Vicilin-like seed storage protein gene promoter and methods of using the same

The present invention relates to isolated vicilin-like gene promoters, promoter-gene constructs, methods of producing polypeptides in plants, methods of monitoring embryo maturity of conifers, and plants per se.

The expression of seed-storage protein (“SSP”) genes in plants is induced during seed development and is restricted to the embryos. SSP gene expression takes place primarily in specialized storage cells located in the embryonic axis, cotyledons, and the endosperm of developing seeds (Goldberg et al., Cell, 56:149-160, 1989). Notably, SSP gene expression does not take place in mature vegetative organs (Thomas, Plant Cell, 5:1401-1410, 1993). In angiosperms and gymnosperms, maximum accumulation of seed-storage proteins and abundant synthesis of oil and starch have been observed during mid to late embryo development.

One example of SSP gene expression is the synthesis of the 2S protein, a seed storage protein encoded by a multi-gene family which synthesis has been shown to be limited to the later stages of seed development. The expression of the 2S protein has been correlated with the accumulation of 2S mRNA (Gander et al., Plant Mol. Biol., 16:437-448, 1991). Regulatory domains within plant promoters found to be necessary for specific expression patterns of SSPs have been determined using deletion and gain of function experiments in transgenic plants (da Silva et al., Plant J., 5:493-505, 1994; de Parter et al., Plant Cell, 5:877-886, 1993; Kawagoe et al., Plant J., 5:885-890,1994; Lessard et al., Plant Mol. Biol., 22:873-885, 1993; Thomas et al., Plant Cell, 2:1171-1180, 1990). Genes that regulate expression of SSPs represent an important model for the study of the mechanisms of developmental-stage and tissue-specific gene expression.

The gene families that encode the main SSPs have been characterized in several plant species (Guerche et al., Plant Cell, 2:469-478, 1990; Higgins, Ann. Rev. Plant Physiol., 35:191-221,1984;Panget al., Plant Mol. Biol., 11:805-820,1988). Seed-storage proteins have been classified into four different groups based on solubility. Albumins, which are water-soluble; globulins, which are salt-soluble; glutelins, which are soluble in acids, alkali ionic detergents, and urea-containing solutions; and prolamins, which are alcohol soluble. The glutelins and prolamins are the major forms of cereal SSPs while the globulins are the most prevalent class of SSPs in legumes and oats (Shotwell et al., The Biochemistry of Plants, Vol. 15: A Comprehensive Treatise, Academic Press, San Diego, Calif., pages 297-345, 1989; Krochko et al., Plant Physiology, 99:46-53, 1992). However, to date there has been no detailed study of the accumulation of seed-storage proteins and the qualitative and quantitative properties of seed-storage proteins in the majority of angiosperms and gymnosperms.

A recent study showed a marked time differential between SSP synthesis and mRNA accumulation during development of zygotic and somatic embryos of alfalfa ( Medicago sativa L. ). Of three storage proteins studied (7S, 11S, and 2S), mRNA for the 2S protein was found early in somatic embryo development (day 3) but the protein associated with the 2S message was not evident until later (day 10). Thus, both transcriptional and post-transcriptional events appear to be important in determining the protein complement of seed tissues. (Krochko et al., 1992).

Superficially, somatic embryos mimic the developmental stages of zygotic seeds, i.e., the globular, heart, torpedo, and cotyledonary stages (Steward et al., Science, 143:20-27, 1964). However, despite the gross morphological similarities, somatic embryos may exhibit other features which suggest aberrant development, including truncated cotyledonary development, precocious germination, recallusing, multiple or fuse cotyledons, or inability to germinate (Ammirato, Bio/Technology, 1:68-74, 1983; Bapat et al., Plant Cell Rep., 7:538-541, 1988).

Comparisons between somatic and zygotic embryos have shown that somatic embryos can accumulate SSPs (Crouch, Planta, 156:64-74, 1982; Shoemaker et al., Plant Cell Rep., 6:12-15, 1987), and can also be induced to become desiccation tolerant under appropriate culture conditions (Senaratna et al., Plant Sci., 65:253-259, 1989). However, few studies have been conducted on physiological and biochemical changes occurring in somatic embryogenesis in parallel with studies in zygotic embryogenesis. In particular, seed-specific storage proteins, because of their nature and abundance, are expected to be useful markers in the study of gene expression in embryogenic systems.

SSP accumulation is thought to be temporally and spatially regulated, primarily at the level of gene transcription (Muntz, Biochem. Physiol. Pflnazen, 182:93-116, 1987). This conclusion is based primarily on the absence of SSP mRNA in non-seed tissues, and the observed coincidence between the period of maximum seed storage protein synthesis in developing seeds and mRNA accumulation, as determined by Northern blot (Walling et al., Proc. Natl. Acad. Sci. USA, 83:2123-2127, 1986). Factors which affect the accumulation of SSPs in somatic and zygotic embryos vary with the specific SSP, the developmental stage, the tissue (axis or cotyledon), and nutritional conditions (Muntz, Biochem. Physiol. Pflnazen, 182:93-116, 1987; Walling et al., Proc. Natl. Acad. Sci. USA, 83:2123-2127, 1986; Laden et al., Plant Physiol., 84:35-41,1987). The relative magnitude of each of these factors remains to be determined.

Many genes have been introduced into plants using a variety of genetic engineering techniques, sometimes using regulatory elements from sources other than the target plant. Often, the gene coding for the desired polypeptide is placed under the control of a constitutive promoter allowing the expression of the desired polypeptide in the plant throughout the entire life of the plant, irrespective of the plant's developmental stage. For example, the 35S promoter from Cauliflower Mosaic Virus (CaMV) has been used extensively for constitutive expression of heterologous genes.

For some applications, however, it is not necessary to continuously express the desired polypeptide throughout the life of the plant. In these instances, it is desirable to limit the expression of the desired polypeptide to specific instances, often linked to the stage of plant development. It is often useful to control the time when the desired polypeptide is to be expressed through the use of inducers. In some situations, it is desirable to maintain baseline expression of a desired polypeptide while allowing over-expression of the desired polypeptide at certain times.

While current compositions and methods may be effective to deliver certain genes to plants, there is a need for improved compositions and methods. There is a need for an inducible promoter that will allow the expression of a desired polypeptide. There is also a need for a method of transforming a target plant with a DNA construct comprising an inducible promoter operatively linked to one or more genes coding for desired polypeptides. There is a further need for a method of assessing the developmental maturity of plant embryos.

The present invention is directed to isolated promoters comprising novel nucleic acid sequences. The promoter is a vicilin-like seed storage gene sequence and may be a nucleic acid sequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5 and SEQ ID NO:6. The isolated promoter of the present invention includes a fragment thereof having substantially the same activity.

Another embodiment of the present invention is a construct of the promoter operably linked to a desired gene. Also provided by the present invention is a nucleic acid sequence having the functional properties of the promoter of the present invention, the complement of which hybridizes under stringent conditions to the nucleic acid sequence of the promoter.

The present invention is also directed to methods of expressing a desired polypeptide in a plant, the method comprising preparing a construct of a vicilin-like gene promoter sequence operably linked to a desired gene, thereby producing a promoter-gene construct, and transforming a plant with the promoter-gene construct which thereby ultimately expresses a desired polypeptide in the plant. Typically, the polypeptide is a SSP or a structural protein. The desired gene may heterologous or non-heterologous and may be a reporter gene, according to the present invention. The method of expressing a desired polypeptide may include constitutive or induced expression of the desired gene. Suitable inducers include abscisic acid (ABA), heat, and light.

Yet another embodiment of the present invention is a method of monitoring embryo development in conifers, the method comprising preparing a construct of a vicilin-like gene promoter sequence operably linked to a reporter gene, thereby producing a promoter-gene construct; and transforming an embryo with the construct, wherein the expression of the reporter gene is indicative of the degree of activation of the vicilin-like gene promoter thereby providing a qualitative marker of embryo development that may be monitored.

The present invention is also directed to transgenic plants, including transgenic plants comprising a vicilin-like gene promoter operably linked to a desired gene. The plant may be an angiosperm or gymnosperm, preferably a conifer.

FIG. 1 depicts the alignment of four promoters isolated from loblolly pine (SEQ ID NOS: 1-4) and the predicted consensus sequence (SEQ ID NO:6) of SEQ ID NOS: 1-4.

FIG. 2 depicts the sequence of vicilin cDNA (SEQ ID NO:16) from loblolly pine. Initiator and termination codons are marked in bold. A polyadenylation-like sequence in the 3′untranslated region is also marked in bold.

FIG. 3 depicts the loblolly pine vicilin-like SSP amino acid sequence (SEQ ID NO:17).

FIG. 4 depicts the amino acid alignment of vicilin-like proteins from loblolly pine (SEQ ID NO:17) and Picea glauca (SEQ ID NO:18), as well as the predicted consensus sequence.

FIG. 5 depicts the alignment of Loblolly pine vicilin-like cDNA (SEQ ID NO: 16) and Picea glauca vicilin-like cDNA. (SEQ ID NO:7)

FIG. 6 depicts a gel showing the presence of vicilin gene transcripts in different stages of somatic embryogenesis. Lanes 4-9 represent stages 4-9, respectively.

FIG. 7 depicts vicilin gene expression in late zygotic embryos. An RT-PCR reaction was carried out with vicilin primers, which produced 1.4 Kb fragments of the vicilin gene. Columns A-J represent stages 9.1-9.10, respectively. Column K represents a 1 Kb ladder.

FIG. 8 depicts a Southern blot hybridization with the vicilin gene. Lane 1 represents tomato genomic DNA. Lane 2 represents Loblolly pine genomic DNA. Lane 3 represents a 1 Kb ladder marker.

FIG. 9A depicts the effect of heat as an inducer on vicilin gene expression.

FIG. 9B depicts the efficiency of transfer from the gel to the blotting media.

FIG. 10 depicts the effect of different concentrations of ABA as an inducer of vicilin gene expression. From left to right, 0.0 μM, 0.1 μM, 1.0 μM, and 10.0 μM ABA were added to the samples.

FIG. 11 depicts the sequence of a fifth promoter (SEQ ID NO:5) isolated from loblolly pine.

The present invention is directed to vicilin-like gene promoters including nucleic acid sequences thereof. The vicilin-like gene promoters were isolated from Loblolly pine genes encoding vicilin-like SSPs. As used herein, the term “vicilin-like” refers to proteins (and polypeptide sequences and nucleic acids encoding therefor) that are SSPs substantially similar to the SSPs found associated with the seeds of leguminous plants. Similarly, vicilin-like promoters are promoters of genes encoding the above mentioned proteins.

As used herein, the term “promoter” refers to a nucleic acid sequence that directs the transcription of a gene. Typically, a promoter is located in the 5′region of a gene and is proximal to the transcriptional start site of the gene. Promoters of the present invention are from about 100 to about 600 nucleotides in length. In a preferred embodiment, the promoters are from about 200 to about 400 nucleotides in length. In a more preferred embodiment, promoters are about 340 nucleotides in length. The present invention includes six vicilin-like gene promoter sequences, including SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and a predicted consensus sequence of SEQ ID NOS: 1-4, SEQ ID NO:6.

All or part of the vicilin-like promoter may be used to express a sequence or gene in accordance with the present invention. All or part of the vicilin-like promoter may be operably linked with a nucleic acid sequence or gene encoding a protein to direct the expression of the sequence or gene. A portion, a part, a fragment, or the like, refers to one or more groups of nucleic acids within the vicilin-like promoter which control the expression of sequences that are operably linked to the promoter. “All”, “part”, and nucleic acid sequences which are “substantially similar” to the nucleic acid sequence of the vicilin-like promoter, for purposes of the present invention, means a nucleic acid molecule the sequence of which is preferably 25%, preferably 50%, more preferably 75%, and most preferably 100% identical to that of the vicilin-like promoter. The portion of the vicilin-like promoter sequence may be in a single consecutive arrangement, or more than one arrangement of consecutive nucleic acids. In addition, the present invention includes sequences which are substantially similar to the sequences of SEQ ID NOS: 1, 2, 3, 4, 5, 6, or portions thereof. Substantially similar, for purposes of the present invention is a sequence which is preferably 25%, preferably 50%, more preferably 75%, and most preferably 100% identical to SEQ ID NO:1, 2, 3, 4, 5, or 6. All or part of SEQ ID NOS: 1, 2, 3, 4, 5, or 6 may be used to express a sequence or gene operably linked thereto.

The vicilin-like gene promoter may be a functional equivalent of any of the nucleic acid sequences set forth in SEQ ID NOS: 1, 2, 3, 4, 5, or 6. As used herein, the term “functional equivalent” refers to any nucleic acid sequence which is complementary to a reference nucleic acid sequence which, under stringent conditions, will hybridize with the reference sequence and has activity similar to the vicilin-like gene promoter.

As used herein, the term “stringent hybridization conditions” are those in which hybridization takes place at 65° C. in 2.5×saline citrate buffer (SSC) followed by a rinse step at 37° C. in a reduced buffer concentration.

As used herein, the term “percent homology” relates to the ratio of the identical nucleotides when two sequences are compared to the total number of nucleotides when the sequences are aligned. Sequence alignments for both DNA and proteins were performed using the National Center for Biotechnology Information's BLAST utility program using default settings.

Other embodiments of the present invention are constructs which include, but are not limited to, nucleic acid sequences including DNA, cDNA, genomic DNA, RNA, mRNA, tRNA, suitable nucleic acid sequences such as the sequences set forth in SEQ ID NOS: 1, 2, 3, 4, 5, 6, and conservative alterations in such nucleic acid sequences including additions, deletions, mutations, and homologues. The sequences within the scope of the present invention include antisense sequences which may alter plant characteristics, including those identified above. Antisense sequences may prevent the translation of certain sequences in plants. Inhibition of expression of certain sequences, such as those responsible for the characteristics identified herein, may be achieved with antisense sequences.

As used herein, the term “desired gene” refers to a nucleic acid sequence which encodes a desired polypeptide. According to the instant invention, “desired genes” include, but are not limited to, SSP genes, structural genes, insecticidal toxins, herbicide resistance genes, drought tolerance genes, anti-microbial genes, anti-fungal genes, anti-viral genes, genes encoding regulatory proteins, and genes encoding enzymes involved in metabolic pathways. While the term “gene” is used throughout the specification to define the sequence operably linked to a vicilin-like promoter, “gene” includes any sequence that has substantially the same activity as the desired gene. One measure of “substantially the same activity”, for example, is a functioning polypeptide or protein resulting from a nucleic acid sequence operably linked to a vicilin-like promoter of the present invention. Accordingly, all or part of a gene is included in the definition of “desired gene”. The definitions set forth above to describe all or part of the vicilin-like promoter are applicable to a desired gene as well. The desired genes may be heterologous or non-heterologous.

As used herein, the term “polypeptide” refers to any translation product of a nucleic acid molecule, regardless of size, whether or not glycosylated, phosphorylated or modified post-translationally. Examples of “polypeptides” contemplated by the instant invention include amino acids, proteins and peptides. “Desired polypeptides” refer to polypeptides encoded by a desired gene, and that may be the result of a promoter-gene construct of the present invention, including polypeptides that are functionally equivalent or that are fragments of the polypeptides or selected polypeptides.

According to the present invention, methods of expressing polypeptides in a plant are provided. Such methods comprise constructing an expression vector construct comprising a vicilin-like gene promoter operably linked to a desired gene and administering the construct to the plant. At least one gene encoding a desired polypeptide may be administered to a plant; however, more than one gene encoding desired polypeptides may be administered to a plant in accordance with the methods of the present invention. The nucleic acid sequence that encodes the polypeptide is operably linked to regulatory elements e.g., a vicilin-like gene promoter) necessary for expression of the sequence in the plant.

As used herein, the term “administration” refers to the mechanism of supplying the expression vector comprising a vicilin-like gene promoter and at least one desired gene to a target plant. Methods of administration of expression vectors to plants are known to those of skill in the art. According to some embodiments of the invention, the expression vector is administered to the target plant by infection with Agrobacterium. One method known in the art for administering the expression vector to a plant is the use of a gene gun. Electroporation and microinjection may also be used to introduce expression vectors into the target plant.

Administration to a plant may comprise transforming the plant with the promoter-gene construct. An expression vector, comprising a vicilin-like gene promoter operably linked to at least one desired gene, may be used to achieve transformation of a plant. As used herein, the term “expression vector” refers to the molecules that comprise a nucleic acid sequence which encode one or more desired polypeptides and which include initiation and termination signals operably linked to regulatory elements including a promoter and polyadenylation signal capable of directing expression in the plant to be treated. Initiation codons and stop codons are generally considered to be part of a nucleic acid sequence that encodes a polypeptide. However, it is necessary that these elements be functional in the cells of the plant to which the gene construct is administered, e.g., the initiation and termination codons are in frame with the coding sequence. Administration to the desired plant may be performed in conjunction with agents which facilitate the uptake and/or expression of the desired gene by the target plant. Such agents include but are not limited to polyethylene glycol, heat treatment of cells, and cold treatment of cells.

Desired genes within expression vectors administered to a plant embryo are expressed under control of the vicilin-like gene promoter, producing the desired polypeptide. Typical expression vectors include, but are not limited to, the pBl series of vectors and derivatives thereof, and green fluorescent protein reporter systems. Other methods of delivering sequences into plants are known in the art, including and not limited to Ti-plasmid vectors, in vitro protoplast transformation, plant virus-mediated transformation, liposome-mediated transformation, gene gun, and ballistic particles.

Target plants to be used in accordance with the present invention are all species of higher and lower plants of the Plant Kingdom. Plant embryos, seedlings, and seeds are included in the scope of the invention. The vicilin-like promoter-gene constructs are useful in plants, plant parts, seeds, and plant culture. A mature plant includes a plant at any stage in development beyond the seedling. A seedling is a very young, immature plant in the early stages of development.

The present invention contemplates target plants including, but not limited to, gymnosperms including and not limited to conifers such as evergreens, trees and shrubs, cycads, oaks, gumwoods, aspens, cottonwoods; angiosperms including but not limited to, annuals, perennials, monocotyledons, and dicotyledons; and the like.

A further aspect of the methods of expressing a desired polypeptide in a plant optionally includes inducing expression of the desired gene with a suitable inducer. The vicilin-like gene promoter is induced by an agent, i.e., an inducer, that initiates transcription, or increases the rate of transcription, of a desired gene. Inducers contemplated by the instant invention include, but are not limited to, abscisic acid, heat, light, and other endogenous signals. Abscisic acid, in accordance with the present invention, includes amounts of abscisic acid not typically found in plants in under natural conditions. For example, in loblolly pine, abscisic acid from about 0.001 μM to about 10 μM can be used as an inducer. Heat, in accordance with the present invention, includes temperatures not typically existing for plants growing under natural conditions. For example, in loblolly pine, heat, from about 30° C. to about 50° C. can be used as an inducer.

Yet another embodiment of the instant invention provides a method of monitoring embryo development in plants, including, but not limited to conifers. The methods of monitoring embryo development include assessing the maturity of seed embryos. One embodiment of the method comprises administering to a target plant a vicilin-like gene promoter operably linked to at least one reporter gene, and transforming an embryo with that construct. Upon uptake by the target plant, the plant's cells express the reporter gene. The expression of the reporter gene under the control of the vicilin-like gene promoter is indicative of the degree of activation of the vicilin-like gene promoter and thereby provides a quantitative marker of embryo development that may be monitored.

As used herein, the term “reporter gene” refers to genes and polypeptides encoded thereby, that are used to monitor gene expression, and, in some cases to allow protein localization in cells. Reporter genes include, but are not limited to, green fluorescent protein (GFP), alkaline phosphatase, chloramphenicol acetyltransferase, B-glucuronidase (GUS), firefly luciferase, bacterial luciferase and β-galactosidase. In a preferred embodiment, the reporter gene encodes GUS. In a more preferred embodiment, the reporter gene encodes green fluorescent protein.

The stage of development of a conifer or other plant embryo can be evaluated by monitoring the expression of a reporter gene functionally linked to a promoter. A non-limiting example of such a promoter is the vicilin-like gene promoter. The reporter gene will only be expressed when the vicilin-like promoter is induced, an event that is coupled to, and therefore an indicator of the maturity of the embryo. The appearance of an easily assayable phenotype, characteristic of the reporter protein, such as color production in an assay, fluorescence of embryos, or the ability of an embryo to grow in the presence of a particular chemical, is dependent on expression of the reporter gene, which is in turn controlled by the vicilin-like promoter in such transgenic embryos. Such phenotypic changes provides a means to assay for embryos having reached a particular stage of development.

Also embodied by the present invention is a “transgenic plant” that refers to a target plant comprising one or more constructs of a vicilin-like gene promoter linked to a desired gene.

Depending upon the nature of the polypeptide produced according to the instant invention, various aspects of the present invention, such as those set forth below, can be exploited. For example, if the polypeptide encoded by the desired gene is a reporter gene, the expression of the polypeptide in the desired plant provides methods of assessing promoter activity. If the polypeptide encoded by the desired gene is a SSP, the expression of the polypeptide provides increased amounts of SSP.

The following examples are meant to illustrate the invention and are not intended to limit the scope of the invention in any way. Those skilled in the art will recognize modifications that are within the spirit and scope of the invention. All references cited herein are hereby incorporated by reference in their entirety.

Pinus taeda

1

actatagggc acgcgtggtc gacggcccgg gctggtaaaa gtgtgtgttt gcagggtgca 60

gataatggca gcatgtatga cattagacat atggcattgg caaatgctgt cgattttggc 120

actcatcact gtaattgttg caacatgtca atcgtctgca acatgccctg gtgatcatag 180

gttatgcaag aactcagacg tgttcaccat tcttcaatac catatgcctc ttctgtctgg 240

ttgcttccac cacgcgtcca tgcatgtgca tgattctctt gtatataaaa gtcccccttg 300

cccattctgt ctagtaccgg acttcaccaa agcaccatca g 341

2

341

Pinus taeda

2

actatagggc acgcgtggtc gaggcccggg ctggtaaaag tgtgtgtttg cagggtgcag 60

ataatggcag catgtatgac attagacata tggcattggc aaatgctgtc gattttggca 120

ctcatcactg taattgttgc aacatgtcaa tcgtccgcaa catgccctgg tgatcatagg 180

ttatgcaaga actcagacgt gttcaccgtt cttcaatacc aaatgcctct tctgtctggt 240

tgcttccacc acgcgtccat gcatgtgcat gattctcttg tatataaaag tcccccttgc 300

ccattctgtc tagtaccgga cttcaccaaa gcaccatcat g 341

3

340

Pinus taeda

3

actatagggc acgcgtggtc gacggcccgg gctggtaaag tgtgtgtttg cagggtgcag 60

ataatggcag catgtatgac attagacata tggcattggc aaatgctatc gattttggca 120

ctcatcactg taattgttgc aacatgtcaa tcgtctgcag catgccctgg cgatcatagg 180

ttatgcaaga actcagatgt gttcaccatt cttcaatacc aaatgcctct tctgtctggt 240

tgcttccacc acgcgtccat gcatgcacat gattctcttg tatataaaag tcccccttgc 300

ccattctgtc tagtaccgac ttcaccaaag caccatcatg 340

4

340

Pinus taeda

4

actatagggc acgcgtggtc gacggcccgg gtggtaaagt gtgtgtttgc agggtgcaga 60

taatggcagc atgtatgaca ttagacatat ggcattggca aatgctatcg attttggcac 120

tcatcactgt aattgttgca acatgtcaat cgtctgcaac atgccctggc gatcataggt 180

tatgcaagaa ctcagatgag ttcaccattc ttcaatacca aatgcctctt ctgtctggtt 240

gcttccacca cgcgtccatg catgcacatg attctcttgt atataaaagt cccccttgcc 300

cattctgtct agtaccagac ttcaccaaag caccatcatg 340

5

565

Pinus taeda

n at 489 is a, c, g, or t

5

actatagggc acgcgtggtc gacggcccgg gctggtaaaa attcatttac taatcaaaac 60

atgatgagat tcataaccaa agtctgttat aaaccatgat tataaccaac agattaacaa 120

tgatagaaca accattaaaa ccacataata acaagtacat ttacacatgg aacacaagag 180

gaaaatagct cttattaaca tatgaaaaat gtaactaggt caaggacttc cacgcaccaa 240

ccaaccatag attgggctga accaaatctt tctttcaact aatcacccct aagccatatt 300

cccagcatga atgtgggact tacaaaaaaa caaacaagga ttccttagga tttaccataa 360

tccaccaagg gattcctagg cccaagccct catctataca actaggattt actgcaatcc 420

caccaaggga ttcctaagcc caaacaagaa acacacacta ccaggattta gataaacccc 480

ctttgtggng ctgctatcag ctnngtttct tttaccttct gnatatcttc tgnggacacc 540

tgnctttana agccgattcc accnt 565

6

342

Artificial Sequence

Description of Artificial Sequenceconsensus of

sequences 1-4

6

actatagggc acgcgtggtc ganggcccgg gntggtaaan gtgtgtgttt gcagggtgca 60

gataatggca gcatgtatga cattagacat atggcattgg caaatgctgt cgattttggc 120

actcatcact gtaattgttg caacatgtca atcgtcngca ncatgccctg gngatcatag 180

gttatgcaag aactcagang tgttcaccat tcttcaatac canatgcctc ttctgtctgg 240

ttgcttccac cacgcgtcca tgcatgnnca tgattctctt gtatataaaa gtcccccttg 300

cccattctgt ctagtaccng acttcaccaa agcaccatca tg 342

7

1590

Picea glauca

7

caagcatcat catggcttta gcttctttac ttatcattct tcttgcaatc tcctcctcct 60

cggctgccct cactgagcca ctagccagca cggccaatcc agaagttttt cctgaatatc 120

tcggccgagg ccgagggaga cgagaagaag agcgagagga gaatccatat gtattccaca 180

gtgacagctt ccggaccaga gcatcatctg aagctggtga aatcagagct ctgccgaact 240

ttggggaggt ctctgaactt cttgaaggga ttagaaaatt cagagttacc tgcattgaaa 300

tgaaacccaa tacggtgatg ctccctcact atattgatgc gacatggatc ttatatgtta 360

ctagaggaag aggttacata gcctatgtgc accagaatga gctggttaaa agaaagttgg 420

aggaaggaga tgtattcggt gttccaagtg gtcatacatt ttatctcgtt aacaacgatg 480

accataacac ccttcgcatt gctagtctcg tgcgtcccgt gtctacggtc cgaggagaat 540

atcagccctt ctacgttgcg ggaggtcgga atcctcagac tgtttactct gcctttagcg 600

atgatgttct cgaggctgca ttcaatacga acgtacagca gcttgaacgt attttcggtg 660

gacacaaaag tggagtcata atccacgcaa atgaagaaca gattagagaa atgatgagga 720

aacggggatt ttcagcagga tctatgtctg cacctgagca ccccaagcct ttcaaccttc 780

ggaaccagaa gccagatttc gagaacgaaa atggcaggtt tactattgct ggtcccaaaa 840

attatccttt tctagacgcg ctcgacgttt ctgttgggct tgccgatttg aatcctggat 900

ccatgacagc cccatctctc aactcgaaat caacgtcaat cggcattgtt acgaatgggg 960

aaggaaggat tgagatggcg tgcccgcacc ttggtcaaca tggttggtct agtccgcgcg 1020

agagaggcga ccaagatatt acctaccaga gagtctgggc aaagctgagg accggcagcg 1080

tttatattgt tcctgctggt catccaatca ccgagatagc ttcaacaaac agccgcctgc 1140

aaatcttgtg gtttgatctt aatacccgcg gcaacgagag acaattcctg gcaggaaaga 1200

acaatgtgct taacacgttg gagagggaga tcaggcagct atccttcaac gtaccacgtg 1260

gggaagagat tgaagaagtg ttgcaggcgc aaaaggatca agtcatcctg agaggccccc 1320

aacgacgaag ccgggacgag gcgaggagct cttcttagat ccatgtcatc atcgcagatc 1380

gcattatgga cgacatgaca agagtttctc gacgttcact cttaatatct acttaaaaat 1440

aagctatcca tatatgaagc ccaataaatg tgttcgaaga tgaactcttt ctgtctaaat 1500

gaatgtatgt atgagtctaa caaagctatc gttgggctct tctcttgcta cttcaatgaa 1560

atggaatgca gatcttctct taaaaaaaaa 1590

8

1413

Zamia furfuracea

8

tggtgggagg ccgcaatcca agggaccatg gcccatctgt gctctctacc gctaatggca 60

gtgctaatgt tgctgcttgc atccgcttgc ttttcggaat tggaaataga agatccctat 120

gtattcgacc aacgcagctt tgtgaccacg gtacagtgta aagccggtca gatcagagct 180

ctacccaact ttagcgccgg cggcagatgt gaattgccac gagggcttgg ggattatagc 240

gttgctcaga taagcttgga accgagatct gtgctgcttc ctcattatat tgaggcagat 300

ttggctttat acgtcacagg aggaagagga agggttgcgt ttgttcatga agagagactg 360

gtagaaaggc agctgcggga cggagatgtg tacgcaattg ctgcaggtat accgttttat 420

attctcaaca cggatgacag tcggcgcctt ttcattcact gtctcctgcg cacgcagtgc 480

tctactaccg gactctatga gtcgttttac gtggtgggag gccgcaatcc gcagaatgtt 540

ttgtcccaat tcagcgagga cgttctgcaa gctgcattca acagttcgaa ggcggtcctg 600

gatcctatgc tggtaagtgg gtttaacaga ggggccataa ttagagtgtc cagagaacaa 660

atggaaaggc tgagcagggg tagaattaag ggattcggag ggtctgagga gccacagccc 720

ttcaacctgc tctacaggaa ccccgacttt tccaacaaca acggtgaaat tttcacagca 780

gacgccgcag atcatcgcgt tttacgccgg ctgaatgttg gagtgcagct tctcaactta 840

aagccgcgct caatgacggc accgcattac gatacaaggt cgaccagaat tggcatcgtt 900

aggaacggga ggggaatcct cgaattagtg cgcccgcaag aacaagaaca acaacaacaa 960

caacaacaag gtcccacata ccagaaacta cgggccaacc ttaaccccgg cactgttttc 1020

ctaacccgcc ccggctaccc ttccactgta attgcctctg gcaatgaggc attgcagata 1080

ttgtacctcg acaattattc ccaaggcagt cgcaggcaat tcctcgcagg aaggagcaat 1140

gtgttgagat atctgcctag ggaaattaag cgattggttt tcccatcttc cgctgaggag 1200

atagaggcaa ccttggaagc gcaggaagat gaggttctcc tcaacgcgca acaagggcgg 1260

gccgaccagt agtagagaaa ccgtggactc cacttggtgc ggctcagtat aaacatatgg 1320

acttctccct agtaccttgc cctgtacagc tcgtcgttct ccatacccac ggaaataaat 1380

aaatgggatc ttgcctcgca tccgcacccc ttt 1413

9

22

Artificial Sequence

Description of Artificial SequencePCR primer

9

gagacgagaa gaagagcgag ag 22

10

22

Artificial Sequence

Description of Artificial SequencePCR primer

10

tgaagtagca agagaagagc cc 22

11

20

Artificial Sequence

Description of Artificial Sequence3′ RACE

primer

11

acatggttgg tctagtccgc 20

12

32

Artificial Sequence

Description of Artificial SequenceGene

Specific Primer

12

cgtagaccac tgtacgcagg agactggtaa tg 32

13

31

Artificial Sequence

Description of Artificial SequenceGene

Specific Primer

13

ctccttcctc caactttctt ttcaccagtt c 31

14

22

Artificial Sequence

Description of Artificial SequencePCR primer

14

gagacgagaa gaagagcgag ag 22

15

23

Artificial Sequence

Description of Artificial SequencePCR primer

15

tgaagtagca agagaaagag ccc 23

16

1581

Pinus taeda

16

aaagcaccat catggctttt gtttctttac ttaccattct tcaagcaatc tcctcctgct 60

ccgttgctct cactgagcca ctagccactg tggccaatca aggagttttt cctgaagatc 120

atgggcgagg gcaccagaga cgagaagaag aacgagagga gaatccgtac gtattccaca 180

gtgacagatt caggatgaga gcgtcatctg acgctggtga aatcagagct ctccccaact 240

ttggtgaggc ctctgaactt cttgaaggga ttagtaaata cagagttacc tgcattgaaa 300

tgagacccaa cacggtcatg ctccctcact atcttgacgc gacatggatt ttatatgtta 360

ctggaggaag aggttacata gcttacgtgc accagaatga actggtgaaa agaaagttgg 420

aggaaggaga tgtatttggt gttccaagtg gtcatacatt ttatctcgtt aacaacgatg 480

accacaacag ccttcgcatt accagtctcc tgcgtacagt gtctacgatg cgaggagaat 540

atgagcccta ctacgttgct ggaggtcgga atcctgagac tgtttactct gcctttagcg 600

atgatgttct cgaagctgca ttcaatacga acgttataga agctagaaca cattttccgg 660

tgcacataga acgggagtca tattccatgg caaatgaaga acagattaga gaaatgttga 720

ggaaacgggg attttcagca gaatccatgt ctgcatctga gcacccaaag ccttttaacc 780

ttcggaacca gaagccagat ttcgagaacg acaatggcag gtttactaga gctggtccca 840

atgaaaatcc tcttcttgac gcggtcgatg ttactgctgg gtttggcgtt ttgaatcctg 900

gaaccatgac agccccatct cacaacacga aagcaacctc aatcgccatt gtcacacagg 960

gggagggaag gattgagatg gcgtgcccgc accttggtca acatggctgg tctagtcggc 1020

gcgagaaagg cgatcaggaa attaattacc agagggtacg ggcaaggctg agaaccggca 1080

ccgtttacgt tgttcctgca ggtcatccaa tcaccgagat agcttgcaca gagggccacc 1140

ttgaaatctt gtggtttgat attaatacga gcggcaacga gagacaattc ctggcaggaa 1200

agtacaatgt gcttcaaacg ctggagaagg aggtcaggca gatatccttc aacataccac 1260

gtggggaaga gctggatgaa gttttacggc ggcaaaagga tcaagtcatc ctcagagggc 1320

cccaaatgca aaggcgagac gagccaagga gctcttctta gatccatgcc atcatcgcag 1380

ctcgcatcat ggacgacaag actagagttt ctccacgttc actctttagt atctacttaa 1440

gaataagtta tgcatatatg aagcccaaaa aatgtgttcg aagatgagct ccttttatct 1500

taatgaatgt atatatgagt ttcaacaaac ctatcgttgg gctcttctct tgctacttca 1560

atgacatgga atgctgatct t 1581

17

523

Pinus taeda

17

Ser Thr Ile Met Ala Phe Val Ser Leu Leu Thr Ile Leu Gln Ala Ile

1 5 10 15

Ser Ser Cys Ser Val Ala Leu Thr Glu Pro Leu Ala Thr Val Ala Asn

20 25 30

Gln Gly Val Phe Pro Glu Asp His Gly Arg Gly His Gln Arg Arg Glu

35 40 45

Glu Glu Arg Glu Glu Asn Pro Tyr Val Phe His Ser Asp Arg Phe Arg

50 55 60

Met Arg Ala Ser Ser Asp Ala Gly Glu Ile Arg Ala Leu Pro Asn Phe

65 70 75 80

Gly Glu Ala Ser Glu Leu Leu Glu Gly Ile Ser Lys Tyr Arg Val Thr

85 90 95

Cys Ile Glu Met Arg Pro Asn Thr Val Met Leu Pro His Tyr Leu Asp

100 105 110

Ala Thr Trp Ile Leu Tyr Val Thr Gly Gly Arg Gly Tyr Ile Ala Tyr

115 120 125

Val His Gln Asn Glu Leu Val Lys Arg Lys Leu Glu Glu Gly Asp Val

130 135 140

Phe Gly Val Pro Ser Gly His Thr Phe Tyr Leu Val Asn Asn Asp Asp

145 150 155 160

His Asn Ser Leu Arg Ile Thr Ser Leu Leu Arg Thr Val Ser Thr Met

165 170 175

Arg Gly Glu Tyr Glu Pro Tyr Tyr Val Ala Gly Gly Arg Asn Pro Glu

180 185 190

Thr Val Tyr Ser Ala Phe Ser Asp Asp Val Leu Glu Ala Ala Phe Asn

195 200 205

Thr Asn Val Ile Glu Ala Arg Thr His Phe Pro Val His Ile Glu Arg

210 215 220

Glu Ser Tyr Ser Met Ala Asn Glu Glu Gln Ile Arg Glu Met Leu Arg

225 230 235 240

Lys Arg Gly Phe Ser Ala Glu Ser Met Ser Ala Ser Glu His Pro Lys

245 250 255

Pro Phe Asn Leu Arg Asn Gln Lys Pro Asp Phe Glu Asn Asp Asn Gly

260 265 270

Arg Phe Thr Arg Ala Gly Pro Asn Glu Asn Pro Leu Leu Asp Ala Val

275 280 285

Asp Val Thr Ala Gly Phe Gly Val Leu Asn Pro Gly Thr Met Thr Ala

290 295 300

Pro Ser His Asn Thr Lys Ala Thr Ser Ile Ala Ile Val Thr Gln Gly

305 310 315 320

Glu Gly Arg Ile Glu Met Ala Cys Pro His Leu Gly Gln His Gly Trp

325 330 335

Ser Ser Arg Arg Glu Lys Gly Asp Gln Glu Ile Asn Tyr Gln Arg Val

340 345 350

Arg Ala Arg Leu Arg Thr Gly Thr Val Tyr Val Val Pro Ala Gly His

355 360 365

Pro Ile Thr Glu Ile Ala Cys Thr Glu Gly His Leu Glu Ile Leu Trp

370 375 380

Phe Asp Ile Asn Thr Ser Gly Asn Glu Arg Gln Phe Leu Ala Gly Lys

385 390 395 400

Tyr Asn Val Leu Gln Thr Leu Glu Lys Glu Val Arg Gln Ile Ser Phe

405 410 415

Asn Ile Pro Arg Gly Glu Glu Leu Asp Glu Val Leu Arg Arg Gln Lys

420 425 430

Asp Gln Val Ile Leu Arg Gly Pro Gln Met Gln Arg Arg Asp Glu Pro

435 440 445

Arg Ser Ser Ser Ile His Ala Ile Ile Ala Ala Arg Ile Met Asp Asp

450 455 460

Lys Thr Arg Val Ser Pro Arg Ser Leu Phe Ser Ile Tyr Leu Arg Ile

465 470 475 480

Ser Tyr Ala Tyr Met Lys Pro Lys Lys Cys Val Arg Arg Ala Pro Phe

485 490 495

Ile Leu Met Asn Val Tyr Met Ser Phe Asn Lys Pro Ile Val Gly Leu

500 505 510

Phe Ser Cys Tyr Phe Asn Asp Met Glu Cys Ser

515 520

18

448

Picea glauca

18

Met Ala Leu Ala Ser Leu Leu Ile Ile Leu Leu Ala Ile Ser Ser Ser

1 5 10 15

Ser Ala Ala Leu Thr Glu Pro Leu Ala Ser Thr Ala Asn Pro Glu Val

20 25 30

Phe Pro Glu Tyr Leu Gly Arg Gly Arg Gly Arg Arg Glu Glu Glu Arg

35 40 45

Glu Glu Asn Pro Tyr Val Phe His Ser Asp Ser Phe Arg Thr Arg Ala

50 55 60

Ser Ser Glu Ala Gly Glu Ile Arg Ala Leu Pro Asn Phe Gly Glu Val

65 70 75 80

Ser Glu Leu Leu Glu Gly Ile Arg Lys Phe Arg Val Thr Cys Ile Glu

85 90 95

Met Lys Pro Asn Thr Val Met Leu Pro His Tyr Ile Asp Ala Thr Trp

100 105 110

Ile Leu Tyr Val Thr Arg Gly Arg Gly Tyr Ile Ala Tyr Val His Gln

115 120 125

Asn Glu Leu Val Lys Arg Lys Leu Glu Glu Gly Asp Val Phe Gly Val

130 135 140

Pro Ser Gly His Thr Phe Tyr Leu Val Asn Asn Asp Asp His Asn Thr

145 150 155 160

Leu Arg Ile Ala Ser Leu Val Arg Pro Val Ser Thr Val Arg Gly Glu

165 170 175

Tyr Gln Pro Phe Tyr Val Ala Gly Gly Arg Asn Pro Gln Thr Val Tyr

180 185 190

Ser Ala Phe Ser Asp Asp Val Leu Glu Ala Ala Phe Asn Thr Asn Val

195 200 205

Gln Gln Leu Glu Arg Ile Phe Gly Gly His Lys Ser Gly Val Ile Ile

210 215 220

His Ala Asn Glu Glu Gln Ile Arg Glu Met Met Arg Lys Arg Gly Phe

225 230 235 240

Ser Ala Gly Ser Met Ser Ala Pro Glu His Pro Lys Pro Phe Asn Leu

245 250 255

Arg Asn Gln Lys Pro Asp Phe Glu Asn Glu Asn Gly Arg Phe Thr Ile

260 265 270

Ala Gly Pro Lys Asn Tyr Pro Phe Leu Asp Ala Leu Asp Val Ser Val

275 280 285

Gly Leu Ala Asp Leu Asn Pro Gly Ser Met Thr Ala Pro Ser Leu Asn

290 295 300

Ser Lys Ser Thr Ser Ile Gly Ile Val Thr Asn Gly Glu Gly Arg Ile

305 310 315 320

Glu Met Ala Cys Pro His Leu Gly Gln His Gly Trp Ser Ser Pro Arg

325 330 335

Glu Arg Gly Asp Gln Asp Ile Thr Tyr Gln Arg Val Trp Ala Lys Leu

340 345 350

Arg Thr Gly Ser Val Tyr Ile Val Pro Ala Gly His Pro Ile Thr Glu

355 360 365

Ile Ala Ser Thr Asn Ser Arg Leu Gln Ile Leu Trp Phe Asp Leu Asn

370 375 380

Thr Arg Gly Asn Glu Arg Gln Phe Leu Ala Gly Lys Asn Asn Val Leu

385 390 395 400

Asn Thr Leu Glu Arg Glu Ile Arg Gln Leu Ser Phe Asn Val Pro Arg

405 410 415

Gly Glu Glu Ile Glu Glu Val Leu Gln Ala Gln Lys Asp Gln Val Ile

420 425 430

Leu Arg Gly Pro Gln Arg Arg Ser Arg Asp Glu Ala Arg Ser Ser Ser

435 440 445

19

1596

Artificial Sequence

Description of Artificial SequenceVicilin-like

gene with TA linkers

19

aattcggctt aaagcaccat catggctttt gtttctttac ttaccattct tcaagcaatc 60

tcctcctgct ccgttgctct cactgagcca ctagccactg tggccaatca aggagttttt 120

cctgaagatc atgggcgagg gcaccagaga cgagaagaag aacgagagga gaatccgtac 180

gtattccaca gtgacagatt caggatgaga gcgtcatctg acgctggtga aatcagagct 240

ctccccaact ttggtgaggc ctctgaactt cttgaaggga ttagtaaata cagagttacc 300

tgcattgaaa tgagacccaa cacggtcatg ctccctcact atcttgacgc gacatggatt 360

ttatatgtta ctggaggaag aggttacata gcttacgtgc accagaatga actggtgaaa 420

agaaagttgg aggaaggaga tgtatttggt gttccaagtg gtcatacatt ttatctcgtt 480

aacaacgatg accacaacag ccttcgcatt accagtctcc tgcgtacagt gtctacgatg 540

cgaggagaat atgagcccta ctacgttgct ggaggtcgga atcctgagac tgtttactct 600

gcctttagcg atgatgttct cgaagctgca ttcaatacga acgttataga agctagaaca 660

cattttccgg tgcacataga acgggagtca tattccatgg caaatgaaga acagattaga 720

gaaatgttga ggaaacgggg attttcagca gaatccatgt ctgcatctga gcacccaaag 780

ccttttaacc ttcggaacca gaagccagat ttcgagaacg acaatggcag gtttactaga 840

gctggtccca atgaaaatcc tcttcttgac gcggtcgatg ttactgctgg gtttggcgtt 900

ttgaatcctg gaaccatgac agccccatct cacaacacga aagcaacctc aatcgccatt 960

gtcacacagg gggagggaag gattgagatg gcgtgcccgc accttggtca acatggctgg 1020

tctagtcggc gcgagaaagg cgatcaggaa attaattacc agagggtacg ggcaaggctg 1080

agaaccggca ccgtttacgt tgttcctgca ggtcatccaa tcaccgagat agcttgcaca 1140

gagggccacc ttgaaatctt gtggtttgat attaatacga gcggcaacga gagacaattc 1200

ctggcaggaa agtacaatgt gcttcaaacg ctggagaagg aggtcaggca gatatccttc 1260

aacataccac gtggggaaga gctggatgaa gttttacggc ggcaaaagga tcaagtcatc 1320

ctcagagggc cccaaatgca aaggcgagac gagccaagga gctcttctta gatccatgcc 1380

atcatcgcag ctcgcatcat ggacgacaag actagagttt ctccacgttc actctttagt 1440

atctacttaa gaataagtta tgcatatatg aagcccaaaa aatgtgttcg aagatgagct 1500

ccttttatct taatgaatgt atatatgagt ttcaacaaac ctatcgttgg gctcttctct 1560

tgctacttca atgacatgga atgctgatct tagccg 1596