Patent Grant
11254947
The present invention provides compositions for attenuating the function of atypical CYS HIS rich thioredoxin 4 (ACHT4), a light-regulated protein expressed in plants and algae that controls starch storage in chloroplast, and methods for increasing plant and algae growth and yield.
Genetically modified plants with improved agronomic traits such as yield, pest resistance, herbicide tolerance, improved seed compositions, and the like are desired by both farmers and consumers. Although considerable efforts in plant breeding have provided significant gains in desired phenotypes, the ability to introduce specific DNA into plant genomes provides further opportunities for generation of plants with improved and/or unique phenotypes. The ability to develop genetically modified plants with improved traits depends in part on the identification of genes that are useful in recombinant DNA constructs for production of transformed plants with improved properties.
One genetic modification that would be economically desirable would be to increase the growth and yield production of the plant. There is a need to develop a method for increasing growth in plants, regardless of the locale or the environmental conditions.
The Arabidopsis thaliana atypical cysteine histidine-rich Trxs (ACHTs) constitute a small family of plant-specific and chloroplast-localized Trxs. They are light-regulated and are good catalysts of 2-Cys Prx reduction.
The expression profile of the ACHT family members suggests that they have distinct roles. The role of ACHT4, a recently identified paralog of ACHT1 in Arabidopsis, was previously unknown.
In one embodiment, the present invention provides compositions for attenuating the function of atypical CYS HIS rich thioredoxin 4 (ACHT4), a light-regulated protein expressed in plants and algae that controls starch storage in chloroplast, and methods for increasing plant and algae growth and yield.
In one embodiment, the present invention provides a recombinant polynucleotide encoding an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation.
In another embodiment, the present invention provides a composition comprising a recombinant polynucleotide encoding an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation.
In another embodiment, the present invention provides an expression vector comprising a recombinant polynucleotide encoding an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation.
In another embodiment, the present invention provides a composition comprising an expression vector comprising a recombinant polynucleotide encoding an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation.
In another embodiment, the present invention provides a cell comprising an expression vector comprising a recombinant polynucleotide encoding an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation.
In another embodiment, the present invention provides a composition comprising a cell comprising an expression vector comprising a recombinant polynucleotide encoding an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation.
In another embodiment, the present invention provides a seed comprising a C-terminal deleted form of an atypical CYS HIS rich thioredoxin 4 (ACHT4) gene.
In another embodiment, the present invention provides a plant, or plant part, comprising a C-terminal deleted form of an atypical CYS HIS rich thioredoxin 4 (ACHT4) gene.
In another embodiment, the present invention provides an algae comprising a C-terminal deleted form of an atypical CYS HIS rich thioredoxin 4 (ACHT4) gene.
In another embodiment, the present invention provides a polypeptide comprising an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation.
In another embodiment, the present invention provides a composition comprising a polypeptide comprising a C-terminal deleted form of atypical CYS HIS rich thioredoxin 4 (ACHT4).
In another embodiment, the present invention provides a method of increasing the yield of a plant or algae comprising contacting a cell from said plant or algae with a polynucleotide encoding a C-terminal deleted form of atypical CYS HIS rich thioredoxin 4 (ACHT4), thereby increasing the yield of said plant or algae.
In another embodiment, the present invention provides a method of increasing the productivity of a plant or algae comprising contacting a cell from said plant or algae with a polynucleotide encoding a C-terminal deleted form of atypical CYS HIS rich thioredoxin 4 (ACHT4), thereby increasing the productivity of said plant or algae.
In another embodiment, the present invention provides a method of increasing the size of a plant or algae comprising contacting a cell from said plant or algae with a polynucleotide encoding a C-terminal deleted form of atypical CYS HIS rich thioredoxin 4 (ACHT4), thereby increasing the size of said plant or algae.
In another embodiment, the present invention provides a method of increasing the biomass of a plant or algae comprising contacting a cell from said plant or algae with a polynucleotide encoding a C-terminal deleted form of atypical CYS HIS rich thioredoxin 4 (ACHT4), thereby increasing the biomass of said plant or algae.
In another embodiment, the present invention provides a method of stimulating the growth of a plant or algae comprising contacting a cell from said plant or algae with a polynucleotide encoding a C-terminal deleted form of atypical CYS HIS rich thioredoxin 4 (ACHT4), thereby stimulating the growth of said plant or algae.
In another embodiment, the present invention provides a method of producing a plant or algae having an enhanced phenotype, wherein said method comprises delivering a recombinant polynucleotide encoding an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein to plant or algae cells, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation, regenerating plants or algae from said cells, and screening said plants or algae to identify a plant having an enhanced phenotype.
In one embodiment, the present invention provides compositions for attenuating the function and/or expression of atypical CYS HIS rich thioredoxin 4 (ACHT4), a light-regulated protein expressed in plants and algae that controls starch storage in chloroplast, and methods for increasing plant and algae growth and yield.
ACHT4
The Arabidopsis thaliana atypical cysteine histidine-rich Trxs (ACHTs) constitute a small family of plant-specific and chloroplast-localized Trxs. They are light-regulated and are good catalysts of 2-Cys Prx reduction.
A recently discovered paralog, AtACHT4, was found by the inventors to attenuate starch synthesis in Arabidopsis thaliana by oxidizing a regulatory disulfide on the small subunit (APS1) of the AGPase (ADP-glucose pyrophosphorylase, which catalyzes the first committed step in the starch synthesis pathway; Examples 1-2). The oxidizing reaction of AtACHT4 with AGPase requires the C-terminus of AtACHT4 (Example 2).
Thus, in one embodiment, the present invention provides compositions comprising ACHT4 proteins and nucleic acids and uses thereof. ACHT4 sequences may be from any species comprising such sequences. Table 1 hereinbelow discloses the amino acid sequences of some ACHT4 paralogs in various species, while Table 2 hereinbelow discloses the nucleic acid sequences of some ACHT4 paralogs in various species.
Arabidopsis
thaliana
Solanum
tuberosum
Solanum
tuberosum
Zea mays
Zea mays
Zea mays
Oryza
sativa
Orysa
sativa
Hordeum
vulgare
Hardeum
vulgare
Triticum
aestivum
Manihot
esculenta
Manihot
esculenta
Sorghum
bicolor
Sorghum
bicolor
Sorghum
bicolor
Brassica
napus
Brassica
napus
Brassica
napus
Brassica
napus
Brassica
napus
Ricinus
communis
Phaseolus
vulgaris
Phaseolus
vulgaris
Gossypium
histrum
Gossypium
histrum
Gossypium
histrum
Glycine
max
Glycine
max
Beta
vulgaris
Musa
acuminate
Musa
acuminate
Capsicum
annuum
Cicer
arietinum
Solanum
lycopersicum
Solanum
lycopersicum
Elaeis
guineensis
Elaeis
guineensis
Setaria
italic
Setaria
italic
Setaria
italic
Chlamydomonas
reinhardiii
Chlorella
In another embodiment, the amino acid sequence of ACHT4 is a homolog of any one of the sequences listed in Table 1. In another embodiment, the amino acid sequence of ACHT4 is a paralog of any one of the sequences listed in Table 1. In another embodiment, the amino acid sequence of ACHT4 is a fragment of any one of the sequences listed in Table 1. In another embodiment, the amino acid sequence of ACHT4 is a variant of any one of the sequences listed in Table 1. In another embodiment, the amino acid sequence of ACHT4 comprises any one of the sequences listed in Table 1. In another embodiment, the amino acid sequence of ACHT4 consists essentially of any one of the sequences listed in Table 1. In another embodiment, the amino acid sequence of ACHT4 consists of any one of the sequences listed in Table 1. In another embodiment, the amino acid sequence of ACHT4 corresponds to any one of the sequences listed in Table 1.
In another embodiment, the amino acid sequence of ACHT4 is a homolog of any one of SEQ ID NOs: 1-43. In another embodiment, the amino acid sequence of ACHT4 is a paralog of any one of SEQ ID NOs: 1-43. In another embodiment, the amino acid sequence of ACHT4 is a fragment of any one of SEQ ID NOs: 1-43. In another embodiment, the amino acid sequence of ACHT4 is a variant of any one of SEQ ID NOs: 1-43. In another embodiment, the amino acid sequence of ACHT4 comprises any one of SEQ ID NOs: 1-43. In another embodiment, the amino acid sequence of ACHT4 consists essentially of any one of SEQ ID NOs: 1-43. In another embodiment, the amino acid sequence of ACHT4 consists of any one of SEQ ID NOs: 1-43. In another embodiment, the amino acid sequence of ACHT4 corresponds to any one of SEQ ID NOs: 1-43.
In one embodiment, there is one paralog of ACHT4 in a species. In another embodiment, there are two paralogs of ACHT4 in a species. In another embodiment, there are three paralogs of ACHT4 in a species. In another embodiment, there are four paralogs of ACHT4 in a species. In another embodiment, there are five paralogs of ACHT4 in a species. In another embodiment, there are six paralogs of ACHT4 in a species. In another embodiment, there are seven or more paralogs of ACHT4 in a species.
In one embodiment, a “corresponding sequence” is an amino acid (or nucleic acid) sequence from a first species for which there is a similar or equivalent sequence in a second species, which may be inferred by sequence alignment, as is well known in the art.
Arabiaiopsis
thaliana
Solarium
tuberosum
Solanum
tuberosum
Zea mays
Zea mays
Zea mays
Oryza
saliva
Oryza
saliva
Horareum
vulgare
Horareum
vulgare
Triticum
aestivum
Manihot
esculenta
Manihot
esculenta
Sorghum
bicolor
Sorghum
bicolor
Sorghum
bicolor
Brassica
napus
Brassica
napus
Brassica
napus
Brassica
napus
Brassica
napus
Ricinus
communis
Phaseolus
vulgaris
Phaseolus
vulgaris
Gossypium
histrum
Gossypium
histrum
Gossypium
histrum
Glycine
max
Glycine
max
Beta
vulgaris
Musa
acuminate
Musa
acuminate
Capsicum
annuum
Cicer
arietinum
Solanum
lycopersicum
Solanum
lycopersicum
Elaeis
guineemis
Elaeis
guineemis
Setaria
italic
Setaria
italic
Setaria
italic
Chlamydomonas
reinhardtii
Chlorella
In another embodiment, the nucleic acid sequence of ACHT4 is a homolog of any one of the sequences listed in Table 2. In another embodiment, the nucleic acid sequence of ACHT4 is a paralog of any one of the sequences listed in Table 2. In another embodiment, the nucleic acid sequence of ACHT4 is a fragment of any one of the sequences listed in Table 2. In another embodiment, the nucleic acid sequence of ACHT4 is a variant of any one of the sequences listed in Table 2. In another embodiment, the nucleic acid sequence of ACHT4 comprises any one of the sequences listed in Table 2. In another embodiment, the nucleic acid sequence of ACHT4 consists essentially of any one of the sequences listed in Table 2. In another embodiment, the nucleic acid sequence of ACHT4 consists of any one of the sequences listed in Table 2. In another embodiment, the nucleic acid sequence of ACHT4 corresponds to any one of the sequences listed in Table 2.
In another embodiment, the nucleic acid sequence of ACHT4 is a homolog of any one of SEQ ID NOs: 44-86. In another embodiment, the nucleic acid sequence of ACHT4 is a paralog of any one of SEQ ID NOs: 44-86. In another embodiment, the nucleic acid sequence of ACHT4 is a fragment of any one of SEQ ID NOs: 44-86. In another embodiment, the nucleic acid sequence of ACHT4 is a variant of any one of SEQ ID NOs: 44-86. In another embodiment, the nucleic acid sequence of ACHT4 comprises any one of SEQ ID NOs: 44-86. In another embodiment, the nucleic acid sequence of ACHT4 consists essentially of any one of SEQ ID NOs: 44-86. In another embodiment, the nucleic acid sequence of ACHT4 consists of any one of SEQ ID NOs: 44-86. In another embodiment, the nucleic acid sequence of ACHT4 corresponds to any one of SEQ ID NOs: 44-86.
ACHT4ΔC
In one embodiment, the present invention provides a recombinant polynucleotide encoding an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation.
The inventors have demonstrated herein that over-expression (OE) of a C-terminal truncated form of AtACHT4 (AtACHT4ΔC, in which there is a 47 amino acid deletion of the C-terminus) has a dominant negative effect in Arabidopsis plants. AtACHT4ΔC overexpression relieves the oxidation of AGPase and boosts starch synthesis. In contrast, the OE of the full length ACHT4 does not, confirming that the C-terminus of AtACHT4 is indeed required for the attenuation of starch synthesis and the authenticity of the stimulating effect of AtACHT4-C-Del.
In addition, as demonstrated in Example 2, the stimulation of starch synthesis by AtACHT4-C-Del results in a significant increase in transitory starch content in Arabidopsis leaves and stimulates growth, indicating that AtACHT4ΔC stimulates the export of photosynthates from the chloroplast which are then directed toward plant metabolism and growth.
There are homologs of the ACHT4 found in Arabidopsis (AtACHT4), and in other species including potato, cassava, maize, rice, barley, wheat, sorghum, rapeseed, castor, bean, cotton, soybean, beet, banana, peppers, chickpea, tomato, oil palm, millet, several species of algae, and other plants and algae (Tables 1-2). As described in Example 3, ACHT4 in potato (paralogs StACHT4-2 and StACHT4-1) is similarly involved in attenuating starch synthesis and growth, and overexpression of the C-terminally deleted forms of StACHT4-1 and StACHT4-2, respectively, disinhibits (i.e. promotes) starch synthesis and nearly doubles tuber yield and plant shoot growth, respectively.
Since ACHT4 is expressed in all major crop and biofuel species, including rice and corn, attenuation of ACHT4 through dominant negative C-terminal deletions or other means, represents a promising new target for increasing plant growth and yield in all major crop and biofuel species.
In one embodiment, the inactivating mutation in the ACHT4 gene as described in the methods and compositions of the present invention is a deletion mutation. In another embodiment, the inactivating mutation is an insertion mutation. In another embodiment, the inactivating mutation is a substitution mutation. In another embodiment, the inactivating mutation is a null mutation. In another embodiment, the inactivating mutation is another type of mutation known in the art. In one embodiment, the insertion, deletion or substitution mutation comprises an insertion, deletion or substitution of a single nucleic acid, while in another embodiment, it comprises an insertion, deletion or substitution of 1-5 nucleic acids, 1-10 nucleic acids, 5-20 nucleic acids, 10-50 nucleic acids, 25-100 nucleic acids, 100-500 nucleic acids, 300-400 nucleic acids, 200-500 nucleic acids, or 500 or more nucleic acids.
In one embodiment, the mutation is a dominant negative mutation. In one embodiment, a dominant negative mutation (also called an antimorphic mutation) comprises an altered gene product that acts antagonistically to, attenuates, or inhibits the function(s) of the wild-type allele.
In one embodiment, the inactivating mutation in the C-terminal portion of ACHT4 is a deletion of the entire C-terminal portion of ACHT4. In another embodiment, the inactivating mutation in the C-terminal is a deletion of a portion of the C-terminal portion of ACHT4.
In one embodiment, a “corresponding sequence” is a nucleic acid (or amino acid) sequence from a first species for which there is a similar or equivalent sequence in a second species, which may be inferred by sequence alignment, as is well known in the art.
In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 141-207 nucleic acids of the ACHT4 gene. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 150-225 nucleic acids of the ACHT4 gene. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 150-300 nucleic acids of the ACHT4 gene. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 75-150 nucleic acids of the ACHT4 gene. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 3-75 nucleic acids of the ACHT4 gene. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 3-30 nucleic acids of the ACHT4 gene. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 75-225 nucleic acids of the ACHT4 gene. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 3-60 nucleic acids of the ACHT4 gene. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 60-120 nucleic acids of the ACHT4 gene. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 120-180 nucleic acids of the ACHT4 gene. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 150-225 nucleic acids of the ACHT4 gene.
In one embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 687-825 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 651-825 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 600-825 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 801-825 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 699-825 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 750-825 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 774-825 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 687-750 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species.
In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 699-903 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 600-903 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 651-903 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 750-903 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 801-903 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 849-903 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 876-903 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 825-903 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 699-801 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 699-849 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species.
In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 486-690 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 501-690 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 549-690 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 600-690 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 651-690 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 486-651 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 486-600 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 486-549 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of nucleic acids 486-501 of any one of the sequences listed in Table 2 or a corresponding nucleic acid sequence from another species.
In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises insertion of a non-native sequence into a portion of the C-terminal of ACHT4 encoding the C-terminal of ACHT4, wherein said the C-terminal of ACHT4 is inactivated as a result.
In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises substitution of nucleic acid residues, as is known to one of skill in the art. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 consists essentially of any of the mutations listed hereinabove. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 consists of any of the mutations listed hereinabove.
In another embodiment, the present invention provides a composition comprising a recombinant polynucleotide encoding an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation.
In another embodiment, the present invention provides an expression vector comprising a recombinant polynucleotide encoding an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation.
In one embodiment, “expression” as used herein refers to transcription of DNA to produce RNA. The resulting RNA may be without limitation mRNA encoding a protein, antisense RNA that is complementary to an mRNA encoding a protein, or an RNA transcript comprising a combination of sense and antisense gene regions, such as for use in RNAi technology. Expression as used herein may also refer to production of encoded protein from mRNA.
In another embodiment, the present invention provides a composition comprising an expression vector comprising a recombinant polynucleotide encoding an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation.
Recombinant Polynucleotides
In one embodiment, “recombinant polynucleotide” refers to a polynucleotide having a genetically engineered modification introduced through manipulation via mutagenesis, restriction enzymes, and the like. Recombinant polynucleotides may comprise DNA segments obtained from different sources, or DNA segments obtained from the same source, but which have been manipulated to join DNA segments which do not naturally exist in the joined form. A recombinant polynucleotide may exist outside of the cell, for example as a PCR fragment, or integrated into a genome, such as a plant genome.
The present invention contemplates the use of polynucleotides effective for imparting an enhanced phenotype to genetically modified plants or algae expressing said polynucleotides. Exemplary polynucleotides for use in the present invention are provided herein in Table 2 (SEQ ID NO: 44 through SEQ ID NO: 86). A subset of the nucleic molecules of this invention includes fragments of the disclosed polynucleotides consisting of oligonucleotides of at least 15, preferably at least 16 or 17, more preferably at least 18 or 19, and even more preferably at least 20 or more, consecutive nucleotides. Such oligonucleotides are fragments of the larger molecules having a sequence provided herein in Table 2 (SEQ ID NO: 44 through SEQ ID NO: 86), and find use, for example as probes and primers for detection of the polynucleotides of the present invention.
Also of interest in the present invention are variants of the polynucleotides provided herein. Such variants may be naturally occurring, including homologous polynucleotides from the same or a different species, or may be non-natural variants, for example polynucleotides synthesized using chemical synthesis methods, or generated using recombinant DNA techniques. Degeneracy of the genetic code provides the possibility to substitute at least one base of the protein encoding sequence of a gene with a different base without causing the amino acid sequence of the polypeptide produced from the gene to be changed. Hence, a polynucleotide useful in the present invention may have any base sequence that has been changed from the sequences in Table 2 (SEQ ID NO: 44 to SEQ ID NO: 86) by substitution in accordance with degeneracy of the genetic code.
Homologs of the polynucleotides provided herein will generally demonstrate significant identity with the polynucleotides provided herein. A polynucleotide of the present invention is substantially identical to a reference polynucleotide if, when the sequences of the polynucleotides are optimally aligned there is about 60% nucleotide equivalence; more preferably 70%; more preferably 80% equivalence; more preferably 85% equivalence; more preferably 90%; more preferably 95%; and/or more preferably 98% or 99% equivalence over a comparison window. A comparison window is preferably at least 50-100 nucleotides, and more preferably is the entire length of the polynucleotide provided herein. Optimal alignment of sequences for aligning a comparison window may be conducted by algorithms; preferably by computerized implementations of these algorithms (such as the Wisconsin Genetics Software Package). The reference polynucleotide may be a full-length molecule or a portion of a longer molecule. Preferentially, the window of comparison for determining polynucleotide identity of protein encoding sequences is the entire coding region.
Promoters
In one embodiment, a polynucleotide of the present invention is operatively linked in a recombinant polynucleotide to a promoter functional in a plant or alga to provide for expression of the polynucleotide in the sense orientation such that a desired polypeptide is produced. Also considered are embodiments wherein a polynucleotide is operatively linked to a promoter functional in a plant to provide for expression of the polynucleotide in the antisense orientation such that a complementary copy of at least a portion of an mRNA native to the target plant host is produced. Such a transcript may contain both sense and antisense regions of a polynucleotide, for example where RNAi methods are used for gene suppression.
In one embodiment, the promoter of the expression vector of the present invention is operably linked to the polynucleotide. In one embodiment, the promoter is a constitutive promoter. In another embodiment, the promoter is an inducible promoter. In another embodiment, the promoter is a tissue-specific promoter.
In one embodiment, a promoter used in the compositions and methods of the present invention is cisgenic, i.e. is a promoter that is native to the plant.
Recombinant polynucleotides of the present invention are assembled in recombinant DNA constructs using methods known to those of ordinary skill in the art. Thus, DNA constructs used for transforming plant cells will comprise a polynucleotide one desires to introduce into a target plant. Such constructs will also typically comprise a promoter operatively linked to said polynucleotide to provide for expression in the target plant. Other construct components may include additional regulatory elements, such as 5′ or 3′ untranslated regions (such as polyadenylation sites), intron regions, and transit or signal peptides.
Numerous promoters that are active in plant cells have been described in the literature. These include promoters present in plant genomes as well as promoters from other sources, including nopaline synthase (NOS) promoter and octopine synthase (OCS) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens, caulimovirus promoters such as the cauliflower mosaic virus or figwort mosaic virus promoters.
These and numerous other promoters that function in plant cells are known to those skilled in the art and available for use in recombinant polynucleotides of the present invention to provide for expression of desired genes in genetically modified plant cells.
Furthermore, the promoters may be altered to contain multiple “enhancer sequences” to assist in elevating gene expression. Such enhancers are known in the art. By including an enhancer sequence with such constructs, the expression of the selected protein may be enhanced. These enhancers often are found 5′ to the start of transcription in a promoter that functions in eukaryotic cells, but can often be inserted in the forward or reverse orientation 5′ or 3′ to the coding sequence. In some instances, these 5′ enhancing elements are introns. Deemed to be particularly useful as enhancers are the 5′ introns of the rice actin 1 and rice actin 2 genes. Examples of other enhancers that can be used in accordance with the invention include elements from the CaMV 35S promoter, octopine synthase genes, the maize alcohol dehydrogenase gene, the maize shrunken 1 gene and promoters from non-plant eukaryotes.
Organ-Specific Promoters
In other aspects of the invention, expression in plant seed tissues is desired to effect improvements in seed composition. In one embodiment, promoters for use for seed composition modification include promoters from seed genes such as napin, globulin 1, glutelin 1, and peroxiredoxin antioxidant.
In still other aspects of the invention, preferential expression in plant green tissues is desired. In one embodiment, promoters for expression in plant green tissues include those from genes such as SSU, aldolase and pyruvate orthophosphate dikinase (PPDK).
Recombinant constructs prepared in accordance with the invention will also in one embodiment, include a 3′ untranslated DNA region that typically contains a polyadenylation sequence following the polynucleotide coding region. Examples of useful 3′ UTRs include those from the nopaline synthase gene of Agrobacterium tumefaciens (nos), a gene encoding the small subunit of a ribulose-1,5-bisphosphate carboxylase-oxygenase (rbcS), and the T7 transcript of Agrobacterium tumefaciens.
Constructs and vectors may also include a transit peptide for targeting of a gene target to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle.
Host Cells
In another embodiment, the present invention provides a cell comprising an expression vector comprising a recombinant polynucleotide encoding an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation.
In one embodiment, the cell is from an Arabidopsis. In one embodiment, the cell is from Arabidopsis thaliana. In one embodiment, the cell is from a crop plant. In one embodiment, the crop plant is Solanum tuberosum (Potato). In another embodiment, the crop plant is Zea mays (Maize). In another embodiment, the crop plant is Oryza sativa (Rice). In another embodiment, the crop plant is Manihot esculenta (Cassava). In another embodiment, the crop plant is Hordeum vulgare (Barley). In another embodiment, the crop plant is Triticum aestivum (Wheat). In another embodiment, the crop plant is Sorghum bicolor. In another embodiment, the crop plant is Brassica napus (Rapeseed). In another embodiment, the crop plant is Ricinus communis (Castor). In another embodiment, the crop plant is Phaseolus vulgaris (Bean). In another embodiment, the crop plant is Gossypium histrum (Cotton). In another embodiment, the crop plant is Glycine max (Soybean). In another embodiment, the crop plant is Beta vulgaris (Beet). In another embodiment, the crop plant is Musa acuminate (Banana). In another embodiment, the crop plant is Capsicum annuum (Sweet and Chili Peppers). In another embodiment, the crop plant is Cicer arietinum (Chick pea). In another embodiment, the crop plant is Solanum lycopersicum (Tomato). In another embodiment, the crop plant is Elaeis guineensis (African oilpalm). In another embodiment, the crop plant is Setaria italic (Foxtail millet).
In another embodiment, the crop plant is a food crop. In another embodiment, the crop plant is a nutritionally enhanced food crop.
In another embodiment, the cell is a moss cell. In one embodiment, the moss is Physcomitrella patens. In another embodiment, the cell is an algae cell. In one embodiment, the algae cell is Chlamydomonas reinhardtii. In another embodiment, the algae cell is Ostreococcus tauri. In another embodiment, the algae cell is a Chlorella.
Provided herein are host cells that contain a vector, e.g., a DNA plasmid and support the replication and/or expression of the vector. Host cells may be prokaryotic cells such as E. coli, or eukaryotic cells such as yeast, plant, insect, amphibian, or mammalian cells. In some embodiments, host cells are monocotyledonous or dicotyledonous plant cells. In other embodiments monocotyledonous host cell is a maize host cell. In certain embodiments, the host cell utilized in the methods of the present invention is transiently transfected with the nucleic acid molecules of the invention.
In some embodiments, the host cell utilized in the methods of the present invention is a plant protoplast. Plant protoplasts are plant cells that had their entire plant cell wall enzymatically removed prior to the introduction of the molecule of interest. The complete removal of the cell wall disrupts the connection between cells producing a homogenous suspension of individualized cells which allows more uniform and large scale transfection experiments. This comprises, but is not restricted to protoplast fusion, electroporation, liposome-mediated transfection, and polyethylene glycol-mediated transfection. Protoplast preparation is therefore a very reliable and inexpensive method to produce millions of cells.
In particular embodiments, the plant protoplast is derived from one of the following genuses: Acorus, Aegilops, Allium, Amborella, Antirrhinum, Apium, Arabidopsis, Arachis, Beta. Betula, Brassica, Capsicum, Ceratopteris, Citrus, Cryptomeria, Cycas, Descurainia, Eschscholzia, Eucalyptus, Glycine, Gossypium, Hedyotis, Helianthus, Hordeum, Ipomoea, Lactuca, Limm, Liriodendron, Lotus, Lupinus. Lycopersicon, Medicago, Mesembryanthemum, Nicotiana, Nuphar, Pennisetum, Persea, Phaseolus, Physcomitrella, Picea, Pinus, Poncirus, Populus, Prunus, Robinia, Rosa, Saccharum, Schedonorus, Secale, Sesamum, Solanum, Sorghum, Stevia, Thellungiella, Theobroma, Triphysaria, Triticum, Vitis, Zea, or Zinnia. In some embodiments, the host cell is derived from a genus that is different from the genus from which the ACHT4 gene is derived.
Also provided herein are plant cells having the nucleotide sequence constructs of the invention. A further aspect of the present invention provides a method of making such a plant cell involving introduction of a vector including the construct into a plant cell. For integration of the construct into the plant genome, such introduction will be followed by recombination between the vector and the plant cell genome to introduce the sequence of nucleotides into the genome. RNA encoded by the introduced nucleic acid construct may then be transcribed in the cell and descendants thereof, including cells in plants regenerated from transformed material. A gene stably incorporated into the genome of a plant is passed from generation to generation to descendants of the plant, so such descendants should show the desired phenotype.
Optionally, germ line cells may be used in the methods described herein rather than, or in addition to, somatic cells. The term “germ line cells” refers to cells in the plant organism which can trace their eventual cell lineage to either the male or female reproductive cell of the plant. Other cells, referred to as “somatic cells” are cells which give rise to leaves, roots and vascular elements which, although important to the plant, do not directly give rise to gamete cells. Somatic cells, however, also may be used. With regard to callus and suspension cells which have somatic embryogenesis, many or most of the cells in the culture have the potential capacity to give rise to an adult plant. If the plant originates from single cells or a small number of cells from the embryogenic callus or suspension culture, the cells in the callus and suspension can therefore be referred to as germ cells. In the case of immature embryos which are prepared for treatment by the methods described herein, certain cells in the apical meristem region of the plant have been shown to produce a cell lineage which eventually gives rise to the female and male reproductive organs. With many or most species, the apical meristem is generally regarded as giving rise to the lineage that eventually will give rise to the gamete cells. An example of a non-gamete cell in an embryo would be the first leaf primordia in corn which is destined to give rise only to the first leaf and none of the reproductive structures.
In another embodiment, the present invention provides a composition comprising a cell comprising an expression vector comprising a recombinant polynucleotide encoding an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation.
Seeds
In another embodiment, the present invention provides a seed comprising a C-terminal deleted form of an atypical CYS HIS rich thioredoxin 4 (ACHT4) gene.
The present invention is directed to seed from a genetically modified plant, wherein the genome of said seed comprises an exogenous polynucleotide comprising a functional portion of an encoding region for a polypeptide provided herein, and wherein plants grown from said seed exhibit an enhanced phenotype as compared to the phenotype of a control plant. In one embodiment, the enhanced phenotype is increased yield. Exogenous polynucleotides of the present invention include recombinant polynucleotides providing for expression of mRNA encoding a polypeptide.
Plants and Plant Parts
In another embodiment, the present invention provides a plant, or plant part, comprising a C-terminal deleted form of an atypical CYS HIS rich thioredoxin 4 (ACHT4) gene.
The present invention provides a plant comprising a C-terminal deleted form of an ACHT4 gene. Transformed seeds and plant parts are also encompassed. In one embodiment, the plant part is a seed. In another embodiment, the plant part is a leaf. In another embodiment, the plant part is a stem. In another embodiment, the plant part is a root. In another embodiment, the plant part is a flower. In another embodiment, the plant part is a tuber. In another embodiment, the plant part is a fruit.
In one embodiment, a plant of the present invention is any plant that comprises an ACHT4 gene or homolog.
In one embodiment, a plant of the present invention is a crop plant. In one embodiment, the crop plant is Solanum tuberosum (Potato). In another embodiment, the crop plant is Zea mays (Maize). In another embodiment, the crop plant is Oryza sativa (Rice). In another embodiment, the crop plant is Manihot esculenta (Cassava). In another embodiment, the crop plant is Hordeum vulgare (Barley). In another embodiment, the crop plant is Triticum aestivum (Wheat). In another embodiment, the crop plant is Sorghum bicolor. In another embodiment, the crop plant is Brassica napus (Rapeseed). In another embodiment, the crop plant is Ricimus communis (Castor). In another embodiment, the crop plant is Phaseolus vulgaris (Bean). In another embodiment, the crop plant is Gossypium histrum (Cotton). In another embodiment, the crop plant is Glycine max (Soybean). In another embodiment, the crop plant is Beta vulgaris (Beet). In another embodiment, the crop plant is Musa acuminate (Banana). In another embodiment, the crop plant is Capsicum annuum (Sweet and Chili Peppers). In another embodiment, the crop plant is Cicer arietinum (Chick pea). In another embodiment, the crop plant is Solanum lycopersicum (Tomato). In another embodiment, the crop plant is Elaeis guineensis (African oilpalm). In another embodiment, the crop plant is Setaria italic (Foxtail millet).
In another embodiment, the crop plant is a food crop. In another embodiment, the crop plant is a nutritionally enhanced food crop.
In another embodiment, a plant of the present invention is an Arabidopsis. In one embodiment, the Arabidopsis plant is Arabidopsis thaliana. In another embodiment, the Arabidopsis plant is Arabidopsis arenicola, Arabidopsis arenosa, Arabidopsis cebennensis, Arabidopsis croatica, Arabidopsis halleri, Arabidopsis lyrata, Arabidopsis neglecta, Arabidopsis pedemontana, or Arabidopsis suecica.
In another embodiment, a plant of the present invention is a moss. In one embodiment, the moss is a Sphagnum. In one embodiment, the Sphagnum species is cristatum or subnitens. In one embodiment, the moss is used for peat. In one embodiment, peat is used for fuel, as a horticultural soil additive, and in smoking malt in the production of Scotch whisky. In another embodiment, the moss is used for decorative purposes, such as in gardens and in the florist trade. In another embodiment, the moss is used as insulation. In another embodiment, the moss is used as an absorber of liquids. In another embodiment, moss is used for first-aid dressings, for diapers or napkins. In another embodiment, the moss is a Physcomitrella patens. In another embodiment, the moss is a Fontinalis antipyretica which, in one embodiment, is used to subdue fires.
In one embodiment, the plant is an ornamental plant.
Plants included in the invention are any plants amenable to transformation techniques, including gymnosperms and angiosperms, both monocotyledons and dicotyledons.
In addition to a plant, the present invention provides any clone of such a plant, seed, selfed or hybrid progeny and descendants, and any part of any of these, such as cuttings, seed. The invention provides any plant propagule, that is any part which may be used in reproduction or propagation, sexual or asexual, including cuttings, seed and so on. Also encompassed by the invention is a plant which is a sexually or asexually propagated off-spring, clone or descendant of such a plant, or any part or propagule of said plant, off-spring, clone or descendant. Plant extracts and derivatives are also provided.
Algae
In another embodiment, the present invention provides an alga comprising a C-terminal deleted form of an atypical CYS HIS rich thioredoxin 4 (ACHT4) gene.
In one embodiment, the alga is a microalga. In one embodiment, the species of the alga is selected from the following species: Ankistrodesmus, Botryococcus braunii, Chlorella, Chlorella protothecoides (autotrophic/heterothrophic), Crypthecodinium cohnii, Cyclotella, Dunaliella tertiolecta, Gracilaria, Hantzschia, Nannochloris, Nannochloropsis, Neochloris oleoabundans, Nitzschia, Phaeodactylum tricornutum, Pleurochrysis carterae (also called CCMP647), Sargassum, Scenedesmus, Schizochytrium, Stichococcus, Tetraselmis suecica, and Thalassiosira pseudonana. In another embodiment, the alga is a Chlamydomonas reinhardtii. In another embodiment, the alga is a Ostreococcus tauri.
In another embodiment, the present invention provides a polypeptide comprising a C-terminal deleted form of atypical CYS HIS rich thioredoxin 4 (ACHT4).
Proteins and Polypeptides
Polypeptides considered in the present invention are entire proteins or at least a sufficient portion of the entire protein to impart the relevant biological activity of the protein, e.g. enhanced plant phenotype. The term “protein” also includes molecules consisting of one or more polypeptide chains. Thus, a polypeptide useful in the present invention may constitute an entire protein having the desired biological activity, or may constitute a portion of an oligomeric protein having multiple polypeptide chains. Polypeptides useful for generation of genetically modified plants having enhanced properties include the polypeptide provided herein as SEQ ID NOs: 1-43, as well as homologs of such polypeptides.
In one embodiment, the inactivating mutation in the C-terminal portion of ACHT4 is a deletion of the entire C-terminal domain of ACHT4. In one embodiment, the C-terminal domain of ACHT4 is the sequence that is downstream of the conserved thioredoxin (Trx) domain (as depicted in
In one embodiment, the amino acid sequence of the C-terminal of ACHT4 comprises:
In another embodiment, the amino acid sequence of the C-terminal of ACHT4 is a homolog of SEQ ID NO: 87. In another embodiment, the amino acid sequence of the C-terminal of ACHT4 is a paralog of SEQ ID NO: 87. In another embodiment, the amino acid sequence of the C-terminal of ACHT4 is a fragment of SEQ ID NO: 87. In another embodiment, the amino acid sequence of the C-terminal of ACHT4 is a variant of SEQ ID NO: 87. In another embodiment, the amino acid sequence of the C-terminal of ACHT4 comprises SEQ ID NO: 87. In another embodiment, the amino acid sequence of the C-terminal of ACHT4 corresponds to SEQ ID NO: 87.
In another embodiment, the inactivating mutation in the C-terminal is a deletion of the 68-69 amino acid C-terminal portion of ACHT4.
In one embodiment, the amino acid sequence of the C-terminal of ACHT4 comprises:
In another embodiment, the amino acid sequence of the C-terminal of ACHT4 comprises:
In another embodiment, the amino acid sequence of the C-terminal of ACHT4 is a homolog any one of SEQ ID NOs: 88-89. In another embodiment, the amino acid sequence of the C-terminal of ACHT4 is a paralog any one of SEQ ID NOs: 88-89. In another embodiment, the amino acid sequence of the C-terminal of ACHT4 is a fragment any one of SEQ ID NOs: 88-89. In another embodiment, the amino acid sequence of the C-terminal of ACHT4 is a variant any one of SEQ ID NOs: 88-89. In another embodiment, the amino acid sequence of the C-terminal of ACHT4 comprises any one of SEQ ID NOs: 88-89. In another embodiment, the amino acid sequence of the C-terminal of ACHT4 corresponds to any one of SEQ ID NOs: 88-89.
In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 47-69 amino acids of the C-terminal portion of ACHT4. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 50-75 amino acids of the C-terminal portion of ACHT4. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 50-100 amino acids of the C-terminal portion of ACHT4. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 25-50 amino acids of the C-terminal portion of ACHT4. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 1-25 amino acids of the C-terminal portion of ACHT4. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 1-10 amino acids of the C-terminal portion of ACHT4. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 25-75 amino acids of the C-terminal portion of ACHT4. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 1-20 amino acids of the C-terminal portion of ACHT4. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 20-40 amino acids of the C-terminal portion of ACHT4. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 40-60 amino acids of the C-terminal portion of ACHT4. In another embodiment, the inactivating mutation in the C-terminal is a deletion of the final 50-70 amino acids of the C-terminal portion of ACHT4.
In one embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 229-275 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 250-275 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 260-275 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 240-275 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 229-240 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 229-250 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 229-260 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 229-270 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species.
In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 233-301 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 240-301 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 250-301 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 260-301 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 275-301 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 290-301 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 233-250 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 233-275 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 233-290 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species.
In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 162-230 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 175-230 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 200-230 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 220-230 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 162-175 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 162-200 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises a deletion of amino acids 162-220 of any one of the sequences listed in Table 1 or a corresponding amino acid sequence from another species.
In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises insertion of a non-native sequence into a portion of the C-terminal of ACHT4 encoding the C-terminal of ACHT4, wherein said the C-terminal of ACHT4 is inactivated as a result.
In another embodiment, an inactivating mutation in the C-terminal of ACHT4 comprises substitution of amino acid residues, such as a substitution of polar for non-polar residues, non-polar for polar residues, charged for uncharged residues, positively charged for negatively charged residues, or vice versa, or a combination thereof, as is known to one of skill in the art. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 consists essentially of any of the mutations listed hereinabove. In another embodiment, an inactivating mutation in the C-terminal of ACHT4 consists of any of the mutations listed hereinabove.
In one embodiment, an ACHT4 protein having an inactivating mutation in the C-terminal portion is a truncated ACHT4. In another embodiment, an ACHT4 protein having an inactivating mutation in the C-terminal portion is represented as ACHT4ΔC. In one embodiment, the truncated form of ACHT4 comprises:
In another embodiment, the truncated form of ACHT4 comprises:
In another embodiment, the truncated form of ACHT4 comprises:
In another embodiment, the amino acid sequence of the C-terminal of ACHT4 is a homolog any one of SEQ ID NOs: 90-92. In another embodiment, the amino acid sequence of the C-terminal of ACHT4 is a paralog any one of SEQ ID NOs: 90-92. In another embodiment, the amino acid sequence of the C-terminal of ACHT4 is a fragment any one of SEQ ID NOs: 90-92. In another embodiment, the amino acid sequence of the C-terminal of ACHT4 is a variant any one of SEQ ID NOs: 90-92. In another embodiment, the amino acid sequence of the C-terminal of ACHT4 comprises any one of SEQ ID NOs: 90-92. In another embodiment, the amino acid sequence of the C-terminal of ACHT4 corresponds to any one of SEQ ID NOs: 90-92.
Homologs of the polypeptides of the present invention may be identified by comparison of the amino acid sequence of the polypeptide to amino acid sequences of polypeptides from the same or different plant sources, e.g. manually or by using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. As used herein, a homolog is a peptide from the same or a different organism that performs the same biological function as the polypeptide to which it is compared. An orthologous relation between two organisms is not necessarily manifest as a one-to-one correspondence between two genes, because a gene can be duplicated or deleted after organism phylogenetic separation, such as speciation. For a given polypeptide, there may be no ortholog or more than one ortholog. Other complicating factors include alternatively spliced transcripts from the same gene, limited gene identification, redundant copies of the same gene with different sequence lengths or corrected sequence. A local sequence alignment program, e.g. BLAST, can be used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) used to measure the sequence base similarity.
A further aspect of the invention comprises functional homolog proteins which differ in one or more amino acids from those of a polypeptide provided herein as the result of one or more of the well-known conservative amino acid substitutions, e.g. valine is a conservative substitute for alanine and threonine is a conservative substitute for serine. Conservative substitutions for an amino acid within the native polypeptide sequence can be selected from other members of a class to which the naturally occurring amino acid belongs. Representative amino acids within these various classes include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conserved substitutes for an amino acid within a native amino acid sequence can be selected from other members of the group to which the naturally occurring amino acid belongs. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Naturally conservative amino acids substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. A further aspect of the invention comprises polypeptides that differ in one or more amino acids from those of a described protein sequence as the result of deletion or insertion of one or more amino acids in a native sequence.
Homologs of the polypeptides provided herein will generally demonstrate significant identity with the polypeptides provided herein. In one embodiment, the present invention provides polypeptides with at least about 50% sequence identity. In one embodiment, the present invention provides polypeptides with at least about 70% sequence identity. In one embodiment, the present invention provides polypeptides with at least about 80% sequence identity with an amino acid sequence of any of the amino acid sequences listed in Table 1. In one embodiment, the present invention provides polypeptides with higher identity to such a polypeptide sequence, e.g. 90% to 990, identity. Identity of protein homologs is determined by optimally aligning the amino acid sequence of a putative protein homolog with a defined amino acid sequence and by calculating the percentage of identical and conservatively substituted amino acids over the window of comparison. Preferentially, the window of comparison for determining identity is the entire polypeptide sequence disclosed herein, e.g. the full sequence of any one of the sequences listed in Table 1.
In another embodiment, the present invention provides a composition comprising a polypeptide comprising a C-terminal deleted form of atypical CYS HIS rich thioredoxin 4 (ACHT4).
Methods of Use for Plants Expressing ACHT4ΔC
In another embodiment, the present invention provides a method of increasing the yield of a plant or algae comprising contacting a cell from said plant or algae with a polynucleotide encoding a C-terminal deleted form of atypical CYS HIS rich thioredoxin 4 (ACHT4), thereby increasing the yield of said plant or algae.
In another embodiment, the present invention provides a method of increasing the productivity of a plant or algae comprising contacting a cell from said plant or algae with a polynucleotide encoding a C-terminal deleted form of atypical CYS HIS rich thioredoxin 4 (ACHT4), thereby increasing the productivity of said plant or algae.
In another embodiment, the present invention provides a method of increasing the size of a plant or algae comprising contacting a cell from said plant or algae with a polynucleotide encoding a C-terminal deleted form of atypical CYS HIS rich thioredoxin 4 (ACHT4), thereby increasing the size of said plant or algae.
In another embodiment, the present invention provides a method of increasing the biomass of a plant or algae comprising contacting a cell from said plant or algae with a polynucleotide encoding a C-terminal deleted form of atypical CYS HIS rich thioredoxin 4 (ACHT4), thereby increasing the biomass of said plant or algae.
In another embodiment, the present invention provides a method of stimulating the growth of a plant or algae comprising contacting a cell from said plant or algae with a polynucleotide encoding a C-terminal deleted form of atypical CYS HIS rich thioredoxin 4 (ACHT4), thereby stimulating the growth of said plant or algae.
In another embodiment, the present invention provides a method of enhancing the starch content of a plant or algae comprising contacting a cell from said plant or algae with a polynucleotide encoding a C-terminal deleted form of atypical CYS HIS rich thioredoxin 4 (ACHT4), thereby enhancing the starch content of said plant or algae.
In one embodiment, the plant or algae has enhanced starch in transitory starch stores. In another embodiment, the plant or algae has enhanced starch in non-transitory starch stores. In one embodiment, the plant has enhanced starch content in one or more leaves. In another embodiment, the plant has enhanced starch content in the roots. In another embodiment, the plant has enhanced starch content in the stem. In another embodiment, the plant has enhanced starch content in one or more seeds. In another embodiment, the plant has enhanced starch content in its tubers. In another embodiment, the plant has enhanced starch content in its fruit. In another embodiment, the plant has enhanced starch content in one or more of its flowers.
In one embodiment, the present invention provides methods comprising the step of “contacting” a cell with a polynucleotide or expression vector as described herein. In one embodiment, plants are genetically modified using a microbial vector comprising ACHT4ΔC. In one embodiment, the microbial vector is Agrobacterium tumefaciens. In another embodiment, plants are genetically modified using microprojectile bombardment. In one embodiment, corn, rice, and other cereal grains are genetically modified using microprojectile bombardment. In another embodiment, plants are genetically modified using electroporation. In another embodiment, plants are genetically modified using microinjection, which in one embodiment, is direct microinjection of genetically modified DNA into anchored cells. In another embodiment, plants are genetically modified using transposons or transposable elements.
In one embodiment, the step of contacting is performed in vitro. In another embodiment, the step of contacting is performed in vivo.
In one embodiment, the ACHT4ΔC is integrated into the plant or algae chromosome. In another embodiment, the ACHT4ΔCis expressed from a vector.
Methods of Producing Genetically Modified Plants
In another embodiment, the present invention provides a method of producing a plant having an enhanced phenotype, wherein said method comprises transforming plant cells with a recombinant polynucleotide encoding an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation, regenerating plants from said cells, and screening said plants to identify a plant having an enhanced phenotype.
In another embodiment, the present invention provides a method of producing an algae having an enhanced phenotype, wherein said method comprises delivering a recombinant polynucleotide encoding an atypical CYS HIS rich thioredoxin 4 (ACHT4) protein to algae cells, wherein the C-terminal portion of said ACHT4 protein comprises an inactivating mutation, growing algae from said cells, and screening said algae to identify a plant having an enhanced phenotype.
Genetically modified plant seed provided by this invention may be grown to generate genetically modified plants having an enhanced phenotype as compared to an appropriate control line. Such seed is obtained by screening transformed plants for enhanced phenotypes resulting from the introduction of a recombinant polynucleotide into the genomic DNA of tissue from a parental line. The recombinant polynucleotide is introduced into the genome to produce genetically modified cells that can be cultured into genetically modified plants having an enhanced phenotype as compared to the parental line or other appropriate control. Such genetically modified cells are cultured into genetically modified plants that produce progeny genetically modified seed. Preferably, multiple genetically modified plants (events) comprising the recombinant polynucleotides are evaluated, e.g. from 2 to 20 or more genetically modified events, to identify a desired enhanced phenotype. Although the design of a recombinant polynucleotide is based on a rational expectation of a phenotypic modification, the present invention also contemplates that unexpected, yet desired enhanced phenotypes may be obtained.
Genetically modified plants grown from genetically modified seed as described herein will have improved phenotypes that contribute to increased yield or other increased plant value, including, for example, improved seed quality. Of particular interest are plants having altered cell division, enhanced plant growth and development, stress tolerance, including tolerance to abiotic and biotic stress, altered seed or flower development, improved light response, and enhanced carbon and/or nitrogen metabolism, transport or utilization properties.
Genetic Modification
In one embodiment, the present invention provides a cisgenic plant. In one embodiment, a cisgenic plant of the present invention is genetically modified, in one embodiment, comprises a second copy of a gene in which a portion of said gene is deleted, but contains no foreign or heterologous genes. In one embodiment, the promoters used in the expression of ACHT4ΔC are cisgenic. In one embodiment, food crops of the present invention are cisgenic.
In another embodiment, the present invention provides a transgenic plant. In one embodiment, a transgenic plant of the present invention is genetically modified with foreign or heterologous genes. In one embodiment, transgenic plants of the present invention are used for biofuel. In another embodiment, transgenic plants of the present invention are food crop plants.
Any method or delivery system may be used for the delivery and/or transformation (plant cells)/transfection (algae cells) of the nucleic acid vectors encoding ACHT4 and homologs, paralogs, etc. in the host cell, e.g., plant protoplast. The vectors may be delivered to the host cell either alone, or in combination with other agents. Transient expression systems may also be used. Homologous recombination may also be used.
Transformation may be accomplished by a wide variety of means, as is known to those of ordinary skill in the art. Such methods include, but are not limited to, Agrobacterium-mediated transformation (e.g., Komari et al., 1998, Curr. Opin. Plant Biol., 1:161), including floral dip transformation, particle bombardment mediated transformation (e.g., Finer et al., 1999, Curr. Top. Microbiol. Immunol., 240:59), protoplast electroporation (e.g., Bates, 1999, Methods Mol. Biol., 111:359), viral infection (e.g., Porta and Lomonossoff, 1996, Mol. Biotechnol. 5:209), microinjection, and liposome injection. Other exemplary delivery systems that can be used to facilitate uptake by a cell of the nucleic acid include calcium phosphate and other chemical mediators of intracellular transport, microinjection compositions, and homologous recombination compositions (e.g., for integrating a gene into a preselected location within the chromosome of the cell). Alternative methods may involve, for example, the use of liposomes, electroporation, or chemicals that increase free (or “naked”) DNA uptake, transformation using viruses or pollen and the use of microprojection. Standard molecular biology techniques are common in the art (e.g., Sambrook et al., 1989, Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, New York).
For the Agrobacterium tumefaciens-based plant transformation system, additional elements present on transformation constructs will, in one embodiment, include T-DNA left and right border sequences to facilitate incorporation of the recombinant polynucleotide into the plant genome.
In one embodiment, DNA is inserted randomly, i.e. at a non-specific location, in the genome of a target plant line. In another embodiment, DNA insertion is targeted in order to achieve site-specific integration, e.g. to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression. Several site specific recombination systems exist which are known to function in plants including cre-lox and FLP-FRT.
Transformation methods of this invention are preferably practiced in tissue culture on media and in a controlled environment. “Media” refers to the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant may be regenerated is useful as a recipient cell. Callus may be initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation. Practical transformation methods and materials for making genetically modified plants of this invention, e.g. various media and recipient target cells, transformation of immature embryos and subsequent regeneration of fertile genetically modified plants are known in the art.
In one embodiment, the method of transformation of algae comprises any of the methods as described hereinabove. In one embodiment, transformation of algae is accomplished using glass bead-assisted transformation, particle gun-mediated (biolistic) transformation, treatment with cellulolytic enzymes to weaken their cell walls, or homologous recombination.
Markers of Genetic Transformation
In one embodiment, DNA is introduced into only a small percentage of target cells in any one experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a genetically modified DNA construct into their genomes. Preferred marker genes provide selective markers that confer resistance to a selective agent, such as an antibiotic or herbicide. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells will be those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells may be tested further to confirm stable integration of the exogenous DNA. Useful selective marker genes include those conferring resistance to antibiotics such as kanamycin (nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS).
Screenable markers which provide an ability to visually identify transformants can also be employed, e.g., a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known. It is also contemplated that combinations of screenable and selectable markers will be useful for identification of transformed cells.
Cells that survive exposure to the selective agent, or cells that have been scored positive in a screening assay, may be cultured in regeneration media and allowed to mature into plants. Developing plantlets can be transferred to soil less plant growth mix, and hardened off, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO2, and 25-250 microeinsteins m−2 s−1 of light, prior to transfer to a greenhouse or growth chamber for maturation. Plants are preferably matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 weeks to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown to plants on solid media at about 19 to 28° C. After regenerating plants have reached the stage of shoot and root development, they may be transferred to a greenhouse for further growth and testing. Plants may be pollinated using conventional plant breeding methods known to those of skill in the art and seed produced.
Progeny may be recovered from transformed plants and tested for expression of the exogenous recombinant polynucleotide. Useful assays include, for example, “molecular biological” assays, such as Southern and Northern blotting and PCR; “biochemical” assays, such as detecting the presence of RNA, e.g. double stranded RNA, or a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
One of skill in the art will be able to select an appropriate vector for introducing the encoding nucleic acid sequence in a relatively intact state. Thus, any vector which will produce a host cell, e.g., plant protoplast, carrying the introduced encoding nucleic acid should be sufficient. The selection of the vector, or whether to use a vector, is typically guided by the method of transformation selected.
Plant Regeneration
Following transformation, plant cells transformed with a plant expression vector may be regenerated, e.g., from single cells, callus tissue or leaf discs according to standard plant tissue culture techniques. Almost any plant can be entirely regenerated from cells, tissues, and organs of the plant using methods that are known in the art.
The transformed plants may then be grown, and either pollinated with the same transformed strain or different strains, and the resulting hybrid having expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure expression of the desired phenotypic characteristic has been achieved.
Normally, a plant cell is regenerated to obtain a whole plant from the transformation process. The term “growing” or “regeneration” as used herein means growing a whole plant from a plant cell, a group of plant cells, a plant part (including seeds), or a plant piece (e.g., from a protoplast, callus, or tissue part).
Regeneration from protoplasts varies from species to species of plants, but generally a suspension of protoplasts is first made. In certain species, embryo formation can then be induced from the protoplast suspension. The culture media will generally contain various amino acids and hormones, necessary for growth and regeneration. Examples of hormones utilized include auxins and cytokinins. Efficient regeneration will depend on the medium, on the genotype, and on the history of the culture. If these variables are controlled, regeneration is reproducible. Regeneration also occurs from plant callus, explants, organs or parts.
In vegetatively propagated crops, the mature genetically modified plants are propagated by utilizing cuttings or tissue culture techniques to produce multiple identical plants. Selection of desirable genetically modified plants is made and new varieties are obtained and propagated vegetatively for commercial use.
In seed propagated crops, mature genetically modified plants can be self-crossed to produce a homozygous inbred plant. The resulting inbred plant produces seed containing the genetic mutation. These seeds can be grown to produce plants that would produce the selected phenotype, e.g., increased lateral root growth, uptake of nutrients, overall plant growth and/or vegetative or reproductive yields.
Parts obtained from the regenerated plant, such as flowers, seeds, leaves, branches, fruit, and the like are included in the invention, provided that these parts comprise cells comprising the isolated nucleic acid of the present invention. Progeny and variants, and mutants of the regenerated plants are also included within the scope of the invention, provided that these parts comprise the introduced nucleic acid sequences. Genetically modified plants expressing a selectable marker can be screened for transmission of the nucleic acid of the present invention by, for example, standard immunoblot and DNA detection techniques. Genetically modified plant cells are also typically evaluated on levels of expression of the genetically modified nucleic acid. Expression at the RNA level can be determined initially to identify and quantitate expression-positive plants. Standard techniques for RNA analysis can be employed and include PCR amplification assays using oligonucleotide primers designed to amplify only the genetically modified RNA templates and solution hybridization assays using genetically modified nucleic acid-specific probes. The RNA-positive plants can then analyzed for protein expression by Western immunoblot analysis using the specifically reactive antibodies of the present invention. In addition, in situ hybridization and immunocytochemistry according to standard protocols can be done using genetically modified nucleic acid specific polynucleotide probes and antibodies, respectively, to localize sites of expression within genetically modified tissue. Generally, a number of genetically modified lines are usually screened for the incorporated nucleic acid to identify and select plants with the most appropriate expression profiles.
In one embodiment, the present invention provides a genetically modified plant that is homozygous for the introduced genetically modified nucleic acid; i.e., a genetically modified plant that contains two added nucleic acid sequences, one gene at the same locus on each chromosome of a chromosome pair. A homozygous genetically modified plant can be obtained by sexually mating (selfing) a heterozygous genetically modified plant that contains a single added genetically modified nucleic acid, germinating some of the seed produced and analyzing the resulting plants produced for altered expression of a polynucleotide of the present invention relative to a control plant (i.e., native, non-genetically modified). Back-crossing to a parental plant and out-crossing with a non-genetically modified plant are also contemplated.
Transformed plant cells which are derived by any of the above transformation techniques can be cultured to regenerate a whole plant which possesses the transformed genotype. Such regeneration techniques often rely on manipulation of certain phytohormones in a tissue culture growth medium.
Products of Genetically Modified Plants and Algae
Engineered plants exhibiting the desired physiological and/or agronomic changes can be used directly in agricultural production.
Thus, provided herein are products derived from the genetically modified plants or methods of producing genetically modified plants provided herein. In certain embodiments, the products are commercial products. Some non-limiting example include genetically engineered trees for e.g., the production of pulp, paper, paper products or lumber; tobacco, e.g., for the production of cigarettes, cigars, or chewing tobacco: crops, e.g., for the production of fruits, vegetables and other food, including grains, e.g., for the production of wheat, bread, flour, rice, corn; and canola, sunflower, e.g., for the production of oils or biofuels.
Biofuels
In one embodiment, biofuels are derived from a genetically engineered plant or algae of the present invention.
In one embodiment, a biofuel is a fuel that is produced through contemporary biological processes, such as agriculture and anaerobic digestion, as opposed to a fuel produced by geological processes such as those involved in the formation of fossil fuels, such as coal and petroleum, from prehistoric biological matter. Biofuels can be derived directly from plants, or indirectly from agricultural wastes.
In one embodiment, plants or plant parts as described herein are used as biofuel.
In one embodiment, algae are used as a biofuel. In one embodiment, the biofuel is selected from the group consisting of: biodiesel, ethanol, biojet fuel, and green gasoline.
In one embodiment, the biofuel is an alcohol fuel, such as bioethanol. In one embodiment, the bioethanol is produced by fermentation of sugars derived from wheat, corn, sugar beets, sugar cane, molasses, or a combination thereof. In another embodiment, the bioethanol is produced by fermentation of any sugar or starch from which alcoholic beverages such as whiskey, can be made (such as barley, potato and fruit waste, etc).
Thus, in one embodiment, the present invention provides a biofuel comprising genetically modified plants or plant parts of the present invention. In one embodiment, the present invention provides a process of producing a biofuel comprising: delivering a recombinant polynucleotide encoding a C-terminal-inactivated ACHT4 to plant or algae cells, regenerating plants or algae from said cells, screening said plants or algae to identify a plant having enhanced yield, extracting sugar or starch from some or all portions of said plant or algae or progeny thereof, fermenting said sugars to produce an alcoholic mixture, and distilling said alcohol from said mixture. Methods for producing biofuels from plants and plant parts are known in the art.
In one embodiment, algae are a sustainable source for essential omega-3 fatty acids.
In another embodiment, algae of the present invention are used as commodity animal feeds. In another embodiment, algae of the present invention are used as a source for foods. In one embodiment, essential omega-3 fatty acids from algae are used in infant formula and other food products and vitamins. In another embodiment, carbohydrates and emulsifiers produced from seaweeds are used in food products. In another embodiment, Spirulina is used in food products.
In another embodiment, algae of the present invention are used as a source for specialty feeds.
In one embodiment, algae contain carbohydrates, proteins, vegetable oils, micronutrients, vitamins, as well as valuable pigments used in animal feeds, such as beta carotene, lutein and astaxanthin. In another embodiment, algae is used as a source of feed in aquaculture operations, including as feed for fish and shellfish like clams, oysters, mussels and scallops.
In another embodiment, algae of the present invention are used as a source for chemicals. In one embodiment, the chemical is a plastic. In one embodiment, the chemical is a fertilizer. In one embodiment, the alga is seaweed. In one embodiment, some microalgae fix atmospheric nitrogen and could be a source of organic fertilizers (“green manure”).
In another embodiment, algae of the present invention are used as a source for cosmetics. In one embodiment, the cosmetic is a skin-care product. In one embodiment, the cosmetic provide UV protection. In another embodiment, algae of the present invention are used as a source for pharmaceuticals.
In one embodiment, multiple products from the same algal biomass are possible.
In certain embodiments, commercial products are derived from a genetically engineered species of woody, ornamental or decorative, crop or cereal, fruit or vegetable plant, and algae (e.g., Chlamydomonas reinhardii), which may be used in the compositions and methods provided herein. Non-limiting examples of plants include plants from the genus Arabidopsis or the genus Oryza. Other examples include plants from the genuses Acorus, Aegilops, Allium, Amborella, Antirrhinum, Apium, Arachis, Beta, Betula, Brassica, Capsicum, Ceratopteris, Citrus, Cryptomeria, Cycas, Descurainia, Eschscholzia, Eucalyptus, Glycine, Gossypium, Hedyotis, Helianthus, Hordeum, Ipomoea, Lactuca, Linum, Liriodendron, Lotus, Lupinus, Lycopersicon, Medicago, Mesembryanthemum, Nicotiana, Nuphar, Pennisetum, Persea, Phaseolus, Physcomitrella, Picea, Pinus, Poncirus, Populus, Prumns, Robinia, Rosa, Saccharum, Schedonorus, Secale, Sesamum, Solanum, Sorghum, Stevia, Thellungiella, Theobroma, Triphysaria, Triticum, Vitis, Zea, or Zinnia.
In some embodiments, commercial products are derived from a genetically engineered gymnosperms and angiosperms, both monocotyledons and dicotyledons. Examples of monocotyledonous angiosperms include, but are not limited to, asparagus, field and sweet corn, barley, wheat, rice, sorghum, onion, pearl millet, rye and oats and other cereal grains. Examples of dicotyledonous angiosperms include, but are not limited to tomato, tobacco, cotton, rapeseed, field beans, soybeans, peppers, lettuce, peas, alfalfa, clover, cole crops or Brassica oleracea (e.g., cabbage, broccoli, cauliflower, brussel sprouts), radish, carrot, beets, eggplant, spinach, cucumber, squash, melons, cantaloupe, sunflowers and various ornamentals.
In certain embodiments, commercial products are derived from a genetically engineered woody species, such as poplar, pine, sequoia, cedar, oak, etc.
In other embodiments, commercial products are derived from a genetically engineered plant including, but are not limited to, wheat, cauliflower, tomato, tobacco, corn, petunia, trees, etc.
In certain embodiments, commercial products are derived from genetically engineered crop plants, for example, cereals and pulses, maize, wheat, potatoes, tapioca, rice, sorghum, millet, cassava, barley, pea, and other root, tuber, or seed crops. In one embodiment, commercial products are derived from a genetically engineered cereal crops, including, but are not limited to, any species of grass, or grain plant (e.g., barley, corn, oats, rice, wild rice, rye, wheat, millet, sorghum, triticale, etc.), non-grass plants (e.g., buckwheat flax, legumes or soybeans, etc.). In another embodiment, commercial products are derived from a genetically engineered grain plants that provide seeds of interest, oil-seed plants and leguminous plants. In other embodiments, commercial products are derived from a genetically engineered grain seed, such as corn, wheat, barley, rice, sorghum, rye, etc. In yet other embodiments, commercial products are derived from a genetically engineered oil seed plants, such as cotton, soybean, safflower, sunflower, Brassica, maize, alfalfa, palm, coconut, etc. In certain embodiments, commercial products are derived from a genetically engineered oil-seed rape, sugar beet, maize, sunflower, soybean, or sorghum. In some embodiments, commercial products are derived from genetically engineered leguminous plants, such as beans and peas (e.g., guar, locust bean, fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea, etc.)
In certain embodiments, commercial products are derived from a genetically engineered horticultural plant of the present invention, such as lettuce, endive, and vegetable brassicas including cabbage, broccoli, and cauliflower, and carnations and geraniums; tomato, tobacco, cucurbits, carrot, strawberry, sunflower, tomato, pepper, chrysanthemum, poplar, eucalyptus, and pine.
In still other embodiments, commercial products are derived from a genetically engineered corn (Zea mays), canola (Brassica napus, Brassica rapa ssp.), alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghum vulgare), sunflower (Helianthus annuus), wheat (Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum, Nicotiana benthamiana), potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton (Gossypium hirsutum), sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musa spp.), avocado (Peryea americana), fig (Ficus casica), guava (Psidium guajava), mango (Mangifera indica), olive (Olea europaea), papaya (Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamia integrifolia), almond (Prinus amygdalus), sugar beets (Beta vulgaris), oats, barley, Arabidopsis spp., vegetables, ornamentals, and conifers.
Enhanced Phenotype
In one embodiment, “enhanced phenotype” as used herein refers to a measurable improvement in a crop trait including, but not limited to, yield increase, including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density. Many agronomic traits can affect “yield”, including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits. Other traits that can affect yield include, efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), seed size, composition of seed (starch, oil, protein) and characteristics of seed fill.
In another embodiment, the present invention also provides genetically modified plants that demonstrate enhanced phenotypic properties that may or may not confer an increase in overall plant yield. Such properties include enhanced plant morphology, plant physiology or enhanced components of the mature seed harvested from the genetically modified plant. In another embodiment, the present invention also provides genetically modified plants with enhancements in seed oil, tocopherol, protein and starch components, including increases in the quantity of any of these components, alterations in the ratios of any of these components, or production of new types of these components that do not exist in the seed from control plants. By way of example, increases in total tocopherol content are desirable, as are increases in the relative percentage of alpha-tocopherol produced by plants.
In one embodiment, “increased yield” of a genetically modified plant of the present invention may be evidenced and measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e. seeds, or weight of seeds, per acre), bushels per acre, tonnes per acre, tons per acre, kilo per hectare. Increased yield may result from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved responses to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Polynucleotides of the present invention may also be used to provide plants having improved growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways.
Use for Gene Suppression
In one embodiment, polynucleotides of the present invention include recombinant polynucleotides providing for expression of mRNA encoding a polypeptide. In another embodiment, polynucleotides of the present invention include recombinant polynucleotides providing for expression of mRNA complementary to at least a portion of an mRNA native to the target plant for use in suppression of the ACHT4 gene.
In one embodiment, “gene suppression” is used herein to refer to reduction or suppression of expression of a target protein in a host cell as the result of transcription of a recombinant polynucleotide provided herein, wherein the polynucleotide is oriented with respect to a promoter to provide for production of RNA having a gene silencing effect, such as antisense RNA or interfering RNA (RNAi).
Other Methods of Down-Regulating ACHT4 Protein Expression in Plants and Algae
In another embodiment, the present invention provides an antibody against a polypeptide comprising a C-terminal deleted form of atypical CYS HIS rich thioredoxin 4 (ACHT4).
In one embodiment, down-regulation of ACHT4 protein is partial. In another embodiment, the down-regulation completely eliminates protein activity by decreasing overall steady state levels of the protein associated therewith.
In one embodiment, down-regulation of ACHT4 protein comprises decreasing the levels of ACHT4 protein. In another embodiment, down-regulation of ACHT4 protein comprises decreasing the activity of ACHT4 protein.
In one embodiment, the down-regulation is achieved by antisense RNA. In another embodiment, the down-regulation is achieved by ribozyme technology, which, in one embodiment, works at the RNA translational level and involves making catalytic RNA molecules which bind to and cleave the mRNA of interest. Both of these were found effective in regulating protein levels in plants. In another embodiment, the down-regulation is achieved by co-suppression.
In another embodiment, the down-regulation is achieved using antibodies. In one embodiment, the antibody is a monoclonal antibody. In another embodiment, the down-regulation is achieved using functional fragments of antibodies, which in one embodiment, is a single chain antibody (SCAb).
In one embodiment, the antibody binds to ACHT4ΔC. In another embodiment, the antibody binds to ACHT4.
In another embodiment, the present invention provides methods for using antibodies to ACHT4 and/or ACHT4ΔC as described herein and functional fragments thereof (e.g., Fv or Fab fragments), for increasing plant or algae yield and/or growth comprising administering said antibody to a plant or alga. Methods for producing antibodies and functional fragments of antibodies are known in the art.
In another embodiment, the present invention provides methods for using antisense RNA, ribozymes, etc for increasing plant or algae yield and/or growth comprising transforming said plant or alga with said RNA or ribozyme.
Combinations of Modified Genetic Traits
The present invention also encompasses genetically modified plants with stacked engineered traits, e.g. a crop having an enhanced phenotype resulting from expression of a recombinant polynucleotide provided herein, in combination with herbicide and/or pest resistance traits. For example, genes of the current invention can be stacked with other traits of agronomic interest, such as a trait providing herbicide resistance, for example a RoundUp Ready trait, or insect resistance, such as using a gene from Bacillus thuringensis to provide resistance against lepidopteran, coliopteran, homopteran, hemiopteran, and other insects. Herbicides for which resistance is useful in a plant include glyphosate herbicides, phosphinothricin herbicides, oxynil herbicides, imidazolinone herbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylurea herbicides, bialaphos herbicides, sulfonamide herbicides and gluphosinate herbicides.
All patents, patent applications, and scientific publications cited herein are hereby incorporated by reference in their entirety.
The following examples are presented in order to more fully illustrate the preferred embodiments of the invention. They should in no way be construed, however, as limiting the broad scope of the invention.
Materials and Methods
Plant Material and Growth Conditions
Arabidopsis thaliana var Columbia were grown under a 8/16 h light/dark cycle at 20° C./18° C., respectively, at 80 μE*m−2*s−1 (unless otherwise stated) for 3-4 weeks. Thylakoid membranes were isolated as previously described.
Generation of Genetically Modified Plants
ACHT4, ACHT4MT (in which the non-nucleophilic cysteine of the active site was replaced with a serine), ACHT4ΔC and ACHT1 open reading frames were ligated upstream and in frame of the HA3 affinity tag and under the control of the 35S promotor into pART7 vector. All four constructs were used to transform Arabidopsis leaves using a standard floral dip transformation protocol (Clough and Bent Plant J. 1998 December; 16(6):735-43, incorporated herein by reference in its entirety). In short, the floral dip transformation method involves simple dipping of developing floral tissues into a solution containing Agrobacterium tumefaciens, 5% sucrose and 500 microliters per litre of surfactant Silwet L-77.
Protein Redox Assays, Immunoblot and Affinity Purification Analyses
The disulfide state of plant-extracted proteins, the identification of intermolecular disulfide complexes, and their isolation by immunoprecipitation were assayed in planta as previously described. The mass-spectrometry (MS) analysis is detailed in Table 3.
The Mass-spectrometry analysis was performed by the Biological Mass Spectrometry Unit at Weizmann Institute of Science by online reversed-phase nano-liquid chromatography, electrospray ionization tandem mass spectrometric analyses. Survey scans were recorded in the FT-mode followed by data-dependent collision-induced dissociation of the 7 most intense ions in the linear ion trap. Raw spectra were processed using open-source software DTA SuperCharge. The data were searched with MASCOT (Matrix Science, London, UK) against a Swissprot or NCBI database. Control samples treated with DTT or derived from wild-type plants, allowed for the subtraction of nonspecific background proteins.
Trapped intermolecular disulfide protein complexes were incubated overnight at 4° C. in RIPA buffer (1% sodium deoxycholate, 0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl, pH 8, and 140 mM NaCl) with either anti-HA (Sigma A2095) resin or anti-2-Cys Prx- or anti-APS1-coated protein G beads (Amersham). The proteins were eluted with either reducing or nonreducing 2× sample buffer and separated on SDS-PAGE gels for immunoblots or for MS analysis. Anti-APS1 polyclonal antibodies were raised in rabbits at GenScript HK Ltd., using a purified peptide (CILGLDDQRAKEMPF (SEQ ID NO: 111)). 2-Cys Prx-specific polyclonal antibodies were as in Dangoor, 2009 Plant Physiol. 149:1240-1250, which is incorporated herein by reference in its entirety. Mouse monoclonal anti-HA antibodies (SIGMA H9658) were used in protein blot assays.
Starch Analysis
Starch content of 0.8 g samples of two month-old rosettes was analyzed using the SIGMA starch assay kit (SA20-1KT). Every replicate included ten rosettes.
Accession Numbers
Sequence data can be found in the Arabidopsis Genome Initiative or GenBank/EMBL databases under the following accession numbers: ACHT4 (AT1G08570), 2-Cys PrxA (At3g11630), and APS1 (AT5G48300).
Results
Reoxidation of ACHT4 by 2-Cys Prx Shortly after Illumination
In order to examine whether other thylakoid-associated members of the ACHT family have similar or unique roles to that of ACHT1, we analyzed the redox state changes of the ACHT4 catalytic site following the onset of growth light (80 to 100 μE*m−2*s−1) after a typical 16 h night period in plants expressing ACHT4 (Dangoor, 2009). The catalytic site of ACHT4 was found to be mostly disulfide-bonded at the end of the night and to undergo rapid reduction within 1 min of exposure to the light (
To investigate the identity of the proteins that ACHT4 exchanges disulfides with in planta, we captured as in (Dangoor, 2012, The Plant Cell 24(5):1894-1906, which is incorporated herein by reference in its entirety) its intermolecular disulfide reaction intermediates. Protein blot analysis of denatured, but not reduced, plant extracts identified three intermolecular disulfide-linked ACHT4-containing protein complexes, verified by their susceptibility to chemical reduction by dithiothreitol (DTT) (
To verify the authenticity of ACHT4-2-Cys Prx intermolecular disulfide complexes, they were pulled down in a reciprocal analysis performed under non-reducing denaturing conditions, with either anti-HA (for ACHT4) or anti-2-Cys Prx sera. Protein blot analysis of denatured and reduced samples identified the 2-Cys Prx in the anti-HA pulled down complexes and ACHT4 in the anti-2-Cys Prx pulled down complexes (
APS1 is a Unique Target of ACHT4
The comparison of the intermolecular disulfide complexes formed in planta by ACHT4 and ACHT1 uncovered a major disulfide linked complex unique to ACHT4 (
ACHT4 Participates in the Diurnal Redox Regulation of AGPase
The trapping in vivo of APS1-ACHT4 and 2-Cys Prx-ACHT4 disulfide exchange reaction intermediates (RIs) opened the possibility of studying the environmental stimuli that influence ACHT4-driven AGPase redox control. First, we analyzed the changes in the 2-Cys Prx-ACHT4 and APS1-ACHT4 RIs, and the corresponding changes in the APS1 redox state upon light onset of plants grown under a 8/16 h light/dark regime. Intriguingly, the low APS1-ACHT4 RI level and the high 2-Cys Prx-ACHT4 level in the dark contrasted each other (
The analysis of the concomitant changes in the redox state of APS1 showed that APS1 was resting in the inactive intermolecular disulfide form in the dark (
Both 2-Cys Prx ACHT4 and APS1 RI levels and APS1 redox state after the light was switched off, i.e. the period in which oxidation is expected to turn off AGPase, were inverse to those observed after the onset of light. The 2-Cys Prx-ACHT4 RI remained stable during the first 5 min and then increased, suggesting increased oxidation by 2-Cys Prx during that time period (
ACHT4 Participates in the Regulation of APS1 During Fluctuations in Light Intensity
We found that ACHT4 participated in the diurnal regulation of APS1, that has been proposed to influence the day and night cycles of starch synthesis and degradation. The reactions of ACHT4 with APS1 and 2-Cys Prx, as judged by the levels of their disulfide exchange reaction intermediates, seemed to reach balanced levels after a transition period in the light (
The C-Terminus of ACHT4 is Important for its Reaction with APS1
We then assessed whether the distinct 47-amino acid-long ACHT4 C-terminus (
Both ACHT4 and ACHT1 are thylakoid-associated proteins. The distinct reaction of ACHT4, and not of ACHT1, with APS1, prompted us to investigate whether the disparity might be due to different thylakoid localization, either the grana, the grana margins, or the stroma lamella. Protein blot analysis showed that ACHT1 was primarily found in both the grana and the stroma lamella domains, whereas ACHT4 was mainly present in the stroma lamella domain and was undetectable in the grana (
The finding that the deletion of the ACHT4 C-terminus diminished its disulfide exchange reaction with APS1 (
Expression of AtACHT4ΔC Also Increases Arabidopsis Biomass by 10%
Stimulation of starch synthesis results not only in increased accumulation of transitory starch in Arabidopsis leaves, it also stimulates growth, indicating that OE of AtACHT4ΔC stimulates the export of photosynthates from the chloroplast which are then directed toward growth and biomass accumulation.
AtACHT4ΔC-OE, AtACHT4-OE and WT lines were grown under long day (18 h/6 h of light/dark cycle at 21° C./18° C.) for 4 weeks. The plants shoot was excised and fresh weight (FW) were recorded. The tissues were dried at 60° C. for 4 days and dry weight (DW) were recorded. The FW of AtACHT4ΔC-OE plants was increased by 10.6% (
Potato Plants
Arabidopsis has one paralog of ACHT4 where other crop plants, including potato, maize, rice, barley, wheat, sorghum, castor, bean, rapeseed, cotton, soybean, beat, banana, chili, chickpea, tomato, African oilpalm, Foxtail millet, cassava and the algae Chlamydomonas and Chlorella have one to five paralogs (
Materials and Methods
Identification and Over Expression of AtACHT Homologs in Potato
We analyzed the potato genome for AtACHT4 homologs. Protein blast (blastP) analysis of AtACHT against the genome database of potato (solgenomics.net) identified two paralogs, StACHT4-1 (XP_006348023.1) and StACHT4-2 (XP_006351368.1). A 69 and 68 amino acid long tail region at the C-terminus of StACHT4-1 and StACHT4-2, correspondingly, were identified. Four constructs of StACHT4-1ΔC and StACHT4-2ΔC, StACHT4-1, and StACHT4-2 were custom synthesized by Hy-Laboratories Ltd., Israel, were subcloned into pART7 vector under control of CaMV35S promoter and OCS terminator. HA (Human influenza hemagglutinin) tag was fused in frame at the C-terminus of each of the protein coding sequences. All four StACHT4::pART7 and StACHT4-2ΔC:: pART7 constructs were digested with NotI and subcloned into pART27 plant expression vector (
Results
The Over Expression of StACHT4-2ΔC in Potato Plants Nearly Doubles the Tubers Yield in Comparison to WT Plants
The StACHT4-2ΔC-OE and WT plants (Solanum tuberosum cv. Desiree) were planted on May 22nd 2016 and were grown for 60 days in the green-house. Plants were harvested, tubers were collected, counted and their fresh weight was recorded (
StACHT4-1ΔC OE Lines Accumulated Increased Level of Transitory Starch in Leaves
To test whether StACHT4-1ΔC OE proteins regulate transitory starch content, young green leaves from 6-weeks old green-house grown plant were collected and analyzed for the starch content by Sigma starch assay kit (Catalog Number SA20) with some modifications. Approximately, 0.25-0.5 g fresh leaves were ground to fine powder using liquid nitrogen and then suspended into 20 ml DMSO and 5 ml of 8M HCl solution. The suspensions were incubated for 30 min at 60° C. and then 50 ml water was added to it. The pH was adjusted between 4 and 5, allowed to cool and volume adjusted till 100 ml. From these samples 400 μl were mixed with same volume of starch assay reagent, incubated for 15 min at 60° C. and then allowed to cool at room temperature. Then 200 μl of starch assay mixture were mixed with same volume of glucose assay reagent, incubated at room temperature for 15 min and then absorbance was recorded at 340 nm. Simultaneously, standard starch powder (provided in kit) at concentration of 0, 2, 4, 6, 8 and 10 mg were also used for starch assay and a standard curve was plotted using the absorbance data. The absorbance recorded from the leaves sample of potato transgenic lines were then used to calculate the starch level in leaves using formula obtained from the standard curve. Notably, the StACHT4-1ΔC OE lines accumulated 19% higher starch content relative to WT plants (
OE of the potato paralogs StACHT4-2ΔC and StACHT4-1ΔC relieves growth and starch synthesis attenuation, as was found for OE of Arabidopsis AtACHT4ΔC. However, in potato, each of the two potato paralogs has a unique function. OE expression of StACHT4-2ΔC stimulated the allocation of photosynthates towards growth and near doubles tuber yield and plants shoot growth (
It will be appreciated by those skilled in the art that changes could be made to the embodiments described above without departing from the broad inventive concept thereof. It is understood, therefore, that this invention is not limited to the particular embodiments disclosed, but it is intended to cover modifications that are within the spirit and scope of the invention, as defined by the appended claims.
This application is a National Phase Application of PCT International Application No. PCT/IL2016/050891, filed on Aug. 16, 2016, claiming priority from U.S. Provisional Patent Application Ser. No. 62/205,768 filed on Aug. 17, 2015, which are all hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/IL2016/050891 | 8/16/2016 | WO | 00 |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2017/029662 | 2/23/2017 | WO | A |
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| 7135616 | Heard et al. | Nov 2006 | B2 |
| 20150067923 | Coruzzi et al. | Mar 2015 | A1 |
| 20150176021 | Vinocur et al. | Jun 2015 | A1 |
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| Number | Date | Country | |
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| 20190119696 A1 | Apr 2019 | US |
| Number | Date | Country | |
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| 62205768 | Aug 2015 | US |
