WO2012100682A1 - Gène bnaox1 issu de brassica napus, associé au métabolisme respiratoire, et son utilisation - Google Patents

Gène bnaox1 issu de brassica napus, associé au métabolisme respiratoire, et son utilisation Download PDF

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WO2012100682A1
WO2012100682A1 PCT/CN2012/070283 CN2012070283W WO2012100682A1 WO 2012100682 A1 WO2012100682 A1 WO 2012100682A1 CN 2012070283 W CN2012070283 W CN 2012070283W WO 2012100682 A1 WO2012100682 A1 WO 2012100682A1
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plant
protein
promoter
seq
nucleic acid
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PCT/CN2012/070283
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Hanzhong Wang
Wei Hua
Jing Liu
Guihua Liu
Xinfa Wang
Zhiyong Hu
Gaomiao ZHAN
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Oil Crops Research Institute, Chinese Academy Of Agricultural Sciences
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Publication of WO2012100682A1 publication Critical patent/WO2012100682A1/fr

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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
    • C12N15/8247—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 lipid metabolism, e.g. seed oil composition
    • 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
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0006—Oxidoreductases (1.) acting on CH-OH groups as donors (1.1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y101/00—Oxidoreductases acting on the CH-OH group of donors (1.1)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the present invention relates to the field of plant genetic engineering, more particularly to a respiratory metabolism-associated enzyme BnAOXl gene and protein, as well as to the use of the respiratory metabolism-associated enzyme BnAOXl gene and protein to increase the yield and oil content of oil crops.
  • Rapeseed is one of the most important sources for edible vegetable oil and forage protein, as well as the most important raw material for biodiesel in Europe. Rape (Brassica napus) is the oil crop of the largest acreage in China, and is the fifth-largest crop in China following rice, wheat, maize, and soybean. Rapeseed oil is the major edible oil for Chinese residents, accounting for more than 40% of the total domestic vegetable oil consumption. As the biggest country in consuming vegetable oil in the world, however, China has a self-supporting vegetable oil of less than 40%. To meet the conflicting need, in addition to expansion of planting area, increasing rape oil content and rape yield are the most efficient ways to increase rape oil yield per unit planting area. High yield of rape can significantly improve the benefit from rape production and increase the total profit of rapeseed.
  • Cyanide-resistant respiration refers to a respiratory pathway insensitive to cyanide.
  • An alternative oxidase is the terminal oxidase of the cyanide-resistant respiratory pathway in plant mitochondrial respiratory chain, and is extensively found in higher plants and some of fungi and algae. While the alternative oxidase is a terminal oxidase of the alternative pathway, its endogenous substrates include the reduced ubiquinone and O2.
  • the respiratory electron flow of the alternative pathway branches off at ubiquinone in the main path (cytochrome pathway) and bypasses two ATP-formation sites of Complex III and IV.
  • Four electrons from the electron flow are transferred directly by AOX to an oxygen molecule, which is reduced to H2O, while the energy not used for ATP synthesis is lost in the form of heat release.
  • the alternative oxidase is a di-iron carboxylate protein. It has structural features common to other di-iron carboxylate proteins and a function of eliminating molecular oxygen. More importantly, it can also regulate actively the operation degree of cyanide resistant respiratory pathway by alternating the structure itself, thus further regulating various aspects of the cellular metabolism and functionality, in order to adapt to variable environmental conditions, enhance the plants' adaptation to various adverse environments and regulate growth rate in plants.
  • the alternative oxidase is involved in cellular apoptosis and photosynthesis. Regulation of the AOX activity is affected by various factors. Environmental stresses, plant injury, frost, drought, osmotic pressure, pathogenic bacteria, etc. may induce the expression of AOX.
  • Salicylic acid SA
  • hydrogen peroxide hydrogen peroxide
  • ethylene ethylene
  • main respiratory chain inhibitors may also induce the expression of AOX in plants.
  • Cyanide-resistant respiration activity and AOX expression level are higher in the spadix of the thermogenic plant.
  • the AOX expression level is associated with the developmental stages. For example, AOX is expressed during senescence and fruit maturation.
  • AOX regulates energy metabolism. When the energy metabolism is saturated, AOX pathway releases the energy by way of heat elimination and maintains the electron transport and TCA.
  • "The energy overflow hypothesis" put forward by Lambers (1982, Physiology Plant 55:478) states that transferring of electrons through AOX may enable TCA cycle and glycolysis to proceed when the main respiratory chain activity is inhibited or the reduction power within a cell is running high.
  • the AOX activity can be activated by pyruvate, a substrate in TCA cycle, indicating that transfer of part of the electrons through the AOX pathway can be induced when the substrate concentration in TCA cycle is too high.
  • Environmental changes result in differences in the AOX expression, which has effects both on mitochondrial functions and on cellular functions outside the mitochondria.
  • Oxygen stress induces the expression of the AOX to effect anti- oxidation, that is, Oxidation mediated by the AOX can decrease reactive oxygen species (ROS) generated by the main respiratory chain when the cytochrome pathway is inhibited.
  • ROS reactive oxygen species
  • the ROS level is decreased when AOX is over-expressed in a transgenic tobacco plant, while the ROS level is increased when AOX is not over-expressed in the transgenic tobacco plant.
  • Hansen et al (2002, Thermochim Acta 388:415) found that environmental changes can alter the growth rate of a plant, that is, AOX plays a balancing role in growth kinetics of a plant.
  • thermogenic plant Heat released through AOX in the thermogenic plant may enable pollens to emanate fragrance, thus attract insect vectors (Meeuse, (1975) Annu Rev Plant Physiol 26:117). Expression of AOX in non-thermogenic plants is not associated to such a thermogenesis (Borecky and Vercesi (2005) Bioscience Reports 25:271). AOX can effectively control respiration rate and maintain energy homeostasis in cells to ensure normal growth of a plant encountering environmental changes (Hansen et al (2002) Thermochim Acta 388:415).
  • AOX not only allows Arabidopsis thaliana to germinate at low temperatures, but also can prevent the generation of peroxides by plant tissues under adverse conditions, to avoid adverse effect on various cellular functions (Fiorani et al (2005) Plant Physiology 139:1795). AOX plays a key role in the cytoplasm and in some of the carbon metabolism pathways (Umbach et al (2005) Plant Physiol 139:1806). A variety of environmental stresses can alter the proceeding of the cyanide-resistant pathway in plants. However, those studies in this regard are still very superficial, and the signal regulation mechanism and physiological significance of said proceeding is unclear yet.
  • the current invention provides methods and means to improve plant seed yield and seed oil content by increasing the expression of AOX1, as will become apparent from the following description, examples, drawings and claims provided herein.
  • a method for increasing oil content, such as seed oil content, and/or seed yield, such as Thousand Kernel Weight in plants comprising increasing expression of a nucleic acid encoding an AOXl protein.
  • said AOXl protein has at least 60% sequence identity to SEQ ID No. 2.
  • said AOXl protein has at least 80% sequence identity to SEQ ID No. 2.
  • said nucleic acid has at least 80% sequence identity to SEQ ID NO. 1.
  • a method for increasing seed oil content and/or seed yield, such as Thousand Kernel Weight in plants comprising increasing expression of a nucleic acid encoding a protein with has at least 80% sequence identity to the AOX1B protein from Arabidopsis thaliana.
  • said nucleic acid has at least 80% sequence identity to the AOX1B coding sequence from Arabidopsis thaliana.
  • a method for increasing oil content and/or seed yield, such as Thousand Kernel Weight in plants comprising increasing expression of a nucleic acid encoding an AOXl protein comprising the steps of
  • nucleic acid encoding an AOXl protein
  • said plant-expressible promoter is a constitutive promoter, and in yet another embodiment, said constitutive promoter is the 35S promoter. In a further embodiment, said plant-expressible promoter is a seed-specific promoter.
  • plants are provided that are obtained by the methods according to the invention.
  • Another embodiment provides seeds from plants obtained by the methods according to the invention.
  • oil from the seeds of plants obtained by the methods according to the invention are provided.
  • a further object of the invention is to provide an isolated DNA encoding the Brassica napus AOXl protein of SEQ ID No. 2, such as an isolated DNA consisting of SEQ ID No. 1. Yet a further object of the invention provides an isolated Brassica napus AOX protein consisting of the nucleotide sequence SEQ ID No. 2.
  • the invention further provides a chimeric gene comprising the following operably linked nucleic acid molecules:
  • nucleic acid encoding an AOXl protein containing at least 60% sequence identity to SEQ ID No. 2; and optionally
  • said AOXl protein contains at least 80% sequence identity to SEQ ID No. 2, whereas in yet another embodiment said nucleic acid encoding an AOXl protein contains at least 80% sequence identity to SEQ ID No. 1.
  • said plant-expressible promoter is a constitutive promoter, such as the 35S promoter, and in yet a further embodiment, said plant-expressible promoter is a seed-specific promoter.
  • the invention further relates to the use of the chimeric genes or of the isolated DNA according to the invention to increase plant seed oil content and/or Thousand Kernel Weight.
  • the invention further provides methods for producing oil, comprising harvesting seeds from the plants according to the invention and extracting the oil from said seeds.
  • the invention provides a method of producing food or feed such as oil, meal, grain, starch, flour or protein, or an industrial product such as biofuel, fiber, industrial chemicals, a pharmaceutical or a neutraceutical, comprising obtaining the plant according to the invention or a part thereof, and preparing the food, feed, or industrial product from the plant or part thereof.
  • Figure 1 Schematic representation of an Arabidopsis expression vector for a rape respiratory metabolism-associated gene BnAOXl.
  • FIG. 3 Comparison of the seed sizes between the transgenic plant carrying a rape respiratory metabolism-associated gene BnAOXl and the wild type control, wherein seeds from the wild type control are shown in Fig.3A, and those from the transgenic line are shown in Fig. 3B.
  • the current invention is based on the finding that overexpression of AOXl in Arabidopsis results in increased seed oil content and increased Thousand Kernel Weight.
  • a method for increasing oil content, such as seed oil content, in plants, and/or seed yield, such as Thousand Kernel Weight comprising increasing expression of a nucleic acid encoding an AOXl protein.
  • said AOXl protein has at least 60% sequence identity to SEQ ID No. 2.
  • said AOXl protein has at least 80% sequence identity to SEQ ID No. 2.
  • said nucleic acid has at least 80% sequence identity to SEQ ID NO. 1.
  • said AOXl protein has at least 80% sequence identity to the AOXIB protein from Arabidopsis thaliana.
  • said nucleic acid has at least 80% sequence identity to the AOXIB coding sequence from Arabidopsis thaliana.
  • Increasing oil content such as seed oil content, in plants, and/or seed yield, such as Thousand Kernel Weight, as used herein, can be increasing oil content, or increasing seed yield, or increasing both oil content and seed yield.
  • At least 60% sequence identity can be, for example, at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity.
  • At least 80% sequence identity can be, for example, at least 80%, or at least 83%, or at least 85%, or at least 87%, or at least 90%, or at least 95%, or at least 97%, or at least 98%, or at least 99%, or 100% sequence identity.
  • the methods and means described herein are believed to be suitable for all plant cells and plants, both dicotyledonous and monocotyledonous plant cells and plants including but not limited to cotton, Brassica, oilseed rape, wheat, corn or maize, barley, sunflowers, rice, oats, sugarcane, soybean, vegetables (including chicory, lettuce, tomato), tobacco, potato, sugarbeet, papaya, pineapple, mango, Arabidopsis thaliana, but also plants used in horticulture, floriculture or forestry.
  • oil producing plants such as rapeseed ⁇ Brassica spp), flax ⁇ Linum usitatissimuni) , safflower ⁇ Carthamus tinctorius), sunflower ⁇ Helianthus annuus), maize or corn ⁇ Zea mays), soybean ⁇ Glycine max), mustard ⁇ Brassica spp.
  • the methods and means described herein can also be used in algae such as Scenedesmus dimorphus, Euglena gracilis, Phaeodactylum tricornutum, Pleurochrysis carterae, Prymnesium parvum, Tetraselmis chui, Tetraselmis suecica, Isochrysis galbana, Nannochloropsis salina, Botryococcus braunii, Dunaliella tertiolecta, Nannochloris spp. or Spirulina spp.
  • algae such as Scenedesmus dimorphus, Euglena gracilis, Phaeodactylum tricornutum, Pleurochrysis carterae, Prymnesium parvum, Tetraselmis chui, Tetraselmis suecica, Isochrysis galbana, Nannochloropsis salina, Botryococcus braunii, Dun
  • a "Brassica plant” is a plant which belongs to one of the species Brassica napus, Brassica rapa (or campestris) , or Brassica juncea. Alternatively, the plant can belong to a species originating from intercrossing of these Brassica species, such as B. napocampestris, or of an artificial crossing of one of these Brassica species with another species of the Cruciferacea.
  • a Brassica oilseed plant refers to any one of the species Brassica napus, Brassica rapa (or campestris), Brassica carinata, Brassica nigra or Brassica juncea.
  • An increase in seed oil content can be an increase with at least 2%, or at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 40%, or at least 50%. Said increase is an increase with respect to levels as obtained in control plants.
  • Thousand Kernel Weight as used herein, also “TKW”, refers to the weight in grams of 1000 seeds. An increased TKW may result from an increase in seed size and/or an increase in seed weight.
  • An increase in Thousand Kernel Weight can be an increase with at least 2%, or at least 5%, or at least 10%, or at least 15%, or at least 20%, or at least 25%, or at least 30%, or at least 40%, or at least 50%. Said increase is an increase with respect to Thousand Kernel Weight as obtained in control plants.
  • control plant as used herein is generally a plant of the same species which has wild-type levels of AOXl.
  • Wild-type levels of AOXl refers to the typical levels of AOXl protein in a plant as it most commonly occurs in nature. Said control plant has thus not been provided either with a nucleic acid molecule encoding AOXl, or with a nucleic acid activating expression of the endogenous AOXl, such as a T-DNA activation tag or a promoter with stronger activity than the endogenous promoter.
  • the AOXl protein refers the Alternative Oxidase protein, which is the terminal oxidase of the cyanide -resistant respiratory pathway in the plant mitochondrial respiratory chain. Endogenous substrates of AOX1 include reduced ubiquinone and O 2 . The respiratory electron flow of the alternative pathway branches off at ubiquinone in the main path (cytochrome pathway) and bypasses two ATP-formation sites of Complex III and IV. Four electrons from the electron flow are transferred directly by AOX1 to an oxygen molecule, which is reduced to H2O, while the energy not used for ATP synthesis is lost in the form of heat release.
  • AOX1 is a di-iron carboxylate protein. It has structural features common to other di-iron carboxylate proteins and a function of eliminating molecular oxygen.
  • An AOX1 protein is a protein that contains at least 50%, or at least 55%, or at least 60%, or at least 65%, or at least 70%, or at least 75%, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or at least 98%, or at least 99% sequence identity to any of the AOX proteins as described in this application.
  • AOX proteins are the Arabidopsis thaliana AOXl proteins AOX1A (AT3G22370), AOXIB (AT3G22360), AOX1C (AT3G27620), and AOX1D (AT1G32350).
  • AOXIB protein from Arabidopsis thaliana also AtAOXlB, as used herein refers to the protein with accession number At3g22360
  • AOXIB coding sequence from Arabidopsis thaliana also AtAOXlB, refers to the coding sequence with accession number At3g22360.
  • homologous nucleotide sequence may be identified and isolated by hybridization under stringent conditions using as probes identified nucleotide sequences.
  • High stringency conditions can be provided, for example, by hybridization at 65°C in an aqueous solution containing 6x SSC (20x SSC contains 3.0 M NaCl, 0.3 M Na-citrate, pH 7.0), 5x Denhardt's (lOOX Denhardt's contains 2% Ficoll, 2% Polyvinyl pyrollidone, 2% Bovine Serum Albumin), 0.5% sodium dodecyl sulphate (SDS), and 20 ⁇ g/ml denaturated carrier DNA (single-stranded fish sperm DNA, with an average length of 120 - 3000 nucleotides) as non-specific competitor. Following hybridization, high stringency washing may be done in several steps, with a final wash (about 30 min) at the hybridization temperature in 0.2-0. lx SSC, 0.1% SDS.
  • Moderate stringency conditions refers to conditions equivalent to hybridization in the above described solution but at about 60-62°C. Moderate stringency washing may be done at the hybridization temperature in lx SSC, 0.1% SDS.
  • Low stringency refers to conditions equivalent to hybridization in the above described solution at about 50-52°C. Low stringency washing may be done at the hybridization temperature in 2x SSC, 0.1% SDS. See also Sambrook et al. (1989) and Sambrook and Russell (2001).
  • sequences encoding AOX1 may also be obtained by DNA amplification using oligonucleotides specific for genes encoding AOXl as primers, such as but not limited to oligonucleotides comprising or consisting of about 20 to about 50 consecutive nucleotides from the known nucleotide sequences or their complement.
  • an AOXl protein which has "at least 80% sequence identity to SEQ ID No. 2" can be an AOXl protein with 80%, or 83%, or 85%, or 87%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% sequence identity to SEQ ID No. 2, or can be SEQ ID No. 2 itself.
  • An AOX protein which has at least 80% sequence identity to SEQ ID NO. 2 can be an AOX protein which has at least 80% sequence identity to both SEQ ID NO. 2 and the AOXIB protein from Arabidopsis thaliana, such as an AOX protein having 87% sequence identity to both SEQ ID NO. 2 and the AOXIB protein from Arabidopsis thaliana.
  • a nucleic acid which has "at least 80% sequence identity to SEQ ID NO. 1" can be a nucleic acid with 80%, or 83%, or 85%, or 87%, or 90%, or 91%, or 92%, or 93%, or 94%, or 95%, or 96%, or 97%, or 98%, or 99% sequence identity to SEQ ID No. 1, or can be SEQ ID No. 1 itself.
  • a nucleic acid which has at least 80% sequence identity to SEQ ID NO. 1 can be a nucleic acid which has at least 80% sequence identity to both SEQ ID NO. 1 and the AOXIB coding sequence from Arabidopsis thaliana, such as a nucleic acid having 83% sequence identity to both SEQ ID NO. 1 and the AOXIB coding sequence from Arabidopsis thaliana.
  • mutant alleles or genes can be obtained by addition, substitution, insertion or deletion of one or more nucleotides with respect to the AOX sequences according to the invention.
  • Increasing expression of a nucleic acid encoding an AOX1 protein can comprise increasing expression on a whole plant level.
  • increasing expression of a nucleic acid encoding an AOX1 protein can comprise increasing expression in specific plant parts or tissues, such as seeds, or specific seed tissues.
  • Increasing expression of a nucleic acid encoding an AOX1 protein can conveniently be achieved by heterologously expressing a nucleic acid encoding AOX1.
  • expression of the endogenous AOX1 encoding gene can be increased through, for example, T-DNA activation tagging, or by targeted genome engineering technologies in which, for example, the endogenous promoter is modified such that it drives higher levels of expression, or in which the endogenous promoter is replaced with a stronger promoter.
  • T-DNA activation tagging is a method to activate endogenous genes by random insertion of a T-DNA carrying promoter or enhancer elements, which can cause transcriptional activation of flanking plant genes.
  • the method can consist of generating a large number of transformed plants or plant cells using a specialized T-DNA construct, followed by selection for the desired phenotype.
  • Targeted genome engineering refers to generate intended and directed modifications into the genome. Such intended modifications can be insertions at specific genomic locations, deletions of specific endogenous sequences, and replacements of endogenous sequences. Targeted genome engineering can be based on homologous recombination. Targeted genome engineering to increase expression of the AOX1 endogene can consist of insertion of a promoter, stronger than the endogenous promoter, in front of the AOX1 coding sequence, or insert an enhancer to increase promoter activity, or insert elements enhancing RNA stability or enhancing translation of the encoded mRNA.
  • a method for increasing oil content, such as seed oil content, and/or seed yield, such as Thousand Kernel Weight, in plants comprising increasing expression of a nucleic acid encoding an AOXl protein comprising the steps of-'
  • nucleic acid encoding an AOXl protein
  • said plant-expressible promoter is a constitutive promoter, and in yet another embodiment, said constitutive promoter is the 35S promoter. In a further embodiment, said plant-expressible promoter is a seed-specific promoter.
  • Said plant cell can be provided with a chimeric gene using methods well-known in the art. Methods to provide plant cells with a chimeric gene are not deemed critical for the current invention and any method to provide plant cells with a chimeric gene suitable for a particular plant species can be used. Such methods are well known in the art and include Agrobacteriumr mediated transformation, particle gun delivery, microinjection, electroporation of intact cells, polyethylene glycol -mediated protoplast transformation, electroporation of protoplasts, liposome-mediated transformation, silicon-whiskers mediated transformation etc. Said chimeric gene may be stably integrated into the genome of said plant cell, resulting in a transformed plant cell. The transformed plant cells obtained in this way may then be regenerated into mature fertile transformed plants.
  • plant-expressible promoter means a DNA sequence that is capable of controlling (initiating) transcription in a plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell, i.e., certain promoters of viral or bacterial origin such as the CaMV35S (Harpster et al. (1988) Mol Gen Genet.
  • the subterranean clover virus promoter No 4 or No 7 (WO9606932), or T-DNA gene promoters but also tissue-specific or organ-specific promoters including but not limited to seed-specific promoters (e.g., WO89/03887), organ-primordia specific promoters (An et al. (1996) Plant CeZ/ 8(l):i5-30), stem-specific promoters (Keller et al, (1988) EMBO J. 7(12): 3625-3633), leaf specific promoters (Hudspeth et al. (1989) Plant Mol Biol.
  • mesophyl-specific promoters such as the light-inducible Rubisco promoters
  • root-specific promoters such as the light-inducible Rubisco promoters
  • tuber-specific promoters such as the tuber-specific promoters
  • vascular tissue specific promoters such as the vascular tissue specific promoters (Peleman et al. (1989) Gene 84: 359-369)
  • stamen-selective promoters WO 89/10396, WO 92/13956
  • dehiscence zone specific promoters WO 97/13865
  • heterologous promoter refers to a promoter which is not normally associated in its natural context with the coding DNA region operably linked to it in the DNA molecules according to the invention. Accordingly, a heterologous promoter excludes the naturally associated promoter. A heterologous promoter to the nucleic acid encoding an AOX1 protein is therefore a promoter other than the AOX1 promoter.
  • a chimeric gene comprising a heterologous promoter and a nucleic acid encoding an AOX1 protein can also be referred to as an artificial construct.
  • Constitutive promoters are well known in the art, and include the CaMV35S promoter (Harpster et al. (1988) Mol Gen Genet. 212(l):l82-90), Actin promoters, such as, for example, the promoter from the Rice Actin gene (McElroy et al., 1990, Plant Cell 2:163), the promoter of the Cassava Vein Mosaic Virus (Verdaguer et al., 1996 Plant Mol. Biol. 31: H29), the GOS promoter (de Pater et al., 1992, Plant J.
  • Seed specific promoters are well known in the art, including the USP promoter from Vicia faba described in DE10211617; the promoter sequences described in WO2009/073738; promoters from Brassica napus for seed specific gene expression as described in WO2009/077478; the plant seed specific promoters described in US2007/0022502; the plant seed specific promoters described in WO03/014347; the seed specific promoter described in WO2009/125826; the promoters of the omega_3 fatty acid desaturase family described in WO2006/005807 and the like.
  • a "transcription termination and polyadenylation region” as used herein is a sequence that drives the cleavage of the nascent RNA, whereafter a poly(A) tail is added at the resulting RNA 3' end, functional in plants.
  • Transcription termination and polyadenylation signals functional in plants include, but are not limited to, 3'nos, 3'35S, 3'his and 3'g7.
  • seed oil plants refers to plants producing oil in their seeds.
  • seed oil plants are Brassica oilseeds (including Brassica napus, Brassica campestris (rapa), Brassica juncea or Brassica carinata), sunflower, safflower, soybean, palm, Jatropha, flax, crambe, camelina, corn, sesame, castor beans.
  • Brassicaceae plants are plants which according to the current botanical standard would be classified into the family Brassicaceae (formerly Cruciferaeae) .
  • Brassicaceae (Mustard) family members are easy to distinguish. They are annual or perannual plants with alternate leaves without stipules and possess simple inflorescence or branched racemes.
  • the flowers are bilaterally symmetrical and hypogynous. With few exceptions, the flowers have 4 petals (free) alternating with 4 sepals (free) >' 6 stamens (4 long and 2 short), an ovary of 2 united carpels with parital placenta, 2 locular through the formation of a membranous false septum ; fruit is a dehiscent capsule opening by 2 valves.
  • Brassicaceae include inter alia the following genera : Sisymbrium, Descurania, Alliaria, Arabidopsis, Myagrum, Isatis, Bunia, Erysium, Hesperis, Malcolmia, Matthiola, Chorispora, Euclidium, Barbarea, Rorippa, Armoracia, Nasturtium, Dentaria, Cardamine, Cardaminopsis, Arabis, Lunaria, Alyssum, Berteroa, Lobularia, Draba, Erophila, Cochlearia, Camelina, Neslia, Capsella, Hornungia, Thlsapi, Iberis, Lepidium, Cardaria, Coronopus, Subularia, Conringia, Diplotaxis, Brassica, Sinapsis, Eruca, Erucastrum, Coincya, Hirschfeldia, Cakile, Rapistum, Crambe, Enarthrocarpus, Rhaphanus and Clausia.
  • plants are provided that are obtained by the methods according to the invention.
  • Another embodiment provides seeds from plants obtained by the methods according to the invention.
  • oil from the seeds of plants obtained by the methods according to the invention is provided.
  • the obtained plants can be used in a conventional breeding scheme to produce more plants with the same characteristics or to introduce the characteristic of increased expression of a nucleic acid encoding an AOXl protein according to the invention in other varieties of the same or related plant species, or in hybrid plants.
  • the obtained plants can further be used for creating propagating material.
  • Plants according to the invention can further be used to produce gametes, seeds (including crushed seeds and seed cakes), embryos, either zygotic or somatic, progeny or hybrids of plants obtained by methods of the invention. Seeds obtained from the obtained plants according to the invention are also encompassed by the invention.
  • Creating propagating material relates to any means know in the art to produce further plants, plant parts or seeds and includes inter alia vegetative reproduction methods (e.g. air or ground layering, division, (bud) grafting, micropropagation, stolons or runners, storage organs such as bulbs, corms, tubers and rhizomes, striking or cutting, twin- scaling), sexual reproduction (crossing with another plant) and asexual reproduction (e.g. apomixis, somatic hybridization).
  • vegetative reproduction methods e.g. air or ground layering, division, (bud) grafting, micropropagation, stolons or runners, storage organs such as bulbs, corms, tubers and rhizomes, striking or cutting, twin- scaling
  • sexual reproduction crossing with another plant
  • asexual reproduction e.g. apomixis, somatic hybridization
  • a further object of the invention is an isolated DNA encoding the Brassica napus AOXl protein of SEQ ID No. 2, such as an isolated DNA consisting of SEQ ID No. I.
  • Yet a further object of the invention provides an isolated Brassica napus AOXl protein consisting of the nucleotide sequence of SEQ ID No. 2.
  • isolated DNA refers to DNA not occurring in its natural genomic context, irrespective of its length and sequence. Isolated DNA can, for example, refer to DNA which is physically separated from the genomic context, such as a fragment of genomic DNA.
  • Isolated DNA can also be an artificially produced DNA, such as a chemically synthesized DNA, or such as DNA produced via amplification reactions, such as polymerase chain reaction (PCR) well-known in the art.
  • Isolated DNA can further refer to DNA present in a context of DNA in which it does not occur naturally.
  • isolated DNA can refer to a piece of DNA present in a plasmid.
  • the isolated DNA can refer to a piece of DNA present in another chromosomal context than the context in which it occurs naturally, such as for example at another position in the genome than the natural position, in the genome of another species than the species in which it occurs naturally, or in an artificial chromosome.
  • isolated protein refers to a protein not occurring in its natural cellular context, irrespective of its length and sequence.
  • An isolated protein can, for example, refer to a protein present in a plant cell in which it does not naturally occur.
  • An isolated protein can also be a protein isolated from a plant cell in which it naturally occurs or in which it does not naturally occur.
  • an isolated protein can be a protein produced in a heterologous expression system well known in the art, such as a plant expression system, a yeast expression system, a microbial expression system, a mammalian expression system, where the isolated protein can reside within the cells of said expression system, can be extracted from the cells of said expression system, or can be purified from said expression system.
  • an isolated protein can be produced in an in vitro system, from which it can be extracted and purified.
  • the invention further provides a chimeric gene comprising the following operably linked nucleic acid molecules:
  • nucleic acid encoding an AOX1 protein containing at least 60% sequence identity to SEQ ID No. % and optionally
  • said AOX1 protein contains at least 80% sequence identity to SEQ ID No. 2
  • said nucleic acid encoding an AOX1 protein contains at least 80% sequence identity to SEQ ID No. 1.
  • said plant-expressible promoter is a constitutive promoter, such as the 35S promoter, and in yet a further embodiment, said plant-expressible promoter is a seed-specific promoter.
  • the invention further relates to the use of the chimeric genes or of the isolated DNA according to the invention to increase plant seed oil content and/or to seed yield such as Thousand Kernel Weight.
  • the invention further provides methods for producing oil, comprising harvesting seeds from the plants according to the invention and extracting the oil from said seeds.
  • the invention provides a method of producing food or feed such as oil, meal, grain, starch, flour or protein, or an industrial product such as biofuel, fiber, industrial chemicals, a pharmaceutical or a neutraceutical, comprising obtaining the plant according to the invention or a part thereof, and preparing the food, feed, or industrial product from the plant or part thereof.
  • nucleic acid or protein comprising a sequence of nucleotides or amino acids
  • a chimeric gene comprising a DNA region which is functionally or structurally defined, may comprise additional DNA regions etc.
  • SEQ ID NO: Brassica napus AOX1 coding sequence.
  • SEQ ID NO: 3 BnAOXl forward primer.
  • SEQ ID NO: 4 BnAOXl reverse primer.
  • SEQ ID NO: 5 AtAOXlB forward primer.
  • SEQ ID NO: 7 T7 primer.
  • SEQ ID NO: 8 35S promoter specific primer.
  • the applicants of the present invention have screened out a set of genes including BnAOXl gene, which are differentially expressed not only between two kinds of Brassica napus parent lines but also between the extremely segregated lines of F2 generation, by analyzing the differences in gene expression between two kinds of parent lines with significantly different oil-content as well as between the mixed samples of the segregated lines of F2 generation exhibiting significantly different oil-content.
  • BnAOXl gene which are differentially expressed not only between two kinds of Brassica napus parent lines but also between the extremely segregated lines of F2 generation, by analyzing the differences in gene expression between two kinds of parent lines with significantly different oil-content as well as between the mixed samples of the segregated lines of F2 generation exhibiting significantly different oil-content.
  • the applicants have cloned the full-length gene, constructed an expression vector comprising said full-length gene, and transformed thereof into the model plant Arabidopsis thaliana.
  • the respiratory metabolism- associated gene BnAOXl from Brassica napus is useful in increasing the oil content and the
  • a high-oil content Brassica napus line zy036 (oil content thereof being 51%) was crossed with a low-oil content Brassica napus line Y817 (oil content thereof being 35%) in order to establish a population of F2 generation.
  • a total of 169 F2 offspring plantlets was used as the starting material.
  • the silique (with pod shells and ovules) were collected respectively from each of F2 plants about 25 days post blossom; and the oil content was determined in mature seeds from the individual plants.
  • the silique from individual plants having a determined oil content of greater than 47% and of less than 38.5% were weighed and mixed in equal amount of 200 mg respectively to constitute two mixed samples with two extreme oil contents, which samples are coded by H and L, respectively.
  • 11 plants had an oil content of greater than 47% and 11 plants had an oil content of less than 38.5%. There was difference of 11.1% between the average oil contents of the two mixed samples.
  • Genes expressed in the two parents and two mixed samples were assayed. The genes whose expression levels were different between the parents as well as between the mixed samples of F2 generations were identified, from which the gene BnAOXl originates.
  • zy036 and Y817 used in the study had been established by the technicians of the biotechnical breeding team in the Oil Crops Research Institute (OCRI) of the Chinese Academy of Agricultural Sciences (CAAS) under the direction of the researcher Wang Hanzhong.
  • the zy036 line is developed by establishing a recurrent selection population consisted of Zhongshuang No.4, Zhongshuang No.7, Zhongshuang No.9, Huashuang No.3, and Youyan No.9, performing recurrent selections of two generations, followed by recurrent selection of the third generation by microspore culture of excellent individual plants therefrom, and eventually conducting high oil content-directed selection.
  • Y817 is a maintenance line for the hybrid cultivar Zhongyou hybrid No.l (Study on techniques about seed production of Zhouyou Hybrid No.l and their utilization, rural Economy and Science -Technology, (10), 2001).
  • Brassica napus genomic sequences homologous to the AOX1 gene have been identified by means of the sequence of Arabidopsis thaliana AOX1B gene (AtAOXlB; Accession Number At3g22360) and an in-house sequence database, then spliced them together, and designed primers flanking the coding regions of the gene of interest on the basis of the spliced sequence.
  • RT-PCR amplification was performed using the cDNA first strand from the parent zy036 as template. The amplified fragment was sequenced, to obtain the coding region sequence of BnAOXl gene.
  • a protein is disclosed, the base sequence encoding said protein is the nucleotide sequence shown in SEQ ID NO: 1.
  • a protein is disclosed, the sequence thereof being the amino acid sequence shown as SEQ ID NO: 2.
  • primers for amplifying the sequence of the homologous gene AtAOXlB in Arabidopsis thaliana were designed by use of gene sequences (At3g22360).
  • Primers for amplification in Arabidopsis thaliana were as follows: AtAOXlB forward primer: [5'-atgatgatgagtcgtcgcta-3'] (SEQ ID No. 5) and reverse primer: [5'-tcaatgatatccaatgggagc-3'] (SEQ ID No. 6), for use in direct amplification from cDNA of the Arabidopsis thaliana 10-dayold siliques.
  • RNA extraction extraction of RNA with TRIZOL TM Kit: grind 100 mg of raw material in liquid nitrogen.
  • step C The mixture from step B is centrifuged at 4°C at 12000g for 15min. The resultant supernatant is transferred into a new tube. 500 ⁇ of isopropanol is added and mixed well. Then, the resulting mixture is left at RT for 15 min.
  • the resulting mixture is centrifuged at 4°C at 7500g for 7 min. The resultant supernatant is discarded and the RNA pellet is air-dried.
  • the air-dried RNA pellet is dissolved in DEPC-H 2 O.
  • Reverse transcription of the first strand cDNA is performed with Revert- Aid H Minus First Strand cDNA Synthesis Kit (Fermentas), with operation following the instruction of the kit used.
  • BnAOXl and AtAOXlB genes are obtained.
  • the sequences of BnAOXl and AtAOXlB genes are as follows: a respiratory metabolism-associated gene BnAOXl from Brassica napus, encoding the BnAOXl protein, the base sequence thereof is the nucleotide sequence shown in SEQ ID NO: V, the BnAOXl protein, the sequence of the protein is the amino acid sequence as shown in SEQ ID NO: 2, and gene sequences of Arabidopsis as published (database accession number At3g22360).
  • the method for using the cloned gene comprises the following steps:
  • the method for cloning the Brassica napus gene according to the present invention is the one commonly used in the art, such as, CTAB protocol is used to extract DNA from plant leaves.
  • CTAB protocol is used to extract DNA from plant leaves.
  • the methods for extracting mRNA are various and have been well-established, such as, TRIzol Reagent Protocol from Invitrogen Co. or
  • cDNA library construction is also a conventional technique in molecular biology. Methods for constructing and transforming the vector according to the present invention into a plant are also those commonly used in the art.
  • the involved plasmids (the entry vector PCR8/GW/TOPO and the expression vector plasmid pEarley gate 100) in the methods, host cells for transformation (e.g., Agrobacterium tumefaciens GV3101), and reagents (sucrose, etc.,) used are commercially available.
  • the method most commonly used for polymorphic analysis of molecular marker is polyacrylamide gel electrophoresis in which the reagents used are commercially available, such as, acrylamide, methylene bisacrylamide, etc.
  • the gene sequence obtained by PCR amplification was ligated into the TOPO entry vector (Invitrogen Co.), then the obtained vector was transformed into DH5a competent cells (Invitrogen Co.). Screening of positive colonies with spectinomycin was performed.
  • the plasmid of interest with the forwardly ligated insert was identified by PCR amplification using a vector-specific primer (T7 primer, TAATACGACTCACTATAGGG) (SEQ ID No. 7) and a gene-specific primer (the forward primer for the target gene, the sequence of which was shown in Example l).
  • T7 primer TAATACGACTCACTATAGGG
  • a gene-specific primer the forward primer for the target gene, the sequence of which was shown in Example l.
  • the plasmid of interest was mini-prepared, then recombined into the vector pEarleygate 100 (Invitrogen Co.), and finally transformed into DH5a competent cells.
  • Example 2 The expression vector prepared in Example 2 was introduced into Agrobacterium tumefaciens GV3101 and further into Arabidopsis thaliana plants, using the following steps ⁇
  • the treated plant was grown overnight with avoidance of light and then cultivated normally until producing seed. The seeds were harvested for further screening and identification.
  • the vernalized Arabidopsis seeds were seeded in artificial soil irrigated with the saturated Hyponex NO.2 (commercially available) nutrient solution and covered with plastic wrap. Two days later, light was given, and three days later, the plastic wrap was removed.
  • Conditions in the artificial cultivation chamber were as follows : Relative humidity: 80%, constant temperature of 20-24°C, light intensity of 80-200 umol M 2 /S, light cycle: 8h of Dark, 16 h of Light. After about one week, a herbicide (glyphosate) was sprayed to screen positive plants.
  • glyphosate glyphosate
  • step C the resulting mixture from step C was centrifuged at 12000 rpm at room temperature for 15 min. DNA pellet was washed by adding 200 ⁇ of 70% (vol/vol) ethanol.
  • step E the resultant from step E was centrifuged at 12000 rpm at room temperature for 15 min. Ethanol was discarded. The tube with the DNA pellet was placed inversely on a paper towel until the ethanol was volatilized completely.
  • the extracted DNA pellet in the tube was dissolved in 100 ⁇ of sterile water.
  • the DNA concentration was estimated with a spectrometer or by electrophoresis.
  • Formulation ratio for the PCR reaction mixture was the same as that in identification of the plasmid by PCR, and based on the sequence of 35S promoter in the plant expression vector and the sequences of the reverse primers for the BnAOXl and AtAOXlB genes: [5'-tcaatgatccaattggag-3'] (SEQ ID No. 4) and [5'-tcaatgatatccaatgggagc-3'] (SEQ ID No. 6), respectively, the time and temperature for the reaction was as follows: 94°C 3 min, 30 cycles of 94°C 45s, 59°C 45s, 72°C 2 min 30s, then 72°C 5 min.
  • Example 5 Determination of seed oil content and thousand kernel weight of the transgenic Arabidopsis thaliana
  • the transgenic homozygous lines were grown at 21-23 °C in a greenhouse. The seeds were harvested and then tested the changes in the oil content and thousand kernel weight (Fig. 3). The overall phenotype of the transgenic Arabidopsis thaliana plant overexpressing BnAOXl gene or AtAOXlB gene was not significantly different from that of the wild type Arabidopsis thaliana control. After harvest of the seeds, it was found that the seeds from the transgenic plant were significantly larger than those from the wild type control ( Figure 3). The seed oil content was determined by pulse nuclear magnetic resonance spectrometer.
  • Table 1 Determination of the oil content and the thousand kernel weight (TKW) in Arabidopsis thaliana overexpressing BnAOXl (BnAOXl- 1 to BnAOXl-4) or AtAOXlB(AtAOXl-l to AtAOXl-4).

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Abstract

Cette invention concerne les séquences du gène AOX1 et de la protéine provenant de Brassica napus. L'invention concerne également l'utilisation du gène AOX1 pour augmenter la teneur en huile des graines et le poids de mille grains chez les plantes.
PCT/CN2012/070283 2011-01-27 2012-01-20 Gène bnaox1 issu de brassica napus, associé au métabolisme respiratoire, et son utilisation WO2012100682A1 (fr)

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