WO2018234191A1 - Résistance accrue à la sécheresse dans des plantes - Google Patents

Résistance accrue à la sécheresse dans des plantes Download PDF

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WO2018234191A1
WO2018234191A1 PCT/EP2018/065981 EP2018065981W WO2018234191A1 WO 2018234191 A1 WO2018234191 A1 WO 2018234191A1 EP 2018065981 W EP2018065981 W EP 2018065981W WO 2018234191 A1 WO2018234191 A1 WO 2018234191A1
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farnesylation
orthologue
drought
orthologues
plant
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PCT/EP2018/065981
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Peter Brodersen
Andrea BARGHETTI
Lars Sjögren
Maïna FLORIS
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University Of Copenhagen
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8291—Hormone-influenced development
    • C12N15/8293—Abscisic acid [ABA]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8218—Antisense, co-suppression, viral induced gene silencing [VIGS], post-transcriptional induced gene silencing [PTGS]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • the present invention relates to drought-resistant plants, as well as methods of obtaining such plants and nucleic acids and expression vectors useful therefor.
  • Post-translational modification is important for the control of cellular protein activity.
  • Lipid modifications influence the hydrophobicity of proteins and may regulate their membrane association and interactions with other proteins.
  • Prenylation a form of Iipidation in which a poly-unsaturated farnesyl (C1 5 H2 5 ) or geranylgeranyl (C 2 oH 35 ) group is attached to the C-terminal part of target proteins, is particularly widespread in eukaryotic organisms. Protein farnesylation and geranylgeranylation may be catalyzed by deeply conserved heterodimeric enzymes that share the same a-subunit, but have distinct ⁇ -subunits.
  • ENHANCED RESPONSE TO ABA 1 (ERA 1, AT5G40280), while the ⁇ -subunit GGB (AT2G39550) confers specificity to protein geranylgeranyl transferase.
  • Protein farnesylation is implicated in abscisic acid (ABA) signaling, because eral and pip mutants exhibit pronounced hypersensitivity to ABA.
  • ABA abscisic acid
  • ABA is required for seed dormancy and for restriction of water loss in response to drought.
  • ABA signal transduction pathway has now been elucidated in which ABA perception by pyrabactin- resistance (PYRI )-like receptors is linked to activation of a set of protein kinases in the SNF1 -related kinase 2 (SnRK2) family by inhibition of the phosphatases encoded by ABSCISIC ACID INSENSITIVE ⁇ ABI)1 and ABI2.
  • SnRK2 kinases essential for ABA signaling, in turn phosphorylate numerous targets including transcriptional activators of an ABA-related gene expression program and ion channels implicated in rapid closure of guard cells.
  • farnesyl transferase mutants In addition to ABA hypersensitivity, several developmental phenotypes have been observed in farnesyl transferase mutants. These include altered phyllotaxis and increased floral organ numbers, both of which may derive from enlarged and disorganized meristems in eral and pip (Running et al. 1998; Yalovsky et al. 2000; Running et al. 2004). Moreover, farnesyl transferase mutants are late flowering, and have rounder leaf shape. While the effect on leaf shape is probably explained by reduced brassinosteroid biosynthesis due to defective CYP85A2 farnesylation (Northey et al. 2016), the molecular basis of the other phenotypes remains unexplained.
  • J2 (AT5G22060) and J3 (AT3G441 10), are two of more than 100 isoforms of Heat Shock Protein 40 (HSP40) proteins encoded in the Arabidopsis genome. HSP40 proteins can initiate a conserved chaperone assembly line that mediates
  • an HSP40 dimer binds a client protein and triggers ATP hydrolysis in HSP70 to drive formation of a high- affinity HSP70-ADP-client complex.
  • the adaptor protein Hop mediates client transfer to HSP90 for final conformational maturation together with a host of HSP90 co- chaperones.
  • Proteins with binding pockets for hydrophobic ligands constitute another well-studied class of clients of the HSP90 assembly line, exemplified by the vertebrate steroid hormone receptors.
  • the HSP90 chaperone pathway plays a crucial role in development and immune signaling.
  • auxin and jasmonate receptors a class of proteins with hydrophobic binding pockets, as well as intracellular immune receptors and effector proteins of small RNA-guided gene regulation, two classes of multidomain proteins whose functions are associated with extensive conformational changes.
  • WO 02/097097 discloses drought-tolerant plants obtained by manipulation of the enzyme catalyzing farnesylation (farnesyl transferase) so that its expression and activity are altered.
  • J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are
  • a drought-resistant plant derived from a parent plant comprising one or more orthologue of J2 of Arabidopsis thaliana (SEQ ID NO: 1 ) and/or one or more orthologue of J3 of Arabidopsis thaliana (SEQ ID NO: 2), wherein the orthologues of J2 and J3 are farnesylation targets in the parent plant, wherein, at least under drought conditions:
  • At least one of a farnesylation-deficient J2 orthologue and a farnesylation-deficient J3 orthologue is active in the drought-resistant plant.
  • Also provided is a method of increasing drought resistance in a plant comprising one or more orthologue of J2 of Arabidopsis thaliana (SEQ ID NO: 1 ) and/or one or more orthologue of J3 of Arabidopsis thaliana (SEQ ID NO: 2), wherein the orthologues of J2 and/or J3 are farnesylation targets, said method comprising:
  • nucleic acid capable of specifically inactivating farnesylation of an orthologue of J2 of Arabidopsis thaliana (SEQ ID NO: 1 ) upon expression, as described herein. Also provided is a nucleic acid capable of specifically inactivating farnesylation of an orthologue of J3 of Arabidopsis thaliana (SEQ ID NO: 2) upon expression, as described herein. Also provided are expression vectors comprising the nucleic acids described herein.
  • polypeptides encoded by the nucleic acids described herein are also provided.
  • antibodies capable of specific binding to the polypeptides described herein.
  • Fig. 1 J3 is farnesylated in planta.
  • A Schematic overview of transient expression of His 6 -J3 in N. benthamiana to test Cys417-dependent incorporation of 14 C-mevalonate- derived label into J3 protein.
  • B Ni 2+ -affinity purified fractions of extracts from N.
  • Fig. 2 Genetic evidence for functional links between ERA1 and HSP40/70/90.
  • Fig. 3 J2/J3 are key farnesylated targets for ABA signalling and drought resistance.
  • Bottom Pictures of plates after 14 days of growth on plates with the indicated concentrations of ABA. The experiment was repeated twice with independent seed lots. Seed lots originated from parental plants that were grown side by side, and mutant and wild type seeds were cleaned at the same time and stored together before use in the experiments.
  • B Water retention in 5-week old rosettes after a 2-week period without watering.
  • J2/J3 are key farnesylation targets in plant development.
  • B Representative micrographs of inflorescence meristems from the indicated genotypes. The width of the meristem was measured as the length of the white line originating at the youngest primordium.
  • Fig.5 Expression of miR397/398/408/857 is impaired in the absence of J2/J3 farnesylation.
  • A Plots of read counts of miRbase-matching small RNAs. Upper panel, Col-0 (abscissa) versus era1-2; middle panel, j2-2/j3-2 expressing J3 WT
  • the plp-3 mutant is a previously uncharacterized exonic T-DNA insertion in the PLP gene (GABI-KAT_386_C07, Fig. S1 ). The same northern membrane was used for consecutive hybridization to miR398, miR408 and U6.
  • C qPCR analysis of pri-miR397a, pri-miR398a/b, pri-miR408 and pri- miR857 abundance. 16-day old sterile grown seedlings were used for RNA extraction.
  • pri-miR397 For pri-miR397, pri-miR398b and pri-miR857, the same pool of oligo(dT)-primed cDNA was used as template.
  • pri-miR408 gene specific reverse transcription was performed to avoid amplification of an overlapping mRNA transcribed from the opposite strand.
  • the figure shows results of one set of biological samples (50 seedlings pooled per RNA preparation) with standard deviation between technical triplicates, and the whole experiment was repeated twice with nearly identical results.
  • D qPCR analysis of pri-miR156a, pri-miR164a, pri-miR166a and SPL7 mRNA abundance. The same RNA samples as those analyzed in (C) were used.
  • Drought-resistant or drought-tolerant A drought-resistant plant, plant cell or plant part refers herein to a plant, plant cell or plant part, respectively, having increased resistance to drought compared to a parent plant from which they are derived. Methods of determining drought resistance are known to the person of skill in the art. Examples of such methods are provided in the examples below.
  • Orthologue two genes from different species are orthologues if they share homology and are thought to have evolved from a common ancestral gene, e.g. by speciation events. Orthologues encode proteins which often, but not always, share the same function. By extension, the term orthologue can be used to refer to proteins sharing the same function. In the present context, the term orthologue preferably refers to proteins having the same function or to genes encoding proteins having the same function. For example, an orthologue of J2 of Arabidopsis thaliana in another plant may preferably also be an isoform of Heat Shock Protein 40 (HSP40). All J2/J3 orthologues comprise a C-terminal farnesylation motif (Ca-ia 2 X).
  • HSP Heat Shock Protein
  • the term refers to proteins capable of performing chaperone functions, in particular in response to stress conditions. The function of many HSPs is to stabilise new proteins to ensure correct folding or to help refolding proteins that were damaged by cell stress.
  • Specific inactivation the term is herein to be construed as the inactivation of one specific gene or gene product, or as the inactivation of one specific reaction on a specific substrate or target.
  • specific inactivation of farnesylation of a J2 orthologue refers to the specific inactivation of the farnesylation of said J2 orthologue; specific inactivation of a farnesylation-proficient J3 orthologue may refer to the specific inactivation of the farnesylation of the J3 orthologue or to the genetic inactivation of the gene encoding the farnesylation-proficient J3 orthologue.
  • specific inactivation is typically achieved by techniques known in the art, as detailed below.
  • Specific inactivation of a reaction on a specific substrate can be performed by inactivating the enzyme responsible for the reaction provided that the enzyme has only one substrate.
  • Prenylation also known as isoprenylation or lipidation
  • Protein prenylation involves the transfer of either a farnesyl or a geranyl-geranyl moiety to C-terminal cysteine(s) of the target protein.
  • farnesyl transferase Caax protease
  • geranylgeranyl transferase There are three enzymes that carry out prenylation in the cell: farnesyl transferase, Caax protease and geranylgeranyl transferase.
  • Farnesylation is a type of prenylation, a post-translational modification of proteins by which an isoprenyl group is added to a cysteine residue. It is an important process to mediate protein-protein interactions and protein-membrane interactions. It is carried out by farnesyltransferase, consisting of an apha-subunit and a beta-subunit.
  • Farnesyltransferase recognises farnesylation sites having a sequence Ca-ia 2 X motif, where C is a cysteine residue, ai and a 2 are each independently selected from the group consisting of aliphatic amino acids, and X is selected from the group consisting of M, S, Q, A and C, wherein M is a methionine residue, S is a serine residue, Q is a glutamine residue, A is an alanine residue and C is a cysteine residue.
  • the farnesylation motif is often CAQQ.
  • Farnesyltransferase and geranylgeranyltransferase I are very similar proteins. They consist of two subunits, the osubunit, which is common to both enzymes, and the ⁇ - subunit, whose sequence identity is just 25%. These enzymes recognise the Ca-ia 2 X box at the C-terminus of the target protein. C is the cysteine that is prenylated, ai and a 2 are independently any aliphatic amino acid, and the identity of X determines which enzyme acts on the protein.
  • farnesylation Such protein may be as described herein, i.e. it can be a mutant, or it can be a protein encoded by a gene having a wild type sequence, but where farnesylation is specifically inactivated, for example by expressing silencing RNAs which prevent or reduce translation of the transcript into a farnesylation-proficient protein.
  • farnesylation-proficient when applied to a protein refers to a protein which can be farnesylated.
  • J2 and J3 are farnesylation targets in planta
  • Farnesyl transferase mutant plants have been generated which have increased drought resistance. However, such mutants display a number of developmental phenotypes, possibly because farnesyl transferase has other substrates than the substrates directly involved in drought resistance.
  • J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are based on the surprising finding that J2 and J3 are
  • J2 and J3 of Arabidopsis thaliana are set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.
  • the methods disclosed herein are useful for obtaining plants which are drought- tolerant, drought-resistant, or generally have increased tolerance to water stress compared to a parent plant. This can be particularly relevant for plants which have suboptimal tolerance to water stress in the region where they are cultivated.
  • the parent plant which it is desirable to modify in order to obtain a drought-resistant or drought-tolerant plant, may be any plant type including crops.
  • the parent plant is a food crop or an industrial crop such as a fibre crop, an energy crop or a medicinal crop.
  • the parent plant may for example be canola, wheat, rice, barley, maize, sugarcane, potato, soybean, millet, linen, cotton, corn, rye, tomato, cucumber or pepper.
  • the plant may belong to any genus, for example, the plant may be a species from the genera Arabidopsis, Brassica, Oryza, Zea, Sorghum, Brachypodium, Miscanthus, Gossypium, Triticum, Glycine, Pisum, Phaseolus, Lycopersicon, Trifolium, Cannabis, Cucurbita, Rosa, Vitis, Juglans, Fragaria, Lotus, Medicago, Onobrychis, Trigonella, Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Nicotiana, Solanum, Petunia, Digitalis, Majorana, Ciahorium, Helianthus, Lactuca, Bromus, Asparagus, Antirrhinum, Heterocallis, Nemesis, Pelargonium, Panieum, Pennisetum, Ranunculus, Sene
  • the plant wherein farnesylation of the J2 and J3 orthologues is specifically inactivated as described herein may be in the form of a protoplast, a plant cell, a plant tissue or a plant (e.g., angiosperm, monocot or dicot).
  • plant is thus meant to include not only a whole plant but also a portion thereof (i.e., cells, and tissues, including for example, leaves, stems, shoots, roots, flowers, fruits and seeds).
  • plant cells including for example, leaves, stems, shoots, roots, flowers, fruits and seeds, plant-derived progeny, tissue cultures, protoplast cultures and somatic embryos.
  • the present disclosure thus relates to plants having increased drought resistance. Such plants are obtained by the methods disclosed therein, and rely on the specific inactivation of farnesylation of J2 and J3 or their orthologues in the plant of interest.
  • the specific inactivation may be transient or permanent.
  • orthologues of J2 and/or J3 in the plant of interest may need to be farnesylation-deficient in order for the desired phenotype to be achieved.
  • Orthologues of J2 and J3 are found in most plant species. In some species, there is one orthologue of J2 and one orthologue of J3. In other species, there are several orthologues of J2 and/or several orthologues of J3. Some plants may have only orthologues of J2, while others may have only orthologues of J3. The skilled person will know how to determine how many orthologues of J2 and/or J3 are present in the plant to be modified, by determining the degree of amino acid conservation and the presence of a C-terminal Ca-ia 2 X farnesylation motif. The motif in plants is often a CAQQ motif.
  • a drought-resistant plant derived from a parent plant, the parent plant comprising an orthologue of J2 of Arabidopsis thaliana (SEQ ID NO: 1 ) and an orthologue of J3 of Arabidopsis thaliana (SEQ ID NO: 2), wherein the orthologues of J2 and J3 are farnesylation targets in the parent plant,
  • the plant preferably expresses at least one J2 or J3 orthologue when not under drought conditions.
  • at least one farnesylation-proficient J2 or J3 orthologue is expressed and active when the plant is not under drought conditions.
  • the farnesylation-deficient J2 or J3 orthologues are expressed and active at least when the plant is under drought conditions.
  • farnesylation-deficient orthologue of J2 or a farnesylation-deficient orthologue of J3 will be considered active if it is capable of performing at least some or all of the functions of farnesylation-proficient orthologues of J2 and J3 which are independent of their farnesylation.
  • farnesylation-deficient orthologues of J2 and J3 are not expected to behave like mutant proteins having a total loss of function. Instead, they preferably retain at least some of the activities of farnesylation-proficient orthologues of J2 and J3.
  • the orthologues of J2 and J3 in the parent plant comprise a farnesylation site.
  • Farnesylation sites comprise a Ca-ia 2 X motif, where C is a cysteine residue, ai and a 2 are each independently selected from the group consisting of aliphatic amino acids, and X is selected from the group consisting of M, S, Q, A and C, wherein M is a methionine residue, S is a serine residue, Q is a glutamine residue, A is an alanine residue and C is a cysteine residue.
  • the Ca-ia 2 X motif is a CAQQ motif.
  • J2/J3 orthologues in a given plant will typically be found, if not already known to the skilled person, by identifying the closest homologue to the sequences of J2 or J3 in A. t aliana as set out in SEQ ID NO: 1 and SEQ ID NO: 2, respectively, having the highest degree of sequence identity.
  • the J2/J3 orthologue has at least 70% sequence identity to SEQ ID NO: 1 , such as at least 75% sequence identity, for example at least 80% sequence identity, such as at least 81 % sequence identity, for example at least 82% sequence identity, such as at least 83% sequence identity, for example at least 84% sequence identity, such as at least 85% sequence identity, for example at least 86% sequence identity, such as at least 87% sequence identity, for example at least 88% sequence identity, such as at least 89% sequence such as at least 90% sequence identity, for example at least 91 % sequence identity, such as at least 92% sequence identity, for example at least 93% sequence identity, such as at least 94% sequence identity, for example at least 95% sequence identity, such as at least 96% sequence identity, for example at least 97% sequence identity, such as at least 98% sequence identity, for example at least 99% sequence identity to SEQ ID NO: 1.
  • the J2/J3 orthologue has at least 70% sequence identity to SEQ ID NO: 2, such as at least 75% sequence identity, for example at least 80% sequence identity, such as at least 81 % sequence identity, for example at least 82% sequence identity, such as at least 83% sequence identity, for example at least 84% sequence identity, such as at least 85% sequence identity, for example at least 86% sequence identity, such as at least 87% sequence identity, for example at least 88% sequence identity, such as at least 89% sequence such as at least 90% sequence identity, for example at least 91 % sequence identity, such as at least 92% sequence identity, for example at least 93% sequence identity, such as at least 94% sequence identity, for example at least 95% sequence identity, such as at least 96% sequence identity, for example at least 97% sequence identity, such as at least 98% sequence identity, for example at least 99% sequence identity to SEQ ID NO: 2.
  • Farnesylation is usually, but not always, followed by proteolytic removal of a-ia 2 X and carboxymethylation of the terminal COOH.
  • Specific inactivation of farnesylation of the J2 and J3 orthologues in a parent plant in order to obtain a drought-tolerant plant may be achieved in various ways, for example mutation, transcriptional regulation, post transcriptional and translational regulation.
  • Such methods include, but are not limited to, antisense methods, RNAi constructs, including all hairpin constructs and RNAi constructs useful for inhibition by dsRNA- directed DNA methylation or inhibition by mRNA degradation or inhibition of translation, microRNA (miRNA), including artificial miRNA (amiRNA) technologies, mutagenesis and TILLING methods, in vivo site specific mutagenesis techniques and
  • the farnesylation sites of the J2 and J3 orthologues are mutated so that the J2 and J3 orthologues comprise a mutated farnesylation site, for example a site which cannot be recognised or bound by farnesyl transferase, or a site which can be recognised and bound by farnesyl transferase but which prevents farnesylation from occurring in a complete manner.
  • the sequence corresponding to the farnesylation site may in the drought-resistant plant have a sequence differing from the consensus sequence Caia 2 X by at least one residue, such as by at least two residues, such as by at least four residues, such as by all four residues.
  • the last four amino acid residues of J2 and J3 of A are mutated so that the J2 and J3 orthologues comprise a mutated farnesylation site, for example a site which cannot be recognised or bound by farnesyl transferase, or a site which can be recognised and bound by farnesyl transferase but which prevent
  • thaliana constitute the farnesylation sites (SEQ ID NO: 1 and SEQ ID NO: 2, respectively) of J2 and J3.
  • the cysteine is mutated to another amino acid residue.
  • ai and a 2 are independently mutated to amino acid residues, which are not aliphatic.
  • X is mutated to an amino acid which is not M, S, Q, A or C.
  • the farnesylation sites of the J2 and J3 orthologues are mutated so that the orthologues are devoid of farnesylation sites altogether. This can be done by deleting one, two, three or all four of the amino acids of which the farnesylation motif consists.
  • the farnesylation site Ca-ia 2 X is mutated by deletion of C, a- ⁇ , a 2 or X.
  • the farnesylation site is mutated by deletion of two of C, a- ⁇ , a 2 or X.
  • the farnesylation site is mutated by deletion of three of C, a ⁇ ⁇ , a 2 or X.
  • the farnesylation site is mutated by deletion of all four of C, a ⁇ ⁇ , a 2 or X.
  • the cysteine residue is mutated to a serine residue.
  • the cysteine residue is mutated to a serine residue.
  • the cysteine residue is mutated to a serine residue
  • farnesylation site has the sequence CAQQ and is mutated by deletion of C, A, or either of the Q residues.
  • the farnesylation site is mutated by deletion of two of C, A, or either of the Q residues.
  • the farnesylation site is mutated by deletion of three of C, A, or either of the Q residues.
  • the farnesylation site is mutated by deletion of all four of C, A, or either of the Q residues.
  • the cysteine residue is mutated to a serine residue.
  • Other methods for mutating the farnesylation site are readily available to the skilled person. For example, the farnesylation site may be disrupted by insertion of an additional amino acid within the Ca-ia 2 X sequence, so that the site can no longer be recognised nor processed by farnesyl transferase.
  • the orthologues of J2 and J3 are not mutated at the genomic level.
  • the genome of the drought-resistant plant may comprise wild-type copies of the genes encoding J2 and J3, which are normally expressed and farnesylated under normal conditions, i.e. normally expressed when not under drought conditions.
  • the plant may additionally have been transformed with an expression vector comprising a nucleic acid which is designed for specifically inactivating the
  • Useful nucleic acids for achieving this include RNAi, antisense RNAs, silencing RNAs and guide RNAs.
  • the nucleic acid may be an artificial RNAi, also known as hairpin construct.
  • a portion of the gene encoding the orthologue to inhibit is used and cloned in a sense and antisense direction having a spacer separating the sense and antisense portions and including well-chosen mismatches, as in natural mRNA precursors, and as is known to the skilled person.
  • the size of the gene portions is preferably at least 21 nucleotides in length and the spacer may be a little as 13 nucleotides (Schwab et al., 2006) in length and may be an intron sequence, a coding or non-coding sequence.
  • Antisense RNA relies on the expression of the target gene, or a portion thereof, in an antisense orientation resulting in inhibition of the endogenous gene expression and activity.
  • the antisense portions need not be a full length gene nor be 100% identical, as is known to the skilled person. Often it is sufficient if the antisense is at least about 70% or more identical to the endogenous target gene and has a sufficient length to allow specific recognition of the target by the encoded antisense RNA.
  • the plant to be modified carries a wild-type copy of the gene or genes encoding the J2 and J3 orthologues and a modified copy encoding the corresponding farnesylation-deficient J2 and J3 orthologues. Any of these genes may be present in the genome, as an extrachromosomal copy or on an expression vector.
  • the plant to be modified may further carry a nucleic acid as described herein above, wherein said nucleic acid is expressed only under drought conditions.
  • the plant when the conditions are not drought conditions, the plant expresses at least active and farnesylation-proficient J2 and J3 orthologues; the farnesylation- deficient J2 and J3 orthologues may however also be expressed.
  • the farnesylation- deficient J2/J3 orthologues may be expressed constitutively, their genes may be operably linked to their native promoter, or to a drought-inducible promoter.
  • the functions performed by the J2 and J3 orthologues in normal conditions are performed by a pool of farnesylation-proficient J2 and J3 orthologues and of farnesylation-deficient J2 and J3 orthologues.
  • nucleic acid When drought conditions are present, expression of a nucleic acid as described above may be induced, wherein the nucleic acid is designed to specifically target the wild-type (farnesylation-proficient) J2 and J3 orthologues, but not the farnesylation-deficient J2 and J3 orthologues.
  • a nucleic acid may be a silencing RNA, an RNAi, or an antisense RNA, which prevent translation of the farnesylation-proficient J2 or J3 transcript into a protein.
  • the nucleic acid may also be a guide RNA; if expressed together with the remaining CRISPR-Cas machinery, such a guide RNA will result in cleavage of the gene or genes encoding the farnesylation-proficient J2 or J3 orthologues and prevent expression of the corresponding farnesylation-proficient proteins. Expression of the farnesylation-deficient J2 and J3 orthologues is not affected. As a result, expression of the wild-type J2 and J3 orthologues is inhibited; only the farnesylation-deficient J2 and J3 orthologues are expressed and active, resulting in practice in a plant wherein farnesylation of J2 and J3 has been specifically inactivated under drought conditions.
  • the plant may be designed in such a way that expression of farnesylation-deficient J2 and J3 orthologues only or mainly occurs under drought conditions.
  • farnesylation-proficient J2 and J3 orthologues are expressed and active under normal conditions, but not under drought conditions. Expression of farnesylation- deficient J2 and J3 orthologues is repressed under normal conditions, but is active under drought conditions. This can be achieved for example by having a copy of genes encoding farnesylation-deficient J2 and J3 orthologues under the control of a drought- inducible promoter, as described herein below. Conversely, expression of
  • J2 and J3 orthologues is down-regulated at least under drought conditions.
  • guide RNAs, RNAis, antisense RNAs and nucleic acid constructs designed to mutate a J2/J3 orthologue, which are capable of recognising the orthologue or orthologues to be specifically inactivated will not be able to recognise and bind the other orthologue or orthologues.
  • guide RNAs, RNAis and antisense RNAs, or nucleic acid constructs designed to mutate a J2/J3 orthologue may be able to recognise and specifically inactive several J2/J3 orthologues at once.
  • the skilled person will know how to identify a target sequence which is common for all the orthologues that it is desirable to specifically inactivate as described herein when the sequence homology allows it.
  • the genes encoding farnesylation-proficient J2 and J3 orthologues are preferably operably linked to a first promoter.
  • the genes encoding farnesylation-deficient J2 and J3 orthologues may be operably linked to a second promoter different from the first promoter.
  • Expression of a transcription factor capable of repressing expression of the genes under control of the first promoter, but not of the genes under the control of the second promoter may be under the control of a drought- inducible promoter.
  • transcription of farnesylation-proficient J2 and J3 orthologues is inactivated under drought conditions, while transcription of farnesylation-proficient J2 and J3 orthologues is not affected by the transcriptional repression.
  • any combination of the above methods may also be envisaged.
  • the plant to be modified comprises several orthologues of J2 and J3, one of these or more may be permanently mutated at the genomic level, while the remaining J2 and J3 orthologues may be transiently inactivated under drought conditions only, as described above. Where nucleic acid constructs are needed, this may require the design of one nucleic acid per J2 or J3 orthologue.
  • nucleic acids capable of specifically inactivating farnesylation of an orthologue of J2 of Arabidopsis thaliana (SEQ ID NO: 1 ) upon expression, as well as nucleic acids capable of specifically inactivating farnesylation of an orthologue of J3 of Arabidopsis thaliana (SEQ ID NO: 2) upon expression.
  • the nucleic acid may be comprised within an expression vector, as is known in the art and as otherwise described herein.
  • the nucleic acid may be DNA.
  • the skilled person knows how to design DNA molecules to be used for specifically knocking out a gene, in particular a J2 or J3 orthologue gene.
  • introduction of the nucleic acid in the plant or plant cell results in knocking out of the J2 or J3 orthologue.
  • Another nucleic acid may be used to knock in genes encoding farnesylation-deficient J2 or J3 orthologues.
  • the nucleic acid not only comprises the gene to be knocked in, but also comprises a promoter, such as a drought-inducible promoter.
  • the nucleic acid encodes a farnesylation-deficient J2 or J3 orthologue, such as any of the orthologues described herein above.
  • the nucleic acid encodes a farnesylation-deficient J2 orthologue, wherein the farnesylation site has been modified, mutated, deleted or otherwise disrupted. In another embodiment, the nucleic acid encodes a farnesylation-deficient J3 orthologue, wherein the farnesylation site has been modified, mutated, deleted or otherwise disrupted.
  • polypeptides encoded by such nucleic acids such as a farnesylation-deficient J2 orthologue or a farnesylation-deficient J3 orthologue.
  • antibodies capable of specifically binding such polypeptides are also provided.
  • the nucleic acid may be an RNAi, an antisense RNA, a silencing RNA or a guide RNA.
  • Gene editing systems such as CRISPR-Cas may be used to obtain drought-resistant plants as described herein.
  • the nucleic acid is a guide RNA.
  • the plant cell also comprises the remaining components of the CRISPR-Cas machinery, for example an endonuclease, such as Cas9 or a Cas9 mutant.
  • the expression of each of the components may be under the control of drought-inducible promoter, whereby these are only expressed under drought conditions.
  • the guide RNA may be
  • the entire set of components of the CRISPR-Cas machinery is expressed only under drought conditions.
  • the guide RNA in this case preferably recognises the sequence or sequences encoding the endogenous J2 and J3 orthologues. In drought conditions, the CRISPR-Cas machinery may specifically bind to said sequences, thereby preventing expression of the endogenous, farnesylation-proficient J2 and J3 orthologues.
  • J2 or J3 orthologues may at the same time be expressed as described herein elsewhere, either transiently or permanently.
  • the nucleic acid is a silencing RNA, an RNAi or an antisense RNA, having a sequence complementary to the sequence of the farnesylation- proficient J2 or J3 orthologues transcripts.
  • the sequence may include a sequence complementary to the farnesylation motif, to ensure that only the farnesylation- proficient copies are targeted by the nucleic acid.
  • expression of the nucleic acid is under the control of a drought-inducible promoter. Upon drought, the nucleic acid is expressed, and is capable of specifically binding the transcripts of the endogenous J2 or J3 orthologues, thereby down-regulating or shutting down expression of the orthologues.
  • expression vectors comprising any of the above nucleic acids. Where several nucleic acids are needed, each may be comprised within an individual vector. However, it is also possible to construct expression vectors comprising several of the above nucleic acids at once.
  • nucleic acid is preferably operably linked to a promoter, for example a constitutive promoter, a tissue specific promoter or an inducible promoter.
  • promoters suitable for expression of a nucleic acid in plants include promoters from plant viruses such as the 35S promoter from cauliflower mosaic virus (CaMV), promoters from genes such as rice actin, ubiquitin; pEMU, MAS, maize H3 histone; and, the 5'- or 3'-promoter derived from T-DNA of Agro bacterium tumefaciens, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter, the Nos promoter, the rubisco promoter, the GRP1 -8 promoter, ALS promoter, a synthetic promoter, such as Rsyn7, SCP and UCP promoters, ribulose-1 ,3-diphosphate carboxylase, fruit- specific promoters, heat shock promoters, seed-specific promoters and other transcription initiation regions from various plant genes, for example, including the various opine initiation regions, such as for example, octopine, mannopine,
  • Additional regulatory elements that may be connected to a PK220 encoding nucleic acid sequence for expression in plant cells include terminators, polyadenylation sequences, and nucleic acid sequences encoding signal peptides that permit localization within a plant cell or secretion of the protein from the cell.
  • Such regulatory elements and methods for adding or exchanging these elements with the regulatory elements gene are known and include, but are not limited to, 3' termination and/or polyadenylation regions such as those of the Agrobacterium tumefaciens nopaline synthase (nos) gene; the potato proteinase inhibitor II (PINII) gene; and the CaMV 19S gene.
  • a preferable inducible promoter is a drought inducible promoter. Drought-inducible promoters are known in the art. The person of skill in the art also knows how to identify drought-inducible promoters in a given plant. This can be done for example by performing whole-genome transcriptome analysis of the same plant under normal conditions and under drought conditions, and comparing the resulting expression profiles. Introduction of nucleic acids in plants
  • Numerous methods for introducing nucleic acids into plants are known and can be used, including biological and physical plant transformation protocols (See, for example, Miki et al., (1993) and Bent wt al. (1998)).
  • the methods chosen vary with the host plant, and include chemical transfection methods such as calcium phosphate, polyethylene glycol (PEG) transformation, microorganism-mediated gene transfer such as Agrobacterium, electroporation, protoplast transformation, micro-injection, flower dipping and biolistic bombardment.
  • the most widely utilized method for introducing an expression vector into plants is based on the natural transformation system of Agrobacterium tumefaciens and A. rhizogenes which are plant pathogenic bacteria which genetically transform plant cells.
  • the Ti and Ri plasmids of A. tumefaciens and A. rhizogenes respectfully, carry genes responsible for genetic transformation of plants.
  • Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer are known to the skilled person.
  • a generally applicable method of plant transformation is microprojectile-mediated transformation, where DNA is carried on the surface of microprojectiles measuring about 1 to 4 ⁇ .
  • the expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate the plant cell walls and membranes.
  • Aerosol Beam Injector ABSI
  • Aerosol beam technology is used to accelerate wet or dry particles to speeds enabling the particles to penetrate living cells. Aerosol beam technology employs the jet expansion of an inert gas as it passes from a region of higher gas pressure to a region of lower gas pressure through a small orifice. The expanding gas accelerates aerosol droplets, containing nucleic acid molecules to be introduced into a cell or tissue. The accelerated particles are positioned to impact a preferred target, for example a plant cell. The particles are constructed as droplets of a sufficiently small size so that the cell survives the penetration.
  • CRISPR-Cas requires the design of a guide RNA, which together with a Cas enzyme such as Cas9 in a cell results in specific recognition and cleavage of a target sequence.
  • the target sequence can be replaced by another gene.
  • This regeneration and growth process typically includes the steps of selection of transformed cells, culturing those
  • transgenic embryos and seeds are similarly regenerated.
  • the resulting transgenic rooted shoots are thereafter planted in an appropriate plant growth medium such as soil.
  • the development or regeneration of plants containing the foreign, exogenous gene that encodes a polypeptide of interest introduced by Agrobacterium from leaf explants can be achieved by methods well known in the art. These procedures vary depending upon the particular plant strain employed, such variations being well known in the art.
  • SPL7 (SQUAMOSA promoter binding protein-like 7) is homologous to Copper response regulator 1 , the transcription factor that is required for switching between plastocyanin and cytochrome c6 in response to copper deficiency in Chlamydomonas reinhardtii.
  • SPL7 binds directly to GTAC motifs in the miR398 promoter in vitro (A. thaliana), and these motifs are essential and sufficient for the response to copper deficiency in vivo.
  • SPL7 is also required for the expression of multiple microRNAs, miR397, miR408, and miR857, involved in copper homeostasis and of genes encoding several copper transporters and a copper chaperone, indicating a central role in response to copper deficiency. Consistent with this idea, the growth of spl7 plants is severely impaired under low-copper conditions (Yamasaki et al. 2009).
  • SPL7-regulated miRNAs are poorly expressed in eral mutants and this effect can also be fully explained by lack of farnesylation of J2/J3.
  • the experimentally validated target mRNAs corresponding to these miRNAs do not encode proteins known to be involved in development (summarized in (Arribas-Hernandez et al. 2016)).
  • sp/7 knockout mutants do not exhibit developmental phenotypes similar to era 7 or pip mutants (Yamasaki et al. 2009).
  • the sequences of miRNAs from different species are available at the miRbase (http://www.mirbase.org).
  • drought-resistant plants in which expression of one or more SPL7-regulated miRNA is down-regulated.
  • the SPL7-regulated miRNA is miR397.
  • the SPL7-regulated miRNA is miR398.
  • the SPL7-regulated miRNA is miR408.
  • the SPL7-regulated miRNA is miR857.
  • the expression of the SPL7-regulated miRNAs is down- regulated by specific inactivation of SPL7. This can be achieved by any of the methods described herein.
  • expression of a nucleic acid targeting SPL7 can be under the control of a drought-inducible promoter. Under normal conditions, SPL7 is expressed and normally active. Under drought conditions, expression of a nucleic acid capable of specifically inactivating SPL7 is induced. As a consequence, expression of miRNAs miR397, miR398, miR408 and miR857 is down-regulated, and a drought-resistant phenotype is achieved.
  • expression of one or more of miR397, miR398, miR408 and miR857 may be down-regulated as is known in the art. This can be done by introducing nucleic acids in the plant, which are capable of specifically inhibiting expression miR397, miR398, miR408 or miR857. Such nucleic acids may either prevent the transcription of the corresponding pre-microRNAs., or prevent the formation of the RNA-induced silencing complex (RISC) in which the miRNAs are integrated after DICER-mediated processing.
  • RISC RNA-induced silencing complex
  • a method of increasing drought resistance in a plant comprising down-regulating expression of one or more of miR397, miR398, miR408 and miR857 at least under drought conditions.
  • expression of one or more of said miRNAs is down-regulated directly, e.g. by targeting each miRNA individually.
  • expression of SPL7 is specifically inactivated at least under drought conditions, thereby reducing expression of said miRNAs at least under drought conditions.
  • one or more of the orthologues of J2 and J3 are knocked out and the plant expresses a mutant J2 or J3 which is farnesylation-deficient.
  • Such mutant J2 or J3 may be obtained by any of the methods described above in the section "specific inactivation of farnesylation of J2 and J3 orthologues", and may for example have a mutated farnesylation motif differing from Ca-ia 2 X by at least one amino acid residue, such as by at least two residues, such as by at least three residues, such as by four residues.
  • the plant comprises a J2 orthologue and the specific
  • the inactivation of farnesylation of the J2 orthologue is performed by knocking out the J2 orthologue in a permanent manner.
  • the plant comprises a J2 orthologue and the specific inactivation of farnesylation of the J2 orthologue is performed by knocking out the J2 orthologue in a transient manner.
  • the plant comprises a J2 orthologue and the specific inactivation of farnesylation of the J2 orthologue is performed by down-regulating expression of the J2 orthologue in a permanent manner.
  • the plant comprises a J2 orthologue and the specific inactivation of farnesylation of the J2 orthologue is performed by down-regulating expression of the J2 orthologue in a transient manner.
  • the plant comprises a J2 orthologue and the specific inactivation of farnesylation of the J2 orthologue is performed by inhibiting the activity of the J2 orthologue in a permanent manner. In other embodiments, the plant comprises a J2 orthologue and the specific inactivation of farnesylation of the J2 orthologue is performed by inhibiting the activity of the J2 orthologue in a transient manner. For plants where more than one J2 orthologue is present, each of the individual
  • orthologues can be independently specifically inactivated as described herein.
  • the plant comprises a J3 orthologue and the specific
  • the inactivation of farnesylation of the J3 orthologue is performed by knocking out the J3 orthologue in a permanent manner.
  • the plant comprises a J3 orthologue and the specific inactivation of farnesylation of the J3 orthologue is performed by knocking out the J3 orthologue in a transient manner.
  • the plant comprises a J3 orthologue and the specific inactivation of farnesylation of the J3 orthologue is performed by down-regulating expression of the J3 orthologue in a permanent manner.
  • the plant comprises a J3 orthologue and the specific inactivation of farnesylation of the J3 orthologue is performed by down-regulating expression of the J3 orthologue in a transient manner.
  • the plant comprises a J3 orthologue and the specific inactivation of farnesylation of the J3 orthologue is performed by inhibiting the activity of the J3 orthologue in a permanent manner. In other embodiments, the plant comprises a J3 orthologue and the specific inactivation of farnesylation of the J3 orthologue is performed by inhibiting the activity of the J3 orthologue in a transient manner. For plants where more than one J3 orthologue is present, each of the individual orthologues can be independently specifically inactivated as described herein.
  • Zea mays also known as maize or corn
  • Maize is a large grain plant first domesticated by indigenous peoples in southern Mexico.
  • the leafy stalk of the plant produces separate pollen and ovuliferous inflorescences or ears, which are fruits, yielding kernels or seeds.
  • Maize has become a staple food in many parts of the world, with total production surpassing that of wheat or rice. However not all of this maize is consumed directly by humans.
  • Some of the maize production is used for corn ethanol, animal feed and other maize products, such as corn starch and corn syrup.
  • the six major types of corn are dent corn, flint corn, pod corn, popcorn, flour corn, and sweet corn.
  • the present methods can be used to obtain drought-resistant maize.
  • a drought-resistant maize plant may thus be a maize plant wherein farnesylation of at least one of the ten J2/J3 orthologues has been specifically inactivated. In some embodiments, farnesylation of at least two of the ten J2/J3 orthologues has been specifically inactivated. In other embodiments, farnesylation of at least three of the ten J2/J3 orthologues has been specifically inactivated. In other embodiments,
  • farnesylation of at least four of the ten J2/J3 orthologues has been inactivated. In some embodiments, farnesylation of at least five of the ten J2/J3 orthologues has been inactivated. In some embodiments, farnesylation of at least six of the ten J2/J3 orthologues has been inactivated. In some embodiments, farnesylation of at least seven of the ten J2/J3 orthologues has been inactivated. In some embodiments, farnesylation of at least eight of the ten J2/J3 orthologues has been inactivated. In some embodiments, farnesylation of at least nine of the ten J2/J3 orthologues has been inactivated. In some embodiments, farnesylation of all ten J2/J3 orthologues has been inactivated.
  • Oryza indica is a variety of Oryza sativa, which is commonly known as Asian rice and is the plant species most commonly referred to in English as rice.
  • Oryza sativa is a grass with a genome consisting of 430Mb across 12 chromosomes. It is renowned for being easy to genetically modify, and is a model organism for cereal biology. Oryza sativa contains two major subspecies: the sticky, short-grained japonica or sinica variety, and the non-sticky, long-grained indica variety.
  • Japonica varieties are usually cultivated in dry fields, in temperate East Asia, upland areas of Southeast Asia, and high elevations in South Asia, while indica varieties are mainly lowland rices, grown mostly submerged, throughout tropical Asia.
  • Oryza sativa which is broad-grained and thrives under tropical conditions, was identified based on morphology and initially called javanica, but is now known as tropical japonica. Examples of this variety include the medium-grain Tinawon' and 'Unoy' cultivars, which are grown in the high-elevation rice terraces of the Cordillera Mountains of northern Luzon, Philippines.
  • the present methods can be used to obtain drought-resistant Oryza indica.
  • Oryza indica counts four J2/J3 orthologues (SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49 and SEQ ID NO: 50), all comprising a farnesylation motif at the C-terminal end.
  • a drought-resistant Oryza indica may thus be an Oryza indica plant wherein
  • farnesylation of at least one of the four J2/J3 orthologues has been specifically inactivated. In some embodiments, farnesylation of at least two of the four J2/J3 orthologues has been specifically inactivated. In other embodiments, farnesylation of at least three of the four J2/J3 orthologues has been specifically inactivated. In other embodiments, farnesylation of all four J2/J3 orthologues has been inactivated.
  • Common wheat Triticum aestivum
  • bread wheat is a cultivated wheat species. About 95% of the wheat produced is common wheat, which is the most widely grown of all crops.
  • the present methods can be used to obtain drought-resistant Triticum aestivum.
  • a drought-resistant Triticum aestivum plant may thus be a Triticum aestivum plant wherein farnesylation of at least one of the seven J2/J3 orthologues has been specifically inactivated.
  • farnesylation of at least two of the seven J2/J3 orthologues has been specifically inactivated.
  • farnesylation of at least three of the seven J2/J3 orthologues has been specifically inactivated.
  • farnesylation of at least four of the seven J2/J3 orthologues has been inactivated.
  • farnesylation of at least five of the seven J2/J3 orthologues has been inactivated.
  • farnesylation of at least six of the seven J2/J3 orthologues has been inactivated.
  • farnesylation of all seven J2/J3 orthologues has been inactivated.
  • Hordeum vulgare also known barley, is a member of the grass family. It is a major cereal grain grown in temperate climates globally. It was one of the first cultivated grains, particularly in Eurasia as early as 10,000 years ago. Barley has been used as animal fodder, as a source of fermentable material for beer and certain distilled beverages, and as a component of various health foods. It is used in soups and stews, and in barley bread of various cultures. Barley grains are commonly made into malt in a traditional and ancient method of preparation. In 2014, barley was ranked fourth among grains in quantity produced (144 million tonnes) behind corn, rice and wheat.
  • the present methods can be used to obtain drought-resistant Hordeum vulgare.
  • Hordeum vulgare counts three J2/J3 orthologues (SEQ ID NO: 23, SEQ ID NO: 24 and SEQ ID NO: 25) all comprising a farnesylation motif at the C-terminal end.
  • a drought-resistant Hordeum vulgare may thus be a Hordeum vulgare plant wherein farnesylation of at least one of the three J2/J3 orthologues has been specifically inactivated. In some embodiments, farnesylation of at least two of the three J2/J3 orthologues has been specifically inactivated. In other embodiments, farnesylation of all three J2/J3 orthologues has been inactivated.
  • Sorghum bicolor commonly called sorghum and also known as great millet, durra, jowari, or milo, is a grass species cultivated for its grain, which is used for food, both for animals and humans, and for ethanol production.
  • Sorghum originated in northern Africa, and is now cultivated widely in tropical and subtropical regions. Sorghum is the world's fifth most important cereal crop after rice, wheat, maize and barley.
  • S. bicolor is typically an annual plant, but some cultivars are perennial. It grows in clumps that may reach over 4 m high. The grain is small, ranging from 2 to 4 mm in diameter.
  • Sweet sorghums are sorghum cultivars that are primarily grown for foliage, syrup production, and ethanol; they are taller than those grown for grain.
  • Sorghum bicolor is the cultivated species of sorghum; its wild relatives make up the botanical genus Sorghum. The present methods can be used to obtain drought-resistant Sorghum bicolor.
  • Sorghum bicolor counts four J2/J3 orthologues (SEQ ID NO: 26, SEQ ID NO: 27, SEQ ID NO: 28 and SEQ ID NO: 29) all comprising a farnesylation motif at the C-terminal end.
  • a drought-resistant Sorghum bicolor may thus be a Sorghum bicolor plant wherein farnesylation of at least one of the four J2/J3 orthologues has been specifically inactivated. In some embodiments, farnesylation of at least two of the four J2/J3 orthologues has been specifically inactivated. In some embodiments, farnesylation of at least three of the four J2/J3 orthologues has been specifically inactivated. In other embodiments, farnesylation of all four J2/J3 orthologues has been inactivated.
  • the tomato is the edible fruit of Solanum lycopersicum, commonly known as a tomato plant, which belongs to the nightshade family, Solanaceae. Numerous varieties of domestic tomato are widely grown in temperate climates across the world, with greenhouses allowing its production throughout the year and in cooler areas. The plants typically grow to 1-3 meters (3-10 ft) in height and have a weak stem that often sprawls over the ground and vines over other plants. It is a perennial in its native habitat, and grown as an annual in temperate climates. An average common tomato weighs approximately 100 grams (4 oz). Including Solanum lycopersicum, there are currently 13 species recognized in the Solanum section Lycopersicon. Three of these species: S.
  • cheesmaniae, galapagense, and pimpinellifolium— are fully cross compatible with domestic tomato.
  • S. chmielewskii, S. habrochaites, S. neorickii, and S. pennelli can be readily crossed with domestic tomato, with some limitations.
  • Five species: S. arcanum, S. chilense, S. corneliomulleri, S. huaylasense, and S. peruvianum, can be crossed with domestic tomato with difficulty and usually require embryo rescue to produce viable plants.
  • the present methods can be used to obtain drought-resistant Solanum lycopersicum.
  • Solanum lycopersicum counts four J2/J3 orthologues (SEQ ID NO: 17, SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20) all comprising a farnesylation motif at the C- terminal end.
  • a drought-resistant Solanum lycopersicum may thus be a Solanum lycopersicum plant wherein farnesylation of at least one of the four J2/J3 orthologues has been specifically inactivated. In some embodiments, farnesylation of at least two of the four J2/J3 orthologues has been specifically inactivated. In some embodiments, farnesylation of at least three of the four J2/J3 orthologues has been specifically inactivated. In other embodiments, farnesylation of all four J2/J3 orthologues has been inactivated.
  • Vitis vinifera (common grape vine) is a species of Vitis, native to the Mediterranean region, central Europe, and southwestern Asia, from Morocco and Portugal north to southern Germany and east to northern Iran. There are currently between 5,000 and 10,000 varieties of Vitis vinifera grapes though only a few are of commercial significance for wine and table grape production.
  • Vitis vinifera counts two J2/J3 orthologues (SEQ ID NO: 21 and SEQ ID NO: 22) all comprising a farnesylation motif at the C-terminal end.
  • a drought-resistant Vitis vinifera may thus be a Vitis vinifera plant wherein farnesylation of at least one of the two J2/J3 orthologues has been specifically inactivated. In some embodiments, farnesylation of both J2/J3 orthologues has been inactivated.
  • Brassica rapa L. is a plant consisting of various widely cultivated subspecies including the turnip (a root vegetable); napa cabbage, bomdong, bok choy, and cime di rapa (leaf vegetables) and (Brassica rapa subsp. oleifera, an oilseed which has many common names, including field mustard, bird rape, keblock, and colza).
  • the oil made from the seed is sometimes also called canola.
  • the present methods can be used to obtain drought-resistant Brassica rapa.
  • Brassica rapa counts four J2/J3 orthologues (SEQ ID NO: 10, SEQ ID NO: 1 1 , SEQ ID NO: 12, SEQ ID NO: 13), all comprising a farnesylation motif at the C-terminal end.
  • a drought-resistant Brassica rapa may thus be a Brassica rapa plant wherein farnesylation of at least one of the four J2/J3 orthologues has been specifically inactivated. In some embodiments, farnesylation of at least two of the four J2/J3 orthologues has been specifically inactivated. In other embodiments, farnesylation of at least three of the four J2/J3 orthologues has been specifically inactivated. In other embodiments, farnesylation of all four J2/J3 orthologues has been inactivated.
  • Glycine is a genus in the bean family Fabaceae. The best known species is the soybean (Glycine max).
  • the present methods can be used to obtain drought-resistant glycine.
  • Glycine counts six J2/J3 orthologues (SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8 and SEQ ID NO: 9) all comprising a farnesylation motif at the C-terminal end.
  • a drought-resistant glycine plant may thus be a glycine plant wherein farnesylation of at least one of the six J2/J3 orthologues has been specifically inactivated. In some embodiments, farnesylation of at least two of the six J2/J3 orthologues has been specifically inactivated. In other embodiments, farnesylation of at least three of the six J2/J3 orthologues has been specifically inactivated. In other embodiments,
  • farnesylation of at least four of the six J2/J3 orthologues has been inactivated. In some embodiments, farnesylation of at least five of the six J2/J3 orthologues has been inactivated. In some embodiments, farnesylation of all six J2/J3 orthologues has been inactivated.
  • Medicago truncatula also known as the barrelclover, strong-spined medick, barrel medic, or barrel medick
  • barrelclover also known as the barrelclover, strong-spined medick, barrel medic, or barrel medick
  • barrelclover strong-spined medick
  • barrel medic barrel medic
  • barrel medick is a small annual legume native to the Mediterranean region that is used in genomic research. It is a low-growing, clover-like plant 10-60 centimetres (3.9-23.6 in) tall with trifoliate leaves. This species is studied as a model organism for legume biology because it has a small diploid genome, is self-fertile, has a rapid generation time and prolific seed production, is amenable to genetic
  • Medicago truncatula counts three J2/J3 orthologues (SEQ I D NO: 14, SEQ I D NO: 15 and SEQ I D NO: 16) all comprising a farnesylation motif at the C-terminal end.
  • a drought-resistant Medicago truncatula may thus be a Medicago truncatula plant wherein farnesylation of at least one of the three J2/J3 orthologues has been specifically inactivated. In some embodiments, farnesylation of at least two of the three J2/J3 orthologues has been specifically inactivated. In other embodiments,
  • ProJ3:J3:terJ3 and ProJ3:J3 C417S :terJ3 constructs were made by a USER cloning strategy. PCR fragments containing 1333 bp of J3 (AT3G441 10) promoter and 482 bp downstream of the stop codon were amplified from BAC F26G5, and cloned into a derivative of pCAMBIA3300 containing a USER cassette (Nour-Eldin et al. 2006). ProJ3:2xFLAG-2xHA-J3:terJ3 and ProJ3:2xFLAG-2xHA-J3 C417S :terJ3 were constructed using appropriate primers.
  • the 2xFLAG-2-HA cassette was amplified from an SDE3- 2xFLAG-2xHA construct (Garcia et al. 2012), and J3 and J3 C417S fragments were amplified from the ProJ3:J3:terJ3 and ProJ3:J3 C417S :terJ3 constructs described above.
  • pEAQ-J3 a J3 cDNA was amplified from oligo(dT)-primed cDNA, and cloned into the Agel-Xhol site of pEAQ (Sainsbury et al. 2009).
  • pEAQ-HT-J3 His 6 -J3
  • a J3 cDNA was amplified, cloned into the entry vector pCR8/GW/TOPO (Invitrogen) and recombined into pEAQ-HT-dest2 (Sainsbury et al. 2009).
  • pEAQ-HT-dest2 pEAQ-HT-dest2
  • Plants were transformed by floral dipping with Agrobacterium tumefaciens strain GV3101 (Clough and Bent 1998).
  • the leaves were transferred to new tubes of 2 ml containing 500 uL (50uCi, 60 Ci/mmol) 14 C-mevalonate and 500uL 0.5xMS until the entire volume had been taken up by the leaf. The leaves were then left in 0.5xMS for 12 h. Total lysates were then prepared and hexahistidine tagged protein was purified by immobilized Ni 2+ affinity chromatography as described below.
  • Uncharacterized Arabidopsis T-DNA insertion mutants (era1-9, plp-3, j2-2, ⁇ 3-2) were genotyped using PCR to confirm the T-DNA insertion sites and select homozygous mutants.
  • the deletion in era1-2 was confirmed by PCR with primers inside the ERA1 gene body, and by the total absence of signal in quantitative RT-PCRs from RNA prepared from era1-2.
  • eral -2/era1 -2, hsp90.2-3/+ individuals were identified by PCR in F2 populations of era1-2 crossed to hsp90.2-3 followed by Ase ⁇ digestion for hsp90.2-3 genotyping. Double homozygous plants were then identified and characterized in the F3 generation.
  • ⁇ 2-2J ⁇ 3-2 double mutants expressing J3 WT or J3 C417S transgenes were constructed as follows: j2-2/j2-2;j3-2/+ was transformed by ProJ3:J3 WT :terJ3 and ProJ3:J3 C417S :terJ3 constructs. PCR screening of 200 primary transformants yielded no j2-2/j3-2 double homozygotes, probably due to the fact that floral dip transformation primarily targets female reproductive tissue and that j2/j3 is transmitted poorly through the pollen. T2 populations of ten independent lines (five J3 WT and five J3 C417S , all with ⁇ 2-2/ ⁇ 2-2; ⁇ 3-2/+ genotypes) were then rescreened by PCR.
  • Double homozygotes were identified in two independent lines for both J3 WT and J3 C417S constructs at the expected frequency of 1/8, taking into account that transgenic J3 must be transmitted through the pollen. Double homozygous plants were amplified, and individuals homozygous for J3 transgenes were identified in subsequent generations before molecular and phenotypic analyses. A CAPS marker using Pfe ⁇ digestion of amplified PCR products was developed to confirm presence of J3 C417S .
  • Arabidopsis inflorescence meristems were isolated from the primary inflorescences of plants at the same developmental stage, such that Col-0 plants were about 8 days younger than farnesyl transferase and J3 mutant lines to compensate for the later flowering of the mutants. After two hours of fixation in Karnovsky's Fixative, material was dehydrated in a graded acetone series (30%, 50%, 70%, 90% and 100%).
  • RNA 5-20 ⁇ g purified total RNA was mixed with 4*loading buffer (20mM HEPES, 1 mM EDTA, 50% formamide, 3% glycerol, bromophenol blue, pH 7.8) and heated at 95°C for 2 minutes before being snap frozen on ice and loaded on pre-heated 18% acrylamide (19:1 , Serva), Tris-borate-EDTA (TBE) gels containing 6M urea. Gels were run at 90V for approximately 3h and then blotted by wet transfer to an Amersham
  • RNA was chemically crosslinked to the membrane with EDC ((1 - ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) at 60 C° for 1.5h following the procedure of ((Pall et al. 2007)).
  • EDC ((1 - ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride) at 60 C° for 1.5h following the procedure of ((Pall et al. 2007)).
  • miRNA-specific probes were produced by PNK- labeling of complementary DNA oligonucleotides (T4 Polynucleotide Kinase,
  • Fermentas with [ ⁇ - 32 ⁇ ]- ⁇ , and were hybridized to membranes in PerfectHyb Plus Hybridization buffer (Sigma) overnight at slow rotation at 42°C. Washed blots (3x20min in 2*SSC, 2% SDS at 42°C) were exposed to imaging plates (BAS-MS, Fujifilm) and visualized using a laser scanner (Typhoon FLA 7000, GE Healthcare).
  • RNA was treated with DNase I (Fermentas), and converted to cDNA with Revert Aid reverse transcriptase (Fermentas) primed by oligo-(dT) according to instructions.
  • Quantitative PCR was performed with the SYBRGreen Mastermix (Fermentas) on a CFX Connect Real-Time System (Bio-Rad). Melting curve analysis of products amplified by each primer pair showed that they amplified a single PCR product. Actin and GAPDH were tested as normalization controls. Actin was found to be more robust across the samples analyzed, and was used as a normalization control.
  • His 6 -J3 was purified by immobilized Ni 2+ affinity chromatography using nitrilo tri-acetic acid-conjugated agarose beads (Protino, Machery-Nagel), following the manufacturer's instructions.
  • FHA-J3 was purified by immunoaffinity chromatography using M2- conjugated agarose beads (Sigma), following the manufacturer's instructions. For Ni 2+ - NTA purification, 5g of N.
  • benthamiana leaf tissue was ground to a fine powder and lysed in 6 ml of lysis buffer (50mM Tris HCI pH7.5, 150mM NaCI, 5mM MgCI 2 , 0.1 %Nonidet P40, Protease Inhibitor cocktail Roche) and filtered through 0.45 ⁇ acrodisc.
  • lysis buffer 50mM Tris HCI pH7.5, 150mM NaCI, 5mM MgCI 2 , 0.1 %Nonidet P40, Protease Inhibitor cocktail Roche
  • a bed volume of 500 ul Ni 2+ -NTA agarose beads was used to bind His 6 - tagged proteins. Beads were collected on 15 ml disposable columns (Biorad), and the flow-through was re-applied once to the column.
  • lysis buffer 50mM Tris HCI pH7.5, 150mM NaCI, 5mM MgCI 2 , 0.1 %Nonidet P40, Protease Inhibitor cocktail (Roche), wash buffer: 50mM Tris HCI pH7.5, 500mM NaCI, 5mM MgCI 2 , 0.1 %Nonidet P40, elution buffer:
  • Ixphosphate-buffered saline FLAG peptide 150 ng/uL.
  • a bed volume of 60 ul M2- agarose beads (Sigma) per ml lysate was used.
  • HSP40 J2/J3
  • HSP70, HSP90, SIP2 and PYR1 antibodies were purchased from Agrisera.
  • TIR1 antibodies were a kind gift from Lionel Navarro, and have been described in (Arribas-Hernandez et al. 2016).
  • Inflorescences were snap frozen and ground to a fine powder.
  • 1.2 ml of microsome buffer 50mM MOPS, 0,5M Sorbitol, 10mM EDTA, 1 % BSA, Roche protease inhibitors version 1 1 (1 tablet/10 ml), pH 7.6 were added to 0.2g ground tissue and vortexed thoroughly. Samples were spun at 8000xg for 10 min at 4°C. Supernatants were transferred to new tubes and repeatedly spun at 8000xg until no pellet was visible. Supernatants ("Total extracts”) were spun at 100000 xg for 30 min at 4°C.
  • Pellets were resuspended in wash buffer (50mM MOPS, 0,5M Sorbitol, 10mM EDTA, Roche protease inhibitors version 1 1 (1 tablet/10 ml), pH 7.6)) and re-pelleted by
  • RNA sequencing was prepared from 1 ug of total seedling RNA. All libraries were generated using the NEBNext Small RNA Library Prep Set (Multiplex) (NEB) following NEB instructions. The quality of purified RNA and of constructed libraries was confirmed using an Agilent Bioanalyzer and sequenced on an Illumina platform (Aros, Denmark).
  • NEB NEBNext Small RNA Library Prep Set
  • Raw illumina sequencing reads were pre-processed by trimming away the sequence of the miRNA ligation adapter (AGATCGGAAGAGCACACGTCTGAACTCC, SEQ ID NO: 52) using the program cutadapt. Trimmed reads shorter than 21 nt were discarded to be able to distinguish between mature miR398a and miR398b/c that only differ in sequence at the 21 st nucleotide. The trimmed reads were aligned against sequences of the 427 A. thaliana miRNAs annotated in miRBase v21 using strand-specific alignment with bowtie2 (Langmead et al. 2009). To take sequencing errors into account, imperfect alignments were allowed. This did not impact the false positive rate as the number of hits to C. elegans let-7 was negligible compared to A. thaliana miRNA sequences.
  • Example 2 - J2 and J3 are farnesylated in vivo
  • J2 and J3 share identical putative farnesylation sites (CAQQ) at the C-terminus.
  • J2/J3 are ubiquitously expressed, that they are tightly co-regulated, and that J3 is the major isoform, because it is expressed to 3-4 fold higher levels than J2.
  • J2/J3 are farnesylated in vivo.
  • transient overexpression of J3 in Nicotiana benthamiana produced a specific 50 kDa band that co-migrated with the only protein detected in microsomal fractions of Arabidopsis lysates (not shown).
  • a transgenic line expressing a dexamethasone-inducible artificial miRNA targeting J3 (amiR-J3) in a y ' 2 knockout background showed reduced intensity of the 50 kDa band specifically upon amiR-J3 induction (not shown).
  • the 50 kDa band detected by the antibodies corresponds to J2/J3 protein.
  • Nicotiana benthamiana to express His 6 -J3 WT and His 6 -J3 C417S with a Cys-Ser mutation in the CaaX motif, precluding prenylation (Zhang and Casey 1996).
  • J2/J3 migration was slower in samples prepared from the ERA 1 deletion mutant era1-2 (Cutler et al. 1996) and from the the T- DNA insertion mutant era1-9 (SAIL_146D09), but not from ggb-1 mutants (Johnson et al. 2005) (Fig. 1 D).
  • the gel mobility analyses and the incorporation of mevalonate-derived 14 C-label into J3 WT , but not into J3 C417S establish that J3 is farnesylated in vivo.
  • Example 5 Farnesylation-deficient J3 mutants have enlarged meristems and developmental phenotypes similar to eral mutants
  • eral mutants exhibit developmental phenotypes including late flowering, altered phyllotaxis, reduced fertility, and a stochastic increase in petal number (Running et al. 1998; Yalovsky et al. 2000; Ziegelhoffer et al. 2000).
  • the underlying cause of some of these phenotypes may relate to the defective control of meristem size.
  • Both eral and pip exhibit enlarged meristems, suggesting that farnesylated targets somehow control the balance between cellular proliferation and differentiation in the meristem (Running et al., 1998; Yalovsky et al., 2000).
  • J2/J3 are particularly important farnesylation targets for several previously described developmental phenotypes of farnesyl transferase mutants, including defective control of meristem size.
  • miR398 accumulation was also weakly affected in y ' 3-2 single, but not in y ' 2-2 mutants (Fig. 5B).
  • miR397/398/408/857 are all controlled by the transcription factor SPL7 (Yamasaki et al. 2009), suggesting that their transcription may be compromised upon loss of HSP40 farnesylation.
  • pri-miR397a, pri-miR398b/c, and pri-miR857 levels were strongly reduced in farnesyl transferase mutants and in farnesylation- defective J3 C417S lines (Fig. 5C), in contrast to other pri-miRNAs which showed levels similar to wild type (Fig. 5C).
  • pri-miR398a encoded by the least expressed and only MIR398 gene whose transcription is not controlled by SPL7, accumulated to higher levels in era1-2 and in farnesylation-defective J3 C417S lines (Fig. 5C).
  • SPL7 function as the cause of reduced miR397-398-408-857 accumulation, and may provide an explanation for residual miR398 levels detected in farnesyl transferase and farnesylation-defective J3 C417S lines.
  • SEQ ID NO: 2 J3 amino acid sequence from Arabidopsis thaliana (AT3G441 10) MFGRGPSKKS DNTKFYEILG VPKSASPEDL KKAYKKAAIK NHPDKGGDPE KFKELAQAYE VLSDPEKREI YDQYGEDALK EGMGGGGGGH DPFDIFSSFF GGGPFGGNTS RQRRQRRGED WHPLKVSLE DVYLGTMKKL SLSRNALCSK CNGKGSKSGA SLKCGGCQGS GMKVSIRQLG PGMIQQMQHA CNECKGTGET INDRDRCPQC KGDKVIPEKK VLEVNVEKGM QHSQKITFEG QADEAPDTVT GDIVFVLQQK EHPKFKRKGE DLFVEHTLSL TEALCGFQFV LTHLDGRSLL IKSNPGEWK PDSYKAISDE GMPIYQRPFM KGKLYIHFTV EFPDSLSPDQ
  • SEQ ID NO: 8 >Glycine_max
  • SEQ ID NO: 10 >Brassica_rapa
  • SEQ ID NO: 1 1 >Brassica_rapa
  • SEQ ID NO: 12 >Brassica_rapa
  • SEQ ID NO: 17 >SolanumJycopersicum
  • SEQ ID NO: 18 SolanumJycopersicum
  • SEQ ID NO: 19 >SolanumJycopersicum
  • SEQ ID NO: 20 >Solanum_lycopersicum
  • SEQ ID NO: 26 >Sorghum_bicolor
  • SEQ ID NO: 27 >Sorghum_bicolor
  • SEQ ID NO: 28 >Sorghum_bicolor
  • SEQ ID NO: 41 Triticum_aestivum
  • SEQ ID NO: 42 Triticum_aestivum
  • SEQ ID NO: 48 >Oryza_indica
  • SEQ ID NO: 50 Oryza_indica
  • SEQ ID NO: 51 antigen against HSP40 (J2/J3) - H 2 N-CNHPDKGGDPEKFKEL-CONH 2
  • SEQ ID NO: 52 AGATCGGAAGAGCACACGTCTGAACTCC
  • Floral dip a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16: 735-743.
  • Cutler S Ghassemian M, Bonetta D, Cooney S, McCourt P. 1996. A protein farnesyl transferase involved in abscisic acid signal transduction in Arabidopsis. Science 273: 1239-1241 .
  • the WIGGUM gene is required for proper regulation of floral meristem size in Arabidopsis. Development 125: 2545-2553.
  • Enlarged meristems and delayed growth in pip mutants result from lack of CaaX prenyltransferases. Proc Natl Acad Sci USA 101 : 7815-7820.

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L'invention concerne des plantes résistantes à la sécheresse, ainsi que des procédés d'obtention de telles plantes, et des acides nucléiques et des vecteurs d'expression utiles à cet effet.
PCT/EP2018/065981 2017-06-19 2018-06-15 Résistance accrue à la sécheresse dans des plantes WO2018234191A1 (fr)

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CN111512961A (zh) * 2020-06-02 2020-08-11 晋中学院 一种抗旱优质谷子的选育方法
CN114763549A (zh) * 2021-12-23 2022-07-19 西藏自治区农牧科学院农业研究所 一种青稞抗旱相关基因及鉴别抗旱青稞的试剂盒和方法

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111512961A (zh) * 2020-06-02 2020-08-11 晋中学院 一种抗旱优质谷子的选育方法
CN114763549A (zh) * 2021-12-23 2022-07-19 西藏自治区农牧科学院农业研究所 一种青稞抗旱相关基因及鉴别抗旱青稞的试剂盒和方法
CN114763549B (zh) * 2021-12-23 2023-09-29 西藏自治区农牧科学院农业研究所 一种青稞抗旱相关基因及鉴别抗旱青稞的试剂盒和方法

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