cell according to claim 1 or 2, wherein the heterologous P 1B The type heavy metal ATPase gene encodes the amino acid sequence shown in SEQ ID NO: 39; the heterologous ABC transporter gene encodes the amino acid sequence shown in SEQ ID NO: 37, and the phytochelatin synthase gene encodes the amino acid sequence shown in SEQ ID NO:

38.

4. The genetically modified rice plant or plant cell of claim 3, wherein the heterologous P 1B The type heavy metal ATPase gene comprises a nucleic acid sequence that, due to the degeneracy of the genetic code, has at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity with the polynucleotide sequence of SEQ ID NO: 22; the heterologous ABC transporter gene comprises a nucleic acid sequence that, due to the degeneracy of the genetic code, has at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity with the polynucleotide sequence of SEQ ID NO: 36; and the heterologous phytochelatin synthase gene comprises a nucleic acid sequence that, due to the degeneracy of the genetic code, has at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity with the polynucleotide sequence of SEQ ID NO:

32.

5. The genetically modified rice plant or plant cell according to any one of claims 1 to 4, wherein the exogenous P 1B The type heavy metal ATPase gene, the exogenous ABC transporter gene and / or the exogenous phytochelatin synthase gene are from cereal crops.

6. The genetically modified rice plant or plant cell according to claim 4 or 5, wherein the heterologous P 1B The type heavy metal ATPase gene is OsHMA3 and comprises the nucleic acid sequence shown in SEQ ID NO: 22, the heterologous ABC transporter gene is OsABCC1 and comprises the nucleic acid sequence shown in SEQ ID NO: 36, and the heterologous phytochelatin synthase gene is OsPCS1 and comprises the nucleic acid sequence shown in SEQ ID NO:

32.

7. The genetically modified rice plant or plant cell of claim 6, comprising a heterologous OsHMA3 gene operably linked to an OsActin1 promoter, a heterologous OsABCC1 gene operably linked to an OsActin1 promoter, and a heterologous OsPCS1 gene operably linked to an OsActin1 promoter.

8. The genetically modified rice plant or plant cell according to any one of claims 1 to 7, which is of the species Oryza sativa L.

9. A method for constructing a genetically modified rice plant having reduced arsenic (As) and cadmium (Cd) in rice grains compared to rice grains of a control rice plant, the method comprising the steps of: a) Producing a heterologous P gene operably linked to an OsActin1 promoter 1B Genetically modified rice plants expressing a heavy metal ATPase gene; b) producing a genetically modified rice plant comprising a heterologous ATP-binding cassette (ABC) transporter gene operably linked to an OsActin1 promoter; c) producing a genetically modified rice plant comprising a heterologous phytochelatin synthase gene operably linked to an OsActin1 promoter; d) Select and overexpress the exogenous P 1B a genetically modified rice plant containing a type 1 heavy metal ATPase gene, the exogenous ATP-binding cassette (ABC) transporter gene, and the exogenous phytochelatin synthase gene; e) crossing two of the three genetically modified rice plants to produce a plant that is doubly homozygous for the exogenous gene; as well as f) crossing the double homozygous plant of step (e) with a third genetically modified rice plant to produce a triple homozygous plant overexpressing the exogenous gene. 10 . The method according to claim 9 , wherein the OsActin1 promoter is as defined in claim 2 .

11. The method according to claim 9 or 10, wherein the heterologous gene is as defined in any one of claims 3 to 6.

12. A kit for constructing a genetically modified rice plant, wherein the rice grains of the genetically modified rice plant have reduced arsenic (As) and cadmium (Cd) compared to rice grains of a control rice plant, wherein the kit comprises: Bacteria comprising a vector comprising a heterologous heavy metal ATPase gene operably linked to an OsActin1 promoter, and / or bacteria comprising a vector comprising a heterologous ATP-binding cassette (ABC) transporter gene operably linked to an OsActin1 promoter, and / or bacteria comprising a vector comprising a heterologous phytochelatin synthase gene operably linked to an OsActin1 promoter. 13 . The kit according to claim 12 , wherein the OsActin1 promoter comprises the nucleic acid sequence shown in SEQ ID NO: 21 or SEQ ID NO: 31 or a functional sequence variant thereof.

14. The kit according to claim 12 or 13, wherein the heterologous P 1B The type heavy metal ATPase gene encodes the amino acid sequence shown in SEQ ID NO: 39; the heterologous ABC transporter gene encodes the amino acid sequence shown in SEQ ID NO: 37, and the phytochelatin synthase gene encodes the amino acid sequence shown in SEQ ID NO:

38.

15. The kit according to claim 14, wherein the heterologous P 1B The type heavy metal ATPase gene comprises a nucleic acid sequence that, due to the degeneracy of the genetic code, has at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity with the polynucleotide sequence of SEQ ID NO: 22; the heterologous ABC transporter gene comprises a nucleic acid sequence that, due to the degeneracy of the genetic code, has at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity with the polynucleotide sequence of SEQ ID NO: 36; and the heterologous phytochelatin synthase gene comprises a nucleic acid sequence that, due to the degeneracy of the genetic code, has at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity, or 100% sequence identity with the polynucleotide sequence of SEQ ID NO:

32.

16. The kit according to any one of claims 12 to 15, wherein the exogenous P 1B The type heavy metal ATPase gene, the exogenous ABC transporter gene and / or the exogenous phytochelatin synthase gene are from cereal crops.

17. The kit according to claim 15 or 16, wherein the heterologous P 1B The type heavy metal ATPase gene is OsHMA3 and comprises the nucleic acid sequence shown in SEQ ID NO: 22, the heterologous ABC transporter gene is OsABCC1 and comprises the nucleic acid sequence shown in SEQ ID NO: 36, and the heterologous phytochelatin synthase gene is OsPCS1 and comprises the nucleic acid sequence shown in SEQ ID NO: 32.

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