Patent Grant
11834666
The present invention relates generally to genetically engineered land plants that express a plant CCP1-like mitochondrial transporter protein, and more particularly, to such genetically engineered land plants comprising a modified gene for the plant CCP1-like mitochondrial transporter protein.
The world faces a major challenge in the next 35 years to meet the increased demands for food production to feed a growing global population, which is expected to reach 9 billion by the year 2050. Food output will need to be increased by up to 70% in view of the growing population. Increased demand for improved diet, concomitant land use changes for new living space and infrastructure, alternative uses for crops and changing weather patterns will add to the challenge.
Major agricultural crops include food crops, such as maize, wheat, oats, barley, soybean, millet, sorghum, pulses, bean, tomato, corn, rice, cassava, sugar beets, and potatoes, forage crop plants, such as hay, alfalfa, and silage corn, and oilseed crops, such as camelina, Brassica species (e.g. B. napus (canola), B. rapa, B. juncea, and B. carinata), crambe, soybean, sunflower, safflower, oil palm, flax, and cotton, among others. Productivity of these crops, and others, is limited by numerous factors, including for example relative inefficiency of photochemical conversion of light energy to fixed carbon during photosynthesis, as well as loss of fixed carbon by photorespiration and/or other essential metabolic pathways having enzymes catalyzing decarboxylation reactions. Crop productivity is also limited by the availability of water. Achieving step changes in crop yield requires new approaches.
One potential approach involves metabolic engineering of crop plants to express carbon-concentrating mechanisms of cyanobacteria or eukaryotic algae. Cyanobacteria and eukaryotic algae have evolved carbon-concentrating mechanisms to increase intracellular concentrations of dissolved inorganic carbon, particularly to increase concentrations of CO2 at the active site of ribulose-1,5-bisphosphate carboxylase/oxygenase (also termed RuBisCO). It has recently been shown by Schnell et al., WO 2015/103074 that Camelina plants transformed to express CCP1 of the algal species Chlamydomonas reinhardtii have reduced transpiration rates, increased CO2 assimilation rates and higher yield than control plants which do not express the CCP1 gene. More recently, Atkinson et al., (2015) Plant Biotechnol. J., doi: 10.1111/pbi.12497, discloses that CCP1 and its homolog CCP2, which were previously characterized as Ci transporters, previously reported to be in the chloroplast envelope, localized to mitochondria in both Chlamydomonas reinhardtii, as expressed naturally, and tobacco, when expressed heterologously, suggesting that the model for the carbon-concentrating mechanism of eukaryotic algae needs to be expanded to include a role for mitochondria. Atkinson et al. (2015) disclosed that expression of individual Ci (bicarbonate) transporters did not enhance growth of the plant Arabidopsis.
In co-pending Patent Application PCT/US2017/016421, to Yield10 Bioscience, a number of orthologs of CCP1 from algal species that share common protein sequence domains including mitochondrial membrane domains and transporter protein domains were shown to increase seed yield and reduce seed size when expressed constitutively in Camelina plants. Schnell et al., WO 2015/103074, also reported a decrease in seed size in higher yielding Camelina lines expressing CCP1.
In U.S. Provisional Patent Application 62/462,074, to Yield10 Bioscience, CCP1 and its orthologs from other eukaryotic algae are referred to as mitochondrial transporter proteins. The inventors tested the impact of expressing CCP1 or its algal orthologs using seed-specific promoters with the unexpected outcome that both seed yield and seed size increased. These inventors also recognized the benefits of combining constitutive expression and seed specific expression of CCP1 or any of its orthologs in the same plant.
Unfortunately, “transgenic plants,” “GMO crops,” and/or “biotech traits” are not widely accepted in some regions and countries and are subject to regulatory approval processes that are very time consuming and prohibitively expensive. The current regulatory framework for transgenic plants results in significant costs (˜$136 million per trait; McDougall, P. 2011, “The cost and time involved in the discovery, development, and authorization of a new plant biotechnology derived trait.” Crop Life International) and lengthy product development timelines that limit the number of technologies that are brought to market. This has severely impaired private investment and the adoption of innovation in this crucial sector. Recent advances in genome editing technologies provide an opportunity to precisely remove genes or edit control sequences to significantly improve plant productivity (Belhaj, K. 2013, Plant Methods, 9, 39; Khandagale & Nadal, 2016, Plant Biotechnol Rep, 10, 327) and open the way to produce plants that may benefit from an expedited regulatory path, or possibly unregulated status.
Given the costs and challenges associated with obtaining regulatory approval and societal acceptance of transgenic crops there is a need to identify, where possible, plant mitochondrial transporter proteins, ideally derived from crops or other land plants, that can be genetically engineered to enable enhanced carbon capture systems to improve crop yield and/or seed yield, particularly without relying on genes, control sequences, or proteins derived from non-land plants to the extent possible.
In accordance with one aspect of the present invention, a genetically engineered land plant that expresses a plant CCP1-like mitochondrial transporter protein is disclosed. The genetically engineered land plant comprises a modified gene for the plant CCP1-like mitochondrial transporter protein. The plant CCP1-like mitochondrial transporter protein is an ortholog of CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1 derived from a source land plant. The plant CCP1-like mitochondrial transporter protein is localized to mitochondria of the genetically engineered land plant based on a mitochondrial targeting signal intrinsic to the plant CCP1-like mitochondrial transporter protein. The modified gene comprises (i) a promoter and (ii) a nucleic acid sequence encoding the plant CCP1-like mitochondrial transporter protein. The promoter is non-cognate with respect to the nucleic acid sequence. The modified gene is configured such that transcription of the nucleic acid sequence is initiated from the promoter and results in expression of the plant CCP1-like mitochondrial transporter protein.
A genetically engineered land plant that expresses a plant CCP1-like mitochondrial transporter protein is disclosed. The genetically engineered land plant comprises a modified gene for the plant CCP1-like mitochondrial transporter protein. The plant CCP1-like mitochondrial transporter protein is an ortholog of CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1 derived from a source land plant. The plant CCP1-like mitochondrial transporter protein is localized to mitochondria of the genetically engineered land plant based on a mitochondrial targeting signal intrinsic to the plant CCP1-like mitochondrial transporter protein. The modified gene comprises (i) a promoter and (ii) a nucleic acid sequence encoding the plant CCP1-like mitochondrial transporter protein. The promoter is non-cognate with respect to the nucleic acid sequence. The modified gene is configured such that transcription of the nucleic acid sequence is initiated from the promoter and results in expression of the plant CCP1-like mitochondrial transporter protein.
Surprisingly, it has been determined that certain land plants encode orthologs of algal CCP1 of Chlamydomonas reinhardtii, herein termed plant CCP1-like mitochondrial transporter proteins. This was surprising because, among other reasons, no plant CCP1-like mitochondrial transporter proteins of land plants were revealed in standard BLAST searches aimed at identifying CCP1 orthologs in land plants, and thus initial attempts to identify plant CCP1-like mitochondrial transporter proteins by conventional means suggested that land plants may not encode such proteins at all. Serendipitously, the plant CCP1-like mitochondrial transporter proteins were identified instead based on analyses of a Transcriptome Shotgun Assembly database, as discussed below.
Also surprisingly, the plant CCP1-like mitochondrial transporter proteins appear to cluster into two distinct groups, herein termed Tier 1 CCP1 orthologs and Tier 2 CCP1 orthologs, based on similarities of predicted amino acid sequence and structure with respect to CCP1. The plant Tier 1 CCP1 orthologs exhibit about 60% sequence identity with respect to CCP1 of Chlamydomonas reinhardtii, cluster narrowly based on the degree of their sequence similarity, and have been identified thus far only in four plant species, Zea nicaraguensis (also termed teosinte), Erigeron breviscapus, Cosmos bipinnatus, and Poa pratensis, none of which are particularly closely related phylogenetically. The plant Tier 2 CCP1 orthologs exhibit about 30% sequence identity with respect to CCP1 of Chlamydomonas reinhardtii, substantially lower than for Tier 1, also cluster narrowly based on the degree of their sequence similarity, and would appear to be more common, having been identified thus far in six major crop species, Zea mays (also termed maize), Triticum aestivum, Solanum tuberosum, Glycine max, Oryza sativa, and Sorghum bicolor. This was surprising because there had not been any apparent reason to expect any clustering of plant CCP1-like mitochondrial transporter proteins, let alone clustering into two distinct groups. This also was surprising because Zea nicaraguensis, again teosinte, is a wild progenitor of Zea mays, again maize, and thus the two are closely related phylogenetically, yet Zea nicaraguensis includes a Tier 1 CCP1, whereas Zea mays includes a Tier 2 CCP1.
Also surprisingly, it has been determined that further clustering occurs within the Tier 1 CCP1 orthologs when various algal CCP1 orthologs are included, specifically several algal Tier 1 CCP1 orthologs, namely those of Gonium pectorale (KXZ50472.1), Gonium pectorale (KXZ50486.1), and Volvox carteri f. nagariensis, herein termed Tier 1A, exhibit about 80% sequence identity in comparison to CCP1 of Chlamydomonas reinhardtii, whereas one algal Tier 1 CCP1 ortholog, namely Ettlia oleoabundans, herein termed Tier 1B, instead exhibits 60% sequence identity and clustering with the plant Tier 1 CCP1 orthologs, also herein termed Tier 1B. Strikingly, the algal and plant Tier 1B CCP1 orthologs seem to be more closely related to each other than to the other algal or plant CCP1 orthologs. This suggests that the intriguing possibility that the plant Tier 1B CCP1 orthologs may have resulted from horizontal gene transfer from Ettlia oleoabundans or related algae. This also suggests that Zea nicaraguensis and the other plant species encoding Tier 1B CCP1 orthologs may serve as sources of CCP1 orthologs that are proximally derived from land plants, rather than from algae, thus decreasing regulatory concerns and risk associated with genetic modification of crops, while still being able to provide increases in crop yield comparable to those observed for CCP1 and CCP1 orthologs derived from algae.
Without wishing to be bound by theory, it is believed that by genetically engineering a land plant to comprise a modified gene for a plant CCP1-like mitochondrial transporter protein, with the plant CCP1-like mitochondrial transporter protein being an ortholog of CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1 derived from a source land plant, the plant CCP1-like mitochondrial transporter protein being localized to mitochondria of the genetically engineered land plant based on a mitochondrial targeting signal intrinsic to the plant CCP1-like mitochondrial transporter protein, the modified gene comprising a promoter and a nucleic acid sequence encoding the plant CCP1-like mitochondrial transporter protein, the promoter being non-cognate with respect to the nucleic acid sequence, and the modified gene being configured such that transcription of the nucleic acid sequence is initiated from the promoter and results in expression of the plant CCP1-like mitochondrial transporter protein, will result in enhanced yield, based for example on an increased CO2 assimilation rate and/or a decreased transpiration rate of the genetically engineered land plant, in comparison to a reference land plant that does not comprise the modified gene. It is believed that the plant CCP1-like mitochondrial transporter protein will enhance transport of malate (also termed MAL) and/or oxaloacetate (also termed OAA) from or into the mitochondria and/or otherwise alter mitochondrial metabolism by transport of bicarbonate and/or other small molecules, thereby enhancing rates of carbon fixation by increasing CO2 recovery from photorespiration and respiration. Alternatively, the increased transport of small molecules may prevent the accumulation of photorespiratory intermediates that may inhibit photosynthesis. Moreover, it is believed that by genetically engineering the land plant to express a plant CCP1-like mitochondrial transporter protein that is localized to mitochondria in particular, it will be possible to stack expression of the plant CCP1-like mitochondrial transporter protein with expression of other proteins in deliberate and complementary approaches to further enhance yield. In addition, it is believed that by modifying the land plant to express a plant CCP1-like mitochondrial transporter protein of a land plant in particular, it will be possible to generate genetically engineered crops that include only genes, control sequences, and proteins that are proximally derived from land plants, and thus are already generally recognized as safe for human consumption.
As noted, a genetically engineered land plant that expresses a plant CCP1-like mitochondrial transporter protein is disclosed. A land plant is a plant belonging to the plant subkingdom Embryophyta, including higher plants, also termed vascular plants, and mosses, liverworts, and hornworts.
The term “land plant” includes mature plants, seeds, shoots and seedlings, and parts, propagation material, plant organ tissue, protoplasts, callus and other cultures, for example cell cultures, derived from plants belonging to the plant subkingdom Embryophyta, and all other species of groups of plant cells giving functional or structural units, also belonging to the plant subkingdom Embryophyta. The term “mature plants” refers to plants at any developmental stage beyond the seedling. The term “seedlings” refers to young, immature plants at an early developmental stage.
Land plants encompass all annual and perennial monocotyledonous or dicotyledonous plants and includes by way of example, but not by limitation, those of the genera Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersicon, Nicotiana, Solarium, Petunia, Digitalis, Majorana, Cichorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Senecio, Salpiglossis, Cucumis, Browaalia, Glycine, Pisum, Phaseolus, Lolium, Oryza, Zea, Avena, Hordeum, Secale, Triticum, Sorghum, Picea, Populus, Camelina, Beta, Solanum, and Carthamus. Preferred land plants are those from the following plant families: Amaranthaceae, Asteraceae, Brassicaceae, Carophyllaceae, Chenopodiaceae, Compositae, Cruciferae, Cucurbitaceae, Euphorbiaceae, Fabaceae, Labiatae, Leguminosae, Papilionoideae, Liliaceae, Linaceae, Malvaceae, Poaceae, Rosaceae, Rubiaceae, Saxifragaceae, Scrophulariaceae, Solanaceae, Sterculiaceae, Tetragoniaceae, Theaceae, Umbelliferae.
The land plant can be a monocotyledonous land plant or a dicotyledonous land plant. Preferred dicotyledonous plants are selected in particular from the dicotyledonous crop plants such as, for example, Asteraceae such as sunflower, tagetes or calendula and others; Compositae, especially the genus Lactuca, very particularly the species sativa (lettuce) and others; Cruciferae, particularly the genus Brassica, very particularly the species napus (oilseed rape), campestris (beet), oleracea cv Tastie (cabbage), oleracea cv Snowball Y (cauliflower) and oleracea cv Emperor (broccoli) and other cabbages; and the genus Arabidopsis, very particularly the species thaliana, and cress or canola and others; Cucurbitaceae such as melon, pumpkin/squash or zucchini and others; Leguminosae, particularly the genus Glycine, very particularly the species max (soybean), soya, and alfalfa, pea, beans or peanut and others; Rubiaceae, preferably the subclass Lamiidae such as, for example Coffea arabica or Coffea liberica (coffee bush) and others; Solanaceae, particularly the genus Lycopersicon, very particularly the species esculentum (tomato), the genus Solanum, very particularly the species tuberosum (potato) and melongena (aubergine) and the genus Capsicum, very particularly the genus annuum (pepper) and tobacco or paprika and others; Sterculiaceae, preferably the subclass Dilleniidae such as, for example, Theobroma cacao (cacao bush) and others; Theaceae, preferably the subclass Dilleniidae such as, for example, Camellia sinensis or Thea sinensis (tea shrub) and others; Umbelliferae, particularly the genus Daucus (very particularly the species carota (carrot)) and Apium (very particularly the species graveolens dulce (celery)) and others; and linseed, cotton, hemp, flax, cucumber, spinach, carrot, sugar beet and the various tree, nut and grapevine species, in particular banana and kiwi fruit. Preferred monocotyledonous plants include maize, rice, wheat, sugarcane, sorghum, oats and barley.
Of particular interest are oilseed plants. In oilseed plants of interest the oil is accumulated in the seed and can account for greater than 10%, greater than 15%, greater than 18%, greater than 25%, greater than 35%, greater than 50% by weight of the weight of dry seed. Oil crops encompass by way of example: Borago officinalis (borage); Camelina (false flax); Brassica species such as B. campestris, B. napus, B. rapa, B. carinata (mustard, oilseed rape or turnip rape); Cannabis sativa (hemp); Carthamus tinctorius (safflower); Cocos nucifera (coconut); Crambe abyssinica (crambe); Cuphea species (Cuphea species yield fatty acids of medium chain length, in particular for industrial applications); Elaeis guinensis (African oil palm); Elaeis oleifera (American oil palm); Glycine max (soybean); Gossypium hirsutum (American cotton); Gossypium barbadense (Egyptian cotton); Gossypium herbaceum (Asian cotton); Helianthus annuus (sunflower); Jatropha curcas (jatropha); Linum usitatissimum (linseed or flax); Oenothera biennis (evening primrose); Olea europaea (olive); Oryza sativa (rice); Ricinus communis (castor); Sesamum indicum (sesame); Thlaspi caerulescens (pennycress); Triticum species (wheat); Zea mays (maize), and various nut species such as, for example, walnut or almond.
Camelina species, commonly known as false flax, are native to Mediterranean regions of Europe and Asia and seem to be particularly adapted to cold semiarid climate zones (steppes and prairies). The species Camelina sativa was historically cultivated as an oilseed crop to produce vegetable oil and animal feed. In addition to being useful as an industrial oilseed crop, Camelina is a very useful model system for developing new tools and genetically engineered approaches to enhancing the yield of crops in general and for enhancing the yield of seed and seed oil in particular. Demonstrated transgene improvements in Camelina can then be deployed in major oilseed crops including Brassica species including B. napus (canola), B. rapa, B. juncea, B. carinata, crambe, soybean, sunflower, safflower, oil palm, flax, and cotton.
As will be apparent, the land plant can be a C3 photosynthesis plant, i.e. a plant in which RuBisCO catalyzes carboxylation of ribulose-1,5-bisphosphate by use of CO2 drawn directly from the atmosphere, such as for example, wheat, oat, and barley, among others. The land plant also can be a C4 plant, i.e. a plant in which RuBisCO catalyzes carboxylation of ribulose-1,5-bisphosphate by use of CO2 shuttled via malate or aspartate from mesophyll cells to bundle sheath cells, such as for example maize, millet, and sorghum, among others.
Accordingly, in some examples the genetically engineered land plant is a C3 plant. Also, in some examples the genetically engineered land plant is a C4 plant. Also, in some examples the genetically engineered land plant is a major food crop plant selected from the group consisting of maize, wheat, oat, barley, soybean, millet, sorghum, potato, pulse, bean, tomato, and rice. In some of these examples, the genetically engineered land plant is maize. Also, in some examples the genetically engineered land plant is a forage crop plant selected from the group consisting of silage corn, hay, and alfalfa. In some of these examples, the genetically engineered land plant is silage corn. Also, in some examples the genetically engineered land plant is an oilseed crop plant selected from the group consisting of camelina, Brassica species (e.g. B. napus (canola), B. rapa, B. juncea, and B. carinata), crambe, soybean, sunflower, safflower, oil palm, flax, and cotton.
The land plant comprises a modified gene for the plant CCP1-like mitochondrial transporter protein.
The plant CCP1-like mitochondrial transporter protein is an ortholog of CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1 derived from a source land plant.
The term “ortholog” means a polynucleotide sequence or polypeptide sequence possessing a high degree of homology, i.e. sequence relatedness, to a subject sequence and being a functional equivalent of the subject sequence, wherein the sequence that is orthologous is from a species that is different than that of the subject sequence. Homology may be quantified by determining the degree of identity and/or similarity between the sequences being compared.
As used herein, “percent homology” of two polynucleotide sequences or of two polypeptide sequences is determined using the algorithm of Karlin and Altschul (1990), Proc. Natl. Acad. Sci., U.S.A. 87: 2264-2268. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al. (1990), J. Mol. Biol. 215: 403-410. BLAST nucleotide searches are performed with the NBLAST program, score=100, word length 12, to obtain nucleotide sequences homologous to a reference polynucleotide sequence. BLAST protein searches are performed with the XBLAST program, score=50, word length=3, to obtain amino acid sequences homologous to a reference polypeptide sequence. To obtain gapped alignments for comparison purposes, Gapped BLAST is utilized as described in Altschul et al. (1997), Nucleic Acids Res. 25: 3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters are typically used.
In the case of polypeptide sequences that are less than 100% identical to a reference sequence, the non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence. Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
Where a particular polypeptide is said to have a specific percent identity to a reference polypeptide of a defined length, the percent identity is relative to the reference peptide. Thus, a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide that is 50% identical to the reference polypeptide over its entire length. Many other polypeptides will meet the same criteria.
For reference, as discussed above CCP1 is a mitochondrial transporter protein of Chlamydomonas reinhardtii. Moreover, CCP1 has an amino acid sequence in accordance with SEQ ID NO: 1. Accordingly, the plant CCP1-like mitochondrial transporter protein is a polypeptide sequence possessing a high degree of sequence relatedness to CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1 and being a functional equivalent thereof.
As noted, the plant CCP1-like mitochondrial transporter protein is an ortholog of CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1 derived from a source land plant.
For reference, Chlamydomonas reinhardtii is a eukaryotic alga. In contrast to a land plant, a eukaryotic alga is an aquatic plant, ranging from a microscopic unicellular form, e.g. a single-cell alga, to a macroscopic multicellular form, e.g. a seaweed, that includes chlorophyll a and, if multicellular, a thallus not differentiated into roots, stem, and leaves, and that is classified as chlorophyta (also termed green algae), rhodophyta (also termed red algae), or phaeophyta (also termed brown algae). Eukaryotic algae include, for example, single-cell algae, including the chlorophyta Chlamydomonas reinhardtii, Chlorella sorokiniana, and Chlorella variabilis. Eukaryotic algae also include, for example, seaweed, including the chlorophyta Ulva lactuca (also termed sea lettuce) and Enteromorpha (Ulva) intenstinalis (also termed sea grass), the rhodophyta Chondrus crispus (also termed Irish moss or carrigeen), Porphyra umbilicalis (also termed nori), and Palmaria palmata (also termed dulse or dillisk), and the phaeophyta Ascophyllum nodosum (also termed egg wrack), Laminaria digitata (also termed kombu/konbu), Laminaria saccharina (also termed royal or sweet kombu), Himanthalia elongata (also termed sea spaghetti), and Undaria pinnatifida (also termed wakame). Eukaryotic algae also include, for example, additional chlorophyta such as Gonium pectorale, Volvox carteri f. nagariensis, and Ettlia oleoabundans.
The source land plant from which the plant CCP1-like mitochondrial transporter protein is derived can be a land plant as described above, i.e. a plant belonging to the plant subkingdom Embryophyta.
In some examples the source land plant is a different type of land plant than the genetically engineered land plant. In accordance with these examples, the plant CCP1-like mitochondrial transporter protein can be heterologous with respect to the genetically engineered land plant. By this it is meant that the particular plant CCP1-like mitochondrial transporter protein derived from the source land plant is not normally encoded, expressed, or otherwise present in land plants of the type from which the genetically engineered land plant is derived. This can be because land plants of the type from which the genetically engineered land plant is derived do not normally encode, express, or otherwise include the particular plant CCP1-like mitochondrial transporter protein, and this can be so whether or not the land plants normally express a different, endogenous CCP1-like mitochondrial transporter protein. The genetically engineered land plant expresses the particular plant CCP1-like mitochondrial transporter protein based on comprising the modified gene for the plant CCP1-like mitochondrial transporter protein. Accordingly, the modified gene can be used to accomplish modified expression of the plant CCP1-like mitochondrial transporter protein, and particularly increased expression of CCP1 ortholog(s), including the plant CCP1-like mitochondrial transporter protein and any endogenous CCP1-like mitochondrial transporter proteins.
Also in some examples the source land plant is the same type of land plant as the genetically engineered land plant. In accordance with these examples, the plant CCP1-like mitochondrial transporter protein can be homologous with respect to the genetically engineered land plant. By this it is meant that the particular plant CCP1-like mitochondrial transporter protein is normally encoded, and may normally be expressed, in land plants of the type from which the genetically engineered land plant is derived. In accordance with these examples, the land plant can be genetically engineered to include additional copies of a gene for the plant CCP1-like mitochondrial transporter protein and/or to express an endogenous copy a gene for the plant CCP1-like mitochondrial transporter protein at higher levels and/or in a tissue-preferred manner based on modification and/or replacement of a promoter for the endogenous copy of the gene. Again, the genetically engineered land plant expresses the particular plant CCP1-like mitochondrial transporter protein based on comprising the modified gene for the plant CCP1-like mitochondrial transporter protein, resulting in modified expression of the plant CCP1-like mitochondrial transporter protein, and particularly increased expression of CCP1 ortholog(s).
As discussed above, it is believed that the plant CCP1-like mitochondrial transporter protein will enhance transport of malate and/or oxaloacetate from or into the mitochondria and/or otherwise alter mitochondrial metabolism by transport of bicarbonate and/or other small molecules. Accordingly, the plant CCP1-like mitochondrial transporter protein may be a protein that transports malate and/or oxaloacetate by any transport mechanism. Mitochondrial transporters useful for practicing the disclosed invention include transporters involved in the transport of dicarboxylic acids into and out of the mitochondria in plant cells. In particular these transporters can be involved in the transport of oxaloacetate (i.e. OAA) and/or malate (i.e. MAL). In the case of the transport of OAA and MAL, the transporter can be antiporters such that OAA and MAL are transported simultaneously in the opposite directions, for example such that OAA is transported in, while MAL is transported out. Basically the mitochondrial transporter acts as a malate/oxaloacetate shuttle. In other cases the shuttle may transport OAA and one or more other dicarboxylic acids or other metabolites. Transporters or shuttles which transport OAA are a preferred embodiment of this invention. The directionality of flow of either metabolite is determined by the growth conditions experienced by the plant at any particular time. The plant CCP1-like mitochondrial transporter protein also may be a protein that otherwise alters mitochondrial metabolism by transport of bicarbonate and/or other small molecules. Classes of bicarbonate transport proteins include anion exchangers and Na+/HCO3−1 symporters. Increased transport of other small molecules may prevent their buildup which might otherwise inhibit photosynthesis.
The plant CCP1-like mitochondrial transporter protein is localized to mitochondria of the land plant based on a mitochondrial targeting signal intrinsic to the plant CCP1-like mitochondrial transporter protein. The plant CCP1-like mitochondrial transporter protein can be localized to mitochondria for example based on being encoded by DNA present in the nucleus of a plant cell, synthesized in the cytosol of the plant cell, targeted to the mitochondria of the plant cell, and inserted into outer membranes and/or inner membranes of the mitochondria. A mitochondrial targeting signal is a portion of a polypeptide sequence that targets the polypeptide sequence to mitochondria. A mitochondrial targeting signal intrinsic to the plant CCP1-like mitochondrial transporter protein is a mitochondrial targeting signal that is integral to the plant CCP1-like mitochondrial transporter protein, e.g. based on occurring naturally at the N-terminal end of the plant CCP1-like mitochondrial transporter protein or in discrete segments along the plant CCP1-like mitochondrial transporter protein. This is in contrast, for example, to fusion of a heterologous mitochondrial targeting signal to a mitochondrial transporter protein that would not otherwise be targeted to mitochondria. For reference, also as discussed above CCP1 is localized to mitochondria in both Chlamydomonas reinhardtii, as expressed naturally, and tobacco, when expressed heterologously. Accordingly, the plant CCP1-like mitochondrial transporter protein can be a mitochondrial transporter protein that is encoded by nuclear DNA, synthesized cytosolically, targeted to the mitochondria, and inserted into outer membranes and/or inner membranes thereof, based on targeting by a portion of the polypeptide sequence integral to plant CCP1-like mitochondrial transporter protein. The plant CCP1-like mitochondrial transporter protein does not have typical plastid targeting signals.
Suitable plant CCP1-like mitochondrial transporter proteins can be identified, for example, based on searching databases of polynucleotide sequences or polypeptide sequences for orthologs of CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1, wherein the polynucleotide sequences or polypeptide sequences are derived from land plants, in view of the disclosure herein, as discussed below. Such searches can be carried out, for example, by use of BLAST, e.g. tblastn, and databases including translated polynucleotides, whole genome shotgun sequences, and/or transcriptome assembly sequences, among other sequences and databases. Potential orthologs of CCP1 may be identified, for example, based on percentage of identity and/or percentage of similarity, with respect to polypeptide sequence, of individual sequences in the databases in comparison to CCP1 of Chlamydomonas reinhardtii. For example, potential orthologs of CCP1 may be identified based on percentage of identity of an individual sequence in a database and CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1 of at least 25%, e.g. at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95%, wherein the individual sequence is derived from a land plant. Also for example, potential orthologs of CCP1 may be identified based on percentage of similarity of an individual sequence in a database and CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1 of at least 10%, e.g. at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95%, wherein the individual sequence is derived from a land plant. Also for example, potential orthologs of CCP1 may be identified based on both percentage of identity of at least 25%, e.g. at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95%, and percentage of similarity of at least 10%, e.g. at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 90%, or at least 95%, wherein the individual sequence is derived from a land plant.
Suitable plant CCP1-like mitochondrial transporter proteins also can be identified, for example, based on functional screens.
For example, some cyanobacterial bicarbonate transporters have previously been shown to functionally localize into the Escherichia coli cytoplasmic membrane, including some bicarbonate transporters, as reported by Du et al. (2014), PLoS One 9, e115905. Expression of six particular cyanobacterial bicarbonate transporters in E. coli using a mutant E. coli strain, termed EDCM636, that is deficient in carbonic anhydrase activity and that is unable to grow on LB or M9 plates without supplementation with high levels of CO2, restored growth of the E. coli mutant at atmospheric levels of CO2, whereas expression of various others did not, as reported by Du et al. (2014). Function of CCP1 and potential orthologs thereof with respect to transport of malate and/or oxaloacetate, bicarbonate, or other metabolites may be tested by an analogous approach, and corresponding functional screens developed, also based on restoring growth of mutant E. coli strains.
Function of CCP1 and potential orthologs thereof with respect to transport of malate and/or oxaloacetate, bicarbonate, or other metabolites also may be tested, and corresponding functional screens developed, based on use of yeast modified to express CCP1 and potential orthologs thereof. Transport of bicarbonate or other metabolites from mitochondria of yeast so modified would indicate that these sequences also enable transport of bicarbonate in yeast.
Following identification of a plant CCP1-like mitochondrial transporter protein, genetic engineering of a land plant to express the plant CCP1-like mitochondrial transporter protein can be carried out by methods that are known in the art, as discussed in detail below.
The genetically engineered land plant can be a genetically engineered land plant that includes no heterologous proteins, e.g. wherein the plant CCP1-like mitochondrial transporter protein is homologous with respect to the genetically engineered land plant, or only one heterologous protein, e.g. wherein the only heterologous plant protein that the genetically engineered land plant comprises is the plant CCP1-like mitochondrial transporter protein. As noted above, Atkinson et al. (2015) also discloses that expression of individual putative Ci transporters did not enhance Arabidopsis growth, and suggests that stacking of further components of carbon-concentrating mechanisms will probably be required to achieve a significant increase in photosynthetic efficiency in this species, albeit without having tested expression of CCP1 in particular. In contrast, without wishing to be bound by theory, it is believed that a genetically engineered land plant that expresses a plant CCP1-like mitochondrial transporter protein as described herein will achieve a significant increase in photosynthetic efficiency in the genetically engineered land plant without need for stacking of further components of carbon-concentrating mechanisms, and thus without heterologous and/or modified expression of any other protein by the genetically engineered land plant. The corresponding genetically engineered land plant will provide advantages relative to plants that are modified to express multiple genes, for example in terms of simpler methods of making the genetically engineered land plant, and lower risk of harmful effects of other proteins subject to heterologous and/or modified expression with respect to use of the genetically engineered land plant as a food crop, a forage crop, or an oilseed crop.
Considering the plant CCP1-like mitochondrial transporter protein in more detail, the plant CCP1-like mitochondrial transporter protein can correspond to a plant CCP1-like mitochondrial transporter protein selected from among specific polypeptide sequences of source land plants. As noted above, mitochondrial transporter proteins include CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1. As also noted, plant CCP1-like mitochondrial transporter protein may be identified based on homology to CCP1. Exemplary CCP1-like mitochondrial transporter proteins identified this way include (a) a plant CCP1-like mitochondrial transporter protein of Zea nicaraguensis of SEQ ID NO: 7, (b) a plant CCP1-like mitochondrial transporter protein of Erigeron breviscapus of SEQ ID NO: 6, (c) a plant CCP1-like mitochondrial transporter protein of Poa pratensis of SEQ ID NO: 8, and (d) a plant CCP1-like mitochondrial transporter protein of Cosmos bipinnatus of SEQ ID NO: 9. These correspond to Tier 1 plant CCP1-like mitochondrial transporter proteins. Exemplary CCP1-like mitochondrial transporter protein identified this way also include (a) a plant CCP1-like mitochondrial transporter protein of Zea mays of SEQ ID NO: 16, (b) a plant CCP1-like mitochondrial transporter protein of Triticum aestivum of SEQ ID NO: 12, (c) a plant CCP1-like mitochondrial transporter protein of Solanum tuberosum of SEQ ID NO: 13, (d) a plant CCP1-like mitochondrial transporter protein of Glycine max of SEQ ID NO: 14, (e) a plant CCP1-like mitochondrial transporter protein of Oryza sativa of SEQ ID NO: 15, and (f) a plant CCP1-like mitochondrial transporter protein of Sorghum bicolor of SEQ ID NO: 17. These correspond to Tier 2 plant CCP1-like mitochondrial transporter proteins.
Accordingly, in some embodiments the plant CCP1-like mitochondrial transporter protein comprises at least one of (a) a plant CCP1-like mitochondrial transporter protein of Zea nicaraguensis, (b) a plant CCP1-like mitochondrial transporter protein of Erigeron breviscapus, (c) a plant CCP1-like mitochondrial transporter protein of Poa pratensis, or (d) a plant CCP1-like mitochondrial transporter protein of Cosmos bipinnatus. For example, in some embodiments the plant CCP1-like mitochondrial transporter protein comprises a plant CCP1-like mitochondrial transporter protein of Zea nicaraguensis.
Also in some embodiments, the plant CCP1-like mitochondrial transporter protein comprises at least one of (a) a plant CCP1-like mitochondrial transporter protein of Zea nicaraguensis of SEQ ID NO: 7, (b) a plant CCP1-like mitochondrial transporter protein of Erigeron breviscapus of SEQ ID NO: 6, (c) a plant CCP1-like mitochondrial transporter protein of Poa pratensis of SEQ ID NO: 8, or (d) a plant CCP1-like mitochondrial transporter protein of Cosmos bipinnatus of SEQ ID NO: 9. For example, in some embodiments the plant CCP1-like mitochondrial transporter protein comprises a plant CCP1-like mitochondrial transporter protein of Zea nicaraguensis of SEQ ID NO: 7.
Also in some embodiments, the plant CCP1-like mitochondrial transporter protein comprises one or more of (a) a plant CCP1-like mitochondrial transporter protein of Zea mays, (b) a plant CCP1-like mitochondrial transporter protein of Triticum aestivum, (c) a plant CCP1-like mitochondrial transporter protein of Solanum tuberosum, (d) a plant CCP1-like mitochondrial transporter protein of Glycine max, (e) a plant CCP1-like mitochondrial transporter protein of Oryza sativa, or (f) a plant CCP1-like mitochondrial transporter protein of Sorghum bicolor. For example, in some embodiments the plant CCP1-like mitochondrial transporter protein comprises a plant CCP1-like mitochondrial transporter protein of Zea mays.
Also in some embodiments, the plant CCP1-like mitochondrial transporter protein comprises one or more of (a) a plant CCP1-like mitochondrial transporter protein of Zea mays of SEQ ID NO: 16, (b) a plant CCP1-like mitochondrial transporter protein of Triticum aestivum of SEQ ID NO: 12, (c) a plant CCP1-like mitochondrial transporter protein of Solanum tuberosum of SEQ ID NO: 13, (d) a plant CCP1-like mitochondrial transporter protein of Glycine max of SEQ ID NO: 14, (e) a plant CCP1-like mitochondrial transporter protein of Oryza sativa of SEQ ID NO: 15, or (f) a plant CCP1-like mitochondrial transporter protein of Sorghum bicolor of SEQ ID NO: 17. For example, in some embodiments the plant CCP1-like mitochondrial transporter protein comprises a plant CCP1-like mitochondrial transporter protein of Zea mays of SEQ ID NO: 16.
The plant CCP1-like mitochondrial transporter protein also can correspond to a plant CCP1-like mitochondrial transporter protein including specific structural features and characteristics shared among various orthologs of CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1. Such structural features and characteristics shared among the various orthologs of CCP1, namely the Tier 1 algal CCP1-like mitochondrial transporter proteins of Gonium pectorale (KXZ50472.1) (SEQ ID NO: 2), Gonium pectorale (KXZ50486.1) (SEQ ID NO: 3), Volvox carteri f. nagariensis (SEQ ID NO: 4), and Ettlia oleoabundans (SEQ ID NO: 5), and Tier 1 plant CCP1-like mitochondrial transporter proteins of Erigeron breviscapus (SEQ ID NO: 6), Zea nicaraguensis (SEQ ID NO: 7), and Cosmos bipinnatus (SEQ ID NO: 9), include (i) (a) a proline residue at position 268, (b) an aspartate residue or glutamine residue at position 270, (c) a lysine residue or arginine residue at position 273, and (d) a serine residue or threonine residue at position 274, with numbering of positions relative to CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1, and (ii) an overall identity of at least 15%. These noted amino acid residues occur at or after the C-terminal portion of a potential transmembrane region of each of CCP1 and the various Tier 1 algal and plant orthologs, namely that of Gonium pectorale (KXZ50472.1) (SEQ ID NO: 2), Gonium pectorale (KXZ50486.1) (SEQ ID NO: 3), Volvox carteri f. nagariensis (SEQ ID NO: 4), and Ettlia oleoabundans (SEQ ID NO: 5), Erigeron breviscapus (SEQ ID NO: 6), Zea nicaraguensis (SEQ ID NO: 7), and Cosmos bipinnatus (SEQ ID NO: 9), as well as among various other orthologs of CCP1. Conservation of the noted amino acid residues, in combination with an overall identity of at least 15%, suggests a structure/function relationship shared among such mitochondrial transporter proteins. Thus, for example, the plant CCP1-like mitochondrial transporter protein can be an ortholog of CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1 based on comprising: (i) (a) a proline residue at position 268, (b) an aspartate residue or glutamine residue at position 270, (c) a lysine residue or arginine residue at position 273, and (d) a serine residue or threonine residue at position 274, with numbering of positions relative to CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1, and (ii) an overall identity of at least 15%.
The plant CCP1-like mitochondrial transporter protein also can correspond to a plant CCP1-like mitochondrial transporter protein including additional specific structural features and characteristics shared among orthologs of CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1. For example, the plant CCP1-like mitochondrial transporter protein can be an ortholog of CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1 based on comprising: (i) (a) a glycine residue at position 301, (b) a glycine residue at position 308, and (c) an arginine residue at position 315, with numbering of positions relative to CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1, and (ii) an overall identity of at least 15%. These noted amino acid residues also are conserved among CCP1 and the various Tier 1 algal and plant orthologs, as well as other CCP1 orthologs.
The plant CCP1-like mitochondrial transporter protein also can correspond to a plant CCP1-like mitochondrial transporter protein including Tier 1 CCP1 signature sequences shared specifically among Tier 1 algal and plant orthologs of CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1. For example, the plant CCP1-like mitochondrial transporter protein can be an ortholog of CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1 based on comprising: (i) one or more Tier 1 CCP1 signature sequences of (a) LLGIHFP (SEQ ID NO: 18) at position 104-110, (b) LRDMQGYAWFF (SEQ ID NO: 19) at position 212-222, (c) AGFGLWGSMF (SEQ ID NO: 20) at position 258-267, or (d) AIPVNA (SEQ ID NO: 21) at position 316-321, with numbering of positions relative to CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1, and (ii) an overall identity of at least 60%. These noted Tier 1 CCP1 signature sequences also are conserved specifically among CCP1 and the various Tier 1 algal and plant orthologs.
The plant CCP1-like mitochondrial transporter protein also can correspond to a plant CCP1-like mitochondrial transporter protein that does not only localize to mitochondria, but that also localizes to chloroplasts. As noted above, Atkinson et al. (2015) discloses that CCP1 and its homolog CCP2, which are characterized as putative Ci transporters previously reported to be in the chloroplast envelope, localized to mitochondria in both Chlamydomonas reinhardtii, as expressed naturally, and tobacco, when expressed heterologously. Without wishing to be bound by theory, it is believed that localization of plant CCP1-like mitochondrial transporter proteins to mitochondria to a greater extent than to chloroplasts promotes enhanced yield. Thus, for example, the plant CCP1-like mitochondrial transporter protein can be localized to mitochondria of the genetically engineered land plant to a greater extent than to chloroplasts of the genetically engineered land plant by a factor of at least 2, at least 5, or at least 10.
The plant CCP1-like mitochondrial transporter protein also can correspond to a plant CCP1-like mitochondrial transporter protein that does not differ in any biologically significant way from a wild-type plant CCP1-like mitochondrial transporter protein. As noted above, the plant CCP1-like mitochondrial transporter protein is localized to mitochondria of the genetically engineered land plant based on a mitochondrial targeting signal intrinsic to the plant CCP1-like mitochondrial transporter protein, and this is in contrast, for example, to fusion of a heterologous mitochondrial targeting signal to a plant protein that would not otherwise be targeted to mitochondria. In some examples, the plant CCP1-like mitochondrial transporter protein also does not include any other modifications that might result in the plant CCP1-like mitochondrial transporter protein differing in a biologically significant way from a wild-type plant CCP1-like mitochondrial transporter protein. Thus, for example the plant CCP1-like mitochondrial transporter protein can consist essentially of an amino acid sequence that is identical to that of a wild-type plant CCP1-like mitochondrial transporter protein. The corresponding genetically engineered land plant will provide advantages, e.g. again in terms of lower risk of harmful effects with respect to use of the genetically engineered land plant as a food crop, a forage crop, or an oilseed crop.
The modified gene comprises (i) a promoter and (ii) a nucleic acid sequence encoding the plant CCP1-like mitochondrial transporter protein.
The promoter is non-cognate with respect to the nucleic acid sequence. A promoter that is non-cognate with respect to a nucleic acid sequence means that the promoter is not naturally paired with the nucleic acid sequence in organisms from which the promoter and/or the nucleic acid sequence are derived. Instead, the promoter has been paired with the nucleic acid sequence based on use of recombinant DNA techniques to create a modified gene. Accordingly, in this case, the promoter is not naturally paired with the nucleic acid sequence in the source land plant, i.e. the land plant from which the nucleic acid sequence encoding the plant CCP1-like mitochondrial transporter protein had been derived, nor in the organism from which the promoter has been derived, whether that organism is the source land plant or another organism. Instead, the promoter has been paired with the nucleic acid sequence based on use of recombinant DNA techniques to create the modified gene.
The modified gene is configured such that transcription of the nucleic acid sequence is initiated from the promoter and results in expression of the plant CCP1-like mitochondrial transporter protein. Accordingly, in the context of the modified gene, the promoter functions as a promoter of transcription of the nucleic acid sequence, and thus of expression of the plant CCP1-like mitochondrial transporter protein.
In some examples, the promoter is a constitutive promoter. In some examples, the promoter is a seed-specific promoter. In some examples, the modified gene is integrated into genomic DNA of the genetically engineered land plant. In some examples, the modified gene is stably expressed in the genetically engineered land plant. In some examples the nucleic acid sequence encodes a wild-type plant CCP1-like mitochondrial transporter protein. In some examples, the nucleic acid sequence encodes a variant, modified, mutant, or otherwise non-wild-type plant CCP1-like mitochondrial transporter protein. These exemplary features, and others, of the promoter, the nucleic acid sequence, and the modified gene are discussed in detail below.
The genetically engineered land plant also can be a genetically engineered land plant that expresses nucleic acid sequences encoding plant CCP1-like mitochondrial transporter proteins in both a seed-specific and a constitutive manner, wherein the nucleic acid sequences encoding the plant CCP1-like mitochondrial transporter proteins may be the same or different nucleic acid sequences, from the same source land plant or from different source land plants. Without wishing to be bound by theory, it is believed that constitutive expression of plant CCP1-like mitochondrial transporter proteins results in much higher numbers of pods, and that seed-specific expression of plant CCP1-like mitochondrial transporter proteins can supply the carbon needed to fill seeds to a full size, and that thus the yield should be higher. Accordingly, in some examples the genetically engineered land plant (i) expresses the plant CCP1-like mitochondrial transporter protein in a seed-specific manner, and (ii) expresses another plant CCP1-like mitochondrial transporter protein constitutively, the other plant CCP1-like mitochondrial transporter protein also corresponding to an ortholog of CCP1 of Chlamydomonas reinhardtii of SEQ ID NO: 1 derived from a source land plant.
The genetically engineered land plant can have a CO2 assimilation rate that is higher than for a corresponding reference land plant not comprising the modified gene. For example, the genetically engineered land plant can have a CO2 assimilation rate that is at least 5% higher, at least 10% higher, at least 20% higher, or at least 40% higher, than for a corresponding reference land plant that does not comprise the modified gene.
The genetically engineered land plant also can have a transpiration rate that is lower than for a corresponding reference land plant not comprising the modified gene. For example, the genetically engineered land plant can have a transpiration rate that is at least 5% lower, at least 10% lower, at least 20% lower, or at least 40% lower, than for a corresponding reference land plant that does not comprise the modified gene.
The genetically engineered land plant also can have a seed yield that is higher than for a corresponding reference land plant not comprising the modified gene. For example, the genetically engineered land plant can have a seed yield that is at least 5% higher, at least 10% higher, at least 20% higher, at least 40% higher, at least 60% higher, or at least 80% higher, than for a corresponding reference land plant that does not comprise the modified gene.
As noted above, following identification of a plant CCP1-like mitochondrial transporter protein of a source land plant, genetic engineering of a land plant to express the plant CCP1-like mitochondrial transporter protein can be carried out by methods that are known in the art, for example as follows.
DNA constructs useful in the methods described herein include transformation vectors capable of introducing transgenes or other modified nucleic acid sequences into land plants. As used herein, “genetically engineered” refers to an organism in which a nucleic acid fragment containing a heterologous nucleotide sequence has been introduced, or in which the expression of a homologous gene has been modified, for example by genome editing. Transgenes in the genetically engineered organism are preferably stable and inheritable. Heterologous nucleic acid fragments may or may not be integrated into the host genome.
Several plant transformation vector options are available, including those described in Gene Transfer to Plants, 1995, Potrykus et al., eds., Springer-Verlag Berlin Heidelberg New York, Genetically engineered Plants: A Production System for Industrial and Pharmaceutical Proteins, 1996, Owen et al., eds., John Wiley & Sons Ltd. England, and Methods in Plant Molecular Biology: A Laboratory Course Manual, 1995, Maliga et al., eds., Cold Spring Laboratory Press, New York. Plant transformation vectors generally include one or more coding sequences of interest under the transcriptional control of 5′ and 3′ regulatory sequences, including a promoter, a transcription termination and/or polyadenylation signal, and a selectable or screenable marker gene.
Many vectors are available for transformation using Agrobacterium tumefaciens. These typically carry at least one T-DNA sequence and include vectors such as pBIN19. Typical vectors suitable for Agrobacterium transformation include the binary vectors pCIB200 and pCIB2001, as well as the binary vector pCIB 10 and hygromycin selection derivatives thereof. See, for example, U.S. Pat. No. 5,639,949.
Transformation without the use of Agrobacterium tumefaciens circumvents the requirement for T-DNA sequences in the chosen transformation vector and consequently vectors lacking these sequences are utilized in addition to vectors such as the ones described above which contain T-DNA sequences. The choice of vector for transformation techniques that do not rely on Agrobacterium depends largely on the preferred selection for the species being transformed. Typical vectors suitable for non-Agrobacterium transformation include pCIB3064, pSOG 19, and pSOG35. See, for example, U.S. Pat. No. 5,639,949. Alternatively, DNA fragments containing the transgene and the necessary regulatory elements for expression of the transgene can be excised from a plasmid and delivered to the plant cell using microprojectile bombardment-mediated methods.
Zinc-finger nucleases (ZFNs) are also useful in that they allow double strand DNA cleavage at specific sites in plant chromosomes such that targeted gene insertion or deletion can be performed (Shukla et al., 2009, Nature 459: 437-441; Townsend et al., 2009, Nature 459: 442-445).
The CRISPR/Cas9 system (Sander, J. D. and Joung, J. K., Nature Biotechnology, published online Mar. 2, 2014; doi;10.1038/nbt.2842) is particularly useful for editing plant genomes to modulate the expression of homologous genes encoding enzymes. All that is required to achieve a CRISPR/Cas edit is a Cas enzyme, or other CRISPR nuclease (Murugan et al. Mol Cell 2017, 68:15), and a single guide RNA (sgRNA) as reviewed extensively by others (Belhag et al. Curr Opin Biotech 2015, 32: 76; Khandagale and Nadaf, Plant Biotechnol Rep 2016, 10:327). Several examples of the use of this technology to edit the genomes of plants have now been reported (Belhaj et al. Plant Methods 2013, 9:39; Zhang et al. Journal of Genetics and Genomics 2016, 43: 251).
TALENs (transcriptional activator-like effector nucleases) or meganucleases can also be used for plant genome editing (Malzahn et al., Cell Biosci, 2017, 7:21)
Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell targeted for transformation. Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome include microinjection (Crossway et al. (1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606), Agrobacterium-mediated transformation (Townsend et al., U.S. Pat. No. 5,563,055; Zhao et al. WO US98/01268), direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), and ballistic particle acceleration (see, for example, Sanford et al., U.S. Pat. No. 4,945,050; Tomes et al. (1995) Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag, Berlin); and McCabe et al. Biotechnology 6:923-926 (1988)). Also see Weissinger et al. Ann. Rev. Genet. 22:421-477 (1988); Sanford et al. Particulate Science and Technology 5:27-37 (1987) (onion); Christou et al. Plant Physiol. 87:671-674 (1988) (soybean); McCabe et al. (1988) BioTechnology 6:923-926 (soybean); Finer and McMullen In Vitro Cell Dev. Biol. 27P:175-182 (1991) (soybean); Singh et al. Theor. Appl. Genet. 96:319-324 (1998)(soybean); Dafta et al. (1990) Biotechnology 8:736-740 (rice); Klein et al. Proc. Natl. Acad. Sci. USA 85:4305-4309 (1988) (maize); Klein et al. Biotechnology 6:559-563 (1988) (maize); Tomes, U.S. Pat. No. 5,240,855; Buising et al., U.S. Pat. Nos. 5,322,783 and 5,324,646; Tomes et al. (1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods, ed. Gamborg (Springer-Verlag, Berlin) (maize); Klein et al. Plant Physiol. 91:440-444 (1988) (maize); Fromm et al. Biotechnology 8:833-839 (1990) (maize); Hooykaas-Van Slogteren et al. Nature 311:763-764 (1984); Bowen et al., U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. Proc. Natl. Acad. Sci. USA 84:5345-5349 (1987) (Liliaceae); De Wet et al. in The Experimental Manipulation of Ovule Tissues, ed. Chapman et al. (Longman, N.Y.), pp. 197-209 (1985) (pollen); Kaeppler et al. Plant Cell Reports 9:415-418 (1990) and Kaeppler et al. Theor. Appl. Genet. 84:560-566 (1992) (whisker-mediated transformation); D'Halluin et al. Plant Cell 4:1495-1505 (1992) (electroporation); Li et al. Plant Cell Reports 12:250-255 (1993) and Christou and Ford Annals of Botany 75:407-413 (1995) (rice); Osjoda et al. Nature Biotechnology 14:745-750 (1996) (maize via Agrobacterium tumefaciens). References for protoplast transformation and/or gene gun for Agrisoma technology are described in WO 2010/037209. Methods for transforming plant protoplasts are available including transformation using polyethylene glycol (PEG), electroporation, and calcium phosphate precipitation (see for example Potrykus et al., 1985, Mol. Gen. Genet., 199, 183-188; Potrykus et al., 1985, Plant Molecular Biology Reporter, 3, 117-128), Methods for plant regeneration from protoplasts have also been described [Evans et al., in Handbook of Plant Cell Culture, Vol 1, (Macmillan Publishing Co., New York, 1983); Vasil, I K in Cell Culture and Somatic Cell Genetics (Academic, Orlando, 1984)].
Recombinase technologies which are useful for producing the disclosed genetically engineered plants include the cre-lox, FLP/FRT and Gin systems. Methods by which these technologies can be used for the purpose described herein are described for example in (U.S. Pat. No. 5,527,695; Dale and Ow, 1991, Proc. Natl. Acad. Sci. USA 88: 10558-10562; Medberry et al., 1995, Nucleic Acids Res. 23: 485-490).
Transformation protocols as well as protocols for introducing nucleotide sequences into plants may vary depending on the type of plant or plant cell, i.e., monocot or dicot, targeted for transformation.
Suitable methods of introducing nucleotide sequences into plant cells and subsequent insertion into the plant genome are described in US 2010/0229256 A1 to Somleva & Ali and US 2012/0060413 to Somleva et al.
The transformed cells are grown into plants in accordance with conventional techniques. See, for example, McCormick et al., 1986, Plant Cell Rep. 5: 81-84. These plants may then be grown, and either pollinated with the same transformed variety or different varieties, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that constitutive expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure constitutive expression of the desired phenotypic characteristic has been achieved.
Procedures for in planta transformation can be simple. Tissue culture manipulations and possible somaclonal variations are avoided and only a short time is required to obtain genetically engineered plants. However, the frequency of transformants in the progeny of such inoculated plants is relatively low and variable. At present, there are very few species that can be routinely transformed in the absence of a tissue culture-based regeneration system. Stable Arabidopsis transformants can be obtained by several in planta methods including vacuum infiltration (Clough & Bent, 1998, The Plant J. 16: 735-743), transformation of germinating seeds (Feldmann & Marks, 1987, Mol. Gen. Genet. 208: 1-9), floral dip (Clough and Bent, 1998, Plant J. 16: 735-743), and floral spray (Chung et al., 2000, Genetically engineered Res. 9: 471-476). Other plants that have successfully been transformed by in planta methods include rapeseed and radish (vacuum infiltration, Ian and Hong, 2001, Genetically engineered Res., 10: 363-371; Desfeux et al., 2000, Plant Physiol. 123: 895-904), Medicago truncatula (vacuum infiltration, Trieu et al., 2000, Plant J. 22: 531-541), camelina (floral dip, WO/2009/117555 to Nguyen et al.), and wheat (floral dip, Zale et al., 2009, Plant Cell Rep. 28: 903-913). In planta methods have also been used for transformation of germ cells in maize (pollen, Wang et al. 2001, Acta Botanica Sin., 43, 275-279; Zhang et al., 2005, Euphytica, 144, 11-22; pistils, Chumakov et al. 2006, Russian J. Genetics, 42, 893-897; Mamontova et al. 2010, Russian J. Genetics, 46, 501-504) and Sorghum (pollen, Wang et al. 2007, Biotechnol. Appl. Biochem., 48, 79-83).
Following transformation by any one of the methods described above, the following procedures can be used to obtain a transformed plant expressing the transgenes: select the plant cells that have been transformed on a selective medium; regenerate the plant cells that have been transformed to produce differentiated plants; select transformed plants expressing the transgene producing the desired level of desired polypeptide(s) in the desired tissue and cellular location.
The cells that have been transformed may be grown into plants in accordance with conventional techniques. See, for example, McCormick et al. Plant Cell Reports 5:81-84 (1986). These plants may then be grown, and either pollinated with the same transformed variety or different varieties, and the resulting hybrid having constitutive expression of the desired phenotypic characteristic identified. Two or more generations may be grown to ensure that constitutive expression of the desired phenotypic characteristic is stably maintained and inherited and then seeds harvested to ensure constitutive expression of the desired phenotypic characteristic has been achieved.
Genetically engineered plants can be produced using conventional techniques to express any genes of interest in plants or plant cells (Methods in Molecular Biology, 2005, vol. 286, Genetically engineered Plants: Methods and Protocols, Pena L., ed., Humana Press, Inc. Totowa, NJ; Shyamkumar Barampuram and Zhanyuan J. Zhang, Recent Advances in Plant Transformation, in James A. Birchler (ed.), Plant Chromosome Engineering: Methods and Protocols, Methods in Molecular Biology, vol. 701, Springer Science+Business Media). Typically, gene transfer, or transformation, is carried out using explants capable of regeneration to produce complete, fertile plants. Generally, a DNA or an RNA molecule to be introduced into the organism is part of a transformation vector. A large number of such vector systems known in the art may be used, such as plasmids. The components of the expression system can be modified, e.g., to increase expression of the introduced nucleic acids. For example, truncated sequences, nucleotide substitutions or other modifications may be employed. Expression systems known in the art may be used to transform virtually any plant cell under suitable conditions. A transgene comprising a DNA molecule encoding a gene of interest is preferably stably transformed and integrated into the genome of the host cells. Transformed cells are preferably regenerated into whole fertile plants. Detailed description of transformation techniques are within the knowledge of those skilled in the art.
Plant promoters can be selected to control the expression of the transgene in different plant tissues or organelles for all of which methods are known to those skilled in the art (Gasser & Fraley, 1989, Science 244: 1293-1299). In one embodiment, promoters are selected from those of eukaryotic or synthetic origin that are known to yield high levels of expression in plants and algae. In a preferred embodiment, promoters are selected from those that are known to provide high levels of expression in monocots.
Constitutive promoters include, for example, the core promoter of the Rsyn7 promoter and other constitutive promoters disclosed in WO 99/43838 and U.S. Pat. No. 6,072,050, the core CaMV 35S promoter (Odell et al., 1985, Nature 313: 810-812), rice actin (McElroy et al., 1990, Plant Cell 2: 163-171), ubiquitin (Christensen et al., 1989, Plant Mol. Biol. 12: 619-632; Christensen et al., 1992, Plant Mol. Biol. 18: 675-689), pEMU (Last et al., 1991, Theor. Appl. Genet. 81: 581-588), MAS (Velten et al., 1984, EMBO J. 3: 2723-2730), and ALS promoter (U.S. Pat. No. 5,659,026). Other constitutive promoters are described in U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463; and 5,608,142.
“Tissue-preferred” promoters can be used to target gene expression within a particular tissue. Tissue-preferred promoters include those described by Van Ex et al., 2009, Plant Cell Rep. 28: 1509-1520; Yamamoto et al., 1997, Plant J. 12: 255-265; Kawamata et al., 1997, Plant Cell Physiol. 38: 792-803; Hansen et al., 1997, Mol. Gen. Genet. 254: 337-343; Russell et al., 199), Transgenic Res. 6: 157-168; Rinehart et al., 1996, Plant Physiol. 112: 1331-1341; Van Camp et al., 1996, Plant Physiol. 112: 525-535; Canevascini et al., 1996, Plant Physiol. 112: 513-524; Yamamoto et al., 1994, Plant Cell Physiol. 35: 773-778; Lam, 1994, Results Probl. Cell Differ. 20: 181-196, Orozco et al., 1993, Plant Mol. Biol. 23: 1129-1138; Matsuoka et al., 1993, Proc. Natl. Acad. Sci. USA 90: 9586-9590, and Guevara-Garcia et al., 1993, Plant J. 4: 495-505. Such promoters can be modified, if necessary, for weak expression.
Seed-specific promoters can be used to target gene expression to seeds in particular. Seed-specific promoters include promoters that are expressed in various tissues within seeds and at various stages of development of seeds. Seed-specific promoters can be absolutely specific to seeds, such that the promoters are only expressed in seeds, or can be expressed preferentially in seeds, e.g. at rates that are higher by 2-fold, 5-fold, 10-fold, or more, in seeds relative to one or more other tissues of a plant, e.g. stems, leaves, and/or roots, among other tissues. Seed-specific promoters include, for example, seed-specific promoters of dicots and seed-specific promoters of monocots, among others. For dicots, seed-specific promoters include, but are not limited to, bean β-phaseolin, napin, β-conglycinin, soybean oleosin 1, Arabidopsis thaliana sucrose synthase, flax conlinin soybean lectin, cruciferin, and the like. For monocots, seed-specific promoters include, but are not limited to, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken 1, shrunken 2, and globulin 1.
Chemical-regulated promoters can be used to modulate the expression of a gene in a plant through the application of an exogenous chemical regulator.
Specific exemplary promoters useful for expression of genes in dicots and monocots are provided in TABLE 1 and TABLE 2, respectively.
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Glycine max
Brassica napus
Brassica napus
Brassica napus
Brassica napus
Brassica napus
Brassica napus
Brassica napus
Brassica napus
Brassica napus
Brassica napus
Brassica napus
Certain embodiments use genetically engineered plants or plant cells having multi-gene expression constructs harboring more than one transgene and promoter. The promoters can be the same or different.
Any of the described promoters can be used to control the expression of one or more of genes, their homologs and/or orthologs as well as any other genes of interest in a defined spatiotemporal manner.
Nucleic acid sequences intended for expression in genetically engineered plants are first assembled in expression cassettes behind a suitable promoter active in plants. The expression cassettes may also include any further sequences required or selected for the expression of the transgene. Such sequences include, but are not restricted to, transcription terminators, extraneous sequences to enhance expression such as introns, vital sequences, and sequences intended for the targeting of the gene product to specific organelles and cell compartments. These expression cassettes can then be transferred to the plant transformation vectors described infra. The following is a description of various components of typical expression cassettes.
A variety of transcriptional terminators are available for use in expression cassettes. These are responsible for the termination of transcription beyond the transgene and the correct polyadenylation of the transcripts. Appropriate transcriptional terminators are those that are known to function in plants and include the CaMV 35S terminator, the tm1 terminator, the nopaline synthase terminator and the pea rbcS E9 terminator. These are used in both monocotyledonous and dicotyledonous plants.
The coding sequence of the selected gene may be genetically engineered by altering the coding sequence for optimal expression in the crop species of interest. Methods for modifying coding sequences to achieve optimal expression in a particular crop species are well known (Perlak et al., 1991, Proc. Natl. Acad. Sci. USA 88: 3324 and Koziel et al., 1993, Biotechnology 11: 194-200).
Individual plants within a population of genetically engineered plants that express a recombinant gene(s) may have different levels of gene expression. The variable gene expression is due to multiple factors including multiple copies of the recombinant gene, chromatin effects, and gene suppression. Accordingly, a phenotype of the genetically engineered plant may be measured as a percentage of individual plants within a population. The yield of a plant can be measured simply by weighing. The yield of seed from a plant can also be determined by weighing. The increase in seed weight from a plant can be due to a number of factors, including an increase in the number or size of the seed pods, an increase in the number of seed and/or an increase in the number of seed per plant. In the laboratory or greenhouse seed yield is usually reported as the weight of seed produced per plant and in a commercial crop production setting yield is usually expressed as weight per acre or weight per hectare.
A recombinant DNA construct including a plant-expressible gene or other DNA of interest is inserted into the genome of a plant by a suitable method. Suitable methods include, for example, Agrobacterium tumefaciens-mediated DNA transfer, direct DNA transfer, liposome-mediated DNA transfer, electroporation, co-cultivation, diffusion, particle bombardment, microinjection, gene gun, calcium phosphate coprecipitation, viral vectors, and other techniques. Suitable plant transformation vectors include those derived from a Ti plasmid of Agrobacterium tumefaciens. In addition to plant transformation vectors derived from the Ti or root-inducing (Ri) plasmids of Agrobacterium, alternative methods can be used to insert DNA constructs into plant cells. A genetically engineered plant can be produced by selection of transformed seeds or by selection of transformed plant cells and subsequent regeneration.
In some embodiments, the genetically engineered plants are grown (e.g., on soil) and harvested. In some embodiments, above ground tissue is harvested separately from below ground tissue. Suitable above ground tissues include shoots, stems, leaves, flowers, grain, and seed. Exemplary below ground tissues include roots and root hairs. In some embodiments, whole plants are harvested and the above ground tissue is subsequently separated from the below ground tissue.
Genetic constructs may encode a selectable marker to enable selection of transformation events. There are many methods that have been described for the selection of transformed plants (for review see (Miki et al., Journal of Biotechnology, 2004, 107, 193-232) and references incorporated within). Selectable marker genes that have been used extensively in plants include the neomycin phosphotransferase gene nptII (U.S. Pat. Nos. 5,034,322, U.S. Pat. No. 5,530,196), hygromycin resistance gene (U.S. Pat. No. 5,668,298, Waldron et al., (1985), Plant Mol Biol, 5:103-108; Zhijian et al., (1995), Plant Sci, 108:219-227), the bar gene encoding resistance to phosphinothricin (U.S. Pat. No. 5,276,268), the expression of aminoglycoside 3″-adenyltransferase (aadA) to confer spectinomycin resistance (U.S. Pat. No. 5,073,675), the use of inhibition resistant 5-enolpyruvyl-3-phosphoshikimate synthetase (U.S. Pat. No. 4,535,060) and methods for producing glyphosate tolerant plants (U.S. Pat. Nos. 5,463,175; 7,045,684). Other suitable selectable markers include, but are not limited to, genes encoding resistance to chloramphenicol (Herrera Estrella et al., (1983), EMBO J, 2:987-992), methotrexate (Herrera Estrella et al., (1983), Nature, 303:209-213; Meijer et al, (1991), Plant Mol Biol, 16:807-820); streptomycin (Jones et al., (1987), Mol Gen Genet, 210:86-91); bleomycin (Hille et al., (1990), Plant Mol Biol, 7:171-176); sulfonamide (Guerineau et al., (1990), Plant Mol Biol, 15:127-136); bromoxynil (Stalker et al., (1988), Science, 242:419-423); glyphosate (Shaw et al., (1986), Science, 233:478-481); phosphinothricin (DeBlock et al., (1987), EMBO J, 6:2513-2518).
Methods of plant selection that do not use antibiotics or herbicides as a selective agent have been previously described and include expression of glucosamine-6-phosphate deaminase to inactive glucosamine in plant selection medium (U.S. Pat. No. 6,444,878) and a positive/negative system that utilizes D-amino acids (Erikson et al., Nat Biotechnol, 2004, 22, 455-8). European Patent Publication No. EP 0 530 129 A1 describes a positive selection system which enables the transformed plants to outgrow the non-transformed lines by expressing a transgene encoding an enzyme that activates an inactive compound added to the growth media. U.S. Pat. No. 5,767,378 describes the use of mannose or xylose for the positive selection of genetically engineered plants.
Methods for positive selection using sorbitol dehydrogenase to convert sorbitol to fructose for plant growth have also been described (WO 2010/102293). Screenable marker genes include the beta-glucuronidase gene (Jefferson et al., 1987, EMBO J. 6: 3901-3907; U.S. Pat. No. 5,268,463) and native or modified green fluorescent protein gene (Cubitt et al., 1995, Trends Biochem. Sci. 20: 448-455; Pan et al., 1996, Plant Physiol. 112: 893-900).
Transformation events can also be selected through visualization of fluorescent proteins such as the fluorescent proteins from the nonbioluminescent Anthozoa species which include DsRed, a red fluorescent protein from the Discosoma genus of coral (Matz et al. (1999), Nat Biotechnol 17: 969-73). An improved version of the DsRed protein has been developed (Bevis and Glick (2002), Nat Biotech 20: 83-87) for reducing aggregation of the protein.
Visual selection can also be performed with the yellow fluorescent proteins (YFP) including the variant with accelerated maturation of the signal (Nagai, T. et al. (2002), Nat Biotech 20: 87-90), the blue fluorescent protein, the cyan fluorescent protein, and the green fluorescent protein (Sheen et al. (1995), Plant J 8: 777-84; Davis and Vierstra (1998), Plant Molecular Biology 36: 521-528). A summary of fluorescent proteins can be found in Tzfira et al. (Tzfira et al. (2005), Plant Molecular Biology 57: 503-516) and Verkhusha and Lukyanov (Verkhusha, V. V. and K. A. Lukyanov (2004), Nat Biotech 22: 289-296). Improved versions of many of the fluorescent proteins have been made for various applications. It will be apparent to those skilled in the art how to use the improved versions of these proteins, including combinations, for selection of transformants.
The plants modified for enhanced yield may have stacked input traits that include herbicide resistance and insect tolerance, for example a plant that is tolerant to the herbicide glyphosate and that produces the Bacillus thuringiensis (BT) toxin. Glyphosate is a herbicide that prevents the production of aromatic amino acids in plants by inhibiting the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSP synthase). The overexpression of EPSP synthase in a crop of interest allows the application of glyphosate as a weed killer without killing the modified plant (Suh, et al., J. M Plant Mol. Biol. 1993, 22, 195-205). BT toxin is a protein that is lethal to many insects providing the plant that produces it protection against pests (Barton, et al. Plant Physiol. 1987, 85, 1103-1109). Other useful herbicide tolerance traits include but are not limited to tolerance to Dicamba by expression of the dicamba monoxygenase gene (Behrens et al, 2007, Science, 316, 1185), tolerance to 2,4-D and 2,4-D choline by expression of a bacterial aad-1 gene that encodes for an aryloxyalkanoate dioxygenase enzyme (Wright et al., Proceedings of the National Academy of Sciences, 2010, 107, 20240), glufosinate tolerance by expression of the bialophos resistance gene (bar) or the pat gene encoding the enzyme phosphinotricin acetyl transferase (Droge et al., Planta, 1992, 187, 142), as well as genes encoding a modified 4-hydroxyphenylpyruvate dioxygenase (HPPD) that provides tolerance to the herbicides mesotrione, isoxaflutole, and tembotrione (Siehl et al., Plant Physiol, 2014, 166, 1162).
The genetically engineered land plant that expresses a plant-CCP1 like mitochondrial transporter protein, as disclosed, can be further modified for further enhanced yield too.
Initial Attempts to Identify CCP1-Like Orthologs in Land Plants
Initial attempts to determine whether land plants encode CCP1 orthologs suggested that land plants do not. Typical BLAST searches do not reveal CCP1 homologs in higher plants. For example, a conventional BLAST search using CCP1 of Chlamydomonas reinhardtii as the query sequence and the standard protein database (nr) did not yield any Tier 1 CCP1 ortholog matches from higher plants. The top hits in that type of search are shown in TABLE 3.
reinhardtii]
reinhardtii]
reinhardtii]
pectorale]
pectorale]
Strikingly, the results reveal only three non-CCP1 hits, corresponding to hypothetical proteins of the algae Gonium pectorale (KXZ50472.1), Volvox carteri f. nagariensis (XP_002951243.1), and Gonium pectorale (KXZ50486.1), respectively, all with 80+% identity to CCP1, then an immediate drop-off to a spinach protein with only 37% identity. Following the spinach protein are hundreds of proteins with 30+% identity that probably derive most of their identity from the mere fact that they are mitochondrial carrier proteins.
Successful Identification of CCP1-Like Orthologs in Land Plants
Serendipitously, higher-plant homologs to CCP1 were found in the Transcriptome Shotgun Assembly (tsa_nr) database based on further sequence comparisons. This revealed that land plants do encode CCP1 orthologs. This also implied that the only higher plants that contain CCP1 homologs have yet to be genome-sequenced.
Results are shown in TABLE 4 and TABLE 5.
Chlamydomonas
reinhardtii
Gonium pectorale
Gonium pectorale
Volvox carteri f.
nagariensis
Ettlia
353a
oleoabundans
Erigeron
352a
breviscapus
Zea nicaraguensis
354a
Poa pratensis
141d
Cosmos
bipinnatus
Talaromyces
stipitatus
e
Saitoella
complicata
e
aSequence from first methionine of deposited transcribed mRNA sequence to first stop codon.
bWebsite: genome.jp/tools/motif
cWebsite: prosite.expasy.org
dPartial protein sequence
eTop two Tier 2 CCP1 orthologs in tblastn search shown for comparison.
Chlamydomonas
reinhardtii
Glycine max
Zea mays
Oryza sativa
Triticum aestivum
Sorghum bicolor
Solanum tuberosum
The results indicate that certain land plants encode orthologs of algal CCP1 of Chlamydomonas reinhardtii. Moreover, the plant CCP1-like mitochondrial transporter proteins encoded by these land plants appear to cluster into two groups, termed Tier 1 CCP1 orthologs and Tier 2 CCP1 orthologs, based on sequence and structural similarity to CCP1. As shown in TABLE 4, the plant Tier 1 CCP1 orthologs exhibit about 60% sequence identity in comparison to CCP1 of Chlamydomonas reinhardtii, cluster narrowly based on their similar degrees of identity, and have been identified thus far only in four plant species, Zea nicaraguensis (also termed teosinte), Erigeron breviscapus, Cosmos bipinnatus, and Poa pratensis, none of which are particularly closely related phylogenetically. As shown in TABLE 5, the plant Tier 2 CCP1 orthologs exhibit about 30% sequence identity in comparison to CCP1 of Chlamydomonas reinhardtii, substantially lower than for Tier 1, also cluster narrowly based on their similar degrees of identity, and would appear to be more common, having been identified thus far in six major crop species, Zea mays (also termed maize), Triticum aestivum, Solanum tuberosum, Glycine max, Oryza sativa, and Sorghum bicolor. This was surprising because there had not been any apparent reason to expect any clustering of plant CCP1-like mitochondrial transporter proteins, let alone clustering into two distinct groups. This was also surprising because Zea nicaraguensis, again teosinte, is a wild progenitor of Zea mays, again maize, and Zea nicaraguensis includes a Tier 1 CCP1 ortholog, whereas Zea mays includes a Tier 2 CCP1 ortholog.
It also has been determined that further clustering occurs within the Tier 1 CCP1 orthologs, with several algal Tier 1 CCP1 orthologs, namely those of Gonium pectorale (KXZ50472.1), Gonium pectorale (KXZ50486.1), and Volvox carteri f. nagariensis, termed Tier 1A, exhibiting about 80% sequence identity in comparison to CCP1 of Chlamydomonas reinhardtii, and with one algal Tier 1 CCP1 ortholog, namely that of Ettlia oleoabundans, termed Tier 1B, instead exhibiting 60% sequence identity and clustering with the plant Tier 1 CCP1 orthologs, also termed Tier 1B. Strikingly, the algal and plant Tier 1B CCP1 orthologs seem to be more closely related to each other than to the other algal or plant CCP1 orthologs, suggesting the intriguing possibility that the plant Tier 1B CCP1 orthologs may have resulted from horizontal gene transfer from Ettlia oleoabundans or related algae. This also suggests that Zea nicaraguensis and the other plant species encoding Tier 1B CCP1 orthologs may serve as sources of CCP1 orthologs that are proximally derived from land plants, rather than from algae, thus decreasing regulatory concerns and risk associated with genetic modification of crops, while providing increases in crop yield comparable to those observed for CCP1 of Chlamydomonas reinhardtii and CCP1 orthologs derived from other algae.
Considering the results in more detail, Tier 1A CCP1 orthologs are very similar to CCP1 and include only the other algae Volvox and Gonium. These algal CCP1 orthologs are 80+% identical to CCP1. Tier 1B identity drops to 60+%, but Phobius plots of transmembrane domains of these proteins continue to look very similar to that of CCP1, whereas Phobius plots of Tier 2 proteins do not.
Tier 1B includes just one alga, Ettlia oleoabundans, and several higher plants, suggesting that Ettlia oleoabundans may be the source of the CCP1 homolog in higher plants, or at least that Ettlia oleoabundans and the higher plants ultimately acquired the CCP1 homolog from a common source.
Plants that Encode Tier 1B CCP1 Orthologs
Considering the plants that encode Tier 1B CCP1 orthologs in more detail, these plants exhibit some distinctive characteristics.
Zea nicaraguensis is a wild progenitor of maize that thrives along often-flooded banks of rivers and streams, so it is tempting to speculate that it acquired its CCP1 ortholog from a species of algae that populates the waters nearby. The original paper that describes Zea nicaraguensis says of it: “Now evidently extremely local and rare, the teosinte at this location is remarkable for its ability to grow in as much as 0.4 m of standing or slowly moving water,” and that “we anticipate that this species will provide maize breeders with a potentially valuable source of germ plasm that may lead to the development of maize capable of growing in water-logged soils” (Iltis et al., Novon 10:382-390 (2000)).
Erigeron breviscapus is a flower used for medicinal purposes found at higher elevations in China. Distribution of Erigeron breviscapus has been described as follows: “Mid-elevation mountains, alpine to montane meadows, forest margins, Pinus forests, streamsides, grasslands, disturbed slopes, roadsides; 1200-3600 m. Guangxi, Guizhou, Hunan, Sichuan, E and S Xizang, Yunnan” (website: efloras.org). So Erigeron breviscapus, like Zea nicaraguensis, is found on stream banks as well.
Cosmos bipinnatus is a large aster that grows in temperate climates. Cosmos bipinnatus is used as an ornamental flower, but can spread as a weed.
Poa pratensis is native to North America, according to the USDA (National Resources Conservation Service, USDA, Plant Guide: Kentucky Blue Grass, Poa pratensis L., website: plants.usda.gov/plantguide/pdf/pg_popr.pdf). Poa pratensis grows preferentially in cool and humid climates and is a common dominant of Midwestern prairies.
Homology Searches
Considering approaches for identifying CCP1 orthologs in land plants in more detail, various BLAST searches (e.g. tblastn; website blast.ncbi.nlm.nih.gov/Blast.cgi) were conducted using a translated nucleotide database, a whole-genome shotgun (also termed WGS) database, and a transcriptome assembly (also termed TSA) database to find sequences highly similar to the CCP1 protein from Chlamydomonas reinhardtii in land plants and inedible algae species (TABLE 4 and TABLE 5). Several sequences with 60% or greater identity to CCP1 were found, followed by a much larger number of sequences with identities of about 30% and below, with no representatives in between. As noted above, these groups were named Tier 1 and Tier 2, respectively. Publicly available internet algorithms were used to predict putative transmembrane regions to further characterize the sequences, including Motif Finder (website: genome.jp/tools/motif/), ProSite (website: prosite.expasy.org/), and Phobius (website: phobius.sbc.su.se/). The Motif Finder program identified Mito_carr (PF00153) domains in each of the Tier 1 proteins (TABLE 4), indicating that they are likely mitochondrial carrier proteins that transport solutes into and out of mitochondria (website: pfam.xfam.org/family/PF00153). The ProSite program predicted that CCP1 and the Tier 1 proteins contain SOLCAR (PS50920) domains (TABLE 4), indicating that they are likely solute carrier proteins involved in energy transfer in the inner mitochondrial membrane (website: prosite.expasy.org/cgi-bin/prosite/nicedoc.pl?PS50920). The Phobius tool (website: phobius.sbc.su.se) was used to compare predicted transmembrane domains of the proteins to those of CCP1 (
Multiple Sequence Alignment
Multiple sequence alignments of CCP1 of Chlamydomonas reinhardtii and the orthologs described above were prepared using a Multiple Sequence Alignment tool (EMBL-EBI; ebi.ac.uk/Tools/msa/clustalo/).
Common Features
There are several features shared by the orthologs that now can be used to identify further representatives as sequence data of additional plants become available. Aside from their high degree of identity to CCP1 (60% or greater), the Tier 1 CCP1 orthologs also share very similar transmembrane architecture (
Chlamydomonas
reinhardtii
Erigeron
breviscapus
Zea
nicaraguensis
Gonium
pectorale 16g646
Gonium
pectorale 16g661
Volvox carteri f.
nagariensis
Ettlia
oleoabundans
Cosmos
bipinnatus
When defining a class of plant genes or proteins such as those with functions complementary to, or similar to, CCP1 of Chlamydomonas reinhardtii, it is beneficial to utilize a screen, selection, or other test that identifies candidates as members or non-members of the useful family. The most thorough screen of such activity is in whole plants over a sustained period to insure that yield and efficiency of carbon capture are indeed improved. However, a more-facile screen in a simpler system that requires less time and still serves as a good predictor of yield improvement by virtue of demonstration of similar function to CCP1 would be valuable. There are many systems in which such a screen could reasonably be conducted, of which some examples are as follows.
Yeast
A useful eukaryotic model system is Saccharomyces cerevisiae, whose genome has been sequenced and for which databases with functional information such as that hosted by Stanford University (website: yeastgenome.org) are available. Knockout mutants and libraries are available for this organism, such as the Yeast Knockout Collection at GE Life Sciences (website: dharmacon.gelifesciences.com). CCP1-like candidates can therefore be expressed in yeast using standard molecular biology techniques to complement various known yeast mitochondrial transporter mutants in order to classify the candidates according to function and identify whether or not they are similar in function to CCP1. An example of this approach is found in Herzig et al., Science 337:93-96 (2012), in which mitochondrial transporters from mouse complemented yeast mutants deficient in the ability to transport pyruvate into the mitochondrion.
Escherichia coli
The Gram-negative bacterium E. coli can serve as a model for mitochondria, because both systems have a double-membrane structure. Using standard techniques of molecular biology and bacterial transformation, CCP1 orthologs can be expressed functionally in E. coli and the resulting phenotype examined. Mutants of E. coli lacking one or more transporter proteins can be especially useful in this regard. E. coli mutants are widely available, such as in the Keio collection, which contains all single-gene mutants producing viable cells (website: cgsc2.biology.yale.edu/KeioList.php). For example, ADP/ATP carrier proteins from various plants were functionally expressed and characterized in E. coli (Haferkamp et al., Eur. J. Biochem. 269:3172 (2002)), in which the transport of radiolabelled ADP and ATP was measured.
Lactococcus lactis
The Gram-positive bacterium Lactococcus lactis has only a single cell membrane and is amenable to genetic manipulation. Therefore, standard molecular biology techniques can be utilized to introduce CCP1 homologs into this organism as a screening platform. An example of this approach can be found in Kunji et al., Biochimica et Biophysica Acta 1610:97 (2003), in which eukaryotic mitochondrial carrier proteins were functionally expressed and characterized using transport of radiolabelled ATP in both intact cells and in membrane vesicles prepared from whole cells.
Isolated Mitochondria
Direct methods for the measurement of mitochondrial solute transport exist, such as those outlined in Palmieri and Klingenberg, Methods Enzymol. 56:279 (1979). Such methods can be used, for example, on yeast mitochondria expressing CCP1 vs. wild-type yeast mitochondria or mitochondria isolated from various yeast mutants. Such tests can also be carried out on mitochondria isolated from Chlamydomonas reinhardtii (wild-type vs. CCP1 mutants).
Liposomes
Mitochondrial carrier proteins can be expressed to high levels in a facile system such as E. coli and reconstituted into liposomes. For example, the Arabidopsis thaliana mitochondrial basic amino acid carrier AtmBAC1 was expressed in E. coli, purified, and reconstituted into phospholipid vesicles and was shown to transport arginine, lysine, ornithine, and histidine (Hoyos et al., Plant J. 33:1027 (2003)).
Chlamydomonas reinhardtii
It has been shown, for example by Pollock et al., Plant Mol. Biol. 56:125 (2004), that Chlamydomonas reinhardtii double mutants in CCP1 and CCP2 suffer growth defects in long-term (>48-hour) cultures. Therefore, a complementation test can be used with such mutants that defines CCP1 complementation as the ability of a gene to complement the loss of CCP1 and CCP2 in Chlamydomonas reinhardtii by restoring long-term growth rates to normal.
For Agrobacterium-mediated transformation of maize, a binary vector containing a promoter, the CCP1 gene, and a terminator is constructed and an expression cassette for a selectable marker, such as the bar gene imparting resistance to the herbicide bialophos, are included.
pYTEN-5 (SEQ ID NO: 49;
In preparation for transformation, pYTEN-5 is transformed into an Agrobacterium tumefaciens strain, such as A. tumefaciens strain EHA101. Agrobacterium-mediated transformation of maize can be performed following a previously described procedure (Frame et al., 2006, Agrobacterium Protocols Wang K., ed., Vol. 1, pp 185 199, Humana Press) as follows.
Plant Material: Plants grown in a greenhouse are used as an explant source. Ears are harvested 9-13 d after pollination and surface sterilized with 80% ethanol.
Explant Isolation, Infection and Co-Cultivation: Immature zygotic embryos (1.2-2.0 mm) are aseptically dissected from individual kernels and incubated in A. tumefaciens strain EHA101 culture (grown in 5 ml N6 medium supplemented with 100 μM acetosyringone for stimulation of the bacterial vir genes for 2-5 h prior to transformation) at room temperature for 5 min. The infected embryos are transferred scutellum side up on to a co-cultivation medium (N6 agar-solidified medium containing 300 mg/l cysteine, 5 μM silver nitrate and 100 μM acetosyringone) and incubated at 20° C., in the dark for 3 d. Embryos are transferred to N6 resting medium containing 100 mg/l cefotaxime, 100 mg/l vancomycin and 5 μM silver nitrate and incubated at 28° C., in the dark for 7 d.
Callus Selection: All embryos are transferred on to the first selection medium (the resting medium described above supplemented with 1.5 mg/l bialaphos) and incubated at 28° C., in the dark for 2 weeks followed by subculture on a selection medium containing 3 mg/l bialaphos. Proliferating pieces of callus are propagated and maintained by subculture on the same medium every 2 weeks.
Plant Regeneration and Selection: Bialaphos-resistant embryogenic callus lines are transferred on to regeneration medium I (MS basal medium supplemented with 60 g/l sucrose, 1.5 mg/l bialaphos and 100 mg/l cefotaxime and solidified with 3 g/l Gelrite) and incubated at 25° C., in the dark for 2 to 3 weeks. Mature embryos formed during this period are transferred on to regeneration medium II (the same as regeneration medium I with 3 mg/l bialaphos) for germination in the light (25° C., 80-100 μE/m2/s light intensity, 16/8-h photoperiod). Regenerated plants are ready for transfer to soil within 10-14 days.
pYTEN-6 (SEQ ID NO: 50;
In DNA fragment pYTEN-6, the CCP1 gene from Z. nicaraguensis is expressed from the hybrid maize cab5 promoter containing the maize HSP70 intron. There is an NPTII gene, encoding neomycin phosphotransferase from Escherichia coli K-12, conferring resistance to kanamycin for selection of transformants. The NPTII gene is expressed from the maize ubiquitin promoter with a 3′ UTR from the maize ubiquitin gene. It will be apparent to those skilled in the art that many selectable markers can be used that are not derived from plant pest sequences for selection purposes. These include maize acetolactate synthase/acetohydroxy acid synthase (ALS/AHAS) mutant genes conferring resistance to a range of herbicides from the ALS family of herbicides, including chlorsulfuron and imazethapyr; a 5-enolpyruvoylshikimate-3-phosphate synthase (EPSPS) mutant gene from maize, providing resistance to glyphosate; as well as multiple other selectable markers that are all reviewed in Que et al., 2014 (Que, Q. et al., Front. Plant Sci. 5 Aug. 2014; doi.org/10.3389/fpls.2014.00379).
DNA fragment pYTEN-6 can be transformed into maize protoplasts, calli, or immature embryos using biolistics as reviewed in Que et al., 2014.
In some cases, it will be advantageous to express CCP1 from a seed-specific promoter. There are many seed-specific promoters known and it will be apparent to those skilled in the art that seed-specific promoters from multiple different sources can be used to practice the invention, including the seed-specific promoters listed in TABLE 2.
DNA fragment pYTEN-7 (SEQ ID NO: 51;
DNA fragment pYTEN-7 can be transformed into maize protoplasts, calli, or immature embryos using biolistics as reviewed in Que et al, 2014.
Transformation of protoplasts of Brassica napus can be performed as follows.
To express the CCP1-like gene from Z. nicaraguensis in canola, a linear DNA fragment, pYTEN-8 (SEQ ID NO: 52;
Protoplast isolation: Seeds of Brassica napus are surface sterilized with 70% ethanol for 2 min followed by gentle shaking in 0.4% hypochlorite solution for 20 min. The seeds are washed three times in double distilled water, and sown on sterilized ½ MS media in Petri plates that are placed without the lids in sterile MAGENTA jars. Protoplasts are isolated from 40 newly expanding leaves of Brassica plants. The mid vein is removed and the abaxial surface of the leaves are gently scored with a sterile scalpel. The leaves are then floated with abaxial side down in Petri plates containing 15 ml of Enzyme B2 solution (B5 salts, 1% Onozuka R 10, 0.2% Macerozyme R 10, 13% sucrose, 5 mM CaCl2·2H2O, 0.5% Polyvinylpyrrolidone, 1 mg/L NAA, 1 mg/L 2, 4-D, 1 mg/L BA, MES 0.05%, pH 6.0). Petri plates are sealed with PARAFILM and leaves incubated overnight at 22° C. in the dark without shaking. Following the overnight incubation the plates are gently agitated by hand and incubation continued for 15-20 min on a rotary shaker set at 20 rpm. The digested material, consisting of a crude protoplast suspension, is then filtered through a funnel lined with 63 μm nylon screen and the filtrate collected in 50 ml falcon centrifuge tubes. An equal volume of 17% B5 wash solution (B5 salts, 5 mM CaCl2·2H2O, 17% sucrose, 0.06% IVIES, pH 6.0) is added to the filtrate and centrifuged at 100 g for 10 minutes. The protoplast enriched fraction (˜4 ml) floating in the form of a ring is carefully removed and transferred to fresh 15 ml FALCON tubes and 11 ml of WW5-2 media (0.1 M CaCl2·2H2O, 0.2 M NaCl, 4 mM KCl, 0.08% Glucose, 0.1% MES, pH 6.0) is added per tube. The resulting suspension is gently mixed by inversion and then centrifuged at 100 g for 5 minutes. After centrifugation the supernatant is carefully decanted and discarded and the pellet consisting of an enriched protoplast fraction is retained. Protoplasts are washed twice with WW5-2 solution followed by centrifugation at 100 g and resuspended in 5 ml of WW5-2 media. The density of protoplasts is counted with a hemocytometer using a small drop of the protoplast suspension. The suspension is cooled in a refrigerator (2-8° C.) for 40-45 min.
Brassica napus protoplast transfection and culture: For protoplast transfection, the protoplasts after cold incubation are pelleted by centrifugation at 100 g for 3 minutes and then resuspended in WMMM media (15 mM MgCl2-6H2O, 0.4 M Mannitol, 0.1 M (CaNO3)2, 0.1% MES, pH 6) to a density of 2×106 protoplasts per ml. 500 μl of protoplast suspension is dispensed into 15 ml FALCON tubes and 50 μl of a mixture consisting of 50 μg DNA of linear DNA fragment pYTEN-8 is added to protoplast suspension and mixed by shaking. 500 μl of PEGB2 (40% PEG 4000, 0.4 M Mannitol, 0.1 M Calcium Nitrate, 0.1% MES, pH 6.0) is added gently to protoplast DNA mixture while continuously shaking the tube. The mixture is incubated for 20 min with periodic gentle shaking. Subsequently WW5-2 media is gradually added in two stages, first a 5 ml aliquot of WW5-2 is added to the protoplast mixture which is then allowed to incubate for 10 minutes followed by addition of a second 5 ml aliquot of WW5-2 solution and incubation for 10 min. After the second incubation, the protoplasts are carefully resuspended and then pelleted by centrifugation. The protoplast pellet is resuspended in 12 ml of WW5-2 solution then pelleted by centrifugation at 100 g for 5 min. The pellet is washed once more in 10 ml of WW5-2 then pelleted by centrifugation at 100 g for 3 min. The protoplast pellet is resuspended in K3P4 medium (Kao's basal salts, 6.8% Glucose, 1% MES, 0.5% Ficoll 400, 2 mM CaCl2·2H2O, 1 mg/L 2, 4-D, 1 mg/L NAA, 1 mg/L Zeatin, pH 5.8, 200 mg/L Carbenicillin, 200 mg/L Cefotaxime) at a density of 1×105 protoplasts per ml and 1.5 ml of the suspension is dispensed per 60×15 mm petri plate. The plates are sealed with PARAFILM and maintained in plastic boxes with opaque lids at 22° C., 16 h photoperiod, under dim fluorescent lights (25 μEm−2 s−1).
Brassica napus, Proliferation of calli and regeneration of lines: After 4-5 days the protoplast cultures are fed with 1-1.25 ml of medium consisting of a 1:1 mixture of K3P4 medium and EmBed BI medium (MS Basal salts, 3.4% sucrose, 0.05% MES, 1 mg/L NAA, 1 mg/L 2,4-D and 1 mg/L BA, pH 6.0). The plates are resealed and placed under dim light for 1-2 days and then under medium light (60-80 μEm−2 s−1). After 4-5 days, the protoplasts are fed with 4.5 ml of a 3:1 mixture of K3P4: Embed BI medium. The plate contents are then transferred to a 100×75 mm plate and 3 ml of lukewarm Embed BI medium containing 2.1% SeaPlaque agarose is added to the protoplast suspension. The contents of the plate are swirled to gently mix the protoplast suspension with the semi-solid media and the plates are allowed to solidify in the tissue culture flow hood. Plates are sealed and cultured under dim light conditions for a week. After 7-9 days, the embedded protoplast cultures in each plate are cut into 6-8 wedges and transferred onto two plates of Proliferation B1 media (MS Basal salts, 3.4% sucrose, 0.05% MES, 1 mg/L NAA, 1 mg/L 2,4-D and 1 mg/L BA, pH 6.0, 0.8% sea plaque agarose, 200 mg/L Carbenicillin, 200 mg/L Cefotaxime) with 60 mg/L L-phosphinothricin for selection. Proliferation plates are incubated under dim light for the first 1-2 days and then moved to bright light (150 μEm−2 s−1). Green surviving colonies are obtained after 3 to 4 weeks at which point they are transferred to fresh Proliferation B 1 plates for an additional 2-3 weeks. Large green calli are transferred to Regeneration B2 Plates (MS Basal salts, 3% sucrose, 30 μM AgNO3, 0.05% polyvinylpyrrolidone, 0.05% MES, 0.1 mg/L NAA, 5 mg/l N6-(2-isopentenyl)adenine (2-iP), 0.1 μg/L GA3, pH 5.8, 0.8% sea plaque agarose, 100 mg/L Carbenicillin, 100 mg/L Cefotaxime) with 10 mg/L L-phosphinothricin for selection. Calli are transferred to fresh Regeneration B2 plates every 3 to 4 weeks. Shoots with normal morphology are transferred to rooting medium (B5 salts+0.1 mg/L NAA) and incubated under dim light conditions. Plantlets are potted in a soilless mix (Sunshine Mix 4) in 6 inch (15 cm) pots and irrigated with NPK (20-20-20) fertilizer. Plantlets are acclimatized under plastic cups for 5-6 days and maintained in growth room at 22° C./18° C. and 16 hour photoperiod under 200-300 μEm−2 s−1 light.
Plants are allowed to set seed (T1 seed). T1 seeds are harvested and planted in soil and grown in a greenhouse. Plants are grown to maturity and T2 seed is harvested. Seed yield per plant and oil content of the seeds is measured.
A vector containing the Z. nicaraguensis CCP1 gene under the control of a seed-specific promoter from the soya bean oleosin isoform A gene is constructed. Plasmid pYTEN-9 (SEQ ID NO: 53;
The purified DNA fragment containing the CCP1 expression cassette is co-bombarded with DNA encoding an expression cassette for the hygromycin resistance gene via biolistics into embryogenic cultures of soybean Glycine max cultivars X5 and Westag97, to obtain transgenic plants. The hygromycin resistance gene is expressed from a plant promoter, such as the soybean actin promoter (SEQ ID NO: 42) and the 3′ UTR from the soybean actin gene (soybean actin Gene ID Glyma.19G147900).
The transformation, selection, and plant regeneration protocol is adapted from Simmonds (2003) (Simmonds, 2003, Genetic Transformation of Soybean with Biolistics. In: Jackson J F, Linskens H F (eds) Genetic Transformation of Plants. Springer Verlag, Berlin, pp 159-174) and is performed as follows.
Induction and Maintenance of Proliferative Embryogenic Cultures: Immature pods, containing 3-5 mm long embryos, are harvested from host plants grown at 28/24° C. (day/night), 15-h photoperiod at a light intensity of 300-400 μmol m−2 s−1. Pods are sterilized for 30 s in 70% ethanol followed by 15 min in 1% sodium hypochlorite [with 1-2 drops of Tween 20 (Sigma, Oakville, ON, Canada)] and three rinses in sterile water. The embryonic axis is excised and explants are cultured with the abaxial surface in contact with the induction medium [MS salts, B5 vitamins (Gamborg O L, Miller R A, Ojima K. Exp Cell Res 50:151-158), 3% sucrose, 0.5 mg/L BA, pH 5.8), 1.25-3.5% glucose (concentration varies with genotype), 20 mg/l 2,4-D, pH 5.7]. The explants, maintained at 20° C. at a 20-h photoperiod under cool white fluorescent lights at 35-75 μmol m−2 s−1, are sub-cultured four times at 2-week intervals. Embryogenic clusters, observed after 3-8 weeks of culture depending on the genotype, are transferred to 125-ml Erlenmeyer flasks containing 30 ml of embryo proliferation medium containing 5 mM asparagine, 1-2.4% sucrose (concentration is genotype dependent), 10 mg/12,4-D, pH 5.0 and cultured as above at 35-60 μmol m−2 s−1 of light on a rotary shaker at 125 rpm. Embryogenic tissue (30-60 mg) is selected, using an inverted microscope, for subculture every 4-5 weeks.
Transformation: Cultures are bombarded 3 days after subculture. The embryogenic clusters are blotted on sterile Whatman filter paper to remove the liquid medium, placed inside a 10×30-mm Petri dish on a 2×2 cm2 tissue holder (PeCap, 1 005 μm pore size, Band SH Thompson and Co. Ltd. Scarborough, ON, Canada) and covered with a second tissue holder that is then gently pressed down to hold the clusters in place. Immediately before the first bombardment, the tissue is air dried in the laminar air flow hood with the Petri dish cover off for no longer than 5 min. The tissue is turned over, dried as before, bombarded on the second side and returned to the culture flask. The bombardment conditions used for the Biolistic PDS-I000/He Particle Delivery System are as follows: 737 mm Hg chamber vacuum pressure, 13 mm distance between rupture disc (Bio-Rad Laboratories Ltd., Mississauga, ON, Canada) and macrocarrier. The first bombardment uses 900 psi rupture discs and a microcarrier flight distance of 8.2 cm, and the second bombardment uses 1100 psi rupture discs and 11.4 cm microcarrier flight distance. DNA precipitation onto 1.0 μm diameter gold particles is carried out as follows: 2.5 μl of 100 ng/μl of insert DNA of pYTEN-9 and 2.5 μl of 100 ng/μl selectable marker DNA (cassette for hygromycin selection) are added to 3 mg gold particles suspended in 50 μl sterile dH2O and vortexed for 10 sec; 50 μl of 2.5 M CaCl2 is added, vortexed for 5 sec, followed by the addition of 20 μl of 0.1 M spermidine which is also vortexed for 5 sec. The gold is then allowed to settle to the bottom of the microfuge tube (5-10 min) and the supernatant fluid is removed. The gold/DNA is resuspended in 200 μl of 100% ethanol, allowed to settle and the supernatant fluid is removed. The ethanol wash is repeated and the supernatant fluid is removed. The sediment is resuspended in 120 μl of 100% ethanol and aliquots of 8 μl are added to each macrocarrier. The gold is resuspended before each aliquot is removed. The macrocarriers are placed under vacuum to ensure complete evaporation of ethanol (about 5 min).
Selection: The bombarded tissue is cultured on embryo proliferation medium described above for 12 days prior to subculture to selection medium (embryo proliferation medium contains 55 mg/l hygromycin added to autoclaved media). The tissue is sub-cultured 5 days later and weekly for the following 9 weeks. Green colonies (putative transgenic events) are transferred to a well containing 1 ml of selection media in a 24-well multi-well plate that is maintained on a flask shaker as above. The media in multi-well dishes is replaced with fresh media every 2 weeks until the colonies are approximately 2-4 mm in diameter with proliferative embryos, at which time they are transferred to 125 ml Erlenmeyer flasks containing 30 ml of selection medium. A portion of the proembryos from transgenic events is harvested to examine gene expression by RT-PCR.
Plant regeneration: Maturation of embryos is carried out, without selection, at conditions described for embryo induction. Embryogenic clusters are cultured on Petri dishes containing maturation medium (MS salts, B5 vitamins, 6% maltose, 0.2% gelrite gellan gum (Sigma), 750 mg/l MgCl2, pH 5.7) with 0.5% activated charcoal for 5-7 days and without activated charcoal for the following 3 weeks. Embryos (10-15 per event) with apical meristems are selected under a dissection microscope and cultured on a similar medium containing 0.6% phytagar (Gibco, Burlington, ON, Canada) as the solidifying agent, without the additional MgCl2, for another 2-3 weeks or until the embryos become pale yellow in color. A portion of the embryos from transgenic events after varying times on gelrite are harvested to examine gene expression by RT-PCR.
Mature embryos are desiccated by transferring embryos from each event to empty Petri dish bottoms that are placed inside MAGENTA boxes (Sigma) containing several layers of sterile Whatman filter paper flooded with sterile water, for 100% relative humidity. The MAGENTA boxes are covered and maintained in darkness at 20° C. for 5-7 days. The embryos are germinated on solid B5 medium containing 2% sucrose, 0.2% gelrite and 0.075% MgCl2 in Petri plates, in a chamber at 20° C., 20-h photoperiod under cool white fluorescent lights at 35-75 μmol m−2 s−1. Germinated embryos with unifoliate or trifoliate leaves are planted in artificial soil (Sunshine Mix No. 3, SunGro Horticulture Inc., Bellevue, WA, USA), and covered with a transparent plastic lid to maintain high humidity. The flats are placed in a controlled growth cabinet at 26/24° C. (day/night), 18 h photoperiod at a light intensity of 150 μmol m−2 s−1. At the 2-3 trifoliate stage (2-3 weeks), the plantlets with strong roots are transplanted to pots containing a 3:1:1:1 mix of ASB Original Grower Mix (a peat-based mix from Greenworld, ON, Canada):soil:sand:perlite and grown at 18-h photoperiod at a light intensity of 300-400 μmolm−2 s−1.
T1 seeds are harvested and planted in soil and grown in a controlled growth cabinet at 26/24° C. (day/night), 18 h photoperiod at a light intensity of 300-400 μmol m−2 s−1. Plants are grown to maturity and T2 seed is harvested. Seed yield per plant and oil content of the seeds is measured.
The following are exemplary embodiments of the genetically engineered land plant that expresses a plant CCP1-like mitochondrial transporter protein as disclosed herein.
The invention has been described with reference to the example embodiments described above. Modifications and alterations will occur to others upon a reading and understanding of this specification. Examples embodiments incorporating one or more aspects of the invention are intended to include all such modifications and alterations insofar as they come within the scope of the appended claims.
The material in the ASCII text file, named “YTEN-57557WO-Sequences_ST25.txt”, created Jun. 12, 2018, file size of 159,744 bytes, is hereby incorporated by reference.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2018/037740 | 6/15/2018 | WO |
| Publishing Document | Publishing Date | Country | Kind |
|---|---|---|---|
| WO2018/232232 | 12/20/2018 | WO | A |
| Number | Name | Date | Kind |
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| 5593874 | Brown | Jan 1997 | A |
| 20050108791 | Edgerton | May 2005 | A1 |
| 20090130710 | Kermode | May 2009 | A1 |
| 20100229256 | Somleva | Sep 2010 | A1 |
| 20110321190 | Akula et al. | Dec 2011 | A1 |
| 20120060413 | Somleva | Mar 2012 | A1 |
| 20120216318 | La Rosa | Aug 2012 | A1 |
| 20130333061 | Wu | Dec 2013 | A1 |
| 20150307890 | Wu et al. | Oct 2015 | A1 |
| 20160186197 | Pankratov | Jun 2016 | A1 |
| 20160326541 | Schnell | Nov 2016 | A1 |
| 20180291352 | Malik | Oct 2018 | A1 |
| 20190338302 | Peoples | Nov 2019 | A1 |
| 20200370063 | Skraly | Nov 2020 | A1 |
| Number | Date | Country |
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| 2014188428 | Nov 2014 | WO |
| 2015103074 | Jul 2015 | WO |
| 2016164810 | Oct 2016 | WO |
| 2017091309 | Jun 2017 | WO |
| 2017136668 | Aug 2017 | WO |
| 2018156686 | Aug 2018 | WO |
| 2019104278 | May 2019 | WO |
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