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WO2009003429A2 - Procédé de régulation de la production de biomasse dans les plantes, séquences d'adn et leur procédé de préparation - Google Patents

Procédé de régulation de la production de biomasse dans les plantes, séquences d'adn et leur procédé de préparation Download PDF

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Publication number
WO2009003429A2
WO2009003429A2 PCT/CZ2008/000075 CZ2008000075W WO2009003429A2 WO 2009003429 A2 WO2009003429 A2 WO 2009003429A2 CZ 2008000075 W CZ2008000075 W CZ 2008000075W WO 2009003429 A2 WO2009003429 A2 WO 2009003429A2
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Prior art keywords
ckil
gene
sequence
expression
homologue
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WO2009003429A3 (fr
Inventor
Jan Hejatko
Ildoo Hwang
Romana Dobesova
Hojin Ryu
Jaroslava Dubova
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Masarykova Univerzita
POSTECH Academy Industry Foundation
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Masarykova Univerzita
POSTECH Academy Industry Foundation
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Publication of WO2009003429A2 publication Critical patent/WO2009003429A2/fr
Publication of WO2009003429A3 publication Critical patent/WO2009003429A3/fr
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    • 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)
    • C12N15/8241—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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A40/00—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production
    • Y02A40/10—Adaptation technologies in agriculture, forestry, livestock or agroalimentary production in agriculture
    • Y02A40/146—Genetically Modified [GMO] plants, e.g. transgenic plants

Definitions

  • the invention relates to a method of regulation of biomass production in plants by means of modification of activity of a gene or a gene product, to DNA sequences and a method of preparation thereof.
  • the European Union has set the targets in increasing the use of renewable resources (RR) in two fundamental documents: careful White paper” has set the fundamental target in increasing the share of RR in primary energy resources consumption in the whole European Union from 6 % in 1995 to 12 % in 2010 and the Directive 2001/77/EC determines the aim of the EU to increase the RR share in electric energy production from 13.9 % in 1997 to 22.1 % in 2010. Biomass should substantially contribute to fulfilling these aims, both waste biomass and biomass cultivated for this purpose.
  • RR renewable resources
  • genes for the herbicide resistance of the plants, the genes for resistance to insect pests and the viral diseases resistance genes predominate among the genes used for the genetic modification of plants (Milos Ondrej, Jaroslav Drobnik: Transgenoze rostlin, Academia, Praha, 2002) - the first genetically modified plant entered the Czech Republic officially on the 14th May 2001, when genetically modified soybean resistant against the herbicide Roundup (soybean line GTS 40-3-2 and all progenies derived from this line by traditional cultivation methods) was approved for processing (not for cultivating).
  • the genetic methods leading solely to the increase of the volume of biomass are rare, some of them were performed only for the underground parts of the plants.
  • the conductive tissues are the place of production of xylem cells, i.e. wood, wherein the lignification of the cell walls and the cellulose deposition occurs. These processes are critical for the production of exploitable biomass and can therefore have an economic impact.
  • the gene CYTOKININ INDEPENDENT! (CKIl) was identified in the year 1996 by activation mutagenesis as a dominant regulator of cytokinin response in Arabidopsis thaliana (Kakimoto T., Science 274, 982-985 (1996)).
  • An increased expression of the gene CKIl (CKIl under the control of a strong constitutive promoter, tetramer of enhancers of the promoter of tobacco mosaic virus for 35S RNA [CaMV 35S RNA promoter]) leads during cultivation on the growth medium without cytokinins to phenotypic features that are dependent on the presence of cytokinins in the cultivation medium (greening and shooting).
  • CKIl Based on the similarity of the amino acid sequence of CKIl with the sensor histidine kinases of bacteria, the function of CKIl was predicted to be a cytokinin receptor. Nevertheless, further experiments did not show any cytokinin binding to CKIl. Later, CKIl homologues were identified, genes AHK2, AHK3 and AHK4/CRE1/WOI, and their function as genuine receptors in cytokinin perception in plants was identified (Inoue et al., Nature 409, 1060-1063 (2001); Yamada et al., Plant Cell Physiol 42, 1017-1023 (2001)).
  • CKIl its constitutive activity, independent of the presence or absence of cytokinins, was found, and therefore, the hypothesis was pronounced that it is not a genuine cytokinin receptor (Yamada et al., Plant Cell Physiol 42, 1017-1023 (2001)).
  • CKIl homologues genes AHK2, AHK3 and AHK4, was identified in vascular tissues of A. thaliana and their function in the response to exogenous cytokinins, root growth, hypocotyl elongation, number of cells in leaves and size of the leaf in A. thaliana was proven (Higuchi et al., Proc Natl Acad Sci U S A 101, 8821-8826 (2004); Nishimura et al., Plant Cell 16, 1365-1377 (2004)).
  • vascular tissues and particularly the stem cells of vascular tissue are place of biomass production in plants
  • changes in the capacity of plant vascular tissues to produce biomass seems to be one of the most efficient ways to modulate biomass production in plants.
  • cytokinin signalling is considered to be essential for formation and differentiation of vascular tissue in the root, it might imply a possibility to develop a technique to increase the production of plant biomass through growth of plant via regulating cytokinin- associated genes in the aerial part of the plants (i.e. shoots), too.
  • Object of the present invention is a method of regulation of biomass production in a plant, wherein the gene CKIl (Sequence ID No. 1) or a homologue or an orthologue thereof is identified in the plant and the expression of the gene CKIl or the homologue or the orthologue thereof or the activity of the gene product (the encoded protein) is modified.
  • a homologue is a gene sequentially similar to the gene CKIl - similarity on the level of nucleotide sequences -, or a gene, the product of which is similar to the CKIl protein, - similarity on the level of amino acid sequence -, which had developed in the past from the same sequence.
  • An orthologue is a gene which has the same or a similar function in a different species, i.e. in this case a gene the function of which in another plant species corresponds to the function of the gene CKIl in A. thaliana.
  • homologues or orthologues are carried out on the basis of the following criteria: i) sequence homology on the level of nucleotides or amino acids, containing particularly the conserved regions of sensor histidine kinases.
  • sequence homology on the level of nucleotides or amino acids, containing particularly the conserved regions of sensor histidine kinases.
  • cDNA of homologous genes e.g. by means of hybridization with cDNA library or genome library of the respective species using the probe obtained from the genomic DNA or cDNA of CKIl or any of the already identified homologues thereof, e.g. the genes AHK2, AHKS or AHK4/CRE1/WOL or by similar methods.
  • ahk2, ahk3, ahk4 or ckil using the encoding sequence of the homologue put under the control of the promoter of the respective gene and in the respective mutant background by methods generally known in the art or by means of analysis of the activity of the gene product in the cytokinin signal transduction using the analysis in plant protoplasts, as described earlier (Hwang and Sheen, Nature 413, 383-389 (2001)) and Hi) expression of the homologue or the orthologue in vascular tissues or tissues adjacent to vascular tissues of the respective plant species.
  • the modification of the expression of the gene CKIl or the homologue or the orthologue thereof comprises the modification of the expression of histidine kinase coding region of said gene.
  • the expression of the gene CKIl or the homologue or the orthologue thereof is modified so that it is decreased or eliminated, e.g. by means of RNA interference, insertion mutagenesis or similar methods.
  • the activity of the gene product of the homologue or the orthologue can be regulated by means of methods of site-directed mutagenesis in selected amino acids, preferably in amino acids participating in the transfer of phosphate by the respective sensor histidine kinase, in potential interactions with other, e.g. regulatory, proteins or in amino acids with a regulatory activity.
  • the thus modified DNA under the control of a constitutively or conditionally active promoter is then used for the preparation of stable transformants of the concerned plant species using the methods and approaches generally known in the art.
  • RNA interference When RNA interference is used for decreasing the activity of the homologue or the orthologue, it is necessary to prepare the recombinant DNA by methods of molecular biology generally known in the art, said recombinant DNA containing parts of cDNA sequence of the homologue or the orthologue in reverse repetition, separated by another DNA sequence, e.g. part of the encoding sequence uidA (Sequence ID No. 5; its part GUSp, Sequence ID No. 6) or a natural intron of the homologue or the orthologue.
  • Suitable part of the cDNA sequence is the sequence of nucleotides, the order of which corresponds at least in 20 % to the nucleotide sequence of CKIp2, Sequence ID No. 4. This construct is then inserted under the control of a constitutively active promoter (e.g.
  • CaMV 35S RNA promoter or a conditionally active promoter (e.g. dexamethasone inducible promoter) and terminated by a transcription terminator.
  • the transcription terminator may be any element capable of terminating transcription in plant cells, such as terminator of the NOPALINE SYNTHASE (NOS) gene.
  • NOS NOPALINE SYNTHASE
  • GV3013::pMP90 strain with rifampicine resistance, bearing a helper plasmid pMP90 resistant to gentamicine as a selection marker [Koncz and Schell, MoI Gen Genet 204, 383-396 (1986)] or similar).
  • This is followed by the preparation of transgenic plants of the concerned species using the methods known in the art, either by infiltration of inflorescence using the transgenic Agrobacterium, carrying the binary vector, part of which is the said gene construct, or by another method suitable for the concerned plant species, e.g. by means of infiltration of tissue explants by the transgenic Agrobacterium, carrying the binary vector, part of which is the said gene construct, and in vitro regenerations of the transgenic plants or similar methods.
  • Object of the invention is further a nucleotide sequence, having the sequence of nucleotides at least in 20 % identical with the nucleotide sequence of CKIp2 (Sequence ID No. 4).
  • Object of the invention is a sequence of recombinant DNA for the regulation of gene expression by means of RNA interference, having the nucleotide sequence at least in 20 % identical with the nucleotide sequence of 35S::CKIi2::pA (Sequence ID No. 7). This sequence is also shown in Fig. 7.
  • Object of the invention is further a method of preparation of the recombinant DNA for the regulation of gene expression by RNA interference, wherein a specific part of the cDNA or the encoding region of said gene is amplified using primers containing a sequence of 18-21 nucleotides specific for the cDNA or the encoding sequence of said gene and regions inserted to the 5 ' end of these primers, designated herein RNAi_up and RNAi_down, RNAi_up:
  • a connecting sequence is subsequently amplified, preferably a part of the encoding sequence of the gene uidA (Sequence ID No. 5; its part GUSp, Sequence ID No. 6), which is amplified using the primers Bgus and Hgus, Bgus:
  • sequences than uidA can be used as the connecting sequence, and in such a case, they are amplified using primers consisting of 18-21 nucleotides, specific for the respective sequence, and further sequences, inserted to the 5 ' end of these primers, designated herein LOOP_up and LOOP_down.
  • sequence of 4 nucleotides at the 5 ' end of the sequences RNAi_up, RNAi_down, LOOP_up and LOOP_down was optimized for the amplification of the sequences CKIp2, resp. GUSp.
  • sequences of 4 nucleotides can optionally be modified so that the amplification of said sequence is enhanced (improvement of the thermodynamic properties of the primers after the insertion of the 18-21 nucleotides specific for the sequence of the homologue or the orthologue and the connecting sequence, respectively, prevention of the formation of secondary structure and mutual similarities etc.), optionally, these 4 nucleotides can be omitted.
  • the regulation of the activity of the gene product (encoded protein) of the homologue or the orthologue can be carried out by means of the preparation of recombinant DNA, consisting of modified cDNA or genomic DNA of the homologue or the orthologue under the control of a suitable promoter (constitutively or conditionally active promoter).
  • a suitable promoter constitutitutively or conditionally active promoter.
  • the modification of the cDNA or the encoding region of the genome DNA consists in the replacement of a part of the nucleotide sequence by methods known in the art (site-directed mutagenesis) in such a way that during the translation of mRNA into protein, the substitution of selected amino acids of the homologue or the orthologue by other selected amino acids occurs.
  • Modified can be the amino acids participating in the transfer of phosphate by the respective histidine kinase, encoded by the homologue or the orthologue and/or it is possible to modify other amino acids, respectively, that can participate in the phosphate transfer as such or in potential interactions with other, e.g. regulatory proteins, or amino acids with a regulatory activity.
  • These amino acids must be identified by methods known in the art, particularly by comparing the amino acid sequence of the homologue or the orthologue with the amino acid sequences of similar proteins and experimentally.
  • the resulting construct is inserted into a binary vector and stable transformant lines of the respective plant species are prepared by the above described methods and by methods known in the art, said lines expressing the modified allele of the homologue or the orthologue.
  • This step is followed by the selection of several independent lines of primary transformants on the basis of the presence of a selection marker which is a part of the inserted construct, by identification of a homozygous line in their progeny and by analysis of their phenotype, all these steps being carried out by methods known in the art of molecular biology of plants.
  • a specific staining e.g. with a mixture of orange GG and aniline blue, on hand-made sections, or morphological and histological analyses on thin section prepared using the methods known in the art.
  • the expression of the gene CKIl or the homologue or the orthologue thereof or the activity of the gene product is modified so that it is increased, e.g. by means of overexpressing said gene.
  • an overexpression vector comprising a promoter for the expression in plant cell; one or more genes selected from the group consisting of CKIl and the homologues and the orthologues thereof, linked operable to the promoter; and a transcriptional terminator for plant cell.
  • the promoter is a CaMV 35S promoter.
  • the plant can be treated with the gene product.
  • Another aspect of this embodiment of the invention is an agent for regulating the growth in volume of a plant comprising one or more active ingredients selected from the group consisting of one or more proteins selected from the group consisting of gene products of CKIl and the homologues and the orthologues thereof; and the overexpression vector.
  • the gene CKIl or a homologue or an orthologue thereof can preferably be selected from the group consisting of CKIl (CYTOKININ- INDEPENDENT 1, SEQ ID NO: 2) coding gene, AHK2 (ARABIDOPSIS HISTIDINE KINASE 2, SEQ ID NO: 8) coding gene, AHK3 (ARABIDOPSIS HISTIDINE KINASE 3, SEQ ID NO: 9) coding gene, and AHK4 (WOL, CYTOKININ RESPONSE 1, SEQ ID NO: 10) coding gene.
  • CKIl CYTOKININ- INDEPENDENT 1
  • AHK2 ARABIDOPSIS HISTIDINE KINASE 2, SEQ ID NO: 8
  • AHK3 ARABIDOPSIS HISTIDINE KINASE 3, SEQ ID NO: 9
  • AHK4 WOL, CYTOKININ RESPONSE 1, SEQ ID NO: 10.
  • the staining by this method allows to distinguish between lignified cell walls, which are stained in yellow to orange together with cytoplasma, and living, i.e. not yet fully lignified cell walls, which are stained in blue. Using fluorescence, it is possible to identify lignified cells and cells with cellulose deposition.
  • This method provides for colour distinguishing of the cell components of vascular tissues, using microscopic observation in transmitted light, particularly differential interference contrast (DIC microscopy): Phloem is stained in blue, metaxylem in orange and protoxylem in deep blue to magenta.
  • DI microscopy differential interference contrast
  • cytokinin signalling pathways are conserved in plants and it can be assumed that the formation of vascular bundles (VB) in other plant species is directed by similar (homologous) genes as in the model plant A. thaliana.
  • model plants for the research of economic utility in wood production are used particularly poplar tree (Populus trichocarp ⁇ ) and birch tree (Betula sp.).
  • poplar tree Populus trichocarp ⁇
  • birch tree Betula sp.
  • homologues of the CKIl gene, participating in osmoregulation were identified ( chefsdor et al., FEBS Lett 580, 77-81 (2006)).
  • the CKIl protein is a positive regulator of the cell division in cambium, therefore increasing the expression of CKIl encoding gene or the homologue or the orthologue thereof or increasing the activity of the gene product increases the number of cells in the cambial region of vascular tissues of a plant. It is at the same time a negative regulator of the cell differentiation and might be involved in the regulation of the width of the VB, leading to formation of wide VB with enlarged area of the metaxylem region.
  • Fig. 1 shows the analysis of the biomass production in transgenic lines bearing the construct for decreasing the expression of CKIl according to example 3.
  • Fig. 2 represents the comparison of several independent transgenic lines with a decreased CKIl expression (lines CJH4T3) with mutants in the genes homologous to
  • OXIDASE/DEHYDROGENASE3 leading to a decreased level of endogenous cytokinins (35S::AtCKX3), with wild-type plants (wt CoI-O). Shown are rossette diameter and number of leaves in a rossette during the plant growth (A, 8 weeks; B, 10 weeks; C, 12 weeks and D, 14 weeks old plants cultivated under the short day conditions).
  • Fig. 3 shows the phenotypic analysis according to example 3 : A faster development of vascular bundles and an increased deposition of cellulose during the secondary thickening in A. thaliana as a result of the repression of the CKIl gene activity.
  • Hand-made sections were stained with a mixture of aniline blue and orange GG and observed using DIC optics (a, c, e, g, i, k, m, o, q, s) or using fluorescence (b, d, f, h, j, 1, n, p, r, t), with the use of triple filter (U-M61000, DAPI/FITC/TRITC triple filter block, Olympus), if, interfascicular arch fibers; mx, metaxylem; p, phloem; px, protoxylem.
  • DIC optics a, c, e, g, i, k, m, o, q, s
  • fluorescence b, d, f, h, j, 1, n, p, r, t
  • triple filter U-M61000, DAPI/FITC/TRITC triple filter block, O
  • Scale bars 100 ⁇ m (a, b, e, f, i, j, m, n, q, r) and 50 ⁇ m (c, d, g, h, k, 1, o, p, s, t).
  • the arrows depict the regions of interfascicular arch fibres, the arrowheads depict the metaxylem region.
  • Fig. 4 represents the quantification and the comparison of the thickness of the layer of lignified cells in interfascicular arcs of two independent transgenic lines with a decreased CKIl expression, CKIl, 3 '5S:: CKl Ii, 1-5 and 5-3 (shortened designations of the lines CJH4T3, 1-5 and CJH4T3, 5-3), in double mutants ahk2 ahJt4, ahk3 ahk4 and ahk2 ahk3 and in the transgenic line with overexpression of the gene AtCKX3 (35S::AtCKX3) for CYTOKINININ OXIDASE/DEHYDROGENASE3 with a decreased endogenous cytokinin levels in comparison with the wild-type line wt CoI- 0. Shown are the dimensions in ⁇ m.
  • Fig. 5 shows the analysis of the effect of CKIl to the cytokinin signal transduction in protoplasts of A. thaliana according to example 5.
  • Fig. 6 shows the phenotypic analysis of transgenic lines with an increased expression of wild-type CKIl gene and of the dominant negative mutant CKI1 H4O5Q .
  • A B. Ectopic expression of CKIl leads to sterility and an increased production of trichomes (A) and thick fasciated inflorescence stems (B).
  • C Changes in the architecture of vascular bundles in the plants with an increased expression of CKIl (transgenic line 35S::CKI1).
  • the arrows indicate the formation of ectopic tissues that resemble vascular bundles.
  • D E. Ectopic expression and overexpression of CKIl leads to the formation of additional vegetative tissues initiated from lateral meristems.
  • the arrow indicates an ectopic axillary bud formed in transgenic lines 35S::CKI1. Scale bars 100 ⁇ m.
  • F Ectopic overexpression of CKIl leads to disruptions in proper development of vascular bundles in A. thaliana.
  • Transverse section of the inflorescence stems of transgenic lines with overexpression of CKIl H405Q 35S::CKI1 H405Q , a, b a c), wild-type (d) and double mutant ahU ahk3 (e). Scale bars 100 ⁇ m.
  • Fig. 7 represents the sequence of recombinant DNA 35S::CKIi2::pA (Sequence ID No. 7).
  • Fig. 8 demonstrates expression of CKIl in vascular tissue. Specificity of CKIl promoter as determined in pCKIl:;uidA transgenic plants.
  • GUS activity is located in cells of the vascular bundle sheath located at the lateral borders of the phloem and cambium (arrowhead) and xylem (arrows, see also figures b and c).
  • b In situ localization of CKIl mRNA in cross-sections of the inflorescence stem.
  • c-h In situ immunolocalization of CKIl using CXCKIIED in cambium of VB (deep purple signal, arrowheads) on the cross sections of inflorescence stem, c, d wt, e, f 35S::CKIi line, g, h 35S::CKI1.
  • CKIl or CREl-CKIl leads to increase of the number of cambium layers.
  • Overexpression of dominant negative allele CKI1 H4O5Q leads to a dramatic reduction of cambium formation.
  • B. Reduction of CKIl expression by T-DNA insertion resembles CKIl RNAi plants. Transverse sections of the inflorescence stems of wild-type (WS-2, a) and the heterozygous CKIl T-DNA insertion lines, ckil-5/CKIl (b) and ckil-6/CKIl (c).
  • Fig. 10 demonstrates that cytokinin signalling via AHK2 and AHK3 affects vascular bundle development in Arabidopsis.
  • A. The loss-of-function ahk2 or ahk3 mutations disrupt proper development of the vascular bundles.
  • Transverse sections of the inflorescence stems of a, f, k, wild-type (Col-0), b, g, 1 ahk2, c, h, m ahk3, d, i, n ahk2, 3 and e, j, o 35$: : AtCKX lines. Note reduction of cambial layers in ahkl, ahk3 and particularly in ahk2, 3 line.
  • Fig. 12 demonstrates that CKIl is functionally conserved with AHK2 and AHK3 in the vasculature development.
  • A The dwarf architecture resulting from deletion of ahk2/ahk3 was rescued in the presence of CKIl.
  • B Expression analysis of HA-tagged CKIl and CKI1H405Q proteins under the control of 35S promoter in the transgenic lines. Total proteins from three-week-old of each designated plants were subjected to 7.5% SDS-PAGE.
  • C, D Ectopic expression of CKIl rescues the abnormal vasculature of the ahk2/ahk3 mutant.
  • the arrow indicates the immature vasculature. Scale bars, 100 ⁇ m.
  • Sequence 1 genomic sequence of CKIl Sequence 2 - amino acid sequence of CKIl
  • Sequence 6 sequence GUSp (part of uidA)
  • Sequence 7 sequence of the recombinant DNA of 35S: : CKIiI: :pA
  • RNA interference The region of cDNA of the CKIl gene designated CKIp2 and delimited by the sequences of CKIp2_up and CKIp2_down primers was amplified by PCR. Similarly, part of the encoding sequence of the uidA gene (Sequence ID No. 5) was amplified with Bgus and Hgus primers, and was designated GUSp (Sequence ID No. 6).
  • the primers used contain, in addition to the nucleotides derived from the CKIl and uidA sequences, respectively, inserted sites that are recognized by specific restriction endonucleases, see the sequences of the primers, wherein the inserted regions are underlined (non-specific to the sequence of the template used for amplification) and the sites recognized by restriction endonucleases are marked in colour.
  • these are BamHI and Hindlll
  • the CKIp2_down primer these are Xbal and Sail specific sites
  • the Hgus primer contains site recognised by Hindlll
  • the Bgus primer contains the recognition sequence for BamHI.
  • the DNA chains obtained by the amplification with the primers Hgus and Bgus were digested by HindIII and BamHI. After purification using QIAquick PCR Purification Kit (Qiagen), all three different fragments were mixed in equimolar amounts and added was the vector pBluescript (Stratagene), cleaved by the enzymes Xbal and Sail and dephosphorylated by thermo labile alkaline phosphatase (Alkaline Phosphatase, shrimp, Roche, as described by the manufacturer) so that the resulting molar ratio between the inserted fragments and the plasmid DNA was 1 : 1 to 3:1.
  • RNA interference can be used universally for the preparation of constructs for silencing genes by RNA interference as far as the parts of the cDNA of the silenced gene are chosen which do not contain recognition sites for the restriction enzymes BamHI, HindIII, Xbal and Sail. Furthermore, it is necessary to prepare primers containing approximately 18-21 nucleotides specific for the selected part of the cDNA and in addition RNAi_up sequence at the 5' end of the ,,left (forward)" primer, and RNAi_down at the 5' end of the ,,right (reverse)” primer, respectively (see the underlined parts of the sequences of the primers CKIp2_up and CKIp2_down).
  • the binary vector pVKH35SGUSpA (Reintantz et al., Plant Cell 13, 351-367 (2001)) was modified by digestion with the restriction enzymes BamHI and Hindlll, creating blunt ends using Klenow enzyme (Roche) and by subsequent religation by T4 DNA polymerase (all enzymes from Roche).
  • the resulting vector which was designated pVKH::(35S::pA), contained the 35S expression cassette (CaMV 35S RNA promoter and transcription terminator).
  • the construct CKIpH was cleaved out by Sail from the vector pBsc::CKIp2i and it was cloned into the binary vector pVKH::(35S::pA), digested by Sail.
  • the resulting construct was designated pVKH::(35S::CKIi2::pA) (Sequence ID No. 7), abbreviated to pJH_006.
  • the construct was checked by sequencing (see Sequence ID No. 7) and transformed by electroporation into Agrobacterium tumefaciens, strain GV3013::pMP90 (Koncz and Schell, MoI Gen Genet 204, 383-396 (1986)).
  • Selected transgenic lines were cultivated in vitro in square-shaped Petri dishes (12x12 cm) for 21 days on MS medium supplemented with 1% sucrose and solidified with 1.5 % phytagel, containing various concentrations of dimethylsulphoxide (DMSO), in vertical position in cultivation chambers with adjustable growth conditions (Percival Scientific, Ltd., relative air humidity 70 %, short day conditions [8 hours light, 16 hours dark], temperature during light period 21 0 C, temperature during dark period 19 0 C).
  • DMSO dimethylsulphoxide
  • Hand-made sections from living plants were prepared and stained with aniline blue and orange GG.
  • the sections were prepared by razor-blade from basal nodes of inflorescence lateral branches, growing from basal node of the main inflorescence stem of A. thaliana and were immediately stained for 15 minutes in a staining solution containing 0.25% aniline blue and 1% orange GG, destained for 10-15 min. in water and mounted into 50% glycerol.
  • the staining solution was prepared by the following procedure: 0.25 g of water-soluble aniline blue and 1 g of orange GG were dissolved in 100 ml of water.
  • This method also allows the colour distinction between particular cell and functional components of vascular tissues in plants: phloem is stained in blue, metaxylem in orange and protoxylem deep blue to magenta (see Fig. 3).
  • the sections were observed on the same day and the documentation was performed by motorized microscope (BX 61, Olympus) equipped with digital camera (DP50, Olympus) and controlled by a special software (AnalySIS, Soft Imaging System, GmbH). It was found on the sections that abnormalities occur in the development of vascular (conductive, vasal) tissues of the inflorescence stem in transgenic lines.
  • FIG. 3 shows the phenotype of vascular bundles in lateral inflorescence branches of wild-type (a-d), 35S::CKIi plants (transgenic plants bearing the construct for silencing the gene CKIl 35S::CKH2::pA, line CJH4T3, 5-3 [e-h]) and in mutants in the genes homologous to the CKIl gene, genes AHK2, AHK3 and AHK4: ahk2 ahk3, (i-1) and ahkl ahk4 (q-t).
  • Figures (m-p) show the phenotype of the transgenic line with overexpression of the gene for CYTOKINININ OXIDASE/DEHYDROGENASE3 (35S::AtCKX3) leading to decreased levels of endogenous cytokinins.
  • Hand-made sections were stained with a mixture of aniline blue and orange GG and observed using DIC optics (a, c, e, g, i, k, m, o, q, s). Phloem is stained in blue, metaxylem in orange and protoxylem in deep blue to magenta.
  • the increased intensity of staining with orange GG in the if region in 35S::CKIli lines (e and g) reflects the increased cellulose deposition and lignification in comparison to wild-type lines, which are in these regions stained in blue (a and c).
  • a and c interfascicular arch fibres; mx, metaxylem; p, phloem; px, protoxylem.
  • Scale bar 100 ⁇ m (a, b, e, f, i, j, m, n, q, r) and 50 ⁇ m (c, d, g, h, k, 1, o, p, s, t).
  • the identified phenotype of vascular tissues was opposite to the phenotype in the lines with a decreased expression of CKIl, as found by the analysis of the phenotype of vascular tissues of the inflorescence stem (see above), it means that in comparison with the wild-type there is a delayed and incomplete differentiation of protoxylem and metaxylem, formation of thinner interfascicular arc fibres among vascular bundles and a decreased deposition of cellulose in both xylem and the region of interfascicular arc fibres ; compare the intensity of the orange GG staining in the region of interfascicular arcs in Fig.
  • ahk2 mutant line In ahk2 mutant line, the number of cell layers in the cambial region was dramatically decreased, and the cell arrangements in the xylem and phloem were markedly changed (Fig. 10b, g, 1). Weaker phenotype was identifiable in the ahk3 line (Fig. 10c, h, m). In the ahk2, ahk3 double mutants, the reduction of the cambium and size of VB was more pronounced in comparison to both single mutants (Fig. 1Od, i, n). Reduction of cambium layer and VB size was identified also as a result of endogenous CK depletion in 35S::AtCKX3 and 35SrAtCKXl lines (Fig. 1Oe, j and 10o, respectively). Taken together, two-component signalling cascade initiated by CKIl, AHK2, and AHK3 is likely to play critical roles in normal vascular bundle development, especially in maintenance of cambial cell
  • CKIl with the modified activity were prepared.
  • the phenotype analysis of these transgenic lines has shown changes in the formation of vascular tissues.
  • the genomic DNA for CKIl was amplified using primers CKIlgen_fwd and CKIlgen rev.
  • the resulting DNA (CKIlge ⁇ ) was digested using BamHI and Stul and cloned into the vector pCB302ES, which was obtained by modifying the vector pCB302-2 (Xiang et al, Plant MoI Biol 40, Il l-Ill (1999)) in such a way that the coding region of CKIl became under the control of the promoter 35SC4PPDK (Hwang and Sheen, Nature 413, 383-389 (2001)).
  • the resulting vector was designated pCB302ES::(35S::CKIlgen).
  • the sequence CKIlgen was modified so that the resulting DNA encoded the mutant CKI1 H4O5Q with the replacement of histidine in the position 405 (nucleotide sequence CAC) by glutamine (CAA).
  • the resulting construct was designated pCB302ES::(35S::CKIlgenH405Q).
  • This DNA was then used for transformation of the protoplasts of A. thaliana and the activity of the encoded proteins was analysed as to their effect to the transduction of a signal in the cytokinin signalling pathway by known methods (Hwang and Sheen,
  • the transduction of the signal in the cytokinin signalling pathway is in the protoplasts of A. thaliana quantified using the luciferase activity measurements, the expression of which is controlled by the cytokinin inducible promoter of the gene ARR6.
  • ARR6 the cytokinin inducible promoter of the gene ARR6.
  • OXIDASE/DEHYDROGENASE2 35S::CKX2-9) were used for the transformation.
  • a decrease of the endogenous level of the active forms of cytokinins (isopentyladenine and zeatine) to approx. 45 % of the wild-type level occurred (Werner et al, Plant Cell 15, 2532-2550 (2003)) as a result of the overexpression of AtCKX2.
  • the wild-type allele CKIl constitutive, i.e. independent of cytokinin presence activation of the cytokinin signalling pathway in A. thaliana occurs.
  • the DNA of pCB302ES::CKIlgen and pCB302ES::CKUgenH405Q was transformed into Agrohacterium tumefaciens, strain GV3101, and by infiltration of the inflorescence (Clough and Bent, Plant J 16, 735-743 (1998)) transgenic plants were obtained, producing increased quantities of CKIl, resp.
  • CKI1 H4O5Q in the form of recombinant proteins (a fusion protein, containing in addition to the amino acid sequence of CKIl and CKI1 H4O5( ⁇ , respectively, also the sequence of two copies of the so-called epitope HA (hemaglutinine), allowing the detection of the fusion protein using the anti-HA epitope antibodies, see Fig. 6Ff).
  • Three independent transgenic lines bearing the construct 35S:: CKIl gen, designated 2-2, 2-13, 1-13 and four independent transgenic lines bearing the construct 35S::CKIlgenH405Q , designated 2, 6, 7 and 9 were analysed.
  • FIG. 6 shows the results of the phenotypic analysis of the transgenic lines with an increased expression of the wild-type CKIl gene and of the dominant negative mutant CKI1 H4O5Q .
  • A, B. Ectopic expression of CKIl leads to sterility and an increased production of trichomes (A) and thick fasciated inflorescence stems (B).
  • C. Changes in the architecture of vascular bundles in the plants with an increased expression of CKIl (transgenic line 3 '5S:: CKIl).
  • the arrows indicate the formation of ectopic tissues that resemble vascular bundles.
  • D E. Ectopic overexpression of CKIl leads to the formation of additional vegetative tissues initiated from lateral meristems. The node structures of wild-type and transgenic lines 35S::CKI1 (D), transverse and longitudinal sections from wild-type plants (E.a) and transgenic lines 35S::CKI1 (E.b). The arrow indicates an ectopic axillary bud formed in transgenic lines 35Sr. CKIl. Scale bars 100 ⁇ m.
  • F Ectopic overexpression of CKIl leads to disruptions of proper development of vascular bundles in A. thaliana.
  • Transverse sections of the inflorescence stems of transgenic lines with overexpression of CKIl H405 ® 35S::CKI1 H405Q , a, b a c), wild-type (d) and double mutant ahk2 ahk3 (e). Scale bars 100 ⁇ m.
  • the phenotype identified in vascular tissues was to a large extent opposite to the phenotype in lines with an overexpression of the wild-type allele of CKIl.
  • the inflorescence stems a smaller number of cell layers, particularly in case of cambium cells, and an uneven size of cells in both xylem and phloem were identified (see Fig. 6).
  • a similar phenotype was identified also in double mutants ahk2 ahk3.
  • Fig. 9 reveal that CKIl plays an important role in development of phloem and xylem consisting in vascular tissue.
  • the wild type line used as a control was ecotype Ws-2 of Arabidopsis thaliana.
  • the amount of protein CKI1 H4O5Q expressed in plant was determined by immunoblotting, and the obtained results are shown in Fig. 6. Further, the decrease of gene expression in CKIl RNAi was determined by Quantitative Real Time
  • Fig. 6 shows the amount of protein CKI1 H4O5Q determined by immunoblotting, indicating the increase of the foreign protein expression in CKI1 H4O5Q overexpressing plant lines Nos. 6 and 9.
  • Fig. 11 shows that the expressed amounts in the plants with decreased expression of gene CKIl by RNA interference or T-DNA insertion is decreased by at least 50% compared with those in wild type.
  • the present invention comprising genetic manipulations of the activity of CKIl and the homologues and the orthologues thereof, provides for targeted obtaining of plants with an increased production of biomass.
  • Another possible uses are in e.g. biotechnological applications allowing the decontamination of contaminated soils by phytoremediation.
  • the use of transgenic plants producing enzymes allowing the decontamination by decomposition of pollutants, e.g. petrol products, is considered and an increased production of biomass can increase the efficiency of their use.
  • An increased deposition of cellulose, observed in transgenic lines, is suitable for use in paper and wood-processing industry, wherein it can particularly facilitate and reduce the prices of the time-consuming wood production.
  • the increase of biomass in some plant species will be advantageous also for the food-processing and pharmaceutical industry.
  • An important area of utility is the production of the so-called energy plants, e.g. Uteusa sorrel, wherein such genetic modification can enhance the energetic balance of the plants.
  • the invention can be used in breeding of plants, wherein the regulation of the formation of lignified tissues and of lignification of vascular tissues and interfascicular arc fibres can be used in breeding of plant varieties with improved stem erectness (e.g. to avoid procumbent-like phenotype of cereals or rapeseed).

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Abstract

L'invention porte sur un procédé de régulation de la production de biomasse dans les plantes, où le gène CKI1 ou un homologue ou un orthologue de celui-ci est identifié dans la plante et l'expression du gène CKI1 ou d'un homologue ou d'un orthologue de celui-ci ou l'activité du produit génique est modifiée. L'identification de l'homologue ou de l'orthologue du gène CKI1 dans l'espèce de plante comprend les étapes suivantes consistant à : i) identifier l'homologie des séquences nucléotidiques ou l'homologie des séquences d'acides aminés, ii) prouver l'activité histidine kinase du produit génique du gène identifié, iii) identifier l'expression dans des tissus vasculaires de l'espèce de plante respective. La modification de l'expression du gène comprend l'augmentation ou la diminution de l'expression.
PCT/CZ2008/000075 2007-07-04 2008-07-02 Procédé de régulation de la production de biomasse dans les plantes, séquences d'adn et leur procédé de préparation Ceased WO2009003429A2 (fr)

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CN110183524A (zh) * 2019-06-11 2019-08-30 扬州大学 一个促进大豆主根伸长的基因GmKRP2a、蛋白及其应用

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