Transgenic plant with reduced heavy metals and methods for preparation thereof

Abstract

Provided are: a plant cell, in which expression or activity of a phytochelatin synthase (PCS) gene or a protein encoded by the PCS gene is reduced as compared with a parent cell; a plant having that has reduced heavy metal absorption, and includes the plant cell; a method of reducing heavy metals in a plant, the method including reducing expression or activity of a PCS gene or a protein encoded by the PCS gene, as compared with a parent cell; a CRISPR-Cas9 recombinant vector including a single guide RNA targeting a PCS gene; and a method of preparing a plant with reduced heavy metals, the method including transforming a plant cell with the recombinant vector.

Claims

1. A cell of a Nicotiana species plant having reduced heavy metal absorption in which expression or activity of a phytochelatin synthase (PCS) gene or a protein encoded by the PCS gene is reduced by a CRISPR/Cas9 system comprising a single guide RNA(sgRNA) targeting the PCS gene, as compared with a wild-type cell, wherein the PCS gene is a PCS gene derived from Nicotiana sylvestris (NtPCSs), a PCS gene derived from Nicotiana tomentosiformis (NtPCSt), or a combination thereof (NtPCSst), wherein the sgRNA targeting the NtPCSs gene consists of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, the sgRNA targeting the NtPCSt gene consists of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, and the sgRNA targeting the NtPCSs gene and the NtPCSt gene consists of the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

2. The cell of claim 1, wherein the plant is Nicotiana tabacum.

3. A plant having reduced heavy metal absorption, comprising the cell of claim 1.

4. A method of reducing heavy metals in a plant of a Nicotiana species, the method comprising reducing expression or activity of a PCS gene or a protein encoded by the PCS gene by a CRISPR/Cas9 system comprising a single guide RNA(sgRNA) targeting the PCS gene, as compared with a wild-type cell of the plant, wherein the PCS gene is a PCS gene derived from Nicotiana sylvestris (NtPCSs), a PCS gene derived from Nicotiana tomentosiformis (NtPCSt), or a combination thereof (NtPCSst), wherein the sgRNA targeting the NtPCSs gene consists of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2, the sgRNA targeting the NtPCSt gene consists of the nucleotide sequence of SEQ ID NO: 3 or SEQ ID NO: 4, and the sgRNA targeting the NtPCSs gene and the NtPCSt gene consists of the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6.

5. The method of claim 4, wherein the heavy metal is one or more selected from the group consisting of cadmium, arsenic, antimony, lead, mercury, chromium, tin, zinc, barium, bismuth, nickel, cobalt, manganese, iron, copper, and vanadium.

Description

BRIEF DESCRIPTION OF DRAWINGS

(1) FIGS. 1A and 1B show alignment of a part of known reference nucleotide sequences and part of a nucleotide sequence of an NtHMA gene of KB108, specifically, FIG. 1A shows a result of comparing exon 2 sites in NtHMA genes (gRNA_A4_e2: a site specific to the NtHMA gene, designed to bind with sgRNA; NtHMA_gDNA_ref is SEQ ID NO: 44; NtHMA_CDS_ref is SEQ ID NO: 45; NtHMA_CDS_ref is SEQ ID NO: 46; NtHMA_gDNA_KF109 is SEQ ID NO: 47; gRNA_A4_e2 is SEQ ID NO: 48) and FIG. 1B shows a result of comparing exon 2 sites in the NtHMA genes (gRNA_B4_e2: a site specific to the NtHMA gene, designed to bind with sgRNA; NtHMA_gDNA_ref is SEQ ID NO: 49; NtHMA_CDS_ref is SEQ ID NO: 50; NtHMA_CDS_ref is SEQ ID NO: 51; tHMA_gDNA_KF109 is SEQ ID NO: 52; gRNA_B4_e2 is SEQ ID NO: 53);

(2) FIG. 2 shows a structure of a genetic scissors expression vector including two sgRNAs;

(3) FIGS. 3A to 3D show photographs of each stage of plant tissue culture after transformation by an Agrobacterium-mediated transformation method, specifically, FIG. 3A shows a stage of cutting leaf tissues and transforming them by culturing with Agrobacterium, FIG. 3B shows a stage of inducing callus differentiation and shoot differentiation, FIG. 3C shows a stage of inducing root differentiation, and FIG. 3D shows a state of a plantlet in which differentiation is completed;

(4) FIG. 4 shows results of electrophoresis by amplifying each target gene site with respect to gDNA of 24 individuals of tissue cultures to examine the occurrence and patterns of mutations (Lane M: 1 kb DNA marker, Lanes 1 to 7: amplification results of NtHMA site, Lanes 9 to 15: amplification results of NtHMA site, Lanes 17 to 21: amplification results of NtPCSs site, Lanes 23 to 27: amplification results of NtPCSt site, Lanes 8, 16, 22, 28: Non-template control (NTC));

(5) FIGS. 5A and 5B show results of sequencing analysis of NtHMA sites of a wild-type KB108 and a variant, specifically, FIG. 5A shows a result of sequencing analysis of the KB108 (SEQ ID NO: 54) and FIG. 5B shows a result of sequencing analysis of the variant, in which a site into which an adenine base is inserted, compared to the nucleotide sequence of the wild-type, is indicated by a red arrow (SEQ ID NO: 55);

(6) FIG. 6 shows results of electrophoresis after performing a gene amplification reaction to examine whether a gene introduced into an F.sub.1 plant is inherited;

(7) FIG. 7 shows a graph showing relative values of the cadmium contents, based on the cadmium content of a control group, by summarizing results of primary and secondary cadmium content analysis performed in a hydroponic culture system;

(8) FIG. 8 shows results of analyzing the cadmium contents of plants cultured in a greenhouse soil environment;

(9) FIG. 9 shows a graph of comparing growth characteristics of an NtHMAa variant and an NtPCSst variant, as compared with the control KB108 (Height: height of plant, # of leaves: number of leaves, Weight: weight of leaves); and

(10) FIG. 10 shows photographs in which the growth status of the NtHMAa variant and the NtPCSst variant, as compared with the control KB108, was observed with the naked eye.

MODE OF DISCLOSURE

(11) Hereinafter, the present disclosure will be described in more detail with reference to exemplary embodiments. However, these exemplary embodiments are only for illustrating, and the scope of the present disclosure is not limited to these exemplary embodiments.

Example 1. Production of Tobacco with Reduced Cadmium Using NtPCS Gene-Targeting CRISPR/Cas9 System

(12) (1) Construction of Gene Vehicle

(13) (1.1) Examination of Nucleotide Sequences of Heavy Metal-Related Genes (NtHMA and NtPCS)

(14) Two types of genes related to cadmium absorption and transport in tobacco were selected; NtHMA and NtHMA, which are genes encoding proteins acting as gates that open and close when transporting divalent metal ions absorbed into roots to other tissues; and NtPCS, which is a gene encoding a protein involved in the storage and transport of metal ions by binding to metal ions in cells.

(15) To examine nucleotide sequences of the NtHMA and NtPCS genes in a burley tobacco variety (KB108) which is a research target plant, primers specific to each gene were prepared, based on the nucleotide sequence information published in the National Center for Biotechnology Information (NCBI) database, and a gene amplification reaction (PCR: Polymerase Chain Reaction) was performed. The sequences of the primers specific to each gene and PCR conditions are shown in Table 1 below.

(16) TABLE-US-00001 TABLE1 Amplifi- cation PCRconditions SEQ Nucleotide size Annealing Extension ID Primername sequence(5.fwdarw.3) (bp) Tmp time Cycles NO: F_HMA GAAACAAAGAAG 2937 62C. 90sec 35 7 TTGAGCAAGAGC TATT RHMA AGCCTTAGTGAG 8 ATGATTTATAACA CAA F_HMA GACACAAAGAAT 2374 62C. 90sec 35 9 CTGAGCAAGAGC TATT R_HMA AGCTAGAGTAGG 10 ACCACACATTAAT TCT F_PCSs AAATGGCGATGG 927 68C. 90sec 35 11 CGGGTTTGTAT R_PCSs GTCGGGAAGGAT 12 TAGAACACAAATT CAC F_PCSt AATGGCGATGGC 2731 68C. 90sec 35 13 GGGTTTATATC R_PCSt AGTCCGGAAGGA 14 TAGGAACACAGA TT

(17) As shown in FIG. 1, as a result of analyzing the nucleotide sequence by amplifying the gDNA region of the KB108 variety, the nucleotide sequence was found to mostly correspond to the published nucleotide sequence. Specifically, as a result of aligning and comparing a part of the published reference nucleotide sequences (Database accession Nos. HF675180.1 and HF937054.1) with the nucleotide sequences of NtHMA genes of KB108, when comparing each exon 2 site in the NtHMA gene (A) and NtHMA gene (B), most of them was confirmed to correspond to each other, and the site was designed such that sgRNA specifically bind to each gene.

(18) The results of examining homology between the NtPCSs gene (only some sequences) of the KB108 variety, a reference genome nucleotide sequence of N. tabacum (TN90), and a reference genome nucleotide sequence of N. sylvestris are shown in Table 2 below, and the results of examining homology between the NtPCSt gene of the KB108 variety, a reference genome nucleotide sequence of N. tabacum (TN90), and a reference genome nucleotide sequence of N. tometosiformis are shown in Table 3 below.

(19) TABLE-US-00002 TABLE 2 Homology comparison of PCSs_KB108 PCSs_TN90 PCSs_Nsyl PCSs gene (KT&G) (NW015845165.1) (NW009518934.1) PCSs_KB108(KT&G) ID 1.000 1.000 PCSs_TN90(NW_015845165.1) 1.000 ID 1.000 PCSsNsyl(NW009518934.1) 1.000 1.000 ID

(20) TABLE-US-00003 TABLE 3 Homology comparison of PCSt_KB108 PCSt_TN90 PCSt_Ntom PCSt gene (KT&G) (NW015825368.1) (NW008939610.1) PCSt_KB108(KT&G) ID 1.000 0.986 PCSt_TN90(NW_015825368.1) 1.000 ID 0.986 PCStNtom(NW008939610.1) 0.986 0.986 ID

(21) The results of comparing nucleotide sequences of the NtPCSs gene derived from Nicotiana sylvestris and the NtPCSt gene derived from Nicotiana tomentosiformis with the reference genome nucleotide sequence of N. tabacum (TN90) showed 100% homology, as confirmed in Tables 2 and 3. Further, the NtPSs gene (only some sequences) showed 100% homology to the reference genome data of N. sylvestris, and the NtPCSt gene showed about 98.6% homology to the reference genome data of N. tometosiformis.

(22) (1.2) Design of Genetic Scissors Block and Recombination of Vehicle

(23) The nucleotide sequences of the NtHMA, NtHMA, NtPCSs and NtPCSt genes obtained in Example 1-(1) were compared, and a part specific to each gene was selected as a genetic scissors guide (sgRNA). A total of 5 types of recombinant vehicles were completed by cloning gene scissors expression blocks selected as gene vehicles expressed in plants.

(24) Information about the gene vehicles including each of six sgRNAs capable of specifically binding to the NtHMA and NtHMA3 genes is shown in Table 4 below.

(25) TABLE-US-00004 TABLE4 SEQID Vector sgRNA Target Site Sequence NO: Vlk_HMAA6 gRNA_A1 HMA Exon1 TCTTTCTTACCAATT 15 TGTTG gRNA_A2 Intron1 TGTTTGTACAAGCTT 16 TTAGA gRNA_A3 Intron1 ATGGTAACTTCAATA 17 ATTATA gRNA_A4 Exon2 AAGCAAGCATAAGA 18 GTGAA gRNA_A5 Exon2 CCACACCTCTAAAAA 19 TAAT gRNA_A6 Intron2 TCATATAAATTGGGA 20 CAAA Vlk_HMAB6 gRNA_B1 HMA Exon1 CAATTTGTTGCTGAG 21 AAATG gRNA_B2 Intron1 AGTGGAGAAAAGAT 22 GAAGAA gRNA_B3 Intron1 ATGGTAACTACAATA 23 ATTATA gRNA_B4 Exon2 AAGCAAGTATAAGA 24 GTGAA gRNA_B5 Exon2 GATTCCTCCAATTAT 25 TTTT gRNA_B6 Exon2 CCACACCCCTAAAAA 26 TAAT

(26) Information about the gene vehicles including each of two sgRNAs capable of specifically binding to the NtPCSs and NtPCSt genes is shown in Table 5 below.

(27) TABLE-US-00005 TABLE5 SEQID Vector sgRNA Target Site Sequence NO: Vlk_PCS_S gRNA_S1 PCSs Exon1 AAGCGAAATCAACA 1 GCCGGAG gRNA_S2 Exon2 GGCATTCAAGACCA 2 TGGAA Vlk_PCS_T gRNA_T1 PCSt Exon1 CGAGTTCTTCCGTC 3 GCCTC gRNA_T2 Exon2 GGCATTCAAGACCA 4 TAGAA Vlk_PCS_ST gRNA_ST1 PCSs Exon1 TAGAAGCGAAATCA 5 PCSt ACAGC gRNA_ST2 Exon2 GCCATCCAGAATGG 6 AACAA

(28) In detail, pBI121, which is a binary vector replicable in E. coli and agrobacterium and is a vector widely used for plant transformation, was digested with HindII and EcoRI and prepared to clone a GE_block which is needed for the CRISPR/Cas9 system. The GE_block consists of, in this order, a CaMV 35S promoter with dual enhancer (P_35Sd), a multi cloning site (MCS) for cloning Cas9 block, a CaMV 35S terminator (T_35S), a linker sequence, and a multi cloning site (MCS) for cloning sgRNA block. HindIII and EcoRI recognition nucleotide sequences were added at both ends thereof. Each block of the GE_block was prepared by DNA synthesis, and sequentially cloned to complete the GE_block. A recombinant vehicle (V1k_GE) was prepared by ligation of pBI121 and GE_block, each digested with HindIII and EcoRI. CRISPR/Cas9_block consists of a block (Cas9_block) consisting of a Cas9 coding sequence (CDS) and a C-terminus nuclear localization sequence (NLS), and BamHI and Sac recognition nucleotide sequences at both ends thereof; and a block capable of expressing sgRNA (sgRNA_PMT) consisting of a U6 promoter (P_U6, SEQ ID NO: 43), sgRNA, and poly T. Two types of sgRNA blocks capable of specifically bind to NtPCS genes (NtPCSs and/or NtPCSt gene) were linked into one contiguous DNA through an overlap extension PCR technique to complete sgRNA_PMT. SaII and SpeI recognition nucleotide sequences exist at both ends of sgRNA_PMT.

(29) As shown in FIG. 2, V1 k_GE and CRISPR/Cas9_block, each digested with BamHI and SacI, were ligated together through a ligation reaction, and then digested with SaII and SpeI, and sgRNA_PMT was inserted to construct a gene vehicle.

(30) (2) Introduction of Recombinant Vehicle into Gene Transfer Microorganism (Agrobacterium)

(31) A plant vehicle was transformed into Agrobacterium LBA4404 strain by a freeze-thaw method.

(32) In detail, the Agrobacterium strain was inoculated in a YEP liquid medium (10 g of yeast extract, 10 g of Bacto peptone, 5 g of NaCl), and then incubated with shaking under conditions of 28 C. and 250 rpm for 16 hours. Cells were separated by centrifuging the culture medium under conditions of a speed of 3,000 g and 4 C. for 20 minutes, and suspended in 20 mM CaCl.sub.2) to prepare competent cells. 5 L of plasmid DNA (vehicle for plants) was added to 100 L of competent cells, and incubated in liquid nitrogen for 5 minutes and at 37 C. for 5 minutes. 1 mL of YEP liquid medium was added and incubated with shaking under conditions of 28 C. and 250 rpm for 2 hours. 100 L of the culture medium was spread on a YEP solid medium containing 100 mg/L of kanamycin, and then incubated at 28 C. for 3 days. Respective single colonies were sub-cultured, and whether the plasmid DNA was transformed was confirmed by PCR.

(33) (3) Plant Tissue Culture

(34) (3.1) Plant Transformation

(35) The Agrobacterium strain, in which transformation of the gene vehicle was confirmed in Example 1-(2) above, was cultured in a YEP liquid medium (including 70 mg/L kanamycin and 70 mg/L streptomycin) under conditions of 28 C. for 24 hours. In addition, the leaves of the plant at 1 month after germination were sterilized with 70% ethanol and Clorox, and cut into 3 mm3 mm slices, which were placed in a Petri dish containing 5 ml of MS liquid medium, and then 1 mL of the culture solution of Agrobacterium strain was evenly sprayed to prepare Tobacco leaf slices. The Tobacco leaf slices were then incubated at 25 C. in dark conditions for 48 hours.

(36) (3.2) Plant Tissue Culture

(37) The leaf slices were washed with sterile distilled water (including 200 ug/ml cefotaxim) four times, and then explanted in a shooting medium (MS medium, 2 mg/L BA, 0.1 mg/L NAA, 200 mg/L cefotaxim, 100 mg/L kanamycin), and incubated under conditions of 25 C., 16 h/8 h photoperiod, and sub-cultured with a fresh medium every 2 weeks to perform washing and explanting on the selection medium.

(38) In addition, shoots differentiated from the leaf slices were cut and explanted in a rooting medium (MS medium, including 200 mg/L cefotaxime), and incubated under conditions of 25 C. and 16 h/8 h photoperiod to perform explanting on the rooting medium.

(39) As a result, as shown in FIG. 3, after transformation into tobacco leaf tissues by the Agrobacterium-mediated transformation method, whether callus differentiation, leaf differentiation, and root differentiation occurred well in sequence was confirmed. Through tissue culture, 102 individuals of tissue culture plantlets with leaves, stems, and roots were obtained.

(40) (4) Selection of Variant

(41) (4.1) Examination of Mutation Occurrence and Patterns Thereof in Target Gene

(42) Each part of the leaves of the tissue cultures obtained in Example 1-(3) was collected, and gDNA was extracted therefrom, and then each target gene site was amplified through a PCR reaction. The nucleotide sequence of the amplified gene product was analyzed, and 50 tissue cultures with mutations in the NtHMA gene and 60 tissue cultures with mutations in the NtPCS gene were selected.

(43) In detail, 100 mg of healthy leaf tissue was sampled and uniformly ground, and gDNA was extracted and purified using a commercial kit (e.g., Nucleospin 96 plant II, Macherey Nagel, Germany) using a silica column. FIG. 4 shows results of electrophoresis by amplifying each target gene site with respect to gDNAs of 24 individuals of tissue cultures to examine the occurrence and patterns of mutations.

(44) FIG. 5 shows an illustration of base sequence analysis by amplifying the target gene site through PCR, after extraction/purification of gDNA from the leaf tissue. As shown in FIG. 5, as a result of nucleotide sequence analysis of the NtHMA sites of the wild-type KB108 and the variant, it was confirmed that an adenine base was inserted in the nucleotide sequence of the variant, as compared with the nucleotide sequence of the wild-type.

(45) The mutation patterns of the NtHMA gene were classified according to the site, and as a result, as shown in Table 6 below, mutation rates were 44% to 48% at the sites of gRNA_HMA_A4 and gRNA_HMA_B4 prepared by targeting exon 2, and it was confirmed that the mutation occurred most frequently at the exon 2 site of the NtHMA gene.

(46) TABLE-US-00006 TABLE 6 Number of Number of tissue Mutation Target Site mutation culture rate (%) gRNA_HMA_A1 HMA Exon 1 1 25 4 gRNA_HMA_A2 Intron 1 3 12 gRNA_HMA_A3 Intron 1 0 0 gRNA_HMA_A4 Exon 2 12 48 gRNA_HMA_A5 Exon 2 2 8 gRNA_HMA_A6 Intron 2 10 40 gRNA_HMA_B1 HMA Exon 1 5 25 20 gRNA_HMA_B2 Intron 1 7 28 gRNA_HMA_B3 Intron 1 0 0 gRNA_HMA_B4 Exon 2 11 44 gRNA_HMA_B5 Exon 2 5 20 gRNA_HMA_B6 Exon 2 2 8

(47) The mutation patterns of the NtPCS gene were classified according to the site, and as a result, as shown in Table 7 below, mutation rates were 45% to 70% at the sites of gRNA_PCSs_e2, gRNA_PCSt_e2, and gRNA_PCSst_e2 prepared by targeting exon 2, and it was confirmed that the mutation occurred most frequently at the exon 2 site of the NtPCS gene.

(48) TABLE-US-00007 TABLE 7 Number of Number of tissue Mutation Target Site mutation culture rate (%) gRNA_PCSs_e1 PCSs Exon 1 0 20 0 gRNA_PCSs_e2 Exon 2 12 60 gRNA_PCSt_e1 PCSt Exon 1 0 20 0 gRNA_PCSt_e2 Exon 2 14 70 gRNA_PCSst_e1 PCSs Exon 1 0 20 0 gRNA_PCSst_e2 PCSt Exon 2 9 45
(4.2) Obtaining of Seeds of F1 Generation and Selecting of Introduced Gene-Removed Plants

(49) 50 individuals of the NtHMA gene variant and 60 individuals of the NtPCS gene variant, in which the mutations were identified, were transplanted into pots filled with bed soil, and grown in a greenhouse. Seeds of F.sub.1 generation were obtained through self-pollination to remove the gene block introduced for CRISPR/Cas9 expression. The seeds of F.sub.1 generation were seeded in a 128-hole tray and grown for 30 days, then the leaves were collected and uniformly grounded, and then gDNA was extracted and purified using a silica column and a commercial kit (e.g., Nucleospin 96 plant II, Macherey Nagel, Germany). PCR was performed using a 35S promoter and primers specifically amplifying the Cas9 DNA block. Sequences of the primers are shown in Table 8 below.

(50) TABLE-US-00008 TABLE8 PCR SEQID Primer Sequence length Note NO: F_C9 GACCATCCTGGACTTCCT 420bp Cas9 27 GAAGAGC detected R_C9 TGCAGGTAGTACAGGTAC 28 AGCTTCTCG F_35S GCTCCTACAAATGCCATC 195bp 35Spromoter 29 A detected R_35S GATAGTGGGATTGTGCGT CA

(51) As a result, as shown in FIG. 6, plants in which the 535S promoter and the Cas9 DNA block were not detected, were selected.

(52) (4.3) Genotyping Information of Final Selected Plants

(53) The final selected F1 plants (108_PMTm_F1) did not include the introduced gene, and were plants, in which homozygous mutation occurred in each of the NtHMA genes and NtPCS genes. The genotypes of the final plants, in which mutations were induced in each of the NtHMA genes and the NtPCS genes, are shown in Table 9 below.

(54) TABLE-US-00009 TABLE 9 Mutation Target Mutation site pattern A6_02 HMA Exon 2 GT del B6_02 HMA Exon 2 T ins AB_01 HMA Exon 2 GTGA del HMA Intron 1 T del Exon 2 A ins Exon 2 T ins T_05 PCSt Exon 2 T ins ST_01 PCSs Exon 2 A ins PCSt Exon 2 A ins

(55) In addition, the gDNA nucleotide sequences, GDS nucleotide sequences, and amino acid sequences of the PCSs genes and PCst genes of the control plant (KB108), and the plant (PCSst), in which mutations were induced in the NtPCSs and NtPCSt genes are summarized and shown in Table 10 below.

(56) TABLE-US-00010 TABLE 10 Plant Type SEQ ID NO: Abbreviation KB108 PCSs_gDNA 31 KB108_PCSs_gDNA PCSs_CDS 32 KB108 PCSs_CDS PCSs_amino acid 33 KB108 PCSs_AA PCSt_gDNA 34 KB108_PCSt_gDNA PCSt_CDS 35 KB108_PCSt_CDS PCSt_amino acid 36 KB108_PCSt_AA PCSst PCSs_gDNA 37 PCSst PCSs_gDNA PCSs CDS 38 PCSst PCSs_CDS PCSs_amino acid 39 PCSst_PCSs_AA PCSt_gDNA 40 PCSst_PCSt_gDNA PCSt_CDS 41 PCSst_PCSt_CDS PCSt_amino acid 42 PCSst_PCSt_AA

(57) (In the amino acid sequence, * represents a stop codon, and the stop codon in the middle of the amino acid sequence represents an early stop codon caused by a mutation in the nucleic acid sequence.)

(58) For reference, amino acid sequences of SEQ ID NO: 39 and SEQ ID NO: 42 are as follows.

(59) TABLE-US-00011 SEQIDNO:39: MAMAGLYRRVLPSPPAVDFASTEGKQLFLEAIQNGNNGRI FQVDLLFSDTV*TGLLWFG*PFHGLECPCY*SRKKMER SEQIDNO:42: MAMAGLYRRVLPSPPAVDFASTEGKQLFLEAIQNGNNGRI FQVDLLFSDTV*TGLLWFG*PFYGLECPCY*SRKKMERAL EMV**IYVGLL*ASGEG*S*RDLFWESGMFGSLCRSEGRS FSL*S*YY**LP*TSHGLHY***LSSDLIIS*RPF*ADRF GPLFAYWWLSRGKGYGTDSRCCEV*ISSSLGSPPSPLGSH EHN**SYRIT*GVYANY*ASQSSCTAIYPEL*T*ELGHYL KAFDG*SSCPVKF*ECEGHKRCSLYCSFKSTFKFC*IHKV DSGSSKARGEWSKFE*RGERKASYQGRGIETSAGHSSL*A CHKHFIFKKFYLPVKSSIRQQFG*CCRKHLLPRSRSFCRK IWFIG*VLLSPNMC*MLQSYRGQFCYSGVWDSCKWEWGAG G*CSGPYISSKD*LLSLRASWLLANAPCK*RCADSTIAGI TSTYMVSNKRYEGLAGNREPCLSREPASFAARRDFAPART VPPPQEMQG*QGRRRFSCTSLL

Example 2. Analysis of Cadmium Content in Tobacco Plant with Mutated NtPCS Gene

(60) (1) Analysis of Cadmium Content Using Hydroponic Culture System

(61) The germinated plants were transplanted to a hydroponic culture system, and then the flower stalk was cut from each plant grown for 80 days, and 2 weeks later, all leaves of each plant were harvested, and the harvested leaves were dried in a dry oven at 65 C. for 48 hours. Then, the leaves were put in a container containing glass beads and ground using a gyro-shaker. Experiments were conducted by dividing primary and secondary experiments according to the time of obtaining the plant variants. The cadmium contents of KB108 (wild-type, control) and variants were quantitatively analyzed by a GC/MS analytical technique. The results of the two experiments were expressed as a relative amount compared to the cadmium absorption amount of the control plant.

(62) As a result, as shown in FIG. 7 and Table 11 below, the cadmium content in the leaves of the plants with the mutated NtHMA or NtHMA gene was reduced by 25% or 2%, as compared with that of the control group, and the cadmium content in the leaves of the plants with both the mutated NtHMA and NtHMA genes was reduced by 64%, as compared with that of the control group. In particular, the cadmium content in the leaves of the plants with both the mutated NtPCSs and NtPCSt genes was reduced by 85%, as compared with that of the control group, indicating a remarkable cadmium reduction effect. These plants show the excellent cadmium reduction effect, even as compared with the plants with both the mutated NtHMA and NtHMA genes.

(63) TABLE-US-00012 TABLE 11 Cadmium Plant (Relative value) KB108 (Control) 1.00 NtHMA 0.75 NtHMA 0.98 NtHMA 0.36 NtPCSt 1.25 NtPCSst 0.15
(2) Analysis of Cadmium Content in Greenhouse Environment

(64) To examine whether the effect of reducing cadmium absorption of plants with the mutated NtPCS gene is maintained in the soil environment, a cadmium absorption experiment was performed in a greenhouse environment. In detail, each of the germinated plants was transplanted to a pot in a greenhouse environment, and then the flower stalk was cut from each plant grown for 60 days, and 2 weeks later, all leaves of each plant (lower leaves, upper leaves, or entire leaves) were harvested, and the harvested leaves were dried in a dry oven at 65 C. for 48 hours. Then, the leaves were put in a container containing glass beads and ground using a gyro-shaker. The cadmium contents of KB108 (wild-type, control) and variants were quantitatively analyzed by a GC/MS analytical technique.

(65) As a result, as shown in FIG. 8 and Table 12 below, cadmium was rarely detected in a control group without cadmium treatment (KB108_NC), whereas a very high level of cadmium above the limit of quantitation (LOQ) of the assay was detected in a control group with cadmium treatment (KB108_PC). The cadmium content in the leaves of the plants with the mutated NtHMA or NtHMA gene was reduced by about 26%, as compared with that of the control group, and the cadmium content in the leaves of the plants with both the mutated NtHMA and NtHMA genes was reduced by about 77%, as compared with that of the control group. In particular, the cadmium content in the leaves of the plants with both the mutated NtPCSs and NtPCSt genes was reduced by about 87%, as compared with that of the control group, indicating a remarkable cadmium reduction effect. These plants show the excellent cadmium reduction effect, even as compared with the plants with both the mutated NtHMA and NtHMA3 genes.

(66) TABLE-US-00013 TABLE 12 Lower leaves Upper leaves Whole leaves Cadmium Cadmium Cadmium content content content Plant (g/g) (g/g) (g/g) KB108_NC 0.08 0.04 0.13 (Negative control) KB108_PC 2.44 2.13 4.57 (Positive control) NtHMA 1.05 2.27 3.31 NtHMA 1.19 1.48 2.67 NtHMA 0.68 0.48 1.16 NtPCSst 0.23 0.26 0.49

(67) Taken together, the cadmium content in the leaves of plants with both the mutated NtPCSs and NtPCSt genes was reduced by 86%, as compared with that of the control, under the hydroponic environment, and reduced by 87%, as compared with the control, under the soil environment, indicating the highest cadmium reduction effect.

Example 3. Examination of Growth Characteristics of Tobacco Plant with Mutated NtPCS Gene

(68) To examine whether the NtPCS mutant plants are able to overcome a growth inhibition phenomenon, which is the limit of the NtHMA mutant plant, the growth characteristics of plants with the mutated NtHMA and/or NtHMA3 gene(s), and plants with both the mutated NtPCSs and NtPCSt genes were compared to those of control groups. In detail, the plant height, the number of leaves, and the weight of leaves of five individuals for each plant type were measured and compared.

(69) As a result, as shown in FIG. 9 and Table 13 below, the plants with both the mutated NtHMA and NtHMA3 genes showed a 30% decrease in the plant height, no significant difference in the number of leaves, and a 16% decrease in the weight of the leaves, as compared with the control group. These results indicate that plants with the mutated NtHMA and NtHMA3 genes exhibit growth inhibition. In contrast, the plants with both the mutated NtPCSs and NtPCSt genes showed a 15% slight decrease in the plant height, but no significant changes in the number and weight of leaves, as compared with the control group, indicating no growth inhibition.

(70) TABLE-US-00014 TABLE 13 Height Number of Weight of No. Plant (cm) leaves leaves (g) 1 KB108 62 12 145.5 2 (Control) 66 13 149.0 3 63 13 142.0 4 68 13 142.0 5 67 12 129.0 Mean 65.2 12.6 141.5 1 NtHMA 45 13 146.0 2 Mutant 55 13 167.0 3 53 13 154.0 4 52 13 156.0 5 53 13 163.0 Mean 51.6 13 157.2 1 NtHMA 58 12 144.6 2 Mutant 51 14 169.0 3 57 13 176.0 4 58 14 172.0 5 54 13 160.0 Mean 55.6 13.2 164.32 1 NtHMA 50 11 108.5 2 Mutant 50 12 157.5 3 40 14 123.0 4 41 12 107.0 5 47 13 99.0 Mean 45.6 12.4 117.0 1 NtPCSst 61 13 145.0 2 Mutant 60 13 158.0 3 57 13 149.0 4 54 13 142.0 5 47 12 138.0 Mean 55.8 12.8 146.4

(71) In addition, as shown in FIG. 10, the NtHMAa variant showed growth inhibition detectable with the naked eye, as compared with the control group, whereas the NtPCSst variant showed no significant difference in the growth, as compared with the control group.