WO2015117265A1 - Longs arn non codants pour la modulation efficace de l'utilisation de phosphate dans les plantes - Google Patents

Longs arn non codants pour la modulation efficace de l'utilisation de phosphate dans les plantes Download PDF

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WO2015117265A1
WO2015117265A1 PCT/CN2014/071873 CN2014071873W WO2015117265A1 WO 2015117265 A1 WO2015117265 A1 WO 2015117265A1 CN 2014071873 W CN2014071873 W CN 2014071873W WO 2015117265 A1 WO2015117265 A1 WO 2015117265A1
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seq
incrna
molecule
rna
plant
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PCT/CN2014/071873
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Zhi Lu
Dong Liu
Chao DI
Yue Wu
Minxuan KAI
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Tsinghua University
Bayer Cropscience Lp
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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
    • C12N15/8271—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
    • 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]

Definitions

  • the present invention relates to the fields of the agriculture and molecular biology. More particularly, the invention is directed at methods for identification of long non- coding RNAs in plants, particularly non-polyadenylated long non-coding RNAs which are differentially expressed under phosphate starvation conditions, such as long non- coding RNAs whose transcription is upregulated in plants during phosphate starvation. Modulation of the expression of such long non-coding RNAs can be used to alter phosphate use efficiency in plants.
  • IncRNAs have been found to fulfill essential roles in different species [8]. Because of their much lower expression level and evolutionary conservation than protein-coding mRNAs [9, 10], as well as uncertain functions for these transcripts, they were initially considered as transcriptional noise [11, 12]. But this hypothesis was soon challenged by the discovery that many IncRNAs were detected during specific developmental stages [13, 14]. Although most IncRNAs have not been well characterized due to difficulties of cloning, some have been extensively studied and are well known for us, such as Xist, an inactive X-specific IncRNA [15, 16], and COLD AIR, an intronic IncRNA [17].
  • Sequencing of small RNA libraries from stressed A. thaliana seedlings has discovered 26 new miRNAs and 102 novel endogenous small RNAs, suggesting that they have roles in stress responses [3].
  • nutrient homeostasis e.g. phosphorus balance
  • nutrient homeostasis e.g. phosphorus balance
  • Pi- dependent miR-399 targets and cleaves PH02 mRNA, which encodes the E2 conjugase that decreases the Pi content and weakens Pi remobilization [5].
  • IPS 1 INDUCED BY PHOSPHATE STARVAYIO 1
  • AT4 act as target mimics to inhibit miRNA- 399 activity [6, 7].
  • RNA-seq RNA-sequencing
  • the invention provides long non-coding RNA( IncRNA) molecules which are differentially expressed in a plant grown under phosphate starvation conditions when compared to a plant grown under normal conditions with adequate phosphate supply and which are unpolyadenylated such as IncRNA molecules comprising a nucleotide sequence having at least 80% percent identity with any one of SEQ ID No. 4; SEQ ID No. 5; SEQ ID No. 6; SEQ ID No. 15; SEQ ID No. 18; SEQ ID No. 26; SEQ ID No. 28; SEQ ID No. 32; SEQ ID No. 34; SEQ ID No. 36; SEQ ID No. 40; SEQ ID No. 42; SEQ ID No. 47; SEQ ID No.
  • the invention provides single stranded DNA molecules which when transcribed yield RNA molecules comprising such IncRNA or the complement of such single stranded DNA molecules or double stranded DNA molecules.
  • the expression of the IncRNA molecule may be upregulated as is the case for IncRNA molecules comprises a nucleotide sequence having at least 80% percent identity with any one of SEQ ID Nos. 34, 107, 139, 194 or 195.
  • the IncRNA molecule may comprise at least two conserved hairpin loops, preferably further comprising two co-variance base pairings as is the case for IncRNA molecules comprises a nucleotide sequence having at least 80% percent identity with SEQ ID No. 149.
  • the invention provides use of such IncRNA molecules or DNA molecules which when transcribed yield such IncRNA molecules, to modulate inorganic phosphate use efficiency in a plant.
  • the invention provides the use of a IncRNA molecule which is differentially expressed in a plant grown under phosphate starvation conditions when compared to a plant grown under normal conditions with adequate phosphate supply, or a DNA molecule which when transcribed yields such IncRNA molecule, or the complement of the DNA molecule, to modulate inorganic phosphate use efficiency in a plant.
  • This may also comprise IncRNA molecules which are polyadenylated such as IncRNA molecules comprising a nucleotide sequence having at least 80 % sequence identity to SEQ ID No. 216; SEQ ID No. 235; SEQ ID No. 240; SEQ ID No. 250; SEQ
  • the IncRNA molecule may also comprise a nucleotide sequence of any one of Tables 5 to 10.
  • the invention further provides a method for modulating the phosphate use efficiency in a plant comprising the step of increasing or decreasing the transcription and/or concentration of an IncRNA molecule as herein described in a plant cell.
  • the method may comprise a step of decreasing transcription and/or concentration of an IncRNA molecule which may be achieved by mutating the DNA region from which the IncRNA molecule is transcribed, or by expression of an inhibitory RNA molecule specifically recognizing the IncRNA molecule.
  • the method may comprise a step of increasing transcription and/or concentration of an IncRNA molecule which may be achieved by mutating the DNA region from which the IncRNA molecule is transcribed, or by introducing a recombinant gene comprising:
  • RNA molecule comprising or consisting of said IncRNA molecule
  • a DNA region which when transcribed encodes an RNA molecule comprising or consisting of said IncRNA molecule; and optionally c. an appropriate transcription termination region, and/or polyadenylation region.
  • the invention also provides modified plant cells comprising a modulated concentration or transcription of an IncRNA molecule as herein described, when compared to an unmodified plant cell and modified plant, or plant part, tissue or organ comprising a multitude of or consisting essentially of modified plant cells, as well as seeds of such plants comprising the mutation, genetic alteration or recombinant leading to or resulting in the modulated concentration or transcription of an IncRNA molecule as herein described.
  • the invention also provides a method for isolating further IncRNA molecules involved in phosphate use efficiency in a plant comprising the step of identifying a nucleotide sequence having a degree of homology to the IncRNA molecules as herein described and isolating or synthesizing such RNA molecule comprising or consisting of such nucleotide sequences.
  • Figure 1 Flowchart of RNA sequencing and data processing suitable to identify non-polyadenylated long non-coding RNAs .
  • Panel (A) Purification of non-polyA RNAs and total RNAs.
  • Panel (B) Construction of strand-specific cDNA libraries and sequencing, sequencing at short and long read length are different at steps of size selection and Illumina sequencing.
  • Figure 2 Comparison of total and non-polyA RNA-seq with short and long read length.
  • Panel (A) Length distribution of clean-read assembled fragments from three sequencing data.
  • Panel (B) Length distribution of novel IncRNAs from three sequencing data.
  • Panel (C) Numbers of novel IncRNAs at different expressed values (RPKM) from non-polyA RNA-seq (short reads), gray bars. Overlap ratio with novel IncRNAs from total RNA-seq, black line.
  • Panel (E) Validation of novel IncRNA candidates from non-polyA RNA-seq by RT-PCR.
  • Non-polyA and PolyA candidates were amplified from two cDNA libraries: non-polyA RNAs and polyA RNAs; RT(-), negative control without reverse transcriptase; Left box, polyA RNA (control); Right box, non-polyA RNA.
  • FIG. 3 Identification of polyA and non-polyA IncRNAs associated with inorganic phosphate (Pi) starvation.
  • Panel (A) Flowchart of polyA and non-polyA IncRNA prediction and characterization.
  • Panel (B) Differential expression of polyA and non-polyA IncRNAs between plants grown under low Pi conditions and the control plants.
  • Panel (C) Comparison of the proportion of differentially expressed polyA and non- polyA IncRNAs. !, !2 test p-value ⁇ 0.01 ; Gray box, differentially expressed polyA IncRNAs; Dotted box, differentially expressed polyA IncRNAs; gray plus dotted boxes, all identified polyA IncRNAs.
  • FIG. 4 Characterization of identified polyA and associated with Pi starvation.
  • Panel (A) DNA conservation of novel IncRNAs in multiple plant species. Compared to coding genes and unexpressed intergenic control regions, IncRNAs show moderate DNA conservation.
  • Panel (B) Comparison of DNA and protein conservation, RNA structure conservation and structure stability (free energy) of IncRNAs and coding genes. RNA structure conservation was normalized by their DNA conservation scores.
  • Panel (C) GO enrichment of non-polyA IncRNA co-located (on cis-regulatory region) with coding genes. Pi-starvation associated GO terms are shown.
  • FIG. 5 Validation of novel non-polyA IncRNAs in Pi starvation.
  • Panel (C) An example of conserved secondary structure of the 5' end of a non-polyA IncRNA (lnc-149). Two conserved short hairpins are displayed in the top and left region, and one variable long hairpin onn the right. Covariance base- pairings are encircled with dark dots and conserved loops are indicted by darker boxes green (darkest means conserved in three genomes and lightest box in two genomes). The multiple alignments from lnc-146 as can be found in three plant genomes (A. thaliana SEQ ID No. 1328; A. lyrata SEQ ID No. 1329; and T. halophile SEQ ID No. 1330) are also provided to illustrate the conserved bases and loops in the primary sequences.
  • Figure 6 Illustration of the Reads quality by FastQC. Raw reads quality is represented by uploading FastQ files of sequencing reads to the FastQC program (see Example 1/Methods).
  • Figure 7 Novel non-polyA and polyA IncRNAs (both total and differentially expressed) were sub-typed as Transgenic elements, pseudogene, antisense, ambiguous and intergenic by aligning to TAIR10 genome (see Example 1/ Methods).
  • Figure 8 GO enrichment of polyA IncRNAs co-located (on the cis-regulatory region) with coding genes.
  • the current invention is based upon the identification of long non coding RNAs, including novel unpolyadenylated long non coding RNAs, which are differentially expressed upon growth of plants under conditions of inorganic phosphate starvation.
  • the IncRNAs have been detected by processing raw sequencing data based upon an integrative computational model. Validation by RT-PCR indicates that the IncRNAs are bona fide transcripts rather than transcriptional noise.
  • the invention provides long non-coding RNA( IncRNA) molecules which are differentially expressed in a plant grown under phosphate starvation conditions when compared to a plant grown under normal conditions with adequate phosphate supply and which are unpolyadenylated.
  • unpolyadenylated or “non-polyadenylated” or “non-polyA” IncRNA molecules refers to IncRNA molecules which usually lack a polyA-tail.
  • a IncRNA molecule may be classified as unpolyadenylated when, upon RNA sequencing according to methods described herein, particularly in Example 1, the maximum expression value (Reads per Kilobase per Million mapped reads or RPKM value) of each IncRNA determined separately in the polyA reads data and non-polyA reads data is four time greater in non-poly A data than in the polyA data.
  • a IncRNA molecule is considered as "differentially expressed" upon Pi starvation compared to normally grown plants when read counts assigned to IncRNAs, e.g. using DEGseq package [30], normalized against the total mapped reads and analyzed using e.g. the MA-plot based method with random sampling with p-value of 0.05 have a fold- change of 2 or more using RNA of the Pi-starved plants compared to normally grown plants.
  • Table 4 lists long non-coding unpolyadenylated RNAs identified in Arabidopsis thaliana and indicates whether these molecules are differentially expressed under Pi starvation conditions.
  • the invention provides long non-coding RNA molecules comprising a nucleotide sequence which has at least 80% percent identity with any one of SEQ ID No. 4; SEQ ID No. 5; SEQ ID No. 6; SEQ ID No. 15; SEQ ID No. 18; SEQ ID No. 26; SEQ ID No. 28; SEQ ID No. 32; SEQ ID No. 34; SEQ ID No. 36; SEQ ID No. 40; SEQ ID No. 42; SEQ ID No. 47; SEQ ID No. 52; SEQ ID No. 55; SEQ ID No. 57; SEQ ID No. 59; SEQ ID No. 60; SEQ ID No. 62; SEQ ID No. 64; SEQ ID No.
  • sequence identity may be larger than at least 80%, such as at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or be identical with any one of SEQ ID No. 4; SEQ ID No. 5; SEQ ID No. 6; SEQ ID No. 15; SEQ ID No. 18; SEQ ID No. 26; SEQ ID No. 28; SEQ ID No. 32; SEQ ID No. 34; SEQ ID No. 36; SEQ ID No. 40; SEQ ID No. 42; SEQ ID No. 47; SEQ ID No. 52; SEQ ID No. 55; SEQ ID No.
  • the invention further provides DNA molecules which when transcribed yield an RNA molecule comprising said IncRNAs as herein described. Also provided by the invention are the complement of such DNA molecules. DNA molecules may be single- stranded or double stranded. Such DNA molecules may be referred herein as encoding the IncRNA molecules.
  • RNA molecules or the encoding DNA molecules whose expression is upregulated during growth under inorganic phosphate starvation conditions such as lnc-34, lnc-107, lnc-139, lnc-194 or lnc-195.
  • the invention provides IncRNA molecules comprising a nucleotide sequence which has at least 80% percent identity with any one of SEQ ID No. 34, SEQ ID No. 107, SEQ ID No. 139, SEQ ID No. 194 or SEQ ID No.
  • RNA molecules of particular interest are those long- noncoding, unpolyadenylated RNA molecules, differentially expressed under Pi starvation conditions and which comprise a secondary structure, particularly which comprise at least two conserved hairpin loops, preferably further comprising two co- variance base pairings, such as lnc-149( SEQ ID No. 149).
  • These non-polyA IncR A molecules, or DNA molecules encoding such non- polyA IncRNA molecules may be used to modulate inorganic phosphate use efficiency in a plant.
  • a IncRNA molecule may be classified as polyadenylated (poly A) when, upon RNA sequencing according to methods described herein, particularly in Example 1 , the maximum expression value (Reads per Kilobase per Million mapped reads or RPKM value) of each IncRNA determined separately in the polyA reads data and non-polyA reads data is four time greater in poly A data than in the non-polyA data, or is only identified in the polyA data.
  • poly A polyadenylated
  • Table 4 also lists long non-coding polyadenylated RNAs identified in Arabidopsis thaliana and indicates whether these molecules are differentially expressed under Pi starvation conditions.
  • the invention also relates to the use of a IncRNA molecule, whether polyadenylated or unpolyadenylated or "bimorphic", which is differentially expressed in a plant grown under phosphate starvation conditions when compared to a plant grown under normal conditions with adequate phosphate supply, or a DNA molecule which when transcribed yields such IncRNA molecule, or the complement of said DNA molecule, to modulate inorganic phosphate use efficiency in a plant.
  • the invention relates to the use of IncRNA molecule (or encoding DNA molecules) wherein a polyadenylated IncRNA molecule is used comprising a nucleotide sequence having at least 80% sequence identity, or at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity or being identical with any one of SEQ ID No. 216; SEQ ID No. SEQ ID No. 240; SEQ ID No. 250; SEQ ID No. 266; SEQ ID No. 267; SEQ ID No.
  • SEQ ID No. . 571 ; SEQ ID No. 572; SEQ ID No. 600; SEQ ID No. 601 ; SEQ ID No.
  • SEQ ID No. 998 SEQ ID No. 1020; SEQ ID No. 1025; SEQ ID No. 1031; SEQ ID 1033; SEQ ID No. 1047; SEQ ID No. 1048; SEQ ID No. 1068 or SEQ ID No. 1080.
  • IncRNA molecules for phosphate use modulation in a plant comprising a nucleotide sequence of any one of Tables 5 to 10, listing similar sequences found in other plant species, including rice, soybean, wheat, millet, sorghum or corn/maize.
  • the invention thus provides a method of modulating the phosphate use efficiency in a plant comprising the step of increasing or decreasing the transcription and/or concentration of an IncRNA molecule as herein described, in a plant cell.
  • mutagenesis refers to the process in which plant cells (e.g., seed or tissues, such as pollen, etc.) are contacted one or more times to a mutagenic agent, such as a chemical substance (such as ethylmethylsulfonate (EMS), ethylnitrosourea (ENU), etc.) or ionizing radiation (neutrons (such as in fast neutron mutagenesis, etc.), gamma rays (such as that supplied by a Cobalt 60 source), X-rays, etc.), or a combination of the foregoing.
  • a mutagenic agent such as a chemical substance (such as ethylmethylsulfonate (EMS), ethylnitrosourea (ENU), etc.) or ionizing radiation (neutrons (such as in fast neutron mutagenesis, etc.), gamma rays (such as that supplied by a Cobalt 60 source), X-rays, etc
  • mutations created by irradiation are often large deletions or other gross lesions such as translocations or complex rearrangements
  • mutations created by chemical mutagens are often more discrete lesions such as point mutations.
  • EMS alkylates guanine bases, which results in base mispairing: an alkylated guanine will pair with a thymine base, resulting primarily in G/C to A/T transitions.
  • plants can be regenerated from the treated cells using known techniques. For instance, the resulting seeds may be planted in accordance with conventional growing procedures and following self-pollination seed is formed on the plants. Alternatively, doubled haploid plantlets may be extracted to immediately form homozygous plants.
  • DeleteageneTM Delete- a- gene; Li et al., 2001, Plant J 27: 235-242
  • PCR polymerase chain reaction
  • the IncRNA expression may be downregulated by introducing a chimeric DNA construct which yields a sense RNA molecule capable of down-regulating IncRNA expression by co-suppression.
  • the transcribed DNA region will yield upon transcription a so-called sense RNA molecule capable of reducing the concentration of the IncRNA molecule in the target plant or plant cell in a transcriptional or post-transcriptional manner.
  • the transcribed DNA region (and resulting RNA molecule) comprises at least 20 consecutive nucleotides having at least 95% sequence identity to the nucleotide sequence of the IncR A-encoding DNA region present in the plant cell or plant.
  • the IncRNA expression may be downregulated by introducing a chimeric DNA construct which yields an anti-sense RNA molecule capable of down-regulating IncRNA expression by co-suppression.
  • the transcribed DNA region will yield upon transcription a so-called antisense RNA molecule capable of reducing the concentration of the IncRNA molecule in the target plant or plant cell in a transcriptional or post-transcriptional manner.
  • the transcribed DNA region (and resulting RNA molecule) comprises at least 20 consecutive nucleotides having at least 95% sequence identity to the complement of the nucleotide sequence of the IncRNA molecule in the plant cell or plant.
  • the minimum nucleotide sequence of the antisense or sense RNA region of about 20 nt of the IncRNA molecule may be comprised within a larger RNA molecule, varying in size from 20 nt to a length equal to the size of the target IncRNA molecule.
  • the mentioned antisense or sense nucleotide regions may thus be about from about 21 nt to about 5000 nt long, such as 21 nt, 40 nt, 50 nt, 100 nt, 200 nt, 300 nt, 500 nt or 1000 nt in length.
  • the nucleotide sequence of the used inhibitory RNA molecule or the encoding region of the transgene is completely identical or complementary to the IncRNA molecule, the expression of which is targeted to be reduced in the plant cell.
  • the sense or antisense regions may have an overall sequence identity of about 40 % or 50 % or 60 % or 70 % or 80 % or 90 % or 100 % to the nucleotide sequence of the endogenous parpl gene or the complement thereof.
  • antisense or sense regions should comprise a nucleotide sequence of 20 consecutive nucleotides having about 95 to about 100 % sequence identity to the nucleotide sequence of the IncRNA molecule.
  • the stretch of about 95 to about 100% sequence identity may be about 50, 75 or 100 nt.
  • IncR A molecule transcription or concentration may be down-regulated by introducing a chimeric DNA construct which yields a double- stranded RNA molecule capable of down-regulating IncRNA expression. Upon transcription of the DNA region the RNA is able to form dsRNA molecule through conventional base paring between a sense and antisense region, whereby the sense and antisense region are nucleotide sequences as hereinbefore described.
  • dsRNA-encoding parpl expression-reducing chimeric genes according to the invention may further comprise an intron, such as a heterologous intron, located e.g. in the spacer sequence between the sense and antisense RNA regions in accordance with the disclosure of WO 99/53050 (incorporated herein by reference).
  • an intron such as a heterologous intron, located e.g. in the spacer sequence between the sense and antisense RNA regions in accordance with the disclosure of WO 99/53050 (incorporated herein by reference).
  • IncRNA molecule transcription or concentration can be down-regulated by introducing a chimeric DNA construct which yields a pre-miRNA RNA molecule which is processed into a miRNA capable of guiding the cleavage of the IncRNA molecule.
  • miRNAs are small endogenous RNAs that regulate gene expression in plants, but also in other eukaryotes. In plants, these about 21 nucleotide long RNAs are processed from the stem-loop regions of long endogenous pre-miRNAs by the cleavage activity of DICERLIKE 1 (DCL1). Plant miRNAs are highly complementary to conserved target mRNAs, and guide the cleavage of their targets. miRNAs appear to be key components in regulating the gene expression of complex networks of pathways involved inter alia in development.
  • a "miRNA” is an RNA molecule of about 20 to 22 nucleotides in length which can be loaded into a RISC complex and direct the cleavage of a target RNA molecule, wherein the target RNA molecule comprises a nucleotide sequence essentially complementary to the nucleotide sequence of the miRNA molecule whereby one or more of the following mismatches may occur:
  • a "pre-miRNA” molecule is an RNA molecule of about 100 to about 200 nucleotides, preferably about 100 to about 130 nucleotides which can adopt a secondary structure comprising a dsRNA stem and a single stranded RNA loop and further comprising the nucleotide sequence of the miRNA and its complement sequence of the miRNA* in the double-stranded RNA stem.
  • the miRNA and its complement are located about 10 to about 20 nucleotides from the free ends of the miRNA dsRNA stem.
  • the length and sequence of the single stranded loop region are not critical and may vary considerably, e.g. between 30 and 50 nt in length.
  • the difference in free energy between unpaired and paired RNA structure is between -20 and -60 kcal/mole, particularly around -40 kcal/mole.
  • the complementarity between the miRNA and the miRNA* do not need to be perfect and about 1 to 3 bulges of unpaired nucleotides can be tolerated.
  • the secondary structure adopted by an RNA molecule can be predicted by computer algorithms conventional in the art such as mFold, UNAFold and RNAFold.
  • the particular strand of the dsRNA stem from the pre-miRNA which is released by DCL activity and loaded onto the RISC complex is determined by the degree of complementarity at the 5' end, whereby the strand which at its 5' end is the least involved in hydrogen bounding between the nucleotides of the different strands of the cleaved dsRNA stem is loaded onto the RISC complex and will determine the sequence specificity of the target RNA molecule degradation.
  • miRNA molecules may be comprised within their naturally occurring pre-miRNA molecules but they can also be introduced into existing pre-miRNA molecule scaffolds by exchanging the nucleotide sequence of the miRNA molecule normally processed from such existing pre-miRNA molecule for the nucleotide sequence of another miRNA of interest.
  • the scaffold of the pre-miRNA can also be completely synthetic.
  • synthetic miRNA molecules may be comprised within, and processed from, existing pre- miRNA molecule scaffolds or synthetic pre-miRNA scaffolds.
  • Increase of transcription and/or concentration of a lncRNA molecule as herein described can be achieved by providing the plant cells with a recombinant gene, wherein the recombinant gene comprises a plant expressible promoter operably linked to a DNA region which when transcribed yields an RNA molecule comprising or consisting of that lncRNA molecule and optionally, an appropriate transcription termination region and/or polyadenylation region.
  • plant- operative promoter or "plant- expressible promoter” means a promoter which is capable of driving transcription in a plant, plant tissue, plant organ, plant part, or plant cell. This includes any promoter of plant origin, but also any promoter of non-plant origin which is capable of directing transcription in a plant cell.
  • Promoters that may be used in this respect are constitutive promoters, such as the promoter of the cauliflower mosaic virus (CaMV) 35S transcript (Hapster et al.,1988, Mol. Gen. Genet. 212: 182-190), the CaMV 19S promoter (U.S. Pat. No. 5,352,605; WO 84/02913; Benfey et al, 1989, EMBO J. 8:2195-2202), the subterranean clover virus promoter No 4 or No 7 (WO 96/06932), the Rubisco small subunit promoter (U.S. Pat. No. 4,962,028), the ubiquitin promoter (Holtorf et al, 1995, Plant Mol.
  • CaMV cauliflower mosaic virus
  • CaMV 19S promoter U.S. Pat. No. 5,352,605; WO 84/02913; Benfey et al, 1989, EMBO J. 8:2195-2202
  • T-DNA gene promoters such as the octopine synthase (OCS) and nopaline synthase (NOS) promoters from Agrobacterium, and further promoters of genes whose constitutive expression in plants is known to the person skilled in the art.
  • OCS octopine synthase
  • NOS nopaline synthase
  • tissue-specific or organ-specific promoters preferably seed-specific promoters, such as the 2S albumin promoter (Joseffson et al, 1987, J. Biol. Chem. 262: 12196-12201), the phaseolin promoter (U.S. Pat. No. 5,504,200; Bustos et al, 1989, Plant Cell l .(9):839-53), the legumine promoter (Shirsat et al, 1989, Mol. Gen. Genet. 215(2):326-331), the "unknown seed protein” (USP) promoter (Baumlein et al, 1991, Mol. Gen. Genet.
  • tissue-specific or organ-specific promoters like organ primordia-specific promoters (An et al., 1996, Plant Cell 8: 15-30), stem- specific promoters (Keller et al, 1988, EMBO J. 7(12): 3625-3633), leaf-specific promoters (Hudspeth et al., 1989, Plant Mol. Biol. 12: 579-589), mesophyl-specific promoters (such as the light-inducible Rubisco promoters), root-specific promoters (Keller et al., 1989, Genes Dev.
  • tuber-specific promoters Keil et al., 1989, EMBO J. 8(5): 1323-1330
  • vascular tissue-specific promoters Pieris et al., 1989, Gene 84: 359-369
  • stamen-selective promoters WO 89/10396, WO 92/13956
  • dehiscence zone-specific promoters WO 97/13865
  • RNA polymerase I In addition to promoters recognized by RNA polymerase I, also promoter recognized by RNA Polymerase I or RNA polymerase III promoters may be used including Type 3 Pol III promoters which can be found e.g. associated with the genes encoding 7SL RNA, U3 snRNA and U6 snRNA. Other nucleotide sequences for type 3 Pol III promoters can be found in nucleotide sequence databases under the entries for the A. thaliana gene AT7SL-1 for 7SL RNA (X72228), A. thaliana gene AT7SL-2 for 7SL RNA (X72229), A.
  • thaliana gene AT7SL-3 for 7SL RNA (AJ290403), Humulus lupulus H17SL-1 gene (AJ236706), Humulus lupulus H17SL-2 gene (AJ236704), Humulus lupulus H17SL-3 gene (AJ236705), Humulus lupulus H17SL-4 gene (AJ236703), A. thaliana U6-1 snRNA gene (X52527), A. thaliana U6-26 snRNA gene (X52528), A. thaliana U6-29 snRNA gene (X52529), A.
  • thaliana U6-1 snRNA gene (X52527), Zea mays U3 snRNA gene (Z29641), Solanum tuberosum U6 snRNA gene (Z17301; X 60506; S83742), Tomato U6 smal nuclear RNA gene (X51447), A. thaliana U3C snRNA gene (X52630), A.
  • thaliana U3B snRNA gene (X52629), Oryza sativa U3 snRNA promoter (X79685), Tomato U3 smal nuclear RNA gene (X14411), Triticum aestivum U3 snRNA gene (X63065), Triticum aestivum U6 snRNA gene (X63066).
  • the recombinant DNA molecules as herein described optionally comprise a DNA region involved in transcription termination and/or polyadenylation.
  • a variety of DNA region involved in transcription termination and/or polyadenylation functional in plants are known in the art and those skilled in the art will be aware of terminator and polyadenylation sequences that may be suitable in performing the methods herein described.
  • the polyadenylation region may be derived from a natural gene, from a variety of other plant genes, from T-DNA genes or even from plant viral genomes.
  • the 3' end sequence to be added may be derived from, for example, the nopaline synthase or octopine synthase genes, or alternatively from another plant gene, or from any other eukaryotic gene.
  • Terminator regions for Pol III promoters include a so-called « oligo dT stretch » which is a stretch of consecutive T-residues that serve as a terminator for the RNA polymerase III activity. It should comprise at least 4 T-residues, but obviously may contain more T-residues.
  • providing a recombinant DNA molecule may refer to introduction of an exogenous DNA molecule to a plant cell by transformation, optionally followed by regeneration of a plant from the transformed plant cell.
  • the term may also refer to introduction of the recombinant DNA molecule by crossing of a transgenic plant comprising the recombinant DNA molecule with another plant and selecting progeny plants which have inherited the recombinant DNA molecule or transgene.
  • Yet another alternative meaning of providing refers to introduction of the recombinant DNA molecule by techniques such as protoplast fusion, optionally followed by regeneration of a plant from the fused protoplasts.
  • Transformation of plants is now a routine technique.
  • any of several transformation methods may be used to introduce the nucleic acid/gene of interest into a suitable ancestor cell. Transformation methods include the use of liposomes, electroporation, chemicals that increase free DNA uptake, injection of the DNA directly into the plant, particle gun bombardment, transformation using viruses or pollen and microprojection. Methods may be selected from the calcium/polyethylene glycol method for protoplasts (Krens et al. (1982) Nature 296: 72- 74 ; Negrutiu et al. (1987) Plant. Mol. Biol.
  • Transgenic rice plants can be produced via Agrobacterium-mQdiatQd transformation using any of the well-known methods for rice transformation, such as described in any of the following: European patent application EP 1 198985 Al ; Aldemita and Hodges (1996) Planta 199: 612-617 ; Chan et al. (1993) Plant. Mol. Biol. 22 (3): 491-506 ; Hiei et al. (1994) Plant J. 6 (2): 271 -282), which disclosures are incorporated by reference herein as if fully set forth.
  • a suitable method is as described in either Ishida et al. (1996) Nat. Biotechnol. 14(6): 745- 50) or Frame et al.
  • the recombinant DNA molecules according to the invention may be introduced into plants in a stable manner or in a transient manner using methods well known in the art.
  • the chimeric genes may be introduced into plants, or may be generated inside the plant cell as described e.g. in EP 1339859.
  • Gametes, seeds, embryos, either zygotic or somatic, progeny or hybrids of plants which comprising such modulated IncRNA molecule concentration, which are produced by traditional breeding methods are also included within the scope of the present invention.
  • the methods and means described herein are believed to be suitable for all plant cells and plants, both dicotyledonous and monocotyledonous plant cells and plants including but not limited to cotton, Brassica vegetables, oilseed rape, wheat, corn or maize, barley, sunflowers, sorghum, rice, oats, sugarcane, soybean, vegetables (including chicory, lettuce, tomato), tobacco, potato, sugarbeet, papaya, pineapple, mango, Arabidopsis thaliana, but also plants used in horticulture, floriculture or forestry.
  • the invention also provides a method for isolating further IncRNA molecules involved in phosphate use efficiency, in a plant or plant cell, comprising the step of identifying a nucleotide sequence having a degree of homology to the IncRNA molecules herein described and isolating or synthesizing such RNA molecule comprising or consisting of such nucleotide sequence.
  • the identification can occur via hybridization under stringent conditions in plants using probes having the nucleotide sequence of the IncRNA molecules.
  • sequence databases (of genomes or transcriptomes) can be searched using software such as BLASTN for sequences that share a defined degree of sequence identity with the sequences of the IncRNA molecules herein described.
  • sequence identity of two related nucleotide or amino acid sequences, expressed as a percentage, refers to the number of positions in the two optimally aligned sequences which have identical residues (xlOO) divided by the number of positions compared.
  • a gap i.e., a position in an alignment where a residue is present in one sequence but not in the other is regarded as a position with non-identical residues.
  • the alignment of the two sequences is performed by the Needleman and Wunsch algorithm (Needleman and Wunsch (1970) J. Mol Biol. 48: 443-453).
  • RNA sequences are to be essentially similar or have a certain degree of sequence identity with DNA sequences, thymine (T) in the DNA sequence is considered equal to uracil (U) in the RNA sequence.
  • nucleic acid comprising a sequence of nucleotides
  • a chimeric gene as will be described further below which comprises a nucleic acid which is functionally or structurally defined may comprise additional nucleic acids etc.
  • the term “comprising” also includes “consisting of.
  • nucleic acid comprising a certain nucleotide sequence
  • terminology relating to a nucleic acid “comprising" a certain nucleotide sequence refers to a nucleic acid or protein including or containing at least the described sequence, so that other nucleotide or amino acid sequences can be included at the 5' (or N-terminal) and/or 3' (or C-terminal) end, e.g. (the nucleotide sequence of) a selectable marker protein, (the nucleotide sequence of) a transit peptide, and/or a 5' leader sequence or a 3' trailer sequence.
  • SEQ ID Nos. 1-211 nucleotide sequence of respectively NonpolyA_lnc 1 to
  • NonpolyA_lnc 211 from Arabidopsis thaliana.
  • SEQ ID Nos. 212-1081 nucleotide sequence of respectively PolyA lnc 1 to
  • PolyA_lnc 870 from Arabidopsis thaliana From Arabidopsis thaliana.
  • SEQ ID Nos. 1082-1096 nucleotide sequence of orhologous IncRNA molecules from
  • SEQ ID Nos. 1097-1 110 nucleotide sequence of orhologous IncRNA molecules from
  • SEQ ID Nos. 111 1-1 121 nucleotide sequence of orhologous IncRNA molecules from
  • SEQ ID Nos. 1122-1 130 nucleotide sequence of orhologous IncRNA molecules from
  • SEQ ID Nos. 1131-1239 nucleotide sequence of orhologous IncRNA molecules from
  • Triticiim aestivum.
  • SEQ ID Nos. 1240-1297 nucleotide sequence of orhologous IncRNA molecules from
  • SEQ ID Nos. 1298-1327 nucleotide sequence of RT-PCR primers listed in Table 3.
  • SEQ ID No. 1328 nucleotide sequence of lnc-149 from Arabidopsis thaliana.
  • SEQ ID No. 1329 nucleotide sequence of lnc-149 like from Arabidopsis lyrata.
  • SEQ ID No. 1330 nucleotide sequence of lnc-149 from Thellungiella halophile.
  • Non-polyA RNA and total RNA (rRNA removed) purification were extracted from thirteen-day-old seedlings using QIAGEN RNeasy Plant Mini Kit and then quantified by NanoDrop 1000 and 1% agarose gel electrophoresis.
  • To enrich non- polyA RNA we chose 10 ⁇ g RNA to capture polyA RNA using Oligo dT 3 o-probe (Oligotex mRNA Mini Kit, QIAGEN) for four times. 5 ⁇ 1 probes and 80 ⁇ 1 binding buffer were used each time. After centrifuging, probes binding polyA RNAs were precipitated and the supernatant mainly consisted of non-polyA RNA and short-polyA RNA.
  • Non-polyA RNAs and total RNAs were quantified using Agilent 2100 Bioanalyzer (allowing to see zonal distribution for total RNAs after rRNA removal) and then stored -80 °C.
  • RNA-seq longest reads
  • 320-620 bp fragments containing 200-500 bp cDNA fragments and 120bp barcodes were purified,amplified and then sequenced by 100 nt single-end using Illumina HiSeq 2000. For each sample, ⁇ 20M raw reads were collected.
  • Mapped reads were assembled by Cufflinks (v2.0.1) and re-assembled by Cuffcompare (v2.0.1) following the protocol from [28].
  • Transcripts labeled CUFF are considered novel transcripts (without annotation). These transcripts were collected and then re-filtered out to remove those overlapping with coding genes and known ncRNAs.
  • CPC tools [29] were used to calculate coding potential and those reads with CPC ⁇ 0 (low coding potential) were retained. After length selection of transcripts longer than 200 nt transcripts, these were designated as novel IncRNA.
  • Novel IncRNAs were re-located to transposable elements (TE) (>1 nt overlap with TE, no strand-specificity), pseudogene (>1 nt overlap with pseudogene, no strand-specificity), antisense (>50% overlap with mRNA, on opposite strand), intronic (100% overlap with intron, on the same strand), ambiguous ( >1 nt overlap with known ncRNAs or coding genes) and intergenic region (the remainder).
  • TE transposable elements
  • pseudogene >1 nt overlap with pseudogene, no strand-specificity
  • antisense >50% overlap with mRNA, on opposite strand
  • intronic 100% overlap with intron, on the same strand
  • ambiguous >1 nt overlap with known ncRNAs or coding genes
  • intergenic region the remainder.
  • DNA conservation analysis A phylogenetic tree based on the plant species' divergent time was constructed (www.timetree.org) using MEGA5.0. DNA conservation scores between Arabidopsis thaliana and 16 other organisms were calculated using BLASTn followed by the calculation of the average DNA conservation score.
  • DNA conservation scores were calculated across 31 plant species (genomes downloaded from PlantGDB) using BLASTn with default parameters. The maximum Bitscore was used as the feature score. Protein conservation scores were calculated using BLASTx in a similar manner. The coding potential of each bin was calculated using R Acode with default parameters [32]. RNA secondary structure stability of each bin was calculated using RandFold [33], with 1000 times of dinucleotide randomshuffling, and the p-value was used as the feature score.
  • RNA structure conservation scores were denoted by SCI (structure conservation index) scores, which were calculated using RNAz based on multiple alignments between Arabidopsis thaliana, Arabidopsis lyrata, Carica papaya, Thellungiella halophile and Citrus Clementina (downloaded from VISTA database).
  • SCI structure conservation index
  • RNA-seq and tiling array data [34-40] Dozens of RNA-seq and tiling array data [34-40], and a set of unexpressed intergenic regions was defined as negative control.
  • the genomic regions with expression level lower than the mean expression level of all genomic element across all RNA-seq and array samples [31] were defined as unexpressed intergenic regions.
  • Example 2 Development of a non-polyA RNA sequencing method to identify IncRNAs in the Arabidopsis thaliana genome
  • RNA-sequencing methods to identify novel IncRNAs in Arabidopsis: (i) Sequencing of total RNAs (rRNA depleted) for short reads with 36nt in length; (ii) Sequencing of non-polyA RNAs separated from total RNAs by a general RNA-seq protocol for short reads with 36 nt in length; (iii) Sequencing of non- polyA RNAs for long reads with 100 nt in length (see Example 1). These three methods were compared by the length of assembled transcripts and the best one was selected for genome -wide IncRNAs identification.
  • the identification of novel IncRNAs comprised three steps: (i) purifying specific RNA components; (ii) constructing strand-specific cDNA library and sequencing; (iii) high- throughput sequencing data processing to identify novel IncRNAs ( Figure 1).
  • LncRNAs IPS1 and At4 act as target mimics to inhibit the activity of miRNA-399, and are involved in response to Pi starvation in Arabidopsis [6].
  • a systematic understanding of the roles of IncRNAs in Pi starvation response is still lacking.
  • We utilized non-polyA RNA-seq (long read) method (described above, see Examples 1 and 2) to identify IncRNAs which are differentially expressed in Arabidopsis seedling under normal and Pi-deficient condition.
  • Table 4 The chromosomal locations of differentially expressed IncRNA candidates are listed in Table 4. Table 4 also includes a cross reference to the corresponding SEQ ID No. entry in the sequence listing. Also included are GO terms associated coding regions co- localized with the IncRNA as described below.
  • Non-polyA IncRNAs are mainly located in antisense and intergenic regions ( Figure ID), with limited clues to infer their potential cellular functions.
  • Previous studies suggested that IncRNAs located around protein coding genes (no matter upstream, internal or downstream of the sense or antisense strand) can regulate the expression of their 'host' coding genes in Arabidopsis [17, 47]. Therefore we analyzed potential IncRNA functions based on genomic co-location with protein coding genes. 1 Kb regions were identified downstream and upstream the protein coding genes as the potential cis- regulatory regions. IncRNAs, differentially expressed under Pi starvation conditions, which are located in the cis-regulatory regions could potentially regulate their 'host' protein coding genes.
  • the lnc-34 is transcribed from a region located upstream of a gene AT1G74670, which encodes a protein whose expression is responsive to gibberellins and sugar, two signals which function in Pi starvation responses [48, 49].
  • AT1G74670 encodes a protein whose expression is responsive to gibberellins and sugar, two signals which function in Pi starvation responses [48, 49].
  • differential expression for lnc-34 can be found between control and low Pi samples, while no similar phenomenon was found in polyA sequencing.
  • the conserved structure is composed of two conserved short hairpins and one variable long hairpin located at the 5' end (3-70 nt) of lnc-149. Moreover, the three parts of the local structure are connected together by a multi-branch loop and an additional stem. The key points of the connection, one in the start region of the first conserved hairpin and the other in the start region of the variable hairpin, are constrained by base pairs with covariance (the bases change and the structures retain), implying the conserved structure is favored during evolution and may have a function in the stress response by low Pi conditions. Furthermore, the loop regions in the two conserved hairpins are also maintained in three or two genomes, demonstrating that the loops may have a function, for instance, to bind R A-binding proteins.
  • Example 5 Identification of orthologues of IncRNA in other crop species.
  • a plant expressible promoter such as a CaMV35S promoter b) a DNA region which when transcribed yields an R A comprising a nucleotide sequence of a IncR A, preferably a non-polyA IncR A upregulated under Pi starvation conditions, including a non-poly IncRNA selected from lnc-24, lnc-107, lnc-139, lnc-194, lnc-195 or lnc-149
  • the recombinant genes are introduced into plants, particularly Arabidopsis plants through transformation methods known in the art and transgenic plants are identified.
  • RNA-seq an assessment of technical reproducibility and comparison with gene expression arrays. Genome research, 2008. 18(9): p. 1509- 17.
  • RNAcode robust discrimination of coding and noncoding regions in comparative sequence data.
  • At-TAX a whole genome tiling array resource for developmental expression analysis and transcript identification in Arabidopsis thaliana. Genome biology, 2008. 9(7): p. Rl 12.
  • AtmtPNPase is required for multiple aspects of the 18S rRNA metabolism in Arabidopsis thaliana mitochondria. Nucleic acids research, 2004. 32(17): p. 5174-82.
  • RNA-seq short reads
  • non-polyA RNA-seq long reads
  • low Pi samples were sequenced at lOOnt single-end. Reads were aligned to TAIRIO rRNA, and remaining were mapped to TAIRIO genome using Tophat with two mismatches.
  • abscisic acid mediated signaling pathway cell communication,cell death,cellular membr fusion,cytoplasm, defense response to bacterium, incompatible interaction, defense res to fungus,embryo development,endoplasmic reticulum unfolded protein response,ethyl mediated signaling pathway,ethylene mediated signaling pathway,glycolysis,Golgi organization,Golgi vesicle transport,hyperosmotic responsejasmonic acid mediated sig pathwayjasmonic acid mediated signaling pathway,lateral root morphogenesis,MAPK cascade,NAD+ ADP-ribosyltransferase activity,NAD+ ADP-ribosyltransferase activity,NA ADP-ribosyltransferase activity, negative regulation of defense response,negative regula of programmed cell death,nitric oxide biosynthetic

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Abstract

L'invention concerne de longs ARN non codants dans des plantes, en particulier, de longs ARN non codants non polyadénylés qui sont exprimés différemment dans des conditions de manque de phosphate, tels que de longs ARN non codants dont la transcription subit une régulation positive dans les plantes pendant le manque de phosphate. La modulation de l'expression de tels longs ARN non codants peut être utilisée pour modifier efficacement l'utilisation du phosphate dans les plantes.
PCT/CN2014/071873 2014-02-07 2014-02-07 Longs arn non codants pour la modulation efficace de l'utilisation de phosphate dans les plantes WO2015117265A1 (fr)

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CN111304197A (zh) * 2018-12-11 2020-06-19 东北农业大学 一种甜菜耐碱胁迫的长链非编码rna基因及其制备方法与应用
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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2019233863A1 (fr) 2018-06-04 2019-12-12 Bayer Aktiengesellschaft Benzoylpyrazoles bicycliques utilisés comme herbicide
CN111304197A (zh) * 2018-12-11 2020-06-19 东北农业大学 一种甜菜耐碱胁迫的长链非编码rna基因及其制备方法与应用
CN110699355A (zh) * 2019-07-30 2020-01-17 中山大学 长链非编码rna基因rovule及其在调节水稻胚乳发育中的应用
CN110699355B (zh) * 2019-07-30 2023-09-22 中山大学 长链非编码rna基因rovule及其在调节水稻胚乳发育中的应用
CN114807137A (zh) * 2021-07-07 2022-07-29 忻州师范学院 马铃薯高温响应lncRNA及其应用
CN114214334A (zh) * 2022-01-12 2022-03-22 山东农业大学 来源于盐芥的基因EsH2A.3在调控植物耐盐性中的应用
CN114214334B (zh) * 2022-01-12 2023-08-04 山东农业大学 来源于盐芥的基因EsH2A.3在调控植物耐盐性中的应用

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