WO2009129743A1 - Gif1启动子及其应用 - Google Patents

Gif1启动子及其应用 Download PDF

Info

Publication number
WO2009129743A1
WO2009129743A1 PCT/CN2009/071419 CN2009071419W WO2009129743A1 WO 2009129743 A1 WO2009129743 A1 WO 2009129743A1 CN 2009071419 W CN2009071419 W CN 2009071419W WO 2009129743 A1 WO2009129743 A1 WO 2009129743A1
Authority
WO
WIPO (PCT)
Prior art keywords
gene
plant
tissue
promoter
interest
Prior art date
Application number
PCT/CN2009/071419
Other languages
English (en)
French (fr)
Inventor
何祖华
王二涛
李群
Original Assignee
中国科学院上海生命科学研究院
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 中国科学院上海生命科学研究院 filed Critical 中国科学院上海生命科学研究院
Publication of WO2009129743A1 publication Critical patent/WO2009129743A1/zh

Links

Classifications

    • 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
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8227—Root-specific
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216—Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/8223—Vegetative tissue-specific promoters
    • C12N15/8226—Stem-specific, e.g. including tubers, beets
    • 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
    • C12N15/8222—Developmentally regulated expression systems, tissue, organ specific, temporal or spatial regulation
    • C12N15/823—Reproductive tissue-specific promoters
    • C12N15/8234—Seed-specific, e.g. embryo, endosperm

Definitions

  • the invention belongs to the field of botany and genetic engineering.
  • the present invention relates to novel plant (preferably crop) tissue-specific expression promoters, and to the use of said promoters, particularly in genetic engineering for initiating specific expression of a gene of interest in plant tissues. . Background technique
  • the research on crop yield improvement mainly focuses on the following aspects: 1. Increasing the source of crops, ie enhancing the photosynthesis of crops; 2. Increasing the size of crop stocks; 3. Increasing the transport of crop photosynthetic products from sources to banks. ability.
  • the roots of plants play an important role in their growth, nutrient absorption, energy transfer and so on. Improving the ability to absorb, transport, and grow crop roots helps to improve overall crop growth and quality.
  • the internodes of plants are the hub of material and energy transportation, and also play a role in the support of plants.
  • tissue-specific transformation of these plants is a useful means of improving their quality.
  • Tissue-specific expression promoters can drive expression of a target gene in a particular tissue or cell without affecting the physiological activities of other tissues of the plant.
  • an isolated polynucleotide selected from the group consisting of:
  • nucleotide sequence consisting of the sequence represented by 1-2379 of SEQ ID NO: 3; (2) having the nucleotide sequence shown by 1-2379 of SEQ ID NO: 3 and capable of initiating the purpose a nucleotide sequence specifically expressed by a tissue in a plant;
  • nucleotide sequence capable of hybridizing to a nucleotide sequence defined by (1), (2) or (3) under stringent conditions and capable of initiating tissue-specific expression of a target gene in a plant;
  • nucleotide sequence defined by (1), (2) or (3) and capable of initiating tissue specificity in plants Expressed nucleotide sequence;
  • the sequence of the polynucleotide is shown as 1-2379 of SEQ ID NO: 3.
  • the plant is a crop, preferably a gramineous plant, and more preferably the grass crop is selected from the group consisting of: rice, wheat, barley, corn, or sorghum.
  • the gene of interest is selected from the group consisting of a crop grain filling protein having the amino acid sequence of SEQ ID NO: 2, and a gene associated with transport and accumulation of crop starch.
  • the gene of interest is a foreign gene.
  • the gene of interest is a structural gene.
  • the gene of interest is a gene associated with the quality, yield, resistance or metabolism of the crop kernel.
  • the gene of interest is a crop grain filling protein having the amino acid sequence of SEQ ID NO: 2.
  • the tissue that specifically expresses the gene of interest is a seed, a root and/or an internode, ie one of the seed, the root, and the internode, two or three.
  • the tissue that specifically expresses the gene of interest is a seed.
  • a polynucleotide of the invention as a promoter for initiating plant tissue-specific expression of a gene of interest.
  • the plant is a crop, preferably a gramineous plant, more preferably the grass
  • the plant is selected from the group consisting of: rice, wheat, barley, corn, or sorghum.
  • the tissue specifically expressed by the gene of interest is a seed, a root and/or an internode, preferably a seed.
  • a vector comprising the polynucleotide of the invention described above as a promoter element.
  • the vector further comprises a gene of interest operably linked to the nucleic acid.
  • the gene of interest is selected from the group consisting of a crop grain filling protein having the amino acid sequence of SEQ ID NO: 2, and a gene associated with transport and accumulation of crop starch.
  • the gene of interest is located downstream of the polynucleotide and is less than 1000 bp from the promoter, preferably less than 500 bp, more preferably less than 100 bp, and most preferably less than 50 bp.
  • a genetically engineered host cell comprising the vector of the invention described above; or a polynucleotide of the invention in which the exogenous source of the invention is integrated into the genome.
  • a method of producing a transgenic plant, the tissue of the transgenic plant specifically expressing a gene of interest comprising:
  • the plant is a crop.
  • the tissue specifically expressed by the gene of interest is a seed, a root and/or an internode, preferably a seed.
  • a transgenic plant is provided.
  • the transgenic plant is made using the foregoing method of the invention.
  • the transgenic plant is a polynucleotide comprising the aforementioned polynucleotide of the present invention or before the present invention
  • the transgenic plant is a crop, more preferably a gramineous plant, and more preferably the gramineous plant is selected from the group consisting of: rice, wheat, barley, corn, or sorghum.
  • the transgenic plant is a crop, more preferably a gramineous plant, and more preferably the gramineous plant is selected from the group consisting of: rice, wheat, barley, corn, or sorghum.
  • Figure 1 shows the sequence of the GIF1 promoter in a preferred embodiment of the invention.
  • FIG. 2 shows the tissue specificity of the GUS gene under the action of the promoter of the present invention. among them,
  • Elongated internodes seeds 2 days after flowering; seeds 4 days after flowering; seeds 6 days after flowering; seeds 10 days after flowering; seeds 15 days after flowering;
  • FIG. 3 shows the expression of the GIF1 gene in different parts of plants under the action of the promoter of the present invention.
  • FIG. 3A Expression of the GIF1 gene in mutant and wild-type seeds.
  • FIG. 3B Expression of GIF1 gene in rice seedlings, leaves, roots, internodes and ears.
  • R roots of rice seedlings
  • L rice leaves
  • YL rice seedling leaves
  • I rice internodes
  • P rice tassel
  • ubi-1 ubiquitin gene
  • Figure 3C Expression of the GIF1 gene at different times after flowering (number refers to the number of days after flowering). detailed description
  • the inventors have extensively and intensively studied for the first time to isolate a promoter capable of directing tissue-specific expression of a target gene in a crop from a plant grain filling gene (GIF1, whose sequence is shown in SEQ ID NO: 3).
  • the promoter was derived from the GIF1 gene (6 ⁇ 7, Grain Incomple te Filling ⁇ ) ⁇ ] I region, so the inventors named it the GIF1 promoter.
  • the promoter is particularly useful for site-by-point improvement of plant quality.
  • plant includes, but is not limited to, crops, other plants of economic value or utility, and the like.
  • grasses includes, but is not limited to, grasses. More preferably, the grasses include, but are not limited to, rice, wheat, barley, corn, sorghum, and the like.
  • isolated means that the substance is separated from its original environment (if it is a natural substance, the original environment is the natural environment).
  • the polynucleotides and polypeptides in the natural state in living cells are not isolated and purified, but the same polynucleotide or polypeptide is separated and purified, such as from other substances existing in the natural state. .
  • operably linked refers to the spatial arrangement of the functionality of two or more nucleic acid regions or nucleic acid sequences.
  • the promoter region is placed at a specific position relative to the nucleic acid sequence of the gene of interest such that transcription of the nucleic acid sequence is directed by the promoter region such that the promoter region is "operably linked" to the nucleic acid sequence.
  • promoter or “promoter region (domain;)” refers to a nucleic acid sequence that is normally present upstream (5' end) of the coding sequence of the gene of interest and is capable of directing transcription of the nucleic acid sequence into mRNA.
  • the promoter or promoter region provides a recognition site for RNA polymerase and other factors necessary for proper initiation of transcription.
  • the promoter or promoter region includes a variant of a promoter which is obtained by inserting or deleting a regulatory region, performing random or site-directed mutagenesis or the like.
  • tissue-specific promoters are also known as “organ-specific promoters”. Under the regulation of such promoters, genes are often expressed only in certain specific organs or tissues and exhibit developmental regulation.
  • tissue-specific promoter is a tissue-specific expression promoter in a plant, which is selected from the group consisting of a grain, a root or an internode.
  • the promoter is considered to be tissue or organ specific.
  • exogenous or “heterologous” refers to the relationship between two or more nucleic acid or protein sequences from different sources. For example, if the combination of a promoter and a gene sequence of interest is generally not naturally occurring, the promoter is foreign to the gene of interest. A particular sequence is “exogenous” to the cell or organism into which it is inserted.
  • genes of interest refers to a gene that can be expressed by a promoter of the present invention. Suitable genes of interest include, but are not limited to, improved crop grain quality, yield, trait or metabolic related genes (eg, crop grain filling proteins encoding the amino acid sequence of SEQ ID NO: 2), and genes involved in crop starch transport and accumulation. . Plant tissue-specific expression promoter
  • the present invention provides a polynucleotide (promoter) specifically expressed in a plant tissue, the nucleic acid having a sequence selected from the group consisting of:
  • nucleotide sequence having a nucleotide sequence represented by 1-2379 in SEQ ID NO: 3 and capable of initiating tissue-specific expression of a gene of interest in a plant;
  • nucleotide sequence capable of hybridizing to a nucleotide sequence defined by (1), (2) or (3) under stringent conditions and capable of initiating tissue-specific expression of a target gene in a plant;
  • nucleotide sequence defined by (1), (2) or (3) and capable of initiating tissue specificity in plants Expressed nucleotide sequence;
  • sequence of the polynucleotide is shown as 1-2379 of SEQ ID NO: 3.
  • the plant is a crop, preferably a gramineous plant, including but not limited to rice, corn, wheat, barley, or sorghum.
  • the tissue that specifically expresses the gene of interest is a seed, a root and/or an internode, preferably a seed.
  • a cis-acting element such as an enhancer can also be added to the promoter by a genetic engineering method to further regulate gene expression.
  • Hybridization of polynucleotides is a technique well known to those skilled in the art, and the hybridization characteristics of a particular pair of nucleic acids indicate their similarity or identity. Accordingly, the present invention also relates to hybridization to the aforementioned specified nucleotide sequence and having at least 50%, preferably at least 70%, more preferably at least 80% (e.g., 85%, 90%, 95%, 96%, 97%, 98%, or 99%) identical nucleotides.
  • the invention particularly relates to polynucleotides that hybridize to the polynucleotides of the invention under stringent conditions.
  • Stringent conditions means: (1) hybridization and elution at lower ionic strength and higher temperature, such as 0.2 X SSC, 0.1% SDS, 60 °C ; or (2) hybridization a denaturant such as 50% (v/v) formamide, 0.1% calf serum/0. l%Ficol l, 42 °C, etc.; or (3) at least the identity between the two sequences is at least Hybridization occurs at 50%, preferably 60% or more, 65% or more, 70% or more, 75% or more, 80% or more, 85% or more, or 90% or more, and more preferably 95% or more.
  • the hybridizable polynucleotide also has a function of directing tissue-specific expression of the gene of interest in plants.
  • the present invention also encompasses nucleic acids having 50% or more (preferably 60% or more, 70% or more, 80% or more, more preferably 90% or more, and most preferably 95% or more) identity with any of the promoter sequences of the present invention.
  • the nucleic acid also has the function of directing tissue-specific expression of the gene of interest in plants. "Identity” refers to the level of similarity between two or more nucleic acids (i.e., sequence homology, similarity, or identity) as a percentage of the same position.
  • the ⁇ -glucosidase (GUS) gene can be specifically expressed in the grains, roots or internodes of grasses such as rice, wheat, barley, maize, and sorghum.
  • GUS ⁇ -glucosidase
  • the promoter of the present invention is a tissue or organ specific promoter. The promoter is particularly useful for the quality of a well-directed plant.
  • ⁇ -Glucosidase catalyzes the cleavage of a series of ⁇ -glucosides to produce chromophores or fluorescent substances.
  • the GUS activity can be quantified and spatially localized by spectrophotometer, fluorometer or histochemistry.
  • the GUS gene has been widely used as a reporter gene for transgenic plants, bacteria and fungi, and in particular, it can be used to study specific cell and tissue sites for expression of foreign genes.
  • the tissue-specific promoter of the invention has important application value in theoretical research and crop improvement. These promoters can be applied to label specific tissues, direct the expression of specific functional genes in specific tissues, and apply to growth and development studies and targeted improvements in specific tissues.
  • the promoter of the present invention is particularly useful for improving the quality or phenotype of plants, especially crop kernels (e.g., rice kernels). target gene
  • the promoter of the present invention may be operably linked to a gene of interest which may be foreign (heterologous) relative to the promoter.
  • the nucleic acid sequence of the gene of interest is not particularly limited (e.g., a structural nucleic acid sequence), and the gene of interest preferably encodes a protein having a specific function, such as some proteins having important properties or functions in agriculture or plant improvement. .
  • Suitable genes of interest include, but are not limited to, genes that improve crop quality, traits, or metabolism.
  • the gene of interest is selected from the group consisting of: a gene associated with the quality, yield, resistance or metabolism of the crop kernel (eg, Such as the crop filling protein gene (GIF1 gene), and genes related to the transport and accumulation of crop starch.
  • the promoter of the present invention may also be operably linked to a modified gene sequence of interest which is exogenous (heterologous) relative to the promoter.
  • the gene of interest can be modified to produce a variety of desirable properties.
  • the gene of interest can be modified to increase the amount of essential amino acids, increase translation of amino acid sequences, alter post-translational modifications (such as phosphorylation sites), transport translation products out of the cell, improve protein stability, insert or delete Cell signal, etc.
  • promoters and genes of interest can be designed to downregulate specific genes. This is typically accomplished by attaching a promoter to a sequence of genes of interest that is directed in antisense orientation. Those of ordinary skill in the art are familiar with such antisense techniques. Any nucleic acid sequence can be modulated in this manner.
  • Vector and host cell
  • the recombinant vector comprises a promoter of the invention comprising a multiple cloning site or at least one cleavage site downstream of the promoter.
  • the gene of interest is ligated into a suitable multiple cloning site or restriction site to operably link the gene of interest to the promoter.
  • the recombinant vector includes (from the 5' to 3' direction;): a promoter that directs transcription of the gene of interest, and a gene of interest.
  • the recombinant vector may also include a 3' transcriptional terminator, a 3' polynucleotideization signal, other non-translated nucleic acid sequences, a transport and targeting nucleic acid sequence, a resistance selection marker, an enhancer or an operator.
  • recombinant expression vector refers to a bacterial plasmid, bacteriophage, yeast plasmid, plant cell virus, mammalian cell virus or other vector well known in the art.
  • any plasmid and vector can be used as long as it is capable of replication and stabilization in the host.
  • expression vectors containing the promoter and/or gene sequences of the invention can be constructed using Methods well known to those skilled in the art. These methods include in vitro recombinant DNA techniques, DNA synthesis techniques, in vivo recombination techniques, and the like.
  • the expression vector also includes a ribosome binding site for translation initiation and a transcription terminator.
  • the expression vector preferably comprises one or more selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green Fluorescent protein (GFP) and the like.
  • selectable marker genes to provide phenotypic traits for selection of transformed host cells, such as dihydrofolate reductase, neomycin resistance, hygromycin resistance, and green Fluorescent protein (GFP) and the like.
  • the recombinant vector may contain one or more other promoters in addition to the promoter of the present invention.
  • Such other promoters are, for example, tissue-specific, constitutive or inducible.
  • tissue-specific, constitutive or inducible For example, mannosine Enzymes of Cauliflower Mosaic Virus 19S and 35S (CaMV19S CaMV35S), Enhanced CaMV, Tobacco RB7 and the like.
  • a vector comprising the appropriate promoter and gene of interest described above can be used to transform a suitable host cell to enable expression of the protein.
  • the host cell can be a prokaryotic cell, such as a bacterial cell; or a lower eukaryotic cell, such as a yeast cell; or a higher eukaryotic cell, such as a plant cell.
  • a prokaryotic cell such as a bacterial cell
  • a lower eukaryotic cell such as a yeast cell
  • a higher eukaryotic cell such as a plant cell.
  • Representative examples are: Escherichia coli, Streptomyces, Agrobacterium; fungal cells such as yeast; plant cells, and the like. It will be apparent to one of ordinary skill in the art how to select the appropriate vector and host cell.
  • Transformation of host cells with recombinant DNA can be carried out using conventional techniques well known to those skilled in the art.
  • the host is a prokaryote such as E. coli
  • competent cells capable of absorbing DNA can be harvested after the exponential growth phase and treated by the CaCl 2 method, and the procedures used are well known in the art.
  • Another method is to use MgCl 2 .
  • Conversion can also be carried out by electroporation if desired.
  • the host is a eukaryote, the following DNA transfection methods can be used: calcium phosphate coprecipitation, conventional mechanical methods such as microinjection, electroporation, liposome packaging, and the like.
  • the transformed plants can also be subjected to methods such as Agrobacterium transformation or gene gun transformation, such as leaf disc method, immature embryo transformation method, flower bud soaking method and the like.
  • methods such as Agrobacterium transformation or gene gun transformation, such as leaf disc method, immature embryo transformation method, flower bud soaking method and the like.
  • plants can be regenerated by conventional methods to obtain transgenic plants.
  • the fusion gene can be introduced into plants by a leaf disc transformation-regeneration procedure (as described by Horsch et al. (1985) Science 227: 1229).
  • Other transformation methods can also be employed, such as the PEG method, the gene gun method, and the electrophoresis method (Horsch et al., 1984, Scienc 223:496, Barton et al., 1983, Cell 32: 1033, Liu et al .: Acta Phytophysiol Sin, 21 : 195-205), which is also within the scope of the invention.
  • these methods have the disadvantages of complicated operation, multiple copy and incompleteness of foreign gene insertion, and a high proportion of sterile plants.
  • a method for preparing a transgenic plant is: transferring a binary vector carrying a promoter and a gene of interest (both operably linked) into Agrobacterium, and Agrobacterium further integrates a vector fragment containing a promoter and a gene of interest Go to the chromosome of the plant.
  • the transgenic recipient plants involved are, for example, rice, wheat, and the like.
  • the present inventors constructed a promoter of the GIF1 gene and cloned the ⁇ -glucuronidase gene (GUS), and transformed the rice ZH1 1 , and observed that the GIF1 promoter initiates the GUS reporter gene and the GIF1 gene. Tissue-specific expression.
  • GUS ⁇ -glucuronidase gene
  • the present inventors discovered a rice mutant from the rice mutant 1 1 (ZH11) mutagenic mutant library, and the seed filling of the mutant was more seriously affected, and the vegetative growth did not change compared with the wild type, but The grain filling is seriously affected, the seed 1000-grain weight is reduced by 15-30%, and the rice quality is deteriorated.
  • the inventors named it ggif (grain incomplete fi ing ing 1), indicating that the GIF1 gene is an important control of yield and quality by grouting. Gene.
  • the present inventors obtained a gene-targeted population by crossing the gif l mutant and Jane 97. Use
  • BSA Breast Segregant Analys is
  • the genomic DNA sequence of wild-type GIF1 can be obtained by sequencing and functional verification by SEQ ID NO: 3 (including the promoter); the sequence of the GIF1 coding region (cDNA sequence) can be found in SEQ ID NO: 1 by fine mapping of GIF1.
  • the sequence of the GIF1 protein is shown in SEQ ID NO: 2.
  • the present inventors based on the already cloned GIF1 gene, at NCBI (http://www.ncbi.nlm.nih.gov/) The database searches for the genomic region of the gene, which is predicted by bio-software (http://www.cbs.dtu.dk/services/Promoter/), and finally A 2379 bp was selected as the promoter sequence.
  • NCBI http://www.ncbi.nlm.nih.gov/
  • the database searches for the genomic region of the gene, which is predicted by bio-software (http://www.cbs.dtu.dk/services/Promoter/), and finally A 2379 bp was selected as the promoter sequence.
  • Example 3 Vector construction and Agrobacterium transformation
  • the present inventors constructed a promoter of the GIF1 gene and cloned the ⁇ -glucuronidase gene (GUS), and transformed the rice ZH11 to observe the tissue-specific expression of the GUS reporter gene and the GIF1 gene by the GIF1 promoter.
  • GUS ⁇ -glucuronidase gene
  • the promoter of GIF1 gene was obtained by PCR (the sequence is shown in Figure 1), and the vector ⁇ ⁇ (purchased from Clonetech with GUS) was used, followed by EcoR I, Hind I II enzyme. The fragment was ligated, and the fragment was ligated into PCAMBIA1300 with the same restriction enzyme to obtain a corresponding clone of pCAMBIA1300+ promoter + GUS (for tissue coloration method, see Jeferenson, RA (1987) Plant Mol Biol Rep).
  • Fig. 2A-K The results are shown in Fig. 2A-K. It can be seen that the GUS gene is specifically expressed in the roots, internodes, and microtubule bundles of the dorsal abdomen of the seeds.
  • mRNA is extracted from the wild-type or mutant plants by RT-PCR, and GIF1 is separately amplified by RT-PCR, and the obtained amplification product is subjected to agarose gel electrophoresis.
  • Gif-F cccgccggcgacgagcaccacat ( SEQ ID NO: 6 )
  • Gif-R ccgccggcctgaacaccctgaaga (SEQ ID NO: 7)
  • the primers used to amplify ubi-1 are:
  • ubi-l-F gacggacgcaccctggctgactac (SEQ ID NO: 8)
  • Ubi-1 -R tgctgccaattaccatataccacgac (SEQ ID NO: 9).
  • Figures 3A-C The results are shown in Figures 3A-C, wherein Figure 3A is the result of RT-PCR detection of mutant gift and wild-type mRNA; Figure 3B is the result of RT-PCR detection of GIF1 mRNA in different tissues of rice; Figure 3C is a different period (DAF) RT-PCR results of GIF1 mRNA in rice panicle tissues. All documents mentioned in the present application are incorporated herein by reference as if each document was Individual references are cited as references. In addition, it is to be understood that various modifications and changes may be made by those skilled in the art in the form of the appended claims.

Landscapes

  • Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biomedical Technology (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Biotechnology (AREA)
  • General Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Molecular Biology (AREA)
  • Cell Biology (AREA)
  • Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Microbiology (AREA)
  • Developmental Biology & Embryology (AREA)
  • Pregnancy & Childbirth (AREA)
  • Reproductive Health (AREA)
  • Botany (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Medicinal Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Breeding Of Plants And Reproduction By Means Of Culturing (AREA)

Description

GIF1启动子及其应用
技术领域
本发明属于植物学和基因工程领域。具体而言， 本发明涉及新的植物（优选作 物）组织特异性表达启动子， 本发明还涉及所述启动子的用途， 尤其在启动目的基 因在植物组织中特异性表达的基因工程中的用途。 背景技术
当前， 对于农作物产量改良的研究主要集中在以下几个方面： 1. 增加作物的 源， 即加强作物的光合作用； 2. 增加作物库的大小； 3. 提高作物光合产物由源 向库运输的能力。
在很多作物中， 籽粒是判断该作物优劣的标准， 直接关系到作物的产量和品 质。 尽管人们采取了许多方式来提高农作物的产量和对农作物进行改良， 然而， 目前还缺乏有效的手段。 例如， 我国的主要粮食作物水稻， 当前的许多水稻高产 栽培品种尤其超级杂交稻和大穗大粒品种存在籽粒灌浆不饱满的情况， 这在很大 程度上影响了水稻产量的进一步提高。
植物的根部对其生长、 营养吸收、 能量传递等均具有重要作用。 改善作物根 部的吸收、 传输、 生长等能力有助于提高作物的总体生长和品质。 植物的节间是 物质、 能量运输的枢纽， 也对植物的支撑起到一定作用。
对于除作物以外的其它植物而言， 种子、 节间或根本身或整株植物可能存在 其经济价值或实用价值， 例如观赏等。 因此， 组织特异性地改造这些植物， 是提 高其品质的有用手段。
组织特异性表达启动子可以驱动靶基因在特定的组织或细胞表达， 而不影响 植物其它组织的生理活动。
因此， 为了改良植物 (尤其是作物)的品质性状或调控作物籽粒次生代谢， 本 领域迫切需要开发新的特异性良好的植物组织 (尤其是在种子、根部、节间等部位;) 特异性表达启动子， 从而实现农作物或其它经济植物产量和品质的提高。 发明内容
本发明的目的在于提供一种新的植物（尤其是作物）组织特异性表达启动子，所 述启动子可指导目的基因特异性地在植物籽粒、 根部或节间等组织中表达。 本发明的目的还在于提供所述启动子的用途。 在本发明的第一方面提供了一种分离的多核苷酸， 其选自下组：
(1) 由 SEQ ID NO : 3的 1-2379位所示的序列构成的核苷酸序列； (2) 具有 SEQ ID NO : 3中 1-2379位所示的核苷酸序列且能启动目的基因 在植物中组织特异性表达的核苷酸序列；
(3) SEQ ID NO : 3中 1-2379位所示的核苷酸序列的片段， 所述片段能启 动目的基因在植物中组织特异性表达；
(4)在严格条件下能够与（1)、 （2)或（3)限定的核苷酸序列杂交且能启动目 的基因在植物中组织特异性表达的核苷酸序列；
(5) 与（1)、 （2)或（3)限定的核苷酸序列有 70%、 80 %、 85 %、 90 %、 95 % 以上相同性且能启动目的基因在植物中组织特异性表达的核苷酸序列； 或
(6) 与（1) - (5)任一项所限定的核苷酸序列互补的核苷酸序列。
在一个优选例中， 所述多核苷酸的序列如 SEQ ID NO: 3的 1-2379位所示。 在一个优选例中， 所述植物是作物， 优选禾本科植物， 更优选所述的禾本科 作物选自： 水稻、 小麦、 大麦、 玉米、 或高粱。
在一个优选实施方式中， 所述目的基因选自： 编码具有 SEQ ID N0 : 2氨基酸 序列的作物籽粒灌浆蛋白、 以及与作物淀粉转运、 积累相关的基因。
在一个优选例中， 所述的目的基因是外源基因。
在另一优选例中， 所述的目的基因是结构基因。
在另一优选例中， 所述的目的基因是与作物籽粒的品质、 产量、 抗性或代谢 相关的基因。
在一个优选例中， 所述目的基因为编码具有 SEQ ID N0: 2氨基酸序列的作物 籽粒灌浆蛋白。
在本发明的另一优选实施方式中， 特异性表达所述目的基因的组织是种子、 根部和 /或节间， 即种子、 根部、 与节间中的一者、 两者或三者。
在一个优选例中， 特异性表达所述目的基因的组织是种子。 在本发明的第二方面， 提供了本发明多核苷酸作为启动植物组织特异性地表 达目的基因的启动子的用途。
在一个优选例中， 所述植物是作物， 优选为禾本科植物， 更优选所述的禾本 科植物选自： 水稻、 小麦、 大麦、 玉米、 或高粱。
在一个优选例中， 所述目的基因特异性表达的组织是种子、 根部和 /或节间， 优选种子。 在本发明的第三方面中， 提供了一种载体， 所述的载体含有本发明前述的多 核苷酸， 作为启动子元件。
在一个优选实施方式中， 所述的载体还含有与所述的核酸可操作地连接的目 的基因。
在另一个优选实施方式中， 所述的目的基因选自： 编码具有 SEQ ID N0 : 2氨 基酸序列的作物籽粒灌浆蛋白， 以及与作物淀粉转运、 积累相关的基因。
在一个优选例中， 所述的目的基因位于所述多核苷酸的下游， 且与所述启动 子的间隔小于 1000bp， 优选小于 500bp， 更优选小于 100bp， 最优选小于 50bp。 在本发明的第四方面中， 提供了一种遗传工程化的宿主细胞， 所述的细胞含 有本发明前述的载体； 或其基因组中整合有外源的本发明前述的多核苷酸。 在本发明的第五方面中， 提供了一种制备转基因植物的方法， 所述转基因植 物的组织特异性表达目的基因， 所述的方法包括：
(a)提供含有作为启动子元件的本发明的多核苷酸以及与所述多核苷酸可操 作地连接的目的基因的构建物；
(b)将所述构建物导入植物细胞、 组织或器官；
(c)选出导入了所述构建物或染色体中整合有所述构建物的植物细胞、组织或 器官； 和
(d)使所述植物细胞、 组织或器官再生成植株。
在一个优选例中， 所述植物为作物。
在一个优选例中， 所述目的基因特异性表达的组织是种子、 根部和 /或节间， 优选种子。 在本发明的第六方面中， 提供了一种转基因植物。
在一个优选例中， 所述转基因植物是用本发明前述方法制得的。
在另一优选例中， 所述转基因植物是含有本发明前述的多核苷酸或本发明前 述的载体的植物， 优选所述转基因植物为作物， 更优选为禾本科植物， 更优选所 述的禾本科植物选自： 水稻、 小麦、 大麦、 玉米、 或高粱。 本发明的其它方面由于本文的公开内容， 对本领域的技术人员而言是显而易 见的。 附图说明
图 1显示了本发明优选实施方式中的 GIF1启动子的序列。
图 2显示了在本发明启动子作用下 GUS基因的组织特异性。 其中，
根； 节间；
伸长的节间； 开花后 2天的种子； 开花后 4天的种子； 开花后 6天的种子； 开花后 10天的种子; 开花后 15天的种子;
开花后 25天的种子; 开花后 10天的颖壳;
开花后 10天种子的横切面。
图 3显示了在本发明启动子作用下 GIF1基因在植物不同部位的表达情况。
图 3A: 突变体和野生型种子中 GIF1基因的表达情况。
图 3B : GIF1基因在水稻幼苗、 叶片、 根、 节间和穗的表达情况。 其中 R: 水稻幼苗的根； L: 水稻叶片；
YL: 水稻幼苗叶片； I： 水稻节间；
P: 水稻穗子； ubi-1 : 泛素基因。
图 3C : GIF1基因在开花后不同时间的表达情况（数字指开花后天数）。 具体实施方式
本发明人经过广泛而深入的研究， 首次从植物籽粒灌浆基因（GIF1，其序列如 SEQ ID NO : 3 所示）中分离到一个能够指导目的基因在作物中组织特异性表达的 启动子， 该启动子来源于 GIF1基因（6 ^7， Grain Incomple te Filling ί) ^] I 区域， 因此本发明人将之命名为 GIF1启动子。所述的启动子对于定点地改良植物 品质是特别有用的。 在此基础上完成了本发明。 如本文所用， 所述的 "植物" 包括但不限于： 作物、 具有经济价值或实用价 值的其它植物等。
如本文所用， 所述的 "作物" 包括但不限于： 禾本科植物。 更优选的， 所述 的禾本科植物包括但不限于： 水稻， 小麦、 大麦、 玉米、 高粱等。
如本文所用， "分离的"是指物质从其原始环境中分离出来 (如果是天然的物 质， 原始环境即是天然环境)。 如活体细胞内的天然状态下的多聚核苷酸和多肽是 没有分离纯化的， 但同样的多聚核苷酸或多肽如从天然状态中同存在的其他物质 中分开， 则为分离纯化的。
如本文所用， 所述的 "可操作地连接" 是指两个或多个核酸区域或核酸序列 的功能性的空间排列。 例如： 启动子区被置于相对于目的基因核酸序列的特定位 置， 使得核酸序列的转录受到该启动子区域的引导， 从而， 启动子区域被 "可操 作地连接" 到该核酸序列上。
如本文所用， 所述的 "启动子" 或 "启动子区 (域；) " 是指一种核酸序列， 其 通常存在于目的基因编码序列的上游 (5'端)， 能够引导核酸序列转录为 mRNA。 —般地，启动子或启动子区提供 RNA聚合酶和正确起始转录所必需的其它因子的 识别位点。 在本文中， 所述的启动子或启动子区包括启动子的变体， 其通过插入 或删除调控区域， 进行随机或定点突变等来获得。
如本文所用，术语 "特异性表达" 是指目的基因在特定的时间和 /或特定的组 织表达。 "组织特异性启动子" 又称 "器官特异性启动子" ， 在这类启动子调控 下， 基因往往只在某些特定的器官或组织部位表达， 并表现出发育调节的特性。 本发明中， 所述的 "组织特异性启动子" 是指导基因在植物中组织特异性表达启 动子， 所述组织选自籽粒、 根部或节间。
通常， 如果在某组织或器官中 mRNA以比在其它组织或器官中高至少 1倍， 优选高至少 5倍， 优选至少高 10倍， 更优选至少高 100倍， 最优选至少高 1000 倍水平被表达， 则该启动子被认为是组织或器官特异性的。
如本文所用， "外源的"或 "异源的"是指来自不同来源的两条或多条核酸或 蛋白质序列之间的关系。 例如， 如果启动子与目的基因序列的组合通常不是天然 存在的， 则启动子对于该目的基因来说是外源的。 特定序列对于其所插入的细胞 或生物体来说是 "外源的" 。
如本文所用， "顺式调控元件" 是指对基因的转录起始和转录效率起调节作 用的保守性碱基序列。 如本文所用， "目的基因" 是指可由本发明的启动子指导表达的基因。 合适 的目的基因包括但不限于： 改良作物籽粒品质、 产量、 性状或代谢相关的基因（例 如编码具有 SEQ ID NO : 2氨基酸序列的作物籽粒灌浆蛋白）、 以及与作物淀粉转 运、 积累相关的基因。 植物组织特异性表达启动子
本发明提供一种植物组织特异性表达的多核苷酸（启动子）， 所述的核酸具有 选自下组的序列：
(1) 由 SEQ ID NO : 3的 1-2379位所示的序列构成的核苷酸序列；
(2) 具有 SEQ ID NO : 3中 1-2379位所示的核苷酸序列且能启动目的基因 在植物中组织特异性表达的核苷酸序列；
(3) SEQ ID NO : 3中 1-2379位所示的核苷酸序列的片段， 所述片段能启 动目的基因在植物中组织特异性表达；
(4)在严格条件下能够与（1)、 （2)或（3)限定的核苷酸序列杂交且能启动目 的基因在植物中组织特异性表达的核苷酸序列；
(5) 与（1)、 （2)或（3)限定的核苷酸序列有 70%、 80 %、 85 %、 90 %、 95 % 以上相同性且能启动目的基因在植物中组织特异性表达的核苷酸序列； 或
(6) 与（1) - (5)任一项所限定的核苷酸序列互补的核苷酸序列。
在本发明的一个实施方式中， 所述多核苷酸的序列如 SEQ ID N0: 3的 1-2379 位所示。
在本发明的另一个实施方式中， 所述植物是作物， 优选禾本科植物， 包括但 不限于水稻、 玉米、 小麦、 大麦、 或高粱等。
在本发明的另一个实施方式中， 特异性表达所述目的基因的组织是种子、 根 部和 /或节间， 优选种子。
也可通过基因工程方法在该启动子中加入增强子等顺式作用元件达到进一 步调控基因表达的作用。
多核苷酸的杂交是本领域技术人员熟知的技术， 特定的一对核酸的杂交特性 指示它们的相似性或同一性。 因此， 本发明还涉及与前述指定的核苷酸序列杂交 且两个序列之间具有至少 50%， 较佳地至少 70%， 更佳地至少 80% (例如 85%、 90%、 95%、 96%、 97%、 98%、 或 99%) 相同性的多核苷酸。
本发明特别涉及在严格条件下与本发明所述多核苷酸可杂交的多核苷酸。 "严格条件"是指： （1)在较低离子强度和较高温度下的杂交和洗脱，如 0. 2 X SSC, 0. 1%SDS， 60 °C ; 或（2)杂交时加有变性剂， 如 50% (v/v)甲酰胺， 0. 1%小牛血清 /0. l%Ficol l , 42 °C等； 或（3)仅在两条序列之间的相同性至少在 50%， 优选 60%以 上、 65%以上、 70%以上、 75%以上、 80%以上、 85%以上或 90%以上， 更优选是 95%以上 时才发生杂交。 并且， 可杂交的多核苷酸也具有指导目的基因在植物中组织特异 性表达的功能。
本发明还包括与本发明的任一种启动子序列具有 50% 或以上（优选 60%以上， 70%以上， 80%以上， 更优选 90%以上， 最优选 95%以上）相同性的核酸， 所述核酸 也具有指导目的基因在植物中组织特异性表达的功能。 "相同性" 是指按照位置 相同的百分比， 两条或多条核酸之间的相似水平（即序列同源性、 相似性或同一 性）。
在本发明启动子的指导下， 可以使 β-葡萄糖苷酶 (GUS)基因特异地在水稻、 小麦、 大麦、 玉米、 高粱等禾本科植物的籽粒、 根部或节间中表达。 因此可见， 本发明的启动子是一种组织或器官特异性的启动子。 所述的启动子对于定点地改 良植物的品质是特别有用的。
β-葡萄糖苷酶 (GUS)能催化裂解一系列的 β-葡萄糖苷， 产生具有发色团或荧 光的物质， 可用分光光度计、 荧光计或组织化学等方法对 GUS活性进行定量和空 间定位分析。 在本技术领域中， GUS基因已被广泛地用作转基因植物、 细菌和真 菌的报告基因， 特别是其可被用于研究外源基因表达的具体细胞和组织部位。
本发明的组织特异性启动子在理论研究和作物改良中具有重要的应用价值。 这些启动子可以被应用于标记特定组织， 引导特定的功能基因在特定的组织中表 达， 以及应用于特有组织的生长发育研究和针对性改良。 本发明的启动子对于改 良植物的品质或表型（尤其是作物籽粒（例如水稻籽粒） )是特别有用的。 目的基因
本发明的启动子可以被可操作地连接到目的基因上， 该目的基因相对于启动 子而言可以是外源（异源）的。对所述目的基因的核酸序列没有特别的限制（如一种 结构性核酸序列）， 所述的目的基因优选编码具有特定功能的蛋白， 例如某些在农 业或植物改良上具有重要特性或功能的蛋白。
合适的目的基因包括但不限于： 改良作物品质、 性状或代谢相关的基因。 例 如， 所述的目的基因选自： 与作物籽粒的品质、 产量、 抗性或代谢相关的基因 (例 如作物灌浆蛋白基因（GIF1基因））， 以及与作物淀粉转运、 积累相关的基因。 本发明的启动子还可以被可操作地连接到被改进的目的基因序列上， 该目的 基因相对于启动子是外源（异源）的。 所述的目的基因可以被改进来产生各种期望 的特性。 例如， 目的基因可以被改进来增加必需氨基酸的含量， 提高氨基酸序列 的翻译， 改变翻译后的修饰（如磷酸化位点）， 将翻译产物转运到细胞外， 改善蛋 白的稳定性， 插入或删除细胞信号等。
此外， 启动子和目的基因可以设计成下调特定基因。 这一般是通过将启动子 连接到目的基因序列上来实现， 该序列以反义反向被引导。 本领域的普通技术人 员熟悉这种反义技术。 任何核酸序列可以以这种方式被调节。 载体和宿主细胞
本发明的任何一种前述的启动子和 /或目的基因序列可被包含在重组载体中。 作为一种方式， 所述的重组载体包括本发明的启动子， 在所述启动子的下游 包含多克隆位点或至少一个酶切位点。 当需要表达目的基因时， 将目的基因连接 入适合的多克隆位点或酶切位点内， 从而将目的基因与启动子可操作地连接。
作为另一种方式， 所述的重组载体包括 (从 5'到 3 '方向；)： 引导目的基因转录 的启动子， 和目的基因。如果需要， 所述的重组载体还可以包括 3'转录终止子， 3' 多聚核苷酸化信号， 其它非翻译核酸序列， 转运和靶向核酸序列、 抗性选择标记、 增强子或操作子。
用于制备重组载体的方法是本领域熟知的。 术语 "重组表达载体" 指本领域 熟知的细菌质粒、 噬菌体、 酵母质粒、 植物细胞病毒、 哺乳动物细胞病毒或其他 载体。 总之， 只要其能够在宿主体内复制和稳定， 任何质粒和载体都是可以被采 用的。
本领域的技术人员熟知的方法能用于构建含有本发明所述的启动子和 /或目 的基因序列的表达载体。 这些方法包括体外重组 DNA技术、 DNA合成技术、 体 内重组技术等。 表达载体还包括翻译起始用的核糖体结合位点和转录终止子。
此外， 表达载体优选地包含一个或多个选择性标记基因， 以提供用于选择转 化的宿主细胞的表型性状， 如二氢叶酸还原酶、 新霉素抗性、 潮霉素抗性以及绿 色荧光蛋白（GFP)等。
重组载体中除了含有本发明的启动子， 还可含有一种或多种其它启动子。 所 述的其它启动子例如是： 组织特异性的、 组成型的或诱导型的。 例如甘露氨酸合 成酶的花椰菜花叶病毒 19S 和 35S (CaMV19S CaMV35S)、 增强的 CaMV、 烟草 RB7 等。 包含上述适当的启动子和目的基因的载体， 可以用于转化适当的宿主细胞， 以使其能够表达蛋白质。
宿主细胞可以是原核细胞， 如细菌细胞； 或是低等真核细胞， 如酵母细胞； 或是高等真核细胞， 如植物细胞。 代表性例子有： 大肠杆菌， 链霉菌属、 农杆菌； 真菌细胞如酵母； 植物细胞等。 本领域一般技术人员都清楚如何选择适当的载体 和宿主细胞。
用重组 DNA转化宿主细胞可用本领域技术人员熟知的常规技术进行。当宿主 为原核生物如大肠杆菌时， 能吸收 DNA的感受态细胞可在指数生长期后收获， 用 CaCl2法处理， 所用的步骤在本领域众所周知。 另一种方法是使用 MgCl2。 如 果需要， 转化也可用电穿孔的方法进行。 当宿主是真核生物， 可选用如下的 DNA 转染方法： 磷酸钙共沉淀法， 常规机械方法如显微注射、 电穿孔、 脂质体包装等。 转化植物也可使用农杆菌转化或基因枪转化等方法， 例如叶盘法、 幼胚转化法、 花芽浸泡法等。 对于转化的植物细胞、 组织或器官可以用常规方法再生成植株， 从而获得转基因的植物。
例如， 可通过叶圆片转化-再生程序 (；如 Horsch等人（1985) Science 227: 1229 所述)将融合基因导入植物内。 也可采用其它转化方法， 如 PEG法、 基因枪法、 电激法 (Horsch等人, 1984, Scienc 223 :496, Barton等人， 1983, Cell 32: 1033, Liu等人 : Acta Phytophysiol Sin, 21 : 195-205)， 这也在本发明范围内。但是这些方法有操作复 杂、 外源基因插入的多拷贝性和不完整性以及较高比例的不育植株等缺点
(Potrykus 1990, Bio/Technol, 8:535-542; Finnegan禾口 McElroy, 1994, Bio/Technol. 12:883-888)。
作为一种方式， 制备转基因植物的方法是： 将携带启动子和目的基因（两者可 操作地连接）的双元载体转入农杆菌，农杆菌再将含启动子和目的基因的载体片段 整合到植物的染色体上。 涉及的转基因受体植物例如是水稻、 小麦等。
在本发明的一个实施方式中， 本发明人构建 GIF1基因的启动子连 β -葡糖醛 酸酶基因（GUS)的克隆， 并转化水稻 ZH1 1 , 观察 GIF1启动子启动 GUS报告基因和 GIF1基因的组织特异性表达。 本发明的主要优点 揭示一种可指导目的基因在植物中组织特异性表达的启动子， 所述的启动子 对于定点地改良植物品质特别有用。 实施例
下面结合具体实施例， 进一步阐述本发明。 应理解， 这些实施例仅用于说明 本发明而不用于限制本发明的范围。 下列实施例中未注明具体条件的实验方法， 通常按照常规条件， 如分子克隆实验手册（Molecular cloning : A laboratory manual , 3rd ed.， Sambrook等， Cold Spring Harbor Laboratory , 2001)禾口植 物分子生物学实验手册 (Plant Molecular Biology- A Laboratory Mannual , Clark 等， Springer-Verlag , 1997)中所述的条件， 或按照制造厂商所建议的条件。 实施例 1. GIF1基因的获得
本发明人从水稻中花 1 1 (ZH11)诱变突变体库中， 发现了一个水稻突变体， 该 突变体的种子的灌浆受到较为严重的影响， 营养生长和野生型相比没有变化， 但 籽粒灌浆受到严重影响， 种子千粒重减少 15-30%， 并且米质变差， 本发明人将之 命名为 gifl (grain incomplete fi l l ing 1)， 说明 GIF1基因是一个重要的通过 灌浆控制产量和米质的基因。
本发明人通过 gif l突变体和珍汕 97杂交得到基因定位群体。 利用
BSA (Bulked Segregant Analys i s)分析法， 以均匀分布在水稻 12 条染色体上的 130对 SSR引物对 GIF1位点进行扫描， 将 GIF1位点初步定位在 4号染色体长臂 SRD5附近， 最终将 GIF1基因定位在 caps-4和 caps-8之间的 32Kb中， 经预测这 段区域共包含三个基因， 本发明人通过对突变体和野生型的 DNA的测序， 发现在 突变体 gif l中有一个碱基的缺失（GIF1基因组 DNA序列第 4588位）。
通过对 GIF1的精细定位， 通过测序和功能验证， 得到野生型 GIF1的基因组 (DNA)序列见 SEQ ID N0 : 3 (包括启动子）； GIF1编码区的序列（cDNA序列）见 SEQ ID NO : 1； GIF1蛋白的序列见 SEQ ID NO : 2。
当发生前述的突变（GIF1基因组 DNA序列第 4588位缺失）后， 突变型不表达 GIF1蛋白。 实施例 2. GIF1启动子序列的获得
本发明人根据已经克隆的 GIF1基因， 在 NCBI (http : //www. ncbi . nlm. nih. gov/)数据库搜索该基因的基因组区域， 经过生物软 件预测（http : //www. cbs. dtu. dk/services/Promoter/)启动子， 最终选择了 2379 bp作为启动子序列。 实施例 3. 载体构建和农杆菌转化
本发明人构建 GIF1基因的启动子连 β -葡糖醛酸酶基因（GUS)的克隆，并转化 水稻 ZH11 , 观察 GIF1启动子启动 GUS报告基因和 GIF1基因的组织特异性表达。
具体的构建方法如下：
采用以下引物：
正向弓 I物 tataagcttgatcggccatactcc (SEQ ID NO : 4)禾口
反向弓 I物 taggatccctttgctctcacacttg (SEQ ID NO : 5)，
以 GIF1的基因组 DNA为模板， PCR得到 GIF1基因的启动子（其序列如图 1所 示），并克隆载体 ρΒΙ ΙΟΙ (购自 Clonetech公司，带有 GUS)，再用 EcoR I、 Hind I II 酶切，回收片断，将该片断连入用相同酶切的 PCAMBIA1300便得到了 pCAMBIA1300+ 启动子 +GUS的相应克隆（组织显色方法见 Jeferenson, RA (1987) Plant Mol Biol Rep)。
结果见图 2A-K， 可知 GUS基因在植株的根部、 节间、 种子的背腹部的微管束 有特异性表达。
此外， 采用 RT-PCR的方法， 从所述的野生型或突变型植株中抽提 mRNA， 通 过 RT-PCR分别扩增 GIF1 , 将获得的扩增产物进行琼脂糖电泳检测。
在该实验中， 用来扩增 GIF1的引物为：
Gif-F: cccgccggcgacgagcaccacat ( SEQ ID NO:6 ) 禾口
Gif-R: ccgccggcctgaacaccctgaaga (SEQ ID NO:7)
用来扩增 ubi-1的引物为：
ubi-l-F: gacggacgcaccctggctgactac (SEQ ID NO: 8) 禾口
ubi-1 -R: tgctgccaattaccatataccacgac (SEQ ID NO: 9)。
结果见图 3A-C， 其中， 图 3A是突变体 gifl和野生型的 mRNA的 RT-PCR检测 结果； 图 3B是水稻不同组织中 GIF1 mRNA的 RT-PCR检测结果； 图 3C是不同时期 (DAF)水稻穗部组织中 GIF1 mRNA的 RT-PCR检测结果。 在本发明提及的所有文献都在本申请中引用作为参考， 就如同每一篇文献被 单独引用作为参考那样。 此外应理解， 在阅读了本发明的上述讲授内容之后， 本 领域技术人员可以对本发明作各种改动或修改， 这些等价形式同样落于本申请所 附权利要求书所限定的范围。

Claims

权 利 要 求
1. 一种分离的多核苷酸， 其选自下组：
(1) 由 SEQ ID NO : 3的 1-2379位所示的序列构成的核苷酸序列； (2) 具有 SEQ ID NO : 3中 1-2379位所示的核苷酸序列且能启动目的基因 在植物中组织特异性表达的核苷酸序列；
(3) SEQ ID NO : 3中 1-2379位所示的核苷酸序列的片段， 所述片段能启 动目的基因在植物中组织特异性表达；
(4)在严格条件下能够与（1)、 （2)或（3)限定的核苷酸序列杂交且能启动目 的基因在植物中组织特异性表达的核苷酸序列；
(5) 与（1)、 （2)或（3)限定的核苷酸序列有 70%、 80 %、 85 %、 90 %、 95 % 以上相同性且能启动目的基因在植物中组织特异性表达的核苷酸序列； 或
(6) 与（1) - (5)任一项所限定的核苷酸序列互补的核苷酸序列。
2. 如权利要求 1所述的多核苷酸， 其特征在于， 所述目的基因选自： 编码 具有 SEQ ID N0 : 2氨基酸序列的作物籽粒灌浆蛋白、 以及与作物淀粉转运、 积累 相关的基因。
3. 如权利要求 1所述的多核苷酸， 其特征在于， 特异性表达所述目的基因 的组织是种子、 根部和 /或节间。
4. 权利要求 1所述的多核苷酸作为启动植物组织特异性地表达目的基因的 启动子的用途。
5. 一种载体， 其特征在于， 所述的载体含有权利要求 1-3中任一项所述的 多核苷酸， 作为启动子元件。
6. 如权利要求 5所述的载体， 其特征在于， 所述的载体还含有与所述的核 酸可操作地连接的目的基因。
7. 如权利要求 6所述的载体， 其特征在于， 所述的目的基因选自： 编码具 有 SEQ ID N0 : 2氨基酸序列的作物籽粒灌浆蛋白， 以及与作物淀粉转运、 积累相 关的基因。
8. 一种遗传工程化的宿主细胞， 其特征在于， 所述的细胞：
含有权利要求 5-7中任一项所述的载体； 或
其基因组中整合有外源的权利要求 1-3中任一项所述的多核苷酸。
9. 一种制备转基因植物的方法， 其特征在于， 所述的方法包括： (a)提供含有作为启动子元件的权利要求 1-3中任一项所述的多核苷酸以及 与所述多核苷酸可操作地连接的目的基因的构建物；
(b)将所述构建物导入植物细胞、 组织或器官；
(c)选出导入了所述构建物或染色体中整合有所述构建物的植物细胞、组织或 器官； 和
(d)使所述植物细胞、 组织或器官再生成植株。
10. 用权利要求 9所述的方法制得的转基因植物。
1 1 . 含有权利要求 1-3中任一项所述的多核苷酸或权利要求 5-7中任一项所 述的载体的植物。
12. 如权利要求 1 1所述的植物， 其特征在于， 所述植物为作物， 优选为禾本 科植物， 更优选所述的禾本科植物选自： 水稻、 小麦、 大麦、 玉米、 或高粱。
PCT/CN2009/071419 2008-04-25 2009-04-23 Gif1启动子及其应用 WO2009129743A1 (zh)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN200810036615.4 2008-04-25
CN200810036615 2008-04-25

Publications (1)

Publication Number Publication Date
WO2009129743A1 true WO2009129743A1 (zh) 2009-10-29

Family

ID=41216438

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2009/071419 WO2009129743A1 (zh) 2008-04-25 2009-04-23 Gif1启动子及其应用

Country Status (2)

Country Link
CN (1) CN101565702B (zh)
WO (1) WO2009129743A1 (zh)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104673793A (zh) * 2013-11-27 2015-06-03 中国科学院上海生命科学研究院 豆科植物根系组织特异性启动子及其应用

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102154298B (zh) * 2011-03-24 2012-09-19 浙江大学 水稻根特异启动子Os02g37190及其应用
CN114990112B (zh) * 2021-02-26 2024-03-12 华中农业大学 一种皮刺特异启动子

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101173000A (zh) * 2006-10-30 2008-05-07 中国科学院上海生命科学研究院 作物籽粒灌浆基因gif1及其应用

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU2001259185A1 (en) * 2000-04-26 2001-11-07 Akkadix Corporation Polynucleotide sequences from rice

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN101173000A (zh) * 2006-10-30 2008-05-07 中国科学院上海生命科学研究院 作物籽粒灌浆基因gif1及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL 11 September 2007 (2007-09-11), Database accession no. Q01IS7 *
LIU QI-HUA ET AL.: "Key Starch Synthase in Rice Grain and Its Relation to Grain Filling and Rice Quality", PLANT PHYSIOLOGY COMMUNICATIONS, vol. 42, no. 6, 31 December 2006 (2006-12-31), pages 1211 - 1215 *
WANG E ET AL.: "Control of rice grain-filling and yield by a gene with a potential signature of domestication.", NAT GENET., vol. 40, no. 11, 28 September 2008 (2008-09-28), pages 1370 - 1374 *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104673793A (zh) * 2013-11-27 2015-06-03 中国科学院上海生命科学研究院 豆科植物根系组织特异性启动子及其应用
CN104673793B (zh) * 2013-11-27 2017-12-08 中国科学院上海生命科学研究院 豆科植物根系组织特异性启动子及其应用

Also Published As

Publication number Publication date
CN101565702B (zh) 2012-01-11
CN101565702A (zh) 2009-10-28

Similar Documents

Publication Publication Date Title
US20190330649A1 (en) Plant Grain Trait-Related Protein, Gene, Promoter and SNPS and Haplotypes
WO2018196709A1 (zh) 一种调控作物矮化及其产量的基因及其应用
JP5938444B2 (ja) コメの生産高の増加方法
US20090083877A1 (en) Transcription Factors, DNA and Methods for Introduction of Value-Added Seed Traits and Stress Tolerance
CN111850030B (zh) 蛋白GmULT1在调控植物种子重量中的应用
CN103172715B (zh) 植物表皮毛调控基因及其用途
WO2019129145A1 (en) Flowering time-regulating gene cmp1 and related constructs and applications thereof
CA2727564A1 (en) Methods and means of increasing the water use efficiency of plants
US20130263328A1 (en) Gene involved in the development of the seed
WO2009129743A1 (zh) Gif1启动子及其应用
CN110777150B (zh) 蛋白GmPLATZ在调控植物种子产量中的应用
CN108864265B (zh) 蛋白质TabZIP60在调控植物根系发育中的应用
CN105504031B (zh) 来源于大豆的粒重相关蛋白及其相关生物材料与应用
CA3103906A1 (en) Thermostable rubisco activase and uses thereof
WO2022057780A1 (zh) 一个调控植物氮素利用效率和产量的蛋白质及其应用
CN112142853B (zh) 真菌病害相关的甘蓝型油菜BnTLK1基因及其应用
JP4755838B2 (ja) アセト乳酸合成酵素遺伝子プロモーター
CN116411018B (zh) TaVRT-A2蛋白及其相关生物材料在调控植物种子蛋白质含量中的应用
CN114539369B (zh) ZmEREB167基因在调控玉米籽粒发育中的应用
CN114752620B (zh) ZmGW3蛋白及其基因在调控玉米籽粒发育中的应用
US7109390B2 (en) Alternative splicing factors polynucleotides polypeptides and uses therof
CN113861279B (zh) 大豆转录因子GmbHLH664及其编码基因在提高种子蛋白质含量中的应用
CN110194791B (zh) Spl3蛋白在调控植物花序或果柄发育中的用途
CN116676331A (zh) ZmST1蛋白及其编码基因在调控植物持绿性、抗病性和产量中的应用
WO2011020163A1 (en) Plant gene expression

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 09734221

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: RULE 112 1 EPC

122 Ep: pct application non-entry in european phase

Ref document number: 09734221

Country of ref document: EP

Kind code of ref document: A1