WO2013130470A1 - L'arni de phya 1 du coton améliore la qualité des fibres, l'élongation des racines, la floraison, la maturité et le potentiel de rendement dans glossypium hirsutum l - Google Patents

L'arni de phya 1 du coton améliore la qualité des fibres, l'élongation des racines, la floraison, la maturité et le potentiel de rendement dans glossypium hirsutum l Download PDF

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WO2013130470A1
WO2013130470A1 PCT/US2013/027801 US2013027801W WO2013130470A1 WO 2013130470 A1 WO2013130470 A1 WO 2013130470A1 US 2013027801 W US2013027801 W US 2013027801W WO 2013130470 A1 WO2013130470 A1 WO 2013130470A1
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plant
cotton
rnai
gene
phya
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PCT/US2013/027801
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Ibrokhim Y ABDURAKHMONOV
Zabardast T. BURIEV
Abdusattor ABDUKARIMOV
Johnie Norton JENKINS
Sukumar Saha
Alan E. PEPPER
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Center Of Genomic Technologies, Institute Of Genetics And Plant Experimental Biology Academy Of Science Of Uzbekistan
The United States Of America, As Reprensented By The Secretary Of Agriculture
The Texas A & M University System
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Priority claimed from US13/445,696 external-priority patent/US9663560B2/en
Application filed by Center Of Genomic Technologies, Institute Of Genetics And Plant Experimental Biology Academy Of Science Of Uzbekistan, The United States Of America, As Reprensented By The Secretary Of Agriculture, The Texas A & M University System filed Critical Center Of Genomic Technologies, Institute Of Genetics And Plant Experimental Biology Academy Of Science Of Uzbekistan
Priority to CN201380022173.7A priority Critical patent/CN104320968B/zh
Priority to RU2014138704A priority patent/RU2665804C2/ru
Publication of WO2013130470A1 publication Critical patent/WO2013130470A1/fr

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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)
    • C12N15/8216—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]
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • 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)
    • 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
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8269—Photosynthesis

Definitions

  • Cotton PHYA 1 RNAi Improves Fiber Quality, Root Elongation, Flowering,
  • This invention relates to the role of phytochrome genes in the regulation of flowering, fiber initiation and elongation, and other characteristics affected by altered photomorphogenesis in Gossypium plants; PHYA 1 gene silencing constructs
  • RNA interference of the phytochrome PHYA1 gene to generate novel transgenic plants exhibiting improved cotton fiber quality, early-flowering and early boll maturity, enhanced root elongation, and increased seed cotton production due to both the suppression of PHYA1 and the several fold increases in the expression of other phytochrome genes.
  • photoreceptor gene family is best characterized in the model plant Arabidopsis, which has five phytochrome genes PHYA, PHYB, PHYC, PHYD, and PHYE (Sharrock and Quail. 1989. Genes and Dev. 3:1745-1757; Clack et al. 1994. Plant Mol. Biol. 25:413- 427; Cowl et al. 1994. Plant Physiol. 106:813-814).
  • the phytochromes interact with cryptochromes, the circadian clock, phytohormones, and other signals to regulate floral initiation (Devlin et al. 1998. Plant Cell 10:1479-1487; Devlin et al. 1999. Plant Physiol.
  • PHYB/D/E overexpression correlates with shortening of hypocotyl length (Clough et al. 1995. Plant Physiol. 109:1039-1045; Devlin et al. 1999, supra; Devlin et al. 1998, supra; Lin, C. 2000. Plant Physiol. 1239:39-50) and an early flowering phenotype, as is observed, for example, in phyb mutants, suggesting more complex action mechanisms for PHYB (Bagnall et al., supra; Lin, supra). PHYC also contributes to photoperiodic flowering and natural phenotypic variation in flowering time in
  • the phytochrome gene family has additional importance because there is evidence that the far red/red (FR/R) photon ratio influences length and diameter of developing fiber.
  • FR/R far red/red
  • cotton fibers that were exposed to a high far red/red photon ratio were longer than those exposed to elevated photosynthetic light (Kasperbauer, M. J. 1994. Physiol. Plantarum 91 :317-321 ; Kasperbauer, M. J. 2000. Crop Sci. 40:1673-1678).
  • Genetic improvement of fiber yield and fiber quality, i.e., fiber length and fiber strength, is the primary objective of cotton breeding programs worldwide (Perkins et ai. 1984. In: Cotton Agron. Monogr.
  • phytochrome gene family expression profile results in a changed plant architecture with elongated leaf petioles, fruit branches, boll peduncles and root system, vigorous vegetative growth, early flowering and early boll maturity, senescence-enhanced anthocyanin pigmentation in stems and leaves, increased fiber quality (length, strength, microniare, etc.) and fiber yield phenotypes; and that the changes are stably expressed in subsequent generations and transferable from the transformed Coker 312 genotype to Upland cultivar through genetic hybridization and selection.
  • SEQ ID NO:1 sense nucleotide portion
  • RNAi RNA interference
  • CaMV Cauliflower mosaic virus
  • transgenic cotton plant produced by the methods of the invention, or the progeny thereof, comprising: the PHYA1 RNAi construct of the invention, said plants exhibiting altered expression of photomorphogenic characteristics including changed plant architecture with longer leaf petioles and fruit branches, enhanced elongation of root system, vigorous vegetative growth, early flowering and early boll maturity, senescence-enhanced anthocyanin pigmentation in stems and leafs compared to wild-type non-transformed cotton plant.
  • transgenic cotton cell comprising the PHYA 1 RNAi construct of the invention.
  • transgenic cotton plant comprising the PHYA1 RNAi construct of the invention, wherein the transgenic plant exhibits cotton fibers of increased length and strength as well as improved microniare, elongation and fiber uniformity relative to the wild-type cotton plant.
  • Figures 1A-1 C depict the effects of PHYA1 RNAi in cotton:
  • Figure 1A is a schematic representation of PHYA gene, RNAi fragment position, and pHellsgate- Q: PHYA1 RNAi plasmid;
  • Figure 1 B depicts shoot and root development;
  • Figure 1 C depicts fiber length characteristics of To- generation PHYA1 RNAi and control cotton plants, somatically regenerated in tissue culture.
  • Figures 2A-2D show phytochrome-associated developmental changes in PHYA1 RNAi plants compared to controls:
  • Figure 2A shows the enhanced vegetative growth and early flowering in the T 0 RNAi plant compared to the same-day planted control plant, regenerated via somatic embryogenesis.
  • Figure 2C shows early flowering in the Ti generation RNAi plant compared to the control plant ( Figure 2B) planted the same day in the same environment.
  • Figure 2D shows the difference in petiole length (T 0 ) and
  • Figure 2E shows the difference in root development (T 3 ) compared to the Coker-312 control.
  • Figure 3 depicts the staple length of fibers from RNAi cotton plants in Ti generations. Green bars are staple length indices for 3 individual Coker-312 plants (marked as K-312); amber bars are staple length indices from individual Ti generation RNAi plants; and yellow bar is staple length indices for Pima cotton. Control and RNAi Coker-312 plants were grown in the same greenhouse environment.
  • Figures 4A-4D show phytochrome-associated RNAi effects in a RNAi line derived from the cross between RNAi Coker 312 and AN-Boyovut-2 (Uzbek variety) cultivar:
  • Figure 4A shows senescence-associated anthocyanin pigmentation in the field grown plants;
  • Figures 4B and 4D show anthocyanin accumulation in leaf plates and cotton bolls and the elongation of leaf petioles and the peduncle of bolls;
  • Figure 4C demonstrates the bush type and productivity of the RNAi line developed using this invention.
  • Figure 5 shows a general trend of changing the major fiber quality traits in second generation RNAi plants of Coker-312, compared to controls grown under the same conditions.
  • Figures 6A-6H depict the histograms for average phenotypic characteristics of selected T 2 -generation PHYA 1 RNAi plant families (T 2 -1_7 and T 2 -31_10) compared to the same environment- and condition-grown control cotton plants:
  • Figure 6A depicts the upper half mean (UHM);
  • Figure 6B the micronaire (MIC);
  • Figure 6C fiber strength (STR);
  • Figure 6D fiber uniformity;
  • Figure 6E fiber elongation (ELO);
  • Figure 6F average hypocotyl length;
  • Figure 6G average number of flowers by July 15, 2009;
  • Figure 6H average number of opened bolls by September 15, 2009.
  • Statistical significance of measured traits between RNAi genotypes and control in Wilcoxon matched-pairs signed-rank test at p ⁇ 0.05 was defined with "a", "b", and "c" letters.
  • Figure 7A depicts fiber length characteristics and Figures 7C and 7D depict root development characteristics of selected T 3 -generation PHYA1 RNAi plant families (T 3 - 1_7 and T 3 -31_10) compared to the same environment- and condition- grown control cotton plants.
  • PCR-verification of these selected plants is shown in Figure 7B: M-100 bp ladder, 1 - T 3 -1_7; 2- T 3 -31_10; 3 - Coker 312; 4 - pHellsgate-8::PHY,47 plasmid; 5 - no DNA template control.
  • These plants were used for copy number identification and relative expression analyses using qPCR .
  • Figures 8A and 8B show the difference in vegetative growth between field grown T 3 RNAi and control plants in the experimentally controlled field test of 2009 (Figure 8A).
  • Figure 8A The transferability of the phytochrome-associated RNAi effects from RNAi Coker-312 to Upland cultivar (AN-Boyovut-2) is shown in Figure 8A and 8B.
  • Figure 8C compares the improvement of fiber samples between the original cultivar (left) and the RNAi F 2 hybrids (right) grown in the same environment.
  • Figure 9 shows a general trend of changing the major fiber quality traits in second generation AN-Boyovut-2 x RNAi Coker-312 hybrids, compared to controls grown under the same conditions.
  • This invention concerns the role of phytochrome genes in the regulation of particular phenotypic traits in cotton.
  • We had hypothesized a role for phytochrome genes in the regulation of cotton fiber elongation (Abdurakhmonov, I. Y. 2001 . Thesis. Texas A& M University, USA) based on the findings that our initial efforts on mapping phytochromes genes in a fiber length segregating bi-parental population suggested a significant association of PHYA 1 gene polymorphisms with the fiber length Quantitative Trait Locus (QTL) (Abdurakhmonov 2001 , supra).
  • Plant Ce// 20(9):2324-2338) there have been several reports on involvement of phytochromes and its signal transduction factors in cold/freezing and drought tolerance in Arabidopsis (Kim ei al. 2002. Plant J. 29(6):693-704; Franklin and Whitelam. 2007. Nat. Genet. 39(1 1 )1410- 1413; Beck et al. 2007. J. Biosci. 32(3):501 -510).
  • RNA interference RNA interference
  • Phytochrome-associated fiber elongation could occur because of phytochrome- mediated plant hormone signaling (Neff et al. 2000. Genes Dev. 3:257-271 ; Colon- Carmona et al. 2000. Plant Physiol. 124(4): 1728-1738; Stamm and Kumar. 2010. J. Exp. Bot. 61 (1 1 ):2889-2903) such as auxin (IAA), abscisic acid (ABA), gibberellic acid (GA), brassinosteroids (BR), ethylene and cytokinin, which are recognized as key factors associated with fiber development (Lee et al. 2007. Ann. Bot. 100:1391 -1401 ).
  • auxin IAA
  • ABA abscisic acid
  • GA gibberellic acid
  • BR brassinosteroids
  • ethylene and cytokinin which are recognized as key factors associated with fiber development (Lee et al. 2007. Ann. Bot. 100:1391 -1401 ).
  • Increased levels of PHYA2 and PHYC in the PHYA1 RNAi families can be the result of such a substitution because in rice plants PHYC responds to constant far red light, as PHYA does (Takano et al. 2005. Plant Cell 17:331 1 -3325; Kneissl et al. 2008. Mol. Plant. 1 (1 ):84-102) although the photosensory specificity of PHYC is similar to that of PHYB/D/E (Monte ei al., supra), which is a weak red-light sensor (Schepens ei al. 2004. Curr. Opin. Plant Biol. 7(5):564-569). Additionally, an observed ⁇ 5 to 20-fold increase in expression of PHYE/B genes in our PHYA1 RNAi plants suggests a possible overlapping of functions between cotton PHYAs and PHYE/Bs that may be specific for cotton phytochrome species.
  • RNAi of cotton PHYA 1 genes results in the suppression of targeted genes and also alters the expression level of remaining phytochromes. Observed RNAi effects in cotton, therefore, are due to both the suppression of PHYA 1 and the several fold increases in the expression of other phytochromes.
  • This alteration in the cotton phytochrome gene family expression profile results in a changed plant architecture with elongated leaf petioles and fruit branches, early flowering, early boll maturity, enhanced fiber quality and fiber yield phenotypes. These changes are stably expressed in subsequent generations and transferable from the transformed Coker 312 genotype to Upland cultivar through genetic hybridization and selection.
  • RNAi cotton plants based on RNAi of Gossypium-der ' wed phytochrome genes, will allow breeders to rapidly improve maturity, major fiber quality traits and yield. Transferred PHYA 1 RNAi construct could result in resistance to abiotic stresses in Upland cultivar. This RNAi strategy not only provides a solution to fundamental problems of conventional cotton breeding, but will also result in significant economic income from cotton production worldwide and will open a new paradigm for Upland cotton breeding.
  • a host cell containing the nucleotide sequences of the invention is a bacterial cell, in particular, an Agrobacterium tumefaciens cell.
  • selectable marker which may provide resistance to an antibiotic (kanamycin, hygromycin or methatrexate) or a herbicide (sulfonylurea, imidazolinone, or basta).
  • antibiotic kanamycin, hygromycin or methatrexate
  • herbicide sulfonylurea, imidazolinone, or basta.
  • nucleic acid molecule As used herein, the terms "nucleic acid molecule”, “nucleic acid sequence”, “polynucleotide”, “polynucleotide sequence”, “nucleic acid fragment”, “isolated nucleic acid fragment” are used interchangeably herein. These terms encompass nucleotide sequences and the like.
  • a polynucleotide may be a polymer of RNA or DNA that is single- or double-stranded and that optionally contains synthetic, non-natural or altered nucleotide bases.
  • a polynucleotide in the form of a polymer of DNA may be comprised of one or more segments of cDNA, genomic DNA, synthetic DNA, or mixtures thereof.
  • isolated polynucleotide refers to a polynucleotide that is substantially free from other nucleic acid sequences, such as other chromosomal and
  • isolated polynucleotides may contain polynucleotide sequences which may have originally existed as extrachromosomal DNA but exist as a nucleotide insertion within the isolated polynucleotide.
  • polynucleotides may be purified from a host cell in which they naturally occur.
  • nucleic acid purification methods known to skilled artisans may be used to obtain isolated polynucleotides.
  • the term also embraces recombinant polynucleotides and chemically synthesized polynucleotides.
  • recombinant refers to a nucleic acid molecule which has been obtained by manipulation of genetic material using restriction enzymes, ligases, and similar genetic engineering techniques as described by, for example, Sambrook et al. 1989. Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY or DNA Cloning: A Practical Approach, Vol. I and II (Ed. D. N. Glover), IRL Press, Oxford, 1985. "Recombinant,” as used herein, does not refer to naturally occurring genetic recombinations.
  • chimeric refers to two or more DNA molecules which are derived from different sources, strains, or species, which do not recombine under natural conditions, or to two or more DNA molecules from the same species, which are linked in a manner that does not occur in the native genome.
  • a "construct” or “chimeric gene construct” refers to a nucleic acid sequence encoding a protein, operably linked to a promoter and/or other regulatory sequences.
  • the term "express” or “expression” is defined to mean transcription alone.
  • “Altered levels” or “altered expression” refers to the production of gene product(s) in transgenic organisms in amounts or proportions that differ from that of normal or non-transformed organisms.
  • nucleic acid comprises the requisite information to guide translation of the nucleotide sequence into a specified protein.
  • the information by which a protein is encoded is specified by the use of codons.
  • a nucleic acid encoding a protein may comprise non-translated sequences (e.g., introns) within translated regions of the nucleic acid or may lack such intervening non-translated sequences (e.g., as in cDNA).
  • operably linked refers to the association of two or more nucleic acid fragments on a single nucleic acid fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of affecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • regulatory sequences refer to nucleotide sequences located upstream (5' non- coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences here may include promoters: T7 promoter, CaMV 35S promoter and sub-genomic promoters (two, on either side of the MCS), translation leader sequences, introns, and polyadenylation recognition sequences.
  • Promoter refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3' to a promoter sequence.
  • the promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers.
  • an “enhancer” is a nucleotide sequence that can stimulate promoter activity and may be an innate element of the promoter or a heterologous element inserted to enhance the level or tissue-specificity of a promoter. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from different promoters found in nature, or even comprise synthetic nucleotide segments.
  • promoters may direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental conditions. Promoters that cause a nucleic acid fragment to be expressed in most cell types at most times are commonly referred to as "constitutive promoters". New promoters of various types useful in plant cells are constantly being discovered; numerous examples may be found in the compilation by Okamuro and Goldberg. 1989. Biochemistry of Plants 15:1 -82. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, nucleic acid fragments of different lengths may have identical promoter activity.
  • RNA transcript refers to the product resulting from RNA polymerase-catalyzed transcription of a DNA sequence.
  • the primary transcript When the RNA transcript is a perfect complementary copy of the DNA sequence, it is referred to as the primary transcript or it may be an RNA sequence derived from posttranscriptional processing of the primary transcript and is referred to as the mature RNA.
  • Messenger RNA (mRNA) refers to the RNA that is without introns and that can be translated into polypeptides by the cell.
  • cDNA refers to a DNA that is complementary to and derived from an mRNA template. The cDNA can be single-stranded or converted to double stranded form using, for example, the Klenow fragment of DNA polymerase I.
  • Sense RNA refers to an RNA transcript that includes the mRNA and so can be translated into a polypeptide by the cell.
  • Antisense when used in the context of a particular nucleotide sequence, refers to the complementary strand of the reference transcription product.
  • Antisense RNA refers to an RNA transcript that is complementary to all or part of a target primary transcript or mRNA and that blocks the expression of a target gene. The complementarity of an antisense RNA may be with any part of the specific nucleotide sequence, i.e., at the 5' non-coding sequence, 3' non-coding sequence, introns, or the coding sequence.
  • “Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA, or other RNA that may not be translated but yet has an effect on cellular processes.
  • Gene suppression means any of the well-known methods for suppressing expression of protein from a gene including anti-sense suppression or RNAi
  • RNAi gene suppression In suppressing genes to provide plants with a desirable phenotype, anti- sense and RNAi gene suppression methods are preferred.
  • anti- sense regulation of gene expression in plant cells see U.S. Patent No. 5,107,065.
  • RNAi gene suppression in plants by transcription of dsRNA see U.S. Patent No. 6,506,559, U.S. Patent Application Publication No. 2002/0168707 A1 and U.S. Patent Application Serial No. 09/423,143 (see WO 98/53083), 09/127,735 (see WO 99/53050) 09/084,942 (see WO 99/61631 ), all of which are hereby incorporated by reference.
  • Suppression of a gene by RNAi can be achieved using a recombinant DNA construct having a promoter operably linked to a DNA element comprising a sense and anti-sense element of a segment of genomic DNA of the gene, e.g., a segment of at least about 23 nucleotides, more preferably about 50 to 200 nucleotides where the sense and anti-sense DNA components can be directly linked or joined by an intron or artificial DNA segment that can form a loop when the transcribed RNA hybridizes to form a hairpin structure.
  • Transformation refers to the transfer of a nucleic acid fragment into the genome of a host organism, resulting in genetically stable inheritance. Host organisms containing the transformed nucleic acid fragments are referred to as "transgenic" organisms. Examples of methods of plant transformation include Agrobacterium- mediated transformation (De Blaere et al. 1987. Meth. Enzymol. 143:277) and particle- accelerated or "gene gun” transformation technology (Klein et al. 1987. Nature (London) 327:70-73; U.S. Pat. No. 4,945,050, incorporated herein by reference). Additional transformation methods are disclosed below.
  • isolated polynucleotides of the present invention can be incorporated into recombinant constructs, typically DNA constructs, capable of introduction into and replication in a host cell.
  • a construct can be a vector that includes a replication system and sequences that are capable of transcription and translation of a polypeptide-encoding sequence in a given host cell.
  • a number of vectors suitable for stable transfection of plant cells or for the establishment of transgenic plants have been described in, e.g., Pouwels et al. 1985. Supp. 1987. Cloning Vectors: A Laboratory Manual; Weissbach and Weissbach. 1989. Methods for Plant Molecular Biology, Academic Press, New York; and Flevin et al. 1990.
  • plant expression vectors include, for example, one or more cloned plant genes under the transcriptional control of 5' and 3' regulatory sequences and a dominant selectable marker.
  • plant expression vectors also can contain a promoter regulatory region (e.g., a regulatory region controlling inducible or constitutive, environmentally- or developmentally-regulated, or cell- or tissue-specific expression), a transcription initiation start site, a ribosome binding site, an RNA processing signal, a transcription termination site, and/or a polyadenylation signal.
  • a “protein” or “polypeptide” is a chain of amino acids arranged in a specific order determined by the coding sequence in a polynucleotide encoding the polypeptide. Each protein or polypeptide has a unique function.
  • the invention includes functional polypeptides and functional fragments thereof, as well as mutants and variants having the same biological function or activity.
  • the terms "functional fragment”, “mutant” and “variant” refers to a polypeptide which possesses biological function or activity identified through a defined functional assay and associated with a particular biologic, morphologic, or phenotypic alteration in the cell.
  • Functional fragments for example, can vary in size from a polypeptide fragment as small as an epitope capable of binding an antibody molecule, to a large polypeptide capable of participating in the characteristic induction or programming of phenotypic changes within a cell.
  • a heterologous coding sequence refers to coding sequences which encode peptides or proteins, unrelated to, or, other than, the polypeptides provided above and which are not intrinsically found in the position provided in the chimeric gene construct.
  • the phytochrome genes PHYA, PHYB, PHYC, PHYD, and PHYE encoding the phytochrome proteins PHYA, PHYB, PHYC, PHYD, and PHYE can be cloned using a variety of techniques according to the invention.
  • the simplest procedure for the cloning of such genes requires the cloning of complementary DNA from viral genomic RNA, or of genomic DNA from an organism identified as producing said protein(s), and the transfer of the cloned DNA on a suitable plasmid or vector to a host organism which does not produce the protein, followed by the identification of transformed hosts to which the ability to produce the protein has been conferred.
  • the transforming protein function - conferring DNA can be cleaved into smaller fragments and the smallest which maintains the protein function -conferring ability can be further characterized.
  • Techniques suitable for cloning by homology include standard library screening by DNA hybridization or polymerase chain reaction (PCR) amplification using primers derived from conserved sequences.
  • two DNA sequences are substantially homologous when at least 80% (preferably at least 85% and most preferably 90%) of the nucleotides match over the defined length of the sequence using algorithms such as CLUSTAL or PILEUP.
  • Sequences that are substantially homologous can be identified in a Southern hybridization experiment under stringent conditions as is known in the art. See, for example, Sambrook et al., supra. Sambrook et al. describe highly stringent conditions as a hybridization temperature 5-10° C below the T m of a perfectly matched target and probe; thus, sequences that are "substantially homologous" would hybridize under such conditions.
  • substantially similar refers to nucleic acid fragments wherein changes in one or more nucleotide bases results in substitution of one or more amino acids, but do not affect the functional properties of the polypeptide encoded by the nucleotide sequence.
  • substantially similar also refers to modifications of the nucleic acid fragments of the instant invention such as deletion or insertion of nucleotides that do not substantially affect the functional properties of the resulting transcript. It is therefore understood that the invention encompasses more than the specific exemplary nucleotide or amino acid sequences and includes functional equivalents thereof.
  • a codon for the amino acid alanine, a hydrophobic amino acid may be substituted by a codon encoding another less hydrophobic residue, such as glycine, or a more hydrophobic residue, such as valine, leucine, or isoleucine.
  • a codon encoding another less hydrophobic residue such as glycine
  • a more hydrophobic residue such as valine, leucine, or isoleucine.
  • changes which result in substitution of one negatively charged residue for another such as aspartic acid for glutamic acid, or one positively charged residue for another, such as lysine for arginine, can also be expected to produce a functionally equivalent product.
  • polynucleotide that affects the level of expression of a polypeptide in a virus or in a host cell may comprise the steps of: constructing an isolated polynucleotide of the present invention or an isolated chimeric gene of the present invention; introducing the isolated polynucleotide or the isolated chimeric gene into a host cell; measuring the level of a polypeptide in the host cell containing the isolated polynucleotide; and comparing the level of a polypeptide in the host cell containing the isolated polynucleotide with the level of a polypeptide in a host cell that does not contain the isolated polynucleotide.
  • substantially similar nucleic acid fragments may also be characterized by their ability to hybridize. Estimates of such homology are provided by either DNA- DNA or DNA-RNA hybridization under conditions of stringency as is well understood by those skilled in the art (1985. Nucleic Acid Hybridization, Hames and Higgins, Eds., IRL Press, Oxford, U.K.). Stringency conditions can be adjusted to screen for moderately similar fragments, such as homologous sequences from distantly related organisms, to highly similar fragments, such as genes that duplicate functional enzymes from closely related organisms.
  • isolated sequences that encode PHYA1 polypeptides and which hybridize under stringent conditions to the sequences disclosed herein, or to fragments thereof, are encompassed by the present invention.
  • Substantially similar nucleic acid fragments of the instant invention may also be characterized by the percent identity of the amino acid sequences that they encode to the amino acid sequences disclosed herein, as determined by algorithms commonly employed by those skilled in this art.
  • Computer implementations of these mathematical algorithms can be utilized for comparison of sequences to determine sequence identity. Such implementations include, but are not limited to: CLUSTAL in the PC/Gene program (available from Intelligenetics, Mountain View, Calif.); the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Version 8 (available from Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis., USA). Alignments using these programs can be performed using the default parameters.
  • sequence identity or “identity” in the context of two nucleic acid or polypeptide sequences makes reference to the residues in the two sequences that are the same when aligned for maximum correspondence over a specified comparison window.
  • percentage of sequence identity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule.
  • percentage of sequence identity means the value determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison, and multiplying the result by 100 to yield the percentage of sequence identity.
  • reference sequence is a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset or the entirety of a specified sequence; for example, as a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • polynucleotide sequences means that a polynucleotide comprises a sequence that has at least 80% sequence identity, preferably at least 85%, more preferably at least 90%, most preferably at least 95% sequence identity compared to a reference sequence using one of the alignment programs described using standard parameters.
  • sequence identity preferably at least 85%, more preferably at least 90%, most preferably at least 95% sequence identity compared to a reference sequence using one of the alignment programs described using standard parameters.
  • amino acid sequences for these purposes normally means sequence identity of at least 80%, preferably at least 85%, more preferably at least 90%, and most preferably at least 95%.
  • optimal alignment is conducted using the homology alignment algorithm of Needleman et al. (1970. J. Mol. Biol. 48:443).
  • nucleotide sequences are substantially identical is if two molecules hybridize to each other under stringent conditions.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
  • Tm thermal melting point
  • stringent conditions encompass temperatures in the range of about 1 ° C to about 20°C, depending upon the desired degree of stringency as otherwise qualified herein.
  • a "substantial portion" of an amino acid or nucleotide sequence comprises an amino acid or a nucleotide sequence that is sufficient to afford putative identification of the protein or gene that the amino acid or nucleotide sequence comprises.
  • Amino acid and nucleotide sequences can be evaluated either manually by one skilled in the art, or by using computer-based sequence comparison and identification tools that employ algorithms such as BLAST. In general, a sequence of ten or more contiguous amino acids or thirty or more contiguous nucleotides is necessary in order to putatively identify a polypeptide or nucleic acid sequence as homologous to a known protein or gene.
  • oligonucleotide probes comprising 30 or more contiguous nucleotides may be used in sequence-dependent methods of gene identification and isolation.
  • short oligonucleotides of 12 or more nucleotides may be used as amplification primers in PCR in order to obtain a particular nucleic acid fragment comprising the primers.
  • a "substantial portion" of a nucleotide sequence comprises a nucleotide sequence that will afford specific identification and/or isolation of a nucleic acid fragment comprising the sequence.
  • the instant specification teaches amino acid and nucleotide sequences encoding polypeptides that comprise a particular plant protein. The skilled artisan, having the benefit of the sequences as reported herein, may now use all or a substantial portion of the disclosed sequences for purposes known to those skilled in this art. Thus, such a portion represents a "substantial portion" and can be used to establish
  • fragment a portion of the nucleotide sequence or a portion of the amino acid sequence and hence protein encoded thereby is intended.
  • Fragments of a nucleotide sequence may encode protein fragments that retain the biological activity of the native protein and hence have PHYA1 protein -like activity.
  • fragments of a nucleotide sequence that are useful as hybridization probes may not encode fragment proteins retaining biological activity.
  • variants substantially similar sequences are intended.
  • conservative variants include those sequences that, because of the degeneracy of the genetic code, encode the amino acid sequence of one of the PHYA1 polypeptides of the invention.
  • Naturally occurring allelic variants such as these can be identified with the use of well-known molecular biology techniques, as, for example, with polymerase chain reaction (PCR), a technique used for the amplification of specific DNA segments.
  • variants of a particular nucleotide sequence of the invention will have generally at least about 90%, preferably at least about 95% and more preferably at least about 98% sequence identity to that particular nucleotide sequence as determined by sequence alignment programs described elsewhere herein.
  • variant protein a protein derived from the native protein by deletion (so- called truncation) or addition of one or more amino acids to the N-terminal and/or C- terminal end of the native protein; deletion or addition of one or more amino acids at one or more sites in the native protein; or substitution of one or more amino acids at one or more sites in the native protein is intended.
  • variant proteins encompassed by the present invention are biologically active, that is they continue to possess the desired biological activity of the native protein. Such variants may result from, for example, genetic polymorphism or from human manipulation.
  • Biologically active variants of a native PHYA1 protein of the invention will have at least about 90%, preferably at least about 95%, and more preferably at least about 98% sequence identity to the amino acid sequence for the native protein as determined by sequence alignment programs described elsewhere herein.
  • a biologically active variant of a protein of the invention may differ from that protein by as few as 1 -15 amino acid residues, or even one amino acid residue.
  • polypeptides of the invention may be altered in various ways including amino acid substitutions, deletions, truncations, and insertions. Novel proteins having properties of interest may be created by combining elements and fragments of proteins of the present invention, as well as with other proteins. Methods for such manipulations are generally known in the art. Thus, the genes and nucleotide sequences of the invention include both the naturally occurring sequences as well as mutant forms.
  • proteins of the invention encompass naturally occurring proteins as well as variations and modified forms thereof. Such variants will continue to possess the desired PHYA1 activity. Obviously, the mutations that will be made in the DNA encoding the variant must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure.
  • transgenic includes any cell, cell line, callus, tissue, plant part, or plant the genotype of which has been altered by the presence of a heterologous nucleic acid including those transgenics initially so altered as well as those created by sexual crosses or asexual propagation from the initial transgenic.
  • the term "transgenic” as used herein does not encompass the alteration of the genome (chromosomal or extra-chromosomal) by conventional plant breeding methods or by naturally occurring events such as random cross-fertilization, non- recombinant viral infection, non-recombinant bacterial transformation, non-recombinant transposition, or spontaneous mutation.
  • cotton as used herein includes any species of the genus Gossypium which is used for commercial fiber production, preferably G. hirsutum or G. barbadense.
  • the term "plant” includes reference to whole plants, plant organs (e.g., leaves, stems, roots, etc.), seeds, plant cells, and progeny of same.
  • Parts of transgenic plants are to be understood within the scope of the invention to comprise, for example, plant cells, protoplasts, tissues, callus, embryos as well as flowers, stems, fruits, leaves, roots originating in transgenic plants or their progeny previously transformed with a DNA molecule of the invention and therefore consisting at least in part of transgenic cells, are also an object of the present invention.
  • plant cell includes, without limitation, seeds
  • the class of plants that can be used in the methods of the invention is generally as broad as the class of higher plants amenable to transformation techniques, including both monocotyledonous and dicotyledonous plants.
  • Plant materials used here in this study were the somatically regenerable cotton genotype Gossypium hirsutum line Coker 312 and its transgenic derivative lines, transformed with pHe ⁇ sgate-8 PHYA 1 vector.
  • Regenerable Coker-312 seeds (provided by Dr. Keerti Rathore, Texas A&M University, College Station, Texas, USA).
  • RNAi Coker-312 plants were used several commercially important Uzbek cotton cultivars, e.g., G. hirsutum cv. AN-Boyovut-2 for the conventional genetic hybridization experiments with RNAi Coker-312 plants.
  • RNAi vectors were transformed into the A. tumefaciens strain LBA4404 and used for plant transformation experiments.
  • the attached gene-specific primers Gos_PHYA1 attB1 -F and Gos_PHYA1 attB2-R (Table 1 ) were designed and purchased from Integrated DNA Technologies Inc. (Iowa, USA). These primer pairs specifically amplified the 213 bp PHYA 1 gene fragment (SEQ ID NO:1 ) of cotton which corresponds to a portion of the hinge region of the cotton phytochrome A genes.
  • tetraploid cottons have two different PHYA 1 gene, one acquired from the diploid D-genome ancestor and the other, from the diploid A genome ancestor (Abdurakhmonov et al. 2010, supra). These two PHYA1 genes have 99% nucleotide identity in the 213 bp RNAi portion; they differ by two single nucleotide polymorphisms at the Y (C or T) and R (G or A) positions in the sequence provided and identified by SEQ ID NO:1.
  • the specific cotton phytochrome gene fragment for PHYA 1 was amplified from cotton genomic DNA with KODHiFi, the high fidelity proof reading DNA polymerase (Novagen, USA) using non attB gene-specific primers according to manufacturer's instructions and protocol.
  • the expected gene- specific PCR products were verified using agarose gel electrophoresis.
  • the attB1 and attB2 sites were then attached to the obtained PCR products, in a second round PCR reaction with attB-flanked gene-specific primers (Table 1 ) with the purified first PCR- amplicon serving as a template. Size and correctness of obtained attB-flanked PCR products were verified using gel electrophoresis.
  • PCR products were purified with polyethylene glycol (PEG)- solution (containing 26% PEG 8000, 6.5 mM MgCI 2 and 0.6mM sodium acetate pH 5.2) to remove the remaining attB primers.
  • PEG polyethylene glycol
  • the site-specific recombination reaction with the attB site-flanked gene product and the vector were conducted as described by Helliwell et al. (supra).
  • DH5-cells (Invitrogen, USA). Cells were grown in LB-plates containing the selective antibiotic spectinomycin. Colonies were picked for further verification of correct recombination with attB-sites. Restriction analyses with Xhol (for sense orientation) and Xba (anti-sense orientation) were carried out; verified clones were selected for further RNAi vector preparation, as described by Helliwell et al.
  • GhPP2A1 F GATCCTTGTGGAGGAGTG 100 Artico
  • T 0 plantlets were transferred into soil in pots and grown in a greenhouse environment in 2008.
  • hypocotyl sections of 5-7 mm were isolated from one-week old seedlings. From these hypocotyl sections, 75% were used for transformation experiments; the remaining 25% of the sections were kept separate as a negative control.
  • the hypocotyl sections were wounded with a laboratory razor in several places and placed on medium P1 [4.31 g/L MS salt, 0.4 mg/L thiamine HCL, 100 mg/L myoinositol, 0,75 mg/L MgCI 2 , 3% glucose, 0,2% Phytagel, 5 mg/L 2 ip, 0,1 mg/L NAA, pH 5.8].
  • LBA4404 suspension (5 ⁇ ), bearing pHe ⁇ sgate-8 PHYA 1 RNAi vector, was applied onto the wounded hypocotyl sections and then incubated in the dark at 22°C for 72 hours.
  • the pHellsgate- 8::PHYA1 RNAi vector was grown in YEP medium [10g/L Bacto peptone, 5g/L NaCI, 10g/L Bacto yeast extract, pH 7.0] containing rifampicin (10 m/L) and spectinomycin (50 mg/L) antibiotics. Bacterial cultures were grown in tubes for 36 h at 26°C with 200 rpm shaking.
  • Cells from 5 tubes were pooled, harvested by centrifugation, and resuspended in 10 ml of pre-induction medium (10 g/L glucose, 14.62 g/L MES, 20 ml/L sodium phosphate buffer pH 5.6, 50ml/L 20x AB salt stock (Chilton et al. 1974. Proc. Nat. Acad. Sci. USA 71 (9):3672-3676) containing 100 ⁇ acetosyringone. For controls, 5 ⁇ sterile water was applied in place of bacterial suspension.
  • pre-induction medium 10 g/L glucose, 14.62 g/L MES, 20 ml/L sodium phosphate buffer pH 5.6, 50ml/L 20x AB salt stock (Chilton et al. 1974. Proc. Nat. Acad. Sci. USA 71 (9):3672-3676) containing 100 ⁇ acetosyringone.
  • 5 ⁇ sterile water was applied in place of bacterial suspension.
  • 3 mm callus tissues grown in selective P1 medium were transferred into new P7 medium [4.31 g/L MS salts, 0.4 mg/L thiamine HCL, 100 mg/L myo-inositol, 0,75 mg/L MgCI, 3% glucose, 0,2% Phytagel, 0.1 mg/L 2ip, 5 mg/L NAA, pH 5.8] and grown in continuous culture, sub-culturing the tissues each month. Callus tissues with less than 3 mm were kept in P1 medium for another three weeks and subsequently transferred to the P7 medium.
  • callus tissues grown on selective P7 medium were transferred to new modified medium R5, containing 4.31 g/L MS salt, 1 ml/L vitamins Gamborg solution, 1.9 g/L KN0 3, 0,75 mg/L MgCI, 3% maltose, 0,2% Phytagel).
  • the somatic embryos were generated in 12- 16 weeks in R5 medium.
  • the somatic embryos of 6-7 mm size were then transferred into modified SH1 medium [10 ml/L 100 x micronutrients, 50ml/L 50 x macronutrients, 1 ml/L vitamin B5, 5 g/L sucrose, 15 g/L bactoagar, 2 g/L phytogel] medium and incubated at dark condition for 10 days. A desiccation and root initiation process occurred during this time period.
  • Embryos were then transferred to new SH-2 medium [10 ml/L 100 x micronutrients, 50ml/L 50 x macronutrients, 1 ml/L vitamin B5, 20 g/L sucrose, 1 g/L Phytagel, and 5 g/L agar] and grown for 10 days under a 16 h photoperiod (10 ⁇ m-2 s-1 ) for the development of roots and leaves.
  • embryo plantlets were transferred to SH-3 medium [10 ml/L 100 x micronutrients, 50ml/L 50 x macronutrients, 1 ml/L vitamin B5, 20 g/L sucrose, 1 g/L Phytagel and 2.25 g/L agar] and grown in increased light (70 ⁇ m-2 s-1 ) for full development of roots and leaves. After 10 days, fully developed embryo plantlets were transferred into plastic containers with SH-3 medium resulting in the development of 4-5 leaves and additional roots.
  • SH-3 medium 10 ml/L 100 x micronutrients, 50ml/L 50 x macronutrients, 1 ml/L vitamin B5, 20 g/L sucrose, 1 g/L Phytagel and 2.25 g/L agar.
  • genomic DNAs were isolated from the frozen leaf tissues using method of Dellaporta et al. (1983. Plant Mol. Biol. Rep. 1 :19-21 ) with minor modification and optimization for frozen tissues. Prepared genomic DNAs were analyzed in 0.9% agarose electrophoresis and DNA concentrations were estimated based on Hind III digested ⁇ -phage DNA. RNAi vector-specific 35S-F/PDK-R or PDK- F/OST-R primers pairs (Table 1 ) were used to verify the positive transgenic plants.
  • Amplification reactions were performed in 50 ⁇ volumes containing 4.5 ⁇ 10 x PCR buffer with MgCI 2 , 1 ⁇ BSA, 0.5 ⁇ 25 mM of a dATP, dGTP, dTTP, and dCTP mix, 2.5 ⁇ 50 ng/ml of each reverse and forward primer, and 1 ⁇ 50 ng/ml template DNA.
  • Taq DNA polymerase 0.5 U (Sigma, USA) was added to the reaction at the annealing temperature of first cycle. Amplifications were carried out with a first denaturation at 94°C for 3 min followed by 45 cycles of 94°C for 1 min, 55°C for 1 min (annealing) and 72°C for 2 min (extension).
  • RNAi plants were evaluated for flowering time and boll maturation as well as fiber staple length
  • T 2 generation Based on preliminary flowering and fiber characteristics, individual ⁇ plants from different transformation events, were selected for subsequent T 2 generation plant evaluation. For this, 40-45 T 2 seeds from each selected PCR-positive, Ti plants were planted, germinated in small paper-soil pots under solar light conditions. When true leaves appeared, they were transplanted into the field station of the Institute of Genetics and Plant Experimental Biology, Tashkent, Uzbekistan in 2009. Forty to 45 T 2 plants, derived from each ⁇ plant (single seed decent) of different transformation event, were grown as a family with 25 non-transgenic control Coker 312 plants in standard field plot design in a two row (60 cm row spacing) plot 10 meters long.
  • the average indices for hypocotyl length, number of opened flowers and opened bolls of each field-grown T 2 RNAi family and control plants were recorded. First flowers opened were tagged with date indication to determine flowering time difference. Fiber quality traits of these field grown individual T 2 generation RNAi and control plants, including upper half mean (UHM), fiber strength (STR), micronaire (MIC), and fiber uniformity was measured using High Volume Instrumentation (HVI) at the fiber testing Center "SIFAT", Tashkent Uzbekistan. All plants were self-pollinated to produce pure T 3 generation seeds, wrapping the petals with cotton threads before flower opening.
  • UHM upper half mean
  • STR fiber strength
  • MIC micronaire
  • HVI High Volume Instrumentation
  • T 3 generation Based on field evaluations in 2009, we selected plants from two different T 2 RNAi plant families (T 2 -1_7 and T 2 -31_10) with improved cotton fiber quality, vigorous shoot and root development and early flowering phenotypes compared to control plants. In 2010, self-pollinated T 3 generation seeds from these two RNAi families were grown in the same field conditions in a 10 row, 10 meter long plot (90 cm row spacing, 0.010 ha), along with side-by-side grown 0.010 ha control plants. We measured yield by weighing the seed cotton from 600 hundred plants/0.010 ha of T 3 RNAi cotton families and control families..
  • Lint percentage the weight of 100 seeds (seed index) and lint index were measured manually and averaged from 24 individual plants of each selected T 3 RNAi plant families and control plants, taking seed cottons from six fully matured bolls per plant.
  • SI FAT fiber testing Center
  • T 3 plants from the field were dug at the flowering and boll maturation stages, roots were washed and root lengths were compared with control plants growing in the same field.
  • the statistical significance of trait differences between RNAi families and control plants were tested with nonparametric two paired sample test (Wilcoxon matched-pairs signed- rank test) using Plainstat ver. 0.2.1 (Retrieved from the Internet: ⁇ URL:. plainstat.com).
  • RNAi genotypes with fiber length of 1 .32 inch, micronaire of 4.6, fiber strength of 35.5 g/tex, and fiber uniformity 88%.
  • Non- transformed control Coker-312 plants grown in the same field had an average UHM of 1.23 inch, MIC 5.2, STR 31 g/tex, and Ul of 87%.
  • T 2 - 1_7 and T 2 -31_10 Based on T 2 phenotypic evaluation, we selected two plant families, namely T 2 - 1_7 and T 2 -31_10, with significantly improved fiber quality such as UHM (p ⁇ 0.001 ), MIC (pO.001 ), Ul (pO.02), ELO (pO.0001 ), flowering (pO.01 ), hypocotyl length
  • Lint index (Lint% x weight of 100 seeds)/seed weight%. Statistical significance of measured traits between two RNAi families (T 3 _1 -7 and T 3 _31 -10) in Wilcoxon matched-pairs signed-rank test was shown as a p ⁇ 0.05.
  • PCR reactions were performed in 12.5 ⁇ volume with the following standard program recommended by the manufacturer: 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min.
  • Each 12.5 ⁇ reaction mixture contained 6.25 ⁇ Master Mix (2x), 0.25 ⁇ (200 nM) of each primer (10 ⁇ ), 1 ⁇ (40nM) of probe (0.5 ⁇ ), 4 ⁇ of template DNA sample (0.2 pg to 20 ng), and 0.75 ⁇ sterile deionized water.
  • 2x PCR Master Mix contained No AmpErase UNG, AmpliTaq Gold DNA polymerase, deoxynucleoside triphosphates with dUTP and Taq Man reaction buffer with magnesium chloride (Applied Biosystems, Foster city, USA).
  • RNAi plant from T 3 -1_7 family have three copies while RNAi plant from T 3 -31_10 family contained 2 copies of pHellsgate-8:: PHYA1 RNAi vector in their genomes.
  • a DNA sample of Bt-cotton that has single copy of cry I transgene inserted in its genome (Table 4).
  • RNAs were isolated from leaf tissues of T 3 -generation RNAi cotton plants and non RNAi control plants using combination of protocols described by Suzuki ei al. (2001. J. Exp. Bot. 52:1575-1579) and Wu ei al. (2002. Plant Mol. Biol. Rep. 20:213-218) with minor modifications.
  • Suzuki ei al. 2001. J. Exp. Bot. 52:1575-1579
  • Wu ei al. 2002. Plant Mol. Biol. Rep. 20:213-218
  • RNAi influence in the expression of PHYA 1 genes as well as other cotton phytochrome genes we utilized quantitative real-time PCR (qRT-PCR) method using SYBR-green based amplicon detection.
  • qRT-PCR quantitative real-time PCR
  • Control and RNAi plants were grown in the same greenhouse environment and under same light conditions.
  • 2-fold dilution series (1 , 2 up to 128x) of 1 :15 diluted cDNA from control Coker 312 was created.
  • Average C t values from at least, 2 were plotted against log of starting amount to obtain standard curves.
  • Relative quantity of the target genes then was calculated by dividing normalized quantity of target gene expression in RNAi plants by the normalized quantity of the same gene expression in control plant, used as a calibrator. Coefficient of variation was calculated from coefficient of variation estimates for GhPP2A1 and each of the phytochromes.
  • RNA pellet was washed by 75% (v/v) ethanol, air dried and dissolved in sterile DEPC-treated water.
  • RNAase free rDNAase I (Ambion, USA) according manufacturer's protocol and re-purified with additional purification steps using acid phenokchloroform (5: 1 ; Ambion, USA) and ethanol precipitation. The concentration of total RNA samples were quantified using spectrophotometer
  • the first strand cDNAs were synthesized from ⁇ 2 ⁇ g total RNAs using Avian RT cDNA kit (Sigma, USA) with random nonamer primers according manufactures protocol.
  • the first strand cDNA synthesized were diluted 1 : 15 with sterile water and used in RT and qRT- PCR analyses.
  • RT-PCR reactions were carried out with intron specific primer pairs (A1341 F/R; Table 1 ; Cronn et al. 2002. Am. J. Bot.
  • RT-PCR reactions were carried out using RT-PCR kit (Sigma, USA) according manufacture's protocol. Samples were subjected to qPCR only if they fail to amplify intronic primer pairs, but not the
  • 2x SYBR GREEN PCR master mix contained No AmpErase UNG, AmpliTaq Gold DNA polymerase, deoxynucleoside triphosphates with dUTP and SYBR Green reaction buffer with magnesium chloride (Applied Biosystems, Foster city, USA). Post real-time PCR dissociation curves were constructed for each primer pairs used to evaluate the primer-dimers, genomic DNA contaminations and misannealing issues. Problematic reaction wells were omitted from the analysis. Analysis of qRT-PCR amplifications was conducted using 7500 System SDS v1 .4 Software(Applied Biosystems, Foster city, USA).
  • Transformation and integration of the pHellsgate-8::P/-/Yy3 ⁇ 4i RNAi vector affected the expression of several cotton phytochrome genes (Table 1 ; Table 7).
  • PHYA 1 gene expression was suppressed by 70% in T 3 -1_7, and 24% in T 3 -31_10 family.
  • the PHYA 1 RNAi construct did not suppress the expression of other phytochrome genes tested with the exception of a slight (10%) down-regulation of PHYB in T 3 -31_10. Rather, we detected 2 to 20-fold overexpression of PHYA2, PHYB (in T 3 -1_7 sample only), PHYC, and PHYE genes in both RNAi plant samples. Intriguingly, high level of overexpression of other phytochromes genes tested was more evident in T 3 -1_7 sample, where the deeper suppression of PHYA 1 gene expression was detected compared to the other RNAi sample T 3 -31_10 (Table 7).
  • T 3 -1_7 1.3 (4.5) 21.37 1.78 1.77 ⁇ 0.23
  • RNAi Coker 312 When we crossed T 0 -generation RNAi Coker 312 plants with four commercial varieties of Uzbekistan cotton (Namangan-77, AN-Boyovut-2, C-6524 and Tashkent-6) and evaluated Fi and F 2 generation hybrids from these crosses we found notably changed plant architecture with elongated petioles and fruiting branches, greater mean number of flowers and bolls and the plants flowered and matured an average of 5-10 days early compared to control plants (original variety) grown side-by-side in the same field conditions. We also observed more anthocyanin pigmentation in RNAi hybrids (Figure 8).

Abstract

Selon l'invention, l'amélioration de la qualité de fibre de cultivars en milieu sec (Glossypium hirsutum), tout en conservant une maturité et une productivité précoces, est un problème fondamental dans la culture de coton classique. Les phytochromes jouent un rôle fondamental dans le développement végétal, la floraison et la longueur des fibres de coton. Des ARN ciblés de gènes PHYA 1 dans l'expression inhibée du coton de PHYA 1 et/ou PHYB, conduisant à la surexpression des gènes PHYA2/B/C/E restants. Cette expression modifiée a induit un nombre de phénotypes associés à un phytochrome, comprenant une longueur et une masse de racine accrues, un pigment anthocyanine accru, le développement de pousses vigoureuses et la croissance végétative, la floraison précoce, la maturité précoce des capsules, la longueur de fibres accrue et le rendement en graines coton accru en comparaison à des plantes témoins. Ces phénotypes ARNi ont été hérités et exprimés de façon stable à travers quatre générations (T0-3) et ont pu être transférés à partir de plantes RNAi Coker-312 à des cultivars en milieu sec par l'intermédiaire d'hybridation classique. Ceci a pour effet dans la culture de coton en milieu sec qui peut offrir un nouveau paradigme dans la culture de coton conduisant à un développement de cultivars en milieu sec productifs, à maturation précoce ayant une longueur de fibre et une résistance de fibre accrues.
PCT/US2013/027801 2012-02-28 2013-02-26 L'arni de phya 1 du coton améliore la qualité des fibres, l'élongation des racines, la floraison, la maturité et le potentiel de rendement dans glossypium hirsutum l WO2013130470A1 (fr)

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RU2014138704A RU2665804C2 (ru) 2012-02-28 2013-02-26 Рнк-интерференция гена phya1 хлопчатника, повышающая качество волокон, удлинение корня, цветение, созревание и потенциал урожайности у хлопчатника мохнатого (gossypium hirsutum l.)

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Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5945579A (en) * 1995-10-05 1999-08-31 The University Of Leicester Modification of crop plant architecture to enhance yield by causing proximity-conditional dwarfing to control shade avoidance reactions
US20030204872A1 (en) * 2002-04-30 2003-10-30 Kim Jeong Il Nucleic acid molecules encoding hyperactive mutant phytochromes and uses thereof
US20060260009A1 (en) * 2005-05-16 2006-11-16 Jeong-Il Kim Nucleic acid molecule encoding bathochromic phytochrome and use thereof
US7566815B2 (en) * 2001-09-03 2009-07-28 National Institute of Agrobiological Sciences and National Agricultural and Bio-oriented Research Organization Control of plant flowering time by regulation of phytochrome c expression

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
GB0029795D0 (en) * 2000-12-06 2001-01-17 Syngenta Ltd Recombinant plants
CN1813067A (zh) * 2003-04-29 2006-08-02 康乃尔研究基金会有限公司 植物光敏素a基因的转基因表达
RU2262834C1 (ru) * 2004-02-02 2005-10-27 ФГУП Российский федеральный ядерный центр - Всероссийский научно-исследовательский институт экспериментальной физики - ФГУП - РФЯЦ - ВНИИЭФ Способ светоимпульсной обработки растений
KR100698954B1 (ko) * 2004-02-27 2007-03-26 제노마인(주) 피토크롬과 상호 결합 작용하는 신규 단백질의 용도

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5945579A (en) * 1995-10-05 1999-08-31 The University Of Leicester Modification of crop plant architecture to enhance yield by causing proximity-conditional dwarfing to control shade avoidance reactions
US7566815B2 (en) * 2001-09-03 2009-07-28 National Institute of Agrobiological Sciences and National Agricultural and Bio-oriented Research Organization Control of plant flowering time by regulation of phytochrome c expression
US20030204872A1 (en) * 2002-04-30 2003-10-30 Kim Jeong Il Nucleic acid molecules encoding hyperactive mutant phytochromes and uses thereof
US20060260009A1 (en) * 2005-05-16 2006-11-16 Jeong-Il Kim Nucleic acid molecule encoding bathochromic phytochrome and use thereof

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ABDURAKHMONOV, IBROKHIM Y. ET AL.: "Duplication, divergence and persistence in the Phytochrome photoreceptor gene family of cottons (Gossypium spp.)", BMC PLANT BIOLOGY, vol. 10, no. 119, 20 June 2010 (2010-06-20) *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113337520A (zh) * 2021-03-31 2021-09-03 甘肃农业大学 陆地棉GhA0749和GhD0744转录因子及其调控开花方面的应用
CN114480421A (zh) * 2022-01-27 2022-05-13 南京农业大学 水稻光敏色素蛋白突变型基因OsPHYA及其应用
CN114480421B (zh) * 2022-01-27 2023-05-26 南京农业大学 水稻光敏色素蛋白突变型基因OsPHYA及其应用
CN115443903A (zh) * 2022-09-30 2022-12-09 石家庄市农林科学研究院 一种适合农机采集的早熟棉花育种结合方法
CN116716316A (zh) * 2023-07-17 2023-09-08 甘肃农业大学 一个响应环境温度调控陆地棉开花的关键基因GhPIF7
CN116716316B (zh) * 2023-07-17 2024-03-15 甘肃农业大学 一个响应环境温度调控陆地棉开花的关键基因GhPIF7

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