WO2009077973A1 - Cassettes d'expression et procédés pour augmenter des rendements en plante - Google Patents

Cassettes d'expression et procédés pour augmenter des rendements en plante Download PDF

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WO2009077973A1
WO2009077973A1 PCT/IB2008/055314 IB2008055314W WO2009077973A1 WO 2009077973 A1 WO2009077973 A1 WO 2009077973A1 IB 2008055314 W IB2008055314 W IB 2008055314W WO 2009077973 A1 WO2009077973 A1 WO 2009077973A1
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plant
promoter
mesophyll
pyrophosphatase
terminator
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Inventor
Chengcai Chu
Lizhen Si
Yafang Zhang
Hongning Tong
Yiqin Wang
Xuebiao Pang
Mohanmad Hajirezai
Uwe Sonnewald
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Institute of Genetics and Developmental Biology of CAS
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Institute of Genetics and Developmental Biology of CAS
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/8245—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving modified carbohydrate or sugar alcohol metabolism, e.g. starch biosynthesis

Definitions

  • the present disclosure relates generally to methods of improving plant yields, and particularly, but not by way of limitation, to expression cassettes containing pyrophosphatase genes and mesophyll-specific expression promoters.
  • the present invention relates to new plasmids and methods for the preparation of transgenic plants, as well as the plants, that are modified through the transfer and the expression of genes which influence the sugar metabolism or the sugar partitioning within a plant, and which are localized on these plasmids.
  • a fraction of the primary photoassimilates are converted into starch in chloroplast, and a second fraction is transported into the cytoplasm for sucrose synthesis and other triose- based metabolic reactions.
  • photoassimilates are distributed within a plant in the form of sugars and preferentially in the form of sucrose.
  • the distribution of sucrose between the source and sink tissues occurs by transport of sucrose via the phloem.
  • One of the important determinants for the strength of a sink is the unloading of the phloem in the sink.
  • the rate of plant photosynthesis depends on light intensity, CO 2 concentration, etc., and may also be influenced by self-metabolic factors such as the activity of various CO 2 -SxUIg Calvin cycle enzymes, e.g., ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCo) and chloroplast fructose- 1,6-diphosphate, partitioning of primary photoassimilates, transportation of carbohydrate assimilates, and the utilization of sink organs.
  • ribulose 1,5-bisphosphate carboxylase/oxygenase RosCo
  • chloroplast fructose- 1,6-diphosphate partitioning of primary photoassimilates
  • transportation of carbohydrate assimilates and the utilization of sink organs.
  • TPT triose phosphate translocator
  • the rate-limiting enzymes for cytoplasmic sucrose synthesis are cytoplasmic fructose- 1, 6-bisphosphatase (cyFBPase) and sucrose phosphate synthase (SPS).
  • the former in combination with phosphofructokinase (PFK) and fructose-6- phosphate-1 -phosphotransferase (PFPase), regulates the conversion of fructose- 1 ,6-bisphosphate (FBP) to fructose 6-phosphate (F6P); the latter catalyzes the final step of sucrose synthesis. Altering the activities of these enzymes, or the balance of their reactions, allows the equilibrium to shift towards increasing sucrose synthesis.
  • PFPase catalyzes the dephosphorization of FBP forming F6P and inorganic pyrophosphate (PPi).
  • PPi is also released by the conversion of glucose- 1 -phosphate (GlP) to UDP-glucose in sucrose synthetic reactions.
  • GlP glucose- 1 -phosphate
  • PPase hydro lyzes one PPi molecule into two Pi molecules.
  • exogenous PPase will shift the equilibrium position of the reactions in the direction of sucrose synthesis.
  • exogenous PPase will provide adequate exchanger-Pi for delivering chloroplastic triose phosphate into the cytoplasm.
  • an E.coli PPase gene has been cloned into an expression cassette downstream of a 35 S promoter, and used to transform potato and tobacco plants. Transcriptional products of this PPase gene are found by Northern blots in both transgenic plants. In leaves of both transgenic plants the ratio of soluble sugars to starch is increased 3-4-fold over wild type. Transgenic tobacco plants show much higher levels of glucose (up to 68-fold), fructose (up to 24-fold), sucrose (up to 12-fold), and starch (up to 8-fold) than non-transgenic plants.
  • Transgenic potato shows a change in assimilate partitioning due to a 2-fold increase in sucrose and a reduction in starch content (Sonnewald U. (1992) Expression of E. coli inorganic pyrophosphatase in transgenic plants alters photoassimilate partitioning, Plant J 2:571-581).
  • prior art transgenic plants exhibit stunted growth, shortened internode distances, and decreased PPi content. Moreover, the soluble sugar content of old source leaves of prior art transgenic plants is 100-fold higher than that of wild type leaves. It is speculated that PPase expression controlled by a constitutive 35 S promoter stunts the growth of transgenic plants. PPase expression in vascular bundles of transgenic plants would lead to decreased PPi content, which in turn blocks the loading of sucrose in phloem as PPi is necessary for long distance transporation of sucrose, thus a large amount of sucroses accumulate in the leaves (Sonnewald U.
  • the inventive expression cassette comprises a mesophyll-specif ⁇ c expression promoter, a pyrophosphatase gene, and, a terminator.
  • the promoter, pyrophosphatase gene, and terminator may be operably-linked.
  • the promoter, pyrophosphatase gene, and terminator are arranged in seriatem.
  • the inventive expression cassette contains a DNA sequence which functions as a mesophyll-specif ⁇ c promoter.
  • the promoter may be a cyFBPase promoter.
  • the promoter comprises the nucleotide bases numbered from 37,511,734 to 37,512,949 from the 5 '-end of the DNA sequence having Genebank Number NC_008394.1.
  • the inventive expression cassette contains a DNA sequence which functions as a terminator.
  • the terminator is an OCS terminator.
  • the terminator comprises the nucleotide bases numbered from 1403 to 1629 from the 5'- end of the DNA sequence having Genebank Number V00088.
  • the inventive expression cassette contains a DNA sequence which encodes a pyrophosphatase enzyme.
  • the pyrophosphatase gene comprises nucleotide bases 1 to 531 from the 5 '-end of the DNA sequence having Genebank Number ABE 10235.
  • the inventive expression cassette may contain a electable marker gene.
  • a mesophyll-specif ⁇ c, pyrophosphatase expression vector, pect The inventive expression vector comprises a plasmid having inserted therein a mesophyll- specif ⁇ c, pyrophosphatase expression cassette.
  • the mesophyll-specif ⁇ c recombinant expression vector is a binary expression vector.
  • the plasmid is pCAMBIA 1300.
  • the mesophyll-specif ⁇ c recombinant expression vector further comprises a selectable marker gene.
  • a transgenic cell comprising a mesophyll-specific, pyrophosphatase expression cassette.
  • the cell may be a bacterium or a plant.
  • the transgenic cell is a plant cell.
  • the transgenic plant cell is a monocotyledon.
  • the transgenic plant cell is a Gramineae.
  • the method includes transforming a plant with a mesophyll-specific, pyrophosphatase expression vector.
  • the vector comprises an expression cassette which comprises a mesophyll-specific expression promoter; a pyrophosphatase gene; and, a terminator.
  • the expression cassette is arranged such that the promoter, pyrophosphatase gene, and terminator are operably-linked.
  • the promoter is a cyFBPase promoter comprising bases 37,511,734 to 37,512,949 from the 5 '-end of Genebank Number NC 008394.1.
  • the terminator is an OCS terminator comprising bases 1403 to 1629 from the 5 '-end of Genebank Number V00088.
  • the pyrophosphatase gene comprises bases 1 to 531 from the 5 '-end of Genebank Number ABE 10235.
  • the method includes a mesophyll-specific, pyrophosphatase expression vector.
  • the inventive expression vector comprises a plasmid having inserted therein a mesophyll-specific, pyrophosphatase expression cassette.
  • the mesophyll-specific recombinant expression vector is a binary expression vector.
  • the plasmid is pCAMBIA 1300.
  • the mesophyll-specific recombinant expression vector further comprises a selectable marker gene.
  • the method for improving plant yield further includes screening for plants having an enhanced level of pyrophosphatase.
  • the method includes screening for enhanced sucrose filling (sink) activity.
  • the method further includes screening for enhanced (number, size, or weight) sink organs.
  • the transformed plant includes a mesophyll-specific, pyrophosphatase expression vector.
  • the vector comprises an expression cassette which comprises a mesophyll-specific expression promoter; a pyrophosphatase gene; and, a terminator.
  • the expression cassette is arranged such that the promoter, pyrophosphatase gene, and terminator are operably-linked.
  • the promoter is a cyFBPase promoter comprising bases 37,511,734 to 37,512,949 from the 5 '-end of Genebank Number NC 008394.1.
  • the terminator is an OCS terminator comprising bases 1403 to 1629 from the 5'-end of Genebank Number V00088.
  • the pyrophosphatase gene comprises bases 1 to 531 from the 5 '-end of Genebank Number ABE 10235.
  • the plant is a monocot.
  • the plant is a Gramineae.
  • the plant is a crop plant.
  • the plant may be rice, wheat, barley or corn.
  • Figure 1 Proposed scheme for carbon partitioning in source leaves and the ectopic expression of PPase in transgenic rice, (a) Proposed scheme for the effects of inorganic pyrophosphatase (PPase) on carbon partitioning in source leaves.
  • PPase inorganic pyrophosphatase
  • Triose-P (triose-phosphate), Fru-1,6-P2 (fructose- 1,6-bisphosphate), Fru-6-P (fructose-6-phosphate), PPi (inorganic pyrophosphate), Pi (inorganic phosphate), Glu-6-P (glucose-6-phosphate), GIu-I -P (glucose- 1- phosphate), UDP-GIu (UDP-glucose), Suc-6-P (sucrose-6-phosphate), FBPase (fructose- 1,6- bisphosphatase), PFK (6-phosphofructokinase), PFP (fructose-6-phosphate- 1- phosphotransferase), UGPase (UDP-glucose pyrophosphorylase), SPS (sucrose phosphate synthase), SPP (sucrose phosphate phosphatase); (b) PPase activities in
  • FIG. 2 is a schematic diagram of plasmid FBP: PPase
  • Figure 3 shows PCR results of partial FBP: PPase-transgenic rice lines with positive resistance screening results
  • Figure 4 shows the test results of Western (a), PPase activity (b), and PPi content (c) for FBP: PPase-transgenic rice;
  • Figure 5 shows the result of sucrose and starch contents, and isotopic tracing in leaves of FBP: PPase-transgenic rice capable of expressing PPase.
  • Figure 6 shows diurnal variation of sucrose and starch contents in FBP: PPase- transgenic rice capable of expressing PPase;
  • Figure 7 shows metabolite analysis of a FBP: PPase-transgenic rice
  • Figure 8 shows photosynthesis variations of a FBP: PPase-transgenic rice capable of expressing PPase;
  • Figure 9 shows increased biomass was found in PPase-expressing plants, (a-b), the photos were taken 60 days after seeding, (c) The photo was taken after harvesting plants.
  • WT wild type
  • 29, 34 and 37 three independent transgenic lines.
  • genes include coding sequences and/or the regulatory sequences required for their expression.
  • gene refers to a nucleic acid fragment that expresses mRNA or functional RNA, or encodes a specific protein, and which includes regulatory sequences. Genes also include nonexpressed DNA segments that, for example, form recognition sequences for other proteins. A gene may also comprise other 5' and 3' untranslated sequences and termination sequences. Further elements that may be present are, for example, introns. Genes can be obtained from a variety of sources, including by cloning from a source of interest or by synthesis using a known or predicted sequence, and may include sequences designed to have desired parameters.
  • Associated With/Operatively Linked refers to two DNA sequences that are related physically or functionally.
  • a promoter or regulatory DNA sequence is said to be "associated with" a DNA sequence that codes for an RNA or a protein if the two sequences are operatively linked, or situated such that the regulator DNA sequence will affect the expression level of the coding or structural DNA sequence.
  • Coding Sequence a nucleic acid sequence that is transcribed into RNA such as mRNA, rRNA, tRNA, snRNA, sense RNA or antisense RNA.
  • RNA is then translated in an organism to produce a protein.
  • Expression refers to the transcription and/or translation of an endogenous gene or a transgene in plants.
  • expression may refer to the transcription of the antisense DNA only.
  • Expression Cassette A nucleic acid sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence.
  • the expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • the expression cassette is heterologous with respect to the host, i.e., the particular nucleic acid sequence of the expression cassette does not occur naturally in the host cell and must have been introduced into the host cell or an ancestor of the host cell by a transformation event.
  • the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus.
  • the promoter can also be specific to a particular tissue, or organ, or stage of development.
  • heterologous DNA sequence refers to a sequence that originates from a source foreign to the particular host cell or, if from the same source, is modified from its original form.
  • a heterologous gene in a host cell includes a gene that is endogenous to the particular host cell but has been modified through, for example, the use of DNA shuffling.
  • the terms also includes non-naturally occurring multiple copies of a naturally occurring DNA sequence.
  • the terms refer to a DNA segment that is foreign or heterologous to the cell, or homologous to the cell but in a position within the host cell nucleic acid in which the element is not ordinarily found. Exogenous DNA segments are expressed to yield exogenous polypeptides.
  • an isolated nucleic acid molecule or an isolated enzyme is a nucleic acid molecule or enzyme that, by the hand of man, exists apart from its native environment and is therefore not a product of nature.
  • An isolated nucleic acid molecule or enzyme may exist in a purified form or may exist in a non-native environment such as, for example, a recombinant host cell.
  • Plant Any whole plant.
  • Plant Cell Structural and physiological unit of a plant, comprising a protoplast and a cell wall.
  • the plant cell may be in form of an isolated single cell or a cultured cell, or as a part of higher organized unit such as, for example, a plant tissue, a plant organ, or a whole plant.
  • Plant Cell Culture Cultures of plant units such as, for example, protoplasts, cell culture cells, cells in plant tissues, pollen, pollen tubes, ovules, embryo sacs, zygotes and embryos at various stages of development.
  • Plant Material Refers to leaves, stems, roots, flowers or flower parts, fruits, pollen, egg cells, zygotes, seeds, cuttings, cell or tissue cultures, or any other part or product of a plant.
  • Plant Organ A distinct and visibly structured and differentiated part of a plant such as a root, stem, leaf, flower bud, or embryo.
  • Plant tissue A group of plant cells organized into a structural and functional unit. Any tissue of a plant in planta or in culture is included. This term includes, but is not limited to, whole plants, plant organs, plant seeds, tissue culture and any groups of plant cells organized into structural and/or functional units. The use of this term in conjunction with, or in the absence of, any specific type of plant tissue as listed above or otherwise embraced by this definition is not intended to be exclusive of any other type of plant tissue.
  • Promoter refers to a nucleotide sequence, usually upstream (5') to its coding sequence, which controls the expression of the coding sequence by providing the recognition for RNA polymerase and other factors required for proper transcription.
  • Promoter includes a minimal promoter that is a short DNA sequence comprised of a TATA box and other sequences that serve to specify the site of transcription initiation, to which regulatory elements are added for control of expression.
  • Promoter also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements that is capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
  • an "enhancer” is a DNA sequence which can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is capable of functioning even when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind sequence-specific DNA-binding proteins that mediate their effects. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors which control the effectiveness of transcription initiation in response to physiological or developmental conditions.
  • the "initiation site” is the position surrounding the first nucleotide that is part of the transcribed sequence, which is also defined as position +1. With respect to this site all other sequences of the gene and its controlling regions are numbered. Downstream sequences (i.e., further protein encoding sequences in the 3' direction) are denominated positive, while upstream sequences (mostly of the controlling regions in the 5' direction) are denominated negative.
  • Promoter elements particularly a TATA element, that are inactive or that have greatly reduced promoter activity in the absence of upstream activation are referred to as "minimal or core promoters.”
  • minimal or core promoters In the presence of a suitable transcription factor, the minimal promoter functions to permit transcription.
  • a “minimal or core promoter” thus consists only of all basal elements needed for transcription initiation, e.g., a TATA box and/or an initiator.
  • Minimal Promoter a promoter element, particularly a TATA element, that is inactive or has greatly reduced promoter activity in the absence of upstream activation. In the presence of a suitable transcription factor, a minimal promoter functions to permit transcription.
  • Expression cassette means a DNA sequence capable of directing expression of a particular nucleotide sequence in an appropriate host cell, comprising a promoter operably linked to the nucleotide sequence of interest which is operably linked to termination signals. It also typically comprises sequences required for proper translation of the nucleotide sequence.
  • the coding region usually codes for a protein of interest but may also code for a functional RNA of interest, for example antisense RNA or a nontranslated RNA, in the sense or antisense direction.
  • the expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.
  • the expression cassette may also be one which is naturally occurring but has been obtained in a recombinant form useful for heterologous expression.
  • the expression of the nucleotide sequence in the expression cassette may be under the control of a constitutive promoter or of an inducible promoter which initiates transcription only when the host cell is exposed to some particular external stimulus.
  • the promoter can also be specific to a particular tissue or organ or stage of development.
  • Vector is defined to include, inter alia, any plasmid, cosmid, phage or Agrobacterium binary vector in double or single stranded linear or circular form which may or may not be self transmissible or mobilizable, and which can transform a prokaryotic or eukaryotic host either by integration into the cellular genome or exist extrachromosomally (e.g. autonomous replicating plasmid with an origin of replication).
  • a plant transformation vector comprises one or more DNA vectors for achieving plant transformation.
  • DNA vectors for achieving plant transformation.
  • Binary vectors as well as vectors with helper plasmids are most often used for Agrobacterium-mQdiatQd transformation, where the size and complexity of DNA segments needed to achieve efficient transformation is quite large, and it is advantageous to separate functions onto separate DNA molecules.
  • Binary vectors typically contain a plasmid vector that contains the cis-acting sequences required for T-DNA transfer (such as left border and right border), a selectable marker that is engineered to be capable of expression in a plant cell, and a polynucleotide of interest (i.e., a polynucleotide engineered to be capable of expression in a plant cell for which generation of transgenic plants is desired).
  • this plasmid vector also present on this plasmid vector are sequences required for bacterial replication.
  • the cis-acting sequences are arranged in a fashion to allow efficient transfer into plant cells and expression therein.
  • the selectable marker sequence and the sequence of interest are located between the left and right borders.
  • a second plasmid vector contains the trans-acting factors that mediate T-DNA transfer from Agrobacterium to plant cells.
  • This plasmid often contains the virulence functions (Vir genes) that allow infection of plant cells by Agrobacterium, and transfer of DNA by cleavage at border sequences and vir-mediated DNA transfer, as in understood in the art (Hellens et al., 2000).
  • the second plasmid vector is not necessary for introduction of polynucleotides into plants by other methods such as microprojection, microinjection, electroporation, polyethylene glycol, etc.
  • shuttle vectors by which is meant a DNA vehicle capable, naturally or by design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria and eukaryotic (e.g. higher plant, mammalian, yeast or fungal cells).
  • the nucleic acid in the vector is under the control of, and operably linked to, an appropriate promoter or other regulatory elements for transcription in a host cell such as a microbial, e.g. bacterial, or plant cell.
  • a host cell such as a microbial, e.g. bacterial, or plant cell.
  • the vector may be a bi-functional expression vector which functions in multiple hosts. In the case of genomic DNA, this may contain its own promoter or other regulatory elements and in the case of cDNA this may be under the control of an appropriate promoter or other regulatory elements for expression in the host cell.
  • Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites at which foreign DNA sequences can be inserted in a determinable fashion without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in the identification and selection of cells transformed with the cloning vector.
  • Marker genes typically include genes that provide tetracycline resistance, hygromycin resistance or ampicillin resistance.
  • Constitutive expression refers to expression using a constitutive or regulated promoter.
  • Constant and regulated expression refer to expression controlled by a regulated promoter.
  • Constitutive promoter refers to a promoter that is able to express the open reading frame (ORF) that it controls in all or nearly all of the plant tissues during all or nearly all developmental stages of the plant. Each of the transcription-activating elements do not exhibit an absolute tissue-specificity, but mediate transcriptional activation in most plant parts at a level of .gtoreq.1% of the level reached in the part of the plant in which transcription is most active.
  • Regulated promoter refers to promoters that direct gene expression not constitutively, but in a temporally- and/or spatially-regulated manner, and includes both tissue- specific and inducible promoters.
  • Typical regulated promoters useful in plants include but are not limited to safener-inducible promoters, promoters derived from the tetracycline- inducible system, promoters derived from salicylate-inducible systems, promoters derived from alcohol-inducible systems, promoters derived from glucocorticoid-inducible system, promoters derived from pathogen-inducible systems, and promoters derived from ecdysome -inducible systems.
  • Tissue-specific promoter refers to regulated promoters that are not expressed in all plant cells but only in one or more cell types in specific organs (such as leaves or seeds), specific tissues (such as embryo or cotyledon), or specific cell types (such as leaf parenchyma or seed storage cells). These also include promoters that are temporally regulated, such as in early or late embryogenesis, during fruit ripening in developing seeds or fruit, in fully differentiated leaf, or at the onset of senescence.
  • Recombinant DNA molecule a combination of DNA molecules that are joined together using recombinant DNA technology.
  • Regulatory Elements Sequences involved in controlling the expression of a nucleotide sequence. Regulatory elements comprise a promoter operably linked to the nucleotide sequence of interest and termination signals. They also typically encompass sequences required for proper translation of the nucleotide sequence.
  • Selectable marker gene a gene whose expression in a plant cell gives the cell a selective advantage.
  • the selective advantage possessed by the cells transformed with the selectable marker gene may be due to their ability to grow in the presence of a negative selective agent, such as an antibiotic or a herbicide, compared to the growth of non-transformed cells.
  • the selective advantage possessed by the transformed cells, compared to non-transformed cells may also be due to their enhanced or novel capacity to utilize an added compound as a nutrient, growth factor or energy source.
  • Selectable marker gene also refers to a gene or a combination of genes whose expression in a plant cell gives the cell both, a negative and a positive selective advantage.
  • Selectable marker genes are utilized for the selection of transformed cells or tissues.
  • Marker genes include genes encoding antibiotic resistance, such as those encoding neomycin phosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), as well as genes conferring resistance to herbicidal compounds, such as glufosinate ammonium, bromoxynil, imidazolinones, and 2,4-dichlorophenoxyacetate (2,4-D).
  • Additional selectable markers include phenotypic markers such as ⁇ -galactosidase and fluorescent proteins such as green fluorescent protein, cyan florescent protein, and yellow florescent protein.
  • Two nucleic acids are “recombined” when sequences from each of the two nucleic acids are combined in a progeny nucleic acid.
  • Two sequences are “directly” recombined when both of the nucleic acids are substrates for recombination.
  • Two sequences are "indirectly recombined” when the sequences are recombined using an intermediate such as a cross-over oligonucleotide.
  • no more than one of the sequences is an actual substrate for recombination, and in some cases, neither sequence is a substrate for recombination.
  • Transformation a process for introducing heterologous DNA into a host cell or organism.
  • Transformed refers to a host organism such as a bacterium or a plant into which a heterologous nucleic acid molecule has been introduced.
  • the nucleic acid molecule can be stably integrated into the genome of the host or the nucleic acid molecule can also be present as an extrachromosomal molecule. Such an extrachromosomal molecule can be auto-replicating.
  • Transformed cells, tissues, or plants are understood to encompass not only the end product of a transformation process, but also transgenic progeny thereof.
  • non-transformed refers to a wild-type organism, e.g., a bacterium or plant, which does not contain the heterologous nucleic acid molecule.
  • an inorganic pyrophosphatase (PPase) gene is expressed in plant mesophyll cells under the regulation of a mesophyll-specific expression promoter.
  • PPase activity by continuously removing PPi from the cytoplasm shifts the equilibrium of sucrose- based reactions in the direction of sucrose synthesis.
  • increased PPase expression in mesophyll (source) cells alters carbohydrate partitioning towards sink organs, and provides adequate exchanger-Pi for delivering triose phosphate into cytoplasm.
  • mesophyll- specific expression of sucrose prevents the loading of sucrose in phloem from being blocked due to PPi content reduction in phloem, particularly in companion cells, then plant yield is improved.
  • the inventive method has been experimentally-demonstrated to significantly improve yields of transgenic plants, especially of transgenic grain plants. It has been showed by experiment, that rice (Indica variety 6547 and Japonica variety 8706), transformed by expression cassettes of the present invention, express PPase localized to mesophyll cells. Moreover, carbohydrate partitioning in transgenic plants is improved and the yields are increased by up to 50 %.
  • a promoter whose controlled expression is limited to plant mesophyll cells (cyFBPase promoter) and a PPase gene are operably-linked in an expression cassette.
  • Expression constructs, cassettes and vectors, containing an operably-linked cyFBPase promoter and a PPase gene are used to transform plant cells.
  • PPase genes direct the expression of PPase functional enzyme in transformed rice under the regulation of a mesophyll- specific expression promoter. Continuously removing PPi in cytoplasm by expressing PPase induces the synthetic equilibrium in the direction of sucrose synthesis, and provides adequate exchanger-Pi for delivering triose phosphate into cytoplasm. As a result, carbohydrate assimilate partitioning in plants is altered and improved plant yields are obtained.
  • a transgenic rice with improved yield is constructed, and carbohydrate assimilates partitioning and content thereof are examined, various method embodiments are also disclosed.
  • Example 1 Construction of PPase-expressing transgenic rice.
  • Plasmid pCambia 1391Z containing a cyFBPase promoter (Cambia, Canberra, Australia) is digested by restriction enzyme EcoRl to obtain a fragment of 1229 bp, i.e. cyFBPase promoter (nucleotide acids 37511734-37512949 from 5' end of Genebank Number NC-008394.1).
  • the resultant cyFBPase promoter is inserted into the EcoRl site of PCAMBIA 1300 to obtain recombinant vectors.
  • the recombinant vectors are validated by enzyme digestion and sequencing, and the recombinant vector pCAMBIA1300 (Cambia, Australia) containing cyFBPase promoter fragment is designated pC A-FBP.
  • a PPase gene fragment of about 530 DNA bp was obtained by PCR amplification using E.coli ⁇ Escherichia coli XLl -blue strain, Labpil Biotech Ltd., Beijing, CN) genome as a template and a forward, 5' primer, Fl : 5' GGATCCATGAGCTTACTCAACGTCCCTGCGGGT 3' (SEQ ID NO: 1) and a reverse 3' primer Rl : 5'
  • the PPase gene fragment was sequenced and shown to comprise nucleotide acids 1-531 from the 5' end of Genebank Number ABE 10235.
  • the amplified PPase gene fragment was digested with BamHl and inserted between the BamHl sites of pUC19 (Labpil Biotech Ltd., Beijing, CN) to obtain recombinant vectors.
  • the recombinant vectors are validated by enzyme digestion and sequencing, and the recombinant vector containing PPase gene is designated as pUC-PPase.
  • Vector pUC-PPase is digested with BamHl, the PPase gene fragment (containing the sequence of nucleotide acids 1-531 from 5' end of Genebank Number ABE10235) is recovered, and is inserted into BamHl sites of pCA-FBP forward, i.e. behind the cyFBpase promoter of pCA-FBP, to obtain recombinant vectors.
  • the recombinant vectors are validated by enzyme digestion and sequencing, and the pCAMBIA1300 recombinant vector correctly containing cyFBpase promoter and forward PPase gene fragment in series is designated as pCA-FBP-PPase.
  • Terminator OCS (227 bp DNA) was obtained by PCR amplification using Agrobacterium tumefaciens AGLl (American Type Culture Collection) as a template, a (forward) 5' primer F2: 5' CAGGGCTCTCAATGGAGTTTGAA 3' (SEQ ID NO: 3), and a (reverse) 3' primer R2: 5' CAATCAGTAAATTGAAC GGAGA 3' (SEQ ID NO: 4).
  • the OCS terminator fragment was sequenced and shown to comprise base pairs 1403-1629 from 5' end of Genebank Number V00088.
  • the OCS terminator fragment was digested with restriction enzymes Sail and HmdIII and inserted between the Sail site and HmdIII sites of pCA-FBP-PPase to obtain recombinant vectors.
  • the recombinant vectors are validated by enzyme digestion and sequencing, and the recombinant vector correctly containing cyFBpase promoter, forward PPase gene fragment, and terminator OCS, in series, is designated FBP:PPase (schematic diagram as shown in Figure 2).
  • 35S-pro is 35S CaMV promoter
  • FBP-pro is cyFBPase promoter
  • t35 is CaMV 35S terminator
  • ⁇ ygromycin is ⁇ ygromycin-resistance selective gene
  • tOCS is OCS terminator
  • FBP PPase transgenic rice
  • Callus of Japonica rice, variety 8706 (The Institute of Genetics and Developmental Biology of the Chinese Academy of Sciences ) is transformed with vector FBP: PPase vector using Agrobacterium tumefaciens AGLl (purchase from A TCC) yielding a T 0 generation of FBP: PPase-transgenic rice lines with positive resistance screening result. Transformation methods are described in Yi Zi-Li 5 CAO Shou-Yun, et al. (2001). Improvement of Transformation Frequency of Rice Mediated by Agrobacterium, Acta Genetica Sinica 28 (4): 352-358.
  • FBP PPase-transgenic rice
  • PCR amplification is performed using genome DNA purified from FBP: PPase-transgenic rice lines obtained above as a template and primers specific for PPase gene encoding region (upstream primer F3: ATGAGCTTACTCAACGTCCCTGCGGGT, SEQ ID NO: 5) and downstream primer R3: TTATTTATTCTTGGCGCGCTCGAA, SEQ ID NO: 6) as primers and wild-type rice (8706) as a control.
  • upstream primer F3 ATGAGCTTACTCAACGTCCCTGCGGGT, SEQ ID NO: 5
  • downstream primer R3 TTATTTATTCTTGGCGCGCTCGAA, SEQ ID NO: 6
  • wild-type rice 8706
  • FBP PPase-transgenic rice
  • Three FBP PPase-transgenic homozygous lines, lines 27, 34, and 73, are obtained by Hygromycin screening and identification in descendants.
  • Total protein (30 ⁇ g ) from fully-expanded mature leaves of non-transgenic rice (rice 8706, negative control, WT) and FBP: PPase-transgenic rice lines 27, 34, and 73 were heat denatured, electrophoreses on SDS- PAGE gels, transferred to nitrocellulose membranes, and Western blotted using PPase-specific antibody (Preparation method of antibody is to inject a rabbit with E.coli PPase protein expressed by E.coli three times (once every 10 days for a month), and collect serum from the rabit).
  • Inorganic pyrophosphate (PPi) content of FBP PPase transgenic rice.
  • the PPi content of FBP:PPase transgenic rice lines 27, 34, and 37 and of non-transgenic rice (rice 8706, negative control, WT) were determined by the method of Sonnewald, U. (1992) Expression of E. coli inorganic pyrophosphates in transgenic plants alters photo assimilate partitioning, Plant J. 2, 571-581.
  • PPi is extracted and loaded on a 5 ⁇ m Hamilton PRP-XlOO (100x4.1 mm) anion exchange column coupled to a 25 ⁇ 2.3 mm guard (Hamilton Inc., USA) with 1.0 ml/min 60 mM ammonia/ 100 mM formic acid as mobile phase and analyzed on a HPLC lOAvp system (Shimadzu, Japan) and a Corona Charged Aerosol Detector (ESA Inc., USA). Sodium pyrophosphate is used as a standard.
  • the PPi content of each transgenic plant line decreased significantly compared to non-transgenic control (WT) ( Figure 4c).
  • FBP:PPase transgenic rice lines 29, 34, and 37 were compared for carbohydrate content against wild-type, non-transgenic rice (rice 8706) by the method of Geigenberger, P. et al. (1998) Overexpression of pyrophosphatase leads to increased sucrose degradation and starch synthesis, increased activities of enzymes for sucrose-starch interconversions, and increased levels of nucleotides in growing potato tubers. Planta 205, 428-437. Sucrose and starch were extracted from fully-expanded, mature leaf.
  • Figure 5 a shows that sucrose content of each of the three FBP:PPase transgenic rice lines does not significant differ from the wild-type control, but starch contents are 4-5 times lower than that of the non-transgenic control ( Figure 5a).
  • NaH 14 COs was placed in the chamber to serve as a source of radiolabeled carbon. Radiocarbon was released by adding 2-3 ml of 10 % 1 mol/L hydrochloric acid to the petri dish. The chamber was immediately closed and sealed following the addition. A 250 ⁇ Einstein light source was placed 60 cm above the chamber. After a 1-hour incubation, leaf discs were collected, extracted with 80 % (v/v) ethanol at 80 0 C for 1 hour; and the radioactivity of the soluble and insoluble fractions was quantified by liquid scintillation counting.
  • Figure 5b shows a significant reduction in the incorporation of newly fixed 14 C into the insoluble fraction and a significant increase in the incorporation into soluble fraction in all three transgenic lines, compared to the non-transgenic control.
  • WT is the average measurement of 5 individual plants of non-transgenic control
  • 29, 34, and 37 are the average measurement result of 5 individual plants from the three FBP:PPase transgenic rice lines 29, 34, and 37 respectively.
  • Metabolite analysis of FBP:PPase transgenic rice [00100] Metabolite analysis wwere determined for FBP:PPase transgenic rice (Ti generation, lines 29, 34, and 37, 5-7 plants per line) and wild-type, non-transgenic control rice (rice 8706, 5- 7 plants) according to the method of Geigenberger, P. et al (1998). As shown in Figure 7, the fructose-6-phosphate and glycerate-3 -phosphate content of FBP:PPase transgenic rice does not differ significantly from control. Glucose-6-phosphate content increases 83 % in transgenic line 29, 26% and 12% in lines 34 and 37 respectively, compared to control.
  • UDP-glucose contents are much higher in the three transgenic lines (29, 3-fold; 34, 2-fold; and 37, 1.18-fold) than those in non-transgenic control.
  • UDP-glucose is one of the reactants for sucrose synthesis and is found only in cytoplasm, so its significant increase in content indicates that metabolic reaction does move towards the direction of sucrose synthesis.
  • WT is the average measurement of non-transgenic control (5-7 plants)
  • 29, 34, and 37 are the average measurement of 5-7 individual plants from FBP:PPase transgenic rice lines 29, 34 and 37.
  • a range of light intensities between 0 and 2000 ⁇ mol-m ⁇ -sec 1 are supplied by an LED source attached on the leaf chamber.
  • the result shows that, compared to non-transgenic control, the net CO 2 assimilate rate of FBP:PPase transgenic rices capable of expressing PPase is significantly increased.
  • photosynthetic rate of the transgenic plant is 20 % higher than non- transgenic control.
  • FBP:PPase transgenic rice (lines 29, 34, and 37) was chosen for small scale field experiment and a non-transgenic line(WT rice 8706) was chosen as control. Sixty plants from each FBP:PPase transgenic and WT control lines were planted. After seeds completely matured, 10 plants were sampled for each line and plant heights, tiller number per plant, fully- filled seed number per panicle, total seed number per panicle, percentage of fully-filled seeds, 1000-seed weight, and plant yields were determined. These data were used to calculate increases in the yields of transgenic lines compared to non-transgenic controls.
  • Yield increase (%) (transgenic lines - non-transgenic contra l)/non-transgenic control x 100%.
  • the result shows that, growth of either aboveground part or underground part of FBP:PPase transgenic rice is more vigorous than that of WT. Improvements can be seen for tiller number per plant, fully- filled seed number per panicle, total seed number per panicle, and percentage of fully- filled seeds, compared to WT; but plant height, 1000-seed weight, and the like have no significant changes. Individual plant yield increases by 24-50 % compared to the control.
  • FBP:PPase transgenic rice lines 29, 34, and 37
  • WT non-transgenic control
  • the FBP:PPase transgenic rice lines and WT control are grown in the field at a distance of 25cm x 15cm. After seeds completely matured, actual yield measurements are performed for the FBP:PPase transgenic rice lines and WT control (rice 8706), and then theory yields thereof and yield increase compared to control are calculated.
  • Example 2 Creation and yield evaluation of PPase-expressing, transgenic Indica rice variety 6547.
  • FBP:PPase in an Indica rice variety 6547 through the Agrobacterium method and perform field experiment of transgenic plant yield.
  • the FBP: PPase vector is transformed into an Indica rice variety 6547 (Yanzhou University) according to the method of example 1, and two FBP:PPase transgenic rice lines capable of expressing PPase (lines 3003 and 3010) are obtained by screening, which are selected for large-scale field plant experiments in 2004 and 2005 respectively.
  • Each experiment is set up in three locations, each location includes two replicates, and each plot is about 133.3 m 2 .

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Abstract

L'invention porte sur un procédé pour améliorer un rendement en plante et sur sa cassette d'expression spécifique. Ledit procédé pour améliorer un rendement en plante comporte la transformation de la cassette d'expression spécifique du mésophylle de PPase en plantes, et le criblage pour des plantes capables d'exprimer PPase, à savoir des plantes avec un rendement amélioré. Ladite cassette d'expression spécifique du mésophylle de PPase comporte un promoteur cyFBPase, un gène PPase et un terminateur OCS ultérieurement en série. Ledit promoteur cyFBPase comporte les acides nucléiques 37511734-37512949 à partir de l'extrémité 5' de la Genebank Numéro NC-008394.1 ; ledit gène PPase comporte les acides nucléotidiques 1-531 à partir de l'extrémité 5' de la Genebank Numéro ABE10235. Ladite cassette d'expression peut également comporter un terminateur connecté à l'extrémité 3' dudit gène PPase, ledit terminateur OSC comportant les acides nucléotidiques 1403-1629 à partir de l'extrémité 5' de la Genebank Numéro V00088.
PCT/IB2008/055314 2007-12-14 2008-12-15 Cassettes d'expression et procédés pour augmenter des rendements en plante Ceased WO2009077973A1 (fr)

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CN112094936B (zh) * 2020-09-27 2023-12-26 南通大学 6-磷酸果糖转移酶TaPFP基因在小麦育种中的应用
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