Transgenic Plants
The invention provides transgenic plants, specifically tobacco plants, which accumulate the amino acid threonine in their leaves. The biosynthetic pathway leading to production of threonine is tightly regulated, and previous attempts to achieve a transgenic plant which overproduces threonine has compromised the fitness of the plants. This invention has overcome these difficulties and found a method of increasing the level of threonine in plant leaves above the corresponding wild type level without compromising plant fitness, comprising altering plant metabolism to achieve increased production of threonine after the initiation of leaf senescence. This invention involves a genetic construct comprising a senescence specific promoter operably linked to a coding sequence encoding a polypeptide having threonine insensitive aspartate kinase activity.
1 . A genetic construct comprising:
a senescence specific promoter operably linked to a coding sequence encoding a polypeptide having threonine insensitive aspartate kinase activity.
2 . The genetic construct according to claim 1 , wherein the senescence specific promoter has been isolated from senescence-associated gene in Arabidopsis.
3 . The genetic construct according to claim 1 , wherein the senescence specific promoter is selected from a group consisting of SAG12, SAG13, SAG101, SAG21 and SAG18, or a functional variant or fragment thereof.
4 . The genetic construct according to claim 3 , wherein the senescence specific promoter is SAG12, or a functional variant or fragment thereof.
5 . The genetic construct according to claim 3 , wherein the promoter comprises a nucleotide sequence substantially as set out in SEQ ID No.1, or a functional variant or fragment thereof.
6 . The genetic construct according to claim 1 , wherein the polypeptide is a threonine insensitive aspartate kinase (AK) or a functional variant or fragment thereof.
7 . The genetic construct according to claim 1 , wherein the polypeptide is a threonine insensitive bifunctional aspartate kinase-homoserine dehydrogenase (AK-HSDH) enzyme.
8 . The genetic construct according to claim 1 , wherein the coding sequence, which encodes the polypeptide having threonine insensitive aspartate kinase activity is derived from Arabidopsis spp., Zea spp., Flaveria spp., or Cleome spp.
9 . The genetic construct according to claim 1 , wherein the coding sequence, which encodes the polypeptide having threonine insensitive aspartate kinase activity is derived from Arabidopsis spp., preferably A. thaliana.
10 . The genetic construct according to claim 7 , wherein at least one threonine binding site of the AK-HSDH is mutated.
11 . The genetic construct according to claim 7 , wherein the Arabidopsis AK-HSDH is mutated at Gln 443 or Gln 524 .
12 . The genetic construct according to claim 7 , wherein the AK-HSDH is mutated at Gln 443 and Gln 524 .
13 . A vector comprising the genetic construct according to claim 1 .
14 . A method of producing a transgenic plant which accumulates a higher level of threonine in the leaves than a corresponding wild type plant cultured under the same conditions comprising:
transforming a plant cell with a genetic construct comprising a senescence specific promoter operably linked to a coding sequence encoding a polypeptide having threonine insensitive aspartate kinase activity; and
regenerating a plant from the transformed cell.
15 . A cell comprising the genetic construct of claim 1 .
16 . A transgenic plant comprising the genetic construct of claim 1 .
17 . The transgenic plant according claim 16 , wherein the plant is from the Brassicaceae family, Poales family or the Solanaceae family.
18 . The transgenic plant according to claim 16 , wherein the plant is a tobacco plant.
19 . A plant propagation product obtainable from the transgenic plant of claim 16 , wherein the plant propagation product comprises the construct of claim 1 .
20 . The plant propagation product according to claim 19 , wherein the plant propagation product is a seed.
21 . A harvested leaf containing a higher level of threonine than harvested leaves than the corresponding level of threonine in a harvested leaf taken from a wild-type plant cultured under the same conditions, wherein the leaf is harvested from the transgenic plant according to claim 16 .
22 . A smoking article comprising:
threonine-enriched tobacco obtained from a mutant tobacco plant, which mutant is capable of over-producing threonine in senescent leaves.
23 . The smoking article according to claim 22 , wherein the mutant tobacco plant has been transformed with a genetic construct that comprises a senescence specific promoter operably linked to a coding sequence encoding a polypeptide having threonine insensitive aspartate kinase activity.
24 . A method of increasing the level of threonine in plant leaves above the corresponding wild type level without compromising plant fitness, comprising:
altering plant metabolism to achieve increased production of threonine after the initiation of leaf senescence.
25 . The method according to claim 24 , comprises transforming the plant with a construct that comprises a senescence specific promoter operably linked to a coding sequence encoding a polypeptide having threonine insensitive aspartate kinase activity.