GENERATION OF LOW-ARSENIC AND LOW-CADMIUM RICE
20250388917 ยท 2025-12-25
Assignee
Inventors
Cpc classification
C12N9/00
CHEMISTRY; METALLURGY
C12Y203/02015
CHEMISTRY; METALLURGY
International classification
C12N15/82
CHEMISTRY; METALLURGY
C12N9/00
CHEMISTRY; METALLURGY
Abstract
The present invention relates to a genetically modified rice plant or plant cell, comprising a heterologous heavy metal ATPase gene operably linked to an OsActin1 promoter: 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 a seed endosperm of the modified rice plant compared to its activity in other vegetative tissues of the modified rice plant; wherein a rice grain of said genetically modified rice plant has reduced arsenic (As) and cadmium (Cd) compared to a control rice plant that has not undergone said genetic modification. The present invention also relates to a method of creating such a genetically modified rice plant or plant cell, and a kit for doing so.
Claims
1. A genetically modified rice plant or plant cell, comprising a heterologous P.sub.1B-type heavy metal ATPase gene operably linked to an OsActin1 promoter; a heterologous ATP-binding cassette (ABC) transporter gene operably linked to an OsActin1 promoter, and a heterologous phytochelatin synthase gene operably linked to an OsActin promoter, wherein the OsActin1 promoter has low activity in a seed endosperm of the modified rice plant compared to its activity in other vegetative tissues of the modified rice plant; wherein a rice grain of said genetically modified rice plant has reduced arsenic (As) and cadmium (Cd) compared to a control rice plant that has not undergone said genetic modification.
2. The genetically modified rice plant or plant cell according to claim 1, wherein the OsActin1 promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 21 or 31 or a functional sequence variant thereof.
3. The genetically modified rice plant or plant cell according to claim 1, wherein the heterologous P.sub.1B-type heavy metal ATPase gene encodes the amino acid sequence set forth in SEQ ID NO: 39; the heterologous ABC transporter gene encodes the amino acid sequence set forth in SEQ ID NO: 37, and the phytochelatin synthase gene encodes the amino acid sequence set forth in SEQ ID NO: 38.
4. The genetically modified rice plant or plant cell according to claim 3, wherein the heterologous P.sub.1B-type heavy metal ATPase gene comprises a nucleic acid sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity or 100% sequence identity, due to the degeneracy of the genetic code, to the polynucleotide sequence set forth in SEQ ID NO: 22, the heterologous ABC transporter gene comprises a nucleic acid sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity or 100% sequence identity, due to the degeneracy of the genetic code, to the polynucleotide sequence set forth in SEQ ID NO: 36, and the heterologous phytochelatin synthase gene comprises a nucleic acid sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity or 100% sequence identity, due to the degeneracy of the genetic code, to the polynucleotide sequence set forth in SEQ ID NO: 32.
5. The genetically modified rice plant or plant cell according to claim 1, wherein the exogenous P.sub.1B-typ heavy metal ATPase gene, the exogenous ABC transporter gene and/or the exogenous phytochelatin synthase gene are from a cereal crop.
6. The genetically modified rice plant or plant cell according to claim 4, wherein the heterologous P.sub.1B-typ heavy metal ATPase gene is OsHMA3 and comprises the nucleic acid sequence set forth in SEQ ID NO: 22, the heterologous ABC transporter gene is OsABCC1 and comprises the nucleic acid sequence set forth in SEQ ID NO: 36, and the heterologous phytochelatin synthase gene is OsPCS1 and comprises the nucleic acid sequence set forth 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 of claim 1, of the species Oryza sativa L.
9. A method of creating a genetically modified rice plant, that has reduced arsenic (As) and cadmium (Cd) in its rice grain compared to a rice grain from a control rice plant, the method comprising the steps of: a) generating a genetically modified rice plant comprising a heterologous P.sub.1B-type heavy metal ATPase gene operably linked to an OsActin/promoter; b) generating a genetically modified rice plant comprising a heterologous ATP-binding cassette (ABC) transporter gene operably linked to an OsActin/promoter; c) generating a genetically modified rice plant comprising a heterologous phytochelatin synthase gene operably linked to an OsActin1 promoter; d) select genetically modified rice plants which, respectively, overexpress said exogenous P.sub.1B-type heavy metal ATPase gene, said exogenous ATP-binding cassette (ABC) transporter gene and said exogenous phytochelatin synthase gene; e) crossing two of the three genetically modified rice plants to generate double homozygote plants with respect to the exogenous genes; and f) crossing a double homozygote plant from step (e) with a third genetically modified rice plant to generate triple homozygote plants that overexpress the exogenous genes.
10. The method according to claim 9, wherein the OsActin1 promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 21 or 31 or a functional sequence variant thereof.
11. The method according to claim 9, wherein: the heterologous P.sub.1B-type heavy metal ATPase gene encodes the amino acid sequence set forth in SEQ ID NO: 39, the heterologous ABC transporter gene encodes the amino acid sequence set forth in SEQ ID NO: 37, and the phytochelatin synthase gene encodes the amino acid sequence set forth in SEQ ID NO: 38.
12. A kit for creating a genetically modified rice plant, having reduced arsenic (As) and cadmium (Cd) in its rice grain compared to a rice grain from a control rice plant, wherein the kit comprises bacteria containing vectors comprising a heterologous heavy metal ATPase gene operably linked to an OsActin1 promoter and/or bacteria containing vectors comprising a heterologous ATP-binding cassette (ABC) transporter gene operably linked to an OsActin1 promoter and/or bacteria containing vectors comprising a heterologous phytochelatin synthase gene operably linked to an OsActin1 promoter.
13. The kit of claim 12, wherein the OsActin1 promoter comprises the nucleic acid sequence set forth in SEQ ID NO: 21 or 31 or a functional sequence variant thereof.
14. The kit according to claim 12, wherein the heterologous P.sub.1B-type heavy metal ATPase gene encodes the amino acid sequence set forth in SEQ ID NO: 39; the heterologous ABC transporter gene encodes the amino acid sequence set forth in SEQ ID NO: 37, and the phytochelatin synthase gene encodes the amino acid sequence set forth in SEQ ID NO: 38.
15. The kit according to claim 14, wherein the heterologous P.sub.1B-type heavy metal ATPase gene comprises a nucleic acid sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity or 100% sequence identity, due to the degeneracy of the genetic code, to the polynucleotide sequence set forth in SEQ ID NO: 22, the heterologous ABC transporter gene comprises a nucleic acid sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity or 100% sequence identity, due to the degeneracy of the genetic code, to the polynucleotide sequence set forth in SEQ ID NO: 36, and the heterologous phytochelatin synthase gene comprises a nucleic acid sequence having at least 80% sequence identity, at least 85% sequence identity, at least 90% sequence identity, at least 95% sequence identity or 100% sequence identity, due to the degeneracy of the genetic code, to the polynucleotide sequence set forth in SEQ ID NO: 32.
16. The kit according to claim 12, wherein the exogenous P.sub.1B-typ heavy metal ATPase gene, the exogenous ABC transporter gene and/or the exogenous phytochelatin synthase gene are from a cereal crop.
17. The kit according to claim 15, wherein the heterologous P.sub.1B-typ heavy metal ATPase gene is OsHMA3 and comprises the nucleic acid sequence set forth in SEQ ID NO: 22, the heterologous ABC transporter gene is OsABCC1 and comprises the nucleic acid sequence set forth in SEQ ID NO: 36, and the heterologous phytochelatin synthase gene is OsPCS1 and comprises the nucleic acid sequence set forth in SEQ ID NO: 32.
Description
BRIEF DESCRIPTION OF THE FIGURES
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DETAILED DESCRIPTION OF THE INVENTION
[0044] Bibliographic references mentioned in the present specification are for convenience listed in the form of a list of references and added at the end of the examples. The whole content of such bibliographic references is herein incorporated by reference.
Definitions
[0045] For convenience, certain terms employed in the specification, examples and appended claims are collected here.
[0046] The term comprising is herein defined to be that where the various components, ingredients, or steps, can be conjointly employed in practising the present invention. Accordingly, the term comprising encompasses the more restrictive terms consisting essentially of and consisting of.
[0047] The term Agrobacterium refers to a soil-borne, Gram-negative, rod-shaped phytopathogenic bacterium which causes crown gall. The term Agrobacterium includes, but is not limited to, the strains Agrobacterium tumefaciens (which typically causes crown gall in infected plants), and Agrobacterium rhizogens (which causes hairy root disease in infected host plants). Infection of a plant cell with Agrobacterium generally results in the production of opines (e.g., nopaline, agropine, octopine etc.) by the infected cell.
[0048] The term expression when used in reference to a nucleic acid sequence, such as a gene, refers to the process of converting genetic information encoded in a gene into RNA (e.g., mRNA, rRNA, tRNA, or snRNA) through transcription of the gene (i.e., via the enzymatic action of an RNA polymerase), and into protein where applicable (as when a gene encodes a protein), through translation of mRNA. Gene expression can be regulated at many stages in the process. Up-regulation or activation refers to regulation that increases the production of gene expression products (i.e., RNA or protein), while down-regulation or repression refers to regulation that decrease production. Molecules (e.g., transcription factors) that are involved in up-regulation or down-regulation are often called activators and repressors, respectively. Overexpression refers to an expression level higher than what would normally be observed for a particular gene.
[0049] 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), the manipulation of which may be deemed desirable for any reason (e.g., treat disease, confer improved qualities, etc.), by one of ordinary skill in the art. Such nucleotide sequences include, but are not limited to, coding sequences of structural genes (e.g., reporter genes, selection marker genes, oncogenes, drug resistance genes, growth factors, etc.), and non-coding regulatory sequences which do not encode an mRNA or protein product (e.g., promoter sequence, polyadenylation sequence, termination sequence, enhancer sequence, etc.).
[0050] The terms amino acid or amino acid sequence, as used herein, refer to an oligopeptide, peptide, polypeptide, or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules. Where amino acid sequence is recited herein to 15 refer to an amino acid sequence of a naturally occurring protein molecule, amino acid sequence and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
[0051] As used herein, the term functional sequence variant refers to a polynucleotide sequence (reference or wild-type sequence) that is altered by one or more nucleic acids without abolishing or substantially altering the polynucleotide activity of the non-variant reference. For example, the OsActin1 promoter defined by SEQ ID NO: 21 or SEQ ID NO: 31 may be truncated or have one or more nucleic acids removed internally and retain activity.
[0052] The term gene encompasses the coding regions of a structural gene and includes sequences located adjacent to the coding region on both the 5 and 3 ends for a distance of about 1 kb on either end such that the gene corresponds to the length of the full-length mRNA. The sequences which are located 5 of the coding region and which are present on the mRNA are referred to as 5 non-translated sequences. The sequences which are located 3 or downstream of the coding region and which are present on the mRNA are referred to as 3 non-translated sequences. The term gene encompasses both cDNA and genomic forms of a gene. A genomic form or clone of a gene contains the coding region termed exon or expressed regions or expressed sequences interrupted with non-coding sequences termed introns or intervening regions or intervening sequences.
[0053] The term seed as used herein includes all tissues that result from the development of a fertilized plant egg; thus, it includes a matured ovule containing an embryo and stored nutrients, as well as an integument or integuments that differentiated into a protective seed coat or testa. The nutrients in seed tissues may be stored in the endosperm or in the body of the embryo, notably in the cotyledons, or both.
[0054] As used herein, the term seed may also refer to a mature and fertilized, i.e. ripened, ovule of a seed plant comprising a plant embryo (i.e. miniature plant) and further comprising an endosperm (i.e. supply of food for the plant embryo) and may be enclosed by a seed coat.
[0055] As used herein, the term rice in reference to a rice plant is an Oryza spp., i.e. cultivated varieties, noncultivated rice plants and ancestral rice plants. Preferably the rice plant of the invention is an Oryza sativa variety.
[0056] The term heterologous when used in reference 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 gene native to an organism that has been altered in some way (e.g., mutated, added in multiple copies, linked to a non-native promoter or enhancer sequence, etc.). Heterologous genes may comprise plant gene sequences that comprise cDNA forms of a plant gene; the cDNA sequences may be expressed in either a sense (to produce mRNA) or anti-sense orientation (to produce an anti-sense RNA transcript that is complementary to the mRNA transcript). Heterologous genes are distinguished from endogenous plant genes in that the heterologous gene sequences are typically joined to nucleotide sequences comprising regulatory elements such as promoters that are not found naturally associated with the gene for the protein encoded by the heterologous gene or with plant gene sequences in the chromosome, or are associated with portions of the chromosome not found in nature (e.g., genes expressed in loci where the gene is not normally expressed).
[0057] The term transgene refers to a foreign gene that is placed into an organism by the process of transfection. The term foreign gene refers to any nucleic acid (e.g., gene sequence) that is introduced into the genome of an organism by experimental manipulations and may include gene sequences found in that organism so long as the introduced gene does not reside in the same location, as does the naturally occurring gene. A transgene may also refer to an exogenous gene such that exogenous genes include but are not limited to reporter genes, marker genes, selection genes, and functional genes. The term endogenous gene refers to a gene naturally encoded and expressed.
[0058] The terms transformants and transformed cells include the primary transformed cell and cultures derived from that cell without regard to the number of transfers. Resulting progeny may not be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that has the same functionality as screened for in the originally transformed cell are included in the definition of transformants.
[0059] The terms in operable combination, in operable order and operably linked refer to the linkage of nucleic acid sequences in such a manner that a nucleic acid molecule capable of directing the transcription of a given gene and/or the synthesis of a desired protein molecule is produced. The term also refers to the linkage of amino acid sequences in such a manner so that a functional protein is produced.
[0060] The terms overexpressed, overexpression and overexpressing and grammatical equivalents, are specifically used in reference to levels of mRNA to indicate a level of expression approximately 3-fold higher than that typically observed in a given tissue in a control or non-transgenic animal. Levels of mRNA are measured using any of a number of techniques known to those skilled in the art including, but not limited to qRT-PCR.
[0061] The terms promoter element, promoter, or promoter sequence refer to a DNA sequence that is located at the 5 end (i.e. precedes) of the coding region of a DNA polymer. The location of most promoters known in nature precedes the transcribed region. The promoter functions as a switch, activating the expression of a gene. If the gene is activated, it is said to be transcribed, or participating in transcription. Transcription involves the synthesis of mRNA from the gene. The promoter, therefore, serves as a transcriptional regulatory element and also provides a site for initiation of transcription of the gene into mRNA.
[0062] The term vector refers to nucleic acid molecules that transfer DNA segment(s). Transfer can be into a cell, cell to cell, etc. The term vehicle is sometimes used interchangeably with vector.
[0063] The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.
Example 1
Materials and Methods
Plant Materials and Growth Conditions
[0064] The rice cultivar used in this study was T5105, an improved aromatic rice in the genetic background of Thai fragrance KTML 105 (Luo and Yin, 2013). Treated or untreated T5105 and transgenic plants were grown in pot soils in a greenhouse at 24-33 C. with a photoperiod of 12 h daylight and 12 h darkness and relative humidity at 80-85%.
Genes, Constructs and Rice Transformation
[0065] Constructs for gene over-expression in rice was made based on binary vector pCAMBIA1305.1 (Accession no. AF304545) as shown schematically in
Southern Blotting Analysis
[0066] Genome DNA from transgenic rice was extracted using E.Z.N.A. HP plant DNA mini kit (Omega BIO-TEK). About 2 g DNA was digested with restriction enzymes Hind II and BamH I (NEB). DNA fragments were separated on a 0.8% (w/v) agarose gel by gel electrophoresis. The fragments then were blotted from the agarose gel onto a Hybond-N.sup.+ membrane (GE Healthcare). Digoxigenin labelled specific nucleic acid probes for hpt gene were amplified by PCR using DIG DNA labelling Mix (Roche) and primer pairs listed in Table 1. Southern blot hybridization and detection of the DIG-labelled probes were performed according to manufacturer's instruction using DIG-High Prime DNA Labeling and Detection Starter Kit II (Roche). ChemiDoc Touch imaging system (Bio-Rad) was used to detect the chemiluminescent signal.
TABLE-US-00001 TABLE1 Oligoprimersusedinthisstudy SEQ Nameofprimer DNAsequences(5to3) Purpose IDNO: ABCC1-F TTGTAGGTAGAAGCCATGGGTTTTGA Making 1 TCCACTG construct ABCC1-R CACCTTACTTTGCCGTCACAAGCTTC Making 2 TACATTTGGTCCCAGTC construct ABCC1-qPCR-F AACAGTGGCTTATGTTCCTCAAG qRT-qPCR 3 ABCC1-qPCR-R AACTCCTCTTTCTCCAATCTCTG qRT-qPCR 4 PCS1-qPCR-F AGCCCAAGTAAAGAGGCTAAC qRT-qPCR 5 PCS1-qPCR-R TACAACAGGGCTGCTTAGAAC qRT-qPCR 6 HMA3-qPCR-F CAGAACAGCAGGTCGAAGAC qRT-qPCR 7 HMA3-qPCR-R CCATTGCTCAAGGCCATCT qRT-qPCR 8 EF-qPCR-F GCACGCTCTTCTTGCTTTC qRT-PCR 9 EF-qPCR-R AGGGAATCTTGTCAGGGTTG qRT-PCR 10 HPT-F AGCCTGAACTCACCGCGACGT DNAprobe 11 HPT-R TACTTCTACACAGCCATCGGTCCA DNAprobe 12 LB-intact-F CATTGCGGACGTTTTTAATGTAC Characterizing 13 transgenein transgenicrice LB-intact-R TCTCGATGAGCTGATGCTTTGG Characterizing 14 transgenein transgenicrice RB-intact-ABCC1-F GGAAGCACAACACCGAAATTG Characterizing 15 transgenein transgenicrice RB-intact-PCS1-F CACCGATATCCTCGACTGAAAC Characterizing 16 transgenein transgenicrice RB-intact-HM3-F ATGGTGTTGGTCGTTGCT Characterizing 17 transgenein transgenicrice RB-intact-R CCCGATCTAGTAACATAGATG Characterizing 18 transgenein transgenicrice
Gene Expression Analyses
[0067] Total RNA was extracted using a Favorprep plant total RNA purification mini kit (FAVORGEN) followed by DNA digestion using DNase I (Roche). The first-strand cDNAs were 5 synthesised from 1 g of total RNA using cDNA synthesis kit (Bio-Rad). Quantitative real-time PCR (qRT-PCR) was performed on CFX96 real-time system (Bio-Rad) using SYBR FAST qPCR Master Mix (KAPA Biosystems). The expression level of the rice elongation factor (EF) gene OsEF-1 (Os03g0178000) gene was used as the internal control. The primers for qRT-PCR of different genes are listed in Table 1.
Test of Rice Seedlings for Tolerance to as and Cd
[0068] Rice seeds were surface-sterilized and germinated on half-strength MS medium in Phytatray II vessels (Sigma-Aldrich) at 25 C. in a tissue culture room with a photoperiod of 16 h light and 8 h darkness. Two-week-old rice seedlings were transferred to half-strength MS medium containing different concentrations of NaAsO.sub.2 (0-100 UM) and/or CdSO.sub.4 (0-40 M) and cultured for another 14 d. The roots of treated seedlings were washed for three times with 5 mM CaCl.sub.2) and deionized water, respectively. They were photographed before the shoot length were measured. The seedling samples were dried at 70 C. in an oven for 7 d and the dry weight of the seedlings was measured. The experiments were conducted with three biological replicates.
Plantation of Rice in Soils Treated with as and/or Cd
[0069] The control soil used in this study contained background levels of As at 2.09 mg/kg and Cd at 0.44 mg/kg. The control soil was supplemented with 10 mg/kg As in the form of NaAsO.sub.2 and/or 3 mg/kg Cd in the form of CdSO.sub.4. Rice seedlings were grown in control soil in nursey for 28 d. There were then transplanted onto soils with or without As and/or Cd treatment and grown to maturity in the greenhouse. Rice seeds and straw were harvested and dried for further analysis. The experiments were conducted with three biological replicates.
Isolation of Intact Protoplasts and Vacuoles from Rice Mesophyll Cells and Treatment of Protoplasts with CdSO.sub.4 or PC2-Cd Complex
[0070] The protoplasts were isolated from rice mesophyll cells as described previously (Trinidad et al., 2021). In brief, the shoots from 10-day-old seedlings of rice germinated and grown on half-strength MS medium were cut into 0.5 cm strips. The protoplasts were released from the strips by adding protoplast isolation buffer [0.6 mannitol, 10 mM methyl ethane sulfonate (MES), 10 mM CaCl.sub.2), 0.1% BSA (w/v), 1.5% (w/v) cellulase RS (C0615, Sigma, USA), and 0.75% (w/V) pectinase RS (P2401, Sigma, USA)] followed by incubation in the dark with gentle shaking for 4 h at 28 C. The 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 setting. The pellet was washed twice with 20 ml W5 buffer (154 mM NaCl, 125 mM CaCl.sub.2, 5 mM KCl and 2 mM MES) and re-collected by spinning at 100 g for 3 min. To isolate the intact vacuoles, 3 ml lysis buffer [0.2M mannitol, 10% Ficoll-400, 15 mM EDTA (pH 8.0), 5 mM sodium phosphate (pH 8.0)] pre-warmed 37 C. was added to the protoplasts. The protoplasts were resuspended gently by being pipetted up and down for 5-8 times and lysed by incubation in a warm water bath at 37 C. for 5-10 min. The vacuoles released from protoplasts were purified by centrifugation on a three-step Ficoll-400 gradient. One volume of lysed protoplast suspension was overlaid with two volumes of Ficoll-400 solution (5% in w/v), prepared by mixing one volume of lysis buffer and one volume vacuole buffer (30 mM KCl, 20 mM HEPES-KOH, pH7.5, 0.4 M betaine, 15 mg mL.sup.1 BSA, and 1 mM DDT). One volume vacuole buffer was then layered on the top of the gradient carefully but quickly. The vacuoles were collected on the interface between 5% Ficoll-400 solution and vacuole buffer after centrifugation at 1,500 g for 20 min. The PC.sub.2Cd complex was formed by mixing CdSO.sub.4, PC.sub.2 and DDT at a molar ratio of 1:1:1 and incubating at 25 C. for 1 h. The protoplasts were resuspended in MMG buffer (0.4 M mannitol, 15 mM MgCl.sub.2, and 4 mM MES, adjust pH to 7.5 with KOH). 10 M CdSO4 or PC.sub.2Cd complex prepared above was added to protoplasts in MMG buffer and then incubated in the dark at room temperature for 1 h. After incubation, the protoplasts were collected by centrifugation at 150 g for 5 min at 20 C. The protoplasts were washed three times with W5 buffer to remove residual Cd in the buffer. Protoplasts and vacuoles isolated from the protoplasts were used for Cd determination by ICP-MS.
Element Analysis by Inductively Coupled Plasma Mass Spectrometry (ICP-MS)
[0071] The concentration of elements in brown rice (de-husked but unpolished rice seeds) and straw was determined by ICP-MS. About 0.1 g dried rice seeds or straw tissues were pre-digested with 3 ml concentrated HNO.sub.3/H.sub.2O.sub.2 mixture (5:1, v:v) overnight at room temperature, and were then digested in a microwave oven (Ethos One, Milestone Technologies). The elements concentration in the digested solution was determined by ICP-MS (7700S, Agilent Technologies, USA) after dilution. The rice flour NIST SRM 1568b was used for certified reference material (CRM) to assess the precision and accuracy of analysis procedures.
Statistical Analysis
[0072] Data were analysed using two-tailed Student's t test (* P<0.05 or ** P<0.01) or one-way ANOVA followed by LSD's test (significance level of P<0.05). All analysis was performed using IBM SPSS statistics 19 software.
Example 2
Generation of OsHMA3, OsABCC1 and OsPCS1-Overexpressed Lines
[0073] Three binary constructs containing the coding regions of cDNA or genomic clones of OsHMA3, OsPCS1 and OsABCC1 genes under the control of OsActin1 promoter were made and used to generate transgenic rice plants in T5105 genetic background via Agrobacterium-mediated rice transformation (
TABLE-US-00002 TABLE 2 Summary of transgenic plants generated in this study No. of T0 plants with No. of single-copy T-DNA and Gene of interest T0 plants overexpression of transgenes P.sub.Actin1:gABCC1:T.sub.Nos 62 10 P.sub.Actin1:cHMA3:T.sub.Nos 22 7 P.sub.Actin1:cPCS1:T.sub.Nos 27 6
Example 3
The Cd Concentration in Grains of the OsHMA3-Overexpressed Lines was Significantly Decreased
[0074] T5105 and the OsHMA3-overexpressed lines (HMA3-L1, HMA3-L3 and HMA3-L12) were grown in control soil and soil containing 3 mg/kg Cd in the form of CdSO.sub.4. After harvest, the seeds and straw were subjected to ICP-MS analysis. The grain Cd concentrations of the OsHMA3-overexpressed lines in the control soil (0.0030.001 mg/kg) and the Cd-treated soil (0.0410.012 mg/kg) was only 2.0% and 2.0%, respectively, to those of T5105 in the control experiments (0.1350.064 mg/kg and 2.0100.813 mg/kg), respectively (
[0075] Seedlings of T5105 and OsHMA3-overexpressed lines were tested on a half-strength MS medium for tolerance to Cd. T5105 plants displayed increased growth retardation to Cd treatment at the concentrations from 10 to 40 M CdSO.sub.4 (
Example 4
The as Concentration in the Rice Grains of the OsABCC1-Overexpressed Lines was Partially Reduced
[0076] The As concentration in seeds and straw of T5105 and the independent OsABCC1-overexpressed lines grown in control soil and soil containing 10 mg/kg As in the form of NaAsO.sub.2 were determined by ICP-MS (
Example 5
The as and Cd Concentrations in the Grains of the OsPCS1-Overexpressed Lines were Partially Reduced
[0077] The overexpression of the OsPCS1 gene in T5105 caused a significant reduction in As and Cd concentrations in rice grain (
Example 6
Co-Expression of the P.sub.Actin1:gABCC1:T.sub.Nos gene and the P.sub.Actin1:cPCS1:T.sub.Nos Gene Showed Synergistic Effect on Reducing as Concentration in Rice Grain
[0078] In rice plants, As (III) was sequestrated into vacuoles in the form of phytochelatin-arsenic (PCAs) complexes through ABCC1 (Hayashi et al., 2017; Song et al., 2014). To further investigate if the co-overexpression of OsABCC1 and OsPCS1 had synergistic effect on decreasing As concentration in rice grain, a AP line was generated to pyramid the P.sub.Actin1:gABCC1:T.sub.Nos gene and the P.sub.Actin1:cPCS1:T.sub.Nos gene into a single line by crossing ABCC1-L27 with PCS1-L1 followed by self-pollination and selection of double homozygotes. Indeed, compared to ABCC1-L27 or PCS1-L1, AP showed an synergistically enhanced tolerance to 25-100 M As (III) at the seedling stages (
Example 7
The Hypersensitivity of the OsPCS1-Overexpressed Lines to Cd Treatment was Relieved by the Co-Overexpression of the OsHMA3 Gene
[0079] To investigate if the co-overexpression of the OsHMA3 gene can relieve the hypersensitivity of the OsPCS1-overexpressed lines to Cd treatment, a transgenic line (HP) carrying both the P.sub.Actin1:cPCS1:T.sub.Nos gene and the P.sub.Actin1:cHMA3:T.sub.Nos gene was generated by crossing PCS1-L1 and HMA3-L3 followed by self-pollination and selection of double homozygotes. Seedlings of HP, PCS1-L1, HMA3-L3 and T5105 were tested on Cd.sup.2+-containing half-strength MS medium. The HP line provided a similar level of enhanced tolerance to Cd treatment to HMA3-L3 with longer shoot length and higher dry weight than those of PCS1-L1 or T5105 (
[0080] To further investigate the effect of OsPCS1 overexpression on Cd sequestration in vacuoles of rice cells, protoplasts and vacuoles were isolated from 10-day-old shoots of T5105 and PCS1-L1 seedlings that were grown on Cd-containing medium and subjected to Cd concentration measurement by ICP-MS analysis. There was no significant difference in the Cd concentration in the protoplasts of T5105 (50.4681.410 ng/10.sup.6 cells) and PCS1-L1 (49.8393.694 ng/10.sup.6 cells) (
Example 8
Generation of Low-As and Low-Cd Rice Grains by Pyramiding P.sub.Actin1:cHMA3:T.sub.Nos, P.sub.Actin1:cPCS1:T.sub.Nos and P.sub.Actin1:gABCC1:T.sub.Nos in a Single Rice Line
[0081] To generate rice grains with low concentration for both As and Cd elements, a rice line (PAH) with the co-overexpression of the OsPCS1, OsABCC1 and OsHMA3 genes was developed through crossing and marker assisted selection with the P.sub.Actin1:cPCS1:T.sub.Nos gene from PCS1-L1, the P.sub.Actin1:gABCC1:T.sub.Nos gene from ABCC1-L27 and the P.sub.Actin1:cHMA3:T.sub.Nos gene from HMA3-L3. Like its parental transgenic lines, the PAH line showed normal growth and development with similar plant architecture, growth duration, panicles and seeds, seed-setting rate and 100-grain weight to those of T5105 (
[0082] In summary, a genetic engineering approach was used to generate transgenic plants that overexpress OsABCC1, OsPCS1 and OsHMA3 under the OsActin1 promoter in the T5105 genetic background. The expression of the P.sub.Actin1:cPCS1:T.sub.Nos gene or P.sub.Actin1:gABCC1:T.sub.Nos gene alone partially reduced As concentration in the grains of transgenic rice plants. The co-expression of the P.sub.Actin1:cPCS1:T.sub.Nos gene and P.sub.Actin1:gABCC1:T.sub.Nos gene significantly reduced As concentration in the grains of the double genes over-expressed transgenic plants. The expression of the P.sub.Actin1:cHMA3:T.sub.Nos gene significantly reduced Cd concentration in the grains of transgenic rice lines. The co-expression of the P.sub.Actin1:cPCS1:T.sub.Nos gene, the P.sub.Actin1:gABCC1:T.sub.Nos gene and the P.sub.Actin1:cHMA3:T.sub.Nos gene significantly reduced both As and Cd concentration in the grains of triple genes over-expressed transgenic plants. All transgenic plants showed similar normal growth and development to the non-transgenic T5105 without any pleiotropic phenotype or yield penalty. The low-As and low-Cd rice will reduce the uptake of two toxic elements by human beings through the consumption of rice and benefit our health.
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