WO2020249108A1 - Up基因及其在植物改良中的应用 - Google Patents

Up基因及其在植物改良中的应用 Download PDF

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WO2020249108A1
WO2020249108A1 PCT/CN2020/095909 CN2020095909W WO2020249108A1 WO 2020249108 A1 WO2020249108 A1 WO 2020249108A1 CN 2020095909 W CN2020095909 W CN 2020095909W WO 2020249108 A1 WO2020249108 A1 WO 2020249108A1
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gene
plant
seq
fruit
protein
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French (fr)
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肖晗
江铸颜
张虹
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中国科学院分子植物科学卓越创新中心
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    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/02—Flowers
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H5/00—Angiosperms, i.e. flowering plants, characterised by their plant parts; Angiosperms characterised otherwise than by their botanic taxonomy
    • A01H5/08—Fruits
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/34—Cucurbitaceae, e.g. bitter melon, cucumber or watermelon 
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/74—Rosaceae, e.g. strawberry, apple, almonds, pear, rose, blackberries or raspberries
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01H—NEW PLANTS OR NON-TRANSGENIC PROCESSES FOR OBTAINING THEM; PLANT REPRODUCTION BY TISSUE CULTURE TECHNIQUES
    • A01H6/00—Angiosperms, i.e. flowering plants, characterised by their botanic taxonomy
    • A01H6/82—Solanaceae, e.g. pepper, tobacco, potato, tomato or eggplant
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    • 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
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    • 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)
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
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    • C12Q2600/00—Oligonucleotides characterized by their use
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention belongs to the field of plant biology. More specifically, the present invention relates to UP genes and their application in plant improvement.
  • the gravity of plants is divided into four stages: Gravity Perception, Signal Transduction, Signal Transmission, and Curvature Response. After plants perceive the gravitational signal, the physical signal formed by the sedimentation of amyloid is transformed into a physiological signal, which is transmitted to the Elongation Zones (EZs) in turn. EZ promotes or inhibits cell elongation to achieve tropism.
  • EZs Elongation Zones
  • auxin is one of the most important plant hormones, and it participates in the regulation of plant organogenesis, morphogenesis, tissue differentiation, apical dominance, and tropism growth. Auxins are mainly synthesized in the active parts of the plant, such as leaf primordia, young leaves, roots and developing seeds. Auxin has the characteristics of Polar auxin transport (PAT).
  • PAT Polar auxin transport
  • the polar transport of auxin is mainly mediated by the asymmetric distribution of auxin transport carriers in cells. Under the stimulation of light, gravity and other factors, the polar positioning of the auxin transporter in the cell will change, causing a change in the distribution of auxin and asymmetric growth.
  • Auxin transport carriers can be divided into input carriers and output carriers.
  • the main input carriers are AUXIN RESISTANT 1/LIKE AUX1 (AUX1/LAX) family members (AUX1, LAX1, LAX2 and LAX3).
  • Members of this family encode membrane proteins with transmembrane domains and are a plant-specific subfamily of the amino acid/auxin permease superfamily.
  • Auxin export vectors mainly include PIN-FORMED (PIN) protein, multi-drug resistance/P-glycoproteins (MDR/PGP) and PILS (PIN-LIKES) family members.
  • the purpose of the present invention is to provide UP gene and its application in plant improvement.
  • a method for regulating the orientation, inflorescence morphology, and yield of a plant which includes: regulating the expression of the UP gene or the protein encoded by it in the plant, thereby regulating the orientation, inflorescence morphology and yield of the plant ;
  • the UP gene or its encoded protein includes its homologues.
  • the method is selected from: (a) down-regulating the expression of the UP gene or the protein encoded by it, so that the plant's fruit stalks face upwards, the top of the inflorescence axis faces upwards, or the plant's fruit weight and single The number of fruit, the number of fruit set or the yield per plant; (b) Up-regulating the expression of the UP gene or the protein encoded by it, so that the fruit stalk of the plant is facing down, the top of the inflorescence axis is growing down, and the aerial part is lacking in weight (making Upside down plants grow towards the ground).
  • the down-regulating the expression of the UP gene or the protein encoded by it includes: knocking out or silencing the UP gene in the plant, or inhibiting the activity of the UP protein; preferably, including: specifically interfering with the UP gene
  • the expressed interference molecule silences the UP gene, uses the CRISPR system for gene editing to knock out the UP gene, and knocks out the UP gene by homologous recombination.
  • the method includes: modifying the UP gene so that the terminator appears in advance (the UP protein it encodes provides termination); the modification includes (but not limited to): site-directed mutation of the UP gene , Gene editing or homologous recombination.
  • the up-regulating the expression of the UP gene or the protein encoded by it includes: transferring the UP gene or an expression construct or vector containing the gene into a plant.
  • the premature occurrence terminator is a codon corresponding to the 130th amino acid in the amino acid sequence shown in SEQ ID NO: 2 or the codon before it is converted to a terminator; or corresponding to SEQ ID NO:
  • the amino acid sequence shown in 6 has an early termination mutation or modification.
  • the regulator is a down-regulator of the UP gene or the protein encoded by it, used to make the plant fruit stalk face up and the top of the inflorescence axis face up, or to increase the fruit weight of the plant and the number of fruits per plant , The number or yield of fruit set per plant.
  • the regulator is the UP gene or the protein encoded by it, an expression construct or vector containing the UP gene, which is used to make the plant fruit stalk face down, the top of the inflorescence axis grows downward or the aerial part faces downward. Loss of severity.
  • a plant UP gene or its encoded protein as a molecular marker for identifying the orientation, inflorescence morphology and yield of a plant's fruit stalk; the UP gene or its encoded protein includes them Homologues.
  • a method for screening regulators that regulate the orientation, inflorescence morphology, and yield of a plant.
  • the method includes: (1) adding a candidate substance to a protein containing the UP gene or the protein encoded by it.
  • the expression or activity of the UP gene or the protein encoded by it is observed in the system of (1); if the candidate substance down-regulates the expression or activity of the UP gene or the protein encoded by it, then It indicates that the candidate substance is a regulator that makes the plant fruit stalk upward and the top of the inflorescence axis upward, or increases the plant fruit weight, the number of fruits per plant, the number of fruit set per plant or the yield; if the candidate substance upregulates the UP gene or The protein encoded by it indicates that the candidate substance is a regulator that makes the plant fruit stalk down, the top of the inflorescence axis down, or the aerial part of the above-ground part is missing; wherein, the UP gene or the protein encoded by it includes their Homologue.
  • a method for directional selection or identification of plant fruit stalk orientation, inflorescence morphology, or yield comprising: identifying the expression of the UP gene or the protein encoded by it in a test plant: The expression of the UP gene or the protein encoded by this type of plant is significantly lower than the average expression value of the UP gene or the protein encoded by this type of plant, then the fruit stalk is upward, and the top of the inflorescence axis grows upward.
  • the fruit weight and the individual fruit are Plants with increased number, fruit-set number or yield per plant; if the expression of the UP gene or the protein encoded by the test plant is significantly higher than the average expression value of the UP gene or the protein encoded by the plant, it is a fruit stalk Facing downwards, the top of the inflorescence axis is growing downwards or the aboveground part is a plant with heaviness missing; wherein, the UP gene or the protein encoded by it includes their homologs.
  • a method for specifically identifying the orientation, inflorescence morphology, or yield phenotype of a plant includes: identifying the UP gene or the protein encoded by the plant to be tested, if not present Gene mutations that cause the terminator to appear prematurely or express the full-length UP protein, it indicates that the plant to be tested is a plant that grows with the fruit stalk down, the top of the inflorescence axis down, or the aerial part has a heaviness loss; if it exists, it causes premature termination.
  • the gene mutation or expression of the UP protein fragment indicates that the plant to be tested is a plant with a fruit stalk facing up and the top of the inflorescence axis facing up, or fruit weight, fruit number per plant, number of fruit set per plant, or increased yield;
  • the UP gene or the protein encoded by it includes their homologs.
  • the pre-occurrence terminator is the codon corresponding to the 130th amino acid in the amino acid sequence shown in SEQ ID NO: 2 is converted to a terminator; or the codon corresponding to the amino acid shown in SEQ ID NO: 6 The sequence appears to be terminated early by mutation or modification.
  • the method includes (but not limited to): sequencing method, PCR amplification method, restriction enzyme analysis method, probe method, hybridization method, chip method, allele polymorphism analysis method for nucleic acid Sequence identification.
  • the nucleic acid sequence is identified by restriction enzyme digestion analysis method according to the sequence position of the terminator in advance and the base sequence adjacent to it; preferably, the restriction enzyme includes (but Not limited to: A restriction endonuclease designed based on the 389th base mutation shown in SEQ ID NO:1, such as EcoRI.
  • the plant is a plant with a fruit stalk; preferably, the plant includes (but not limited to): Solanaceae, melon and fruit plants, and fruit viewing plants; preferably ,
  • the Solanaceae plants include: tomato, pepper, eggplant, medlar, physalis, nightshade; preferably, the melon and fruit plants include: such as cucumber, loofah, pumpkin, wax gourd, watermelon, various fruit trees such as apple Tree, peach tree.
  • the plant is tomato, the amino acid sequence of the protein encoded by the UP gene is shown in SEQ ID NO: 2; after the terminator appears in advance, the amino acid sequence of the protein encoded by the UP gene is shown in SEQ ID NO: 2 is shown in positions 1 to 129 (truncated body); or (b) the plant is pepper, and the amino acid sequence of the protein encoded by the UP gene is shown in SEQ ID NO: 6.
  • (a) in (a), it also includes: passing the amino acid sequence shown in SEQ ID NO: 2 or its truncated body through one or more (such as 1-20, 1-10, 1-5 Or 1 or 2) A protein derived from the substitution, deletion or addition of amino acid residues and having the function of the protein; or the amino acid sequence is 70% of the amino acid sequence defined by SEQ ID NO: 2 or its truncation
  • the above more preferably 80% or more, 85% or more, 90% or more, 95% or more
  • identical polypeptide with the function of the protein or SEQ ID NO: 2 with the function of the protein shown in SEQ ID NO: 2 Fragments.
  • it further includes: passing the amino acid sequence shown in SEQ ID NO: 6 through one or more (such as 1-20, 1-10, 1-5 or 1-2 ) A protein derived from the substitution, deletion or addition of amino acid residues and having the function of the protein; or the amino acid sequence is more than 70% of the amino acid sequence defined by SEQ ID NO: 6 or its truncated body (more preferably For example, more than 80%, more than 85%, more than 90%, more than 95%) polypeptides that are identical and have the function of the protein; or fragments of SEQ ID NO: 6 that have the function of the protein shown in SEQ ID NO: 6.
  • an isolated protein is provided, which is a fragment of the UP protein.
  • it is a fragment produced by the UP gene premature appearance terminator; the premature appearance terminator is corresponding to SEQ ID NO :
  • the codon corresponding to or before the 130th amino acid in the amino acid sequence shown in 2 is converted to a terminator; or it corresponds to the codon corresponding to the 139th amino acid or before in the amino acid sequence shown in SEQ ID NO: 6 It is converted into a terminator; more preferably, its amino acid sequence is shown in SEQ ID NO: 2 from 1 to 129, or as SEQ ID NO: 6 in 1 to 138.
  • an isolated polynucleotide is provided, which encodes the protein.
  • the use of the aforementioned isolated protein or isolated polynucleotide is provided as a molecular marker for specifically identifying the orientation, inflorescence morphology, or yield phenotype of plants.
  • an isolated polynucleotide is provided, the nucleotide sequence of which is shown in SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence fragment of SEQ ID NO: 3 or SEQ ID NO: 4 , This sequence fragment contains the 294th base.
  • the use of the polynucleotide is provided as a molecular marker for identifying the stalk orientation, inflorescence morphology or yield of Solanaceae plant tomato.
  • the nucleotide sequence of the polynucleotide is shown in SEQ ID NO: 3 or SEQ ID NO: 4, or a sequence fragment of SEQ ID NO: 3 or SEQ ID NO: 4, which contains the 294th position Base.
  • a method for identifying the Solanaceae plant tomato by using the polynucleotide comprising: amplifying the nucleosides of SEQ ID NO: 3 and/or SEQ ID NO: 4 in the genome sequence of tomato Acid sequence or sequence fragment; analysis of which corresponds to the 294th base sequence in SEQ ID NO: 3 and/or SEQ ID NO: 4.
  • the nucleotide sequence of SEQ ID NO: 3 and/or SEQ ID NO: 4 is amplified with primers of the sequence shown in SEQ ID NO: 7 and SEQ ID NO: 8.
  • a method for identifying peppers in the Solanaceae family includes: amplifying the genome sequence of peppers containing a sequence fragment corresponding to the 491th base in the sequence of SEQ ID NO: 9; In the base sequence at position 491 in SEQ ID NO: 9, if it is C, it is a phenotype with fruit stalk facing down; if it is G, it is a phenotype with fruit stalk facing up.
  • Figure 1 The difference in morphology of tomato and pepper stalk and inflorescence.
  • A Tomato wild-type fruit stalk and inflorescence morphology
  • B Tomato up mutant fruit stalk and inflorescence morphology phenotype
  • C Pepper downward pedicle and inflorescence phenotype
  • D Pepper upward pedicle and inflorescence Phenotype.
  • FIG. 1 SlUP overexpression transgenic plasmid map and phenotype map of transgenic plants.
  • A SlUP overexpression transgenic plasmid map;
  • B SlUP overexpression transgenic plant phenotype map, the picture shows the SlUP OE transgenic plant cultured upside down, the top can not respond to gravity signals and grow toward the center of the earth.
  • FIG. 3 A map of the transgenic plasmid complementary to the SlUP gene.
  • the plasmid uses pCAMBIA1300 as the backbone, inserts the pUP::SlUP sequence (including the 8.7kb promoter, 1.1kb intron-containing coding sequence, and 2kb 3'UTR sequence) between HindIII and SalI, followed by the difference between SacI and EcoRI Insert NOS termination sequence in between.
  • FIG. 4 Phenotype map of the transgenic line complementary to the SlUP gene.
  • A the overall picture of the transgenic line complementary to the SlUP gene;
  • B the stalk phenotype of the transgenic line complementary to the SlUP gene after pollination.
  • FIG. 1 The homology comparison diagram of SlUP and CaUP genes.
  • FIG. 6 SNP sequence analysis diagram of introns of CaUP gene.
  • the CaUP gene introns of 423 pepper varieties were sequenced, and it was found that there was a SNP site in the intron that was significantly related to the orientation of the pepper fruit.
  • SNP locus is the C base, 251 pepper cultivars out of 255 pepper varieties have stalk down; when the SNP locus is G, 167 pepper cultivars out of 176 pepper varieties have stalk up of.
  • FIG. 7 CaUP gene expression in pepper. Quantitative PCR was used to detect the expression of CaUP gene in 26 pepper varieties. The present inventors found that the expression level of CaUP gene in 16 pepper varieties with the stalk facing down was significantly higher than that of the 10 pepper stalks facing up.
  • Figure 8 Molecular markers of UP gene are used to identify tomato stalk orientation and inflorescence morphology. Description of the drawings: The smaller bands in the figure are peppers with the stalk facing up (only one band is clearly visible after being cut by the enzyme); the larger bands are electrophoresed, which is the pepper with the stalk facing down Variety.
  • Figure 9 Statistics of total fruit yield, fruit number, fruit weight, and fruit set number per plant of wild-type NIL (UP) and mutant NIL (up).
  • A statistics of total fruit yield per plant of wild-type NIL (UP) and mutant NIL (up);
  • B statistics of the number of individual fruit of wild-type NIL (UP) and mutant NIL (up);
  • C statistics of wild-type NIL ( UP) and mutant NIL (up) the average weight of a single fruit;
  • D wild-type NIL (UP) and mutant NIL (up) the number of fruit set.
  • FIG. 1 Individual fruit pictures of wild-type NIL (UP) and mutant NIL (up). A, NIL (UP) individual fruit photos; B, NIL (up) individual fruit photos.
  • the present invention studies and reveals for the first time a new type of plant regulatory gene, called UP gene, and its encoded protein is called UP protein, which can regulate the orientation of plant fruit stalk, inflorescence morphology and yield.
  • the present invention also provides the application of using UP gene or its encoded protein as the target point of plant character regulation; UP gene is an important functional gene that affects plant agronomic characters, and can be used to increase crop yield and design and transform plant organ morphology.
  • the present invention also provides variants of the UP gene.
  • the UP protein is terminated prematurely due to the generation of a stop codon. Therefore, the UP gene, its variants and the proteins encoded by them can also be used as molecules for identifying plant phenotype or yield. mark.
  • the UP protein may be a polypeptide having SEQ ID NO: 2 (SlUP, derived from tomato) or SEQ ID NO: 6 (CaUP, derived from pepper). Sequence variants of the same function.
  • the variant forms include (but are not limited to): several (usually 1-50, preferably 1-30, more preferably 1-20, most preferably 1-10, still more preferably Such as 1-8, 1-5) amino acid deletion, insertion and/or substitution, and addition or deletion of one or several (usually within 20, preferably 10) at the C-terminal and/or N-terminal Within, more preferably within 5) amino acids.
  • Any high homology with the UP protein for example, the homology with the polypeptide sequence shown in SEQ ID NO: 2 or 3 is 70% or higher; preferably the homology is 80% or higher; more Preferably, the homology is 90% or higher, such as 95%, 98%, or 99% homology
  • the homology is 90% or higher, such as 95%, 98%, or 99% homology
  • Polypeptides derived from other species other than tomato or pepper that have higher homology with the sequence shown in SEQ ID NO: 2 or 6, or play the same or similar roles in the same or similar signaling pathways are also included in this Inventing.
  • the "UP” also includes their homologs. It should be understood that although SlUP or CaUP obtained from specific species of tomato or pepper is preferably studied in the present invention, those obtained from other species, especially Solanaceae plants, are highly homologous with said SlUP or CaUP (for example, having more than 60%, Such as 70%, 80%, 85%, 90%, 95%, even 98% sequence identity) other polypeptides or genes are also within the scope of the present invention.
  • the present invention also provides an isolated protein, which is a fragment of the UP protein, preferably, it is a fragment produced by the terminator appearing in advance of the UP gene; the present invention also provides an isolated polynucleotide, which encodes the aforementioned protein.
  • the plant is a plant having a homologue (homologous gene) of the UP gene of the present invention; preferably, the plant has fruit, and the fruit is connected to the stalk, and the fruit is supported by the stalk. And to deliver nutrients.
  • the plant is a plant with a fruit stalk; preferably, the plant includes (but not limited to): Solanaceae, melon and fruit plants, and fruit viewing plants; more specifically, for example: tomato, pepper, eggplant , Wolfberry, physalis, nightshade, cucumber, loofah, pumpkin, winter melon, watermelon, apple tree, peach tree.
  • Solanaceae plant is a Solanaceae plant that contains/expresses the "UP gene” or a homologue thereof; such as tomato or pepper. Tomatoes and peppers are two important horticultural crops, and increasing yield is an important breeding goal.
  • the inventors identified a tomato pedicel (upright pedicel (up)) which is a variant material. Different from the downward curved growth of the fruit stalk of a normal plant, the fruit stalk of this material grows upright after fertilization, and the fruit orientation is similar to that of Chaotian pepper of the same family Solanaceae.
  • the present invention isolates tomato SlUP gene through map-based cloning. In addition, through homology comparison and gene collinearity analysis, the candidate gene CaUP that regulates pepper fruit orientation was cloned. The cloning of SlUP and CaUP genes is of great significance for tomato and pepper genetic breeding and quality improvement.
  • Capsicum is an annual or perennial plant belonging to the genus Capsicum of the Solanaceae family (Solanaceae) and is an important vegetable crop worldwide.
  • Solanaceae Solanaceae
  • 13 scientific research institutes at home and abroad initiated the genome sequencing of pepper.
  • the genome sequencing of the annual pepper Zunla-1 (Zunla-1) and Mexican wild pepper Chiltepin was completed.
  • Seoul University and other institutions completed the genome sequencing of the annual pepper CM334.
  • the pepper genome sequence provides important help for the cloning and molecular breeding of pepper genes.
  • Chaotian pepper (Capsicum annuum var frutescens L) is a variant of pepper, which has the advantages of small fruit, high spiciness, and easy drying. Chaotian pepper is rich in capsaicin and capsaicin. Capsaicin was first separated from capsicum by Thres in 1876. Capsaicin is mainly composed of capsaicin and dihydrocapsaicin, which is the main source of spicy flavor. Capsaicins also have antibacterial, anti-tumor and analgesic effects, and are of great value in medical and health care. An important phenotypic difference between Chaotian pepper and common cultivated pepper lies in the orientation of the stalk. The stalk of Chaotian pepper is facing upward, while the stalk of ordinary pepper is drooping.
  • the method includes: regulating the UP gene or the protein encoded by the plant in order to regulate the orientation, inflorescence morphology and yield of the plant’s fruit stalk, specifically including The fruit stalk faces up/down, the top of the inflorescence axis faces up/down growth, fruit weight, number of fruits per plant, number of inflorescences per plant, and yield.
  • the present invention provides a method for increasing the fruit weight, the number of fruits per plant, the number of inflorescences per plant, or the yield of plants, which comprises: down-regulating the expression of the UP gene or the protein encoded by it.
  • the present invention provides a method to make the plant fruit stalk face down, the top of the inflorescence axis face down, and the aboveground part to be gravitious (make the upside down plant grow towards the ground), including: up-regulating the UP gene or its code The expression of the protein. Up-regulation of the UP gene or the protein it encodes, the stem-to-heavy response of tomato plants changes, and the upside down plants appear to grow towards the ground. This can be used in the molecular design of landscape garden plants to create specific plant morphology.
  • the UP gene or down-regulator of the protein encoded by it refers to any agent that can reduce the activity of the UP protein, reduce the stability of the UP gene or its encoded protein, down-regulate the expression of the UP protein, and reduce the effective effect of the UP protein
  • Time or substances that inhibit the transcription and translation of the UP gene can be used in the present invention as substances useful for down-regulating the UP gene or the protein encoded by it. They can be compounds, small chemical molecules, or biological molecules.
  • the biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.
  • the down-regulator is: interfering RNA molecules (such as siRNA, shRNA) or antisense nucleotides that specifically interfere with the expression of UP genes; or gene editing reagents that specifically edit UP genes, and so on.
  • interfering RNA molecules such as siRNA, shRNA
  • antisense nucleotides that specifically interfere with the expression of UP genes
  • gene editing reagents that specifically edit UP genes, and so on.
  • the UP gene or the up-regulator of the protein encoded by it includes promoters, agonists and activators.
  • the "up-regulation” and “promotion” include the “up-regulation” and “promotion” of protein activity or the "up-regulation” and “promotion” of protein expression. Any substance that can increase the activity of the UP protein, increase the stability of the UP gene or the protein encoded by it, up-regulate the expression of the UP gene, and increase the effective action time of the UP protein, these substances can be used in the present invention, as for up-regulation of the UP gene or its
  • the encoded protein is a useful substance. They can be compounds, small chemical molecules, or biological molecules. The biomolecules can be at the nucleic acid level (including DNA and RNA) or at the protein level.
  • the present invention also provides a method for down-regulating the expression of the UP gene or the protein encoded by it in a plant, which includes targeted mutation, gene editing or gene recombination of the UP gene to achieve down-regulation.
  • the CRISPR/Cas (such as Cas9, Cas13) system is used for gene editing, thereby knocking out or down-regulating the UP gene.
  • Appropriate sgRNA target sites will bring higher gene editing efficiency, so before proceeding with gene editing, you can design and find a suitable target site. After designing specific target sites, in vitro cell activity screening is also needed to obtain effective target sites for subsequent experiments.
  • a method for down-regulating the expression of the UP gene or the protein encoded by it in a plant including: (1) transferring interfering molecules that interfere with the expression of the UP gene (including mRNA) into plant cells, tissues, Organs or seeds to obtain plant cells, tissues, organs or seeds transformed into the interfering molecule; (2) to regenerate plants from the plant cells, tissues, organs or seeds transformed into the interfering molecule obtained in step (1) .
  • the method further includes: (3) selecting the plant cells, tissues or organs transferred into the vector; and (4) regenerating the plant cells, tissues or organs in step (3) into plants.
  • the UP gene and the truncated body encoded by its early termination (for example, it has amino acids corresponding to the amino acids 1 to 129 in the amino acid sequence shown in SEQ ID NO: 2; or has amino acids corresponding to SEQ ID NO: 6
  • the difference between amino acids 1 to 138 in the amino acid sequence shown is clear, and the functions are opposite.
  • it specifically targets the codon corresponding to the 130th position or the amino acid before it in the amino acid sequence shown in SEQ ID NO: 2, and turns it into a terminator; Or targeting the codon corresponding to the 139th position or the amino acid before it in the amino acid sequence shown in SEQ ID NO: 6, so that it is converted into a terminator.
  • a method for up-regulating the expression of the UP gene or the protein encoded by it in a plant includes: converting the expression construct or vector of the UP gene or the protein encoded by the UP gene Transfer to plants.
  • the UP gene and its variants are used to regulate the stalk orientation, inflorescence morphology, and yield of plants, showing a relatively significant regulating effect, providing theoretical guidance and high quality for the rapid promotion of excellent plant varieties Genetic resources.
  • simulating the mutation characteristics of the UP gene provides theoretical guidance and high-quality genetic resources for the rapid improvement of plant varieties using gene editing methods.
  • the present invention provides a gene suitable for identifying the orientation of a plant stalk, namely the UP gene.
  • the invention also provides specific molecular markers designed for the genes, primers for identifying the molecular markers, and identification strategies.
  • the present invention provides a method for specifically identifying the fruit stalk orientation, inflorescence morphology, or yield phenotype of a plant, including: identifying the UP gene of the plant to be tested or the protein encoded by it. Gene mutation or expression of the full-length UP protein indicates that the plant to be tested has a phenotype with the fruit stalk facing down, the top of the inflorescence axis growing downward, or the aerial part of the phenotype is missing.
  • the expression of the UP protein fragment indicates that the plant to be tested is a plant whose fruit stalk is facing upwards, the top of the inflorescence axis is facing upwards, or the fruit weight, the number of fruit per plant, the number of fruit set per plant, or the yield of the plant is increased; wherein, the UP gene The protein encoded by it includes their homologs.
  • the plant is a Solanaceae plant tomato, and the position where the terminator appears in advance in the UP gene is the codon corresponding to the 130th amino acid in the amino acid sequence shown in SEQ ID NO: 2. Mutation from TCA to TGA.
  • the plant is a pepper of the Solanaceae plant, and the position where the terminator appears in advance in the UP gene is the codon corresponding to the first 139 amino acids in the amino acid sequence shown in SEQ ID NO: 6,
  • the mutation is TGA or TAA.
  • nucleic acid sequence analysis Identification any of the various techniques known in the art or being developed to analyze nucleic acid sequences, and these techniques can be included in the present invention.
  • Said methods include, but are not limited to: sequencing method, PCR amplification method, probe method, hybridization method, restriction enzyme digestion analysis method, allelic polymorphism analysis method (such as melting curve method) for nucleic acid sequence analysis Identification, etc.
  • the genomic sequence of the amplified tomato includes the nucleotide sequence or sequence fragment of SEQ ID NO: 3 and/or SEQ ID NO: 4; analysis of which corresponds to SEQ ID NO: 3 and/or SEQ ID NO: the 294th base sequence in 4, if it is C, it is a phenotype with the fruit stalk facing down, the top of the inflorescence axis growing down, and the aerial part lacking heaviness; if it is G, it is a fruit The stalk is upward, the top of the inflorescence axis grows upward, and the phenotype of fruit weight, fruit number per plant, fruit set number per plant, or yield is increased; preferably, it is shown in SEQ ID NO: 7 and SEQ ID NO: 8
  • the sequence primers amplify the nucleotide sequence of SEQ ID NO: 3 and/or SEQ ID NO: 4.
  • the amplified genome sequence of the Solanaceae plant pepper contains a sequence fragment corresponding to the 491th base in the SEQ ID NO: 9 sequence; analysis of which corresponds to the 491th in SEQ ID NO: 9 If the base sequence is C, it is the phenotype with the stalk facing down; if it is G, it is the phenotype with the stalk facing up.
  • a gene fragment derived from a Solanaceae plant is also provided, such as the gene fragment shown in SEQ ID NO: 3 or SEQ ID NO: 4 derived from the tomato UP gene.
  • the present invention also includes gene fragments corresponding to the fragments shown in SEQ ID NO: 3 or SEQ ID NO: 4 in other Solanaceae plants.
  • a method for identifying tomato UP genes which includes PCR amplification with primers shown in SEQ ID NO: 7 or SEQ ID NO: 8, and EcoRI digestion of the amplified product.
  • the amplified product can be cut into less than 321bp by EcoRI enzyme, which is the plant with the fruit stalk facing up, the top of the inflorescence axis facing up, or the fruit weight, the number of fruits per plant, the number of inflorescences per plant, or the plant with increased yield; it cannot be cut by enzyme and Plants with a size of about 321bp are plants with the fruit stalk facing down, the top of the inflorescence axis facing down, or the aerial part of the plant is lacking in gravity.
  • This identification method only needs to perform PCR reaction and/or agarose gel electrophoresis, and by judging the length of the corresponding PCR product, the phenotype or yield of the sample to be tested can be accurately and quickly judged, with low cost and suitable for Large-scale identification, and the amount of sample required is small.
  • the method of obtaining the DNA of the sample to be tested is a technique well known to those skilled in the art.
  • the traditional phenol/chloroform/isoamyl alcohol method can be used, or some commercially available DNA extraction kits can be used.
  • Those skilled in the art are familiar.
  • PCR polymerase chain reaction
  • the present invention also relates to a kit for identifying tomato stalk orientation, inflorescence morphology and yield, said kit containing primers shown in SEQ ID NO: 7 and SEQ ID NO: 8.
  • the kit also includes: restriction endonuclease EcoRI. It should be understood that, according to the disclosure of the present invention, the reagents that can be used for identification are not limited to this.
  • kit may also contain identified instructions for use and/or standard operating procedures.
  • the kit can realize the purpose of rapid detection and batch detection of the orientation of pepper fruit stems.
  • the present invention overcomes the existing shortcomings of the inability to identify plant fruit stalk orientation and inflorescence morphology, and provides simple and effective identification methods, molecular markers and preferred primers thereof, thereby providing a feasible method for rapid and effective identification of plant fruit stalk orientation and inflorescence morphology
  • the method provides a powerful tool for plant breeding and screening.
  • the invention has important application prospects in the molecular design and breeding of plant type and yield traits and the improvement of crop varieties by using genetic engineering technology.
  • the targeted screening of plants can be carried out based on its function or using UP as a molecular marker. Based on this new discovery, it is also possible to screen for substances or potential substances that can regulate the orientation of plant fruit stalks, inflorescence morphology and yield by regulating the UP gene or the protein encoded by it.
  • the present invention provides a method for directed selection or identification of plant regulators, the method comprising: identifying the expression of the UP gene or the protein encoded by the test plant: if it is the UP gene or the protein encoded by the test plant The expression is lower than the average expression value of the UP gene or the protein encoded by this type of plant, or the UP expression is higher than the average expression value of the UP of this type of plant, then the fruit stalk is upward, the top of the inflorescence axis grows upward, and the fruit weight , The number of fruits per plant, the number of inflorescences per plant or the plant with increased yield; if the expression of the UP gene or the protein encoded by the test plant is higher than the average expression value of the UP gene or the protein encoded by the plant, it It is a plant with the fruit stalk facing down, the top of the inflorescence axis facing down, or the aerial part of the plant has lack of heaviness.
  • the present invention provides a method for screening regulators that regulate plant fruit stalk orientation, inflorescence morphology and yield, including: (1) adding candidate substances to a system containing UP genes or their encoded proteins; (2) testing institutes In the system, the expression or activity of the UP gene or its encoded protein in the system of (1) is observed; if the candidate substance down-regulates the expression or activity of the UP gene or its encoded protein, it indicates that the candidate substance is a plant fruit The stalk is upward, the top of the inflorescence axis is upward, or a regulator that increases plant fruit weight, fruit number per plant, fruit set number per plant, or yield; if the candidate substance up-regulates the UP gene or the protein encoded by it, it indicates that Candidate substances are regulators that make plants grow with the fruit stalk down, the top of the inflorescence axis down, or the aboveground part of the plant is lacking in gravity.
  • the method of screening for substances acting on the target by using protein or gene or a specific region as a target is well known to those skilled in the art, and these methods can be used in the present invention.
  • the candidate substance can be selected from peptides, polymeric peptides, peptidomimetics, non-peptide compounds, carbohydrates, lipids, antibodies or antibody fragments, ligands, small organic molecules, small inorganic molecules, nucleic acid sequences, and the like. According to the types of substances to be screened, those skilled in the art know how to choose an appropriate screening method.
  • the inventors have identified an upright pedicel (up) material with morphological variations in tomato fruit stalk and inflorescence. Compared with the wild type, the inflorescence of this material has the top of the inflorescence axis growing upward, and the top of the wild type inflorescence axis will appear.
  • the phenotype with a large downward curve ( Figure 1A, B).
  • the stalk of the up plant was slightly curved downwards as in the wild type on the day of flowering. After fertilization, the wild-type stalk further bends downwards, while the up stalk gradually becomes straight after pollination, forming an upright stalk in 3 to 4 days ( Figure 1A, B).
  • the gene cloning of the present invention is based on the tomato reference genome sequence SL2.40 (https://solgenomics.net/).
  • the up (LA2397) material with the stalk facing up was crossed with the gooseberry tomato LA1589 to obtain F1 seeds.
  • the F2 generation segregating population obtained by F1 selfing was used for genetic analysis and gene mapping.
  • a total of 17 F2 individual plants were found to have a stalk-up phenotype.
  • the upward phenotype is controlled by a recessive single gene.
  • the cDNA sequence of the wild-type SlUP gene is as follows (SEQ ID NO: 1 (642bp), where the position of the base mutation in the box):
  • the wild-type SlUP protein sequence is as follows (SEQ ID NO: 2 (213aa), where the underlined part (positions 130 to 213) disappears after mutation):
  • the overexpression vector pHZ009 of the SlUP gene is a modified vector with pCAMBIA1300 as the backbone.
  • the 35S promoter sequence is inserted between HindIII and XbaI, and the SlUP gene sequence (SEQ ID NO:1) is inserted between XbaI and SacI. Subsequently, a NOS termination sequence was inserted between SacI and EcoRI, as shown in Figure 2A.
  • the present inventors introduced the vector into wild-type tomato LA1781 through the Agrobacterium-mediated method, and the aboveground part of the transgenic tomato obtained was completely lacking in gravity (as shown in Figure 2B).
  • transgene complementary plasmid pHZ037 of SlUP was constructed.
  • This plasmid uses pCAMBIA1300 as the backbone, inserts the pUP::UP sequence (including the 8.7kb promoter, 1.1kb intron-containing coding sequence, and 2kb 3'UTR) between HindIII and SalI, and is followed by the difference between SacI and EcoRI Insert the NOS termination sequence between ( Figure 3).
  • the vector was introduced into the up mutant tomato plant by Agrobacterium-mediated method.
  • the obtained transgenic plants have the phenotype with the stalk facing down, confirming that the mutation of this gene leads to the performance of the stalk facing up ( Figure 4).
  • the cDNA sequence of the wild-type CaUP gene is as follows (SEQ ID NO: 5 (771bp)):
  • the wild-type CaUP protein sequence is as follows (SEQ ID NO: 6; 256aa):
  • the genome sequence of CaUP is as follows (Pepper.v.1.55.chr12_199941045..199939610; 1436bp) (SEQ ID NO: 9; where the underline is the intron region):
  • the inventors sequenced the CaUP genes of 423 pepper varieties, and determined that one SNP in the CaUP intron was significantly related to the orientation of the stalk of the pepper.
  • SNP at position 491 of the CaUP genome sequence corresponding to SEQ ID NO: 9 is a C base
  • 251 pepper varieties out of 255 pepper varieties have the phenotype with the stalk down
  • SNP site is When it is the G base, 167 of the 176 pepper varieties have stalks facing upwards (Figure 6).
  • the quantitative PCR method was used to detect the expression of CaUP gene in 26 pepper stalk cultivars, and it was found that the CaUP gene expression in 16 pepper cultivars with stalk down was significantly higher than that in 10 stalk up ( Figure 7 and table). 1).
  • Primer design for molecular markers Aiming at the mutation site on the second exon of the SlUP gene, the present invention has developed a PCR-based dCAPS marker to detect the SlUP genotype in tomato varieties. The specific steps are as follows, using the following primers to perform a PCR reaction to amplify a 321bp fragment (for the sequence of the fragment, see SEQ ID NO: 3 (used to identify mutant types) and SEQ ID NO: 4 (used to identify wild type). .
  • the DNA sequence of the amplification primer pair is as follows:
  • Upstream primer Primer1 5'-AGGTCCCAGTTGGTCTCGTTTA-3' (SEQ ID NO: 7);
  • Downstream primer Primer2 (SEQ ID NO: 8),
  • the PCR amplification system described in step (1) is a 30 ⁇ L system, including the following components: 100ng/ ⁇ L genomic DNA 1 ⁇ L, 10 ⁇ M Primer1 0.6 ⁇ L, 10 ⁇ M Primer2 0.6 ⁇ L, 10X EasyTaq buffer 3 ⁇ L, 2.5mM dNTP 2.4 ⁇ L, EasyTaq DNA Polymerase 0.3 ⁇ L, ddH 2 O 22.1 ⁇ L.
  • the Easy Taq DNA Polymerase used in this experiment is a reagent from Quanjin Gold Company.
  • the PCR amplification reaction procedure described in step (1) is: 94°C pre-denaturation for 5 minutes; 94°C for 30s, 55°C for 30s, 72°C for 30s, 35 cycles; 72°C for final extension for 10 minutes.
  • the PCR product obtained in step (1) is digested with EcoRI (5'G ⁇ AATTC 3').
  • the digestion reaction system includes the following components: 10X CutSmart buffer 3 ⁇ L, PCR product 10 ⁇ L, EcoRI enzyme (NEB) 1 ⁇ L, ddH 2 O16 ⁇ L. After digestion at 37°C for 1 h, electrophoresis analysis was performed on a 4% agarose gel.
  • the PCR product can be cut into two DNA fragments by EcoRI, it is a mutant with the stalk facing up;
  • PCR product cannot be digested by EcoRI and is still a 321 bp DNA fragment, it is a wild type with the stalk down;
  • test materials are 21 materials from a tomato heterozygous group.
  • the young leaves of a single tomato plant are selected, and the genomic DNA of tomato is extracted by the CTAB method.
  • the specific steps are as follows:
  • PCR amplification was performed using the identification primers Primer1 (SEQ ID NO: 7)/Primer2 (SEQ ID NO: 8).
  • the reaction system was 30 ⁇ L, including the following components: 100ng/ ⁇ L genomic DNA 1 ⁇ L, 10 ⁇ M Primer1 0.6 ⁇ L, 10 ⁇ M Primer2 0.6 uL, 10X Easy Taq buffer 3 ⁇ L, 2.5mM dNTPs 2.4 ⁇ L, Easy Taq DNA Polymerase 0.3 ⁇ L, ddH 2 O 22.1 ⁇ L.
  • the PCR amplification reaction program is: 94°C pre-denaturation 5min; 94°C 30s, 55°C 30s, 72°C 30s, 35 cycles; 72°C final extension 10min.
  • the PCR product was digested with restriction enzyme EcoRI, and the reaction system included the following components: 10X CutSmart buffer 3 ⁇ L, PCR product 10 ⁇ L, EcoRI enzyme (NEB company) 1 ⁇ L, ddH 2 O 16 ⁇ L. Digestion at 37°C for 1h.
  • the digested products were separated by agarose gel electrophoresis to distinguish different genotypes.
  • EcoRI digested PCR products were separated by 4% agarose gel electrophoresis (0.5XTBE, 120V, 1.5-2 hours), EcoRI digested into less than 321bp, which is fruit-stalk-up genotype and cannot be digested. And the size of about 321bp is the fruit stalk down genotype.
  • the samples 1, 3, 5, 7, 8, 9, 10, 11, 12, 13, 14, 16, 17, 18, and 19 in Figure 8 are the material with the stem facing down, and the samples 2, 4, 6, 15 and 20 is the material with the stalk facing upward.
  • Primer1 SEQ ID NO: 7
  • Primer2 SEQ ID NO: 8
  • the primer sequences Primer1 SEQ ID NO: 7
  • Primer2 SEQ ID NO: 8
  • the obtained sequence and PCR fragment size may be different, but the EcoRI enzyme The cut reaction can still identify allelic variants.
  • SEQ ID NO:1 is the cDNA sequence of SlUP gene, the sequence length is 642bp.
  • SEQ ID NO: 2 is the protein sequence encoded by the SlUP gene, which encodes 213 amino acids.
  • SEQ ID NO: 3 is a nucleotide sequence identifying the molecular marker with the tomato stalk and inflorescence facing upward, and the sequence length is 321 bp.
  • SEQ ID NO: 4 is the nucleotide sequence of the molecular marker for identifying tomato fruit stalk and inflorescence downwards, and the sequence length is 321 bp.
  • SEQ ID NO: 5 is the cDNA sequence of the CaUP gene, the sequence length is 771bp.
  • SEQ ID NO: 6 is the protein sequence encoded by the CaUP gene, which encodes 256 amino acids.
  • SEQ ID NO: 7 is the upstream primer Primer1 used to identify the orientation of tomato stalk and the inflorescence morphology.
  • the sequence length is 22bp.
  • SEQ ID NO: 8 is a molecular marker downstream primer Primer2 used to identify tomato stalk orientation and inflorescence morphology, with a sequence length of 27 bp.
  • SEQ ID NO: 9 is the genome sequence of CaUP (Pepper.v.1.55.chr12_199941045..199939610; 1436bp).

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Abstract

提供了一种UP基因及其在植物改良中的应用。揭示了一种新型植物调控基因，称为UP基因，其可以调控植物果柄朝向、花序形态和产量。还提供了以UP基因或其编码的蛋白作为植物性状调控靶点的应用；UP基因是影响植物农艺性状的重要功能基因，可用于提高作物产量，设计改造植物器官形态。还提供了UP基因的变体，由于终止密码子产生而导致UP蛋白提前终止，因此所述的UP基因、其变体以及它们编码的蛋白，还可以作为鉴别植物表型或产量的分子标记。

Description

UP基因及其在植物改良中的应用 技术领域
本发明属于植物生物学领域，更具体地，本发明涉及UP基因及其在植物改良中的应用。
背景技术
植物的向重性分为重力感知(Gravity perception)、信号转导(Signal transduction)、信号传递(Signal transmission)和弯曲反应(Curvature response)四个阶段。植物感知重力信号后，通过淀粉体沉降形成的物理信号，并转化成生理学信号，依次传递到伸长区(Elongation zones，EZs)。EZ通过促进或抑制细胞的伸长实现向性生长。
1900年，Haberlandt和Nemec分别在紫鸭趾草茎节细胞和两栖焊菜根尖细胞中发现了能移动的淀粉体，并把这种能移动的淀粉体叫做耳石或平衡石，建立了“淀粉粒-平衡石假说”，认为重力的识别是由含淀粉体细胞沉降介导。在根中，位于根冠的最外两层柱状细胞内含有淀粉体，是对重力感知敏感的细胞。在茎中，围绕维管束系统的内皮层细胞含有淀粉体。
生长素是植物最重要的激素之一，参与调控植物的器官发生、形态建成、组织分化、顶端优势、向性生长等。生长素主要在植物体内生长活跃的地方合成，如叶原基、幼叶、根以及发育的种子等部位合成。生长素具有极性运输(Polar auxin transport，PAT)特点。植物体内的生长素运输途径主要有两条：一是通过成熟维管束组织的被动运输；另一条是依赖于运输载体蛋白的极性运输。生长素的极性运输主要通过生长素运输载体在细胞中的不对称分布介导。在光、重力等因子的刺激下，生长素运输载体在细胞内的极性定位会发生改变，引起生长素分布变化，发生不对称生长。
生长素运输载体可分为输入载体和输出载体。输入载体主要有AUXIN RESISTANT 1/LIKE AUX1(AUX1/LAX)家族成员(AUX1，LAX1，LAX2和LAX3)。该家族成员编码具有跨膜结构域的膜蛋白，是氨基酸/生长素通透酶超家族中植物所特有的亚家族。生长素输出载体主要有PIN-FORMED(PIN)蛋白、多重抗药性/磷酸糖蛋白家族(Multi-drug Resistance/P-glycoproteins，MDR/PGP)和PILS(PIN-LIKES)家族成员。
近些年关于植物地上部分下胚轴和茎的向重性研究报道较多，调控机制也比较清晰，但花果器官的向重性研究较少。果柄是连接果实和花柄或茎的重要组织器官，参与果实和种子的养分运输。最近研究发现果实中合成的生长素经果柄运输到下部组织，而茎顶端等部位合成的生长素不经果柄向果实运输。果实的生长发育受生长素驱动，施用生长素于未受精子房能诱导单性结实，促进果实的形成和生长。但果实和果柄的生长素极性运输如何影响果实形成和生长发育并不清楚。
综上，克隆控制果柄和果实生长素运输的重要功能基因在利用分子设计育种、基因改良作物产量上具有巨大的经济价值。
发明内容
本发明的目的在于提供UP基因及其在植物改良中的应用。
在本发明的第一方面，提供一种调节植物果柄朝向、花序形态和产量的方法，包括：调节植物中UP基因或其编码的蛋白的表达，从而调节植物果柄朝向、花序形态和产量；所述的UP基因或其编码的蛋白包括其同源物。
在一个优选例中，所述方法选自：(a)下调UP基因或其编码的蛋白的表达，从而使植物果柄朝上，花序轴的顶端朝上生长，或提高植物的果重、单株果实数、单株坐果花序数或产量；(b)上调UP基因或其编码的蛋白的表达，从而使植物果柄朝下，花序轴的顶端朝下生长，地上部分向重性缺失(使得倒挂植株向地生长)。
在另一优选例中，所述下调UP基因或其编码的蛋白的表达包括：在植物中敲除或沉默UP基因，或抑制UP蛋白的活性；较佳地，包括：以特异性干扰UP基因表达的干扰分子来沉默UP基因，以CRISPR系统进行基因编辑从而敲除UP基因，以同源重组的方法敲除UP基因。
在另一优选例中，所述方法包括：对UP基因进行改造使其提前出现终止子(其编码的UP蛋白提供终止)；所述的改造包括(但不限于)：对UP基因进行定点突变，基因编辑或同源重组。
在另一优选例中，所述上调UP基因或其编码的蛋白的表达包括：将UP基因或含有该基因的表达构建物或载体转入植物中。
在另一优选例中，所述的提前出现终止子为相应于SEQ ID NO:2所示氨基酸序列中第130位氨基酸对应的密码子或其之前的密码子转为终止子；或相应于SEQ ID NO:6所示氨基酸序列出现提前终止突变或改造。
在本发明的另一方面，提供一种UP基因、其编码的蛋白或它们的调节剂的用途，用于调节植物果柄朝向、花序形态和产量；所述的UP基因或其编码的蛋白包括其同源物。
在一个优选例中，所述调节剂为UP基因或其编码的蛋白的下调剂，用于使植物果柄朝上，花序轴的顶端朝上生长，或提高植物的果重、单株果实数、单株坐果花序数或产量。
在另一优选例中，所述调节剂为UP基因或其编码的蛋白、含有UP基因的表达构建物或载体，用于使植物果柄朝下，花序轴的顶端朝下生长或地上部分向重性缺失。
在本发明的另一方面，提供一种植物UP基因或其编码的蛋白的用途，用于作为鉴定植物果柄朝向、花序形态和产量的分子标记；所述UP基因或其编码的蛋白包括 它们的同源物。
在本发明的另一方面，提供一种筛选调节植物果柄朝向、花序形态和产量的调节剂的方法，所述方法包括：(1)将候选物质加入到含有UP基因或其编码的蛋白的体系中；(2)检测所述体系中，观测(1)的体系中UP基因或其编码的蛋白的表达或活性；若所述候选物质下调UP基因或其编码的蛋白的表达或活性，则表明该候选物质是使植物果柄朝上、花序轴的顶端朝上生长或提高植物果重、单株果实数、单株坐果花序数或产量的调节剂；若所述候选物质上调UP基因或其编码的蛋白，则表明该候选物质是使植物果柄朝下、花序轴的顶端朝下生长或地上部分向重性缺失的调节剂；其中，所述UP基因或其编码的蛋白包括它们的同源物。
在本发明的另一方面，提供一种定向选择或鉴定植物果柄朝向、花序形态或产量的方法，所述方法包括：鉴定测试植物中UP基因或其编码的蛋白的表达：若是该测试植物的UP基因或其编码的蛋白的表达显著低于该类植物UP基因或其编码的蛋白的平均表达值，则其为果柄朝上，花序轴的顶端朝上生长，果重、单株果实数、单株坐果花序数或产量提高的植物；若是该测试植物的UP基因或其编码的蛋白的表达显著高于该类植物UP基因或其编码的蛋白的平均表达值，则其为果柄朝下，花序轴的顶端朝下生长或地上部分向重性缺失的植物；其中，所述UP基因或其编码的蛋白包括它们的同源物。
在本发明的另一方面，提供一种特异性鉴定植物的果柄朝向、花序形态或产量表型的方法，所述方法包括：鉴定待测植物的UP基因或其编码的蛋白，若不存在导致提前出现终止子的基因突变或表达全长UP蛋白，则表明该待测植物为果柄朝下、花序轴的顶端朝下生长或地上部分向重性缺失的植物；若存在导致提前出现终止子的基因突变或表达UP蛋白片段，则表明该待测植物为果柄朝上、花序轴的顶端朝上生长或果重、单株果实数、单株坐果花序数或产量提高的植物；其中，所述UP基因或其编码的蛋白包括它们的同源物。
在一个优选例中，所述的提前出现终止子为相应于SEQ ID NO:2所示氨基酸序列中第130位氨基酸对应的密码子转为终止子；或相应于SEQ ID NO:6所示氨基酸序列出现提前终止突变或改造。
在另一优选例中，采用包括(但不限于)：测序法，PCR扩增法，限制性酶切分析法，探针法，杂交法，芯片法，等位基因多态性分析法进行核酸序列的鉴定。
在另一优选例中，根据提前出现终止子的序列位置及其邻近位置的碱基序列，通过限制性酶切分析法进行核酸序列的鉴定；较佳地，所述的限制性酶包括(但不限于)：基于SEQ ID NO:1中所示的第389个碱基突变设计的限制性内切酶，如EcoRI。
在另一优选例中，所述的植物是具有果柄的植物；较佳地，所述的植物包括(但不限于)：茄科植物，瓜果类植物，观果类植物；较佳地，所述茄科植物包括：番茄， 辣椒，茄子、枸杞、酸浆、龙葵；较佳地，所述瓜果类植物包括：如黄瓜，丝瓜，南瓜，冬瓜，西瓜，各种果树如苹果树、桃树。
在另一优选例中，(a)所述植物为番茄，UP基因编码的蛋白的氨基酸序列如SEQ ID NO:2所示；提前出现终止子后，UP基因编码的蛋白的氨基酸序列如SEQ ID NO:2中第1～129位所示(截短体)；或(b)所述植物为辣椒，UP基因编码的蛋白的氨基酸序列如SEQ ID NO:6所示。
在另一优选例中，(a)中，还包括：将SEQ ID NO:2或其截短体所示氨基酸序列经过一个或多个(如1～20个、1～10个、1～5或1～2个)氨基酸残基的取代、缺失或添加而形成的，且具有该蛋白功能的衍生的蛋白；或氨基酸序列与SEQ ID NO:2或其截短体限定的氨基酸序列有70％以上(更佳地如80％以上，85％以上，90％以上，95％以上)相同性且具有该蛋白功能的多肽；或具有SEQ ID NO:2所示蛋白的功能的SEQ ID NO:2的片段。
在另一优选例中，(b)中，还包括：将SEQ ID NO:6所示氨基酸序列经过一个或多个(如1～20个、1～10个、1～5或1～2个)氨基酸残基的取代、缺失或添加而形成的，且具有该蛋白功能的衍生的蛋白；或氨基酸序列与SEQ ID NO:6或其截短体限定的氨基酸序列有70％以上(更佳地如80％以上，85％以上，90％以上，95％以上)相同性且具有该蛋白功能的多肽；或具有SEQ ID NO:6所示蛋白的功能的SEQ ID NO:6的片段。
在本发明的另一方面，提供一种分离的蛋白，其是UP蛋白的片段，较佳地，其是UP基因提前出现终止子产生的片段；所述提前出现终止子为相应于SEQ ID NO:2所示氨基酸序列中第130位氨基酸对应或其前面的密码子转为终止子；或相应于SEQ ID NO:6所示氨基酸序列中在第139位氨基酸对应的密码子或之前的密码子转为终止子；更佳地，其氨基酸序列如SEQ ID NO:2中第1～129位所示，或如SEQ ID NO:6中第1～138位所示。
在本发明的另一方面，提供分离的多核苷酸，其编码所述的蛋白。
在本发明的另一方面，提供前述分离的蛋白或分离的多核苷酸的用途，用于作为特异性鉴定植物的果柄朝向、花序形态或产量表型的分子标记。
在本发明的另一方面，提供分离的多核苷酸，其核苷酸序列如SEQ ID NO:3或SEQ ID NO:4所示，或SEQ ID NO:3或SEQ ID NO:4的序列片段，该序列片段包含有其中第294位的碱基。
在本发明的另一方面，提供多核苷酸的用途，用于作为鉴定茄科植物番茄的果柄朝向、花序形态或产量的分子标记。该多核苷酸的核苷酸序列如SEQ ID NO:3或SEQ ID NO:4所示，或SEQ ID NO:3或SEQ ID NO:4的序列片段，该序列片段包含有其中第294位的碱基。
在本发明的另一方面，提供利用所述的多核苷酸鉴定茄科植物番茄的方法，包括：扩增番茄的基因组序列中包含SEQ ID NO:3和/或SEQ ID NO:4的核苷酸序列或序列片段；分析其中对应于SEQ ID NO:3和/或SEQ ID NO:4中第294位碱基序列，若为C，则其为果柄朝下，花序轴的顶端朝下生长，地上部分向重性缺失的表型；若为G，则其为果柄朝上，花序轴的顶端朝上生长，果重、单株果实数、单株坐果花序数或产量提高的表型；较佳地，以SEQ ID NO:7和SEQ ID NO:8所示序列的引物扩增SEQ ID NO:3和/或SEQ ID NO:4的核苷酸序列。
在本发明的另一方面，提供鉴定茄科植物辣椒的方法，包括：扩增辣椒的基因组序列中包含有相应于SEQ ID NO:9序列中第491位的碱基的序列片段；分析其中对应于SEQ ID NO:9中第491位碱基序列，若为C，则其为果柄朝下的表型；若为G，则其为果柄朝上的表型。
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、番茄、辣椒果柄和花序形态的差异。A，番茄野生型果柄和花序形态表型；B，番茄up突变体果柄和花序形态表型；C，辣椒朝下果柄和花序形态的表型；D，辣椒朝上果柄和花序表型。
图2、SlUP过表达转基因质粒图谱和转基因植株表型图。A，SlUP过表达转基因质粒图谱；B，SlUP过表达转基因植株表型图，图为倒挂着培养的SlUP OE转基因植株，顶端不能响应重力信号而朝向地心的方向生长。
图3、SlUP基因互补的转基因质粒图谱。质粒以pCAMBIA1300为骨架，在HindIII和SalI之间插入pUP::SlUP序列(包括8.7kb启动子、1.1kb包含内含子的编码序列以及2kb 3’UTR序列)，并在其后SacI和EcoRI之间插入NOS终止序列。
图4、SlUP基因互补的转基因株系的表型图。A，SlUP基因互补的转基因株系的整体图；B，SlUP基因互补的转基因株系授粉后的果柄表型。
图5、SlUP与CaUP基因的同源比对图。
图6、CaUP基因内含子的SNP位点测序分析图。对423份辣椒品种的CaUP基因内含子进行测序，发现该内含子部位有一个SNP位点与辣椒果实朝向显著相关。当SNP位点为C碱基时，255个辣椒品种中有251个辣椒品种是果柄朝下的；当SNP位点为G时，176个辣椒品种中有167个辣椒品种是果柄朝上的。
图7、辣椒CaUP基因的表达。定量PCR检测CaUP基因在26个辣椒品种中的表达量。本发明人发现16个果柄朝下的辣椒品种中CaUP基因的表达量要显著高于10个果柄朝上的。
图8、UP基因的分子标记用于鉴定番茄果柄朝向和花序形态。附图说明：图中电泳出较小的条带为果柄朝上的辣椒品种(被酶切开后仅有一条带明显可见)；电泳出较大的条带，为果柄朝下的辣椒品种。
图9、野生型NIL(UP)和突变体NIL(up)单株果实总产量、果实数目、单果重以及坐果花序数目统计。A，野生型NIL(UP)和突变体NIL(up)单株果实总产量统计；B，野生型NIL(UP)和突变体NIL(up)单株果实的数目统计；C，野生型NIL(UP)和突变体NIL(up)单个果实平均重量统计；D，野生型NIL(UP)和突变体NIL(up)坐果花序数目统计。
图10、野生型NIL(UP)和突变体NIL(up)单株果实图片。A，NIL(UP)单株果实照片；B，NIL(up)单株果实照片。
具体实施方式
本发明首次研究及揭示了一种新型植物调控基因，称为UP基因，其编码的蛋白称为UP蛋白，其可以调控植物果柄朝向、花序形态和产量。本发明还提供了以UP基因或其编码的蛋白作为植物性状调控靶点的应用；UP基因是影响植物农艺性状的重要功能基因，可用于提高作物产量，设计改造植物器官形态。本发明还提供了UP基因的变体，由于终止密码子产生而导致UP蛋白提前终止，因此所述的UP基因、其变体以及它们编码的蛋白，还可以作为鉴别植物表型或产量的分子标记。
UP基因及其编码蛋白
在本发明中，除非特别说明，所述的UP蛋白可以是具有SEQ ID NO:2(SlUP，番茄来源)或SEQ ID NO:6(CaUP，辣椒来源)序列的多肽，还包括具有与UP蛋白相同功能的序列变异形式。
所述的变异形式包括(但并不限于)：若干个(通常为1-50个，较佳地1-30个，更佳地1-20个，最佳地1-10个，还更佳如1-8个、1-5个)氨基酸的缺失、插入和/或取代，以及在C末端和/或N末端添加或缺失一个或数个(通常为20个以内，较佳地为10个以内，更佳地为5个以内)氨基酸。任何与所述的UP蛋白同源性高(比如与SEQ ID NO:2或3所示的多肽序列的同源性为70％或更高；优选地同源性为80％或更高；更优选地同源性为90％或更高，如同源性95％，98％或99％)的、且具有所述UP蛋白相同功能的蛋白也包括在本发明内。
来源于番茄或辣椒以外其它物种的与SEQ ID NO:2或6所示序列的多肽序列的同源性较高、或在同样或相近的信号通路中发挥同样或相近作用的多肽也包括在本发明中。
本发明中，所述的“UP”也包括它们的同源物。应理解，虽然本发明中优选研 究了获自特定物种番茄或辣椒的SlUP或CaUP，但是获自其它物种、特别是茄科植物的与所述SlUP或CaUP高度同源(如具有60％以上，如70％，80％，85％、90％、95％、甚至98％序列相同性)的其它多肽或基因也在本发明考虑的范围之内。
本发明还提供了分离的蛋白，其是UP蛋白的片段，较佳地，其是UP基因提前出现终止子产生的片段；本发明还提供了分离的多核苷酸，其编码前述的蛋白。
植物改良
如本文所用，所述的植物是具有本发明的UP基因的同源物(同源基因)的植物；较佳地，所述的植物具有果实，且其果实连接果柄，通过果柄支撑果实以及输送营养成分。所述的植物是具有果柄的植物；较佳地，所述的植物包括(但不限于)：茄科植物，瓜果类植物，观果类植物；更具体地例如：番茄，辣椒，茄子、枸杞、酸浆、龙葵，黄瓜，丝瓜，南瓜，冬瓜，西瓜，苹果树、桃树。
如本文所用，“茄科植物(作物)”是包含/表达“UP基因”或其的同源物的茄科植物；如番茄或辣椒。番茄和辣椒是两个重要的园艺作物，提高产量是重要的育种目标。
本发明人在研究工作中，鉴定到一个番茄果柄朝向变异材料upright pedicel(up)。不同于正常植株果柄的向下弯曲生长，该材料的果柄在受精后朝上直立生长，果实朝向与同属茄科的朝天椒相类似。本发明通过图位克隆分离到番茄SlUP基因。此外，通过同源比对和基因共线性分析，克隆了调控辣椒果实朝向的候选基因CaUP，SlUP与CaUP基因的克隆对于番茄和辣椒遗传育种和品质改良具有重要的意义。
辣椒(Capsicum)，是茄科(Solanaceae)辣椒属，一年生或多年生植物，是世界性的重要蔬菜作物。2011年由遵义市农业科学研究院牵头，联合国内外13家科研院所启动了辣椒基因组测序，并于2014年完成了一年生辣椒遵辣1号(Zunla-1)和墨西哥野生辣椒Chiltepin的基因组测序。同年，韩国首尔大学等单位完成了一年生辣椒CM334的基因组测序。辣椒基因组序列为辣椒基因的克隆和分子育种提供了重要帮助。朝天椒(Capsicum annuum var frutescens L)是辣椒的一个变种，具有果小、辣度高、易干制等优点。朝天椒富含辣椒碱、辣椒红素。辣椒素最早在1876年由Thres从辣椒中分离出来，辣椒碱主要由辣椒素和二氢辣椒素组成，是辣味的主要来源。辣椒素类物质还具有抗菌、抗肿瘤和镇痛等作用，在医疗保健上具有重要价值。朝天椒和普通栽培辣椒的一个重要表型差异在于果柄的朝向，朝天椒果柄朝上，而普通辣椒果柄下垂。
因此，基于本发明人的新发现，提供了一种改良植物的方法，所述方法包括：调控植物体内UP基因或其编码的蛋白，进而调控植物体果柄朝向、花序形态和产量，具体包括果柄朝上/下、花序轴的顶端朝上/下生长、果重、单株果实数、单株坐果花 序数、产量。
一方面，本发明提供了一种提高植物的果重、单株果实数、单株坐果花序数或产量的方法，包括：下调UP基因或其编码的蛋白的表达。另一方面，本发明提供了一种使植物果柄朝下，花序轴的顶端朝下生长，地上部分向重性缺失(使得倒挂植株向地生长)的方法，包括：上调UP基因或其编码的蛋白的表达。上调UP基因或其编码的蛋白，番茄植株的茎向重性反应发生变化，倒挂植株表现为向地生长，这可以用于景观园林植物的分子设计，创造出特定植株形态。
应理解，在得知了所述UP基因或其编码的蛋白与其提前终止的突变体的关联性或调控机制后，可以采用本领域人员熟知的多种方法来调节所述的UP基因的表达，这些方法均被包含在本发明中。
本发明中，所述的UP基因或其编码的蛋白的下调剂是指任何可降低UP蛋白的活性、降低UP基因或其编码的蛋白的稳定性、下调UP蛋白的表达、减少UP蛋白有效作用时间、或抑制UP基因的转录和翻译的物质，这些物质均可用于本发明，作为对于下调UP基因或其编码的蛋白有用的物质。它们可以是化合物、化学小分子、生物分子。所述的生物分子可以是核酸水平(包括DNA、RNA)的，也可以是蛋白水平的。例如，所述的下调剂是：特异性干扰UP基因表达的干扰RNA分子(如siRNA、shRNA)或反义核苷酸；或是特异性编辑UP基因的基因编辑试剂，等等。
本发明中，所述的UP基因或其编码的蛋白的上调剂包括了促进剂、激动剂、激活剂。所述的“上调”、“促进”包括了蛋白活性的“上调”、“促进”或蛋白表达的“上调”、“促进”。任何可提高UP蛋白的活性、提高UP基因或其编码的蛋白的稳定性、上调UP基因的表达、增加UP蛋白有效作用时间的物质，这些物质均可用于本发明，作为对于上调UP基因或其编码的蛋白有用的物质。它们可以是化合物、化学小分子、生物分子。所述的生物分子可以是核酸水平(包括DNA、RNA)的，也可以是蛋白水平的。
本发明还提供了一种下调植物中UP基因或其编码的蛋白的表达的方法，包括对UP基因进行靶向性地突变、基因编辑或基因重组，从而实现下调。
作为一种实施方式，采用CRISPR/Cas(如Cas9，Cas13)系统进行基因编辑，从而敲除或下调UP基因。合适的sgRNA靶位点，会带来更高的基因编辑效率，所以在着手进行基因编辑前，可以设计并找到合适的靶位点。在设计特异性靶位点后，还需要进行体外细胞活性筛选，以获得有效的靶位点用于后续实验。
作为另一种实施方式，提供了一种下调植物中UP基因或其编码的蛋白的表达的方法，包括：(1)将干扰UP基因(包括mRNA)表达的干扰分子转入植物细胞、组织、器官或种子，获得转化入所述干扰分子的植物细胞、组织、器官或种子；(2)将步骤(1)获得的转入了所述干扰分子的植物细胞、组织、器官或种子再生成植物。较佳地，所 述方法还包括：(3)选择出转入了所述载体的植物细胞、组织或器官；和(4)将步骤(3)中的植物细胞、组织或器官再生成植物。
本发明中，经精细比较，UP基因与其提前终止所编码的截短体(如具有相应于SEQ ID NO:2所示氨基酸序列中第1～129位氨基酸；或具有相应于SEQ ID NO:6所示氨基酸序列中第1～138位氨基酸)的差异较为明确，功能相反。通过定向地靶向于特定的密码子或其邻近区域，可以实现表型或产量的调控。因此，作为一种更为具体的实施例方式，特异性靶向于相应于SEQ ID NO:2所示氨基酸序列中第130位或其前面的氨基酸对应的密码子，使之转为终止子；或靶向于相应于SEQ ID NO:6所示氨基酸序列中第139位或其前面的氨基酸对应的密码子，使之转为终止子。
作为本发明的另一种实施方式，还提供了一种上调植物中UP基因或其编码的蛋白的表达的方法，所述的方法包括：将UP基因或其编码的蛋白的表达构建物或载体转入植物中。
本发明中，利用UP基因及其变体(包括截短体)调控植物的果柄朝向、花序形态和产量，表现出较为显著的调节作用，为优良的植物品种快速推广提供了理论指导以及优质的基因资源。
并且，模拟UP基因的突变特点，为利用基因编辑手段快速改良植物品种提供了理论指导以及优质的基因资源。
分子标记
本领域中，技术人员对于植物果柄朝向由哪个或哪些基因控制一直没有清晰的了解，现有技术中缺乏鉴定植物果柄朝向的分子标记。基于本发明人的新发现，本发明提供了适用于鉴定植物果柄朝向的基因，即UP基因。本发明还提供了针对所述基因而设计的特异性分子标记，鉴定所述分子标记的引物，以及鉴定策略。
因此，本发明提供了一种特异性鉴定植物的果柄朝向、花序形态或产量表型的方法，包括：鉴定待测植物的UP基因或其编码的蛋白，若不存在导致提前出现终止子的基因突变或表达全长UP蛋白，则表明该待测植物为果柄朝下、花序轴的顶端朝下生长或地上部分向重性缺失的表型，若存在导致提前出现终止子的基因突变或表达UP蛋白片段，则表明该待测植物为果柄朝上、花序轴的顶端朝上生长或果重、单株果实数、单株坐果花序数或产量提高的植物；其中，所述UP基因或其编码的蛋白包括它们的同源物。
作为本发明的优选方式，所述的植物为茄科植物的番茄，其UP基因中提前出现终止子的位置为相应于SEQ ID NO:2所示氨基酸序列中第130位氨基酸对应的密码子，由TCA突变为TGA。
作为本发明的优选方式，所述的植物为茄科植物的辣椒，其UP基因中提前出现 终止子的位置为相应于SEQ ID NO:6所示氨基酸序列中前139位氨基酸对应的密码子，突变为TGA或TAA。
根据本发明的新发现，本领域技术人员可以采用任何本领域公知的或正在发展的多种技术来进行核酸序列的分析，这些技术均可被包含在本发明中。所述的方法例如包括但不限于：测序法，PCR扩增法，探针法，杂交法，限制性酶切分析法，等位基因多态性分析法(如溶解曲线法)进行核酸序列的鉴定，等等。
在优选的实施方式中，扩增番茄的基因组序列中包含SEQ ID NO:3和/或SEQ ID NO:4的核苷酸序列或序列片段；分析其中对应于SEQ ID NO:3和/或SEQ ID NO:4中第294位碱基序列，若为C，则其为果柄朝下，花序轴的顶端朝下生长，地上部分向重性缺失的表型；若为G，则其为果柄朝上，花序轴的顶端朝上生长，果重、单株果实数、单株坐果花序数或产量提高的表型；较佳地，以SEQ ID NO:7和SEQ ID NO:8所示序列的引物扩增SEQ ID NO:3和/或SEQ ID NO:4的核苷酸序列。
在优选的实施方式中，扩增茄科植物辣椒的基因组序列中包含有相应于SEQ ID NO:9序列中第491位的碱基的序列片段；分析其中对应于SEQ ID NO:9中第491位碱基序列，若为C，则其为果柄朝下的表型；若为G，则其为果柄朝上的表型。
在优选的实施方式中，还提供了来自于茄科植物的基因片段，如来自于番茄UP基因的如SEQ ID NO:3或SEQ ID NO:4所示的基因片段。本发明也包括在其它茄科植物中与所述SEQ ID NO:3或SEQ ID NO:4所示片段相应的基因片段。
在更优选的方式中，提供了一种鉴定番茄UP基因的方法，包括以SEQ ID NO:7或SEQ ID NO:8所示的引物进行PCR扩增，对扩增产物进行EcoRI酶切。扩增产物能被EcoRI酶切成小于321bp的为果柄朝上、花序轴的顶端朝上生长或果重、单株果实数、单株坐果花序数或产量提高的植物；不能被酶切且大小约321bp的则为果柄朝下、花序轴的顶端朝下生长或地上部分向重性缺失的植物。这一鉴定方法，只需进行PCR反应和/或琼脂糖凝胶电泳，并通过判断相应的PCR产物的长度，就可以准确、快速地判断待测样品的表型或产量，成本低廉，适合于大规模鉴定，而且所需的样品量很少。
获取待测样品的DNA的方法是本领域技术人员所熟知的技术，例如可采取传统的酚/氯仿/异戊醇法，或者可采用一些商购的DNA提取试剂盒，这类试剂盒是本领域技术人员熟知的。
聚合酶链反应(PCR)技术是本领域技术人员熟知的技术，其基本原理是体外酶促合成特异DNA片段的方法。本发明的方法可采用常规的PCR技术进行。
根据上述，本发明还涉及用于鉴定番茄果柄朝向、花序形态和产量的试剂盒，所述试剂盒中含有SEQ ID NO:7和SEQ ID NO:8所示的引物。在优选的方式中，所述试剂盒中还包括：限制性内切酶EcoRI。应理解，根据本发明所揭示的内容，可以用 于鉴定的试剂并不限于此。
此外，所述的试剂盒中还可含有鉴定的使用说明书和/或标准操作程序。所述的试剂盒可实现快速检测、批量检测辣椒果柄朝向的目的。
本发明克服了现有不能鉴定植物果柄朝向、花序形态的不足，提供了简单、有效的鉴定方法、分子标记及其优选引物，从而为植物果柄朝向、花序形态的快速有效鉴定提供了可行的方法，为植物的育种筛选提供了有力的工具。
在种植早期就能够鉴定出植物的果柄朝向、花序形态和产量，为植物育种工作带来极大的便利。
本发明在植物株型和产量性状的分子设计育种及利用基因工程技术进行农作物品种改良等方面具有重要的应用前景。
植物定向筛选或靶向性筛选调控分子
在得知了UP基因或其编码的蛋白的功能及其分子机制以后，可以基于其功能或以UP为分子标记物，来进行植物的定向筛选。也可基于该新发现来筛选通过调节UP基因或其编码的蛋白，从而定向调控植物果柄朝向、花序形态和产量的物质或潜在物质。
因此，本发明提供了一种定向选择或鉴定植物调节剂的方法，所述方法包括：鉴定测试植物中UP基因或其编码的蛋白的表达：若是该测试植物的UP基因或其编码的蛋白的表达低于该类植物UP基因或其编码的蛋白的平均表达值，或UP表达高于该类植物的UP平均表达值，则其为果柄朝上，花序轴的顶端朝上生长，果重、单株果实数、单株坐果花序数或产量提高的植物；若是该测试植物的UP基因或其编码的蛋白的表达高于该类植物UP基因或其编码的蛋白的平均表达值，则其为果柄朝下，花序轴的顶端朝下生长或地上部分向重性缺失的植物。
本发明提供了一种筛选调节植物果柄朝向、花序形态和产量的调节剂的方法，包括：(1)将候选物质加入到含有UP基因或其编码的蛋白的体系中；(2)检测所述体系中，观测(1)的体系中UP基因或其编码的蛋白的表达或活性；若所述候选物质下调UP基因或其编码的蛋白的表达或活性，则表明该候选物质是使植物果柄朝上、花序轴的顶端朝上生长或提高植物果重、单株果实数、单株坐果花序数或产量的调节剂；若所述候选物质上调UP基因或其编码的蛋白，则表明该候选物质是使植物果柄朝下、花序轴的顶端朝下生长或地上部分向重性缺失的调节剂。
以蛋白或基因或其上特定的区域作为靶点，来筛选作用于该靶点的物质的方法是本领域人员所熟知的，这些方法均可用于本发明。所述的候选物质可以选自：肽、聚合肽、拟肽、非肽化合物、碳水化合物、脂、抗体或抗体片段、配体、有机小分子、无机小分子和核酸序列等。根据待筛选的物质的种类，本领域人员清楚如何选择适用 的筛选方法。
经过大规模的筛选，可以获得一类特异性作用于UP基因或其编码的蛋白、对植物果柄朝向、花序形态和产量的有调控作用的潜在物质。
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J.萨姆布鲁克等编著，分子克隆实验指南，第三版，科学出版社，2002中所述的条件，或按照制造厂商所建议的条件。
实施例1、番茄和辣椒up突变体的表型描述和番茄UP基因的定位
本发明人鉴定到一个番茄果柄和花序形态发生变异的材料upright pedicel(up)，该材料的花序与野生型相比，花序轴的顶端朝上生长，而野生型的花序轴的顶端都会出现较大弯曲向下的表型(图1A，B)。up植株的果柄在开花当天与野生型一样，略为向下弯曲。受精后野生型果柄进一步向下弯曲，而up果柄在授粉后逐渐变直，在3～4天形成直立向上的果柄(图1A，B)。
茄科的辣椒中，本发明人也发现一些品种如野生和观赏辣椒的花柄直立朝上，而有别于多数栽培辣椒的俯垂向下(图1C，D)。
本发明的基因克隆基于番茄参考基因组序列SL2.40(https://solgenomics.net/)。为了分离克隆UP基因(SlUP)，将果柄朝上的up(LA2397)材料与醋栗番茄LA1589杂交，得到F1种子。F1自交获得的F2代分离群体用于遗传分析和基因定位。从94个F2单株中共发现17株具有果柄朝上的表型，卡方测验值χ 2＝2.397小于χ 2 0.05＝3.841，p＝0.7264>0.05，分离比符合3:1，说明果柄朝上的表型受隐性单基因控制。然后利用3730株F2群体进行精细定位，将SlUP基因定位于Marker XPS868与XP0942之间的32.2kb区域内。该区间只有一个蛋白编码基因，经测序比较发现果柄朝上的up突变体在该蛋白编码基因上(第二个外显子)存在一个点突变，即其第389个碱基C突变为G，导致蛋白翻译提前终止。
野生型SlUP基因的cDNA序列如下(SEQ ID NO:1(642bp)，其中方框中碱基位突变位置)：
Figure PCTCN2020095909-appb-000001
Figure PCTCN2020095909-appb-000002
野生型SlUP蛋白序列如下(SEQ ID NO:2(213aa)，其中下划线部分(第130～213位)在发生突变后消失)：
Figure PCTCN2020095909-appb-000003
实施例2、SlUP基因的过表达植株的获得和表型描述
SlUP基因的过表达载体pHZ009是以pCAMBIA1300为骨架的经改造的载体，在HindIII和XbaI之间插入35S启动子序列，在XbaI和SacI之间插入SlUP基因序列(SEQ ID NO:1)，并在其后SacI和EcoRI之间插入NOS终止序列，如图2A所示。
本发明人将该载体通过农杆菌介法导入到野生型番茄LA1781，获得的转基因番茄的地上部分向重性完全缺失(如图2B所示)。
实施例3、SlUP基因的转基因互补实验
为了进一步验证定位到的SlUP基因，构建了SlUP的转基因互补质粒pHZ037。该质粒以pCAMBIA1300为骨架，在HindIII和SalI之间插入pUP::UP序列(包括8.7kb启动子、1.1kb含内含子的编码序列以及2kb 3’UTR)，并在其后SacI和EcoRI之间插入NOS终止序列(图3)。
并将该载体通过农杆菌介导法导入up突变体番茄植株中。获得的转基因植株具有果柄朝下的表型，确认了该基因的突变导致果柄朝上的表现(图4)。
实施例4、辣椒CaUP基因的分离
通过共线性分析和序列同源比对，克隆了调控辣椒果柄朝向的基因CaUP(图5)。根据图5可见，来自辣椒的CaUP与来自番茄的SlUP基因是高度同源的，并且，对应于野生型番茄SlUP氨基酸序列第130位的S，野生型辣椒CaUP中相应位置(第139位)也是S。请见图5中虚线框所示。
野生型CaUP基因的cDNA序列如下(SEQ ID NO:5(771bp))：
Figure PCTCN2020095909-appb-000004
Figure PCTCN2020095909-appb-000005
野生型CaUP蛋白序列如下(SEQ ID NO:6；256aa)：
Figure PCTCN2020095909-appb-000006
CaUP的基因组序列如下(Pepper.v.1.55.chr12_199941045..199939610；1436bp)(SEQ ID NO:9；其中下划线为内含子区域)：
Figure PCTCN2020095909-appb-000007
本发明人对423份辣椒品种的CaUP基因测序，确定CaUP内含子中有一个SNP与辣椒的果柄朝向显著相关。当对应于SEQ ID NO:9的CaUP基因组序列的第491位的SNP位点为C碱基时，255个辣椒品种中有251个辣椒品种具有果柄朝下的表型；而当SNP位点为G碱基时，176个辣椒品种中有167个品种的果柄朝上(图6)。
使用定量PCR方法检测CaUP基因在26个辣椒品种果柄的表达量，发现16个果柄朝下的辣椒品种中CaUP基因的表达量要显著高于10个果柄朝上的(图7和表1)。
表1、CaUP基因的表达量分析中使用的材料品种及果柄表型统计表
Figure PCTCN2020095909-appb-000008
实施例5、针对番茄SlUP基因的分子标记开发和应用
(1)分子标记的引物设计：针对SlUP基因第二个外显子上的突变位点，本发明开发了一个基于PCR的dCAPS标记，用于检测番茄品种中SlUP基因型。具体步骤如下所述，使用以下引物进行PCR反应，扩增321bp片段(该片段的序列见序列表SEQ ID NO:3(用于鉴定突变型)和SEQ ID NO:4(用于鉴定野生型)。
SEQ ID NO:3(下划线区域为内含子)
Figure PCTCN2020095909-appb-000009
SEQ ID NO:4(下划线区域为内含子)
Figure PCTCN2020095909-appb-000010
扩增引物对的DNA序列如下：
上游引物Primer1：5’-AGGTCCCAGTTGGTCTCGTTTA-3’(SEQ ID NO:7)；
下游引物Primer2：
Figure PCTCN2020095909-appb-000011
(SEQ ID NO:8)、
该下游引物中，基于所述突变位点，引入错配碱基使之可以被EcoRI识别。
步骤(1)所述的PCR扩增体系为30μL体系，包括如下组分：100ng/μL基因组DNA 1μL，10μM Primer1 0.6μL，10μM Primer2 0.6μL，10X EasyTaq buffer 3μL，2.5mM dNTP 2.4μL，EasyTaq DNA Polymerase 0.3μL，ddH 2O 22.1μL。本实验所用Easy Taq DNA Polymerase为全式金公司试剂。
步骤(1)所述的PCR扩增反应程序为：94℃预变性5min；94℃ 30s，55℃ 30s，72℃ 30s，35个循环；72℃终延伸10min。
对步骤(1)得到的PCR产物使用EcoRI酶切(5'G↓AATTC 3')，酶切反应体系包括如下组分：10X CutSmart buffer 3μL，PCR产物10μL，EcoRI酶(NEB公司)1μL，ddH 2O16μL。37℃酶切1h后用4％的琼脂糖凝胶进行电泳分析。
如果PCR产物能被EcoRI酶切为两条DNA片段，则是果柄朝上的突变体；
如果PCR产物不能被EcoRI酶切，仍为一条大小为321bp的DNA片段，则是果柄朝下的野生型；
(2)分子标记的应用
1)番茄叶片基因组DNA的提取
试验材料为一个番茄杂合群体的21个材料。
选取番茄单株的幼嫩叶片，采用CTAB法提取番茄的基因组DNA，具体步骤如下：
①取约0.2g植物材料置于2ml离心管中，加入400μL DNA抽提液，加入一枚钢珠，使用组织破碎研磨仪震荡1min，充分研磨材料；
②65℃放置20min，期间晃动材料使充分裂解；
③材料冷却至室温后，加入400μl的氯仿，剧烈混匀；
④室温离心，12000rpm离心10min后，吸取上清至新离心管；
⑤加入等体积的异丙醇，颠倒混匀后室温离心，12000rpm离心10min；
⑥弃上清溶液，加入1ml 75％乙醇，室温离心12000rpm离心5min；
⑦弃上清溶液，加入1ml 75％乙醇，室温离心12000rpm离心5min；
⑧弃上清溶液，放在室温干燥，然后加入30-50uL的1x TE溶液，65℃放置10min助溶。
⑨提取的DNA检测质量后，可放-20℃暂时保存，也可放-80℃长期保存。
2)利用鉴定番茄果柄朝向和花序形态的分子标记分析番茄杂合群体21个样本的果柄朝向和花序形态。
利用鉴定引物Primer1(SEQ ID NO:7)/Primer2(SEQ ID NO:8)进行PCR扩增，反应体系为30μL，包括如下组分：100ng/μL基因组DNA 1μL，10μM Primer1 0.6μL，10μM Primer2 0.6uL，10X Easy Taq buffer 3μL，2.5mM dNTPs 2.4μL，Easy Taq DNA Polymerase 0.3μL，ddH 2O 22.1μL。PCR扩增反应程序为：94℃预变性5min；94℃ 30s，55℃ 30s，72℃ 30s，35个循环；72℃终延伸10min。
PCR产物经限制性内切酶EcoRI消化，反应体系包括如下组分:10X CutSmart buffer 3μL，PCR产物10μL，EcoRI酶(NEB公司)1μL，ddH 2O 16μL。37℃酶切1h。
酶切产物通过琼脂糖凝胶电泳来区分不同的基因型。经EcoRI消化的PCR扩增产物经4％的琼脂糖凝胶电泳(0.5XTBE，120V，1.5-2小时)分离后，EcoRI酶切成小于321bp的为果柄朝上基因型，不能被酶切且大小约321bp的则为果柄朝下基因型。图8中的样品1，3，5，7，8，9，10，11，12，13，14，16，17，18和19为果柄朝下材料，样品2，4，6，15和20为果柄朝上材料。
利用本发明公布的引物序列Primer1(SEQ ID NO:7)，Primer2(SEQ ID NO:8)对不同番茄品种基因组DNA进行扩增，所获得的序列和PCR片段大小可能存在一定差异，但EcoRI酶切反应依然可鉴定等位变异。
实施例6、近等基因系NIL(UP)和NIL(up)的单株果实产量、果实数目、单果重和坐果花序数统计
在农场大棚中分别种植番茄up突变体与LA3242(https://tgrc.ucdavis.edu)回交5次的近等基因系NIL(UP)和NIL(up)各50株。计入统计的存活NIL(UP)植株43株，NIL(up)41株。
结果如图9和图10所示，NIL(up)单株果实产量和单株果实数目分别是NIL(UP)的3～4倍和2～3倍，果实平均重量和坐果花序数也增加了50％和1～2倍。根据统计检验(T-test)，差异达到极显著水平(P<0.001)。
序列表说明：
SEQ ID NO:1是SlUP基因的cDNA序列，序列长度为642bp。
SEQ ID NO:2是SlUP基因编码的蛋白序列，编码了213个氨基酸。
SEQ ID NO:3是鉴定番茄果柄和花序朝上的分子标记的核苷酸序列，序列长度为321bp。
SEQ ID NO:4是鉴定番茄果柄和花序朝下的分子标记的核苷酸序列，序列长度为321bp。
SEQ ID NO:5是CaUP基因的cDNA序列，序列长度为771bp。
SEQ ID NO:6是CaUP基因编码的蛋白序列，编码了256个氨基酸。
SEQ ID NO:7是用于鉴定番茄果柄朝向和花序形态的分子标记的上游引物Primer1，序列长度为22bp。
SEQ ID NO:8是用于鉴定番茄果柄朝向和花序形态的分子标记的下游引物Primer2，序列长度为27bp。
SEQ ID NO:9是CaUP的基因组序列(Pepper.v.1.55.chr12_199941045..199939610；1436bp)。
上述实施案例是本发明较佳的实施方式，但本发明的实施方式并不受上述实施例的限制，其他的任何未背离本发明的精神实质和原理下所做的改变、修饰、替代、简化等均应为等效的置换方式，都包含在本发明的保护范围内。

Claims (24)

  1. 一种调节植物果柄朝向、花序形态和产量的方法，其特征在于，包括：调节植物中UP基因或其编码的蛋白的表达，从而调节植物果柄朝向、花序形态和产量；所述的UP基因或其编码的蛋白包括其同源物。
  2. 如权利要求1所述的方法，其特征在于，所述方法选自：
    (a)下调UP基因或其编码的蛋白的表达，从而使植物果柄朝上，花序轴的顶端朝上生长，或提高植物的果重、单株果实数、单株坐果花序数或产量；
    (b)上调UP基因或其编码的蛋白的表达，从而使植物果柄朝下，花序轴的顶端朝下生长，地上部分向重性缺失。
  3. 如权利要求2所述的方法，其特征在于，下调UP基因或其编码的蛋白的表达包括：在植物中敲除或沉默UP基因，或抑制UP蛋白的活性；较佳地，包括：以特异性干扰UP基因表达的干扰分子来沉默UP基因，以CRISPR系统进行基因编辑从而敲除UP基因，以同源重组的方法敲除UP基因。
  4. 如权利要求2所述的方法，其特征在于，对UP基因进行改造使其提前出现终止子；所述的改造包括：对UP基因进行定点突变，基因编辑或同源重组。
  5. 如权利要求2所述的方法，其特征在于，上调UP基因或其编码的蛋白的表达包括：将UP基因或含有该基因的表达构建物或载体转入植物中。
  6. 如权利要求4或5所述的方法，其特征在于，所述的提前出现终止子为相应于SEQ ID NO:2所示氨基酸序列中第130位氨基酸对应的密码子或其之前的密码子转为终止子；或相应于SEQ ID NO:6所示氨基酸序列出现提前终止突变或改造。
  7. 一种UP基因、其编码的蛋白或它们的调节剂的用途，用于调节植物果柄朝向、花序形态和产量；所述的UP基因或其编码的蛋白包括其同源物。
  8. 如权利要求7所述的用途，其特征在于，所述调节剂为UP基因或其编码的蛋白的下调剂，用于使植物果柄朝上，花序轴的顶端朝上生长，或提高植物的果重、单株果实数、单株坐果花序数或产量；或
    所述调节剂为UP基因或其编码的蛋白、含有UP基因的表达构建物或载体，用于使植物果柄朝下，花序轴的顶端朝下生长或地上部分向重性缺失。
  9. 一种植物UP基因或其编码的蛋白的用途，用于作为鉴定植物果柄朝向、花序形态和产量的分子标记；所述UP基因或其编码的蛋白包括它们的同源物。
  10. 一种筛选调节植物果柄朝向、花序形态和产量的调节剂的方法，其特征在于，所述方法包括：
    (1)将候选物质加入到含有UP基因或其编码的蛋白的体系中；
    (2)检测所述体系中，观测(1)的体系中UP基因或其编码的蛋白的表达或活性；若所述候选物质下调UP基因或其编码的蛋白的表达或活性，则表明该候选物质是使 植物果柄朝上、花序轴的顶端朝上生长或提高植物果重、单株果实数、单株坐果花序数或产量的调节剂；若所述候选物质上调UP基因或其编码的蛋白，则表明该候选物质是使植物果柄朝下、花序轴的顶端朝下生长或地上部分向重性缺失的调节剂；
    其中，所述UP基因或其编码的蛋白包括它们的同源物。
  11. 一种定向选择或鉴定植物果柄朝向、花序形态或产量的方法，其特征在于，所述方法包括：鉴定测试植物中UP基因或其编码的蛋白的表达：
    若是该测试植物的UP基因或其编码的蛋白的表达显著低于该类植物UP基因或其编码的蛋白的平均表达值，则其为果柄朝上，花序轴的顶端朝上生长，果重、单株果实数、单株坐果花序数或产量提高的植物；
    若是该测试植物的UP基因或其编码的蛋白的表达显著高于该类植物UP基因或其编码的蛋白的平均表达值，则其为果柄朝下，花序轴的顶端朝下生长或地上部分向重性缺失的植物；
    其中，所述UP基因或其编码的蛋白包括它们的同源物。
  12. 一种特异性鉴定植物的果柄朝向、花序形态或产量表型的方法，其特征在于，所述方法包括：鉴定待测植物的UP基因或其编码的蛋白，若不存在导致提前出现终止子的基因突变或表达全长UP蛋白，则表明该待测植物为果柄朝下、花序轴的顶端朝下生长或地上部分向重性缺失的植物；若存在导致提前出现终止子的基因突变或表达UP蛋白片段，则表明该待测植物为果柄朝上、花序轴的顶端朝上生长或果重、单株果实数、单株坐果花序数或产量提高的植物；其中，所述UP基因或其编码的蛋白包括它们的同源物。
  13. 如权利要求12所述的方法，其特征在于，所述的提前出现终止子为相应于SEQ ID NO:2所示氨基酸序列中第130位氨基酸对应的密码子或其之前的密码子转为终止子；或相应于SEQ ID NO:6所示氨基酸序列出现提前终止突变或改造。
  14. 如权利要求11～13任一所述的方法，其特征在于，采用包括：测序法，PCR扩增法，限制性酶切分析法，探针法，杂交法，芯片法，等位基因多态性分析法进行核酸序列的鉴定。
  15. 如权利要求14所述的方法，其特征在于，根据提前出现终止子的序列位置及其邻近位置的碱基序列，通过限制性酶切分析法进行核酸序列的鉴定；较佳地，所述的限制性酶包括：基于SEQ ID NO:1中所示的第389个碱基突变设计的限制性内切酶，如EcoRI。
  16. 如权利要求1～6、10～15任一所述的方法或权利要求7～9任一所述的用途，其特征在于，所述的植物是具有果柄的植物；较佳地，所述的植物包括：茄科植物，瓜果类植物，观果类植物；较佳地，所述茄科植物包括：番茄，辣椒，茄子、枸杞、酸浆、龙葵；较佳地，所述瓜果类植物包括：如黄瓜，丝瓜，南瓜，冬瓜，西瓜，各 种果树如苹果树、桃树。
  17. 如权利要求1～6、10～15任一所述的方法或权利要求7～9任一所述的用途，其特征在于，
    (a)所述植物为番茄，UP基因编码的蛋白的氨基酸序列如SEQ ID NO:2所示；提前出现终止子后，UP基因编码的蛋白的氨基酸序列如SEQ ID NO:2中第1～129位所示；或
    (b)所述植物为辣椒，UP基因编码的蛋白的氨基酸序列如SEQ ID NO:6所示。
  18. 一种分离的蛋白，其是UP蛋白的片段，较佳地，其是UP基因提前出现终止子产生的片段；所述提前出现终止子为相应于SEQ ID NO:2所示氨基酸序列中第130位氨基酸对应的密码子或其之前的密码子转为终止子；或相应于SEQ ID NO:6所示氨基酸序列中在第139位氨基酸对应的密码子或之前的密码子转为终止子；更佳地，其氨基酸序列如SEQ ID NO:2中第1～129位所示，或如SEQ ID NO:6中第1～138位所示。
  19. 分离的多核苷酸，其编码权利要求18所述的蛋白。
  20. 权利要求18的蛋白或权利要求19的多核苷酸的用途，用于作为特异性鉴定植物的果柄朝向、花序形态或产量表型的分子标记。
  21. 分离的多核苷酸，其核苷酸序列如SEQ ID NO:3或SEQ ID NO:4所示，或SEQ ID NO:3或SEQ ID NO:4的序列片段，该序列片段包含有其中第294位的碱基。
  22. 权利要求21所述的多核苷酸的用途，用于作为鉴定茄科植物番茄的果柄朝向、花序形态或产量的分子标记。
  23. 鉴定茄科植物番茄的方法，其特征在于，包括：扩增番茄的基因组序列中包含SEQ ID NO:3和/或SEQ ID NO:4的核苷酸序列或序列片段；分析其中对应于SEQ ID NO:3和/或SEQ ID NO:4中第294位碱基序列，若为C，则其为果柄朝下，花序轴的顶端朝下生长，地上部分向重性缺失的表型；若为G，则其为果柄朝上，花序轴的顶端朝上生长，果重、单株果实数、单株坐果花序数或产量提高的表型；较佳地，以SEQ ID NO:7和SEQ ID NO:8所示序列的引物扩增SEQ ID NO:3和/或SEQ ID NO:4的核苷酸序列。
  24. 鉴定茄科植物辣椒的方法，其特征在于，包括：扩增辣椒的基因组序列中包含有相应于SEQ ID NO:9序列中第491位的碱基的序列片段；分析其中对应于SEQ ID NO:9中第491位碱基序列，若为C，则其为果柄朝下的表型；若为G，则其为果柄朝上的表型。
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