Plants containing bromoform-producing genes

Abstract

The present invention provides a system of recombinant nucleic acid molecules comprising two or more genes encoding two or more enzymes involved in bromoform production for expression in plants, a bromoform-producing plant or plant part prepared by transforming a plant using the system, and a method for using the transformed plant or plant part for feeding to ruminants in order to reduce methane emissions caused by ruminants.

Description

Technical Field

[0001] The present disclosure generally relates to transgenic plants. Specifically, the present invention relates to transgenic plants for use in reducing greenhouse gas emissions in ruminant animals.

Background Art

[0002] Agriculture plays an important role in food production worldwide and is a major component of the gross domestic product in several countries. Livestock production is essential for the production of high-quality protein foods and the delivery of food in regions where animal-based foods are the main food source. The environmental impact of livestock production has been investigated for decades, but recently, methane emissions from enteric fermentation have been targeted as a substantial source of greenhouse gases.

[0003] Methane (CH4) is recognized as the second most important greenhouse gas emitted from anthropogenic sources and is mainly due to the microbial activity of methanogenic bacteria in the rumen of ruminant animals. Ruminant animals are animals with a multi-chambered stomach that rely on bacteria to break down plant-based foods. In this digestive process, fully grown cows can emit up to 500 liters of methane into the atmosphere per day, which contributes approximately 17% of the total global anthropogenic methane emissions.

[0004] Asparagopsis taxiformis is a type of red algae. Researchers have demonstrated that feeding dairy cows a diet containing 1-2% of this algae reduces methane emissions by more than 90%. As further shown, this effect was achieved without impairing other fermentation parameters (i.e., volatile fatty acid production) and was observed at Asparagopsis taxiformis organic matter content up to 5%. This effect was found to be due to the inhibition of fermentation by methanogenic bacteria in the rumen by brominated organic hydrocarbons such as bromoform present in Asparagopsis taxiformis. However, bromoform-producing seaweeds like Asparagopsis taxiformis grow relatively slowly in deep-sea water, and therefore, their large-scale production is cost-ineffective.

[0005] Several attempts have been made to clone vanadate-dependent haloperoxidases (VHPOs) derived from algae and bacteria into various organisms, but this has been difficult due to low yields and toxicity issues.

[0006] Therefore, there is still a need for efficient systems to feed livestock while reducing methane emissions. [Overview of the project]

[0007] The following embodiments and aspects are described and illustrated in conjunction with compositions and methods, meaning to be illustrative and not limiting in scope. While some of the above-described problems are reduced or eliminated in various embodiments, other embodiments cover other advantages or improvements.

[0008] The present invention relates to the preparation of transgenic forage plants and fodder plants possessing bromoform-producing genes from red or brown algae in order to reduce methane emissions caused by ruminants.

[0009] Advantageously, genetically modified bromoform-producing pasture plants can be sown in pastures for consumption by ruminants. Alternatively, feed can be prepared from genetically modified bromoform-producing plants and fed to ruminants.

[0010] In some embodiments, the present invention provides a system for gene expression in plants comprising one or more recombinant nucleic acid molecules comprising one or more nucleotide sequences comprising two or more genes, each gene being operably linked to a promoter for expression in plants, and two or more genes being involved in bromoform production and encoding two or more enzymes not encoded by natural plants.

[0011] In some embodiments, two or more genes are contained within a single nucleic acid molecule. In some embodiments, two or more genes are contained within two or more nucleic acid molecules.

[0012] In some embodiments, at least one of two or more genes is derived from an organism selected from Asparagopsis taxiformis, Asparagopsis armata, Condorus crispus, Macrocystis pirifera, Medicago sativa, and Medicago truncatula.

[0013] In some embodiments, at least two of the two or more genes originate from at least one non-plant organism.

[0014] In some embodiments, two or more enzymes include at least one haloperoxidase.

[0015] In some embodiments, two or more enzymes are included in NAD(P)H oxidase (NOX).

[0016] In some embodiments, two or more enzymes include at least one enzyme involved in fatty acid synthesis.

[0017] In some embodiments, at least one enzyme involved in fatty acid synthesis is selected from β-ketoacyl-[acyl-carrier-protein] synthase III (FabH), phosphopantetheinyltransferase (sfp), acetyl-CoA carboxylase (ACCase), and malonyl-CoA synthase (MatB).

[0018] In some embodiments, two or more enzymes include haloperoxidase, NOX, and at least one enzyme involved in fatty acid synthesis selected from FabH, sfp, ACCase, and MatB.

[0019] In some embodiments, two or more enzymes include haloperoxidase, NOX, FabH, sfp, and ACCase.

[0020] In some embodiments, two or more genes are selected from Mbb1, Mbb2, Mbb3, Mbb4, CcVHPO1, CcVHPO2, CcVHPO3, CcVHPO4, CcVHPO5, CcMbb2, Sfp, FaBH, ACCase, and MatB.

[0021] In some embodiments, two or more genes include Mbb1, Mbb2, Mbb3, Mbb4, and at least one gene selected from FaBH, Sfp, ACCase, and MatB.

[0022] In some embodiments, two or more genes include Mbb1, Mbb2, Mbb3, Mbb4, FaBH, Sfp, and ACCase.

[0023] In some embodiments, haloperoxidase has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more to SEQ ID NOX has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more to SEQ ID NOX 2 (Mbb2); FabH is SEQ ID NOX 5 has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more; sfp has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more, to sequence number 6; and / or ACCase has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more to sequence number 7.

[0024] In some embodiments, two or more genes are operably linked to the same promoter. In some embodiments, two or more genes are operably linked to different promoters. In some embodiments, at least one of the promoters is a constitutive promoter. In some embodiments, at least one of the promoters is an inductive promoter. In some embodiments, the inductive promoter is a tissue-specific promoter that is activated in plant organs that develop later in the plant life cycle, such as leaves or flowers. In some embodiments, at least one of the two or more genes further encodes a chloroplast transport peptide for enzyme targeting to chloroplasts.

[0025] In some embodiments, the plant is a pasture plant or a fodder plant. In some embodiments, the plant is a grass, cereal, or legume. In some embodiments, the plant is selected from signal grass (Brachiaria), alfalfa (Medicago sativa, Lucerne), ryegrass, tall fescue, coxsfoot, clover, oats, millet, chrysanthemum, chicory, corn, soybean, and plantain.

[0026] In some embodiments, the present application provides a composition comprising the system disclosed herein.

[0027] In some embodiments, the present application provides a method for preparing a genetically engineered plant or plant part, the method comprising transforming a plant or plant part using the system disclosed herein or using a composition disclosed herein comprising the system disclosed herein.

[0028] In some embodiments, the present application provides a genetically engineered plant or plant part comprising the system disclosed herein or prepared by a method for preparing a genetically engineered plant or plant part disclosed herein.

[0029] In some embodiments, the present application provides a genetically engineered plant or plant part comprising one or more exogenous nucleotide sequences comprising two or more genes, each gene being operably linked to a promoter for expression in a plant, the two or more genes together encoding two or more enzymes involved in bromoform production and not encoded by a native plant.

[0030] In some embodiments, the present application provides a genetically engineered plant cell comprising the system disclosed herein or prepared by a method for preparing a genetically engineered plant or plant part disclosed herein.

[0031] In some embodiments, the present application provides a genetically engineered plant cell comprising one or more exogenous nucleotide sequences comprising two or more genes, each gene being operably linked to a promoter for expression in a plant, the two or more genes together encoding two or more enzymes involved in bromoform production and not encoded by a native plant.

[0032] In some embodiments, the plant is selected from signal grass (Brachiaria), alfalfa (Medicago sativa, Lucerne), ryegrass, tall fescue, coxsfoot, clover, oats, millet, chrysanthemum, chicory, corn, soybean, and plantain.

[0033] In some embodiments, plant parts are selected from seeds, cells, leaves, stems, roots, tubers, culms, flowers, bark, fruits, bulbs, root hairs, and rhizomes.

[0034] In some embodiments, the present application provides compositions comprising genetically modified plants or plant parts disclosed herein, mixed with plants or plant parts that are unable to produce bromoform, the compositions comprising up to about 10% genetically modified plants or plant parts.

[0035] In some embodiments, this application provides a method for reducing methane gas emissions from ruminants, the method being as follows: A step of producing seeds of genetically modified pasture plants containing two or more genes involved in bromoform production; The steps include sowing genetically modified seeds in ruminant pastures; and The process includes the step of growing a pasture plant that produces bromoform from the said seeds, This causes ruminants to consume the bromoform-producing pasture plants.

[0036] In some embodiments, genetically modified seeds are produced by methods for preparing genetically modified plants or plant parts disclosed herein.

[0037] In some embodiments, the ruminant is a cow.

[0038] In some embodiments, this application provides a method for preparing food to reduce methane gas emissions from ruminants, the method being as follows: A step of producing seeds of genetically modified forage plants that contain two or more genes involved in bromoform production; Steps for sowing genetically modified seeds; Steps for growing bromoform-producing forage plants from seeds; and The process includes the step of preparing a food containing a bromoform-producing forage plant or a portion thereof.

[0039] In some embodiments, genetically modified seeds are produced by methods for preparing genetically modified plants or plant parts disclosed herein.

[0040] In some embodiments, the ruminant is a cow.

[0041] Certain embodiments of this disclosure may include some, all, or any of the above-described advantages. One or more technical advantages will be readily apparent to those skilled in the art from the drawings, description, and claims contained herein. Furthermore, while certain advantages have been listed above, various embodiments may include all or some of the listed advantages, or none at all.

[0042] In addition to the exemplary embodiments and models described above, further embodiments and models will become apparent by referring to the drawings and by considering the following detailed description. [Brief explanation of the drawing]

[0043] [Figure 1A]Figures 1A–1C show RT-PCR analysis of transgenic tobacco plants using specific primers to identify the integration and transcription of 7 or 4 bromoform-producing genes per transgenic plant (the primers used are for genes encoding resistance to kanamycin, spectinomycin, and basta used for selection; see Table 2). Plants 1–3 show transcription in plants of kanamycin (Figure 1A), spectinomycin (Figure 1B), and basta (Figure 1C) resistance genes, providing evidence of expression of all seven genes involved in bromoform production: mbb1–4, sfp, fabH, and ACCase (from plasmids 3–5). Plants 4–6 show transcription in plants of kanamycin (Figure 1A) and spectinomycin (Figure 1B) resistance genes (but no transcription of the Bar (basta resistance) gene (Figure 1C)), providing evidence of expression of four genes from plasmids 1 and 2: mbb1–4. MW - molecular weight marker, wt - wild-type tobacco plant, NTC - negative control. [Figure 1B] Figures 1A–1C show RT-PCR analysis of transgenic tobacco plants using specific primers to identify the integration and transcription of 7 or 4 bromoform-producing genes per transgenic plant (the primers used are for genes encoding resistance to kanamycin, spectinomycin, and basta used for selection; see Table 2). Plants 1–3 show transcription in plants of kanamycin (Figure 1A), spectinomycin (Figure 1B), and basta (Figure 1C) resistance genes, providing evidence of expression of all seven genes involved in bromoform production: mbb1–4, sfp, fabH, and ACCase (from plasmids 3–5). Plants 4–6 show transcription in plants of kanamycin (Figure 1A) and spectinomycin (Figure 1B) resistance genes (but no transcription of the Bar (basta resistance) gene (Figure 1C)), providing evidence of expression of four genes from plasmids 1 and 2: mbb1–4. MW - molecular weight marker, wt - wild-type tobacco plant, NTC - negative control. [Figure 1C]Figures 1A–1C show RT-PCR analysis of transgenic tobacco plants using specific primers to identify the integration and transcription of 7 or 4 bromoform-producing genes per transgenic plant (the primers used are for genes encoding resistance to kanamycin, spectinomycin, and basta used for selection; see Table 2). Plants 1–3 show transcription in plants of kanamycin (Figure 1A), spectinomycin (Figure 1B), and basta (Figure 1C) resistance genes, providing evidence of expression of all seven genes involved in bromoform production: mbb1–4, sfp, fabH, and ACCase (from plasmids 3–5). Plants 4–6 show transcription in plants of kanamycin (Figure 1A) and spectinomycin (Figure 1B) resistance genes (but no transcription of the Bar (basta resistance) gene (Figure 1C)), providing evidence of expression of four genes from plasmids 1 and 2: mbb1–4. MW - molecular weight marker, wt - wild-type tobacco plant, NTC - negative control. [Modes for carrying out the invention]

[0044] The following description explains various aspects of the Disclosure. For explanatory purposes, and to provide a complete understanding of the different aspects of the Disclosure, specific configurations and details are described. However, it will also be apparent to those skilled in the art that the Disclosure can be implemented without the presentation of specific details herein. Furthermore, well-known features may be omitted or simplified in order to avoid ambiguity in the Disclosure.

[0045] The problem of methane emissions from ruminants has attracted much attention, and various solutions have been developed. Many of these solutions are based on the fact that red algae (Asparagopsis taxiformis) produce bromoform, which interferes with methane production in the stomachs of cattle. For example, one approach involved feeding Asparagopsis taxiformis to dairy cows, while another involved transferring the gene encoding Asparagopsis taxiformis haloperoxidase, a key enzyme involved in bromoform production, into the genomes of various organisms such as yeast and bacteria in order to produce feed containing these bromoform-producing organisms. Each approach had several drawbacks, including cost, the toxicity of bromoform, and low yields.

[0046] This invention presents a novel approach to this problem, which involves transcribing a combination of bromoform-producing genes from Asparagopsis taxiformis and additional organisms into plants, thereby sowing them in pastures and consuming them by cattle. One advantage of this method is that by providing bromoform through grazing rather than feed supply, it may be possible to achieve sufficient methane inhibition with small amounts of bromoform.

[0047] [System for generating bromoform-producing plants] Accordingly, in some embodiments, a system for gene expression in plants is provided, comprising one or more recombinant nucleic acid molecules comprising one or more nucleotide sequences containing two or more genes, each gene being operably linked to a promoter for expression in the plant, and two or more genes involved in bromoform production, together encoding two or more enzymes not encoded by the natural plant.

[0048] The phrase "one or more nucleotide sequences containing two or more genes" is intended to clarify that the total number of genes in the system is two or more (at least two), and that this total number of genes may be contained in one nucleotide sequence or two or more nucleotide sequences. Similarly, it is clarified that the sum of two or more genes may be contained on one recombinant nucleic acid molecule or on two or more recombinant nucleic acid molecules. Each nucleic acid molecule and each nucleotide sequence may contain one gene, two or more genes, a part of a gene, a non-gene or a part thereof, or any combination of these genes and / or parts thereof. As a whole, the system of recombinant nucleic acid molecules contains at least two genes involved in bromoform production and encodes at least two enzymes involved in bromoform production.

[0049] In some embodiments, two or more genes are contained within a single nucleic acid molecule. In some embodiments, two or more genes are contained within two or more nucleic acid molecules.

[0050] The term "natural plant" refers to the original plant, that is, the plant before the system of the present invention is added. In some embodiments, "natural plant" is a naturally occurring plant that does not involve any genetic modification.

[0051] As used herein, the term “operatably linked” means that each element is positioned such that there is a functional relationship between them. For example, with respect to a gene operatably linked to a promoter, it means that the position and orientation of the gene and promoter are appropriate so that the promoter drives the expression of the gene. It is understood that one promoter may be operatably linked to two or more genes. It is also understood that a promoter may be operatably linked to a gene via a transactivator, and as a result, the promoter may drive the expression of the transactivator that binds to a regulatory element that induces gene expression.

[0052] In some embodiments, two or more genes are operably linked to a single promoter. In some embodiments, each gene is operably linked to a separate promoter.

[0053] Bromoform (CHBr3) is a brominated organic solvent, a colorless liquid at room temperature, and, like chloroform, has a high refractive index, is very dense, and has a sweet odor. Bromoform is known as an inhibitor of methane production and is a common component of seaweed. The mechanism of inhibition is hypothesized to be by reducing the efficiency of cobamide-dependent methyltransferase by interfering with vitamin B12 binding, a crucial step in methane production in the rumen.

[0054] Several organisms are known as bromoform producers, and are not limited to them, but include the algae Asparagopsis taxiformis (red sheep plume or limkov, formerly Asparagopsis sanfordiana), Asparagopsis armata, Condorus crispus (Ireland moss or carrageenan moss), and Macrocystis pirifera (giant kelp).

[0055] Asparagopsis taxiformis is a species of red algae widely distributed in tropical to temperate waters. Researchers have demonstrated that feeding ruminants a diet containing as little as 0.2% Asparagopsis taxiformis seaweed reduces methane emissions by approximately 99%. When additional types of seaweed were tested, many reduced methane emissions by more than 90%. The active ingredient in the seaweed was found to be bromoform. Asparagopsis armata is another species of red algae that has been shown to reduce methane production in dairy cows. Condorus crispus is a species of red algae that is also used as a source of the thickening agent carrageenan. Macrocystis pirifera is a brown macroalga.

[0056] In some embodiments, the two or more genes are three or more, four or more, five or more, six or more, or seven genes.

[0057] In some embodiments, at least one of two or more genes is derived from an organism selected from Asparagopsis taxiformis, Asparagopsis armata, Condorus crispus, Macrocystis pirifera, Medicago sativa, and Medicago truncatula.

[0058] In some embodiments, at least two of the two or more genes originate from at least one non-plant organism. In some embodiments, the at least one non-plant organism is an alga. In some embodiments, the alga is a red or brown alga. In some embodiments, the alga is a red alga. In some embodiments, the alga is selected from Asparagopsis taxiformis, Asparagopsis armata, Condorus crispus, and Macrocystis pirifera. In some embodiments, the alga is Asparagopsis taxiformis.

[0059] Several enzymes have been identified as being involved in bromoform production. These enzymes include haloperoxidases (vanadate-dependent haloperoxidases, or VHPO, etc.) and NAD(P)H oxidase (NOX). Haloperoxidases catalyze the conversion of halide anions to hypohalites using hydrogen peroxide, which acts as an oxidizing agent (Br - +H2O2=>HOBr+OH - Hypohalous acids supply electrophilic halogen ions, halogenating hydrocarbon substrates to yield natural products such as bromoform (3HOBr + CH => CHBr3 + 3OHOC). VHPO uses vanadium as a cofactor. The need for hydrogen peroxide in the peroxidase reaction links this pathway to the enzymatic production of reactive oxygen species (ROS) by NOX. NOX is a membrane-bound enzyme that catalyzes the transfer of electrons from the electron donor NAD(P)H to molecular oxygen via flavin and heme cofactors, producing ROS such as hydrogen peroxide and superoxide anion (invariant to hydrogen peroxide).

[0060] Therefore, in some embodiments, two or more enzymes comprise at least one haloperoxidase. In some embodiments, two or more enzymes comprise NOX. In some embodiments, two or more enzymes comprise at least one haloperoxidase and NOX.

[0061] Furthermore, as shown by Thapa 2020 (ACS Chem Biol. 15(6):1662-1670), fatty acid biosynthesis intermediates such as malonyl-CoA can provide substrates for VHPO enzymes and are therefore thought to be involved in bromoform production. The enzymes involved in the relevant fatty acid synthesis reactions are encoded by the genes FabH (β-ketoacyl-[acyl-carrier-protein] synthase III), MatB (malonyl-CoA synthetase), and Sfp (phosphopantetheinyltransferase). Briefly, malonate and CoA-SH are condensed into malonyl-coenzyme A by MatB. Sfp modifies ACP (acyl carrier protein) to malonyl-S-ACP using malonyl-coenzyme A. After incubation with FabH, it is converted to acetoacetyl-S-ACP. Bromoform can be produced by incubating acetoacetyl-S-ACP with haloperoxidase.

[0062] Malonyl-CoA is often produced from acetyl-CoA by acetyl-CoA carboxylase (ACCase) rather than by malonyl-CoA synthetase. ACCase is a biotin-dependent carboxylase that catalyzes the irreversible carboxylation of acetyl-CoA, generating malonyl-CoA via its two catalytic activities: biotin carboxylase and carboxyltransferase. The most important function of ACCase is to provide a malonyl-CoA substrate for fatty acid biosynthesis.

[0063] In some embodiments, the two or more enzymes are three or more, four or more, five or more, six or more, or seven enzymes.

[0064] Therefore, in some embodiments, two or more enzymes include at least one enzyme involved in fatty acid synthesis. In some embodiments, the at least one enzyme involved in fatty acid synthesis is selected from Spp, FabH, ACCase, and MatB.

[0065] In some embodiments, the two or more enzymes include at least one haloperoxidase and at least one enzyme involved in fatty acid synthesis, selected from FabH, sfp, ACCase, and MatB.

[0066] In some embodiments, two or more enzymes include at least one haloperoxidase, NOX, and at least one enzyme involved in fatty acid synthesis selected from FabH, sfp, ACCase, and MatB.

[0067] In some embodiments, the two or more enzymes include at least one haloperoxidase, NOX, and FabH. In some embodiments, the two or more enzymes include at least one haloperoxidase, NOX, and sfp. In some embodiments, the two or more enzymes include at least one haloperoxidase, NOX, and ACCase.

[0068] In some embodiments, the two or more enzymes include at least one haloperoxidase, NOX, FabH, and sfp. In some embodiments, the two or more enzymes include at least one haloperoxidase, NOX, FabH, and ACCase. In some embodiments, the two or more enzymes include at least one haloperoxidase, NOX, sfp, and ACCase.

[0069] In some embodiments, the two or more enzymes include at least one haloperoxidase, FabH, sfp, and ACCase.

[0070] In some embodiments, at least one haloperoxidase is selected from Mbb1, Mbb3, and Mbb4, as well as combinations thereof. In some embodiments, at least one haloperoxidase comprises Mbb1, Mbb3, and Mbb4. In some embodiments, NOX is Mbb2.

[0071] In some embodiments, Mbb1 has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with respect to sequence number 1. In some embodiments, Mbb1 has the sequence of sequence number 1.

[0072] In some embodiments, Mbb2 has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with respect to sequence number 2. In some embodiments, Mbb2 has the sequence of sequence number 2.

[0073] In some embodiments, Mbb3 has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with respect to sequence number 3. In some embodiments, Mbb3 has the sequence of sequence number 3.

[0074] In some embodiments, Mbb4 has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with respect to sequence number 4. In some embodiments, Mbb4 has the sequence of sequence number 4.

[0075] In some embodiments, FabH has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with respect to SEQ ID NO: 5. In some embodiments, FabH has the sequence of SEQ ID NO: 5.

[0076] In some embodiments, SFP has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with respect to SEQ ID NO: 6. In some embodiments, SFP has the sequence of SEQ ID NO: 6.

[0077] In some embodiments, the ACCase has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with respect to SEQ ID NO: 7. In some embodiments, the ACCase has the sequence of SEQ ID NO: 7.

[0078] In Asparagopsis taxiformis, the relevant genes were identified and localized to a single locus called the marine bromoform biosynthesis locus (GenBank accessions MN966723 and MN893468). The genes identified at the Mbb locus include both VHPOs-Mbb1, Mbb3, and Mbb4, as well as NOX-Mbb2. Note that Mbb2 is not the only NOX gene identified in Asparagopsis taxiformis. Corresponding genes were also identified in the algal species Condorus crispus, including the VHPO genes CcVHPO1-5 and the NOX gene CcMbb2 (Thapa et al., ACS Chem Biol. 15(6):1662-1670). The VHPOs identified above in Asparagopsis taxiformis and Ccrispus are bromine-specific.

[0079] Therefore, in some embodiments, two or more genes are selected from Mbb1, Mbb2, Mbb3, Mbb4, CcVHPO1, CcVHPO2, CcVHPO3, CcVHPO4, CcVHPO5, CcMbb2, Sfp, FaBH, ACCase, and MatB.

[0080] Mbb1-4 were successfully expressed in E. coli, but attempts to express Mbb2 in bacteria and yeast were unsuccessful, and CcMbb2 was expressed in yeast instead. Furthermore, it should be noted that Mbb3 did not produce bromoform and showed low expression levels, and CcVHPO4 and CcVHPO5 were not tested (Thapa et al., ACS Chem Biol. 15(6):1662-1670).

[0081] In some embodiments, two or more genes include at least mbb2 or CcMbb2.

[0082] Furthermore, it should be noted that some genes, particularly NOX and fatty acid synthases, may be endogenously present in plants, and therefore it may be sufficient to provide only haloperoxidases or any other partial list of the genes mentioned above.

[0083] In some embodiments, two or more genes include genes Mbb1 and Mbb2.

[0084] In some embodiments, two or more genes include genes Mbb3 and Mbb4.

[0085] In some embodiments, two or more genes include genes Mbb1, Mbb2, Mbb3, and Mbb4.

[0086] In some embodiments, two or more genes include the genes Mbb1, Mbb3, Mbb4, and at least one gene selected from sfp, FabH, and ACCase.

[0087] In some embodiments, two or more genes include Mbb1, Mbb2, Mbb3, Mbb4, and sfp. In some embodiments, two or more genes include Mbb1, Mbb2, Mbb3, Mbb4, and FabH. In some embodiments, two or more genes include Mbb1, Mbb2, Mbb3, Mbb4, and ACCase.

[0088] In some embodiments, two or more genes include Mbb1, Mbb2, Mbb3, Mbb4, sfp, and FabH, and in some embodiments, two or more genes include Mbb1, Mbb2, Mbb3, Mbb4, sfp, and ACCase.

[0089] In some embodiments, two or more genes include Mbb1, Mbb3, Mbb4, sfp, FabH, and ACCase.

[0090] In some embodiments, the Mbb1, Mbb2, Mbb3, and / or Mbb4 genes are derived from Asparagopsis taxiformis, or encode the same enzyme sequence encoded by the Mbb1, Mbb2, Mbb3, and / or Mbb4 genes of Asparagopsis taxiformis. In some embodiments, the sfp gene is derived from Medicago truncatula, or encodes the same enzyme sequence encoded by the sfp gene of Medicago truncatula. In some embodiments, the FabH gene is derived from Condorus crispus, or encodes the same enzyme sequence encoded by the FabH gene of Condorus crispus. In some embodiments, the ACCase gene is derived from Medicago sativa, or encodes the same enzyme sequence encoded by the ACCase gene of Medicago sativa. In some embodiments, at least one of two or more genes is an ortholog of the gene described above, derived from a different species.

[0091] It is understood that any one of the genes of the present invention may encode the same enzyme sequence encoded by a reference gene (for example, the genes of the present invention encode the same enzyme sequence encoded by the FabH gene of Condorus crispus), but may have a different or modified coding sequence compared to each of the respective genes. In some embodiments, the coding sequence is modified by using codon optimization. In some embodiments, the coding sequence is modified using a codon usage scheme of a different organism. In some embodiments, the coding sequence is modified by using a codon usage scheme of Medicago sativa.

[0092] In some embodiments, the Mbb1 gene has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with respect to the sequence encoding Sequence ID No. 1.

[0093] In some embodiments, the Mbb2 gene has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with respect to the sequence encoding Sequence ID No. 2.

[0094] In some embodiments, the Mbb3 gene has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with respect to the sequence encoding SEQ ID NO: 3.

[0095] In some embodiments, the Mbb4 gene has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with respect to the sequence encoding sequence number 4.

[0096] In some embodiments, the FabH gene has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with respect to the sequence encoding SEQ ID NO: 5.

[0097] In some embodiments, the SFP gene has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with respect to the sequence encoding Sequence ID No. 6.

[0098] In some embodiments, the ACCase gene has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with respect to the sequence encoding SEQ ID NO: 7.

[0099] In some embodiments, two or more genes are operably ligated to the same promoter. In some embodiments, at least two of the two or more genes are operably ligated to different promoters. In some embodiments, one of each of the two or more genes is operably ligated to a separate promoter. In some embodiments, two or more genes are operably ligated to a single promoter.

[0100] One of the problems associated with bromoform production is that bromoform itself can be harmful to the ozone layer. Therefore, the advantage of the present invention is that it makes it possible to control the amount of bromoform produced in various ways, such as by controlling the expression of bromoform-producing genes to specific stages of development, specific states, or specific tissues. This can be achieved, for example, by using inductive promoters that restrict the expression of these genes to specific times or places.

[0101] The promoter can be any promoter suitable for expression in a plant. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inductive promoter. In some embodiments, at least one of the genes is operably ligated to the inductive promoter.

[0102] Non-exclusive examples of constitutive promoters include, for example, the cauliflower mosaic virus 35S promoter, the Agrobacterium NOS promoter, and promoters of housekeeping genes, such as actin (e.g., act8 promoter) or ubiquitin (ubq10 promoter).

[0103] Non-limiting examples of tissue-specific promoters include, for example, the leaf-specific promoter of sedoheptulose-1,7-bisphosphatase (SBPase, e.g., from Brachypodium distachyon), or the fructose-1,6-bisphosphate aldolase (FBPA) promoter, which is highly expressed in leaves.

[0104] The term "constitutive promoter" refers to a promoter that is always "on," i.e., one that drives gene transcription. It is generally not affected by regulatory factors such as temporal or tissue-specific factors. An example of a constitutive promoter is the promoter of housekeeping genes.

[0105] The terms "inducible promoter" or "regulatory promoter" refer to promoters that are, by default, in an "off" state, i.e., do not drive transcription, but are turned on in response to specific regulatory factors, such as tissue-specific or development-specific factors. Non-exclusive examples of inducible promoters include tissue-specific promoters and developmentally regulated promoters.

[0106] Inducible promoters can be used to minimize bromoform production and thereby reduce excess bromoform, which is harmful to both the plant itself and the environment, by ensuring that relevant genes are expressed only when needed.

[0107] For example, according to some embodiments, bromoform is only required when animals are grazing. Therefore, the expression of at least some bromoform-producing genes may be activated only when the plants have reached an appropriate developmental stage and are ready to be eaten. Alternatively, bromoform production may be activated only when cattle are grazing and are turned on by a stress response caused by damage inflicted on plants, for example, by cattle bites.

[0108] Therefore, in some embodiments, the inductive promoter is a developmentally regulated promoter that is induced by factors that are induced in later stages of plant development. In some embodiments, the inductive promoter is a tissue-specific promoter that is activated in specific tissues that occur in later stages of the plant life cycle, such as leaves or flowers.

[0109] In some embodiments, the inducible promoter is induced by a factor activated by consumption by ruminants, such as a stress-induced regulator, such as jasmonic acid. In some embodiments, the inducible promoter is a jasmonic acid promoter or a jasmonic acid-responsive promoter.

[0110] One of the problems with gene targeting in plants is the suppression of foreign genes by plant defense systems. One approach that may help overcome this problem is to target organelles within plant cells, such as plastids like chloroplasts. Targeting chloroplasts may have two advantages: firstly, it isolates foreign genes, potentially protecting them from silencing by the host plant; and secondly, fatty acid synthesis occurs in chloroplasts, which can provide precursors for haloperoxidases.

[0111] Therefore, in some embodiments, at least one of the two or more genes further encodes a plastid-targeting motif or a chloroplast-targeting motif (such as a chloroplast transport peptide). In some embodiments, all of the two or more genes further encode a chloroplast transport peptide.

[0112] Chloroplast transport peptides are short peptides that instruct the transport of proteins to chloroplasts. Transport peptides are typically cleaved upon import. Chimeric chloroplast transport peptides are disclosed in WO2012 / 161982, which is incorporated herein by reference.

[0113] Another approach involves using a P19 viral RNA silencing suppressor, which can prevent the silencing of foreign genes.

[0114] Therefore, in some embodiments, the system further includes a sequence encoding the P19 viral RNA silencing suppressor gene.

[0115] It is understood that the recombinant nucleic acid molecules of the present invention may further include additional elements, as necessary, such as transcription terminators, regulatory elements, and selective markers, for transformation and expression in plants. Non-limiting examples of selective markers include kanamycin resistance, spectinomycin resistance, and / or glufosinate or phosphinothricin herbicide resistance (e.g., trademark Basta).

[0116] In some embodiments, one or more recombinant nucleic acid molecules are contained in at least one vector suitable for transformation into plants and / or expression in plants. In some embodiments, the vector is a binary vector. In some embodiments, the binary vector system is a Ti plasmid-based system in which the gene of interest is cloned into the T-DNA region of the plasmid and transformed into Agrobacterium containing a viral helper plasmid. In some embodiments, the vector is Ti plasmid-based. In some embodiments, the vector is a pAB binary vector. In some embodiments, the vector is a pPA binary vector.

[0117] The recombinant nucleic acid molecules of the present invention can be prepared by any suitable genetic engineering method, including, for example, plasmid cloning, DNA synthesis, restriction enzyme digestion and ligation, T-DNA manipulation, clustered regular-spaced short palindromic repeat (CRISPR) technology, polymerase chain reaction (PCR), and the like.

[0118] In some embodiments, compositions comprising the systems disclosed herein are provided. In some embodiments, the compositions further comprise reagents suitable for transforming plants. Suitable reagents are well known in the art and are also disclosed in the examples.

[0119] In some embodiments, compositions comprising the system of the present invention for use in plant transformation are provided. In some embodiments, the use of compositions comprising the system of the present invention for plant transformation is provided.

[0120] [Method for preparing bromoform-producing plants] In some embodiments, methods are provided for preparing genetically modified plants or plant parts, which include transforming the plants or plant parts with the systems or compositions disclosed herein.

[0121] The definitions and embodiments relating to the methods of the preparation embodiments described above also apply herein, and vice versa. Some particularly relevant embodiments may be noted or explicitly repeated.

[0122] Transgenic plants using the nucleic acids of the present invention can be carried out by any suitable method known in the art. Non-limiting examples of such methods include transformation mediated by Agrobacterium and / or transformation by the floral dip method (e.g., as described in WO2018 / 178975).

[0123] In some embodiments, the method includes the step of transforming the system of the present invention into plant leaves. In some embodiments, the method includes the step of transforming the system of the present invention into plant seeds.

[0124] In some embodiments, the system of the present invention comprises two or more (at least two) recombinant nucleic acid molecules, and at least two of the recombinant nucleic acid molecules are transformed together in the plant. In some embodiments, the system of the present invention comprises two or more recombinant nucleic acid molecules, and at least two of the recombinant nucleic acid molecules are transformed separately in the plant. In some embodiments, the system of the present invention comprises two or more recombinant nucleic acid molecules, and all of the recombinant nucleic acid molecules are transformed together in the plant (simultaneous transformation).

[0125] It is understood that the genes of the present invention may be inserted into plants together or separately by any suitable method, including using any genome editing system or method, which includes systems using manipulated nucleases selected from the group consisting of meganucleases, zinc finger nucleases (ZFNs), transcription activator-like effector-based nucleases (TALENs), clustered regularly spaced short palindromic repeat (CRISPR) systems, and any combination thereof.

[0126] In some embodiments, the method further includes the step of growing a plant from a plant portion transformed by the system of the present invention. In some embodiments, the method further includes the step of obtaining a plant portion from the grown plant.

[0127] For example, plant leaves may be transformed using the system of the present invention, and the plant will grow from the transformed leaves. Seeds can then be obtained from the grown transformed plants to produce further transformed plants.

[0128] In some embodiments, the method further includes a step of selecting transformed plants. Selection may be carried out by any suitable method known in the art, including a vector transformed into plants that encodes a selectable marker such as kanamycin resistance, spectinomycin resistance, and / or glufosinate or phosphinothricin herbicide resistance (e.g., trademark Basta), and the transformants are grown under appropriate selective pressure.

[0129] [Genetically modified bromoform-producing plants and plant parts] In some embodiments, genetically modified plants or plant parts are provided, which include a system disclosed herein or are prepared by a method for preparing genetically modified plants or plant parts disclosed herein.

[0130] In some embodiments, a genetically engineered plant or plant part is provided that comprises one or more exogenous nucleotide sequences containing two or more genes, each gene being operably linked to a promoter for expression in the plant, and the two or more genes together encode two or more enzymes involved in bromoform production that are not encoded by the natural plant.

[0131] In some embodiments, this application provides genetically modified plant cells, which are prepared by a system disclosed herein or by a method for preparing a genetically modified plant or plant part disclosed herein.

[0132] In some embodiments, the present application provides genetically engineered plant cells comprising one or more exogenous nucleotide sequences containing two or more genes, each gene operably linked to a promoter for expression in the plant, and the two or more genes together encode two or more enzymes not encoded by the natural plant that are involved in bromoform production.

[0133] It is understood that the genetically modified plants or plant parts disclosed herein are intended to be bromoform-producing or capable of developing into bromoform-producing plants or plant parts. However, since at least some of the genes involved in bromoform production can be controlled in a tissue-specific or developmental stage manner, bromoform production may be limited to specific tissues or plant parts. Therefore, as used herein, the term “plant part” includes plant parts (and bromine sources) that produce bromoform after being transformed with the construct used, or plant parts that cannot produce bromoform after being transformed but can develop into bromoform-producing plants or plant parts. Examples of the latter include cells or seeds.

[0134] The terms “genetically modified” and “transgenic” are used interchangeably herein and relate to plants or plant parts containing an exogenous nucleotide sequence encoding a bromoform-producing gene, as described herein.

[0135] As used herein, the term “exogenous nucleotide sequence” refers to a nucleotide sequence that is not part of a natural plant.

[0136] In some embodiments, a genetically engineered plant or plant part is provided that contains two or more genes, each gene operably linked to a promoter for expression in the plant, and together the two or more genes encode two or more enzymes involved in bromoform production.

[0137] The definitions and embodiments described above, and those that may relate to embodiments of genetically modified plants, also apply herein, and vice versa. Several particularly relevant embodiments may be pointed out or explicitly repeated.

[0138] The phrase "one or more exogenous nucleotide sequences containing two or more genes" is understood to mean that there may be one exogenous sequence containing two or more genes, or that there may be two or more exogenous sequences if all exogenous sequences together contain two or more genes. If two or more exogenous nucleotide sequences exist, each exogenous sequence does not have to contain a gene, as long as it contains two or more genes together, or it does not have to contain one gene or two or more genes.

[0139] In some embodiments, recombinant nucleic acid molecules are extrachromosomal. In some embodiments, recombinant nucleic acid molecules are integrated into plant chromosomes.

[0140] In some embodiments, the plant is a pasture plant or a fodder plant. In some embodiments, the plant is a pasture plant. In some embodiments, the plant is a fodder plant.

[0141] The terms “pasture plants” or “forage plants” are used herein to refer to plants that are planted or sown in pastures for grazing animals.

[0142] Non-specific examples of grasses and pasture plants include the genus Agrostis (bentgrass), Agrostis capillaris (common bentgrass), Agrostis stronifera (creeping bentgrass), Andropogon harrii (sand blue stem), Arenatherum eratius (false oat grass), Botryochloa blodii (Australian blue stem), Botryochloa pertusa (hurricane grass), Brachiaria (signal grass), Brachiaria decumbensis (Suriname grass), and Brachiaria humide Icola (Colonivia grass), Brachiaria ruziensis, or hybrids of the genus Brachiaria, Bromus (Brom grass), Senculus sialis (Buffalo grass), Chloris gayana (Rose grass), Synodon dactylon (Bermuda grass), Dactylis glomerata (Orchard grass), Echinocloa pyramidalis (Antelope grass), Entracia imbricata (Bungoma grass), Festuca (Fescu), Festuca arundinacea (Tall fescue), Festuca pratensis (Meadow grass) Ufescue, Festuca rubra (red fescue), Heteropogon contortus (black spear grass), Hymenacne amplexicaulis (West Indian wetland grass), Hyparrenia rufa (Jagua), Lisia hexandra (Southern cut grass), Lolium (ryegrass), Lolium multiflora (Italian ryegrass), Lolium perenne (perennial ryegrass), Megatylus maximus (Guinea grass), Melinis miniutiflora (molasses grass), Pasparam conjugatum (carabaograss) Examples include Paspalum dillatum (Dallis grass), Phalaris arundinacea (Reed canary grass), Flameum pratens (Timothy grass), Strawberry grass (Bluegrass, Meadowgrass), Poa arachnifera (Texas bluegrass), Poa pratensis (Kentucky bluegrass), Poa trivialis (Rough bluegrass), Setaria sfacerata (African bristle grass), Temeda triandra (Kangaroo grass), and Tinopyrum intermedium (Intermediate wheatgrass).

[0143] Non-exclusive examples of leguminous plants include Arachs pintoi (pinto peanut), Astragalus sissel (chickpea), Chamaecrista rotundifolia (round-leaved sensitive pea), Clitoria ternatea (butterfly pea), Cumelowia (annual respedesa), Cumelowia stiprasea (Korean clover, Korean respedesa), and Cumelowia striata (Japanese clover). , (commonly known as respedes), Lotus corniculatus (chickweed), Macroptilium atropurpureum (purple bush bean), Macroptilium bracteatum (burgundy bean), Medicago genus (medicago species), Medicago sativa (alfalfa, russern), Medicago truncatula (barrel medicago), Melilothos genus (sweet clover species), Neonotonia y Examples include Tii (perennial soybean), Onoblicis viscifolia (common cephalosa), Stylosanthes (Stylo), Stylosanthes humilis (Townsville Stylo), Stylosanthes scabra (shrub-like Stylo), Trifolium (clover), Trifolium hybridum (Arsice clover), Trifolium incarnatum (crimson clover), Trifolium pratens (red clover), Trifolium repens (white clover), Vicia (vetch), Vicia articulata (one-flower vetch), Vicia elvillia (bitter vetch), Vicia narbonensis (narbon vetch), Vicia sativa (common vetch, tarre), Vicia virosa (hairy vetch), and Vigna parkeri (creeping Vigna).

[0144] The term “forage plants” is used herein to refer to plants that can be used to prepare animal feed. Non-limiting examples of forage plants include alfalfa, barley, duckweed, bird's foot trefoil, brassica (e.g., kale, rapeseed, rutabaga, and turnip), clover, grasses (e.g., Bermuda grass, false oatgrass, fescue, ryegrass), maize (corn), millet, oats, sorghum, soybeans, and wheat.

[0145] The terms "pasture grass" and "forage plants" are not mutually exclusive, as many plants fall under both definitions.

[0146] In some embodiments, the plants are selected from grasses, cereals, and legumes.

[0147] Examples of grasses include signal grass (Brachiaria), ryegrass, tall fescue, coxsfoot, and chrysanthemum; examples of cereals include corn, oats, and millet; and examples of legumes include alfalfa (Medicago sativa, Lucerne), clover, and soybeans.

[0148] In some embodiments, the plant is selected from signal grass (Brachiaria), alfalfa (Medicago sativa, Lucerne), ryegrass, tall fescue, coxsfoot, clover, oats, millet, chrysanthemum, chicory, corn, soybean, and plantain.

[0149] In some embodiments, plant parts are selected from seeds, cells, leaves, stems, roots, tubers, culms, flowers, bark, fruits, bulbs, root hairs, rhizomes, and any suitable part of a plant.

[0150] In some embodiments, the plant portion is selected from seeds or cells. In some embodiments, the plant portion is an explant derived from any plant tissue.

[0151] In some embodiments, at least two of the two or more genes originate from a species different from the plant species. In some embodiments, at least one of the two or more genes originates from an organism selected from Asparagopsis taxiformis, Asparagopsis armata, Condorus crispus, Macrocystis pirifera, Medicago sativa, and Medicago truncatula. In some embodiments, at least two, three, four, five, six, or seven of the two or more genes originate from at least one organism selected from Asparagopsis taxiformis, Asparagopsis armata, Condorus crispus, Macrocystis pirifera, Medicago sativa, and Medicago truncatula.

[0152] In some embodiments, at least one of two or more genes originates from a non-plant organism. In some embodiments, at least two, three, four, or five of two or more genes originate from at least one non-plant organism. In some embodiments, the non-plant organism is an alga. In some embodiments, the alga is a red or brown alga. In some embodiments, the alga is a red alga. In some embodiments, the alga is selected from Asparagopsis taxiformis, Asparagopsis armata, Condorus crispus, and Macrocystis pirifera. In some embodiments, the alga is Asparagopsis taxiformis.

[0153] In some embodiments, genetically modified plants or plant parts are obtained directly by transformation using the system of the present invention.

[0154] In some embodiments, a plant or plant part containing the system of the present invention is not directly obtained by transformation in the system of the present invention, but is derived from a plant or plant part transformed in the system of the present invention, or from a descendant of a transformed plant.

[0155] A further method for reducing the amount of bromoform is to dilute or mix the genetically modified plant or plant part with a plant or plant part that cannot produce bromoform, for example, a plant or plant part that does not contain the system of the present invention. For example, the genetically modified seeds of the present invention may be mixed with non-genetically modified seeds to provide the amount of bromoform required by ruminants.

[0156] Accordingly, in some embodiments, compositions are provided comprising a mixture of the genetically modified plant or plant part (e.g., seeds) of the present invention and a plant or plant part that is unable to produce bromoform. In some embodiments, the composition comprises only plant parts such as seeds and does not contain any additional components.

[0157] In some embodiments, up to approximately 5% w / w, 10% w / w, 15% w / w, 20% w / w, 25% w / w, or 30% w / w of the composition is the genetically modified plant or plant part of the present invention.

[0158] [Methods to reduce methane emissions] The plants or plant parts of the present invention containing the bromoform-producing genes described herein are intended to be consumed by ruminants such as cattle. For this purpose, there are two general approaches: the preparation of livestock feed based on the transgenic plants, or the sowing of transgenic pasture plants in pastures to be consumed by ruminants.

[0159] Therefore, in some embodiments, methods are provided for reducing methane gas emissions from ruminants, the methods being as follows: A step of producing seeds of genetically modified pasture plants that contain two or more genes involved in bromoform production; The steps include sowing genetically modified seeds in ruminant pastures; and The process includes the step of growing a pasture plant that produces bromoform from the said seeds, This causes ruminants to consume the bromoform-producing pasture plants.

[0160] In some embodiments, the method further includes the step of adding a bromine source to bromoform-producing grass plants. In some embodiments, the method does not include adding a bromine source to bromoform-producing grass plants.

[0161] A suitable source of bromine may include a bromine-containing solution, where the bromine is in any suitable form, such as bromine or bromide, for example, a calcium bromide solution. The concentration of bromine (e.g., bromide) in the bromine-containing solution may be about 1–100 ppm, about 5–100 ppm, or about 5–50 ppm. The bromine source can be added by any suitable method, including spraying, irrigating, or immersing plant parts (such as plant leaves or roots) in the bromine-containing solution.

[0162] In some embodiments, genetically modified seeds are produced by methods for preparing genetically modified plants or plant parts disclosed herein.

[0163] In some embodiments, the bromoform-producing plant includes the system disclosed herein.

[0164] Ruminants (suborder Ruminantia) or ruminants (zoan) are herbivorous mammals with hooves that graze (graze) or brows (browse) leaves and branches, obtaining nutrients from plant-based foods primarily through microbial action by fermentation in a specialized stomach before digestion. The process, which occurs in the anterior part of the digestive system and is therefore called foregut fermentation, typically requires the reflux of fermented ingested material (known as cude) and subsequent chewing. The process of re-chewing the cude to further break down the plant material and aid digestion is called rumination. Digestion of food in the rumen (first chamber and the main site of microbial fermentation) is primarily carried out by the rumen microbiome, which contains a high-density population of several species of bacteria, protists, and sometimes yeasts and other fungi. The hydrolysis of cellulose results in sugars, which are further fermented into acetates, lactates, propions, butyrates, carbon dioxide, and methane. Methane is produced in the rumen by a type of archaea called methanogenic bacteria, and this methane is released into the atmosphere. The rumen is the primary site of methane production in ruminants.

[0165] In some embodiments, the ruminant is selected from cattle, domesticated or wild bovines (e.g., cows), goats, sheep, giraffes, deer, gazelles, and antelopes.

[0166] In some embodiments, the ruminant is a cow.

[0167] In some embodiments, a method is provided for preparing food to reduce methane gas emissions from ruminants, the method being as follows: A step of producing seeds of genetically modified forage plants that contain two or more genes involved in bromoform production; Steps for sowing genetically modified seeds; Steps for growing bromoform-producing forage plants from seeds; and The process includes the step of preparing a food containing a bromoform-producing forage plant or a portion thereof.

[0168] The definitions and embodiments described above, and those relating to the embodiments of the method, also apply herein, and vice versa. Several particularly relevant embodiments can be pointed out or explicitly repeated.

[0169] In some embodiments, genetically modified seeds are produced by methods for preparing genetically modified plants or plant parts disclosed herein.

[0170] In some embodiments, the bromoform-producing plant includes the system disclosed herein.

[0171] In some embodiments, the ruminant is a cow.

[0172] It should be noted that the preparation of animal feed from plants is well known in the art. Preparation can be carried out by wet or dry processing methods, including grinding, dry rolling, flaking, popping, micronization, pressure cooking, roasting, extrusion, and pelletizing. Various nutrients can be added to the plant raw materials.

[0173] In some embodiments, the ruminant is selected from cattle, domesticated or wild bovids (e.g., cattle), goats, sheep, giraffes, deer, gazelles, and antelopes.

[0174] Unless otherwise defined, all scientific and technical terms used herein have the meanings that are commonly understood by those skilled in the art to which this invention pertains.

[0175] The terms "a" and "an" refer to one or more grammatical objects of the article (i.e., at least one or more). For example, "an element" means one or more elements.

[0176] The term "approximately" means, when referring to measurable values ​​such as quantities or ratios, to include a variation of ±10% of the indicated value, and such variation is also suitable for carrying out the disclosed invention. Any numerical values ​​appearing in this application are intended to be interpreted as being preceded by "approximately" unless otherwise indicated.

[0177] When used herein, the term “nucleotide sequence” or “nucleic acid sequence” refers to a sequence of nucleotides and may include different types of nucleotides, such as DNA nucleotides, RNA nucleotides, and synthetic nucleotides.

[0178] The term "nucleic acid molecule" refers to a molecule containing at least one nucleotide sequence. Nucleic acid molecules may be linear, circular, or branched, and the nucleotides may be modified or unmodified. In some embodiments, the nucleic acid molecule is a nucleic acid vector (usually a DNA vector) containing elements such as genes, promoters, and linkers.

[0179] When the term “recombinant” is used herein in relation to nucleic acid molecules or sequences, it refers to nucleic acid molecules or sequences that include a combination of nucleotide sequences that cannot be directly isolated from natural organisms and do not exist together in the same molecule in natural organisms.

[0180] As used herein, the term “gene” refers to a portion of a nucleic acid sequence that is transcribed into RNA and, optionally, translated into a protein. Typically, a gene is operably ligated to a promoter that drives transcription, and possibly to additional regulatory elements. In some embodiments, two or more genes are operably ligated to a single promoter, such as a prokaryotic operon. A gene may or may not contain introns.

[0181] As used herein, the term "bp" means one or more base pairs.

[0182] As used herein, the term "amino acid" means one or more amino acids or amino acids.

[0183] While specific embodiments of the present invention have been illustrated and described, it will be clear that the present invention is not limited to the embodiments described herein. Many modifications, changes, variations, substitutions, and equivalents will be apparent to those skilled in the art without departing from the spirit and scope of the invention as described by the following claims.

[0184] The following embodiments are presented to more fully illustrate some embodiments of the present invention. However, they should not be construed as limiting the broad scope of the invention. Those skilled in the art can readily devise many variations and modifications of the principles disclosed herein without departing from the scope of the invention.

[0185] Herein, the present invention will be explained by the following non-limiting embodiments. [Examples]

[0186] [Table 1] [Table 1-1]

[0187] [Table 2]

[0188] [Example 1: Cloning of algal bromoform-producing genes] Gene transcripts encoding mbb1-mbb4 (Asparagopsis taxiformis), FabH (Condurus crispus), sfp (Medicago truncatula), and ACCase (Medicago sativa) were synthesized using Medicago sativa codons and amplified by PCR using the indicated primers. The amplified DNA was cloned into binary vectors containing a selection marker (spectinomycin, kanamycin, or basta) and transformed into Agrobacterium host cells. Genes were screened by colony PCR performed on transformed cells grown on rifampicin and kanamycin (Agrobacterium and vector-selective antibiotics). Several clones were characterized by whole plasmid sequencing to obtain complete coding sequences.

[0189] [Example 2: Implant screening of plants expressing bromoform] The genes were grouped into two different combinations as follows:

[0190] Group 1 (two plasmids containing four genes) Plasmid 1: mbb1 + mbb2 (Selective marker: spectinomycin) Plasmid 2: mbb3 + mbb4 (selective marker: kanamycin) Group 2 (3 plasmids with 7 genes) Plasmid 3: sfp+mbb1+mbb2 (selective marker: spectinomycin) Plasmid 4: FabH + mbb3 + mbb4 (selective marker: kanamycin) Plasmid 5: ACCase (selective marker: BASTA)

[0191] Different coding regions were cloned into pAB binary vectors under the control of CaMV35S, act8, or ubq10 constitutive promoters and HSP, EFIA2, or GmPPO1 terminators, respectively. Different combinations of plasmids 1–5 (including each plasmid from 1–4 individually, as well as combinations including 1+2, 3+4, 1+2+5, and 3+4+5) were transformed into Arabidopsis thaliana plants using Agrobacterium according to the dipping flower method essentially described in WO 2018 / 178975. Briefly, the plants were grown in a soil consisting of 75% peat and 25% perlite and routinely irrigated with water supplemented with fertilizer (e.g., Shefer 5.3.8, ICL Israel) as needed, according to the manufacturer's instructions. The plants began flowering after 3–4 weeks and were ready for transformation. Agrobacterium cultures transformed with the aforementioned plasmid were suspended in a suspension buffer (5% sucrose and 0.03% L-77 silwet), and flowers were immersed in the mixture for 1 minute. After 5–6 weeks of continuous growth, when the plants were dry, the seeds were harvested and kept in a cool, dry place for 2 weeks or until use. Successful transformation of the plants is shown in Figure 1.

[0192] [Table 3]

[0193] T1 generation transgenic seeds were selected with glufosinate ammonium (Basta, Bayer), kanamycin, spectinomycin, or a combination of these selections, according to the manufacturer's instructions. The plants were irrigated with 10 ppm CaBr2. The selected resistant plants were tested for bromoform production three weeks after germination, as described in Example 7 below.

[0194] Plants that can grow on a selection marker and whose transformation by the bromoform-producing gene is confirmed are expected to be able to produce bromoform.

[0195] [Example 3: Bromoform-producing tobacco plant] Tobacco plants (Nicotiana tabacum, cv. Little Dutch) were transformed with the above plasmids (combinations of plasmids 1-5, including 1+2, 3+4, 1+2+5, and 3+4+5) using Agrobacterium tumefaciens strain GV3101. Each of the above plasmids was independently transformed into bacteria by electroporation. Co-transformation with mixed bacteria was used for the expression of several genes in tobacco cells and plants. Tobacco leaf explants were immersed in Agrobacterium tumefaciens suspension for 10 minutes, then placed in petri dishes on solid co-culture medium (Gumborg B5 basal salt with vitamins, 0.1 mM acetosyringone, 2 mg / ml BAP, 0.2 mg / ml NAA, 0.8% plant agar) and incubated at 25C for 2-3 days under dark conditions. After co-culture, the explants were transferred to solidified shoot induction medium (Gumborg B5 basic salts (with vitamins), 2 mg / L BAP, 0.2 mg / L NAA, 250 mg / L cefotaxime, plasmid-mediated selective antibiotic; 100 mg / L kanamycin, 50 mg / L spectinomycin, 2 mg / L basta, 0.8% plant agar) and incubated at 25°C under 16 hours light / 8 hours dark conditions. Every 14 days, the explants were transferred to fresh medium. Fully developed viable shoots were planted in root induction medium (Gumborg B5 basic salts (with vitamins), 2 mg / L IBA, 250 mg / L cefotaxime, plasmid-mediated selective antibiotic; 100 mg / L kanamycin, 50 mg / L spectinomycin, 2 mg / L basta, 0.8% plant agar) and grown until roots developed. After acclimatization in the breeding stock, transgenic plants were transplanted into potting soil and maintained in a greenhouse. Successful plant transformation is shown in Figure 1. The plants were irrigated with 10 ppm CaBr2. Selected tolerant 3-week-old plants were tested for bromoform production as described in Example 7 below.

[0196] [Example 4: Test of bromine uptake by wild tobacco plants] Wild-type tobacco plants were used to test bromine uptake from the soil through their root systems. Tobacco plants were grown in a soil consisting of 75% peat and 25% perlite, routinely irrigated with water supplemented with fertilizer (e.g., Scheffer 5.3.8, ICL Israel), and periodically watered with plain water. From one month of age, irrigation with 5 ppm or 50 ppm CaBr2 was applied twice a week for three consecutive weeks, while control plants continued irrigation without CaBr2. 3 cm 2 Three discs were collected from each plant and used for bromine content analysis to evaluate bromine uptake.

[0197] [Table 4]

[0198] The results shown in Table 4 indicate that plants can absorb bromine from the soil and accumulate it in their leaves. Even these high concentrations of bromine accumulation do not cause any detectable harm to the plants or their respective growth and development.

[0199] [Example 5: Bromoform-producing alfalfa plant] The above coding region was cloned into a pPA binary vector as described above, and alfalfa plants (Medicago sativa spp.) were transformed by Agrobacterium-mediated transformation using combinations of vectors 1-5, including 1+2, 3+4, 1+2+5, and 3+4+5. Seeds were collected from the transformants, planted, and screened for the presence of constructs.

[0200] The seeds are surface-sterilized by immersion in 70% ethanol for 30 seconds. After removing the ethanol, the seeds are treated with 20% sodium hypochlorite and thoroughly washed three times with sterile water. Then, individual seeds are placed in a magenta box containing MS basal medium (Murashige and Skoog 1962), 30 g / L sucrose, and 0.8% plant agar. Cotyledons and fully developed leaves are removed from 2-3 week old plants and cut in half on moistened filter paper.

[0201] Approximately 100 leaf and cotyledon explants are transferred to Agrobacterium tumephasinus suspensions (OD 0.5 at 600 nm) in infection medium (MS basal medium (Murashige and Skoog 1962), 30 g / L sucrose, and 346 μM acetosyringone) at pH 5.8 and incubated at room temperature for 20 minutes. The explants are then transferred to filter paper to prevent overgrowth of bacteria during co-culture access, and then transferred to co-culture medium (Gamborg B5 medium (Gamborg 1984), 30 g / L sucrose, 2 mg / L 2,4-D, 0.1 mg / L kinetin, and 346 μM acetosyringone) and incubated in the dark for 3 days.

[0202] Seven days after inoculation, wash the explants with sterile water to remove bacteria and drain briefly on filter paper. Transfer 10-12 explants to callus induction medium (Gumborg B5 medium, 30 g / L sucrose, 0.8% plant agar, 2 mg / L 2,4-D, 0.1 mg / L kinetin, 500 mg / L cefotaxime sodium, and selective reagents (by construct: 50 mg / L kanamycin, 4 mg / L PPT, 25 mg / L spectinomycin)). Transfer the grafts to fresh medium every 14 days until somatic embryos are formed. Dark green mature embryos are placed in a plant growth medium (Gumborg B5 medium, 30 g / L sucrose, 0.8% plant agar, 0.1 mg / L myo-inositol, 500 mg / L cefotaxime sodium, and selective reagents (construct: 50 mg / L kanamycin, 4 mg / L PPT, 25 mg / L spectinomycin)). The callus is transferred to fresh medium every 14 days until shoots are formed. The green, developed plantlets are transferred to a rooting medium (Gumborg B5 medium, 30 g / L sucrose, 0.8% plant agar, 0.1 mg / L myo-inositol, 500 mg / L cefotaxime sodium). After acclimatization in the reproductive system, the transgenic plants are transplanted into potting soil and maintained in a greenhouse.

[0203] Transgenic seeds of the T1 generation should be germinated at the time of selection according to the manufacturer's instructions (as described above). Three to four days after germination, the plants should be irrigated with 5 ppm or 50 ppm CaBr2. Selected resistant plants should be tested for bromoform production three weeks after germination.

[0204] Plants that can grow on a selection marker and whose transformation by the bromoform-producing gene is confirmed are also expected to be able to produce bromoform.

[0205] [Example 6: Genetic modification of Brachiaria plants] The above plasmids (combinations of plasmids 1-5, including 1+2, 3+4, 1+2+5, and 3+4+5) were transformed into Brachiaria (signaling grass) via Agrobacterium-mediated transformation. Seeds were then collected from the transformants, planted, and screened for the presence of constructs.

[0206] The seeds are surface-sterilized in 20% sodium hypochlorite for 20 minutes and then thoroughly washed three times with sterile water. The disinfected seeds are placed on a solid agar medium containing 5 g / L sucrose and 6 g / L agar for 5 hours, and then immersed in distilled sterile water overnight at room temperature in the dark. The embryo explants are isolated using a scalpel blade.

[0207] Approximately 150 embryos are transferred to Agrobacterium tumephasinus suspensions (OD 0.5 at 600 nm) in infection medium (MS basal medium (Murashige and Skoog 1962), 30 g / L sucrose, 346 μM acetosyringone, and 1 mM dithiothreitol) at pH 5.8 and sonicated for 30 seconds. After sonication, the explants are incubated at room temperature for 2 hours and then transferred to co-culture medium (MS basal medium, 30 g / L sucrose, 100 mg / L casein hydrosylate, 2 mg / L 2,4-D, 0.2 mg / L BAP, and 346 μM acetosyringone) using a single layer of autoclavable filter paper and left in the dark for 3 days.

[0208] After co-culture, the base of each hypocotyl is embedded in callus induction medium (MS basal medium, 30 g / L sucrose, 0.8% plant agar, 300 mg / L casein hydrosylate, 2 mg / L 2,4-D, 0.2 mg / L BAP, 500 mg / L cefotaxime sodium, and a selective reagent (constructed: 50 mg / L kanamycin / 4 mg / L PPT, 25 mg / L spectinomycin)). The explants are transferred to fresh medium every 14 days. Embryonic callus is transferred to a regeneration medium (MS basal medium, 30 g / L sucrose, 0.8% plant agar, 300 mg / L casein hydrosylate, 100 mg / L myo-inositol, 4 mg / L kinetin, 500 mg / L cefotaxime sodium, and selective reagents (constructs: 50 mg / L kanamycin, 4 mg / L PPT, 25 mg / L spectinomycin). Developed shoots are rooted in the presence of IAA mg / L and 15 g / L sucrose. After acclimatization in the reproductive system, transgenic plants are transplanted into potting soil and maintained in a greenhouse.

[0209] Plants that can grow on a selection marker and whose transformation by the bromoform-producing gene is confirmed are also expected to be able to produce bromoform.

[0210] [Example 7: Bromoform Manufacturing Test] Leaves from transformed plants are harvested, crushed, and extracted as detailed above, sometimes after irrigation with bromine (e.g., CaBr2). The extract's bromoform-producing capacity is assayed as follows by testing for bromoform and its precursors by GC / LC-MS: Approximately 500 mg of plant leaves are weighed into an HS-20 vial, and the vial is cryogenically frozen in a beaker containing acetone and dry ice; the leaves are then crushed until a fine powder is obtained, and 13 mL of water, 5.0 g of NaCl, and 10 uL of DCB (1,4-dichlorobenzene) (approximately 0.13 mg / mL) are added to the vial; the vial is sealed and inserted into a headspace gas chromatography / mass spectrometry (GC / MS) analysis system.

Claims

1. A system for gene expression in a plant comprising one or more recombinant nucleic acid molecules, wherein the recombinant nucleic acid molecule comprises one or more nucleotide sequences comprising two or more genes together, each gene being operably linked to a promoter for expression in the plant, and the two or more genes are involved in bromoform production and encode two or more enzymes not encoded by the natural plant.

2. The system according to claim 1, wherein the two or more genes are contained in a single nucleic acid molecule.

3. The system according to claim 1, wherein the two or more genes are contained in two or more nucleic acid molecules.

4. The system according to any one of claims 1 to 3, wherein at least one of the two or more genes is derived from an organism selected from Asparagopsis taxiformis, Asparagopsis armata, Condorus crispus, Macrocystis pirifera, Medicago sativa, and Medicago truncatula.

5. The system according to any one of claims 1 to 4, wherein at least two of the two or more genes are derived from at least one non-plant organism.

6. The system according to any one of claims 1 to 5, wherein the two or more enzymes include at least one haloperoxidase.

7. The system according to any one of claims 1 to 6, wherein the two or more enzymes include NAD(P)H oxidase (NOX).

8. The system according to any one of claims 1 to 7, wherein the two or more enzymes include at least one enzyme involved in fatty acid synthesis.

9. The system according to claim 8, wherein the at least one enzyme involved in fatty acid synthesis is selected from β-ketoacyl-[acyl-carrier-protein] synthase III (FabH), phosphopantetheinyltransferase (sfp), acetyl-CoA carboxylase (ACCase), and malonyl-CoA synthase (MatB).

10. The system according to claim 9, wherein the two or more enzymes include haloperoxidase, NOX, and at least one enzyme involved in fatty acid synthesis selected from FabH, sphp, ACCase, and MatB.

11. The system according to claim 10, wherein the two or more enzymes include haloperoxidase, NOX, FabH, sfp, and ACCase.

12. The system according to any one of claims 1 to 11, wherein two or more of the genes are selected from Mbb1, Mbb2, Mbb3, Mbb4, CcVHPO1, CcVHPO2, CcVHPO3, CcVHPO4, CcVHPO5, CcMbb2, Sfp, FaBH, ACCase, and MatB.

13. The system according to claim 12, wherein the two or more genes include Mbb1, Mbb2, Mbb3, Mbb4, and at least one gene selected from FaBH, Sfp, ACCase, and MatB.

14. The system according to claim 13, wherein the two or more genes include Mbb1, Mbb2, Mbb3, Mbb4, FaBH, Sfp, and ACCase.

15. The haloperoxidase has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence identity with SEQ ID NOX has 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more sequence identity with SEQ ID NOX 2 (Mbb2); the FabH has at least 70% or more, 75% or more sequence identity with SEQ ID NOX 5 The system according to any one of claims 10 to 14, wherein the above has sequence identity of 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more; the sphp has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more with respect to sequence number 6; and / or the ACCase has sequence identity of 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, 95% or more, or 99% or more.

16. The system according to any one of claims 1 to 15, wherein two or more genes are operably linked to the same promoter.

17. The system according to any one of claims 1 to 15, wherein two or more genes are operably linked to different promoters.

18. The system according to any one of claims 1 to 17, wherein at least one of the promoters is a constitutive promoter.

19. The system according to any one of claims 1 to 17, wherein at least one of the promoters is an inductive promoter.

20. The system according to claim 19, wherein the inducible promoter is a tissue-specific promoter that is activated in plant organs that develop in the later stages of the plant life cycle, such as leaves or flowers.

21. The system according to any one of claims 1 to 20, wherein at least one of the two or more genes further encodes a chloroplast transport peptide for targeting the enzyme to the chloroplast.

22. The system according to any one of claims 1 to 21, wherein the plant is a pasture plant or a fodder plant.

23. The system according to any one of claims 1 to 22, wherein the plant is a grass, a cereal, or a legume.

24. The system according to any one of claims 1 to 23, wherein the plant is selected from signal grass (Brachiaria), alfalfa (Medicago sativa, Lucerne), ryegrass, tall fescue, coxsfoot, clover, oats, millet, chrysanthemum, chicory, corn, soybean, and plantain.

25. A composition comprising the system described in any one of claims 1 to 24.

26. A method for preparing a genetically modified plant or plant part, comprising transforming the plant or plant part using the system described in any one of claims 1 to 24 or the composition described in claim 25.

27. Genetically modified plant cells comprising the system described in any one of claims 1 to 24, or prepared by the method described in claim 26.

28. A genetically modified bromoform-producing plant or plant part comprising the system described in any one of claims 1 to 24, or prepared by the method described in claim 26.

29. A genetically engineered bromoform-producing plant or plant part comprising one or more exogenous nucleotide sequences containing two or more genes, each gene operably linked to a promoter for expression in the plant, wherein the two or more genes together encode two or more enzymes involved in bromoform production that are not encoded by the natural plant.

30. A genetically modified bromoform-producing plant or plant part, wherein the plant part is a plant cell.

31. The bromoform-producing plant or plant part according to any one of claims 28 to 30, wherein the plant is selected from signal grass (Brachiaria), alfalfa (Medicago sativa, Lucerne), ryegrass, tall fescue, coxsfoot, clover, oats, millet, chrysanthemum, chicory, corn, soybean, and plantain.

32. The bromoform-producing plant or plant part according to any one of claims 28 to 31, wherein the plant part is selected from seeds, cells, leaves, stems, roots, tubers, sculpts, flowers, bark, fruits, bulbs, furs, and rhizomes.

33. A composition comprising a bromoform-producing plant or plant part according to any one of claims 28 to 32, mixed with a plant or plant part that cannot produce bromoform, the composition comprising up to about 10% of the bromoform-producing plant or plant part.

34. A method for reducing methane gas emissions from ruminants: A step of producing seeds of genetically modified pasture plants containing two or more genes involved in bromoform production; The steps include: sowing genetically modified seeds in ruminant pastures; and A method comprising the step of growing a pasture plant that produces bromoform from the said seeds, thereby causing the bromoform-producing pasture plant to be consumed by ruminants.

35. A method for preparing food to reduce methane gas emissions from ruminant animals, comprising: A step of producing seeds of a genetically modified forage plant that contains two or more genes involved in bromoform production; Steps for sowing genetically modified seeds; The step of growing bromoform-producing forage plants from the said seeds; and A method comprising the step of preparing a food containing a bromoform-producing forage plant or a portion thereof.

36. The method according to claim 34 or 35, wherein the genetically modified seeds are produced by the method according to claim 26.

37. The method according to any one of claims 34 to 36, wherein the ruminant is a cattle.

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