WO2015055045A1 - Protéine de résistance à la pourriture des tiges de maïs provoquée par gibberella et gène codant pour celle-ci ainsi que leurs utilisations - Google Patents

Protéine de résistance à la pourriture des tiges de maïs provoquée par gibberella et gène codant pour celle-ci ainsi que leurs utilisations Download PDF

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WO2015055045A1
WO2015055045A1 PCT/CN2014/084415 CN2014084415W WO2015055045A1 WO 2015055045 A1 WO2015055045 A1 WO 2015055045A1 CN 2014084415 W CN2014084415 W CN 2014084415W WO 2015055045 A1 WO2015055045 A1 WO 2015055045A1
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plant
sequence
seq
protein
stalk rot
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PCT/CN2014/084415
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WO2015055045A9 (fr
Inventor
Mingliang Xu
Qin Yang
Chao Wang
Dongfeng Zhang
Yanling Guo
Jianrong Ye
Yongjie LIU
Guangming Yin
Yipu LI
Nan Zhang
Shaojiang Chen
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China Agricultural University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8279—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance
    • C12N15/8282—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for biotic stress resistance, pathogen resistance, disease resistance for fungal resistance

Definitions

  • the present invention relates to a Gibberella stalk rot resistance protein and a gene encoding the same as well as their use.
  • Stalk rot of corn is a common soil-borne disease in various corn production regions all over the world.
  • a corn plant infected with this disease enters the milk stage, the whole plant wilt, and leaves turn grayish green and later tan.
  • the plant exhibits rotting of the roots and stalk base, leading to early browning, impaired grain filling and reduced thousand-kernel weight.
  • stalk rot disease also causes corn lodging and affects the harvest. Furthermore, the grain quality is affected to a certain degree.
  • the pathogenic cause of stalk rot of corn complicated. Regarding the causal pathogens and predominant races, there are different reports both in China and abroad.
  • the main causal pathogens in China are Fusarium graminearum Schw. and Pythium inflatum Matth.
  • stalk rot of corn has become the number one disease in corn production in China and occurs in the Jiangsu, Henan, Shandong, Sichuan, and Guangxi provinces.
  • the disease in Sichuan province and Xinxiang region of Henan province are severe.
  • the heavy autumn waterlogging in the Yellow-Huai river region causes severe stalk rot disease, resulting in great yield loss in the region.
  • the incidence of disease is generally 10%-20%, and may reach 70% or more in particular years and regions, resulting in yield reduction of 25%-30%, or in the worst cases, no harvest at all.
  • Since the stalk rot of corn is complex and is influenced greatly by the environment, it is very difficult to accurately evaluate resistance to the disease. Hence, it is difficult to conduct a study on the genetic basis of the corn stalk rot disease. Considering that the economic and ecological importance of corn stalk rot disease, development of disease-resistant varieties provides an effective means for controlling the disease.
  • the protein has an amino acid sequence selected from the group consisting of:
  • sequence of SEQ ID NO: 1 consists of 238 amino acids.
  • the protein according to the present invention is associated with resistance to Gibberella stalk rot.
  • the protein is derived from the Zea mays L. inbred 1145 which is highly resistant to Gibberella stalk rot.
  • a tag sequence may be attached to the amino- or the carboxyl-end of the protein (Table 1). Table 1.
  • the ZmCCT protein of the present disclosure can be synthesized artificially or may be expressed biologically by synthesizing its coding gene.
  • the protein of the present disclosure may also be obtained by deleting or adding codons for one or more amino acids from a coding gene of the ZmCCT protein (such as, the DNA sequence of SEQ ID NO: 2), and/or by introducing one or more missense mutations into the coding gene of the ZmCCT protein, and/or by linking a tag coding sequence from Table 1 to the 5' and/or 3' terminus of the coding gene of the ZmCCT protein.
  • nucleic acid molecules for coding the protein or its variants are within the scope of the invention.
  • the invention provides an isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
  • the above nucleic acid molecule encodes a protein exhibiting a biological activity against Gibberella stalk rot.
  • the gene for coding the above ZmCCT protein is designated as ZmCCT.
  • the above stringent hybridization condition may consist of hybridization in a solution of 6xSSC, 0.5% SDS under 65°C, followed by washing with 2*SSC, 0.1% SDS and l xSSC, 0.1% SDS, once for each.
  • the nucleic acid molecules may be a DNA molecule, such as a cDNA, a genomic DNA or a recombinant DNA.
  • the nucleic acid molecules may also be a RNA molecule, such as an mRNA, or an hnRNA.
  • the sequence of SEQ ID NO: 2 consists of 717 nucleotides with an open reading frame (ORF) of nucleotides 1-717 from 5' end for encoding the protein of SEQ ID NO: 1 (ZmCCT protein).
  • the sequence of SEQ ID NO: 3 consists of 8,147 nucleotides with a promoter sequence at nucleotides 1-5,135, a genomic sequence for the ZmCCT gene at nucleotides 5,136-7,682 (including a first exon sequence at nucleotides 5,136-5,609, a second exon sequence at nucleotides 7,440-7,682 and an intron sequence at nucleotides 5,610-7,439), and an untranslated region at nucleotides 7,683-8,147.
  • a DNA sequence comprising the whole sequence of SEQ ID NO: 3 are also within the scope of the invention.
  • the present invention provides an isolated recombinant DNA molecule comprising the ZmCCT gene or a variant thereof, or a fragment thereof, which exhibits the biological activity against Gibberella stalk rot.
  • a recombinant vector, an expression cassette, a transgenic cell line or recombinant bacteria comprising the recombinant nucleic acid molecule or a DNA fragment thereof is also within the scope of the invention.
  • the present invention provides a recombinant vector comprising the nucleic acid molecule of the disclosure.
  • the recombinant vector may be a recombinant expression vector or a recombinant cloning vector.
  • the recombinant expression vector is a carrier for expressing the ZmCCT gene and may be constructed using an existing expression vector.
  • the recombinant expression vector may comprise a 3' -untranslated region of an exogenous gene, in other words, a polyA signal or any DNA fragment involved in the mRNA processing or gene expression.
  • any one of enhanced, constitutive, tissue-specific or inducible promoters may be incorporated before the transcription initiation sequence. They may be used alone or in combination with other promoters.
  • an enhancer may be used, including a translation enhancer or a transcription enhancer.
  • the plant expression vector may be further comprise a marker gene, for example, a gene encoding an enzyme that causes a color change when expressed in a plant in the presence of a substrate (such as GUS) or a gene of a luminescent agent (such as GFP gene, luciferase gene and the like), an antibiotic-resistant marker (such as gentamycin marker, kanamycin marker and the like), or a chemical-resistant marker gene (such as a herbicide-resistant gene).
  • a marker gene for example, a gene encoding an enzyme that causes a color change when expressed in a plant in the presence of a substrate (such as GUS) or a gene of a luminescent agent (such as GFP gene, luciferase gene and the like), an antibiotic-resistant marker (such as gentamycin marker, kanamycin marker and the like), or a chemical-resistant marker gene (such as a herbicide-resistant gene).
  • Transgenic plants can also be screened directly for stress tolerance without the use of any selectable
  • the promoter for initiating the transcription of the ZmCCT gene in the recombinant expression vector comprises a nucleotide sequence as set forth in nucleotides 1 -5,135 of SEQ ID NO: 3.
  • the recombinant expression vector is obtained by inserting the DNA sequence as set forth in SEQ ID NO: 3 in the multiple cloning sites in the plasmid pCAMBIA3301.
  • the multiple cloning site is a Sacl site.
  • the expression cassette comprises a promoter for initiating the expression of the ZmCCT gene, the ZmCXT gene and a transcription termination sequence.
  • the invention provides a recombinant DNA construct comprising the nucleic acid molecule of the disclosure.
  • the invention provides a host cell comprising the protein, the gene, the DNA molecule, and/or the recombinant expression vector or expression cassette as above.
  • the invention provides a transgenic plant comprising the protein, the gene, the DNA molecule, the recombinant expression vector or expression cassette, and/or the host cell as above.
  • the transgenic plant is a monocotyledon or dicotyledon plant. In another embodiment, the transgenic plant is a Gramineae plant. In yet another embodiment, the transgenic plant is a corn plant.
  • the present disclosure also contemplates the use of the above protein, the gene, the DNA molecule, the recombinant expression vector or expression cassette, and/or the host cell in 1) regulating the resistance of a plant against Gibberella stalk rot, or 2) breeding a plant variety having an improved resistance against Gibberella stalk rot.
  • said regulating the resistance against Gibberella stalk rot is to improve the resistance of a plant against Gibberella stalk rot.
  • said breeding a plant variety having an improved resistance against Gibberella stalk rot may comprise selecting a plant with a higher expression of the ZmCCT protein or selecting a plant with increased resistance to Gibberella stalk rot as a parent for breeding.
  • the invention provides a method for producing a transgenic plant, comprising introducing the above gene into an acceptor plant to obtain the transgenic plant, wherein the transgenic plant exhibits the resistance against Gibberella stalk rot.
  • the disclosure provides a method for producing a transgenic plant, comprising introducing the above gene into an acceptor plant to obtain the transgenic plant, and selecting the transgenic plant with improved resistance to against Gibberella stalk rot
  • the plant may be a monocotyledon or dicotyledon plant.
  • the plant is a Gramineae plant.
  • the plant is a corn plant.
  • the invention provides a method for breeding a plant for an improved resistance against Gibberella stalk rot, comprising introducing a gene encoding the ZmCCT protein into an acceptor plant to obtain the transgenic plant which exhibits improved resistance against Gibberella stalk rot when comparing with the acceptor plant without the transgene.
  • the ZmCCT protein is expressed in the transgenic plant (a positive plant identified via PCR) at a higher level than that in the acceptor plant without the transgene.
  • the recombinant DNA encoding the ZmCCT protein comprises a nucleotide sequence selected from the group consisting of: (1) a sequence set forth in SEQ ID NO: 2, or a complementary sequence thereof;
  • the nucleotide sequence encodes a protein exhibiting resistance against Gibberella stalk rot.
  • the above said stringent hybridization condition may consist of hybridization in a solution of 6xSSC, 0.5% SDS under 65°C, followed by washing with 2xSSC, 0.1% SDS and 1 SSC, 0.1% SDS, once for each.
  • the ZmCCT gene is introduced into the acceptor plant via a recombinant expression vector comprising the DNA sequence as shown in SEQ ID NO: 3.
  • the invention provides use of the above protein, gene, DNA molecule, and/or recombinant expression vector or expression cassette in improving the resistance of a plant against Gibberella stalk rot.
  • the plant may be a monocotyledon or dicotyledon plant.
  • the plant is a Gramineae plant.
  • the plant is a corn plant.
  • the improvement in resistance against Gibberella stalk rot manifests as an increase in the percentage of Gibberella stalk rot resistant plants in progeny of the ZmCXT-transgenic plants ⁇ ZmCCT transgene positive plants identified via PCR). In one embodiment, the improvement in resistance against Gibberella stalk rot manifests as an increase in the percentage of Gibberella stalk rot resistant plants in progeny of the ZmCCT transgene positive plants identified via PCR in relative to the acceptor plant not comprising the transgene.
  • the plant is a monocotyledon or dicotyledon plant.
  • the plant is a Gramineae plant.
  • the plant is a corn plant, for example, the Zea mays L. variety Hi II.
  • the present disclosure also contemplates a primer pair for the amplification of full length ZmCCT gene or a fragment thereof, and/or a probe for identification of a transgenic event.
  • the ZmCCT gene according to the invention can increase the percentage of Gibberella stalk rot resistant plants in progeny of ZmCCT- transgenic plants and further improves the resistance to Gibberella stalk rot of ZmCCT gene positive progeny in comparison with negative or acceptor plants not comprising the transgene.
  • Figure 1 is an electrophoretogram showing the identification through PCR of ZmCCT- transgenic T 0 corn plants.
  • Lane 1 is D2000 marker with bands in the order from bottom to top of 100 bp, 250 bp, 500 bp, lkb, and 2 kb; lanes 2, 3, 4, 5, 8, 9, 10, 12, 13, 14, 15, 17, 18, 19, 21, 23, 24, 25, and 26 represent negative transgenic individuals; and lanes 7, 11, 16, 20, and 22 represent positive transgenic individuals (with the band of 518 bp in size).
  • Figure 2 shows the manifestations of the ZmCCT transgene positive plants (P) and negative plants (N) for resistance to the disease, wherein A represents plant surface and B represents a split stem.
  • P represents a positive transgenic plant
  • N represents a negative transgenic plant.
  • Figure 3 is an electrophoretogram showing the identification through qRT-PCR of ZmCXT-transgenic To corn plants.
  • Lane 1 is D2000 marker with a upper and lower bands of 250 bp and 100 bp, respectively; lanes 2, 3, 4, 5, 6, and 7 represent positive transgenic individuals (P); and lanes 8, 9, 10, 11, 12, and 13 represent negative transgenic individuals (N); 28 and 32 indicate the number of PCR cycles, and GADPH is an internal reference gene used as a control.
  • Figure 4 shows the results of statistical analysis on the resistance percentage of Ti plants, wherein P represents a positive transgenic plant and N represents a negative transgenic plant.
  • Figure 5 shows the results of statistical analysis on the resistance percentage of T 2 /T 3 plants, wherein P represents a positive transgenic plant and N represents a negative transgenic plant.
  • isolated refers to at least partially separating a molecule from other molecules normally associated with it in its native or natural state.
  • isolated gene or “isolated DNA molecule” refers to a gene or DNA molecule that is at least partially separated from the nucleic acids which normally flank the gene or DNA molecule in its native or natural state.
  • isolated genes or DNA molecules fused to regulatory or coding sequences with which they are not nomially associated are herein considered isolated. Such molecules are considered isolated even when integrated into the chromosome of a host cell or present in a nucleic acid solution with other DNA molecules.
  • stringency conditions are those described by Sambrook et al., 1989, and by Haymes et al., In: Nucleic Acid Hybridization, A Practical Approach, IRL Press, Washington, DC (1985).
  • Appropriate stringency conditions which promote DNA hybridization for example, 6.0 x sodium chloride/sodium citrate (SSC) at about 45°C, followed by a wash of 2.0 x SSC at 50°C, are known to those skilled in the art or can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • the salt concentration in the wash step can be selected from a low stringency of about 2.0 x SSC at 50°C to a high stringency of about 0.2 x SSC at 50°C.
  • the temperature in the wash step can be increased from low stringency conditions at room temperature, about 22°C, to high stringency conditions at about 65°C. Both temperature and salt may be varied, or either the temperature or the salt concentration may be held constant while the other variable is changed.
  • moderately stringent conditions are at about 2.0 x SSC and about 65 °C.
  • the stringent hybridization condition may consist of hybridization with a solution of 6xSSC, 0.5% SDS at 65°C, followed by washing with 2xSSC, 0.1% SDS and l SSC, 0.1% SDS, once for each.
  • the gene of the present invention comprises the nucleic acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or a fragment thereof.
  • the gene of the present invention shares at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and at least 99% sequence identity with the nucleic acid sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or a fragment thereof.
  • the gene of the present invention shares 95% 96%, 97%, 98%, and 99% sequence identity with the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or a fragment thereof.
  • sequence identity refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g. nucleotides or amino acids.
  • An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e. the entire reference sequence or a smaller defined part of the reference sequence. "Percent identity” is the identity fraction times 100.
  • the gene of the present invention also comprises a variant sequence derived from the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3 via deletion, substitution, insertion and/or addition of one or more codons.
  • a gene mutation refers to a sudden and inheritable variation of a genomic DNA molecule.
  • a gene mutation means a change of base pair composition or arrangement sequence in structure.
  • the generation of a gene mutation may be spontaneous or induced.
  • the methods for artificially inducing a gene mutation include physical factors (such as ⁇ ray, x ray, UV, neutron beam and the like), chemical factors (such as an alkylation agent, a base analogue, an antibiotic and the like) and biological factors (such as certain viruses, bacteria, etc.).
  • a specific variation may be introduced at a specified site in a DNA molecule using a recombinant DNA technique so as to carry out a site-directed mutagenesis.
  • Those skilled in the art may use any of these well-known mutagenesis methods to obtain a variant sequence of the sequence set forth in SEQ ID NO: 2 or SEQ ID NO: 3 or a fragment thereof comprising the deletion, substitution, insertion and/or addition of one or more nucleotides or codons.
  • recombinant refers to a form of DNA and/or protein and/or an organism that would not normally be found in nature and as such was created by human intervention. Such human intervention may produce a recombinant DNA molecule and/or a recombinant plant.
  • a "recombinant DNA molecule” is a DNA molecule comprising a combination of DNA molecules that would not naturally occur together and is the result of human intervention, e.g., a DNA molecule that comprises a combination of at least two DNA molecules heterologous to each other, and/or a DNA molecule that is artificially synthesized and comprises a polynucleotide sequence that deviates from the polynucleotide sequence that would normally exist in nature, and/or a DNA molecule that comprises a transgene artificially incorporated into a host cell's genomic DNA and the associated flanking DNA of the host cell's genome.
  • a recombinant DNA molecule is a DNA molecule described herein resulting from operably linking a heterologous regulatory sequence (such as a promoter, an intron or a 3' untranslated region) to the coding sequence of ZmCCT, or resulting from the insertion of the transgene into the Arabidopsis thaliana, corn or rice genome, which may ultimately result in the expression of a recombinant RNA and/or protein molecule in that organism.
  • a heterologous regulatory sequence such as a promoter, an intron or a 3' untranslated region
  • transgene refers to a polynucleotide molecule artificially incorporated into a host cell's genome. Such transgene may be heterologous to the host cell.
  • transgenic plant refers to a plant comprising such a transgene.
  • DNA sequence refers to the sequence of nucleotides of a DNA molecule, usually presented from the 5' (upstream) end to the 3' (downstream) end.
  • a “probe” is an isolated nucleic acid to which is attached a conventional detectable label or reporter molecule, e.g. , a radioactive isotope, ligand, chemiluminescent agent, or enzyme. Such a probe is complementary to a strand of a target nucleic acid, in the case of the present invention, such as to a strand of SEQ ID NO: 2 or SEQ ID NO: 3. Probes according to the present invention include not only deoxyribonucleic or ribonucleic acids but also polyamides and other probe materials that bind specifically to a target DNA sequence and such binding can be used to detect the presence of that target DNA sequence.
  • Primer pairs of the present invention refer to their use for amplification of a target nucleic acid sequence, e.g., by the polymerase chain reaction (PCR) or other conventional nucleic acid amplification methods.
  • Probes and primers are generally 11 nucleotides or more in length, preferably 18 nucleotides or more, more preferably 24 nucleotides or more, and most preferably 30 nucleotides or more.
  • probes and primers hybridize specifically to a target sequence under high stringency hybridization conditions.
  • probes and primers according to the present invention have complete sequence similarity with the target sequence, although probes differing from the target sequence and that retain the ability to hybridize to target sequences may be designed by conventional methods.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge, MA).
  • Primers and probes based on the nucleotide sequences disclosed herein can be used to identify a transgenic plant and/or confirm the transformation of a plant, to screen/select transgenic plants with stalk rot resistance and to breed plants for talk rot resistance. Conventional methods known in the art can be used for these purposes.
  • DNA molecules, or a fragment thereof can also be obtained by other techniques such as by directly synthesizing the fragment by chemical method, such as using an automated oligonucleotide synthesizer.
  • the inventors isolated the ZmCCT gene from the Zea mays L. inbred 1145 and determined its sequence.
  • the inventors After constructing the recombinant expression vector pCAMBIA3301-ZmCCr, the inventors obtained transformed corn plants via Agrobacterium-mediated transformation and further assessed the resistance to Gibberella stalk rot. The results show that the transgene ZmCCT significantly improve the resistance of corn plant to Gibberella stalk rot.
  • polypeptide comprises a plurality of consecutive polymerized amino acid residues e.g., at least about 15 consecutive polymerized amino acid residues.
  • the polypeptide optionally comprises modified amino acid residues, naturally occurring amino acid residues not encoded by a codon, non-naturally occurring amino acid residues.
  • protein refers to a series of amino acids, oligopeptide, peptide, polypeptide or portions thereof whether naturally occurring or synthetic.
  • an "isolated protein”, whether a naturally occurring or a recombinant polypeptide, is more enriched in (or out of) a cell than the polypeptide in its natural state in a wild-type cell, e.g., more than about 5% enriched, more than about 10% enriched, or more than about 20%, or more than about 50%, or more, enriched, i.e., alternatively denoted: 105%, 110%, 120%, 150% or more, enriched relative to wild type standardized at 100%.
  • the isolated polypeptide is separated from other cellular components with which it is typically associated, e.g., by any of the various protein purification methods.
  • amino acids within these various classes include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine.
  • conserveed substitutes for an amino acid within a native protein or polypeptide can be selected from other members of the group to which the naturally occurring amino acid belongs.
  • a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine
  • a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine
  • a group of amino acids having amide-containing side chains is asparagine and glutamine
  • a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan
  • a group of amino acids having basic side chains is lysine, arginine, and histidine
  • a group of amino acids having sulfur-containing side 30 chains is cysteine and methionine.
  • Naturally conservative amino acids substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine.
  • a further aspect of the disclosure includes proteins that differ in one or more amino acids from those of a described protein sequence as a result of deletion, substitution, or insertion of one or more amino acids in a native sequence.
  • the ZmCCT gene according to the invention may be introduced into a plant by various commonly used plant transformation methods. For example, by Agrobacterium-mediated method, gene gun method, PEG-mediated method, ultrasonic method, ovary injection method, pollen-tube pathway method and the like.
  • Agrobacterium is a gram-negative bacterium commonly found in soil and can chemotactically infect injured sites of most dicotyledonous plants under natural conditions to induce the generation of crown gall and fairy root.
  • Cells of Agrobacterium tumifaciens and Agrobacterium rhizogenes comprise a Ti plasmid or a Ri plasmid, respectively, which has a T-DNA segment.
  • Agrobacterium After entering plant cells by infecting plant wounds, Agrobacterium can insert the T-DNA into the plant genome.
  • Agrobacterium represents a natural plant genetic transformation system.
  • the Agrobacterium-mediated transformation could only be used in dicotyledonous plants. In recent years, it has also been used in some monocotyledon plants (especially in rice and corn).
  • the gene gun-mediated transformation method uses gunpowder explosion or a high-pressure gas to accelerate (this accelerating device is called as a gene gun) micro-projectiles, thereby delivering the high-speed micro-projectiles coated with a target gene into an intact plant tissue and cell, then a transgenic plant is regenerated through a cell and tissue culture technique.
  • the transgene positive plants are screened out, i.e. the transgenic plants.
  • one main advantage of the gene gun transformation is that it is not restricted by plant genotype or species.
  • the construction of the transformation/expression vector plasmid is relatively simple.
  • the gene gun transformation is one of the broadly used methods in transgene studies.
  • a recombinant expression vector Prior to introduction of the transgene into a recipient plant, it is necessary to construct a recombinant expression vector.
  • the recombinant vector for introducing a recombinant DNA molecule into a plant according to the invention.
  • various plasmids for example, a Ti plasmid, an artificial chromosome, a plant virus including a RNA virus, a single-strain DNA virus and the like may be used as the recombinant expression vector of the invention.
  • a number of common vector constructing techniques are known in the art, for example, a conventional vector constructing technique through enzymatic cleave of a DNA plasmid.
  • a DNA plasmid is cleaved by a restriction enzyme and then ligated with a target gene with a ligase to form an expression vector.
  • This technique has a limitation in that it requires multiple cloning intermediates and thus has a low efficiency.
  • the Gateway technology developed by the Invitrogen Inc. does not need a restriction enzyme and a ligase.
  • An entry vector is first constructed, and then a target recombinant DNA sequence is repeatedly introduced into different destination vectors for expression. This method has the characteristics of simplicity, speediness; high cloning efficiency and high specificity (while the positions of reading frame and gene remain unchanged).
  • a recombinant DNA vector can be designed that either does not contain a selection marker, or is a binary or ternary expression vector comprising two or three T-DNAs.
  • Zea mays L. inbred line 1145 obtained from China National Crop Seed Storage
  • Plasmid pCAMBIA3301 as reported in Huixia Shou, Reid G. Palmer, and Kan Wang. Irreproducibility of the Soybean Pollen-Tube Pathway Transformation Procedure. Plant Molecular Biology Reporter, 2002(20): 325-334.
  • Agrobacterium strain LBA4404 Agrobacterium tumefaciens LBA4404 Electro-Cells from Clontech Company, lot No. 91 15.
  • Fusarium graminearum Schw. as reported in Qin Yang, Guangming Yin, Yanling Guo, et al. A major QTL for resistance to Gibberella stalk rot in corn. Theor. Appl. Genet., (2010)121 : 673-687.
  • Example 1 Obtaining and characterizing the ZmCCT gene I. Acquisition of full length cDNA sequence of the ZmCCT gene Using the method of Zuoheng SONG, et al. (Study on the effects of health cultivation measurements on the controlling of corn stalk rot, Liaoning Agricultural Sciences, 1993, No. 5), corn plants of the inbred line 1145 were inoculated with the conidia of Fusarium graminearum Schw. at the tasseling stage. Leaves were collected 16h after inoculation. Total RNA was extracted using the TriZol reagent provided by Invitrogen Company.
  • 5 '-RACE product and 3 '-RACE product were amplified with the gene specific primers - 5'-RACE primer (5'GSPB) and 3'-RACE primer (3'GSPA) (see Table 2) as well as general primers provided in the kit by following the instructions of the kit, and then sequenced.
  • 5 '-untranslated sequence and 3 '-untranslated sequence of the obtained target ZmCCT gene were removed to obtain a full length cDNA sequence of the ZmCCT gene which has the nucleotide sequence as set forth in SEQ ID NO: 2.
  • Primer 1 5*-ATGTCGTCGGGGCCAGCAGC-3' (corresponding to nucleotides 1-20 in SEQ ID NO: 2)
  • Primer 2 5'-TTGCCAAGGTAACCGAATGA-3' (a complementary sequence corresponding to nucleotides 698-717 of SEQ ID NO: 2)
  • amplification was performed with the above primer 1 and primer 2 and the amplified products were detected by electrophoresis on a 1% agarose gel. The results showed that a fragment of about 720 bp was obtained through PCR amplification. After recovering and purifying, this product was ligated into the pEASY-Tl vector (Beijing TransGen Biotech Ltd.) for sequencing and confirmation.
  • the sequencing result showed that the PCR amplification product was identical to the sequence of SEQ ID NO: 2 obtained by the above, that is, the full length cDNA sequence of the ZmCCT gene.
  • This sequence encodes a protein as set forth in SEQ ID NO: 1 which is designated as ZmCCT.
  • Zuohen Corn plants of the inbred line 1145 which is highly resistant to Gibberella stalk rot, were inoculated with the conidia of Fusarium graminearum Schw. at tasseling stage according to the method of Zuoheng SONG et al. (Study about effects of health cultivation measurements on the controlling of corn stalk rot, Liaoning Agricultural Sciences, 1993, No. 5). Leaves were collected 16h after inoculation. The genomic DNA was extracted using SDS alkaline lysis method.
  • nucleotides 5,136-7,682 of SEQ ID NO: 3 wherein nucleotides 5,136-5,609 represented a sequence of a first exon, nucleotides 5,610-7,439 represented a sequence of an intron, and nucleotides 7,440-7,682 represented a sequence of a second exon.
  • genomic region sequence containing the genomic DNA sequence of ZmCCT gene having restriction site Sad at both ends, i.e. "GAGCTC+SEQ ID NO: 3+ GAGCTC” was prepared, which was designated as DNA fragment A.
  • the DNA fragment A was ligated into the pEASY-Tl vector (Beijing TransGen Biotech Ltd.) resulting in a recombinant plasmid, designated as pEASY-Zm CCT.
  • nucleotides 1-5,135 of SEQ ID NO: 3 represent a promoter sequence
  • nucleotides 5,136-7,682 represent the genomic sequence of ZmCCT gene (nucleotides 5,136-5,609 represent a sequence of a first exon
  • nucleotides 5,610-7,439 represent a sequence of an intron
  • nucleotides 7,440-7,682 represent a sequence of a second exon
  • nucleotides 7,683-8,147 represent an untranslated region.
  • the recombinant vector pEASY-ZmCCT obtained in the step III of Example 1 was treated with restriction endonuclease Sad and the target fragment (a genomic region comprising the genomic sequence of ZmCCT gene, about 8. IK) was recovered.
  • This target fragment was inserted into the backbone sequence of pCAMBIA3301 vector digested with Sad, resulting in the recombinant plasmid.
  • the obtained recombinant plasmid was digested with Sad and subjected to electrophoresis. A band of about 8.1Kb was recovered and subjected to sequencing. The result confirmed that the fragment inserted at the Sad site has the sequence of SEQ ID NO: 3.
  • the recombinant vector was therefore, designated as pCAMBIA3301 -ZmCCT.
  • the promoter for initiating the transcription of the genomic sequence of ZmCCT gene consisted of nucleotides 1-5,135 of SEQ ID NO: 3.
  • the recombinant expression vector pCAMBIA3301 -ZmCCT constructed in the above step I was introduced into the Agrobacterium strain LBA4404 following the procedure as below: (1) adding 5 ⁇ of pCAMBIA3301-ZmCCr plasmid DNA at a concentration of
  • Agrobacterium bacteria After transformation, recombinant Agrobacterium bacteria were identified through PCR reactions with a primer pair consisting of primer 3 and primer 4.
  • the Agrobacterium bacteria LBA4404 identified as having the genomic region of ZmCCT as set forth in SEQ ID NO: 3 (the target band in the PCR is of about 8.1 kb) were designated as LBA4404/pCAMBIA3301 -ZmCCT.
  • An Agrobacterium control transformed with a pCAMBIA3301 empty vector was provided and was designated as LBA4404/pCAMBIA3301.
  • Primer 3 5 '-GAGCTCTTGTTGCGACTTGT-3 ' (corresponding to nucleotides 1-20 in SEQ ID NO: 3)
  • Primer 4 5'-GAGCTCGACAAACAGTACAT-3' (a complementary sequence corresponding to nucleotides 8,128-8,147 of SEQ ID NO: 3)
  • the recombinant Agrobacterium LBA4404/pCAMBIA3301 -ZmCCT (or the empty vector control LBA4404/pCAMBIA3301) obtained above was used to transform plants of the Zea mays L. variety Hi II.
  • Corn calli were dipped in a suspension of induced recombinant Agrobacterium cells to obtain a transformed callus. Following transformation, corn calli were cultured on media containing an herbicide to regenerate transgenic plants, i.e. corn plants transformed with the pCAMBIA3301-Z/nCC7Or corn plants transformed with the empty vector pC AMBI A3301.
  • the regenerated transgenic corn plants (To) were further characterized by PCR to screen out ZmCCT positive transgenic plants. Genomic DNA was extracted from the transgenic corn plants and was used as a template for PCR reaction to identify the plants with incorporated pCAMBIA3301 -ZmCCT, wherein the genomic region of ZmCCT as set forth in SEQ ID NO: 3 and the pCAMBIA3301 vector sequence itself were used as the targets for PCR amplification with the primer pair (LBCCT FP/RP). In addition, the plants of non-transformed parent Zea mays L. variety Hi II were provided as a control.
  • LBCCT FP 5'-TAGCTAGCTCCACCACAGCA-3' (corresponding to nucleotides 7,693-7,712 of SEQ ID NO: 3);
  • LBCCT RP 5 ' -TGTGGAATTGTGAGCGGATA-3 ' (corresponding to a sequence on the pCAMBIA3301 vector).
  • Results from PCR characterization of the corn plants transformed with pCAMBIA3301-ZmCC showed that 5 transgenic events exhibited the target band with the expected size of 518 bp, wherein the 5 T 0 positive transgenic plants were designated as Y3-1, Y3-14, Y3-18, Y3-23, and Y3-25, respectively.
  • the 5 To ZmCCT transgenic plants Y3-1 , Y3-14, Y3-18, Y3-23, and Y3-25 obtained from the above Step II were self-crossed to obtain 5 transgenic T ! populations. These T] populations were planted in an experimental plot in Shangzhuang, Beijing. 137 plants were cultivated for each transgenic T 1 population. Each individual plant was assessed for resistance against Gibberella stalk rot, and for the presence/absence of the Zm CCT- transgene. The experiment was repeated three times and the results were averaged.
  • the specific procedure was as below: (1) Genotype analysis and determination of expression level of the ZmCCT gene a) Genotype analysis
  • the progeny plants can be divided into two categories: those positive for the transgene (P) and those negative for the transgene (N).
  • a qRT-PCR reaction was performed following the instructions provided in the kit to determine the expression level of the ZmCCT gene.
  • Reverse primer 5 ' -GACGACTGATCTACCGGCAT-3 ' (a complementary sequence corresponding to nucleotides 553-572 of SEQ ID NO: 2)
  • Reaction procedure 94 ° C 3 min, 94 ° C 30s, 60 ° C 30s, 72 ° C 30s, for 28 or 32 cycles; 72 ° C 10 min, maintaining at 5 ° C .
  • the amplification primers for the Internal reference gene were 5 '-ATCAACGGCTTCGGAAGGAT-3 ' and 5 ' -CCGTGGACGGTGTCGTACTT-3 ' .
  • the regenerated corn plants transformed with the empty vector pCAMBIA3301 as identified in Step II were used as an empty vector control (CK), and non-transgenic parent acceptor plants from Zea mays L. variety Hi II were provided as a parent control (WT).
  • CK empty vector control
  • WT parent control
  • step a) percentage of resistant plant was calculated for the ZmCCT transgene positive plant population (P) and the ZmCCT transgene negative plant population (N) identified in step a) as follows: about 45 days after artificial inoculation of Fusarium graminearum Schw. , the stalks of the inoculated corn plants were split for assessment. Plants with hollow stalk bases and roots, and displaying rot were characterized as diseased plants, whereas the plants with intact and normal stalk bases and roots were characterized as resistant plants ( Figure 2). The percentage of resistant plants was calculated for the ZmCCT transgene positive plant population (P) and the ZmCCT transgene negative plant population (N). Corn plants transformed with the empty vector pCAMBIA3301 as identified in
  • Step II were used as an empty vector control (CK), and non-transgenic parent acceptor plants from Zea mays L. variety Hi II were used as a parent control (WT).
  • T 2 populations were obtained; and by self-crossing the T 2 plants identified as transgenic positive according to the above genotype analysis, T 3 populations were obtained.
  • the genotype analysis and resistance trait assessment were conducted on the T 2 and T 3 populations following the same procedures.
  • the positive transgenic plants (P) had a much higher expression level of the ZmCCT gene than the negative transgenic plants (N).
  • the expression level of the ZmCCT gene was substantially identical to that of the negative transgenic plants (N).
  • the percentage of resistant plant increased significantly for the positive plants (P) of Y3-1 , Y3-18, Y3-23, and Y3-25 populations when compared with the negative plants (N), wherein the percentage of resistant plant increased by 35% for the positive plants (P) of Y3-1 population, by 17% for the positive plants (P) of Y3-18 population, by 25% for the positive plants (P) of Y3-23 population, and by 9% for the positive plants (P) of Y3-25 population, respectively, when compared with their corresponding negative plants (N) of the same population.
  • T 2 positive transgenic plants from Y3-23 population were selected for self-crossing to obtain T 3 populations.

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Abstract

La présente invention concerne une protéine résistante à la pourriture des tiges de maïs provoquée par Gibberella et un gène codant pour celle-ci ainsi que leurs utilisations. Ladite protéine est (a) une protéine comprenant la séquence d'acides aminés de la SEQ ID No. ： 1; ou (b) une protéine comprenant une séquence d'acides aminés dérivée de la SEQ ID No. ： 1 en introduisant une ou plusieurs substitutions et/ou délétions et/ou additions d'acides aminés. La protéine présente une résistance contre la pourriture des tiges de maïs provoquée par Gibberella. La protéine et le gène codant pour la protéine selon la présente invention peuvent améliorer sensiblement la résistance d'une plante contre la pourriture des tiges provoquée par Gibberella.
PCT/CN2014/084415 2013-10-14 2014-08-14 Protéine de résistance à la pourriture des tiges de maïs provoquée par gibberella et gène codant pour celle-ci ainsi que leurs utilisations WO2015055045A1 (fr)

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107354215A (zh) * 2017-08-04 2017-11-17 中国农业大学 一种玉米分子辅助育种方法
CN113061565A (zh) * 2021-04-12 2021-07-02 东北农业大学 一种禾谷镰孢分生孢子快速形成方法

Families Citing this family (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107325161B (zh) * 2016-04-29 2020-04-07 中国农业大学 一种与耐低氮胁迫和高盐胁迫相关的蛋白及其编码基因与应用
CN107653262B (zh) * 2017-11-03 2019-09-27 中国农业大学 ZmCCT9在调控玉米开花期性状中的应用
CN112410347A (zh) * 2019-08-21 2021-02-26 中国农业大学 玉米ZmHsf21基因及其应用
CN112126658A (zh) * 2020-09-30 2020-12-25 四川轻化工大学 植物过表达荧光素酶报告基因重组载体及构建方法、应用

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6169234B1 (en) * 1999-03-01 2001-01-02 Pioneer Hi-Bred International, Inc. Hybrid maize plant and seed 36B08

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6169234B1 (en) * 1999-03-01 2001-01-02 Pioneer Hi-Bred International, Inc. Hybrid maize plant and seed 36B08

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE GENBANK 29 September 2013 (2013-09-29), YANG, Q. ET AL., accession no. F575628 *
DATABASE GENBANK 29 September 2013 (2013-09-29), YANG, Q. ET AL., accession no. GW42769 *
YANG, Q. ET AL.: "A major QTL for resistance to Gibberella stalk rot in maize", THEORETICAL AND APPLIED GENETICS, vol. 4, no. 121, 31 August 2010 (2010-08-31), pages 673 - 687 *
ZHANG, D.F. ET AL.: "Fine-mapping of qRfg2, a QTL for resistance to Gibberella stalk rot in maize", THEORETICAL AND APPLIED GENETICS, vol. 124, no. 3, 29 February 2012 (2012-02-29), pages 585 - 596 *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107354215A (zh) * 2017-08-04 2017-11-17 中国农业大学 一种玉米分子辅助育种方法
CN113061565A (zh) * 2021-04-12 2021-07-02 东北农业大学 一种禾谷镰孢分生孢子快速形成方法
CN113061565B (zh) * 2021-04-12 2023-03-24 东北农业大学 一种禾谷镰孢分生孢子快速形成方法

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