WO2019090496A1 - 小麦蓝粒基因及其应用 - Google Patents

小麦蓝粒基因及其应用 Download PDF

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WO2019090496A1
WO2019090496A1 PCT/CN2017/109818 CN2017109818W WO2019090496A1 WO 2019090496 A1 WO2019090496 A1 WO 2019090496A1 CN 2017109818 W CN2017109818 W CN 2017109818W WO 2019090496 A1 WO2019090496 A1 WO 2019090496A1
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sequence
gene
blue
promoter
plant
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PCT/CN2017/109818
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English (en)
French (fr)
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马力耕
王峥
陈卓
衡燕芳
邓兴旺
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首都师范大学
未名兴旺系统作物设计前沿实验室（北京）有限公司
北京大学现代农业研究院
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Priority to CA3080642A priority Critical patent/CA3080642A1/en
Priority to PCT/CN2017/109818 priority patent/WO2019090496A1/zh
Priority to US16/759,750 priority patent/US11390877B2/en
Priority to CN201780054212.XA priority patent/CN112218951B/zh
Priority to CN202211123928.XA priority patent/CN116064580A/zh
Publication of WO2019090496A1 publication Critical patent/WO2019090496A1/zh

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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
    • 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
    • C12N15/8242—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits
    • C12N15/8243—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine
    • C12N15/825—Phenotypically and genetically modified plants via recombinant DNA technology with non-agronomic quality (output) traits, e.g. for industrial processing; Value added, non-agronomic traits involving biosynthetic or metabolic pathways, i.e. metabolic engineering, e.g. nicotine, caffeine involving pigment biosynthesis
    • 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
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material

Definitions

  • the invention relates to the fields of plant molecular biology, biochemistry, genetics and plant breeding, in particular to a gene regulating wheat blue granule traits.
  • the present invention provides nucleic acid molecules and plant recombinant vectors of four wheat blue granule trait related genes, and methods of using the genes or vectors.
  • the outer end of the endosperm of wheat grain is surrounded by three layers of tissue: peel, seed coat and aleurone layer.
  • the grain color of wheat is determined by different anthocyanins accumulated in different tissues.
  • Purple grain wheat is derived from the purple anthocyanin in the outermost skin.
  • the skin is developed from the female parent, so the purple grain traits show the maternal inheritance pattern.
  • Blue-brown wheat is derived from the aleurone in the aleurone layer, which is mainly produced by distant hybridization between common wheat and other species. There are two main sources: Thinopyrum ponticum and wild.
  • One wheat Triticum monococcum).
  • the Blaukom series derived from a single wheat was formed by replacing the 4A or 4B chromosome of common wheat with a pair of 4A chromosomes of a wheat, and the 4A m chromosome and tetraploid from a wheat.
  • the 4A chromosome in hexaploid wheat is non-homologous.
  • Kuspiral et al. named the blue aleurone layer gene derived from wild one wheat as the Ba2 gene, and the Ba2 gene was located at the near centromere of the long arm of the 4A m chromosome (Dubcovsky et al, 1996).
  • purple grain wheat Genetic analysis of purple grain wheat showed that the purple grain trait was controlled by the Pp-1 (purple pericarp) site located in the short arm of chromosome 7 and the Pp3 site located on the 2AL chromosome, where Pp-1 encodes the Myb family transcription factor, while Pp3
  • the code contains the bHLH (basic helix-loop-helix) structure Domain Myc family transcription factors (Khlestkina et al, 2013; Shoeva et al, 2014).
  • Anthocyanins which determine the color of wheat grains, are water-soluble secondary metabolites, flavonoids, which are widely distributed in the roots, stems, leaves, flowers, fruits and seeds of higher plants.
  • Plant anthocyanin synthesis is a branch of the flavonoid synthesis pathway.
  • the main synthetase in this pathway includes chalcone synthase (CHS), chalcone isomerase (CHI), and flavanone- 3-hydroxylase (flavanone-3-hydroxylase, F3H), dihydroflavonol-4-reductase (DFR), etc. (Miyazaki et al., 2011), all of which are anthocyanin synthesis Structural genes.
  • anthocyanin synthesis is mainly done by three types of transcription factors: MYB, bHLH and WD40.
  • MYB three types of transcription factors
  • bHLH bHLH
  • WD40 The anthocyanins of most species are directly regulated by a protein complex composed of these three transcription factors, and a few anthocyanin synthesis requires only a single regulatory factor to activate.
  • Blue-grain wheat is an ideal morphological feature of wheat marker traits and wheat cytogenetics research, and an important basic material for wheat chromosome engineering research.
  • the present invention utilizes differential expression analysis of blue and white wheat to obtain four genes controlling wheat blue granule traits: two MYB family transcription factors and two bHLH family transcription factors.
  • the invention is useful for studying the synthetic route of the blue-grain wheat aleurone pigment, and can be used as a screening marker in the plant transformation process, and the expression of the gene in the plant can increase the synthesis of anthocyanins, thereby improving the plant's Nutritional value.
  • the present invention provides a blue-grain gene having an anthocyanin synthesis regulating function, and the names of the blue-granular genes are ThMYB1, ThMYB2, ThR1 and ThR2, respectively, and the blue-granular gene can change the tissue or organ of the expression site. It is blue.
  • the genomic nucleotide sequence of the ThMYB1 gene from the start codon to the stop codon is as shown in SEQ ID NO: 1, and the nucleotide sequence of the coding region (CDS) is shown in SEQ ID NO: 2, and its CDS
  • the encoded amino acid sequence is shown in SEQ ID NO: 3.
  • the genomic nucleotide sequence of the ThMYB2 gene from the initiation codon to the stop codon is shown in SEQ ID NO: 4, and the coding region (CDS) nucleotide sequence is shown in SEQ ID NO: 5, and its CDS
  • the encoded amino acid sequence is set forth in SEQ ID NO: 6.
  • the genomic nucleotide sequence of the ThR1 gene from the start codon to the stop codon is as shown in SEQ ID NO: 7, and the coding region (CDS) nucleotide sequence is shown in SEQ ID NO: 8, and the CDS is The encoded amino acid sequence is set forth in SEQ ID NO:9.
  • the genomic nucleotide sequence of the ThR2 gene from the start codon to the stop codon is shown in SEQ ID NO: 10, and the coding region (CDS) nucleotide sequence is shown in SEQ ID NO: 11, and the CDS is The encoded amino acid sequence is set forth in SEQ ID NO: 12.
  • the blue particle gene of the present invention further comprises a nucleotide sequence or a protein sequence homologous to the ThMYB1, ThMYB2, ThR1 and ThR2 genes, and has the same regulation of anthocyanin synthesis.
  • the homologous gene which is highly homologous and has an anthocyanin synthesis regulatory function includes DNA capable of reacting with a sequence having the sequence shown in SEQ ID NO: 1, 2, 4, 5, 7, 8, 10 or 11 under stringent conditions. Hybrid DNA sequence. Or a nucleotide sequence whose encoded amino acid sequence has an affinity of 85% or more with the amino acid sequence of the protein represented by SEQ ID NO: 3, 6, 9, or 12.
  • stringent conditions include, for example, hybridization in a hybridization solution containing 400 mM NaCl, 40 mM PIPES (pH 6.4) and 1 mM EDTA, preferably at a temperature of 53 ° C to 60 ° C, hybridization time. Preferably, it is 12-16 hours, and then washed with a washing liquid containing 0.5 x SSC, and 0.1% SDS, the washing temperature is preferably 62 ° C - 68 ° C, and the washing time is 15 - 60 minutes.
  • the above homologous gene further comprises at least 80%, 85%, 90%, 95%, 98%, or the full length of the sequence represented by SEQ ID NO: 1, 2, 4, 5, 7, 8, 10 or 11. 99% sequence similarity, and has a DNA sequence that regulates the anthocyanin synthesis function of plants, and can be isolated from any plant.
  • the percentage of sequence similarity can be obtained by well-known bioinformatics algorithms, including Myers and Miller algorithm, Needleman-Wunsch global alignment method, Smith-Waterman local alignment method, Pearson and Lipman similarity search method, Karlin and Altschul's algorithm. This is well known to those skilled in the art.
  • the present invention also provides an expression cassette comprising the DNA sequence of the blue particle gene disclosed in the present invention, the nucleotide sequence of the blue particle gene being selected from one of the following group of sequences:
  • the blue-granule gene in the above expression cassette is operably linked to a promoter capable of driving expression thereof, including but not limited to a constitutive promoter, an inducible promoter, and a tissue-specific promoter. Or a spatiotemporal specific expression promoter.
  • a promoter capable of driving expression thereof including but not limited to a constitutive promoter, an inducible promoter, and a tissue-specific promoter. Or a spatiotemporal specific expression promoter.
  • the gene expression of the constitutive promoter of the present invention is not tissue- and time-specific, and external factors have little effect on the expression of the foreign gene initiated by the constitutive promoter.
  • the constitutive promoters include, but are not limited to, CaMV35S, FMV35S, the rice actin (Actin1) promoter, the maize ubiquitin promoter, and the like.
  • the tissue-specific promoter of the present invention has the characteristics of an enhancer and a silencer in addition to the general promoter element as it is, and the promoter has the advantage that the promoter can be expressed in a specific tissue part of the plant, thereby avoiding Unnecessary expression of foreign genes, thereby saving plants The overall energy consumption of the body.
  • the tissue-specific promoter includes, but is not limited to, an LTP2 seed-specific expression promoter, an END2 seed-specific expression promoter, an aleurone-specific expression promoter, and the like.
  • the inducible promoter of the present invention refers to a promoter which can greatly increase the transcription level of a gene under the stimulation of certain specific physical or chemical signals, and the inducible promoter which has been isolated so far includes, but is not limited to, adversity.
  • Inducible expression promoter light-inducible expression promoter, heat-inducible expression promoter, wound-inducible expression promoter, fungal-inducible expression promoter and symbiotic bacteria-induced expression promoter, and the like.
  • the above expression cassette of the present invention may further comprise a screening gene which can be used for screening plants, plant tissue cells or vectors containing the expression cassette.
  • the screening gene includes, but is not limited to, an antibiotic resistance gene, or a herbicide resistance gene, or a fluorescent protein gene.
  • the screening gene includes, but is not limited to, a chloramphenicol resistance gene, a hygromycin resistance gene, a streptomycin resistance gene, a spectinomycin resistance gene, a sulfonamide resistance gene, and a glyphosate resistance gene. Sex gene, glufosinate resistance gene, bar gene, red fluorescent gene DsRED, mCherry gene, cyan fluorescent protein gene, yellow fluorescent protein gene, luciferase gene, green fluorescent protein gene, and the like.
  • the invention also discloses a method for increasing the anthocyanin content of a plant, which can improve the plant by co-expressing the ThMYB1 or ThMYB2 gene provided by the invention with any bHLH transcription factor in the tissues and organs of the plant. Anthocyanin content in tissues and organs.
  • the bHLH transcription factor can be isolated from any plant including, but not limited to, the ThR1 and ThR2 genes provided by the present invention, and the ZmR and ZmB genes derived from maize (Ahmed N, et al. Transient expression of anthocyanin in developing wheat coleoptile by maize). C1 and B-peru regulatory genes for anthocyanin synthesis. Breeding Sci. 2003; 53(1): 29-34.).
  • the above method for increasing the anthocyanin content of a plant can be used to increase the anthocyanin content of any tissue or organ of the plant. Specifically, if it is desired to increase the anthocyanin content in each tissue of the plant as a whole, both the ThMYB1 or ThMYB2 gene and the bHLH transcription factor can be expressed by a constitutive promoter. If you only want to increase the anthocyanin content in a certain tissue or organ, both the ThMYB1 or ThMYB2 gene and the bHLH transcription factor can be used to initiate expression using a promoter specifically expressed in the tissue or organ.
  • the invention also discloses a method for increasing the anthocyanin content of a plant, which can be improved by co-expressing the ThR1 or ThR2 gene provided by the invention with any MYB transcription factor in the tissues and organs of the plant. Anthocyanin content in plant tissues and organs.
  • the MYB-type transcription factor can be isolated from any plant, including but not limited to the ThMYB1 and ThMYB2 genes provided by the present invention, and the ZmC1 gene derived from maize (Ahmed N, et al. Transient expression of anthocyanin in developing wheat coleoptile by maize C1and). B-peru regulatory genes for anthocyanin synthesis. Breeding Sci. 2003; 53(1): 29-34.).
  • the above method for increasing the anthocyanin content of a plant can be used to increase the anthocyanin content of any tissue or organ of the plant.
  • both the ThR1 or ThR2 gene and the MYB transcription factor can be expressed by a constitutive promoter.
  • both the ThR1 or ThR2 gene and the MYB transcription factor can be expressed by a promoter specifically expressed in the tissue or organ.
  • the present invention also provides a visual screening marker gene which produces a macroscopic blue marker in the tissues and organs of a plant by co-expression of the ThMYB1 or ThMYB2 gene with any of the bHLH transcription factors. . Or by co-expressing the ThR1 or ThR2 gene provided by the present invention with any of the MYB-type transcription factors in the tissues and organs of the plant, a macroscopic blue marker mark is produced in the tissues and organs of the plant.
  • the screening marker genes disclosed in the present invention can be used to distinguish between transgenic and non-transgenic materials.
  • the screening marker gene provided by the invention can be used as a screening marker in the breeding process of the male sterile line, and the fertility restoring gene, the pollen inactivating gene and the screening marker gene provided by the invention are transferred to the male sterile line.
  • the fertility restorer gene can restore the fertility of the male sterile line, and the pollen inactivating gene can inactivate the pollen containing the transformed foreign gene, that is, the ability to inseminate, and the screening marker gene provided by the present invention can be used.
  • the sorted non-transgenic seeds are used as hybrid lines for the production of sterile lines, and the transgenic seeds are used as a source to maintain the stable and stable production of the sterile lines.
  • the screening marker gene provided by the invention can also be used as a screening marker in the breeding process of the female sterile line, and the female fertility gene, the pollen inactivating gene and the screening marker gene provided by the invention are transferred into the female sterile line.
  • the female fertility gene can restore the fertility of the female sterile transformed receptor material
  • the pollen inactivating gene can inactivate the pollen containing the transformed exogenous construct, ie, lose the fertilizing ability
  • the screening marker provided by the present invention The gene can be used for sorting of transgenic seeds and non-transgenic seeds, and the sorted non-transgenic seeds are used as hybrids for female sterile lines, and the transgenic seeds are used as a source to maintain a steady and stable production of female sterile lines.
  • Female infertility maintainer can be used as a screening marker in the breeding process of the female sterile line, and the female fertility gene, the pollen inactivating gene and the screening marker gene provided by the invention are transferred into the female sterile line.
  • the present invention also provides a promoter of a blue-grain gene having a characteristic of aleurone-specific expression, the nucleotide sequence of which is shown in SEQ ID NO: 13, 14, 15 or 16.
  • SEQ ID NO: 13, 14, 15 or 16 was ligated to the reporter gene GUS, and the vector was constructed to transform rice and wheat, and the GUS expression activity and expression pattern in the transgenic plants were detected and analyzed, and the roots, stems, leaves and flowers of the transgenic plants were passed. GUS staining analysis was performed with the seeds, and it was found that the promoter-driven GUS gene provided by the present invention was expressed in the aleurone layer of plant seeds.
  • the blue particle gene promoter SEQ ID NO: 13, 14, 15 or 16 provided by the present invention is a promoter for aleurone-specific expression.
  • the aleurone layer-specific expression promoter provided by the present invention comprises the nucleotide sequence shown in SEQ ID NO: 13, 14, 15 or 16 in the sequence listing, or comprises SEQ ID NO: 13, 14, 15 or a nucleotide sequence having a nucleotide sequence of more than 90% similarity, or comprising 500 and more than 500 consecutive nucleotide fragments derived from the sequence of SEQ ID NO: 13, 14, 15 or 16, And can drive the nucleotide sequence operably linked to the promoter in the aleurone of the plant seed Expressed in layers.
  • Expression vectors, transgenic cell lines, host bacteria and the like containing the above sequences are all within the scope of the present invention.
  • Primer pairs that amplify any of the nucleotide fragments of the SEQ ID NO: 13, 14, 15 or 16 promoters disclosed herein are also within the scope of the invention.
  • a “promoter” as used herein refers to a DNA regulatory region that typically comprises a TATA box that directs RNA polymerase II to initiate RNA synthesis at a suitable transcription initiation site for a particular coding sequence. Promoters may also contain additional recognition sequences, which are typically located upstream or 5' of the TATA box, commonly referred to as upstream promoter elements, which function to regulate transcription efficiency. It will be appreciated by those skilled in the art that while nucleotide sequences for the promoter regions disclosed herein have been identified, other regulatory elements for isolating and identifying upstream regions of the TATA box of a particular promoter region identified in the present invention are also Within the scope of the invention.
  • the promoter regions disclosed herein are generally further defined as comprising upstream regulatory elements, such as those elements, enhancers, and the like, for regulating tissue expression and temporal expression functions of the coding sequences.
  • upstream regulatory elements such as those elements, enhancers, and the like
  • promoter elements that enable expression in a target tissue can be identified and isolated for use with other core promoters to verify the preferential expression of male tissues.
  • the core promoter refers to the minimal sequence required for initiation of transcription, such as the sequence known as the TATA box, which is commonly found in the promoters of genes encoding proteins.
  • the aleurone layer-specific expression promoter provided by the present invention can be used in association with a core promoter of its own or other origin.
  • the core promoter may be any known core promoter, such as the cauliflower mosaic virus 35S or 19S promoter (U.S. Patent No. 5,352,605), the ubiquitin promoter (U.S. Patent No. 5,510,474), IN2 core promoter. Son (U.S. Patent No. 5,364,780) or the Scrophularia mosaic virus promoter.
  • the function of the gene promoter of the present invention can be analyzed by operably linking a promoter sequence to a reporter gene to form a transformable vector, and then transferring the vector into a plant, and in obtaining a transgenic progeny, Observing the expression of the reporter gene in various tissues and organs of the plant to confirm its expression characteristics; or subcloning the above vector into an expression vector for transient expression experiments, and detecting the function of the promoter or its regulatory region by transient expression experiments.
  • the choice of appropriate expression vector for testing the function of the promoter or regulatory region will depend on the host and the method by which the expression vector is introduced into the host, such methods being well known to those of ordinary skill in the art.
  • the regions in the vector include regions that control transcription initiation and control processing. These regions are operably linked to a reporter gene, including the YFP, UidA, GUS gene or luciferase.
  • An expression vector comprising a putative regulatory region located in a genomic fragment can be introduced into a complete tissue, such as a staged pollen, or introduced into a callus for functional verification.
  • nucleotide sequence of the aleurone layer-specific promoter provided by the present invention or a fragment or variant thereof can be assembled together with the heterologous nucleotide sequence in an expression cassette for expression in the plant of interest, More specifically, it is expressed in the seeds of the plant.
  • the expression cassette has suitable restriction sites for insertion of the promoter and heterologous nucleotide sequences.
  • the aleurone layer-specific expression promoter disclosed in the invention can be used to drive the expression of the following genes, so that the transformed plants obtain corresponding phenotypes, including but not limited to genes related to yield increase, and enhancing seed nutritional value. Genes, genes that increase anthocyanin content, fluorescent protein genes, and the like.
  • the invention also provides an expression cassette, vector or engineering strain, wherein the expression cassette, vector or engineering strain comprises the aleurone layer specific expression promoter provided by the invention, SEQ ID NO: 13, 14, 15 or 16 Or comprising 500 and more than 500 consecutive nucleotide fragments derived from the sequence of SEQ ID NO: 13, 14, 15 or 16.
  • the provided aleurone layer-specific expression promoter of the present invention can be used for specific expression of a foreign gene in a seed, thereby avoiding the adverse effects of the sustained expression of the foreign gene in other tissues of the plant, in plant genetic engineering.
  • the research has important application value.
  • the nucleotide sequence and promoter sequence or expression cassette of the blue particle gene provided by the present invention can be inserted into a vector, a plasmid, a yeast artificial chromosome, a bacterial artificial chromosome or any other vector suitable for transformation into a host cell.
  • Preferred host cells are bacterial cells, especially bacterial cells for cloning or storing polynucleotides, or for transforming plant cells, such as Escherichia coli, Agrobacterium tumefaciens and Agrobacterium rhizogenes.
  • the expression cassette or vector can be inserted into the genome of the transformed plant cell. Insertions can be either positioned or randomly inserted.
  • Transferring a nucleotide sequence, vector or expression cassette into a plant or introducing a plant or transforming a plant according to the present invention means transferring a nucleotide sequence, a vector or an expression cassette to a receptor by a conventional transgenic method.
  • Any transgenic method known to those skilled in the art of plant biotechnology can be used to transform a recombinant expression vector into a plant cell to produce a transgenic plant of the invention. Transformation methods can include direct and indirect transformation methods. Suitable direct methods include polyethylene glycol-induced DNA uptake, liposome-mediated transformation, introduction using a gene gun, electroporation, and microinjection.
  • the transformation method also includes Agrobacterium-mediated plant transformation methods and the like.
  • the present invention Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a blue-grain gene and a promoter thereof, which can increase the content of anthocyanins in plants, since anthocyanins have resistance
  • the function of oxidation in the era of increasing environmental pollution and people's increasingly pursuit of healthy living, the discovery of genes related to anthocyanin synthesis undoubtedly increased the nutritional value and medical value of edible parts of plants.
  • the blue grain gene can also be used as a screening marker while improving the anthocyanin content of the plant, thereby eliminating the process of screening marker elimination in the transgenic process, saving the time and steps of the genetically modified organism engineering, in actual It has great application value in production applications.
  • Red grain colour gene(R) of wheat is a Myb-type transcription factor. Euphytica 143, 239-242.
  • Khlestkina E.K. Genes determining coloration of different organs in wheat.Russ.J.Genet.Appl.
  • Figure 1 shows the grain color of blue grain wheat and white grain wheat.
  • the picture on the left is the dark blue grain of the blue grain wheat 3114BB, and the picture on the right is the white grain of the parent white grain wheat 3114.
  • Figure 2 is a source of four blue-granule genes verified by genomic PCR for ThMYB1, ThMYB2, ThR1 and ThR2.
  • the left panel shows the agarose gel electrophoresis pattern of the PCR products of the ThMYB1 and ThMYB2 genes
  • the right panel shows the agarose gel electrophoresis pattern of the PCR products of the ThR1 and ThR2 genes.
  • the expression of four genes was not detected in the genome of white wheat 3114, and the expression of four genes was detected in the genomes of blue wheat 3114BB and long white wheat.
  • Figure 3 is a diagram showing the source of four blue-granule genes using semi-quantitative RT-PCR.
  • the agarose gel electrophoresis of the PCR product showed that no expression of four genes was detected in the cDNA of the white wheat 3114, but the expression of four genes was detected in the cDNA of the blue grain wheat 3114BB, among which ACTIN was the housekeeper. Protein gene.
  • Figure 4 is a graph showing the expression pattern of four blue-granule genes in Blue-grain wheat 3114BB by semi-quantitative RT-PCR.
  • the left picture shows the agarose gel electrophoresis pattern of the PCR product of the blue grain gene in different plant organs or tissues, and the right picture shows the blue grain gene in the aleurone layer.
  • the expression levels of different development days are not identical.
  • DPA refers to days post anthesis, which is “the number of days after flowering”.
  • Figure 5 is a simulation of four blue-grain genes using a gene gun transient transformation assay to induce red anthocyanin spots in wheat coleoptiles.
  • the blue-grain genes were combined and transferred to the wheat coleoptiles. After 16 hours of incubation in a light incubator, the wheat coleoptiles were observed by microscopy.
  • the combination of ThMYB1+ThR1 and ThMYB2+ThR1 induced the red cyanine in most cells.
  • the prime spot, ThMYB1+ThR2 combination can only induce anthocyanins in a very small number of cells, and the ThMYB2+ThR2 combination cannot induce the production of anthocyanins.
  • Figure 6 is a schematic representation of the construction of a plant transformation vector for wheat transgenic experiments.
  • LB and RB are the left and right borders of T-DNA, respectively;
  • the expression of Bar resistance gene is driven by Ubip (the promoter of Ubi gene) and terminated by Nos terminator;
  • the expression of ThMYB1 and ThR1 genes are respectively by the respective promoters and Terminator regulation.
  • FIG 7 is T 1 progenies of Grain color using Agrobacterium Wheat fielder background ThMYB1 + ThR1 transgenic wheat. Left fielder non-transgenic varieties of grain white wheat gene, the graph T 1 transgenic plants dark blue lines of grain, the right is T 1 transgenic plants lines were blue grain.
  • the present invention differentially expressed the blue and white wheat (see Fig. 1).
  • the differentially expressed genes of blue and white wheat contain Two types: 1) a gene expressed by the 4Ag chromosome of E. longissima, which contains the blue-granular gene to be cloned by the present invention; 2) a downstream gene change caused by the expression of the 4Ag chromosome of E. longissima, which is derived from Wheat genome.
  • the present invention contemplates the analysis of the blue particle gene described in 1).
  • the material selected blue grain wheat 3114BB and its parent white grain wheat 3114 because the blue color of the aleurone layer of the wheat grain begins about 20 days after flowering, and after about 25 days, the grain aleurone layer all turns blue, so the stripping of the present invention
  • the present invention employs a three-step exclusion method, first excluding the exact alignment to the double-ended sequence on the wheat reference genome, and secondly in the remaining pairs In the short-length sequence reassembling gene, genes highly expressed in white wheat were excluded, and finally genes unrelated to anthocyanin metabolism were excluded from the eligible differentially expressed genes.
  • the present invention obtained differentially expressed genes on 139 non-wheat reference genomes, 35 of which were highly expressed in blue-grain wheat and almost not expressed in white wheat, and were characterized by gene function annotation to exclude anthocyanins.
  • the present invention obtains the coding region genomic sequences of the two genes by PCR amplification.
  • the present invention finds that whether it is ThMYB1 or ThR1, another one is obtained during PCR amplification.
  • the highly homologous sequence thus the two homologous sequences of the invention were cloned and named as ThMYB2 and ThR2, respectively.
  • the promoter sequences and terminator sequences of the four genes are obtained by the present invention by genome walking.
  • genomic nucleotide sequence of the ThMYB1 gene from the initiation codon to the stop codon is as shown in SEQ ID NO: 1
  • nucleotide sequence of the coding region (CDS) is as shown in SEQ ID NO: 2
  • the amino acid sequence encoded by CDS is shown in SEQ ID NO: 3
  • the promoter sequence thereof is shown in SEQ ID NO: 13
  • the terminator sequence thereof is shown in SEQ ID NO: 17.
  • the genomic nucleotide sequence of the ThMYB2 gene from the initiation codon to the stop codon is shown in SEQ ID NO: 4, and the coding region (CDS) nucleotide sequence is shown in SEQ ID NO: 5, and its CDS
  • CDS coding region
  • the encoded amino acid sequence is set forth in SEQ ID NO: 6, the promoter sequence of which is set forth in SEQ ID NO: 14, and the terminator sequence thereof is set forth in SEQ ID NO: 18.
  • the genomic nucleotide sequence of the ThR1 gene from the start codon to the stop codon is as shown in SEQ ID NO: 7, and the coding region (CDS) nucleotide sequence is shown in SEQ ID NO: 8, and the CDS is The encoded amino acid sequence is shown in SEQ ID NO: 9, the promoter sequence is shown in SEQ ID NO: 15, and the terminator sequence is shown in SEQ ID NO: 19.
  • the genomic nucleotide sequence of the ThR2 gene from the start codon to the stop codon is shown in SEQ ID NO: 10
  • the coding region (CDS) nucleotide sequence is shown in SEQ ID NO: 11
  • the CDS is The encoded amino acid sequence is set forth in SEQ ID NO: 12, the promoter sequence of which is set forth in SEQ ID NO: 16, and the terminator sequence thereof is set forth in SEQ ID NO: 20.
  • the present invention verifies the sources of these four genes by genomic PCR and semi-quantitative RT-PCR.
  • Genomic PCR showed that ThMYB1, ThMYB2, ThR1 and ThR2 could not be amplified in B. hirsutum 3114, but these four genes could be amplified in the blue-grain wheat 3114BB and E. longissima genomes.
  • the genes are indeed from the 4Ag chromosome of E. longissima but not from common wheat (see Figure 2).
  • the present invention verifies the expression patterns of these four genes by semi-quantitative RT-PCR.
  • the result is shown in Figure 4.
  • no expression of 4 genes was detected in the roots, stems and leaves of vegetative organs of 3114BB.
  • 4 genes were specifically in the aleurone layer.
  • Tissue expression was not detected in embryo and endosperm tissues, indicating that these four genes are genes specifically expressed in the aleurone tissue, and the promoters are promoters specifically driving the gene in the aleurone layer.
  • the present invention analyzes the expression patterns of ThMYB1, ThMYB2, ThR1 and ThR2 in the blue-grain wheat seed aleurone layer on different days after flowering, and finds that the expression patterns of two MYB and two bHLH genes are different: ThMYB1 and ThMYB2 are in 10DPA And 15DPA aleurone layer expressed at a very low level, abruptly high expression in 20DPA expression, and then gradually decreased in 25DPA and 30DPA; ThR1 and ThR2 were not detected in 10DPA and 15DPA aleurone layers, starting from 20DPA to 30DPA Volume expression.
  • ThMYB1, ThMYB2, ThR1 and ThR2 are all wheat aleurone-specific expression genes, and their expression has space-time specificity.
  • the promoter sequences of the above four genes, SEQ ID NO: 13, 14, 15 and 16 were driven to functionally verify the GUS gene in rice, maize and other plants. It was found that the above promoters specifically drive GUS expression in the aleurone layer, indicating The above promoters provided by the present invention are all aleurone layer-specific expression promoters.
  • the present invention constructs the gene gun transient transformation vector of these four genes.
  • the NOS terminator was cloned into the pEASY-T1simple (full-scale gold company) vector, and then the coding frames of the four genes ThMYB1, ThMYB2, ThR1 and ThR2 were inserted into the NOS, and finally the Ubi from corn was in-fusion.
  • the (Ubiquitin) promoter drives gene expression before insertion into the gene coding frame to form four vectors: Ubi::ThMYB1, Ubi::ThMYB2, Ubi::ThR1 and Ubi::ThR2.
  • the experimental method is referred to (Ahmed et al, 2003), and the transformation vectors of MYB and bHLH genes are respectively combined into two, that is, the combination of ThMYB1+ThR1, ThMYB1+ThR2, ThMYB2+ThR1 and ThMYB2+ThR2 is transferred to the wheat coleoptile. After incubation for 16 hours in a light incubator, the cells were observed under a microscope and photographed. The results are shown in Figure 5. The combination of ThMYB1+ThR1 and ThMYB2+ThR1 can induce the production of anthocyanins, and ThMYB1+ThR2 can also induce cells to produce cyanine. Prime.
  • ThMYB1, ThMYB2, ThR1 and ThR2 are blue-granular genes
  • the present invention selects ThMYB1.
  • ThR1 two genes were constructed to construct a stable transformation vector for wheat transgenic experiments.
  • the binary expression vector pCAMBIA1300 as a backbone
  • the hygromycin resistance expression cassette driven by the 35S promoter of the plant resistance screening gene on pCAMBIA1300 was first replaced with the Bar resistance gene expression cassette driven by the Ubi promoter on the pAHC20 vector.
  • the present invention will have a 3215 bp ThMYB1 genomic sequence (containing a 1952 bp promoter sequence, a 822 bp genomic sequence and a 441 bp terminator sequence) and a 4422 bp ThR1 genomic sequence (including a 2084 bp promoter sequence, a 1720 bp CDS sequence and a 618 bp terminator sequence).
  • This inserts a multiple cloning site to form a plant expression vector (see Figure 6).
  • Example 5 Obtainment of transgenic blue grain wheat
  • the plant expression vector constructed in Example 4 was transformed into Agrobacterium strain C58C1 by electroporation.
  • the Agrobacterium-mediated wheat transformation system was used to transfer the vector into the wheat variety fielder, and 96 transgenic positive T 0 plants were obtained. When the T 0 generation plants were harvested, the color of wheat seeds was observed. Some of the lines were separated by dark blue particles, and some of the lines were separated by light blue particles (about 34%).
  • the specific results are shown in Figure 7.
  • the transgenic results indicate that the ThMYB1 and ThR1 genes are co-expressed in plants and can increase the anthocyanin content in plants.
  • the ThMYB1, ThMYB2, ThR1 and ThR2 provided by the present invention are blue-granular genes in wheat.
  • ThMYB1 or ThMYB2 gene provided by the present invention is combined with the ThR1, ThR2, ZmR and ZmB genes by a MYB gene plus an HLH gene, and transferred into rice, Arabidopsis, maize and other plants for co-expression. It was found to have the same function of increasing the anthocyanin content in the plants.

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Abstract

一种小麦蓝粒基因及其应用，属于植物分子生物学、生物化学、遗传学和植物育种领域。利用蓝粒和白粒小麦的差异表达分析，获得了控制小麦蓝粒性状的四个基因：两个MYB家族转录因子和两个bHLH家族转录因子，并且提供了上述基因的植物重组表达载体和一种调控植物花青素合成的方法，对于研究蓝粒小麦糊粉层色素的合成途径、用作植物转化过程中的筛选标记和提高植株的营养价值具有重要的理论和实践意义。

Description

小麦蓝粒基因及其应用 技术领域
本发明涉及植物分子生物学、生物化学、遗传学和植物育种领域，特别涉及一种调控小麦蓝粒性状的基因。本发明提供四种小麦蓝粒性状相关基因的核酸分子和植物重组载体，以及所述基因或载体的应用方法。
技术背景
普通小麦的籽粒天然存在两种颜色：白粒或红粒，除此之外还有极少数为蓝粒或紫粒。蓝粒或紫粒小麦可以作为重要的遗传标记应用于小麦的遗传育种，特别是在我国，蓝粒小麦的应用研究比较深入，比如“4E-ms杂交小麦生产体系”成功地利用了蓝粒特征进行小麦核雄性不育的有效保持(Zhou et al，2006)。
小麦籽粒的胚乳外面自外向内包裹着三层组织，分别是：果皮、种皮和糊粉层，小麦的籽粒颜色是由不同的组织中积累的不同花青素所决定的。紫粒小麦源于最外层果皮中的紫色花青素，果皮是由母本发育而成的，因此紫粒性状呈现母系遗传模式。而蓝粒小麦源于糊粉层的蓝色花青素，主要是由普通小麦与其他物种之间的远源杂交产生的，其来源主要有两个：长穗偃麦草(Thinopyrum ponticum)和野生一粒小麦(Triticum monococcum)。上世纪六十年代到八十年代，国内外多位学者，如Knott、Sharman、李振声等均从长穗偃麦草与普通小麦的杂交后代中获得了蓝粒小麦，并且证明长穗偃麦草的4E(又名4Ag)染色体携有蓝粒基因。李振声等通过遗传分析，认为蓝粒基因遗传稳定而独立、具有明显的剂量效应，其蓝粒性状很像受一对基因遗传控制(李振声等，1982)。对一系列不同的蓝粒易位系的GISH和FISH实验分析进一步将蓝粒基因定位在4Ag染色体的长臂0.71-0.80区段内(与着丝粒的距离)(Zheng et al，2006)。1990年Keppenne把来源于偃麦草的蓝色糊粉层基因命名Blue aleurone(Ba)基因，也有国外学者认为蓝色性状由两个互补基因共同控制。1982年Joppa等证明由一粒小麦衍生出的Blaukom系列是普通小麦的4A或4B染色体被一粒小麦的一对4A染色体代换后形成的，来自一粒小麦的4Am染色体与四倍体和六倍体小麦中的4A染色体是非同源的。1989年Kuspiral等把来源于野生一粒小麦的蓝色糊粉层基因命名为Ba2基因，Ba2基因定位在4Am染色体长臂的近着丝粒处(Dubcovsky et al，1996)。
目前为止，在小麦中控制红粒和紫粒性状的基因都已经克隆，关于蓝粒性状尚没有报道。2005年Himi等报道了定位于3AL、3BL和3DL染色体的红粒性状主效基因R1、R2和R3编码3个高度同源的Myb家族转录因子(Himi et al，2005)。紫粒小麦的遗传分析显示紫粒性状由位于7号染色体组短臂的Pp-1(purple pericarp)位点和位于2AL染色体的Pp3位点控制，其中Pp-1编码Myb家族转录因子，而Pp3编码含有bHLH(basic helix-loop-helix)结构 域的Myc家族转录因子(Khlestkina et al，2013；Shoeva et al，2014)。
决定小麦籽粒颜色的花青素是一种水溶性的次生代谢产物—类黄酮化合物，广泛分布于高等植物的根、茎、叶、花、果实和种子中。植物花青素合成属于类黄酮合成途径的一个分支，该途径中主要合成酶包括查尔酮合成酶(chalcone synthase,CHS)、查尔酮异构酶(chalcone isomerase,CHI)、黄烷酮-3-羟化酶(flavanone-3-hydroxylase,F3H)、二氢黄酮醇-4-还原酶(dihydroflavonol-4-reductase,DFR)等(宫硖等,2011)，这些基因均为花青素合成的结构基因。而对花青素合成的调控主要是由三类转录因子完成的：MYB、bHLH和WD40。大部分物种的花青素都是由这三类转录因子复合而成的蛋白复合体直接调控激活的，也有少数花青素合成只需要单个调控因子就能激活。
蓝粒小麦是小麦标记性状、小麦细胞遗传学研究的理想形态学特征，也是小麦染色体工程研究中的重要基础材料。本发明利用蓝粒和白粒小麦的差异表达分析，获得了控制小麦蓝粒性状的4个基因：两个MYB家族转录因子和两个bHLH家族转录因子。本发明有助于研究蓝粒小麦糊粉层色素的合成途径，可用作植物转化过程中的筛选标记，同时将该基因在植物中进行表达，可以增加花青素的合成，从而提高植株的营养价值。
发明内容
本文提到的所有参考文献都通过引用并入本文。
除非有相反指明，本文所用的所有技术和科学术语都具有与本发明所属领域普通技术人员通常所理解的相同的含义。除非有相反指明，本文所使用的或提到的技术是本领域普通技术人员公知的标准技术。材料、方法和例子仅作阐述用，而非加以限制。
本发明提供了一种蓝粒基因，具有花青素合成调控功能，所述蓝粒基因的名称分别是ThMYB1、ThMYB2、ThR1和ThR2，所述蓝粒基因可以使其表达部位的组织或器官变为蓝色。其中ThMYB1基因的从起始密码子到终止密码子的基因组核苷酸序列如SEQ ID NO:1所示，其编码区(CDS)的核苷酸序列如SEQ ID NO:2所示，其CDS所编码的氨基酸序列如SEQ ID NO:3所示。其中ThMYB2基因的从起始密码子到终止密码子的基因组核苷酸序列如SEQ ID NO:4所示，其编码区(CDS)核苷酸序列如SEQ ID NO:5所示，其CDS所编码的氨基酸序列如SEQ ID NO:6所示。其中ThR1基因的从起始密码子到终止密码子的基因组核苷酸序列如SEQ ID NO:7所示，其编码区(CDS)核苷酸序列如SEQ ID NO:8所示，其CDS所编码的氨基酸序列如SEQ ID NO:9所示。其中ThR2基因的从起始密码子到终止密码子的基因组核苷酸序列如SEQ ID NO:10所示，其编码区(CDS)核苷酸序列如SEQ ID NO:11所示，其CDS所编码的氨基酸序列如SEQ ID NO:12所示。
本领域技术人员应该知晓，本发明所述的蓝粒基因还包括与ThMYB1、ThMYB2、ThR1和ThR2基因的核苷酸序列或蛋白序列高度同源，并且具有同样的调控植物花青素合成功能的核苷酸或蛋白序列。所述高度同源且具有花青素合成调控功能的的同源基因包括在严谨条件下能够与具有SEQ ID NO:1、2、4、5、7、8、10或11所示序列的DNA杂交的DNA序列。或是其编码的氨基酸序列与SEQ ID NO:3、6、9或12所示的蛋白氨基酸序列具有85％以上相似性的核苷酸序列。本文中使用的“严谨条件”是公知的，包括诸如在含400mM NaCl、40mM PIPES(pH6.4)和1mM EDTA的杂交液中杂交，所述杂交的温度优选是53℃-60℃，杂交时间优选为12-16小时，然后用含0.5×SSC、和0.1％SDS的洗涤液洗涤，洗涤温度优选为62℃-68℃，洗涤时间为15-60分钟。
上述同源基因还包括与SEQ ID NO:1、2、4、5、7、8、10或11所示序列的全长有至少80％、85％、90％、95％、98％、或99％序列相似性，且具有调控植物花青素合成功能的DNA序列，可以从任何植物中分离获得。其中，序列相似性的百分比可以通过公知的生物信息学算法来获得，包括Myers和Miller算法、Needleman-Wunsch全局比对法、Smith-Waterman局部比对法、Pearson和Lipman相似性搜索法、Karlin和Altschul的算法。这对于本领域技术人员来说是公知的。
本发明还提供了一种表达盒，所述表达盒含有本发明所公开的蓝粒基因的DNA序列，所述蓝粒基因的核苷酸序列选自下列组的序列之一：
(a)如SEQ ID NO：1、2、4、5、7、8、10或11所示的核苷酸序列；
(b)其编码氨基酸序列如SEQ ID NO：3、6、9或12所示的核苷酸序列；
(c)在严谨条件下能够与(a)或(b)中所述序列的DNA杂交的DNA序列；或
(d)与(a)-(c)所述序列有至少80％(优选为至少85％)序列相似性，且具有花青素合成调控功能的DNA序列；或
(e)与(a)-(d)之任一所述序列互补的DNA序列。
具体地，上述表达盒中的蓝粒基因还可操作性的连有一个可驱动其表达的启动子，所述启动子包括但不限于组成型启动子、诱导型启动子、组织特异性启动子、或时空特异表达启动子。本发明所述的组成型启动子的基因表达不具组织和时间特异性，外界因素对组成型启动子启动的外源基因表达几乎没有影响。所述组成型启动子包括但不限于CaMV35S、FMV35S、水稻肌动蛋白(Actin1)启动子、玉米泛素(Ubiquitin)启动子等。本发明所述的组织特异性启动子除包含应有的一般启动子元件外，还具有增强子以及沉默子的特性，该类启动子的优点在于可启动基因在植物特定组织部位的表达，避免外源基因的不必要表达，从而节约植物 体的整体能量消耗。所述组织特异性启动子包括但不限于LTP2种子特异表达启动子、END2种子特异表达启动子、糊粉层特异表达启动子等。本发明所述的诱导型启动子是指在某些特定的物理或化学信号的刺激下，可以大幅度地提高基因的转录水平的启动子，目前已经分离的诱导型启动子包括但不限于逆境诱导表达启动子、光诱导表达启动子、热诱导表达启动子、创伤诱导表达启动子、真菌诱导表达启动子和共生细菌诱导表达启动子等。
本发明上述表达盒，还进一步的可以包含一个筛选基因，所述筛选基因可以用于将含有该表达盒的植株、植物组织细胞或载体筛选出来。所述筛选基因包括但不限于抗生素抗性基因、或是抗除草剂基因、或是荧光蛋白基因等。具体地，所述筛选基因包括但不限于：氯霉素抗性基因、潮霉素抗性基因、链霉素抗性基因、奇霉素抗性基因、磺胺类抗性基因、草甘磷抗性基因、草丁膦抗性基因、bar基因、红色荧光基因DsRED、mCherry基因、青色荧光蛋白基因、黄色荧光蛋白基因、荧光素酶基因、绿色荧光蛋白基因等。
本发明还公开了一种提高植物花青素含量的方法，所述方法通过将本发明所提供的ThMYB1或ThMYB2基因与任一个bHLH转录因子在植物的组织器官中进行共表达，即可提高植物组织器官中的花青素含量。
所述bHLH转录因子可分离自任一植物，包括但不限于本发明所提供的ThR1和ThR2基因，及来自玉米的ZmR、ZmB基因(Ahmed N,et al.Transient expression of anthocyanin in developing wheat coleoptile by maize C1and B-peru regulatory genes for anthocyanin synthesis.Breeding Sci.2003；53(1):29-34.)。
上述提高植物花青素含量的方法，可以用于提高植物任一组织或器官的花青素含量。具体地，如果想整体提高植物各个组织中的花青素含量，可以将ThMYB1或ThMYB2基因与bHLH转录因子均用组成型启动子启动表达。如果只想提高某个组织或器官中的花青素含量，可以将ThMYB1或ThMYB2基因与bHLH转录因子均用该组织或器官中特异性表达的启动子启动表达。
本发明还公开了一种提高植物花青素含量的方法，所述方法通过将本发明所提供的ThR1或ThR2基因与任一个MYB类转录因子在植物的组织器官中进行共表达，即可提高植物组织器官中的花青素含量。
所述MYB类转录因子可分离自任一植物，包括但不限于本发明所提供的ThMYB1和ThMYB2基因，及来自玉米的ZmC1基因(Ahmed N,et al.Transient expression of anthocyanin in developing wheat coleoptile by maize C1and B-peru regulatory genes for anthocyanin synthesis.Breeding Sci.2003；53(1):29-34.)。
上述提高植物花青素含量的方法，可以用于提高植物任一组织或器官的花青素含量。具 体地，如果想整体提高植物各个组织中的花青素含量，可以将ThR1或ThR2基因与MYB类转录因子均用组成型启动子启动表达。如果只想提高某个组织或器官中的花青素含量，可以将ThR1或ThR2基因与MYB类转录因子均用该组织或器官中特异性表达的启动子启动表达。
本发明还提供了一种可视的筛选标记基因，所述筛选标记通过将ThMYB1或ThMYB2基因与与任一个bHLH转录因子进行共表达，从而在植物的组织器官中产生肉眼可见的蓝色筛选标记。或是通过将本发明所提供的ThR1或ThR2基因与任一个MYB类转录因子在植物的组织器官中进行共表达，从而在植物的组织器官中产生肉眼可见的蓝色筛选标记。
本发明所公开的筛选标记基因，可以用于区分转基因与非转基因材料。
具体地，本发明所提供的筛选标记基因，可以用作雄性不育系繁殖过程中的筛选标记，将育性恢复基因、花粉失活基因和本发明提供的筛选标记基因转入到雄性不育系中后，育性恢复基因可使雄性不育系育性恢复，花粉失活基因可使含有转化的外源基因的花粉失活，即失去授精能力，本发明所提供的筛选标记基因可以用于转基因种子或组织和非转基因种子或组织的分拣，分拣出的非转基因种子用作不育系生产杂交种，转基因种子用作保持系来源源不断地、稳定地生产不育系。
本发明所提供的筛选标记基因，也可以用作雌性不育系繁殖过程中的筛选标记，将雌性育性基因、花粉失活基因和本发明所提供的筛选标记基因转入到雌性不育系中。其中，雌性育性基因可使雌性不育的转化受体材料恢复育性，花粉失活基因可使含有转化的外源构建体的花粉失活，即失去授精能力，本发明所提供的筛选标记基因可以用于转基因种子和非转基因种子的分拣，分拣出的非转基因种子用作雌性不育系生产杂交种，转基因种子用作保持系来源源不断地、稳定地生产雌性不育系和雌性不育保持系。
本发明还提供了蓝粒基因的启动子，所述启动子具有糊粉层特异表达的特性，其核苷酸序列如SEQ ID NO:13、14、15或16所示。将SEQ ID NO:13、14、15或16与报告基因GUS相连，构建载体转化水稻和小麦，检测分析转基因植株中的GUS表达活性和表达模式，通过对转基因植株的根、茎、叶、花和种子进行GUS染色分析，结果发现本发明所提供的启动子驱动GUS基因在植物种子的糊粉层中表达。说明本发明所提供的蓝粒基因启动子SEQ ID NO:13、14、15或16是一个糊粉层特异性表达的启动子。
本发明所提供的糊粉层特异表达启动子，含有序列表中如SEQ ID NO:13、14、15或16所示的核苷酸序列，或包含与SEQ ID NO:13、14、15或16中所列核苷酸序列具有90％以上相似性的核苷酸序列，或包含来源于SEQ ID NO:13、14、15或16序列上的500个及500以上连续的核苷酸片段，并且可以驱动与该启动子操作性连接的核苷酸序列在植物种子的糊粉 层中表达。含有上述序列的表达载体、转基因细胞系以及宿主菌等均属于本发明的保护范围。扩增本发明所公开的SEQ ID NO:13、14、15或16启动子的任一核苷酸片段的引物对也在本发明的保护范围之内。
本发明所述的“启动子”是指一种DNA调控区域，其通常包含能指导RNA聚合酶II在特定编码序列的合适转录起始位点起始RNA合成的TATA盒。启动子还可包含其它识别序列，这些识别序列通常位于TATA盒的上游或5’端，通常被称为上游启动子元件，起调控转录效率的作用。本领域技术人员应该知晓，虽然已经鉴定了针对本发明公开的启动子区域的核苷酸序列，但是分离和鉴定处于本发明鉴定的特定启动子区域的TATA盒上游区域的其它调控元件也在本发明的范围内。因此，本文公开的启动子区域通常被进一步界定为包含上游调控元件，例如用于调控编码序列的组织表达性和时间表达功能的那些元件、增强子等。以相同的方式，可以鉴定、分离出使得能在目标组织(例如雄性组织)中进行表达的启动子元件，将其与其它核心启动子一起使用，以验证雄性组织优先的表达。核心启动子指起始转录所需的最小限度的序列，例如被称为TATA盒的序列，这是编码蛋白质的基因的启动子通常都具有的。因此，可选地，本发明所提供的糊粉层特异表达启动子可与其自身的或其它来源的核心启动子关联使用。所述核心启动子可以是任何一种已知的核心启动子，例如花椰菜花叶病毒35S或19S启动子(美国专利No.5,352,605)、泛素启动子(美国专利No.5,510,474)、IN2核心启动子(美国专利No.5,364,780)或玄参花叶病毒启动子。
本发明所述基因启动子的功能可以通过以下方法进行分析：将启动子序列与报告基因可操作性连接，形成可转化的载体，再将该载体转入植株中，在获得转基因后代中，通过观察报告基因在植物各个组织器官中的表达情况来确认其表达特性；或者将上述载体亚克隆进用于瞬时表达实验的表达载体，通过瞬时表达实验来检测启动子或其调控区的功能。
用来测试启动子或调控区域功能的适当表达载体的选择将取决于宿主和将该表达载体引入宿主的方法，这类方法是本领域普通技术人员所熟知的。对于真核生物，在载体中的区域包括控制转录起始和控制加工的区域。这些区域被可操作地连接到报告基因，所述报告基因包括YFP、UidA、GUS基因或荧光素酶。包含位于基因组片段中的推定调控区的表达载体可以被引入完整的组织，例如阶段性花粉，或引入愈伤组织，以进行功能验证。
此外，可以将本发明所提供的糊粉层特异表达启动子的核苷酸序列或其片段或变体与异源核苷酸序列一起组装在一个表达盒中，用于在目的植株中表达，更具体地，在该植株的种子中表达。所述表达盒有合适的限制性酶切位点，用于插入所述启动子和异源核苷酸序列。这些表达盒可用于对任何植株进行遗传操作，以获得想要的相应表型。
本发明所公开的糊粉层特异表达启动子，可用于驱动下列基因的表达，以使转化的植株获得相应的表型，所述基因包括但不限于与增产相关的基因、提高种子营养价值的基因、提高花青素含量的基因、荧光蛋白基因等。
本发明还提供了一种表达盒、载体或工程菌株，所述表达盒、载体或工程菌株中包含了本发明所提供的糊粉层特异表达启动子SEQ ID NO:13、14、15或16，或包含来源于SEQ ID NO:13、14、15或16序列上的500个及500以上连续的核苷酸片段。
本发明的所提供的糊粉层特异表达启动子可用于外源基因在种子中的特异性表达，从而避免该外源基因在植物其他组织中持续表达所带来的不利影响，在植物基因工程研究中具有重要的应用价值。
本发明所提供的蓝粒基因的核苷酸序列和启动子序列或表达盒可被插入载体、质粒、酵母人工染色体、细菌人工染色体或其他适合转化进宿主细胞中的任何载体中。优选的宿主细胞是细菌细胞，尤其是用于克隆或储存多核苷酸、或用于转化植物细胞的细菌细胞，例如大肠杆菌、根瘤土壤杆菌和毛根土壤杆菌。当宿主细胞是植物细胞时，表达盒或载体可插入至被转化的植物细胞的基因组中。插入可以是定位的或随机的插入。
本发明所述的将核苷酸序列、载体或表达盒转入植株或引入植株或对植株进行转化，均指通过常规的转基因方法，将核苷酸序列、载体或表达盒转入到受体细胞或受体植株中。植物生物技术领域技术人员已知的任何转基因方法均可被用于将重组表达载体转化进植物细胞中，以产生本发明的转基因植物。转化方法可包括直接和间接的转化方法。合适的直接方法包括聚乙二醇诱导的DNA摄入、脂质体介导的转化、使用基因枪导入、电穿孔、以及显微注射。所述转化方法也包括农杆菌介导的植物转化方法等。
与现有技术相比，本发明具有如下的有益效果：本发明提供了一种蓝粒基因及其启动子，所述蓝粒基因可以提高植物中花青素的含量，由于花青素具有抗氧化的功能，在环境污染加剧，人们日益追求健康生活的时代，该花青素合成相关基因的发现，无疑增加了植物可食用部位的营养价值和医学价值。同时，该蓝粒基因在提高植物的花青素含量的同时，还可以用作筛选标记，免去了转基因过程中的筛选标记剔除的过程，节约了转基因生物工程的时间和步骤，在实际的生产应用中具有重大的应用价值。
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附图用来提供对本发明的进一步理解，并且构成说明书的一部分，与下面的具体实施方式一起用于解释本发明，但并不构成对本发明的限制。
附图说明
图1是蓝粒小麦和白粒小麦的籽粒颜色。左图为蓝粒小麦3114BB的深蓝色籽粒，右图为其亲本白粒小麦3114的白色籽粒。
图2是利用基因组PCR验证ThMYB1、ThMYB2、ThR1和ThR2四个蓝粒基因的来源。左图为ThMYB1和ThMYB2基因的PCR产物的琼脂糖凝胶电泳图，右图为ThR1和ThR2基因的PCR产物的琼脂糖凝胶电泳图。在白粒小麦3114的基因组中检测不到四个基因的表达，而在蓝粒小麦3114BB和长穗偃麦草的基因组中均可检测到四个基因的表达。
图3是利用半定量RT-PCR验证四个蓝粒基因的来源。PCR产物的琼脂糖凝胶电泳图显示，在白粒小麦3114的cDNA中检测不到四个基因的表达，但在蓝粒小麦3114BB的cDNA中可检测到四个基因的表达，其中ACTIN为管家蛋白基因。
图4是利用半定量RT-PCR验证四个蓝粒基因在蓝粒小麦3114BB中的表达模式。左图为蓝粒基因在不同植物器官或组织中的PCR产物的琼脂糖凝胶电泳图，右图为蓝粒基因在糊粉层的 各个发育时期的PCR产物的琼脂糖凝胶电泳图，其中ACTIN为管家蛋白基因。在根、茎、叶、胚和胚乳的cDNA中均检测不到四个基因的表达，而在糊粉层中可检测到四个基因的高量表达，且不同蓝粒基因在糊粉层的不同发育天数表达量不完全相同。DPA是指days post anthesis，即“开花后天数”。
图5是利用基因枪瞬时转化实验验证四个蓝粒基因能够诱导小麦胚芽鞘产生红色花青素斑点。将蓝粒基因两两组合后分别共转小麦胚芽鞘，光照培养箱中培养16小时后，利用显微镜观察小麦胚芽鞘发现，ThMYB1+ThR1组合和ThMYB2+ThR1组合能够诱导大部分细胞产生红色花青素斑点，ThMYB1+ThR2组合只能诱导极少数细胞产生花青素，ThMYB2+ThR2组合不能诱导产生花青素。
图6是用于小麦转基因实验的植物转化载体的构建示意图。其中，LB和RB分别为T-DNA的左右边界；Bar抗性基因的表达由Ubip(Ubi基因的启动子)驱动，由Nos终止子终止；ThMYB1和ThR1基因的表达分别由各自的启动子和终止子调控。
图7是利用农杆菌介导获得小麦品种fielder背景下ThMYB1+ThR1转基因小麦的T1代植株的籽粒颜色。左图为非转基因小麦品种fielder的白色籽粒，中图为T1代转基因植株中呈深蓝色籽粒的株系，右图为T1代转基因植株中呈浅蓝色籽粒的株系。
具体实施方式
下面对本发明的实施例作详细说明，本实施例在以本发明技术方案为前提下进行实施，给出了详细的实施方式和具体的操作过程，但本发明的保护范围不限于下述的实施例。
实施例1、蓝粒基因的克隆
为了克隆来自长穗偃麦草4Ag染色体上的蓝粒基因，本发明对蓝粒和白粒小麦进行了差异表达分析(见图1)，理论上说，蓝粒与白粒小麦的差异表达基因包含了两种类型：1)由长穗偃麦草4Ag染色体表达的基因，其中包含本发明想要克隆的蓝粒基因；2)由长穗偃麦草4Ag染色体表达所引起的下游基因变化，这些基因来自小麦基因组。本发明希望从中分析获得1)中所述的蓝粒基因。材料选取蓝粒小麦3114BB与其亲本白粒小麦3114，因为小麦籽粒的糊粉层蓝色出现始于开花后约20天，到约25天后籽粒糊粉层全部变为蓝色，因此本发明剥取了开花后25天的糊粉层样品，蓝粒和白粒材料各取2份，分别记为blue 1、blue 2、white 1和white 2，提取RNA并进行高通量测序(PE125)，每个样品各测9G数据量。
由于发布的小麦参考基因组只覆盖小麦整个基因组的61％，并且基因注释的完整性和组装序列(scaffolds)片段化，因此直接的序列比对和差异基因表达分析并不能找到目标基因。本发明运用三步排除法，首先排除准确比对到小麦参考基因组上的双端序列，其次在剩余双 端短序列重组装基因中，排除在白粒小麦中高表达基因，最后在符合条件的差异表达基因中排除与花青素代谢无关的基因。
经过上述分析，本发明得到139个非小麦参考基因组上的差异表达基因，其中35个为蓝粒小麦中高表达、白粒小麦中几乎不表达的基因，经过基因功能注释分析，排除与花青素代谢无关的基因后，只有两个目标基因，分别编码MYB蛋白与bHLH蛋白，本发明将其命名为ThMYB1和ThR1。在已知cDNA序列的基础上，本发明通过PCR扩增获得了这两个基因的编码区基因组序列，在此过程中，本发明发现不管是ThMYB1还是ThR1，PCR扩增时会获得另外一个与之高度同源的序列，因此本发明克隆了这两个同源序列并将其分别命名为ThMYB2和ThR2。通过染色体步移技术(genome walking)，本发明分别获得了这4个基因的启动子序列和终止子序列。
其中，ThMYB1基因的从起始密码子到终止密码子的基因组核苷酸序列如SEQ ID NO:1所示，其编码区(CDS)的核苷酸序列如SEQ ID NO:2所示，其CDS所编码的氨基酸序列如SEQ ID NO:3所示，其启动子序列如SEQ ID NO:13所示,其终止子序列如SEQ ID NO:17所示。其中ThMYB2基因的从起始密码子到终止密码子的基因组核苷酸序列如SEQ ID NO:4所示，其编码区(CDS)核苷酸序列如SEQ ID NO:5所示，其CDS所编码的氨基酸序列如SEQ ID NO:6所示，其启动子序列如SEQ ID NO:14所示，其终止子序列如SEQ ID NO:18所示。其中ThR1基因的从起始密码子到终止密码子的基因组核苷酸序列如SEQ ID NO:7所示，其编码区(CDS)核苷酸序列如SEQ ID NO:8所示，其CDS所编码的氨基酸序列如SEQ ID NO:9所示，其启动子序列如SEQ ID NO:15所示，其终止子序列如SEQ ID NO:19所示。其中ThR2基因的从起始密码子到终止密码子的基因组核苷酸序列如SEQ ID NO:10所示，其编码区(CDS)核苷酸序列如SEQ ID NO:11所示，其CDS所编码的氨基酸序列如SEQ ID NO:12所示，其启动子序列如SEQ ID NO:16所示，其终止子序列如SEQ ID NO:20所示。
本发明通过基因组PCR和半定量RT-PCR验证了这4个基因的来源。基因组PCR显示：在白粒小麦3114中，扩增不到ThMYB1、ThMYB2、ThR1和ThR2，而在蓝粒小麦3114BB和长穗偃麦草基因组中则都可以扩增到这4个基因，说明这四个基因确实是来自于长穗偃麦草4Ag染色体而非来自普通小麦(见图2)。在开花后25天的糊粉层组织cDNA中的半定量RT-PCR结果也显示：ThMYB1、ThMYB2、ThR1和ThR2均不在白粒小麦糊粉层中表达而只在蓝粒小麦糊粉层中高量表达(见图3)。上述结果表明，ThMYB1、ThMYB2、ThR1和ThR2这四个基因均来自长穗偃麦草4Ag染色体，并且在蓝粒小麦糊粉层中高量表达，很可能是本发明寻找的蓝粒基因。
实施例2、蓝粒基因的表达模式
本发明用半定量RT-PCR验证了这4个基因的表达模式。结果如图4所示。首先，在蓝粒小麦3114BB营养器官根、茎和叶中，均检测不到4个基因的表达，在开花后20天(20DPA)种子的不同组织中，4个基因均特异地在糊粉层组织表达，而在胚和胚乳组织中均检测不到，表明这4个基因均为糊粉层组织特异表达的基因，其启动子均为驱动基因在糊粉层中特异表达的启动子。进一步本发明分析了开花后不同天数的蓝粒小麦种子糊粉层中ThMYB1、ThMYB2、ThR1和ThR2的表达模式，发现2个MYB与2个bHLH基因的表达模式不尽相同：ThMYB1和ThMYB2在10DPA和15DPA糊粉层中以很低水平表达，在20DPA表达突然高量表达，此后在25DPA和30DPA逐渐降低；ThR1和ThR2在10DPA和15DPA糊粉层中检测不到，从20DPA开始到30DPA持续高量表达。以上结果显示，ThMYB1、ThMYB2、ThR1和ThR2均为小麦糊粉层特异表达基因，并且其表达具有时空特异性。
将上述4个基因的启动子序列SEQ ID NO:13、14、15和16驱动GUS基因在水稻、玉米等植株中进行功能验证，发现上述启动子均驱动GUS在糊粉层中特异表达，表明本发明所提供的上述启动子均为糊粉层特异表达启动子。
实施例3、基因枪瞬时转化小麦胚芽鞘实验
在之前的研究中，采用基因枪轰击的方法将玉米MYB家族转录因子C1和bHLH家族转录因子B1共同瞬时转化小麦胚芽鞘，能够在小麦胚芽鞘诱导产生红色花青素斑点(Ahmed N,Maekawa M,Utsugi S,Himi E,Ablet H,Rikiishi K,et al.Transient expression of anthocyanin in developing wheat coleoptile by maize C1and B-peru regulatory genes for anthocyanin synthesis.Breeding Sci.2003；53(1):29-34)。为了验证本发明所获得的2个MYB基因和2个bHLH基因是否具有同样的功能，本发明将这4个基因构建了基因枪瞬时转化载体。首先将NOS终止子克隆进入pEASY-T1simple(全式金公司)载体，然后将ThMYB1、ThMYB2、ThR1和ThR2这4个基因的编码框插入NOS前，最后用in-fusion的方法将来自玉米的Ubi(Ubiquitin)启动子插到基因编码框前驱动基因表达，形成Ubi::ThMYB1、Ubi::ThMYB2、Ubi::ThR1和Ubi::ThR2四个载体。实验方法参照(Ahmed et al，2003)，分别将MYB和bHLH基因的转化载体做两两组合，即将ThMYB1+ThR1、ThMYB1+ThR2、ThMYB2+ThR1和ThMYB2+ThR2四个组合共转小麦胚芽鞘，光照培养箱中培养16小时后，在显微镜下观察并拍照，结果如图5所示：ThMYB1+ThR1和ThMYB2+ThR1组合能够诱导红色花青素的产生，ThMYB1+ThR2也能诱导细胞产生花青素。
实施例4、植物表达载体的构建
为了进一步验证ThMYB1、ThMYB2、ThR1和ThR2就是蓝粒基因，本发明选取ThMYB1 和ThR1两个基因构建了稳定转化载体用于小麦转基因实验。以双元表达载体pCAMBIA1300为骨架，首先将pCAMBIA1300上的植物抗性筛选基因35S启动子驱动的潮霉素抗性表达框用pAHC20载体上的Ubi启动子驱动的Bar抗性基因表达框取代。在此基础上，本发明将3215bp ThMYB1基因组序列(包含1952bp启动子序列、822bp基因组序列和441bp终止子序列)和4422bp ThR1基因组序列(包含2084bp启动子序列、1720bp CDS序列和618bp终止子序列)以此插入多克隆位点，从而形成植物表达载体(见图6)。
实施例5、转基因蓝粒小麦的获得
将实施例4构建好的植物表达载体利用电击法转化农杆菌菌株C58C1。利用农杆菌介导的小麦转化体系将载体转入小麦品种fielder，获得96株转基因阳性的T0代植株。T0代植株收种时观察小麦籽粒颜色，发现其中有部分株系有深蓝粒分离，部分株系有浅蓝粒分离(约占34％)，具体结果见图7所示。该转基因结果表明，ThMYB1和ThR1基因在植株中共表达，可以增加植物中的花青素含量，本发明所提供的ThMYB1、ThMYB2、ThR1和ThR2是小麦中的蓝粒基因。
实施例6、蓝粒基因在水稻、拟南芥、玉米植株中的功能验证
将本发明所提供的ThMYB1或ThMYB2基因，与ThR1、ThR2、ZmR和ZmB基因，按一个MYB基因加一个HLH基因的方式两两组合，转入水稻、拟南芥、玉米等植株中进行共表达，发现也具有相同的提高植株中的花青素含量的功能。

Claims (10)

  1. 一种提高植物花青素含量的蓝粒基因，其特征在于，所述基因的核苷酸序列选自下列组的序列之一：
    (a)如SEQ ID NO：1、2、4、5、7、8、10或11所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：3、6、9或12所示的核苷酸序列；
    (c)在严谨条件下能够与(a)或(b)中所述序列的DNA杂交的DNA序列；或
    (d)与(a)或(c)所述序列有至少95％序列相似性，且具有提高植物花青素含量功能的核苷酸序列；或
    (e)与(a)-(d)之任一所述序列互补的DNA序列。
  2. 一种表达盒，其特征在于所述表达盒含有一个蓝粒基因，所述蓝粒基因的核苷酸序列选自下列组的序列之一：
    (a)如SEQ ID NO：1、2、4、5、7、8、10或11所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：3、6、9或12所示的核苷酸序列；
    (c)在严谨条件下能够与(a)或(b)中所述序列的DNA杂交的DNA序列；或
    (d)与(a)或(c)所述序列有至少95％序列相似性，且具有提高植物花青素含量功能的核苷酸序列；或
    (e)与(a)-(d)之任一所述序列互补的DNA序列。
  3. 根据权利要求2所述的表达盒，其特征在于所述蓝粒基因还可操作性的连有一个可驱动其表达的启动子，所述启动子包括但不限于组成型启动子、诱导型启动子、组织特异性启动子、或时空特异表达启动子。
  4. 权利要求1所述的蓝粒基因、权利要求2-3所述的表达盒在提供植物花青素含量中的应用。
  5. 一种提高植物组织或器官的花青素含量的方法，所述方法包含将MYB类转录因子与任一个bHLH转录因子在植物的组织器官中进行共表达的步骤，其特征在于，所述MYB类转录因子为ThMYB1或ThMYB2基因，其核苷酸序列选自下列组的序列之一：
    (a)如SEQ ID NO：1、2、4或5所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：3或6所示的核苷酸序列；
    (c)在严谨条件下能够与(a)或(b)中所述序列的DNA杂交的DNA序列；或
    (d)与(a)-(c)所述序列有至少95％序列相似性，且具有花青素合成调控功能的DNA序列；或
    (e)与(a)-(d)之任一所述序列互补的DNA序列。
  6. 根据权利要求5所述的方法，其中所述的bHLH转录因子包括但不限于ThR1、ThR2、ZmR 和ZmB基因。
  7. 根据权利要求6所述的方法，其中所述的ThR1和ThR2基因的核苷酸序列选自下列组的序列之一：
    (a)如SEQ ID NO：7、8、10或11所示的核苷酸序列；
    (b)其编码氨基酸序列如SEQ ID NO：9或12所示的核苷酸序列；
    (c)在严谨条件下能够与(a)或(b)中所述序列的DNA杂交的DNA序列；或
    (d)与(a)-(c)所述序列有至少95％序列相似性，且具有花青素合成调控功能的DNA序列；或
    (e)与(a)-(d)之任一所述序列互补的DNA序列。
  8. 权利要求5-7之任一所述的方法在提高植物组织或器官的花青素含量中的应用。
  9. 权利要求5-7之任一所述的方法在转基因过程中用于区分转基因与非转基因材料的应用。
  10. 一种糊粉层特异表达启动子，其特征在于所述启动子的核苷酸序列如SEQ ID NO：13、14、15或16所示。
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