# Patent

## Title
Transgenic plants modified for reduced cadmium transport, derivative products, and related methods

## Abstract

Various embodiments are directed to transgenic plants, including transgenic tobacco plants and derivative seeds, genetically modified to impede the transport of Cadmium (Cd) from the root system to aerial portions of transgenic plants by reducing the expression levels of HMA-related transporters. Various embodiments are directed to transgenic tobacco plants genetically modified to stably express a RNAi construct encoding RNAi polynucleotides that enable the degradation of endogenous NtHMA RNA variants. Reduced expression of NtHMA transporters in transgenic plants results in substantially reduced content of Cadmium (Cd) in the leaf lamina. Various consumable products that are substantially free or substantially reduced in Cd content can be produced by incorporating leaves derived from transgenic tobacco plants modified to reduce the expression of NtHMA transporters.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a Divisional Application of U.S. patent application Ser. No. 13/770,702, filed on Feb. 19, 2013, which is a Divisional Application of U.S. patent application Ser. No. 12/333,681, filed on Dec. 12, 2008, which issued on Feb. 26, 2013 as U.S. Pat. No. 8,383,889, which claims priority under 35 U.S.C. §119 to U.S. Provisional Application Ser. No. 60/996,982, filed on Dec. 13, 2007, and the content of each is hereby expressly incorporated by reference.


SEQUENCE LISTING
This application hereby incorporates by reference the text file filed electronically herewith having the name “1021238-000933 sequence listing.txt” created on Nov. 20, 2008 with a file size of 125,091 bytes.
TECHNICAL FIELD
Compositions, expression vectors, polynucleotides, polypeptides, transgenic plants, transgenic cell lines, and transgenic seeds, and methods for making and using these embodiments to produce various plants that can reduce the transport of heavy metals into aerial portions.
BACKGROUND
Plants obtain essential heavy metals, such as Zn, Ni, and Cu, by absorbing metal ion substrates from their environment by various transport mechanisms mediated by transmembrane transporters expressed on the surface of root cells and other vascular tissues. Transporters classified as P-type ATPases, such as P1B-type ATPases, are transporters that translocate positively charged substrates across plasma membranes by utilizing energy liberated from exergonic ATP hydrolysis reactions. P1B-type ATPases are also referred to as heavy metal ATP-ases (“HMAs”) or CPx-type ATPases. HMAs have been grouped by substrate specificity into two subclasses, the Cu/Ag and Zn/Co/Cd/Pb groups. The first P1B-type ATPase to be characterized in plants is AtHMA4, cloned from Arabidopsis. Substrate selectivity by HMAs is not strictly limited to the transport of essential metals in that several non-essential metals can be recognized indiscriminantly as substrates, resulting in the accumulation of many non-essential metals, such as Cd, Pb, As, and Hg.
SUMMARY
Various embodiments are directed to compositions and methods for producing transgenic plants, including transgenic tobacco plants, genetically modified to impede Cadmium (Cd) transport from the root system to the leaf lamina by reducing the expression levels of transporters of the HMA family. A HMA homologue (“NtHMA”) has been identified in tobacco, which can be utilized for constructing various RNAi constructs, encoding NtHMA RNAi polynucleotides of interest that can facilitate the degradation of endogenous NtHMA RNA transcripts. Transgenic plants that can express NtHMA RNAi polynucleotides according to this disclosure can be utilized for reducing steady-state levels of NtHMA RNA transcripts, and consequently, for reducing the number of functionally active NtHMA transporters available for transporting metals across cellular membranes.
Various embodiments are directed to recombinant expression vectors comprising various NtHMA RNAi constructs, transgenic plants and seeds genetically modified to exogenously express NtHMA RNAi polynucleotides, cell lines derived from transgenic plants and seeds, and consumable products incorporating leaves derived from transgenic plants produced according to the disclosed methods.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1A is a schematic of a NtHMA genomic clone comprising 11 exons. FIG. 1B provides a list of nucleotide positions mapped to each exon within the isolated NtHMA genomic clone (“Table 1”).
FIG. 2A illustrates an exemplary subcloning strategy for constructing a NtHMA RNAi expression vector that enables the constitutive expression of NtHMA RNAi polynucleotides of interest, as described in Example 2.
FIG. 2B illustrates a hypothetical double-stranded RNA duplex formed (as “stem-loop-stem” structure) from intra-molecular, base-pair interactions within NtHMA RNAi polynucleotide produced as a product transcribed from an exemplary NtHMA RNAi construct.
FIG. 3A shows an exemplary RNAi sequence, NtHMA (660-915), for producing NtHMA RNAi polynucleotides of interest, as described in Example 2.
FIGS. 3B-3D show Cd reduction in leaf lamina of multiple first generation (T0) transgenic lines, representing three varieties, that have been genetically modified to express NtHMA RNAi polynucleotides (660-915), as described in Example 5.
FIG. 4A shows an exemplary RNAi sequence, NtHMA (1382-1584), for producing NtHMA RNAi polynucleotides of interest, as described in Example 3.
FIGS. 4B-4D show Cd reduction in leaf lamina of multiple first generation (T0) transgenic lines, representing three varieties, that have been genetically modified to express NtHMA RNAi polynucleotides (1382-1584), as described in Example 5.
FIGS. 5A-C show normalized NtHMA RNA transcript levels in various first generation (T0) transgenic lines that have been genetically modified to express NtHMA RNAi polynucleotides of interest, as determined by quantitative realtime PCR analysis of leaf lamina extracts, as described in Example 6.
FIG. 6 shows the distribution of Cd and Zn between the leaf lamina and the root of various first generation (T0) transgenic lines that have been genetically modified to express NtHMA RNAi polynucleotides of interest, as presented in Table 2 and described in Example 7.
FIG. 7 shows Cd distribution among the bark, leaf lamina, pith, and root tissues of various first generation (T0) transgenic lines that have been genetically modified to express NtHMA RNAi polynucleotides of interest, as presented in Table 3 and described in Example 8.
FIG. 8 shows Cd distribution between the leaf lamina and the root of various second generation (T1) transgenic lines that have been genetically modified to express NtHMA RNAi polynucleotides of interest, as described in Example 9.



DETAILED DESCRIPTION
I. Isolation of Tobacco NtHMA Genes and Gene Products
FIG. 1A is a schematic of a NtHMA genomic clone comprising 11 exons encoding a heavy metal transporter related to the HMA family of transporters. Example 1 further describes the identification of the NtHMA genomic clone (_HO-18-2) and 4 NtHMA cDNA clones. FIG. 1B. provides nucleotide positions corresponding to exon and intron subregions mapped within the NtHMA genomic clone (_HO-18-2).
A. NtHMA Polynucleotides
The term “polynucleotide” refers to a polymer of nucleotides comprising at least 10 bases in length. The polynucleotides may be DNA, RNA or a DNA/RNA hybrid, comprising ribonucleotides, deoxyribonucleotides, combinations of deoxyribo- and ribo-nucleotides, and combinations of bases and/or modifications, including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine, hypoxanthine, isocytosine, and isoguanine. The term includes single and double-stranded forms of DNA or RNA. The term “DNA” includes genomic DNAs, cDNAs, chemically-synthesized DNAs, PCR-amplified DNAs, and combinations/equivalents thereof. The term “isolated polynucleotide” refers to a polynucleotide not contiguous with any genome of origin, or separated from a native context. The term includes any recombinant polynucleotide molecule such as NtHMA RNAi constructs, NtHMA RNAi expression vectors, NtHMA genomic clones, and fragments and variants thereof.
As shown in FIG. 1A, the NtHMA genomic clone, designated as SEQ ID NO:1, comprises: intron 1 (SEQ ID NO:4), exon 1 (SEQ ID NO:5), intron 2 (SEQ ID NO:6), exon 2 (SEQ ID NO:7), intron 3 (SEQ ID NO:8), exon 3 (SEQ ID NO:9), intron 4 (SEQ ID NO:10), exon 4 (SEQ ID NO:11), intron 5 (SEQ ID NO:12), exon 5 (SEQ ID NO:13), intron 6 (SEQ ID NO:14), exon 6 (SEQ ID NO:15), intron 7 (SEQ ID NO:16), exon 7 (SEQ ID NO:17), intron 8 (SEQ ID NO:18), exon 8 (SEQ ID NO:19), intron 9 (SEQ ID NO:20), exon 9 (SEQ ID NO:21), intron 10 (SEQ ID NO:22), exon 10 (SEQ ID NO:23), intron 11 (SEQ ID NO:24, exon 11 (SEQ ID NO:25), and intron 12 (SEQ ID NO:26). Various embodiments are directed to isolated polynucleotides representing genomic fragments isolated at the NtHMA locus, comprising SEQ ID NO:1, fragments of SEQ ID NO:1, or variants thereof. Various embodiments are directed to isolated NtHMA polynucleotide variants comprising at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:1, or fragments of SEQ ID NO:1.
Various embodiments are directed to isolated polynucleotides having sequences that complements that of NtHMA polynucleotide variants comprising at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:1, or fragments of SEQ ID NO:1. Various embodiments are directed to isolated polynucleotides that can specifically hybridize, under moderate to highly stringent conditions, to polynucleotides comprising SEQ ID NO:1, or fragments of SEQ ID NO:1.
Various embodiments are directed to isolated polynucleotides of NtHMA cDNA (Clone P6663), comprising SEQ ID NO:3, fragments of SEQ ID NO:3, or variants thereof. Various embodiments are directed to isolated NtHMA polynucleotide variants comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:3, or fragments of SEQ ID NO:3. Various embodiments are directed to isolated NtHMA polyribonucleotide variants comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:3, or fragments of SEQ ID NO:3, and in which Ts have been substituted with Us (e.g., RNAs). Various embodiments are directed to isolated polynucleotides that can specifically hybridize, under moderate to highly stringent conditions, to polynucleotides comprising SEQ ID NO:3, or fragments of SEQ ID NO:3. Various embodiments are directed to isolated polynucleotides having a sequence that complements that of NtHMA polynucleotide variants comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:3, or fragments of SEQ ID NO:3.
Various embodiments are directed to isolated polynucleotides of NtHMA cDNA (Clone P6643), comprising SEQ ID NO:47, fragments of SEQ ID NO:47, or variants thereof. Various embodiments are directed to isolated NtHMA polynucleotide variants comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:47, fragments of SEQ ID NO:47. Various embodiments are directed to isolated NtHMA polyribonucleotide variants comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:47, fragments of SEQ ID NO:47, and in which Ts have been substituted with Us (e.g., RNAs). Various embodiments are directed to isolated polynucleotides that can specifically hybridize, under moderate to highly stringent conditions, to polynucleotides comprising SEQ ID NO:47, fragments of SEQ ID NO:47. Various embodiments are directed to isolated polynucleotides having a sequence that complements that of NtHMA polynucleotide variants comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:47, fragments of SEQ ID NO:47.
Various embodiments are directed to biopolymers that are homologous to NtHMA polynucleotides and NtHMA polypeptides (“NtHMA homologues”), which can be identified from different plant species. For example, NtHMA homologues can be experimentally isolated by screening suitable nucleic acid libraries derived from different plant species of interest. Alternatively, NtHMA homologues may be identified by screening genome databases containing sequences from one or more species utilizing a sequence derived from NtHMA polynucleotides and/or NtHMA polypeptides. Such genomic databases are readily available for a number of species (e.g., on the world wide web (www) at tigr.org/tdb; genetics.wisc.edu; stanford.edu/.about.ball; hiv-web.lan1.gov; ncbi.nlm.nig.gov; ebi.ac.uk; and pasteur.fr/other/biology). For example, degenerate oligonucleotide sequences can be obtained by “back-translation” from NtHMA polypeptide fragments. NtHMA polynucleotides can be utilized as probes or primers to identify/amplify related sequences, or to obtain full-length sequences for related NtHMAs by PCR, for example, or by other well-known techniques (e.g., see PCR Protocols: A Guide to Methods and Applications, Innis et. al., eds., Academic Press, Inc. (1990)).
B. NtHMA Polypeptides
The term “NtHMA polypeptide” refers to a polypeptide comprising an amino acid sequence designated as SEQ ID NO:2; polypeptides having substantial homology (i.e., sequence similarity) or substantial identity to SEQ ID NO:2; fragments of SEQ ID NO:2; and variants thereof. The NtHMA polypeptides include sequences having sufficient or substantial degree of identity or similarity to SEQ ID NO:2, and that can function by transporting heavy metals across cell membranes.
NtHMA polypeptides include variants produced by introducing any type of alterations (e.g., insertions, deletions, or substitutions of amino acids; changes in glycosylation states; changes that affect refolding or isomerizations, three-dimensional structures, or self-association states), which can be deliberately engineered or isolated naturally. NtHMA polypeptides may be in linear form or cyclized using known methods (e.g., H. U. Saragovi, et al., Bio/Technology 10, 773 (1992); and R. S. McDowell, et al., J. Amer. Chem. Soc. 114:9245 (1992), both incorporated herein by reference). NtHMA polypeptides comprise at least 8 to 10, at least 20, at least 30, or at least 40 contiguous amino acids.
Various embodiments are directed to isolated NtHMA polypeptides encoded by polynucleotide sequence, SEQ ID NO:1, comprising SEQ ID NO:2, fragments of SEQ ID NO:2, or variants thereof. Various embodiments are directed to isolated NtHMA polypeptide variants comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:2, or fragments of SEQ ID NO:2.
Various embodiments are directed to isolated NtHMA polypeptides (Clone P6663), comprising SEQ ID NO:2, fragments of SEQ ID NO:2, or variants thereof. Various embodiments are directed to isolated NtHMA polypeptide variants comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:2, or fragments of SEQ ID NO:2.
Various embodiments are directed to isolated NtHMA polypeptides (Clone P6643), comprising SEQ ID NO:49, fragments of SEQ ID NO:49, or variants thereof. Various embodiments are directed to isolated NtHMA polypeptide variants comprising at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:49, or fragments of SEQ ID NO:49.
II. Compositions and Related Methods for Reducing NtHMA Gene Expression and/or NtHMA-Mediated Transporter Activity
Suitable antagonistic compositions that can down-regulate the expression and/or the activity of NtHMA and NtHMA variants include sequence-specific polynucleotides that can interfere with the transcription of one or more endogenous NtHMA gene(s); sequence-specific polynucleotides that can interfere with the translation of NtHMA RNA transcripts (e.g., dsRNAs, siRNAs, ribozymes); sequence-specific polypeptides that can interfere with the protein stability of NtHMA, the enzymatic activity of NtHMA, and/or the binding activity of NtHMA with respect to substrates and/or regulatory proteins; antibodies that exhibit specificity for NtHMA; and small molecule compounds that can interfere with the protein stability of NtHMA, the enzymatic activity of NtHMA, and/or the binding activity of NtHMA. An effective antagonist can reduce heavy metal (e.g., Cd) transport into leaf lamina structures by at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%.
A. Definitions
Throughout this disclosure and the appended claims, the terms “a” and “the” function as singular and plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to “an RNAi polynucleotide” includes a plurality of such RNAi polynucleotides, and a reference to “the plant” includes reference to one or more of such plants.
The term “orientation” refers to a particular order in the placement of a polynucleotide relative to the position of a reference polynucleotide. A linear DNA has two possible orientations: the 5′-to-3′ direction and the 3′-to-5′ direction. For example, if a reference sequence is positioned in the 5′-to-3′ direction, and if a second sequence is positioned in the 5′-to-3′ direction within the same polynucleotide molecule/strand, then the reference sequence and the second sequence are orientated in the same direction, or have the same orientation. Typically, a promoter sequence and a gene of interest under the regulation of the given promoter are positioned in the same orientation. However, with respect to the reference sequence positioned in the 5′-to-3′ direction, if a second sequence is positioned in the 3′-to-5′ direction within the same polynucleotide molecule/strand, then the reference sequence and the second sequence are orientated in anti-sense direction, or have anti-sense orientation. Two sequences having anti-sense orientations with respect to each other can be alternatively described as having the same orientation, if the reference sequence (5′-to-3′ direction) and the reverse complementary sequence of the reference sequence (reference sequence positioned in the 5′-to-3′) are positioned within the same polynucleotide molecule/strand.
The term “NtHMA RNAi expression vector” refers to a nucleic acid vehicle that comprises a combination of DNA components for enabling the transport and the expression of NtHMA RNAi constructs. Suitable expression vectors include episomes capable of extra-chromosomal replication such as circular, double-stranded DNA plasmids; linearized double-stranded DNA plasmids; and other functionally equivalent expression vectors of any origin. A suitable NtHMA RNAi expression vector comprises at least a promoter positioned upstream and operably-linked to a NtHMA RNAi construct, as defined below.
The term “NtHMA RNAi construct” refers to a double-stranded, recombinant DNA fragment that encodes “NtHMA RNAi polynucleotides” having RNA interference activity. A NtHMA RNAi construct comprises a “template strand” base-paired with a complementary “sense or coding strand.” A given NtHMA RNAi construct can be inserted into a NtHMA RNAi expression vector in two possible orientations, either in the same (or sense) orientation or in the reverse (or anti-sense) orientation with respect to the orientation of a promoter positioned within a NtHMA RNAi expression vector.
The term “NtHMA RNAi polynucleotides” can target NtHMA RNA for enzymatic degradation, involving the formation of smaller fragments of NtHMA RNAi polynucleotides (“siRNAs”) that can bind to multiple complementary sequences within the target NtHMA RNA. Expression levels of one or more NtHMA gene(s) can be reduced by the RNA interference activity of NtHMA RNAi polynucleotides.
The term “template strand” refers to the strand comprising a sequence that complements that of the “sense or coding strand” of a DNA duplex, such as NtHMA genomic fragment, NtHMA cDNA, or NtHMA RNAi construct, or any DNA fragment comprising a nucleic acid sequence that can be transcribed by RNA polymerase. During transcription, RNA polymerase can translocate along the template strand in the 3′-to-5′ direction during nacent RNA synthesis.
The terms “sense strand” or “coding strand” refer to the strand comprising a sequence that complements that of the template strand in a DNA duplex. For example, the sequence of the sense strand (“sense sequence”) for the identified NtHMA genomic clone is designated as SEQ ID NO:1. For example, the sense sequence for NtHMA cDNA, identified as clone P6663, is designated as SEQ ID NO:3. For example, the sense sequence for NtHMA cDNA, identified as clone P6643, is designated as SEQ ID NO:46. For example, if the sense strand comprises a hypothetical sequence 5′-TAATCCGGT-3′ (SEQ ID NO:50), then the substantially identical corresponding sequence within a hypothetical target mRNA is 5′-UAAUCCGGU-3′ (SEQ ID NO:51).
The term “reverse complementary sequence” refers to the sequence that complements the “sense sequence” of interest (e.g., exon sequence) positioned within the same strand, in the same orientation with respect to the sense sequence. For example, if a strand comprises a hypothetical sequence 5′-TAATCCGGT-3′ (SEQ ID NO:52), then the reverse complementary sequence 5′-ACCGGATTA-3′ (SEQ ID NO:53) may be operably-linked to the sense sequence, separated by a spacer sequence.
The terms “NtHMA RNA transcript” or “NtHMA RNA,” in the context of RNA interference, refer to polyribonucleic acid molecules produced within a host plant cell of interest, resulting from the transcription of endogenous genes of the HMA family, including the isolated NtHMA gene (SEQ ID NO:1). Thus, these terms include any RNA species or RNA variants produced as transcriptional products from HMA-related genes that may be distinct from the isolated NtHMA gene (SEQ ID NO:1) but having sufficient similarity at structural and/or functional levels to be classified within the same family. For example, if a host plant cell selected for genetic modification according to the disclosed methods is tobacco, then target NtHMA RNA transcripts include: (1) pre-mRNAs and mRNAs produced from the transcription of the isolated NtHMA gene (SEQ ID NO:1); (2) pre-mRNAs and mRNAs produced from the transcription of any genes having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to the sequence of the isolated NtHMA gene (SEQ ID NO:1) (i.e. other distinct genes substantially identical to the identified NtHMA gene and encoding related isoforms of HMA transporters); and (3) pre-mRNAs and mRNAs produced from the transcription of alleles of the NtHMA gene (SEQ ID NO:1). The NtHMA RNA transcripts include RNA variants produced as a result of alternative RNA splicing reactions of heteronuclear RNAs (“hnRNAs”) of a particular NtHMA gene, mRNA variants resulting from such alternative RNA splicing reactions, and any intermediate RNA variants.
The terms “homology” or “identity” or “similarity” refer to the degree of sequence similarity between two polypeptides or between two nucleic acid molecules compared by sequence alignment. The degree of homology between two discrete nucleic acid sequences being compared is a function of the number of identical, or matching, nucleotides at comparable positions. The percent identity may be determined by visual inspection and mathematical calculation. Alternatively, the percent identity of two nucleic acid sequences can be determined by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG). Typical default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res. 14:6745, 1986, as described by Schwartz and Dayhoff, eds., Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp. 353-358, 1979; (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for end gaps. Various programs known to persons skilled in the art of sequence comparison can be alternatively utilized.
The term “upstream” refers to a relative direction/position with respect to a reference element along a linear polynucleotide sequence, which indicates a direction/position towards the 5′ end of the polynucleotide sequence. “Upstream” may be used interchangeably with the “5′ end of a reference element.”
The term “operably-linked” refers to the joining of distinct DNA elements, fragments, or sequences to produce a functional transcriptional unit or a functional expression vector.
The term “promoter” refers to a nucleic acid element/sequence, typically positioned upstream and operably-linked to a double-stranded DNA fragment, such as a NtHMA RNAi construct. For example, a suitable promoter enables the transcriptional activation of a NtHMA RNAi construct by recruiting the transcriptional complex, including the RNA polymerase and various factors, to initiate RNA synthesis. “Promoters” can be derived entirely from regions proximate to a native gene of interest, or can be composed of different elements derived from different native promoters and/or synthetic DNA segments. Suitable promoters include tissue-specific promoters recognized by tissue-specific factors present in different tissues or cell types (e.g., root-specific promoters, shoot-specific promoters, xylem-specific promoters), or present during different developmental stages, or present in response to different environmental conditions. Suitable promoters include constitutive promoters that can be activated in most cell types without requiring specific inducers. Examples of suitable promoters for controlling NtHMA RNAi polypeptide production include the cauliflower mosaic virus 35S (CaMV/35S), SSU, OCS, lib4, usp, STLS1, B33, nos or ubiquitin- or phaseolin-promoters. Persons skilled in the art are capable of generating multiple variations of recombinant promoters, as described in a number of references, such as Okamuro and Goldberg, Biochemistry of Plants, Vol. 15:pp 1-82 (1989).
Tissue-specific promoters are transcriptional control elements that are only active in particular cells or tissues at specific times during plant development, such as in vegetative tissues or reproductive tissues. Tissue-specific expression can be advantageous, for example, when the expression of polynucleotides in certain tissues is preferred. Examples of tissue-specific promoters under developmental control include promoters that can initiate transcription only (or primarily only) in certain tissues, such as vegetative tissues, e.g., roots or leaves, or reproductive tissues, such as fruit, ovules, seeds, pollen, pistols, flowers, or any embryonic tissue. Reproductive tissue-specific promoters may be, e.g., anther-specific, ovule-specific, embryo-specific, endosperm-specific, integument-specific, seed and seed coat-specific, pollen-specific, petal-specific, sepal-specific, or combinations thereof.
Suitable leaf-specific promoters include pyruvate, orthophosphate dikinase (PPDK) promoter from C4 plant (maize), cab-m1 Ca+2 promoter from maize, the Arabidopsis thaliana myb-related gene promoter (Atmyb5), the ribulose biphosphate carboxylase (RBCS) promoters (e.g., the tomato RBCS 1, RBCS2 and RBCS3A genes expressed in leaves and light-grown seedlings, RBCS1 and RBCS2 expressed in developing tomato fruits, and/or ribulose bisphosphate carboxylase promoter expressed almost exclusively in mesophyll cells in leaf blades and leaf sheaths at high levels).
Suitable senescence-specific promoters include a tomato promoter active during fruit ripening, senescence and abscission of leaves, a maize promoter of gene encoding a cysteine protease. Suitable anther-specific promoters can be used. Such promoters are known in the art or can be discovered by known techniques; see, e.g., Bhalla and Singh (1999) Molecular control of male fertility in Brassica, Proc. 10th Annual Rapeseed Congress, Canberra, Australia; van Tunen et al. (1990) Pollen- and anther-specific chi promoters from petunia: tandem promoter regulation of the chiA gene. Plant Cell 2:393-40; Jeon et al. (1999) Isolation and characterization of an anther-specific gene, RA8, from rice (Oryza sativa L). Plant Molecular Biology 39:35-44; and Twell et al. (1993) Activation and developmental regulation of an Arabidopsis anther-specific promoter in microspores and pollen of Nicotiana tabacum. Sex. Plant Reprod. 6:217-224.
Suitable root-preferred promoters known to persons skilled in the art may be selected. See, for example, Hire et al. (1992) Plant Mol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetase gene); Keller and Baumgartner (1991) Plant Cell 3(10):1051-1061 (root-specific control element in the GRP 1.8 gene of French bean); Sanger et al. (1990) Plant Mol. Biol. 14(3):433-443 (root-specific promoter of the mannopine synthase (MAS) gene of Agrobacterium tumefaciens); and Miao et al. (1991) Plant Cell 3(1):11-22 (full-length cDNA clone encoding cytosolic glutamine synthetase (GS), which is expressed in roots and root nodules of soybean). See also Bogusz et al. (1990) Plant Cell 2(7):633-641, where two root-specific promoters isolated from hemoglobin genes from the nitrogen-fixing nonlegume Parasponia andersonii and the related non-nitrogen-fixing nonlegume Trema tomentosa are described.
Suitable seed-preferred promoters include both seed-specific promoters (those promoters active during seed development such as promoters of seed storage proteins) and seed-germinating promoters (those promoters active during seed germination). See, e.g., Thompson et al. (1989) BioEssays 10: 108, herein incorporated by reference. Such seed-preferred promoters include, but are not limited to, Cim1 (cytokinin-induced message); cZ19B1 (maize 19 kDa zein); milps (myo-inositol-1-phosphate synthase); mZE40-2, also known as Zm-40 (U.S. Pat. No. 6,403,862); nucic (U.S. Pat. No. 6,407,315); and celA (cellulose synthase) (see WO 00/11177). Gama-zein is an endosperm-specific promoter. Glob-1 is an embryo-specific promoter. For dicots, seed-specific promoters include, but are not limited to, bean .beta.-phaseolin, napin, .beta.-conglycinin, soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, a maize 15 kDa zein promoter, a 22 kDa zein promoter, a 27 kDa zein promoter, a g-zein promoter, a 27 kD .gamma.-zein promoter (such as gzw64A promoter, see Genbank Accession #S78780), a waxy promoter, a shrunken 1 promoter, a shrunken 2 promoter, a globulin 1 promoter (see Genbank Accession # L22344), an Itp2 promoter (Kalla, et al., Plant Journal 6:849-860 (1994); U.S. Pat. No. 5,525,716), cim1 promoter (see U.S. Pat. No. 6,225,529) maize end1 and end2 promoters (See U.S. Pat. No. 6,528,704 and application Ser. No. 10/310,191, filed Dec. 4, 2002); nuc1 promoter (U.S. Pat. No. 6,407,315); Zm40 promoter (U.S. Pat. No. 6,403,862); eep1 and eep2; lec1 (U.S. patent application Ser. No. 09/718,754); thioredoxinH promoter (U.S. provisional patent application 60/514,123); mlip15 promoter (U.S. Pat. No. 6,479,734); PCNA2 promoter; and the shrunken-2 promoter. (Shaw et al., Plant Phys 98:1214-1216, 1992; Zhong Chen et al., PNAS USA 100:3525-3530, 2003).
Examples of inducible promoters include promoters responsive to pathogen attack, anaerobic conditions, elevated temperature, light, drought, cold temperature, or high salt concentration. Pathogen-inducible promoters include those from pathogenesis-related proteins (PR proteins), which are induced following infection by a pathogen (e.g., PR proteins, SAR proteins, beta-1,3-glucanase, chitinase). See, for example, Redolfi et al. (1983) Neth. J. Plant Pathol. 89:245-254; Uknes et al. (1992) Plant Cell 4:645-656; and Van Loon (1985) Plant Mol. Virol. 4:111-116. See also the application entitled “Inducible Maize Promoters”, U.S. patent application Ser. No. 09/257,583, filed Feb. 25, 1999.
In addition to plant promoters, other suitable promoters may be derived from bacterial origin (e.g., the octopine synthase promoter, the nopaline synthase promoter and other promoters derived from Ti plasmids), or may be derived from viral promoters (e.g., 35S and 19S RNA promoters of cauliflower mosaic virus (CaMV), constitutive promoters of tobacco mosaic virus, cauliflower mosaic virus (CaMV) 19S and 35S promoters, or figwort mosaic virus 35S promoter).
The term “enhancer” refers to a nucleic acid molecule, or a nucleic acid sequence, that can recruit transcriptional regulatory proteins such as transcriptional activators, to enhance transcriptional activation by increasing promoter activity. Suitable enhancers can be derived from regions proximate to a native promoter of interest (homologous sources) or can be derived from non-native contexts (heterologous sources) and operably-linked to any promoter of interest within NtHMA RNAi expression vectors to enhance the activity and/or the tissue-specificity of a promoter. Some enhancers can operate in any orientation with respect to the orientation of a transcription unit. For example, enhancers may be positioned upstream or downstream of a transcriptional unit comprising a promoter and a NtHMA RNAi construct. Persons skilled in the art are capable of operably-linking enhancers and promoters to optimize the transcription levels of NtHMA RNAi constructs.
B. RNAi Expression Vectors Comprising NtHMA RNAi Constructs encoding NtHMA RNAi Polynucleotides
RNA Interference (“RNAi”) or RNA silencing is an evolutionarily conserved process by which specific mRNAs can be targeted for enzymatic degradation. A double-stranded RNA (dsRNA) must be introduced or produced by a cell (e.g., dsRNA virus, or NtHMA RNAi polynucleotides) to initiate the RNAi pathway. The dsRNA can be converted into multiple siRNA duplexes of 21-23 bp length (“siRNAs”) by RNases III, which are dsRNA-specific endonucleases (“Dicer”). The siRNAs can be subsequently recognized by RNA-induced silencing complexes (“RISC”) that promote the unwinding of siRNA through an ATP-dependent process. The unwound antisense strand of the siRNA guides the activated RISC to the targeted mRNA (e.g., NtHMA RNA variants) comprising a sequence complementary to the siRNA anti-sense strand. The targeted mRNA and the anti-sense strand can form an A-form helix, and the major groove of the A-form helix can be recognized by the activated RISC. The target mRNA can be cleaved by activated RISC at a single site defined by the binding site of the 5′-end of the siRNA strand. The activated RISC can be recycled to catalyze another cleavage event.
FIG. 2A illustrates the construction of an exemplary NtHMA RNAi expression vector. Various embodiments are directed to NtHMA RNAi expression vectors comprising NtHMA RNAi constructs encoding NtHMA RNAi polynucleotides exhibiting RNA interference activity by reducing the expression level of NtHMA mRNAs, NtHMA pre-mRNAs, and related NtHMA RNA variants. The expression vectors comprise a promoter positioned upstream and operably-linked to a NtHMA RNAi construct, as further defined below. NtHMA RNAi expression vectors comprise a suitable minimal core promoter, a NtHMA RNAi construct of interest, an upstream (5′) regulatory region, a downstream (3′) regulatory region, including transcription termination and polyadenylation signals, and other sequences known to persons skilled in the art, such as various selection markers.
The NtHMA polynucleotides can be produced in various forms, including as double-stranded hairpin-like structures (“dsRNAi”). The NtHMA dsRNAi can be enzymatically converted to double-stranded NtHMA siRNAs. One of the strands of the NtHMA siRNA duplex can anneal to a complementary sequence within the target NtHMA mRNA and related NtHMA RNA variants. The siRNA/mRNA duplexes are recognized by RISC that can cleave NtHMA RNAs at multiple sites in a sequence-dependent manner, resulting in the degradation of the target NtHMA mRNA and related NtHMA RNA variants.
FIG. 2B illustrates the formation of a hypothetical double-stranded RNA duplex formed (as “stem-loop-stem” structure) as a product transcribed from an exemplary NtHMA RNAi construct. In FIG. 2B, a hypothetical NtHMA RNAi construct 10 is shown, comprising 3 double-stranded DNA fragments, such as fragments 1-3. Fragment 1 is positioned upstream and operably-linked to fragment 2, which is positioned upstream and operably-linked to fragment 3, for which DNA strands/sequences 4, 6, and 8 are liked together in tandem to form strand 11, as shown. Alternatively, a NtHMA RNAi construct comprises “a sense sequence” 5, which is positioned upstream and operably-linked to “a spacer sequence” 7, which is positioned upstream and operably-linked to “a reverse complementary sequence” 9. The strands/sequences 5, 7, and 9 can be liked together in tandem to form strand/sequence 12. Alternatively, a NtHMA RNAi construct comprises “a sense sequence” 8, which is positioned upstream and operably-linked to “a spacer sequence” 6, which is positioned upstream and operably-linked to “a reverse complementary sequence” 4. The strands/sequences 8, 6, and 4 can be liked together in tandem to form strand/sequence 11. Strand 12 is complementary to strand 11. Strand 11 is a template strand that can be transcribed into a NtHMA RNAi polynucleotide 13. The NtHMA RNAi polynucleotide 13 forms a hair-pin (“stem-loop-stem”) structure, in which the stem 16 is a complementary region resulting from intra-molecular base-pair interactions of the NtHMA RNAi polynucleotide 15 and the loop 17 represents a non-complementary region encoded by a spacer sequence, such as strands/sequences 6 or 7.
Any NtHMA RNA polynucleotide of interest can be produced by selecting a suitable sequence composition, loop size, and stem length for producing the NtHMA hairpin duplex. A suitable range for designing stem lengths of a hairpin duplex, includes stem lengths of 20-30 nucleotides, 30-50 nucleotides, 50-100 nucleotides, 100-150 nucleotides, 150-200 nucleotides, 200-300 nucleotides, 300-400 nucleotides, 400-500 nucleotides, 500-600 nucleotides, and 600-700 nucleotides. A suitable range for designing loop lengths of a hairpin duplex, includes loop lengths of 4-25 nucleotides, 25-50 nucleotides, or longer if the stem length of the hair duplex is substantial. In certain context, hairpin structures with duplexed regions longer than 21 nucleotides may promote effective siRNA-directed silencing, regardless of loop sequence and length.
Exemplary NtHMA RNAi constructs for down-regulating the expression level of the NtHMA gene (SEQ ID NO:1) and other NtHMA-related genes include the following:
Various embodiments are directed to NtHMA RNAi expression vectors comprising a NtHMA RNAi construct that comprises one or more of: intron 1 (SEQ ID NO:4), exon 1 (SEQ ID NO:5), intron 2 (SEQ ID NO:6), exon 2 (SEQ ID NO:7), intron 3 (SEQ ID NO:8), exon 3 (SEQ ID NO:9), intron 4 (SEQ ID NO:10), exon 4 (SEQ ID NO:11), intron 5 (SEQ ID NO:12), exon 5 (SEQ ID NO:13), intron 6 (SEQ ID NO:14), exon 6 (SEQ ID NO:15), intron 7 (SEQ ID NO:16), exon 7 (SEQ ID NO:17), intron 8 (SEQ ID NO:18), exon 8 (SEQ ID NO:19), intron 9 (SEQ ID NO:20), exon 9 (SEQ ID NO:21), intron 10 (SEQ ID NO:22), exon 10 (SEQ ID NO:23), intron 11 (SEQ ID NO:24, exon 11 (SEQ ID NO:25), and intron 12 (SEQ ID NO:26), fragments thereof, and variants thereof.
Various embodiments are directed to NtHMA RNAi expression vectors comprising: a NtHMA RNAi construct having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to a sequence selected from the group consisting of: exon 1 (SEQ ID NO:5), a fragment of exon 1 (SEQ ID NO:5), exon 2 (SEQ ID NO:7), a fragment of exon 2 (SEQ ID NO:7), exon 3 (SEQ ID NO:9), a fragment of exon 3 (SEQ ID NO:9), exon 4 (SEQ ID NO:11), a fragment of exon 4 (SEQ ID NO:11), exon 5 (SEQ ID NO:13), a fragment of exon 5 (SEQ ID NO:13), exon 6 (SEQ ID NO:15), a fragment of exon 6 (SEQ ID NO:15), exon 7 (SEQ ID NO:17), a fragment of exon 7 (SEQ ID NO:17), exon 8 (SEQ ID NO:19), a fragment of exon 8 (SEQ ID NO:19), exon 9 (SEQ ID NO:21), a fragment of exon 9 (SEQ ID NO:21), exon 10 (SEQ ID NO:23), a fragment of exon 10 (SEQ ID NO:23), exon 11 (SEQ ID NO:25), and a fragment of exon 11 (SEQ ID NO:25).
Various embodiments are directed to NtHMA RNAi expression vectors comprising: a NtHMA RNAi construct encoding NtHMA RNAi polynucleotides capable of self-annealing to form a hairpin structure, in which the RNAi construct comprises (a) a first sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:3 or SEQ ID NO:47; (b) a second sequence encoding a spacer element of the NtHMA RNAi polynucleotide that forms a loop of the hairpin structure; and (c) a third sequence comprising a reverse complementary sequence of the first sequence, positioned in the same orientation as the first sequence, wherein the second sequence is positioned between the first sequence and the third sequence, and the second sequence is operably-linked to the first sequence and to the third sequence.
Various embodiments are directed to NtHMA RNAi expression vectors comprising: a NtHMA RNAi construct encoding NtHMA RNAi polynucleotides capable of self-annealing to form a hairpin structure, in which the RNAi construct comprises (a) a first sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:3 or SEQ ID NO:47; (b) a second sequence encoding a spacer element of the NtHMA RNAi polynucleotide that forms a loop of the hairpin structure; and (c) a third sequence comprising a reverse complementary sequence of the first sequence (SEQ ID NO:46 or SEQ ID NO:48), positioned in the same orientation as the first sequence, wherein the second sequence is positioned between the first sequence and the third sequence, and the second sequence is operably-linked to the first sequence and to the third sequence.
Various embodiments are directed to NtHMA RNAi expression vectors comprising: a NtHMA RNAi construct that comprises a first sequence having “substantial similarity,” or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:3, or portions of SEQ ID NO:3. Various embodiments are directed to NtHMA RNAi expression vectors comprising a NtHMA RNAi construct that comprises a first sequence having “substantial similarity,” or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:47, or portions of SEQ ID NO:47.
Various embodiments are directed to a NtHMA RNAi expression vectors comprising: a NtHMA RNAi construct that comprises a second sequence having “substantial similarity,” or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to a sequence selected from the group consisting of: intron 1 (SEQ ID NO:4), a fragment of intron 1 (SEQ ID NO:4), intron 2 (SEQ ID NO:6), a fragment of intron 2 (SEQ ID NO:6), intron 3 (SEQ ID NO:8), a fragment of intron 3 (SEQ ID NO:8), intron 4 (SEQ ID NO:10), a fragment of intron 4 (SEQ ID NO:10), intron 5 (SEQ ID NO:12), a fragment of intron 5 (SEQ ID NO:12), intron 6 (SEQ ID NO:14), a fragment of intron 6 (SEQ ID NO:14), intron 7 (SEQ ID NO:16), a fragment of intron 7 (SEQ ID NO:16), intron 8 (SEQ ID NO:18), a fragment of intron 8 (SEQ ID NO:18), intron 9 (SEQ ID NO:20), a fragment of intron 9 (SEQ ID NO:20), intron 10 (SEQ ID NO:22), a fragment of intron 10 (SEQ ID NO:22), intron 11 (SEQ ID NO:24), a fragment of intron 11 (SEQ ID NO:24), intron 12 (SEQ ID NO:26), and a fragment of intron 12 (SEQ ID NO:26). Alternatively, the second sequence of the NtHMA RNAi construct can be randomly generated without utilizing an intron sequence derived from the NtHMA gene (SEQ ID NO:1).
Various embodiments are directed to NtHMA RNAi expression vectors comprising: a NtHMA RNAi construct that comprises a third sequence having “substantial similarity,” or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:46, or portions of SEQ ID NO:46. Various embodiments are directed to NtHMA RNAi expression vectors comprising a NtHMA RNAi construct that comprises a third sequence having “substantial similarity,” or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to SEQ ID NO:48, or portions of SEQ ID NO:48.
Various embodiments are directed to NtHMA RNAi expression vectors comprising: a NtHMA RNAi construct that comprises a third sequence having “substantial similarity,” or having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99% sequence identity to a reverse complementary sequence selected from the group consisting of: SEQ ID NO:27 (exon 1), a fragment of SEQ ID NO:27 (exon 1), SEQ ID NO:28 (exon 2), a fragment of SEQ ID NO:28 (exon 2), SEQ ID NO:29 (exon 3), a fragment of SEQ ID NO:29 (exon 3), SEQ ID NO:30 (exon 4), a fragment of SEQ ID NO:30 (exon 4), SEQ ID NO:31 (exon 5), a fragment of SEQ ID NO:31 (exon 5), SEQ ID NO:32 (exon 6), a fragment of SEQ ID NO:32 (exon 6), SEQ ID NO:33 (exon 7), a fragment of SEQ ID NO:33 (exon 7), SEQ ID NO:34 (exon 8), a fragment of SEQ ID NO:34 (exon 8), SEQ ID NO:35 (exon 9), a fragment of SEQ ID NO:35 (exon 9), SEQ ID NO:36 (exon 10), a fragment of SEQ ID NO:36 (exon 10), SEQ ID NO:37 (exon 11), and a fragment of SEQ ID NO:37 (exon 11).
Various embodiments are directed to NtHMA RNAi expression vectors comprising a NtHMA RNAi construct that comprises: SEQ ID NO:38 (“sense sequence/fragment”), the second sequence comprises SEQ ID NO:39 (“spacer sequence/fragment”) and the third sequence comprises SEQ ID NO:40 (“anti-sense sequence/fragment”).
Various embodiments are directed to NtHMA RNAi expression vectors comprising a NtHMA RNAi construct that comprises: SEQ ID NO:42 (“sense sequence/fragment”), the second sequence comprises SEQ ID NO:43 (“spacer sequence/fragment”), and the third sequence comprises SEQ ID NO:44 (“anti-sense sequence/fragment”).
Alternatively, the disclosed sequences can be utilized for constructing various NtHMA polynucleotides that do not form hairpin structures. For example, a NtHMA long double-stranded RNA can be formed by (1) transcribing a first strand of the NtHMA cDNA by operably-linking to a first promoter, and (2) transcribing the reverse complementary sequence of the first strand of the NtHMA cDNA fragment by operably-linking to a second promoter. Each strand of the NtHMA polynucleotide can be transcribed from the same expression vector, or from different expression vectors. The NtHMA RNA duplex having RNA interference activity can be enzymatically converted to siRNAs to reduce NtHMA RNA levels.
C. Expression Vectors for Reducing NtHMA Gene Expression by Co-suppression
Various compositions and methods are provided for reducing the endogenous expression levels for members of the NtHMA gene family by promoting co-suppression of NtHMA gene expression. The phenomenon of co-suppression occurs as a result of introducing multiple copies of a transgene into a plant cell host. Integration of multiple copies of a transgene can result in reduced expression of the transgene and the targeted endogenous gene. The degree of co-suppression is dependent on the degree of sequence identity between the transgene and the targeted endogenous gene. The silencing of both the endogenous gene and the transgene can occur by extensive methylation of the silenced loci (i.e., the endogenous promoter and endogenous gene of interest) that can preclude transcription. Alternatively, in some cases, co-suppression of the endogenous gene and the transgene can occur by post transcriptional gene silencing (“PTGS”), in which transcripts can be produced but enhanced rates of degradation preclude accumulation of transcripts. The mechanism for co-suppression by PTGS is thought to resemble RNA interference, in that RNA seems to be both an important initiator and a target in these processes, and may be mediated at least in part by the same molecular machinery, possibly through RNA-guided degradation of mRNAs.
Co-suppression of members of the NtHMA gene family can be achieved by integrating multiple copies of the NtHMA cDNA or fragments thereof, as transgenes, into the genome of a plant of interest. The host plant can be transformed with an expression vector comprising a promoter operably-linked to NtHMA cDNA or fragments thereof. Various embodiments are directed to expression vectors for promoting co-suppression of endogenous genes of the NtHMA family comprising: a promoter operably-linked to NtHMA cDNA identified as Clone P6663 (SEQ ID NO:3) or a fragment thereof, or NtHMA cDNA identified as Clone P6643 (SEQ ID NO:47) or a fragment thereof. Various embodiments are directed to expression vectors for promoting co-suppression of endogenous genes of the NtHMA family comprising: a promoter operably-linked to NtHMA cDNA, or a fragment thereof, having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3 or SEQ ID NO:47.
Various embodiments are directed to methods for reducing the expression level of endogenous genes of the NtHMA family by integrating multiple copies of NtHMA cDNA or a fragment thereof into a plant genome, comprising: transforming a plant cell host with an expression vector that comprises a promoter operably-linked to SEQ ID NO:3, or a fragment thereof; or SEQ ID NO:47, or a fragment thereof. Various embodiments are directed to methods for reducing the expression level of endogenous genes of the NtHMA family by integrating multiple copies of NtHMA cDNA, or a fragment thereof, into a plant genome, comprising: transforming a plant cell host with an expression vector that comprises a promoter operably-linked to NtHMA cDNA, or a fragment thereof, having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3 or SEQ ID NO:47.
D. Expression Vectors for Reducing NtHMA Gene Expression by Inhibition of Translation by Anti-sense Agents
Various compositions and methods are provided for reducing the endogenous expression level of the NtHMA gene family by inhibiting the translation of NtHMA mRNA. A host plant cell can be transformed with an expression vector comprising: a promoter operably-linked to NtHMA cDNA or a fragment thereof, positioned in anti-sense orientation with respect to the promoter to enable the expression of RNA polynucleotides having a sequence complementary to a portion of NtHMA mRNA. Various expression vectors for inhibiting the translation of HMA mRNA comprise: a promoter operably-linked to NtHMA cDNA identified as Clone P6663 (SEQ ID NO:3) or a fragment thereof; or NtHMA cDNA identified as Clone P6643 (SEQ ID NO:47) or a fragment thereof, in which the NtHMA cDNA, or the fragment thereof, is positioned in anti-sense orientation with respect to the promoter. Various expression vectors for inhibiting the translation of HMA mRNA comprise: a promoter operably-linked to a NtHMA cDNA, or a fragment thereof, having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3 or SEQ ID NO:47, in which the NtHMA cDNA, or the fragment thereof, is positioned in anti-sense orientation with respect to the promoter. The lengths of anti-sense NtHMA RNA polynucleotides can vary, including 15-20 nucleotides, 20-30 nucleotides, 30-50 nucleotides, 50-75 nucleotides, 75-100 nucleotides, 100-150 nucleotides, 150-200 nucleotides, and 200-300 nucleotides.
Various embodiments are directed to methods for reducing the expression level of endogenous genes of the NtHMA family by inhibiting NtHMA mRNA translation, comprising: transforming a plant cell host with an expression vector that comprises a promoter operably-linked to NtHMA cDNA identified as Clone P6663 (SEQ ID NO:3) or a fragment thereof; or NtHMA cDNA identified as Clone P6643 (SEQ ID NO:47) or a fragment thereof, in which the NtHMA cDNA, or the fragment thereof, is positioned in anti-sense orientation with respect to the promoter. Various embodiments are directed to methods for reducing the expression level of endogenous genes of the NtHMA family by inhibiting NtHMA mRNA translation, comprising: transforming a plant cell host with an expression vector that comprises a promoter operably-linked to a NtHMA cDNA, or a fragment thereof, having at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO:3 or SEQ ID NO:47, in which the NtHMA cDNA, or the fragment thereof, is positioned in anti-sense orientation with respect to the promoter.
E. Other Compositions and Methods for Reducing NtHMA Gene Expression
Methods for obtaining conservative variants and more divergent variants of NtHMA polynucleotides and polypeptides are known to persons skilled in the art. Any plant of interest can be genetically modified by various methods known to induce mutagenesis, including site-directed mutagenesis, oligonucleotide-directed mutagenesis, chemically-induced mutagenesis, irradiation-induced mutagenesis, and other equivalent methods. For example, site-directed mutagenesis is described in, e.g., Smith (1985) “In vitro mutagenesis,” Ann. Rev. Genet. 19:423-462, and references therein, such as Botstein & Shortle (1985) “Strategies and Applications of in vitro Mutagenesis,” Science 229:1193-1201; and in Carter (1986) “Site-directed mutagenesis,” Biochem. J. 237:1-7. Oligonucleotide-directed mutagenesis is described in, e.g., Zoller & Smith (1982) “Oligonucleotide-directed Mutagenesis using M13-derived Vectors: an Efficient and General Procedure for the Production of Point mutations in any DNA Fragment,” Nucleic Acids Res. 10:6487-6500. Mutagenesis utilizing modified bases is described in, e.g., Kunkel (1985) “Rapid and Efficient Site-specific Mutagenesis without Phenotypic Selection,” Proc. Natl. Acad. Sci. USA 82:488-492, and in Taylor et al. (1985) “The Rapid Generation of Oligonucleotide-directed Mutations at High Frequency using Phosphorothioate-modified DNA,” Nucl. Acids Res. 13: 8765-8787. Mutagenesis utilizing gapped duplex DNA is described in, e.g., Kramer et al. (1984) “The Gapped Duplex DNA Approach to Oligonucleotide-directed Mutation Construction,” Nucl. Acids Res. 12: 9441-9460). Point-mismatch mutagenesis is described in, e.g., Kramer et al. (1984) “Point Mismatch Repair,” Cell 38:879-887). Double-strand break mutagenesis is described in, e.g., Mandecki (1986) “Oligonucleotide-directed Double-strand Break Repair in Plasmids of Escherichia coli: A Method for Site-specific Mutagenesis,” Proc. Natl. Acad. Sci. USA, 83:7177-7181, and in Arnold (1993) “Protein Engineering for Unusual Environments,” Current Opinion in Biotechnology 4:450-455). Mutagenesis utilizing repair-deficient host strains is described in, e.g., Carter et al. (1985) “Improved Oligonucleotide Site-directed Mutagenesis using M13 Vectors,” Nucl. Acids Res. 13: 4431-4443. Mutagenesis by total gene synthesis is described in, e.g., Nambiar et al. (1984) “Total Synthesis and Cloning of a Gene Coding for the Ribonuclease S Protein,” Science 223: 1299-1301. DNA shuffling is described in, e.g., Stemmer (1994) “Rapid Evolution of a Protein in vitro by DNA Shuffling,” Nature 370:389-391, and in Stemmer (1994) “DNA shuffling by random fragmentation and reassembly: In Vitro Recombination for Molecular Evolution,” Proc. Natl. Acad. Sci. USA 91:10747-10751.
Alternatively, NtHMA genes can be targeted for inactivation by introducing transposons (and IS elements) into the genomes of plants of interest. These mobile genetic elements can be introduced by sexual cross-fertilization and insertion mutants can be screened for loss in NtHMA activity, such as reduced Cd transport. The disrupted NtHMA gene in a parent plant can be introduced into other plants by crossing the parent plant with plant not subjected to transposon-induced mutagenesis by, e.g., sexual cross-fertilization. Any standard breeding techniques known to persons skilled in the art can be utilized. In one embodiment, one or more NtHMA-related genes can be inactivated by the insertion of one or more transposons. Mutations can result in homozygous disruption of one or more NtHMA genes, in heterozygous disruption of one or more NtHMA genes, or a combination of both homozygous and heterozygous disruptions if more than one NtHMA gene is disrupted. Suitable transposable elements can be selected from two broad classes, designated as Class I and Class II. Suitable Class I transposable elements include retrotransposons, retroposons, and SINE-like elements. Such methods are known to persons skilled in the art as described in Kumar and Bennetzen (1999), Plant Retrotransposons in Annual Review of Genetics 33:479.
Alternatively, NtHMA genes can be targeted for inactivation by a method referred to as Targeting Induced Local Lesions IN Genomics (“TILLING”), which combines high-density point mutations with rapid sensitive detection of mutations. Typically, plant seeds are exposed to mutagens, such as ethylmethanesulfonate (EMS) or EMS alkylates guanine, which typically leads to mispairing. Suitable agents and methods are known to persons skilled in the art as described in McCallum et al., (2000), “Targeting Induced Local Lesions IN Genomics (TILLING) for Plant Functional Genomics,” Plant Physiology 123:439-442; McCallum et al., (2000) “Targeted screening for induced mutations,” Nature Biotechnology 18:455-457; and Colbert et al., (2001) “High-Throughput Screening for Induced Point Mutations,” Plant Physiology 126:480-484.
Alternatively, NtHMA genes can be targeted for inactivation by introducing ribozymes derived from a number of small circular RNAs that are capable of self-cleavage and replication in plants. These RNAs can replicate either alone (viroid RNAs) or with a helper virus (satellite RNAs). Examples of suitable RNAs include those derived from avocado sunblotch viroid and satellite RNAs derived from tobacco ringspot virus, lucerne transient streak virus, velvet tobacco mottle virus, solanum nodiflorum mottle virus, and subterranean clover mottle virus. Various target RNA-specific ribozymes are known to persons skilled in the art as described in Haseloff et al. (1988) Nature, 334:585-591.
III. Transgenic Plants, Cell Lines, and Seeds Comprising NtHMA RNAi Polynucleotides and Related Methods
Various embodiments are directed to transgenic plants genetically modified to reduce the NtHMA gene expression level by various methods that can utilized for silencing NtHMA gene expression, and thereby, producing transgenic plants in which the expression level of NtHMA transporters can be reduced within plant tissues of interest. Rates of heavy metal transport and distribution patterns of heavy metal transport, in particular, cadmium transport, can be altered in transgenic plants produced according to the disclosed methods and compositions. Plants suitable for genetic modification include monocots and dicots.
Various embodiments are directed to transgenic tobacco plants genetically modified to reduce the NtHMA gene expression level by various methods, known to persons skilled in the art, that can be utilized for down-regulating NtHMA gene expression, and thereby, producing transgenic tobacco plants in which the expression level of NtHMA transporters can be reduced within plant tissues of interest. Various expression vectors have been provided to produce transgenic lines of tobacco of any variety exhibiting reduced levels of NtHMA gene expression. The disclosed compositions and methods can be applied to any plant species of interest, including plants of the genus Nicotiana, various species of Nicotiana, including N. rustica and N. tabacum (e.g., LA B21, LN KY171, TI 1406, Basma, Galpao, Perique, Beinhart 1000-1, and Petico). Other species include N. acaulis, N. acuminata, N. acuminata var. multiflora, N. africana, N. alata, N. amplexicaulis, N. arentsii, N. attenuata, N. benavidesii, N. benthamiana, N. bigelovii, N. bonariensis, N. cavicola, N. clevelandii, N. cordifolia, N. corymbosa, N. debneyi, N. excelsior, N. forgetiana, N. fragrans, N. glauca, N. glutinosa, N. goodspeedii, N. gossei, N. hybrid, N. ingulba, N. kawakamii, N. knightiana, N. langsdorffii, N. linearis, N. longiflora, N. maritima, N. megalosiphon, N. miersii, N. noctiflora, N. nudicaulis, N. obtusifolia, N. occidentalis, N. occidentalis subsp. hesperis, N. otophora, N. paniculata, N. pauciflora, N. petunioides, N. plumbaginifolia, N. quadrivalvis, N. raimondii, N. repanda, N. rosulata, N. rosulata subsp. ingulba, N. rotundifolia, N. setchellii, N. simulans, N. solanifolia, N. spegazzinii, N. stocktonii, N. suaveolens, N. sylvestris, N. thyrsiflora, N. tomentosa, N. tomentosiformis, N. trigonophylla, N. umbratica, N. undulata, N. velutina, N. wigandioides, and N. x sanderae. Suitable plants for transformation include any plant tissue capable of transformation by various methods of transforming plants known by persons skilled in the art, including electroporation, micro-projectile bombardment, and Agrobacterium-mediated transfer as described, for example, in U.S. Pat. No. 4,459,355 that discloses a method for transforming susceptible plants, including dicots, with an Agrobacterium strain containing a Ti plasmid; U.S. Pat. No. 4,795,855 that discloses transformation of woody plants with an Agrobacterium vector; U.S. Pat. No. 4,940,838 that discloses a binary Agrobacterium vector; U.S. Pat. No. 4,945,050; and U.S. Pat. No. 5,015,580.
Various embodiments are directed to transgenic tobacco plants genetically modified to exogenously express a RNAi construct encoding NtHMA RNAi polynucleotides that facilitate the degradation of NtHMA RNA transcripts, and consequently, that reduce the number of RNA transcripts available for translation into NtHMA transporters. Various embodiments are directed to transgenic plants comprising an expression vector that enable the expression of NtHMA polynucleotides produced according to the disclosed methods. Various embodiments are directed to cell lines derived from transgenic plants produced according to the disclosed methods. Various embodiments are directed to transgenic seeds derived from transgenic plants produced according to the disclosed methods.
Various embodiments are directed to methods for reducing the NtHMA gene expression levels in plants, the method comprising reducing the expression level of a NtHMA gene, which can be accomplished by various methods known to persons skilled in the art. As examples, this disclosure described: (1) RNA interference method for reducing steady-state level of endogenous NtHMA RNA variants available for translation by expression of NtHMA RNAi polynucleotides; (2) co-suppression method for reducing transcription of NtHMA gene(s) by integrating multiple copies of the NtHMA cDNA or fragments thereof, as transgenes, into a plant genome; (3) anti-sense method for reducing the NtHMA translation by the expression of anti-sense polynucleotides that can target NtHMA RNA; and (4) various methods for inducing mutagenesis.
Various embodiments are directed to transgenic tobacco plants genetically modified to reduce the NtHMA gene expression level by various methods, known to persons skilled in the art, and further modified either to reduce the expression of a second endogenous gene of interest (i.e., not NtHMA-related) or to enhance the expression of an exogenous gene of interest (i.e., not NtHMA-related). For example, the down-regulation of a second endogenous gene of interest encoding an enzyme involved in the biosynthesis of alkyloids may be desirable. In other situations, the enhancement in the expression level of a transgene encoding a recombinant protein of interest, such as a human hormone for therapeutic use, may be desirable. Persons skilled in the art are capable of producing various transgenic plants that can be modified, for example, to exogenously express NtHMA RNAi polynucleotides and at least one recombinant gene product of interest, such as a recombinant human growth factor or RNAi polynucleotides that can target a second gene of interest not related to the NtHMA family.
Producing transgenic plants according to the disclosed methods provides a number of advantages. Transgenic plants, including transgenic tobacco plants, can be grown in soils containing variable Cd concentrations, or in soils containing less than desirable Cd concentrations. These transgenic plants and derivative seeds can provide more options for cultivating them in a broader range of soil environments, which may increase the amount of cultivatable soils available to practitioners (e.g., farmers). Furthermore, these transgenic plants, exhibiting reduced Cd content, compared to non-transgenic counterparts can be consumed directly as edible products. The consumption of edible portions of these transgenic plants can be a healthier option compared to the consumption of non-transgenic counterparts. Suitable plants that can be genetically modified according to the disclosed methods, include plants cultivatable for agricultural use, including rice, corn, squash, soybeans, lettuce, potatoes, beats, herbs, wheat, barley, carrots, etc. The % Cd reduction in these transgenic plants, including the leaf lamina portion, can be approximately at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, and 99%, when compared to non-transgenic counterparts. The Cd content of these transgenic plants, including the leaf lamina portion, is a value from a range from about 0.01 to about 0.05 ppm, from about 0.01 to about 0.1 ppm, from about 0.01 to about 0.5 ppm, from about 0.01 to about 1.0 ppm, and from about 0.01 to about 5 ppm.
IV. Consumable Products Incorporating Tobacco Leaves Genetically Modified to Contain Reduced Cd Content
Various embodiments provide transgenic plants, in which the expression level of members of the NtHMA gene family is substantially reduced to curtail or impede Cd transport into the leaf lamina. The leaf lamina derived from transgenic tobacco plants, produced according to the disclosed methods, can be incorporated into various consumable products containing Cd at a level substantially below that of consumable products made by incorporating tobacco leaves derived from plants of the same genotype that were grown under identical conditions, but not genetically modified with respect to the reduced expression level of members of the NtHMA gene family (“non-transgenic counterparts”).
In some embodiments, these transgenic plants exhibiting reduced Cd content compared to non-transgenic counterparts can be incorporated into consumable products, including various smokable articles, such as cigars, cigarettes, and smokeless tobacco products (i.e., non-combustible). Smokable articles and smokeless tobacco products, produced by incorporating tobacco leaves derived from tobacco plants genetically modified to contain reduced Cd levels according to the disclosed methods, can provide healthier options compared to non-transgenic counterparts.
Smokeless tobacco products incorporating tobacco plants genetically modified according to the disclosed methods can be manufactured in any format suitable for comfort in a consumer's oral cavity. Smokeless tobacco products contain tobacco in any form, including as dried particles, shreds, granules, powders, or a slurry (i.e., tobacco extract), deposited on, mixed in, surrounded by, or otherwise combined with other ingredients in any format, such as flakes, films, tabs, foams, or beads. Smokeless tobacco products may be wrapped with a material, which may be edible (i.e., orally disintegrable) or nonedible. Liquid contents of smokeless tobacco products can be enclosed in a form, such as beads, to preclude interaction with a water-soluble wrapper. The wrapper may be shaped as a pouch to partially or completely enclose tobacco-incorporating compositions, or to function as an adhesive to hold together a plurality of tabs, beads, or flakes of tobacco. A wrapper may also enclose a moldable tobacco composition that conforms to the shape of a consumer's mouth. An orally disintegrable wrapper may enclose smokeless tobacco, e.g., as dry snuff or soluble tobacco, and may be formed on continuous thermoforming or horizontal form/fill/seal equipment or other suitable packaging equipment using edible films (which may or may not contain tobacco). Exemplary materials for constructing a wrapper include film compositions comprising HPMC, CMC, pectin, alginates, pullulan, and other commercially viable, edible film-forming polymers. Other wrapping materials may include pre-formed capsules produced from gelatin, HPMC, starch/carrageenan, or other commercially available materials. Such wrapping materials may include tobacco as an ingredient. Wrappers that are not orally disintegrable may be composed of woven or nonwoven fabrics, of coated or uncoated paper, or of perforated or otherwise porous plastic films. Wrappers may incorporate flavoring and/or coloring agents. Smokeless products can be assembled together with a wrapper utilizing any method known to persons skilled in the art of commercial packaging, including methods such as blister packing and stik-paking, in which a small package can be formed by a vertical form/fill/seal packaging machine.
The % Cd reduction in these smokable articles and smokeless products, produced by incorporating tobacco leaves derived from tobacco plants genetically modified to contain reduced Cd levels, is a value of at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, and 100%, when compared to consumable products derived from non-transgenic counterparts. In some embodiments, the Cd content of these smokable articles and smokeless products, produced by incorporating tobacco leaves derived from tobacco plants genetically modified to contain reduced Cd levels, is a value from a range from about 0.01 to about 0.05 ppm, from about 0.01 to about 0.1 ppm, from about 0.01 to about 0.5 ppm, from about 0.01 to about 1.0 ppm, and from about 0.01 to about 5 ppm.
The degree of Cd accumulation in plants can be substantially variable depending on several parameters attributed to the complexity of the genotype and the growth environment. For example, Cd concentrations in field-grown tobacco leaves can be extremely variable depending on factors such as the agro-climate, soil parameters, and cultivars. Furthermore, the relative Cd distribution patterns within different portions of a tobacco plant can vary according to the species, the organ/tissue, and growth conditions (i.e., field-grown vs. hydroponically-grown). On average, the Cd concentrations measured in field-grown tobacco leaves (including midribs and veins) can be in the range from approximately 0.5 to 5 ppm (parts per million, or ug/g of dry weight of tobacco leaves). However, many published Cd levels typically do not define the tobacco maturity stage, the tobacco variety, or the particular leaf portions (i.e., removal from leaf stalk position) harvested for analysis. In some varieties, the lower leaves may accumulate higher Cd levels than the medium and upper leaves. At the intracellular level, Cd can be found in various cell components of a plant cell, including the cell wall, cytoplasm, chloroplast, nucleus, and vacuoles.
Furthermore, Cd content measured in tobacco leaves can vary substantially depending on the Cd levels in the soil environment where the tobacco plants were grown. The leaves of tobacco grown in Cd-contaminated areas can accumulate Cd from about 35 ppm or higher, compared to the leaves of genetically identical counterparts grown in non-contaminated areas, which can accumulate Cd at a range from approximately 0.4 to approximately 8 ppm. The vacuoles within the leaves of plants grown in Cd-contaminated areas can accumulate very high Cd concentrations. Methods for modifying the disclosed compositions to be suitable for a given plant species of interest are known to persons skilled in the art.
EXAMPLES
Example 1
Cloning and Exon Mapping of a Full-Length Nicotiana NtHMA Genomic Clone
Two partial genomic clones representing different portions of an endogenous NtHMA gene were independently identified, referred to as “CHO_OF96xf01.ab1” and “CHO_OF261xo09c1.ab1.” Based on sequence information obtained from the partial genomic clones, a full-length genomic clone (_HO-18-2) and 4 full-length NtHMA cDNAs were subsequently identified, including clone P6663 (SEQ ID NO:3) and clone P6643 (SEQ ID NO:47). The exon and intron subregions of full-length genomic clone (_HO-18-2) (17,921 bp) were mapped. As shown in FIG. 1A, the full-length, endogenous NtHMA gene cloned from Nicotiana comprises 11 exons consisting of 3392 nucleotides in total.
Example 2
Construction of NtHMA RNAi Expression Vector PBI121-NtHMA (660-915) Encoding RNAi Polynucleotides
FIG. 1B provides a list of nucleotide positions mapped to each exon within the isolated NtHMA genomic clone (SEQ ID NO: 1) (“Table 1”). The partial genomic clone CHO_OF96xf01.ab1 includes a part of intron 4, exon 4, intron 5, exon 5, intron 6, and a part of exon 6, as shown in FIG. 1A, and listed under Table 1 of FIG. 1B. The partial genomic clone CHO_OF261 xo09c1 includes a part of intron 7, exon 7, intron 8, exon 8, and a part of exon 9, as shown in FIG. 1A. To produce transgenic plants that can stably produce recombinant NtHMA RNAi polynucleotides of interest that can facilitate the degradation of endogenous RNA transcripts encoding NtHMA polypeptides, two sets of NtHMA RNAi expression vectors, the PBI121-NtHMA (660-915) RNAi expression vector as further described below, and the PBI121-NtHMA (1382-1584) RNAi expression vector as further described in Example 3.
FIG. 2 illustrates an exemplary subcloning strategy for constructing a NtHMA RNAi expression vector that enables the constitutive expression of NtHMA RNAi polynucleotides of interest. Based on exon mapping and sequence analysis of genomic clone CHO_OF96xf01.ab1, RNAi constructs were designed.
FIG. 3A shows an exemplary RNAi sequence, NtHMA (660-915), for producing NtHMA RNAi polynucleotides of interest. In FIG. 3A, NtHMA RNAi RNAi construct comprises a sense fragment (272 bp) (SEQ ID NO:38) composed of exon 4 (272 bp), which is positioned upstream and operably-linked to a spacer fragment (80 bp) (SEQ ID NO:39) composed of intron 5, which is positioned upstream and operably-linked to a reverse complementary fragment (272 bp) (SEQ ID NO:40) composed of exon 4 positioned in anti-sense orientation. RNAi constructs encoding NtHMA RNAi polynucleotides of interest were inserted into the PBKCMV cloning vector, and were placed downstream and operably-linked to a cytomegalovirus (CMV) promoter. XbaI and HindIII sites were incorporated into the 5′ and 3′ ends of the 352 bp NtHMA sense fragment, which included the 80 bp intron fragment by utilizing PCR primers modified to incorporate these restriction enzyme sites (PMG783F: ATTCTAGACTGCTGCTATGTCATCACTGG (SEQ ID NO:54) and PMG783R: ATAAGCTTAGCCTGAAGAATTGAGCAAA (SEQ ID NO:55)). Similarly, SpeI and SacI sites were incorporated into the 5′ and 3′ ends of the corresponding NtHMA reverse complementary fragment by utilizing PCR primers (PMG 785F: ATGAGCTCTGGTTATGTAGGCTACTGCTGCT (SEQ ID NO:56) and PMG 786R: ATACTAGTATTTGTAGTGCCAGCCCAGA (SEQ ID NO:57)) to produce the PBKCMV-NtHMA RNAi plasmid. The PBI121-NtHMA RNAi expression vectors were constructed by (a) excising the β-glucuronidase ORF from the binary expression vector (“pBI121” from CLONTECH), and (b) substituting the NtHMA RNAi construct, excised from the PBKCMV-NtHMA RNAi plasmid, into XbaI/SacI sites of the PBI121 plasmid in place of the removed β-glucuronidase ORF. The PBI121-NtHMA RNAi expression vectors comprise: (i) 352 bp XbaI-HindIII NtHMA sense fragment that includes (ii) 80 bp intron fragment, operably-linked to the (iii) 272 bp SpeII-SacI NtHMA reverse complementary fragment.
Example 3
Construction of NtHMA RNAi Expression Vector PBI121-NtHMA (1382-1584) Encoding RNAi Polynucleotides
FIG. 4A shows an exemplary RNAi sequence, NtHMA (1382-1584), for producing NtHMA RNAi polynucleotides of interest. Based on exon mapping and sequence analysis of genomic clone CHO_OF261xo09c1, a RNAi construct was designed that includes a sense fragment (191 bp) (SEQ ID NO:42) comprising sequences of exon 7, which is positioned upstream and operably-linked to a spacer DNA fragment (139 bp) (SEQ ID NO:43) comprising sequences of intron 8, which is positioned upstream and operably-linked to a reverse complementary fragment (196 bp) (SEQ ID NO:44) comprising sequences of exon 7 positioned in anti-sense orientation. These RNAi constructs encoding NtHMA RNAi polynucleotides of interest were inserted into the PBKCMV cloning vector, and were placed downstream and operably-linked to a cytomegalovirus (CMV) promoter. XbaI and HindIII sites were incorporated into the 5′ and 3′ ends of the 330 bp NtHMA sense fragment, which included the 139 bp intron fragment by utilizing PCR primers modified to incorporate these restriction enzyme sites (PMG754F: ATTCTAGATGAGAGCAAGTCAGGTCATCC (SEQ ID NO:58) and PMG754R: ATAAGCTTTTCAAACATCCACCGCATTA (SEQ ID NO:59)). Similarly, PstI and SacI sites were incorporated into the 5′ and 3′ ends of the corresponding NtHMA reverse complementary fragment by utilizing PCR primers PMG757F: ATGAGCTCGCATTGAGAGCAAGTCAGGTC (SEQ ID NO:60) and PMG757R: ATCTGCAGCCTGTGGTACATCCAGCTCTT (SEQ ID NO:61)) to produce the PBKCMV-NtHMA RNAi expression vector.
The PBI121-NtHMA RNAi expression vectors were constructed by (a) excising the β-glucuronidase ORF from the binary expression vector (“pBI121” from CLONTECH), and (b) substituting the NtHMA RNAi construct, excised from the PBKCMV-NtHMA RNAi plasmid, into XbaI/SacI sites of the PBI121 plasmid in place of the removed β-glucuronidase ORF. The PBI121-NtHMA RNAi expression vectors comprise: (i) 330 bp XbaI-HindIII NtHMA sense fragment that includes (ii) 139 bp intron fragment, operably-linked to the (iii) 196 bp SpeII-SacI NtHMA reverse complementary fragment. The PBI121-NtHMA RNAi expression vectors, such as those described in Examples 2 and 3, can be introduced into any host plant cell of interest by various methods known to persons skilled in the art.
Example 4
Transformation of Burley (TN90), Flue-Cured (K326), and Dark (VA359) Tobacco Varieties with NtHMA RNAi Expression Vectors
Tobacco seeds from three different varieties, Burley (TN90), Flue-cured (K326), and Dark (VA359), were sterilized and germinated in a petridish containing MS basal media supplemented with 5 ml/L plant preservative mixture (PPM). Seedlings, at approximately 7 to 10 days post-germination, were selected for transformation with various NtHMA RNAi expression vectors. A single colony of Agrobacterium tumefaciens LBA4404 was inoculated into a liquid LB medium containing 50 mg l−1 kanamycin (kanamycin mono sulphate), and were incubated for 48 h at 28° C. with reciprocal shaking (150 cycles min−1). Cultured bacterial cells were collected by centrifugation (6000×g, 10 min), and were suspended to a final density of 0.4-0.7 OD600, with 20 ml liquid MS medium containing 20 g−1 sucrose. The 7-10 day seedling explants were immersed into a bacterial suspension for 5 mins, and were blotted on sterile filter papers. Fifty explants were placed onto 40 ml aliquots of REG agar medium (MS basal medium supplemented with 0.1 mg l−1 NAA and 1 mg l−1 BAP) in 100 mm×20 mm petri dishes. The explants were co-cultivated with Agrobacterium at 25° C. After 3 days of co-cultivation, the explants were washed and transferred to RCPK medium (REG medium with 100 mg−1 kanamycin, 500 mg l−1 carbenicillin, and 5 ml PPM) to select for transformants. The explants were subcultured every 2 weeks. After 8-12 weeks of growth under selective conditions, the surviving plants, representing transformants that have integrated the NtHMA RNAi expression constructs into their genomes, were transferred to a rooting medium (MS basal medium supplemented with 100 mg l−1 Kanamycin). Rooted plants were transferred to pots to promote further growth.
Example 5
Cd Reduction in Leaf Lamina of First Generation Transgenics Genetically Modified to Express NtHMA RNAi Polynucleotides
To determine the effect of NtHMA RNAi polynucleotide expression on Cd transport from the root to aerial portions of transgenic plants, the Cd levels were determined for several transgenic lines that have been genetically modified to express either the NtHMA (660-915) or the (1382-1584) RNAi polynucleotides.
Approximately 40 independent transgenic plants, representing three tobacco varieties, were transformed with various PBI121-NtHMA RNAi expression vectors. Initially, transformants were grown in floating trays containing Hoaglands medium for 4 weeks. PCR positive plants for NPT II were selected and potted in 10′′ pots with a hydroponic system containing Hoaglands medium containing 5 μM CdCl2. After 4-8 weeks, two middle leaves samples were harvested and freeze-dried for metal analysis, or were frozen in liquid nitrogen for gene expression analysis. Approximately 500 mg of tobacco was weighed and digested in 10 ml of concentrated HNO3 by microwave-accelerated, reaction system 5 digestion system (CEM corporation, Mathews, N.C.). Heavy metal concentrations were analyzed utilizing inductively coupled plasma-mass spectrophotometry (“ICP-MS,” Agilent 7500A; Agilent Technologies, Palo Alto, Calif.). As non-transgenic tobacco control, a sample consisting of polish-certified, Virginia tobacco leaves, CTA-VTL-2, was prepared under comparable conditions (Dybczynski et al., 1997).
FIGS. 3B-3D show Cd reduction in leaf lamina of multiple first generation (T0) transgenic lines, representing three varieties, that have been genetically modified to express NtHMA RNAi polynucleotides (660-915).
FIGS. 4B-4D show Cd reduction in leaf lamina of multiple first generation (T0) transgenic lines, representing three varieties, that have been genetically modified to express NtHMA RNAi polynucleotides (1382-1584).
Example 6
Reduction in NtHMA RNA Transcripts in Transgenic Tobacco Leaf by the Expression of NtHMA RNAi Polynucleotides
To determine the effect of NtHMA RNAi polynucleotide expression on the steady-state levels of endogenous NtHMA RNA transcripts, the relative change in NtHMA RNA transcripts was measured by isolating total cellular RNA from leaf lamina portions of various transgenic lines, representing three tobacco varieties.
Total RNA was isolated from middle leaves of T0 plants using TRIO Reagent (Sigma-Aldrich, St. Louis, Mo.). To remove DNA impurities, purified RNA was treated with RNase-free DNase (TURBO DNA-free, Ambion, Austin Tex.). To synthesize the first cDNA strand, approximately 10 μg of total RNA was reverse transcribed utilizing the High Capacity cDNA Archive Kit (Applied Biosystems, Foster City, Calif.). To measure the level of NtHMA transcripts in the samples, a quantitative 2-step RT-PCR was performed according to the Taqman MGB probe-based chemistry. The RT mixture contained 4 μM dNTP mix, 1× random primers, 1×RT Buffer, 10 g cDNA, 50 U Multiscribe Reverse transcriptase (Applied Biosystems), 2 U Superase-In RNase Inhibitor (Ambion), and nuclease-free water. The PCR mixture contained 1× Taqman Universal PCR Master Mix (Applied Biosystems, Foster City, Calif.), 400 nM forward primer, 400 nM reverse primer, 250 nM Taqman MGB probe, 2 ng of cDNA, and nuclease-free water. RT-PCR was performed utilizing an ABI 7500 Real-Time System (Applied Biosystems, Foster City, Calif.) and under amplification conditions: 50° C. for 2 min.; 95° C. for 10 min.; 40 cycles of 95° C. for 15 sec.; and 60° C. for 1 min. For normalizing the measured NtHMA RNA transcript levels, the Glyceraldehyde-3-Phosphate Dehydrogenase (G3PDH) was selected as a control endogenous RNA transcript, whose expression level is not responsive to the sequence-specific RNA interference activity of the NtHMA RNAi polynucleotides under analysis. The fold change in NtHMA RNA transcript level caused by NtHMA-RNAi-polynucleotide expression was calculated by determining the ratio of (a)/(b), in which (a) represents the normalized value of NtHMA RNA transcript level determined for samples derived from transgenic plants transformed with a NtHMA RNAi expression vector, and (b) represents the normalized value of NtHMA RNA transcript level determined for samples derived from transgenic plants transformed with a control expression vector deficient in the NtHMA RNAi RNAi construct.
FIGS. 5A-C show normalized NtHMA RNA transcript levels in various first generation (T0) transgenic lines that have been genetically modified to express NtHMA RNAi polynucleotides of interest, as determined by quantitative realtime PCR analysis of leaf lamina extracts. FIG. 5A shows that for multiple independently derived K326 transgenic lines, the RNA transcript levels were reduced by the RNA interference activity of NtHMA (660-915) RNAi polynucleotides. FIG. 5B shows that for multiple, independently derived TN90 transgenic lines, the RNA transcript levels were reduced by the RNA interference activity of NtHMA (660-915) RNAi polynucleotides. FIG. 5C shows that for multiple independently derived VA359 transgenic lines, the RNA transcript levels were reduced by the RNA interference activity of NtHMA (660-915) RNAi polynucleotides. The reduction in NtHMA RNA transcript levels is consistent with the reduction in Cd content measured in the middle leaves for the same transgenic lines tested. “PBI121” represents an expression vector deficient in the RNAi construct encoding NtHMA (660-915) RNAi polynucleotides.
Example 7
Distribution of Cd and Zn in Transgenic Lines Genetically Modified to Express NtHMA RNAi Polynucleotides
To determine the effect of NtHMA (660-915) RNAi polynucleotide expression on the distribution of Cd and Zn within the leaf lamina and the root, the metal content of transgenic plants of three varieties were analyzed. Five transgenic lines of each variety, i.e., Flue-cured (K326), Burley (TN90), and Dark (VA359), were selected for exhibiting Cd content at the lowest range in the leaf lamina. The middle leaves and roots of these transgenic plants and control plants were harvested for metal analysis by ICP_MS. For 8 weeks, all plants were grown in Hoaglands medium supplemented with 5 μM CdCl2 prior to harvesting.
Table 2 lists Cd and Zn levels measured in the leaf lamina and the root of several transgenic lines, representing three tobacco varieties, as provided below. In Table 2, the Cd distribution between the leaf lamina and the root were substantially modified by the expression of NtHMA (660-915) RNAi polynucleotides for all three varieties, Flue-cured (K326), Burley (TN90), and Dark (VA359). For the K326 transgenic lines, the % Cd reduction ranged from 97.16-98.54% when compared to Cd levels observed in K326 Control plants. For TN90 transgenic lines, the % Cd reduction ranged from 85.12-90.96% when compared to Cd levels observed in the TN90 Control. For VA359 transgenic lines, the % Cd reduction ranged from 93.24-99.07% when compared to Cd levels observed in the VA359 Control. The VA359 NtHMA-11 transgenic line exhibited the lowest Cd level (1.62 μg/g) and the highest % Cd reduction (99.07%), when compared against two NtHMA RNAi transgene-deficient control lines (“VA359 PBI121”) that exhibited Cd levels at 158.3-205.96 μg/g. Comparable root analysis of the transgenic lines showed, that a substantial amount of Cd can accumulate in the root, resulting in fold increase in root Cd levels ranging from 6.90-15.38, relative to the Cd levels observed in the respective controls.
In contrast to the significant Cd reduction in the leaf lamina of transgenic lines, the Zn content of the leaf lamina was not substantially reduced, although some reduction was observed in most transgenic lines, caused by the expression of NtHMA (660-915) RNAi polynucleotides. The Zn content within the root (last column of Table 3) increased in all transgenic lines, resulting in a 4-6 fold increase in the transgenic lines of the K326 and VA359 varieties, and a 3-5 fold increase in the TN90 variety.
FIG. 6 shows the distribution of Cd and Zn between the leaf lamina and the root of various first generation transgenic lines that have been genetically modified to express NtHMA RNAi polynucleotides of interest, as presented in Table 2.









TABLE 2








Leaf
Root











Transgenic
Cd
Zn
Cd
Zn


Variety
μg/g
μg/g
μg/g
μg/g














K326 06T458
7.09
22.2
703
201


K326 06T459
4.97
24.1
696
225


K326 06T473
3.7
34
929
215


K326 06T480
3.93
38.6
989
224


K326 06T482
2.55
36.3
520
126


K326 Control
174.7
36.3
64.3
35.7


TN90 06T428
26.3
48.6
626
184


TN90 06T430
16.08
37.2
684
213


TN90 06T444
15.98
28.1
738
234


TN90 06T445
20.72
32.6
618
186


TN90 06T455
17.87
24.4
582
157


TN90 PBI121
181.2
35.5
62.6
44.3


TN90 Control
172.4
32.3
72.9
46.6


VA359 06T493
7.59
23.1
543
148


VA359 06T498
1.62
26.2
706
175


VA359 06T506
5.72
28.8
351
109


VA359 06T542
7.03
27.1
738
136


VA359 06T543
11.78
29.3
547
106


VA359 PBI121
206
47.5
35.3
27.6


VA359 Control 
158.5
32.6
37.6
26.2









Example 8
Cd Distribution in Various Tissues of Transgenic Lines Genetically Modified to Express NtHMA RNAi Polynucleotides
To determine the effect of NtHMA (1382-1584) RNAi polynucleotide expression on Cd distribution within various tissues (i.e., the bark, lamina, pith, and root), the metal content of several transgenic lines representing two varieties, Burley (TN90) and Flue-cured (K326), were analyzed. Fully matured transgenic plants and control plants were harvested for metal analysis by ICP_MS. For 8 weeks, all plants were grown in 5 μM CdCl2 in Hoaglands medium prior to harvesting.
Table 3 lists Cd content in the bark, lamina, pith, and root tissues of several transgenic lines, as provided below. In Table 3, Cd levels were substantially reduced in the bark, lamina, and pith tissues of all transgenic lines tested when compared that of control plants. The “Control” represents non-transgenic plants. The “PBI121” represents transgenic plants transformed with an expression vector deficient in NtHMA RNAi RNAi construct. The extent of Cd reduction in the bark, pith, and leaf lamina of K326 transgenic lines was significantly greater than that observed in TN90 transgenic lines. The expression of RNAi (1382-1584) polynucleotides in K326 transgenic plants resulted in a 9-11 fold Cd reduction in the bark, a 6-13 fold Cd reduction in the pith, and a 31-32 fold Cd reduction in the leaf lamina. The expression of RNAi (1382-1584) polynucleotides in TN90 transgenic plants resulted in a 4-7 fold Cd reduction in the bark, a 5-8 fold Cd reduction in the pith, and a 6-20 fold Cd reduction in the leaf lamina. In contrast, more modest increases (5-6 fold) in Cd content in the root of these transgenic lines were observed when compared to that of control plants.
FIG. 7 shows Cd distribution among the bark, leaf lamina, pith, and the root of various first generation transgenic lines that have been genetically modified to express NtHMA RNAi polynucleotides of interest, as presented in Table 3.













TABLE 3







Transgenic Seed







Variety
Bark Cd
Lamina Cd
Pith Cd
Root Cd






















TN90 06T619
7.36
31.1
4.67
557



TN90 06T658 
3.76
8.89
2.89
727



TN90 Control
30.9
151
25.3
115



TN90 PBI121 
23.1
201
20
124



K326 06T682 
2.02
4.32
1.97
1020



K326 06T696 
2.53
4.48
4.25
1030



K326 Control
19.5
133
25.3
145



K326 PBI121
25.5
143
26.2
253










Example 9
Cd Reduction in Leaf Lamina of Second Generation Transgenic Lines Genetically Modified to Express NtHMA RNAi Polynucleotides
To determine the effect of NtHMA (660-915) RNAi polynucleotide expression on Cd content in leaf lamina, the metal content of two (T1) transgenic lines of VA359 variety were grown in soil containing variable Cd concentrations for 4 weeks. Two transgenic lines, 06T498 and 06T506, selected as kanamycin positives were screened by PCR. Several 10′′ Pots filled with sand:soil mixture were saturated with either 0, 0.1, 0.5, or 5 μM CdCl2. Three plants per treatment per transgenic line were grown for 4 weeks by adding Hoaglands medium to the saucer. Total number of leaves, leaf area index, leaf weight, stalk weight, and root weight were observed. Two middle leaves and root samples were freeze-dried and were subjected to heavy metal analysis.
FIG. 8 shows Cd distribution between the leaf lamina and the root of various second generation (T1) transgenic lines that have been genetically modified to express NtHMA RNAi polynucleotides of interest. In FIG. 8, the Cd content of the transgenic plants was consistently lower than that of control plants at all Cd concentrations tested (0, 0.1, 0.5, and 5 μM). A reduction in Cd content of the leaf lamina (2-4.7 fold) was observed in various transgenic lines tested. The Cd level for the line 06T498 was only  ̃20% of control plants at 5 μM CdCl2. An increase in root Cd content (4-16 fold) was observed in various transgenic lines tested. The highest root Cd content (a 16 fold increase) was observed for line 06T498 at 5 μM CdCl2. Thus, the reduced heavy metal content in the leaf lamina/shoots in transgenic lines, expressing NtHMA (660-915) RNAi polynucleotide, suggested that the translocation of a substantial amount of heavy metals from the root to the leaf lamina/shoots can be interrupted by RNAi interference. The results are consistent with Cd reduction observed in the leaf lamina of first generation transgenic lines, in that the second generation transgenic lines also demonstrated (a) reduced Cd levels in the leaf lamina, and (b) increased Cd in the roots. The transgenic lines did not demonstrate phenotypical differences in general appearance, growth, and development relative to that of control plants.
Example 10
NtHMA Polynucleotides
A NtHMA polynucleotide will generally contain phosphodiester bonds, although in some cases, nucleic acid analogs are included that may have alternate backbones, comprising, e.g., phosphoramidate, phosphorothioate, phosphorodithioate, or O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press); and peptide nucleic acid backbones and linkages. Other analog nucleic acids include those with positive backbones; non-ionic backbones, and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, Carbohydrate Modifications in Antisense Research, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids. Modifications of the ribose-phosphate backbone may be done for a variety of reasons, e.g. to increase the stability and half-life of such molecules in physiological environments or as probes on a biochip. Mixtures of naturally occurring nucleic acids and analogs can be made; alternatively, mixtures of different nucleic acid analogs, and mixtures of naturally occurring nucleic acids and analogs may be made.
A variety of references disclose such nucleic acid analogs, including, for example, phosphoramidate (Beaucage et al., Tetrahedron 49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem. 35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977); Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem. Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate (Mag et al., Nucleic Acids Res. 19:1437 (1991); and U.S. Pat. No. 5,644,048), phosphorodithioate (Briu et al., J. Am. Chem. Soc. 111:2321 (1989), O-methylphophoroamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practical Approach, Oxford University Press), and peptide nucleic acid backbones and linkages (see Egholm, J. Am. Chem. Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl. 31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature 380:207 (1996), all of which are incorporated by reference). Other analog nucleic acids include those with positive backbones (Denpcy et al., Proc. Natl. Acad. Sci. USA 92:6097 (1995); non-ionic backbones (U.S. Pat. Nos. 5,386,023, 5,637,684, 5,602,240, 5,216,141 and 4,469,863; Kiedrowshi et al., Angew. Chem. Intl. Ed. English 30:423 (1991); Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988); Letsinger et al., Nucleoside & Nucleotide 13:1597 (1994); Chapters 2 and 3, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook; Mesmaeker et al., Bioorganic & Medicinal Chem. Lett. 4:395 (1994); Jeffs et al., J. Biomolecular NMR 34:17 (1994); Tetrahedron Lett. 37:743 (1996)) and non-ribose backbones, including those described in U.S. Pat. Nos. 5,235,033 and 5,034,506, and Chapters 6 and 7, ASC Symposium Series 580, “Carbohydrate Modifications in Antisense Research”, Ed. Y. S. Sanghui and P. Dan Cook. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev. (1995) pp 169-176). Several nucleic acid analogs are described in Rawls, C & E News Jun. 2, 1997 page 35. These references are hereby expressly incorporated by reference.
Other analogs include peptide nucleic acids (PNA) which are peptide nucleic acid analogs. These backbones are substantially non-ionic under neutral conditions, in contrast to the highly charged phosphodiester backbone of naturally occurring nucleic acids. This results in two advantages. First, the PNA backbone exhibits improved hybridization kinetics. PNAs have larger changes in the melting temperature (Tm) for mismatched versus perfectly matched basepairs. DNA and RNA typically exhibit a 2-4° C. drop in Tm for an internal mismatch. With the non-ionic PNA backbone, the drop is closer to 7-9° C. Similarly, due to their non-ionic nature, hybridization of the bases attached to these backbones is relatively insensitive to salt concentration. In addition, PNAs are not degraded by cellular enzymes, and thus can be more stable.
Among the uses of the disclosed NtHMA polynucleotides, and combinations of fragments thereof, is the use of fragments as probes or primers or in the development of RNAi molecules. Such fragments generally comprise at least about 17 contiguous nucleotides of a DNA sequence. In other embodiments, a DNA fragment comprises at least 30, or at least 60 contiguous nucleotides of a DNA sequence. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook et al., 1989 and are described in detail above. Using knowledge of the genetic code in combination with the amino acid sequences set forth above, sets of degenerate oligonucleotides can be prepared. Such oligonucleotides are useful as primers, e.g., in polymerase chain reactions (PCR), whereby DNA fragments are isolated and amplified. In certain embodiments, degenerate primers can be used as probes for non-human genetic libraries. Such libraries would include but are not limited to cDNA libraries, genomic libraries, and even electronic EST (express sequence tag) or DNA libraries. Homologous sequences identified by this method would then be used as probes to identify non-human homologues of the NtHMA sequence identified herein.
The disclosure also includes polynucleotides and oligonucleotides that hybridize under reduced stringency conditions, typically moderately stringent conditions, and commonly highly stringent conditions, to an NtHMA polynucleotide described herein. The basic parameters affecting the choice of hybridization conditions and guidance for devising suitable conditions are set forth by Sambrook, J., E. F. Fritsch, and T. Maniatis (1989, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., chapters 9 and 11; and Current Protocols in Molecular Biology, 1995, F. M. Ausubel et al., eds., John Wiley & Sons, Inc., sections 2.10 and 6.3-6.4, incorporated herein by reference), and can be readily determined by those having ordinary skill in the art based on, for example, the length and/or base composition of the polynucleotide. One way of achieving moderately stringent conditions involves the use of a prewashing solution containing 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0), hybridization buffer of about 50% formamide, 6×SSC, and a hybridization temperature of about 55° C. (or other similar hybridization solutions, such as one containing about 50% formamide, with a hybridization temperature of about 42° C.), and washing conditions of about 60° C., in 0.5×SSC, 0.1% SDS. Generally, highly stringent conditions are defined as hybridization conditions as above, but with washing at approximately 68° C., 0.2×SSC, 0.1% SDS. SSPE (1×SSPE is 0.15M NaCl, 10 mM NaH2PO4, and 1.25 mM EDTA, pH 7.4) can be substituted for SSC (1×SSC is 0.15M NaCl and 15 mM sodium citrate) in the hybridization and wash buffers; washes are performed for 15 minutes after hybridization is complete. It should be understood that the wash temperature and wash salt concentration can be adjusted as necessary to achieve a desired degree of stringency by applying the basic principles that govern hybridization reactions and duplex stability, as known to those skilled in the art and described further below (see, e.g., Sambrook et al., 1989). When hybridizing a nucleic acid to a target polynucleotide of unknown sequence, the hybrid length is assumed to be that of the hybridizing nucleic acid. When nucleic acids of known sequence are hybridized, the hybrid length can be determined by aligning the sequences of the nucleic acids and identifying the region or regions of optimal sequence complementarity. The hybridization temperature for hybrids anticipated to be less than 50 base pairs in length should be 5 to 10° C. less than the melting temperature (Tm) of the hybrid, where Tm is determined according to the following equations. For hybrids less than 18 base pairs in length, Tm (° C.)=2(# of A+T bases)+4(# of G+C bases). For hybrids above 18 base pairs in length, Tm (° C.)=81.5+16.6(log 10 [Na+])+0.41(% G+C)−(600/N), where N is the number of bases in the hybrid, and [Na+] is the concentration of sodium ions in the hybridization buffer ([Na+] for 1×SSC=0.165M). Typically, each such hybridizing nucleic acid has a length that is at least 25% (commonly at least 50%, 60%, or 70%, and most commonly at least 80%) of the length of a polynucleotide of the disclosure to which it hybridizes, and has at least 60% sequence identity (e.g., at least 70%, 75%, 80%, 85%, 90%, 95%, 97.5%, or at least 99%) with a polynucleotide of the disclosure to which it hybridizes.
Example 11
NtHMA Polypeptides
A polypeptide of the disclosure may be prepared by culturing transformed or recombinant host cells under culture conditions suitable to express a polypeptide of the disclosure. The resulting expressed polypeptide may then be purified from such culture using known purification processes. The purification of the polypeptide may also include an affinity column containing agents which will bind to the polypeptide; one or more column steps over such affinity resins as concanavalin A-agarose, heparin-Toyopearl® or Cibacrom blue 3GA Sepharose®; one or more steps involving hydrophobic interaction chromatography using such resins as phenyl ether, butyl ether, or propyl ether; or immunoaffinity chromatography. Alternatively, the polypeptide of the disclosure may also be expressed in a form that will facilitate purification. For example, it may be expressed as a fusion polypeptide, such as those of maltose binding polypeptide (MBP), glutathione-5-transferase (GST) or thioredoxin (TRX). Kits for expression and purification of such fusion polypeptides are commercially available from New England BioLab (Beverly, Mass.), Pharmacia (Piscataway, N.J.), and InVitrogen, respectively. The polypeptide can also be tagged with an epitope and subsequently purified by using a specific antibody directed to such epitope. Finally, one or more reverse-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, e.g., silica gel having pendant methyl or other aliphatic groups, can be employed to further purify the polypeptide. Some or all of the foregoing purification steps, in various combinations, can also be employed to provide a substantially homogeneous recombinant polypeptide. The polypeptide thus purified is substantially free of other mammalian polypeptides and is defined in accordance with the invention as an “substantially purified polypeptide”; such purified polypeptides include NtHMA polypeptide, fragment, variant, and the like. Expression, isolation, and purification of the polypeptides and fragments of the disclosure can be accomplished by any suitable technique, including but not limited to the methods described herein.
It is also possible to utilize an affinity column such as a monoclonal antibody generated against polypeptides of the disclosure, to affinity-purify expressed polypeptides. These polypeptides can be removed from an affinity column using conventional techniques, e.g., in a high salt elution buffer and then dialyzed into a lower salt buffer for use or by changing pH or other components depending on the affinity matrix utilized, or be competitively removed using the naturally occurring substrate of the affinity moiety, such as a polypeptide derived from the disclosure.
A polypeptide of the disclosure may also be produced by known conventional chemical synthesis. Methods for constructing the polypeptides of the disclosure or fragments thereof by synthetic means are known to those skilled in the art. The synthetically-constructed polypeptide sequences, by virtue of sharing primary, secondary or tertiary structural and/or conformational characteristics with a native polypeptides may possess biological properties in common therewith, including biological activity.
Example 12
Anti-NtHMA Antibodies
In another embodiment, antibodies that are immunoreactive with the polypeptides of the disclosure are provided herein. The NtHMA polypeptides, fragments, variants, fusion polypeptides, and the like, as set forth herein, can be employed as “immunogens” in producing antibodies immunoreactive therewith. Such antibodies specifically bind to the polypeptides via the antigen-binding sites of the antibody. Specifically binding antibodies are those that will specifically recognize and bind with NtHMA family polypeptides, homologues, and variants, but not with other molecules. In one embodiment, the antibodies are specific for polypeptides having an NtHMA amino acid sequence of the disclosure as set forth in SEQ ID NO:2 and do not cross-react with other polypeptides.
More specifically, the polypeptides, fragment, variants, fusion polypeptides, and the like contain antigenic determinants or epitopes that elicit the formation of antibodies. These antigenic determinants or epitopes can be either linear or conformational (discontinuous). Linear epitopes are composed of a single section of amino acids of the polypeptide, while conformational or discontinuous epitopes are composed of amino acids sections from different regions of the polypeptide chain that are brought into close proximity upon polypeptide folding. Epitopes can be identified by any of the methods known in the art. Additionally, epitopes from the polypeptides of the disclosure can be used as research reagents, in assays, and to purify specific binding antibodies from substances such as polyclonal sera or supernatants from cultured hybridomas. Such epitopes or variants thereof can be produced using techniques known in the art such as solid-phase synthesis, chemical or enzymatic cleavage of a polypeptide, or using recombinant DNA technology.
Both polyclonal and monoclonal antibodies to the polypeptides of the disclosure can be prepared by conventional techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyses, Kennet et al. (eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., (1988); Kohler and Milstein, (U.S. Pat. No. 4,376,110); the human B-cell hybridoma technique (Kosbor et al., Immunology Today 4:72, 1983; Cole et al., Proc. Natl. Acad. Sci. USA 80:2026, 1983); and the EBV-hybridoma technique (Cole et al., 1985, Monoclonal Antibodies And Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). Hybridoma cell lines that produce monoclonal antibodies specific for the polypeptides of the disclosure are also contemplated herein. Such hybridomas can be produced and identified by conventional techniques. For the production of antibodies, various host animals may be immunized by injection with an NtHMA polypeptide, fragment, variant, or mutants thereof. Such host animals may include, but are not limited to, rabbits, mice, and rats, to name a few. Various adjutants may be used to increase the immunological response. Depending on the host species, such adjutants include, but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, dinitrophenol, and potentially useful human adjutants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. The monoclonal antibodies can be recovered by conventional techniques. Such monoclonal antibodies may be of any immunoglobulin class including IgG, IgM, IgE, IgA, IgD, and any subclass thereof.
The antibodies of the disclosure can also be used in assays to detect the presence of the polypeptides or fragments of the disclosure, either in vitro or in vivo. The antibodies also can be employed in purifying polypeptides or fragments of the disclosure by immunoaffinity chromatography.
Example 13
Double-Stranded RNAs
In one embodiment, the disclosure provides double-stranded ribonucleic acid (dsRNA) molecules for inhibiting the expression of the NtHMA gene in a cell (e.g., a plant cell), wherein the dsRNA comprises an antisense strand comprising a region of complementarity which is complementary to at least a part of an mRNA formed in the expression of the NtHMA gene, and wherein the region of complementarity is less than 30 nucleotides in length and wherein said dsRNA, upon contact with a cell expressing said NtHMA gene, inhibits the expression of said NtHMA gene by at least 20%. The dsRNA comprises two RNA strands that are sufficiently complementary to hybridize to form a duplex structure. One strand of the dsRNA (the antisense strand) comprises a region of complementarity that is substantially complementary, and typically fully complementary, to a target sequence, derived from the sequence of an mRNA formed during the expression of the NtHMA gene, the other strand (the sense strand) comprises a region which is complementary to the antisense strand, such that the two strands hybridize and form a duplex structure when combined under suitable conditions. The duplex structure is between about 15 and 30 (e.g., between about 18 and 25), typically between about 19 and 24 (e.g., between 21 and 23) base pairs in length. Similarly, the region of complementarity to the target sequence is between 15 and 30 (e.g., between about 18 and 25), typically between about 19 and 24 (e.g., between 21 and 23) base pairs in length. The dsRNA of the disclosure may further comprise one or more single-stranded nucleotide overhang(s). The dsRNA can be synthesized by standard methods known in the art as further discussed below, e.g., by use of an automated DNA synthesizer, such as are commercially available from, for example, Biosearch, Applied Biosystems, Inc. In another aspect, an expression vector can be used to express an RNAi molecule in vivo.
The dsRNA of the disclosure can contain one or more mismatches to the target sequence. In one embodiment, the dsRNA of the disclosure contains more than 3 mismatches. If the antisense strand of the dsRNA contains mismatches to a target sequence, it is typical that the area of mismatch not be located in the center of the region of complementarity. If the antisense strand of the dsRNA contains mismatches to the target sequence, it is typical that the mismatch be restricted to 5 nucleotides from either end, for example 5, 4, 3, 2, or 1 nucleotide from either the 5′ or 3′ end of the region of complementarity. For example, for a 23 nucleotide dsRNA strand which is complementary to a region of the NtHMA gene, the dsRNA preferably does not contain any mismatch within the central 13 nucleotides. The methods described within the disclosure can be used to determine whether a dsRNA containing a mismatch to a target sequence is effective in inhibiting the expression of the NtHMA gene.
In one embodiment, at least one end of the dsRNA has a single-stranded nucleotide overhang of 1 to 4 (e.g., 1 or 2 nucleotides). dsRNAs having at least one nucleotide overhang have inhibitory properties. The dsRNA may also have a blunt end, typically located at the 5′-end of the antisense strand.
In yet another embodiment, the dsRNA is chemically modified to enhance stability. The nucleic acids of the disclosure may be synthesized and/or modified by methods well established in the art, such as those described in “Current protocols in nucleic acid chemistry”, Beaucage, S. L. et al. (Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is hereby incorporated herein by reference. Chemical modifications may include, but are not limited to 2′ modifications, introduction of non-natural bases, covalent attachment to a ligand, and replacement of phosphate linkages with thiophosphate linkages. In this embodiment, the integrity of the duplex structure is strengthened by at least one, and typically two, chemical linkages. Chemical linking may be achieved by any of a variety of well-known techniques, for example by introducing covalent, ionic or hydrogen bonds; hydrophobic interactions, van der Waals or stacking interactions; by means of metal-ion coordination, or through use of purine analogues.
In yet another embodiment, the nucleotides at one or both of the two single strands may be modified to prevent or inhibit the activation of cellular enzymes, such as, for example, without limitation, certain nucleases. Techniques for inhibiting the activation of cellular enzymes are known in the art including, but not limited to, 2′-amino modifications, 2′-fluoro modifications, 2′-alkyl modifications, uncharged backbone modifications, morpholino modifications, 2′-O-methyl modifications, and phosphoramidate (see, e.g., Wagner, Nat. Med. (1995) 1:1116-8). Thus, at least one 2′-hydroxyl group of the nucleotides on a dsRNA is replaced by a chemical group. Also, at least one nucleotide may be modified to form a locked nucleotide. Such locked nucleotide contains a methylene or ethylene bridge that connects the 2′-oxygen of ribose with the 4′-carbon of ribose. Oligonucleotides containing the locked nucleotide are described in Koshkin, A. A., et al., Tetrahedron (1998), 54: 3607-3630) and Obika, S. et al., Tetrahedron Lett. (1998), 39: 5401-5404). Introduction of a locked nucleotide into an oligonucleotide improves the affinity for complementary sequences and increases the melting temperature by several degrees (Braasch, D. A. and D. R. Corey, Chem. Biol. (2001), 8:1-7).
Conjugating a ligand to a dsRNA can enhance its cellular absorption. In certain instances, a hydrophobic ligand is conjugated to the dsRNA to facilitate direct permeation of the cellular membrane. Alternatively, a ligand conjugated to the dsRNA is a substrate for receptor-mediated endocytosis. These approaches have been used to facilitate cell permeation of antisense oligonucleotides. In certain instances, conjugation of a cationic ligand to oligonucleotides often results in improved resistance to nucleases. Representative examples of cationic ligands are propylammonium and dimethylpropylammonium. Interestingly, anti-sense oligonucleotides were reported to retain their high binding affinity to mRNA when the cationic ligand was dispersed throughout the oligonucleotide. See M. Manoharan Antisense & Nucleic Acid Drug Development 2002, 12, 103 and references therein.
Example 15
Methods for Identifying NtHMA Modulatory Agents
The disclosure provides methods for identifying agents that can modulate NtHMA expression level and/or activity. Candidates (“a test agent”) that may be screened to identify NtHMA-specific modulatory activity include small molecules, chemicals, peptidomimetics, antibodies, peptides, polynucleotides (e.g., RNAi, siRNA, antisense or ribozyme molecules), and agents developed by computer-based design. Modulation of NtHMA includes an increase or decrease in activity or expression. For example, a method for identifying candidates that can modulate NtHMA expression and/or activity, comprises: contacting a sample containing an NtHMA polypeptide or polynucleotide with a test agent under conditions that allow the test agent and the NtHMA polypeptide or polynucleotide to interact, and measuring the expression and/or activity of the NtHMA polypeptide in the presence or absence of the test agent.
In one embodiment, a cell containing an NtHMA polynucleotide is contacted with a test agent under conditions such that the cell and test agent are allowed to interact. Such conditions typically include normal cell culture conditions consistent with the particular cell type being utilized, known in the art. It may be desirable to allow the test agent and the cell to interact under conditions associated with increased temperature or in the presence of regents that facilitate the uptake of the test agent by the cell. A control is treated similarly but in the absence of the test agent. Alternatively, the NtHMA activity or expression may be measured prior to contact with the test agent (e.g., the standard or control measurement) and then again following contact with the test agent. The treated cell is then compared to the control and a difference in the expression or activity of NtHMA compared to the control is indicative of an agent that modulates NtHMA activity or expression.
When NtHMA expression is being measured, detecting the amount of mRNA encoding an NtHMA polypeptide in the cell can be quantified by, for example, PCR or Northern blot. Where a change in the amount of NtHMA polypeptide in the sample is being measured, detecting NtHMA by use of anti-NtHMA antibodies can be used to quantify the amount of NtHMA polypeptide in the cell using known techniques. Alternatively the biological activity (e.g., heavy metal transport) can be measured before and after contact with the test agent.
It will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and the scope of the invention. Accordingly, the invention is not limited except as by the appended claims. Unless defined otherwise, all technical and scientific terms have standard meaning as commonly understood to persons skilled in the art. Although exemplary methods, devices, and materials have been described with particularity, alternative methods and materials, that may be similar or equivalent to those described herein, are applicable for making the disclosed compositions and for practicing the disclosed methods.
Any publication cited or described herein provides relevant information disclosed prior to the filing date of the present application. Statements herein are not to be construed as an admission that the inventors are not entitled to antedate such disclosures.

## Claims

1. An NtHMA RNAi construct that inhibits the expression of an NtHMA messenger RNA to which it corresponds, wherein the construct comprises a heterologous promoter and:
(a) a first sequence having at least 95% sequence identity to a sequence selected from the group consisting of:
exon 1 (SEQ ID NO: 5), a fragment of exon 1 (SEQ ID NO: 5),
exon 2 (SEQ ID NO: 7), a fragment of exon 2 (SEQ ID NO: 7),
exon 3 (SEQ ID NO: 9), a fragment of exon 3 (SEQ ID NO: 9),
exon 4 (SEQ ID NO: 11), a fragment of exon 4 (SEQ ID NO: 11),
exon 5 (SEQ ID NO: 13), a fragment of exon 5 (SEQ ID NO: 13),
exon 6 (SEQ ID NO: 15), a fragment of exon 6 (SEQ ID NO: 15),
exon 7 (SEQ ID NO: 17), a fragment of exon 7 (SEQ ID NO: 17),
exon 8 (SEQ ID NO: 19), a fragment of exon 8 (SEQ ID NO: 19),
exon 9 (SEQ ID NO: 21), a fragment of exon 9 (SEQ ID NO: 21),
exon 10 (SEQ ID NO: 23), a fragment of exon 10 (SEQ ID NO:23),
exon 11 (SEQ ID NO: 25), and a fragment of exon 11 (SEQ ID NO:25);

(b) a second sequence having at least 95% sequence identity to a sequence selected from the group consisting of:
intron 1 (SEQ ID NO: 4), a fragment of intron 1 (SEQ ID NO:4),
intron 2 (SEQ ID NO: 6), a fragment of intron 2 (SEQ ID NO:6),
intron 3 (SEQ ID NO: 8), a fragment of intron 3 (SEQ ID NO:8),
intron 4 (SEQ ID NO: 10), a fragment of intron 4 (SEQ ID NO: 10),
intron 5 (SEQ ID NO: 12), a fragment of intron 5 (SEQ ID NO: 12),
intron 6 (SEQ ID NO: 14), a fragment of intron 6 (SEQ ID NO: 14),
intron 7 (SEQ ID NO: 16), a fragment of intron 7 (SEQ ID NO: 16),
intron 8 (SEQ ID NO: 18), a fragment of intron 8 (SEQ ID NO: 18),
intron 9 (SEQ ID NO: 20), a fragment of intron 9 (SEQ ID NO: 20),
intron 10 (SEQ ID NO: 22), a fragment of intron 10 (SEQ ID NO: 22),
intron 11 (SEQ ID NO: 24), a fragment of intron 11 (SEQ ID NO: 24),
intron 12 (SEQ ID NO: 26), and a fragment of intron 12 (SEQ ID NO: 26); and

(c) a third sequence having at least 95% sequence identity to a sequence selected from the group consisting of:
SEQ ID NO: 27, a fragment of SEQ ID NO: 27,
SEQ ID NO: 28, a fragment of SEQ ID NO: 28,
SEQ ID NO: 29, a fragment of SEQ ID NO: 29,
SEQ ID NO: 30, a fragment of SEQ ID NO: 30,
SEQ ID NO: 31, a fragment of SEQ ID NO: 31,
SEQ ID NO: 32, a fragment of SEQ ID NO: 32,
SEQ ID NO: 33, a fragment of SEQ ID NO: 33,
SEQ ID NO: 34, a fragment of SEQ ID NO: 34,
SEQ ID NO: 35, a fragment of SEQ ID NO: 35,
SEQ ID NO: 36, a fragment of SEQ ID NO: 36,
SEQ ID NO: 37, and a fragment of SEQ ID NO: 37;

wherein the third sequence is the reverse complementary sequence of the first sequence,
wherein said fragments are at least 30 contiguous nucleotides of the respective SEQ ID NO; and
wherein the second sequence is positioned between the first sequence and the third sequence, and the second sequence is operably-linked to the first sequence and to the third sequence.

2. An NtHMA RNAi construct capable of inhibiting the expression of an NtHMA messenger RNA to which it corresponds, wherein the construct comprises:
a first sequence comprising SEQ ID NO:38,
a second sequence comprising SEQ ID NO:39, and
a third sequence comprising SEQ ID NO:40;

wherein the second sequence is positioned between the first sequence and the third sequence, and the second sequence is operably-linked to the first sequence and to the third sequence.

3. An NtHMA RNAi construct capable of inhibiting the expression of an NtHMA messenger RNA to which it corresponds, wherein the construct comprises:
a first sequence comprising SEQ ID NO:42,
a second sequence comprising SEQ ID NO:43, and
a third sequence comprising SEQ ID NO:44;

wherein the second sequence is positioned between the first sequence and the third sequence, and the second sequence is operably-linked to the first sequence and to the third sequence.

4. An NtHMA RNAi construct that inhibits the expression of an NtHMA messenger RNA to which it corresponds, wherein the construct comprises a heterologous promoter and:
(a) and (c); or (a) and (b) and (c),
wherein (a) is a first sequence having at least 95% sequence identity to a sequence selected from the group consisting of:
exon 1 (SEQ ID NO: 5), a fragment of exon 1 (SEQ ID NO: 5),
exon 2 (SEQ ID NO: 7), a fragment of exon 2 (SEQ ID NO: 7),
exon 3 (SEQ ID NO: 9), a fragment of exon 3 (SEQ ID NO: 9),
exon 4 (SEQ ID NO: 11), a fragment of exon 4 (SEQ ID NO: 11),
exon 5 (SEQ ID NO: 13), a fragment of exon 5 (SEQ ID NO:13),
exon 6 (SEQ. ID NO:15), a fragment of exon 6 (SEQ ID NO:15),
exon 7 (SEQ ID NO:17), a fragment of exon 7 (SEQ ID NO:17),
exon 8 (SEQ ID NO:19), a fragment of exon 8 (SEQ ID NO:19),
exon 9 (SEQ ID NO:2), a fragment of exon 9 (SEQ ID NO:21),
exon 10 (SEQ ID NO:23), a fragment of exon 10 (SEQ ID NO:23),
exon 11 (SEQ ID NO:25), and a fragment of exon 11 (SEQ ID NO:25);
wherein (b) is a second sequence having at least 95% sequence identity to a sequence selected from the group consisting of:
intron 1 (SEQ ID NO: 4), a fragment of intron 1 (SEQ ID NO: 4),
intron 2 (SEQ ID NO: 6), a fragment of intron 2 (SEQ ID NO:6),
intron 3 (SEQ ID NO: 8), a fragment of intron 3 (SEQ ID NO: 8),
intron 4 (SEQ ID NO: 10), a fragment of intron 4 (SEQ ID NO: 10),
intron 5 (SEQ ID NO:12), a fragment of intron 5 (SEQ ID NO: 12),
intron 6 (SEQ ID NO:14), a fragment of intron 6 (SEQ ID NO: 14),
intron 7 (SEQ ID NO:16), a fragment of intron 7 (SEQ ID NO:16),
intron 8 (SEQ ID NO: 18), a fragment of intron 8 (SEQ ID NO: 18),
intron 9 (SEQ ID NO: 20), a fragment of intron 9 (SEQ ID NO:20),
intron 10 (SEQ ID NO: 22), a fragment of intron 10 (SEQ ID NO:22),
intron 11 (SEQ ID NO: 24), a fragment of intron 11 (SEQ ID NO: 24),
intron 12 (SEQ ID NO: 26), and a fragment of intron 12 (SEQ ID NO: 26); and

wherein (c) is a third sequence having at least 95% sequence identity to a sequence selected from the group consisting of:
SEQ ID NO: 27, a fragment of SEQ ID NO: 27,
SEQ ID NO: 28, a fragment of SEQ ID NO: 28,
SEQ ID NO: 29, a fragment of SEQ ID NO: 29,
SEQ ID NO: 30, a fragment of SEQ ID NO: 30,
SEQ ID NO: 31, a fragment of SEQ ID NO: 31,
SEQ ID NO: 32, a fragment of SEQ ID NO: 32,
SEQ ID NO: 33, a fragment of SEQ ID NO: 33,
SEQ ID NO: 34, a fragment of SEQ ID NO: 34,
SEQ ID NO: 35, a fragment of SEQ ID NO: 35,
SEQ ID NO: 36, a fragment of SEQ ID NO: 36,
SEQ ID NO: 37, and a fragment of SEQ ID NO: 37;


wherein the third sequence is the reverse complementary sequence of the first sequence,
wherein said fragments are at least 30 contiguous nucleotides of the respective SEQ ID NO; and further wherein when said construct comprises (a) and (b) and (c), the second sequence is positioned between the first sequence and the third sequence, and the second sequence Is operably-linked to the first sequence and to the third sequence.