Plant-gene promoter and methods of using the same

Details Plant-gene promoter and methods of using the same Plant-gene promoter and methods of using the same

2000-08-18

2004-07-06

Fredman, Jeffrey (Department: 1637)

Organic compounds -- part of the class 532-570 series

Organic compounds

Carbohydrates or derivatives

C435S006120, C435S069100, C435S320100, C435S419000, C800S287000

Reexamination Certificate

active

06759529

ABSTRACT:

FIELD
This invention relates to an isolated plant-gene promoter and to methods for using such a promoter. More specifically, the promoter was obtained from a gene encoding a metallothionein-like protein.
BACKGROUND
Metallothionein-like Genes and Their Expression Patterns
Genes encoding Metallothionein-like proteins (i.e., “metallothionein-like genes” or “MT-like genes”) can be categorized into two classes based on the pattern of cysteine distribution within their predicted translation products (Robinson et al.,
Biochem. J
. 295:1-10, 1993). Class I MT-like proteins contain two cysteine-rich domains, as found in animal metallothioneins, and class II MT-like proteins include an additional cysteine-rich domain within the protein. Class I MT-like proteins are further classified into three types (types 1-3) distinguished by the characteristics of their cysteine-containing domains. Each type of MT-like protein also has similar amino acid sequences within spacer regions between the cysteine-rich domains.
To date, Arabidopsis is the only plant species in which metallothionein-like genes of all categories (classes I, II, and types 1-3 of of class I) have been identified. The presence of representatives of each category within a single species (e.g., Arabidopsis) or within closely related species (e.g. wheat, barley, and rice) is significant, as it suggests that plant MT-like proteins may have distinct functions in relation to their structure, patterns of expression, and response to stresses.
The published data on expression of various MT-like genes from a variety of plant species, is summarized in Table 1.
TABLE 1
Metallotheionein-like gene products identified in plants and their
expression patterns. (+) indicates up-regulation, (−) down-regulation
and (+/−) no change in preferentially expressed tissue except as
cited in parenthesis. ABA, absicisic acid; GA
3
, gibberellic acid.
MT-like gene products
Transcript accumulation Response to factors
CLASS I, TYPE 1
Arabidopsis—AtMT1
roots, seedlings
Cu, Zn, Cd
(+, in leaves)
Canola—LSC54
senescent leaves, flowers
—
Mimulus—MT
roots
Cu (+/−); Cd, Zn (−)
Cotton—MT1
root
—
Chickpea—CanMT-1
etiolated epicoty
1
—
White cover—TrMT1B
stolon internode
—
Pea—PsMT
A
roots, etiolated leaves
—
Bean—MT1a, Mt1b
root, stem, aged leaves
Cd, Cu, Zn (+/−)
Grass—pmcMT1
Cu-treated shoots
Cu (+)
Wheat—wali1
roots>leaves
Al (+)
Barley—Ids1
Fe-deficient roots
—
Rice—OsMT-1
roots, sucrose straved
Cu, Heat shock (+)
tissues, senescent leaves
Maize—MT1
roots
—
CLASS I, TYPE 2
Citrus—CitMT36
leaves, fruit
Zn, Cu (+/−)
Apple—AMT1
flower, young fruit
cool storage (+)
Kiwi—pKIWI504
roots, cell division stage
—
fruit
Soybean—KC9-10
leaves>roots
Cu (−)
Tomato—LeMTB
leaves
—
Castor bean—RCMIT
cotyledons
—
Chickpea—CanMT-2
etiolated epicotyl
—
Bean—MT2
trichromes, leaves, stem,
Cu, ZN, Cd, (+/−)
flower
White cover—TrMT1A
stolon node
—
Cabbage—MT
inflorescence
—
Coffee—CAMETAL1
leaves
—
Strawberry—FMET1
fruit
—
Arabidopsis—AtMT2b
leaves
Cu, Cd, Zn
(+, in seedlings)
Tomato—LeMT-A
leaves>roots
—
Tobacco—MT
leaves
wound (+), Cu (+)
Elder—JET12
leaflets
abscission, ethylene
Rice—Ose712, OsMT-2
embryos, sucrose straved
—
tissue
Rice—RicMT
stems>shoots, roots
Cu, Zn, Cd, Fe,
Pb, Al
(+, in shoots)
(−, in roots)
Barley—B22E
embryos, aleurone layer
ABA, GA3 (+/−)
CLASS I, TYPE 3
Papaya—MT
ripe fruit
—
Citrus—CitMT45
fruit
Zn, Cu (+/−)
Kiwi—pKIWI503
ripe fruit
—
Apple—AMT2
fruit, aged leaves
cool storage (+)
Raspberry—RAS2
ripening fruit
—
Strawberry—MT
ripening fruit
—
Cherry—PAMT1
fruit
—
Arabidopsis—AtMT3
leaves
—
Banana—pBAN3-6
fruit, leaves
—
Rice—EST
—
—
White spruce—EMB30
somatic embryos, leaves
—
CLASS II
Soybeen—MT
cotyledons
—
Arabidopsis
dry seeds
—
Wheat—EcI
embryos
ABA (+); Zn (−)
Maize—pMEC
embryos
ABA (+)
From the data in Table 1, it is clear that each MT-like gene type exhibits characteristic developmental and tissue-specific expression patterns. The expression of class II MT genes, such as for the wheat and maize EcMT, is restricted to immature embryos (Kawashima et al.,
Euro. J. Biochem
. 209:971-976, 1992; White and Rivin,
Plant Physiol
. 108:831-832, 1995; Reynolds and Crawford,
Plant Mol. Biol
. 32:823-829, 1996). Type 1 MT-like transcripts have been detected primarily in roots (de Miranda et al.,
FEBS Lett
. 260:277-280, 1990; Evans et al.,
FEBS Lett
. 262:29-32, 1990; Zhou and Goldsbrough,
Plant Cell
6:875-884, 1994; Hsieh et al.,
Plant Mol. Biol
. 32:525-529, 1996) and senescent leaves (Buchanan-Wollaston,
Plant Physiol
. 105:839-846, 1994, Buchanan-Wollaston,
Plant Mol. Biol
. 33:821-834, 1997; Hsieh et al.,
Plant Mol. Biol
. 32:525-529, 1996; Foley et al.,
Plant Mol. Biol
. 33:583-591, 1997). Type 2 MT-like transcripts accumulate in the aerial portions such as leaves, stems, and flowers (Snowden and Gardner,
Plant Physiol
. 103:855-861, 1993; Foley and Singh,
Plant Mol. Biol
. 26:435-444, 1994; Coupe et al.,
Planta
197:442-447, 1995; Zhou and Goldsbrough,
Mol. Gen. Genet
. 248:318-328, 1995; Choi et al.,
Plant Physiol
. 112:353-359, 1996; Whitelaw et al.,
Plant Mol. Biol
. 33:503-511, 1997). Transcripts of type 3 MT-like genes have been detected in fruits, and show differential expression during fruit development (Ledger and Gardner,
Plant Mol. Biol
. 25:877-886, 1994; Lam and Abu Baker,
Plant Physiol
. 112:1735, 1996; and Reid and Ross,
Physiologia Planatrum
100:183-189, 1997). Type 3 MT-like transcripts are also present in leaves (Dong and Dunstan,
Planta
199:459-466, 1996; Bundithya and Goldsbrough,
Plant Physiol
. 114:S-251, 1997; Clendennen and May,
Plant Physiol
. 115:463-469, 1997). Some class I MT genes show programmed expression during embryogenesis. Transcripts of barley pZE40, rice Ose712 (both type 2) and white spruce EMB30 (type 3) genes are expressed temporally during embryo maturation (Smith et al.,
Plant Mol. Biol
. 20:255-266, 1992; Chen and Chen,
Plant Physiol
. 114:1568, 1997; and Dong and Dunstan,
Planta
199:459-466, 1996).
SUMMARY
The invention provides, inter alia, an isolated promoter (as defined herein) from a metallothionein-like gene (i.e., the “dfMTP” promoter; SEQ ID NO: 17). The promoter is useful for expressing heterologous proteins either transiently in host cells or transgenically in stably transformed cells. The dfMTP promoter (SEQ ID NO: 17) can allow for developmental-specific expression of genes placed under its control.
Another aspect of the invention provides fragments and deletions of the promoter, such as those shown in SEQ ID NOS: 22, 23, 24, 25, and variants thereof. The variant promoters are characterized by their retention of at least 50% sequence identity with the disclosed promoter sequences (SEQ ID NOS: 17, 22, 23, 24, and 25), or by their retention of at least 20, 30, 40, 50, or 60 consecutive nucleic acid residues of the disclosed promoter sequences (SEQ ID NOS: 17, 22, 23, 24, and 25). In each case these promoters at least retain promoter activity and, in some cases, these promoters exhibit native dfMTP promoter activity.
It is also contemplated that promoters such as the CaMV35S promoter may be altered through the introduction of one or more sequences found in the dfMTP promoter. The resulting promoter is characterized by its retention of at least 20, 30, 40, 50, or 60 consecutive nucleic acid residues of the disclosed promoter sequences (SEQ ID NOS: 17, 22, 23, 24, and 25).
Another aspect of the invention provides vectors containing the above-described promoters and variants thereof. The vectors can be transformed into host cells. In some cases the resulting host cell can give rise to a transgenic plant.
The invention also provides transgenes. These transgenes include one of the above-described promoter sequences operably linked to one or more open reading frames (ORFs). The transgenes can be cloned into vectors and subsequently used to transform host cells, such as bacterial, insect, mammalian,

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