WO2023216907A1 - Method for shortening length of main stem of cucurbitaceous plant and related product - Google Patents

Method for shortening length of main stem of cucurbitaceous plant and related product Download PDF

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WO2023216907A1
WO2023216907A1 PCT/CN2023/091275 CN2023091275W WO2023216907A1 WO 2023216907 A1 WO2023216907 A1 WO 2023216907A1 CN 2023091275 W CN2023091275 W CN 2023091275W WO 2023216907 A1 WO2023216907 A1 WO 2023216907A1
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plant
sequence
cucurbitaceae
utr
plants
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PCT/CN2023/091275
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French (fr)
Chinese (zh)
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王深浩
杨学勇
黄三文
李海真
李征
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中国农业科学院蔬菜花卉研究所
中国农业科学院深圳农业基因组研究所
北京市农林科学院
西北农林科技大学
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Publication of WO2023216907A1 publication Critical patent/WO2023216907A1/en

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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
    • 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
    • A01H5/04—Stems
    • 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
    • A01H5/10—Seeds
    • 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 
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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
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    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8201—Methods for introducing genetic material into plant cells, e.g. DNA, RNA, stable or transient incorporation, tissue culture methods adapted for transformation
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    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
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    • C12R2001/00—Microorganisms ; Processes using microorganisms
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    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
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    • Y02P60/87—Re-use of by-products of food processing for fodder production

Definitions

  • the present invention relates to the technical fields of molecular biology, biotechnology and plant improvement, and in particular to 5'UTR fragments and biological materials and their application in shortening the length of the main stem of Cucurbitaceae plants, as well as in Cucurbitaceae plant materials or related products. Applications.
  • Plant type is the main factor in "yield per unit area" and is of great significance to improving crop yields and agricultural mechanization management.
  • Dwarf crop varieties have the characteristics of high-density planting, adaptability to mechanization and high yield increase potential. Therefore, dwarf plant type breeding has always been an important direction in crop breeding and improvement.
  • Cucurbitaceous plants such as cucumbers, melons, watermelons and pumpkins are important vegetable crops in the world. Their planting area is second only to solanaceous vegetable crops and is the second largest category of vegetable crops in the world.
  • the output per unit area of important cucurbit crops such as cucumbers, melons, watermelons and pumpkins has only increased by 10%; this is mainly due to the fact that most cucurbits currently have longer main stems and occupy larger areas. , which greatly limits the output per unit area and causes management to be time-consuming and labor-intensive.
  • the moderately dwarfed plant type has the advantages of compact plants, suitable for dense planting, high yield per unit area, saving labor, etc. It is suitable for dense planting of plants, high yield and simplified cultivation needs. Therefore, cultivating dwarf plant varieties with moderately shortened main stems has become a new direction for the plant type breeding and improvement of Cucurbitaceae plants.
  • the present invention provides 5' UTR fragments and biological materials and their application in shortening the main stem length of Cucurbitaceae plants, and their application in Cucurbitaceae plants or related products.
  • the purpose of shortening the main stem length of plants is achieved.
  • One aspect of the present invention provides a 5'UTR fragment (B-region sequence of YABBY1 gene 5'UTR), which 5'UTR fragment includes at least one of the following nucleotide sequences:
  • nucleotide sequence structure is: B 1 -XB 2
  • B 1 is the sequence shown in SEQ ID NO: 1
  • X is any base or no base
  • B 2 is the sequence shown in SEQ ID NO: 2.
  • B 1 sequence is NNNNAAGGCAGATCCAAGGA, where N is G or A;
  • the B 2 sequence is GGTGGTTAATCTGTCAATCCCATCAATCAGTC.
  • the base is selected from adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U).
  • the base is adenine (A).
  • the YABBY1 gene has the function of negatively regulating the shoot apical meristem.
  • the plant shoot apical meristem in the Arabidopsis yabby1 mutant is larger than the control wild-type shoot apical meristem; in Arabidopsis, overexpression of the YABBY1 gene from the 35S promoter can inhibit the shoot apical meristem, making Arabidopsis plants become dwarfed.
  • the present invention clones the Chinese pumpkin dwarf gene Bu through forward genetics.
  • This gene encodes a YABBY transcription factor (YABBY1).
  • YABBY1 YABBY transcription factor
  • the CRISPR-Cas9 gene editing tool is used to delete or partially delete the B-region sequence of the 5'UTR of the YABBY1 gene of pumpkin, cucumber, watermelon and melon.
  • various B-region sequences are displayed. -region sequence The deletion improved the translation efficiency of YABBY1 to varying degrees and proportionally inhibited the main stem length of plants in a dose-dependent manner.
  • the homologous sequence is about 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% with the original nucleotide sequence. % or above, 88% or above, 89% or above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% or above, 96% or above, 97 % or above, 98% or above, 99% or above identity of the nucleotide sequence, or its corresponding cDNA molecule.
  • the "stringent conditions” refer to conditions under which a probe will hybridize to its target sequence to a detectable extent that exceeds hybridization to other sequences (eg, at least 2 times the background). Stringent conditions are sequence dependent and vary depending on the environment. By controlling the stringency of hybridization and/or wash conditions, target sequences that are at least 80% complementary to the probe can be identified. Alternatively, stringent conditions can be adjusted to allow for some sequence mismatches such that lower degrees of similarity are detected.
  • the nucleotide sequence in a) includes at least one of the sequences shown in SEQ ID NO: 3 to SEQ ID NO: 5.
  • SEQ ID NO:3 sequence is:
  • SEQ ID NO:4 sequence is:
  • SEQ ID NO:5 sequence is:
  • the 5'UTR fragment also includes a 5'-end flanking sequence A 1 and/or a 3'-end flanking sequence A 2 , where A 1 or A 2 is a sequence of any length and any combination of bases. .
  • the 5' end flanking sequence A1 sequence is AN(2)CN(4)CAN(7)N(8)AN(10)N(11)AN(13)N(14 )N(15)N(16)N(17)N(18)N(19)N(20)N(21)N(22)N(23)N(24)N(25)AAN(28)N (29)N(30)AAAAN(35), where the 2nd and 28th-30th N is G or A, the 4th N is G or T, the 7th, 8th and 10th N is A or T, and the 7th, 8th and 10th N is A or T.
  • N at position 13 is A or C
  • N at position 13 is A
  • N at position 14 is C or without any base
  • N at positions 15-17, 19, 21-23 is A or without any base
  • Bases, N at positions 18, 20, 24, 25 and 35 are A, G or no base.
  • Another aspect of the present invention provides a biological material, which is any one of the following 1) to 5):
  • Another aspect of the present invention provides the application of the above-mentioned 5'UTR fragment or biological material in shortening the length of plant main stems.
  • the plant is a Cucurbitaceae plant.
  • Cucurbitaceae plants include but are not limited to pumpkin (Cucurbita moschata), cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo), gourd (Lagenaria siceraria), winter melon (Benincasa) hispida), Luffa cylindrica, Momordica charantia, Sechium edule, Gynostemma pentaphyllum, Hemsleya chinensis, Trichosanthes kirilowii, Momordica cochinchinensis, Luo Han Guo (Siraitia grosvenorii) etc.
  • Other Cucurbitaceae plants not listed are also within the protection scope of the present invention.
  • the present invention provides a method for cultivating Cucurbitaceae plant materials.
  • the above-mentioned 5'UTR fragment or partial fragment thereof is deleted in the recipient plant to obtain the target plant; compared with the recipient plant, the main stem length of the target plant is shorten.
  • gene editing tools are used to delete the conserved B-region sequence in the 5'UTR of the YABBY1 gene in cucurbit crops, so as to improve the protein translation efficiency of the gene and achieve the purpose of shortening the main stem length of cucurbit plants. .
  • gene editing CRISPR-Cas9 technology is used to delete the target sequence.
  • the expression cassette, or recombinant vector, or recombinant microorganism introduced into the CRISPR-Cas9 system is applied to gene editing of cucurbit plants such as pumpkins, cucumbers, watermelons, and melons.
  • the B-region sequence can be deleted, thereby shortening these cucurbits. The length of the main stem of the plant.
  • vectors include but are not limited to PKSE401, PKSE402, pGREB32, pHRO4, and pCXUN-BE3.
  • PKSE402 is selected as the vector.
  • the target sequence is deleted using microorganisms or plant cells introduced with the above-mentioned expression cassette and/or vector.
  • microorganisms can be introduced into microorganisms or plant cells.
  • microorganism refers to the expression cassette and/or vector introduced into the CRISPR-Cas9 system, and also includes the progeny of such microorganism carrying the expression cassette and/or vector.
  • Microorganisms can be any prokaryotic or eukaryotic cell organism.
  • microorganisms include, but are not limited to, Agrobacterium, Bacillus subtilis, Bacillus subtilis, Escherichia coli, lactic acid bacteria, insect cells, or other microorganisms commonly used by those skilled in the art as suitable host cells.
  • microorganism is preferably Agrobacterium.
  • Agrobacterium strains include but are not limited to: Agrobacterium EHA105, Agrobacterium LBA440, Agrobacterium EHA101, and Agrobacterium GV3101.
  • reagents and/or kits containing expression cassettes and/or vectors of the CRISPR-Cas9 system are used to cultivate materials with shortened main stems of Cucurbitaceae plants.
  • Another aspect of the present invention provides a Cucurbitaceae plant material or its related products, which is a plant grown from the above-mentioned plant cell line, plant protoplast, cell or callus tissue or its related products; or the above-mentioned Cucurbitaceae plant is used Plants or their related products obtained by cultivation methods of materials;
  • the relevant products are plant parts, plant cells, pollen or seeds.
  • plant materials include germplasm, strains, lines, and commercial varieties.
  • Another aspect of the present invention provides a breeding method for Cucurbitaceae plant materials, which includes self-crossing the above-mentioned Cucurbitaceae plant materials or crossing them with other varieties to obtain Cucurbitaceae plant materials.
  • the above-mentioned breeding method for Cucurbitaceae plants involves selfing diploid materials with shortened main stems or hybridizing with other diploid materials to obtain offspring of materials with shortened main stems.
  • the breeding method of the above-mentioned Cucurbitaceae plants is to double the diploid material with shortened main stem into tetraploid, cross it with other tetraploid materials, and then reduce the doubling into tetraploid. Body material descendants.
  • the above-mentioned breeding method for Cucurbitaceae plants involves doubling diploid material with shortened main stems into tetraploid and then hybridizing with other tetraploid materials to obtain tetraploid material.
  • plant materials include germplasm, strains, lines, and commercial varieties.
  • Another aspect of the present invention provides a Cucurbitaceae plant material or its related products, which is the progeny formed by the above-mentioned Cucurbitaceae plant material being self-crossed or hybridized with other varieties, as well as the plant material or its related products formed by the growth of the progeny;
  • the relevant products are plant parts, plant cells, pollen or seeds.
  • Another aspect of the present invention provides a commercial plant product made from the above-mentioned Cucurbitaceae plant material or its related products.
  • Commercial plant products include food, medicine, cosmetics/skin care products, feed or other products.
  • foods include but are not limited to: fresh fruits, dried fruits, frozen fruits, pickled fruits, honeyed fruits, fried fruits or fruits in various processed forms thereof.
  • Fruits in various processed forms include but are not limited to fruit strips, fruit pieces, fruit slices, fruit granules, fruit powder, fruit paste, fruit juice, and fermented products.
  • medicines include: various dosage forms made from medicinal parts of plants; the medicinal parts of plants can be roots, stems, leaves, flowers, fruits, seeds, melon stems, etc.; the dosage forms can be this Any dosage form recognized in the field.
  • cosmetics/skin care products include but are not limited to: facial cleanser, toner, lotion, cream, essence, facial mask, eye cream, eye mask, isolation lotion, sunscreen lotion, liquid foundation, and BB cream.
  • feed includes but is not limited to: liquid feed, solid feed, semi-solid feed or feed raw materials.
  • Another aspect of the present invention provides a method for manufacturing commercial plant products, including obtaining the above-mentioned Cucurbitaceae plant materials or related products and manufacturing commercial plant products.
  • the present invention uses gene editing tools to delete or partially delete the conserved B-region sequence of the 5'UTR of the YABBY1 gene of Cucurbitaceae plants (pumpkins, cucumbers, watermelons, melons, etc.), thereby improving the translation efficiency of YABBY1, thereby shortening
  • the length of the main stem of Cucurbitaceae plants can achieve the purpose of dwarfing Cucurbitaceae plants and enabling dense planting and high yields, while also greatly saving labor.
  • Figure 1 shows the map-based cloning of Chinese pumpkin dwarfing gene Bu
  • a mixed pool sequencing analysis of dwarf pool and creeping pool in F2 segregating population, the signal site is located on Chinese pumpkin chromosome 15; b, fine mapping of dwarf gene Bu; c, there are 8 genes in the fine mapping interval candidate genes; d, gene structure of Bu candidate gene; e, evolutionary tree analysis of Bu gene;
  • Figure 2 shows that there is a conserved sequence region “B-region” in the 5’UTR of the YABBY1 gene of Cucurbitaceae plants;
  • Plant species include Arabidopsis thaliana, tomato (Solanum lycopersicum), and six cucurbit species, namely Chinese pumpkin (Cucurbita moschata). Cucumber (Cucumis sativus), melon (Cucumis melo), watermelon (Citrullus lanatus), winter melon (Benincasa hispida) and gourd (Lagenaria siceraria); the underlined region is the conserved region present in the 5'UTR of the YABBY1 gene of Cucurbitaceae plants "B-region";
  • Figure 3 shows that B-region deletion of the 5’UTR of the Chinese pumpkin CmoYABBY1 gene results in shorter main stems of pumpkin plants;
  • a CRISPR dual target site design of the B-region of the 5'UTR of the Chinese pumpkin CmoYABBY1 gene, and the obtained editing sequence results
  • b Chinese pumpkin T1 homozygous edited plants show shortening of the main stem
  • c After removing the leaves from plants homozygous for the T1 generation editor, the shortened internode and main stem can be more clearly displayed;
  • Figure 4 shows that B-region deletion of the 5’UTR of the cucumber CsYABBY1 gene causes the main stem of the cucumber plant to become shorter;
  • Figure 5 shows that B-region deletion of the 5’UTR of the watermelon ClYABBY1 gene causes the main stem of the watermelon plant to become shorter;
  • Figure 6 shows that B-region deletion of the 5’UTR of the melon CmYABBY1 gene causes the main stem of the melon plant to become shorter;
  • a CRISPR dual target site design of the B-region of the 5'UTR of the muskmelon CmYABBY1 gene, and the obtained editing sequence results
  • b plants homozygous for editing in the muskmelon T1 generation show shortening of the main stem
  • c muskmelon T1 Statistics on main stem length of plants homozygous for editing.
  • the invention discloses 5'UTR fragments and biological materials and their application in regulating the length of plant main stems.
  • Cucurbitaceae For plants or related products, those skilled in the art can learn from the content of this article and appropriately improve the process parameters. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention.
  • the methods and applications of the present invention have been described through preferred embodiments. Relevant persons can obviously make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of this invention.
  • UTR Untranslated Region
  • 5'-UTR 5'-untranslated region
  • leader sequence leader
  • 3' untranslated region 3'-untranslated region
  • Translated region 3'-untranslated region, 3'-UTR
  • cDNA The full name is complementary DNA, which is a kind of complementary deoxyribonucleic acid
  • CRISPR/Cas9 is an adaptive immune defense formed by bacteria and archaea during the long-term evolution process. It can be used to fight against invading viruses and foreign DNA.
  • the CRISPR/Cas9 system provides immunity by integrating fragments of invading phage and plasmid DNA into CRISPR and using corresponding CRISPR RNAs (crRNAs) to direct the degradation of homologous sequences.
  • crRNA CRISPR-derived RNA
  • tracrRNA trans-activating RNA
  • This complex guides the nuclease Cas9 protein to pair with the sequence target site of crRNA. Cut double-stranded DNA.
  • sgRNA single-guide RNA
  • sgRNA single-guide RNA
  • sgRNA small guide RNA
  • sgRNA small guide RNA
  • It is a guide RNA that guides the insertion or deletion of uridine residues into kinetoplasts during the RNA editing process. It is a small non-coding RNA that can be paired with pre-mRNA.
  • gRNA edits RNA molecules, approximately 60-80 nucleotides in length, transcribed by individual genes;
  • Hybridization The crossing of two individuals with different genotypes. Also refers to mating between different breeds. Plants can refer to cross-pollination between different species;
  • Plant refers to self-pollinating
  • F2 It is the second generation of hybrid or selfing
  • Chromosome ploidy refers to the number of chromosome sets or genomes contained in a cell.
  • the number of chromosomes contained in normal gametocytes or half of the number of chromosomes in somatic cells is called a set of haploid chromosomes, represented by the symbol n.
  • the complete set of haploid chromosomes of an organism is called the genome or chromosome set of that organism.
  • Cells with one set of chromosomes and individuals composed of such cells are called haploids (n)
  • cells or individuals with two sets of chromosomes are called diploids (2n)
  • cells with more than two complete sets of chromosomes are called diploids (2n).
  • polyploids including triploid (3n), tetraploid (4n), etc.
  • Polyploidy formed from chromosome sets from the same source is called autopolyploidy
  • polyploidy formed from different chromosome sets from different sources is called allopolyploidy;
  • Sanger sequencing The Sanger method is based on the nucleotide starting at a fixed point, randomly ending at a specific base, and fluorescently labeling behind each base, resulting in A, T, C, G.
  • InDel insertion-deletion insertion and deletion markers refer to the differences in the whole genome between two parents. Compared with the other parent, a certain number of nucleotides are inserted or deleted in the genome of one parent. According to the indel sites in the genome, design some PCR primers that amplify these indel sites. This is the InDel marker;
  • Single nucleotide polymorphism mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. It is one of the most common heritable variations in humans. Accounts for more than 90% of all known polymorphisms;
  • Genome structural variations usually refer to large-length sequence changes and positional relationship changes on the genome. There are many types of genome structural variations, including insertion or deletion of long fragments of more than 50 bp (Big Indel), tandem repeats (Tandem repeats), chromosomal inversions (Inversion), and sequence translocations within or between chromosomes (Translocation). ), copy number variations (CNV), and more complex forms of mosaic variation.
  • the YABBY1 gene in pumpkin is expressed as CmoYABBY1, and the gene number corresponding to pumpkin is CmoCh15G012090;
  • the YABBY1 gene in cucumber is expressed as CsYABBY1, and the gene number corresponding to cucumber is CsaV3_5G003950;
  • the YABBY1 gene in watermelon is expressed as ClYABBY1, and the gene number corresponding to watermelon The number is Cla97C01G003950;
  • the YABBY1 gene in melon is expressed as CmYABBY1, and the gene number corresponding to melon is MELO3C000087.2.
  • the reagents, instruments, bacterial strains or biological materials used in the present invention can all be purchased from the market.
  • Example 2 Deletion of the B-region fragment or part of the 5’UTR of the CmoYABBY1 gene causes the main stem of pumpkin plants to become shorter
  • Dual target site sequences were designed on the B-region of the CmoYABBY1 gene, both with a length of 19 bp.
  • the position of the dual target site design is shown in Figure 3a.
  • the nucleotide sequence of target site 1 is AAAAGGGAAGGCAGATCCA (5'-3') (SEQ ID NO: 6)
  • the nucleotide sequence of target site 2 is TGTTTGGTTGACTGATTGA (5 '-3') (SEQ ID NO: 7).
  • the PKSE402-CRISPR/Cas9 vector obtained above was transferred into Agrobacterium competent cells EHA105 through heat shock transformation to obtain the recombinant strain EHA105/PKSE402-CRISPR/Cas9.
  • the recombinant strain EHA105/PKSE402-CRISPR/Cas9 was transformed using Agrobacterium to infect cotyledons. Remove the embryo from the germinated pumpkin seeds, cut the distal end of the cotyledons and remove 1/3, and use the remaining cotyledons as explants. These explants were transferred to triangular flasks containing 20 mL of Agrobacterium EHA105 suspension and sonicated, and then the pumpkin explants were vacuum infiltrated in the Agrobacterium suspension. Explants were co-cultured with Agrobacterium on moist filter paper in the dark for 3 days and then transferred to shoot induction medium. After 3-4 weeks of culture, since the PKSE402 vector carries the tagged green fluorescent protein, the plants with green fluorescence are selected as T0 generation transgenic pumpkin plants.
  • the genomic DNA of T0 generation transgenic pumpkin plants was extracted as a template, and the B-region region was amplified by PCR to obtain PCR amplification products of different strains.
  • the PCR amplification products of different strains were subjected to Sanger sequencing, and the sequencing results were compared with the B-region of wild-type pumpkin.
  • the CRISPR/Cas9 tool was used to design dual target sites in the B-region of the cucumber CsYABBY1 (CsaV3_5G003950) gene for gene editing.
  • Dual target site sequences were designed on the B-region of the CsYABBY1 gene, both with a length of 19 bp. Dual target site design The calculated positions are shown in Figure 4a.
  • the nucleotide sequence of target site 1 is AAAAGAAAAGGCAGATCCA (5'-3') (SEQ ID NO: 8), and the nucleotide sequence of target site 2 is ATTGGTAGTGACTGATTGA (5'-3'). ) (SEQ ID NO: 9).
  • Example 4 B-region deletion of the 5’UTR of the ClYABBY1 gene results in shorter main stems of watermelon plants
  • the CRISPR/Cas9 tool was used to design dual target sites in the B-region of the watermelon ClYABBY1 (Cla97C01G003950) gene for gene editing.
  • Double target site sequences were designed on the B-region of the ClYABBY1 gene, both with a length of 19 bp.
  • the position of the dual target site design is shown in Figure 5a.
  • the nucleotide sequence of target site 1 is AAAAGAAAAGGCAGATCCA (5'-3') (SEQ ID NO: 10)
  • the nucleotide sequence of target site 2 is TTGGGGTTTGACTGATTGA (5 '-3') (SEQ ID NO: 11).
  • the process of genetic transformation of watermelon is as follows: watermelon seeds are germinated, then the embryo is removed, and the cotyledons without embryos are cut into 1.5 ⁇ 1.5-mm pieces.
  • the explants were infected with Agrobacterium containing PKSE402-CRISPR/Cas9.
  • the infected cotyledon explants were cultured in the dark for 4 days, and then transferred to the selective induction medium. After regenerated adventitious buds appeared, the adventitious buds were cut out and transferred to the selective extension medium. After cultivating for several weeks, the plants with green fluorescence are selected as T0 generation transgenic watermelon plants.
  • Example 5 B-region deletion of the 5’UTR of the CmYABBY1 gene causes the main stem of the melon plant to become shorter
  • the CRISPR/Cas9 gene editing tool was used to design dual target sites in the B-region of the melon gene CmYABBY1 (MELO3C000087.2).

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Abstract

The present invention relates to the technical field of biology, in particular to a 5'UTR fragment, a biological material, a method for shortening the length of a main stem of a cucurbitaceous plant, and a related product. The 5'UTR fragment comprises: a nucleotide sequence with a structure of B1-X-B2, wherein B1 is a sequence set forth in SEQ ID NO: 1, X is any base or no base, and B2 is a sequence set forth in SEQ ID NO: 2; a complementary sequence, a degenerate sequence or a homologous sequence thereof; a nucleotide sequence hybridized therewith or a complementary sequence thereof; and a cDNA sequence of a sequence described above. By means of utilizing a gene editing tool, a conserved B-region fragment of a YABBY1 gene 5'UTR of the cucurbitaceous plant or a partial fragment thereof is deleted, such that the translation efficiency of YABBY1 is improved, thereby shortening the length of the main stem of the cucurbitaceous plant, achieving the purposes of plant type dwarfing and high-yield close planting of the cucurbitaceous plant, and meanwhile, allowing for significant labor savings.

Description

一种缩短葫芦科植物主茎长度的方法及相关产品A method for shortening the length of main stems of cucurbit plants and related products
本申请要求于2022年05月07日提交中国专利局、申请号为202210493626.5、发明名称为“一种缩短葫芦科植物主茎长度的方法及相关产品”的中国专利申请的优先权，同时要求于2023年04月20日提交中国专利局、申请号为202310430770.9、发明名称为“一种缩短葫芦科植物主茎长度的方法及相关产品”的中国专利申请的优先权，其全部内容通过引用结合在本申请中。This application claims the priority of the Chinese patent application submitted to the China Patent Office on May 7, 2022, with the application number 202210493626.5 and the invention title "A method of shortening the main stem length of Cucurbitaceae plants and related products", and also claims the priority on The priority of the Chinese patent application submitted to the China Patent Office on April 20, 2023, with the application number 202310430770.9 and the invention title "A method for shortening the length of the main stem of Cucurbitaceae plants and related products", the entire content of which is incorporated by reference in in this application.
技术领域Technical field
本发明涉及分子生物学、生物技术和植物改良的技术领域，特别涉及5’UTR片段和生物材料及其在缩短葫芦科植物主茎长度中的应用、以及在葫芦科植物材料或其相关产品中的应用。The present invention relates to the technical fields of molecular biology, biotechnology and plant improvement, and in particular to 5'UTR fragments and biological materials and their application in shortening the length of the main stem of Cucurbitaceae plants, as well as in Cucurbitaceae plant materials or related products. Applications.
背景技术Background technique
随着世界人口的迅速增加和耕地的逐渐减少，如何实现高效农业生产已成为全球最重要的科学挑战之一。植物株型是“单位面积产量”的主要因素，对提高农作物产量和农业机械化管理等具有重要意义。农作物矮化品种具有高密植、适应机械化和增产潜力大等特点，因此矮化株型育种一直是农作物育种改良的重要方向。With the rapid increase in the world's population and the gradual reduction of arable land, how to achieve efficient agricultural production has become one of the most important scientific challenges in the world. Plant type is the main factor in "yield per unit area" and is of great significance to improving crop yields and agricultural mechanization management. Dwarf crop varieties have the characteristics of high-density planting, adaptability to mechanization and high yield increase potential. Therefore, dwarf plant type breeding has always been an important direction in crop breeding and improvement.
葫芦科植物如黄瓜、甜瓜、西瓜和南瓜等是世界上重要的蔬菜作物，其种植面积在世界范围内仅次于茄科类蔬菜作物，是世界上第二大类蔬菜作物。但是近十年来，重要的葫芦科作物如黄瓜、甜瓜、西瓜和南瓜的单位面积产量仅仅增长了10％；这主要是由于目前绝大多数葫芦科植物的主茎较长、占地面积较大，这大大限制了单位面积产量，同时造成管理上费时费工。适度矮化的株型由于具有植株紧凑，适于密植，单位面积产量高，节约劳动力等优点，适合植物的密植高产和轻简化栽培需求。因此培育主茎适度缩短的矮化株型品种成为葫芦科植物株型育种改良的新方向。 Cucurbitaceous plants such as cucumbers, melons, watermelons and pumpkins are important vegetable crops in the world. Their planting area is second only to solanaceous vegetable crops and is the second largest category of vegetable crops in the world. However, in the past ten years, the output per unit area of important cucurbit crops such as cucumbers, melons, watermelons and pumpkins has only increased by 10%; this is mainly due to the fact that most cucurbits currently have longer main stems and occupy larger areas. , which greatly limits the output per unit area and causes management to be time-consuming and labor-intensive. The moderately dwarfed plant type has the advantages of compact plants, suitable for dense planting, high yield per unit area, saving labor, etc. It is suitable for dense planting of plants, high yield and simplified cultivation needs. Therefore, cultivating dwarf plant varieties with moderately shortened main stems has become a new direction for the plant type breeding and improvement of Cucurbitaceae plants.
发明内容Contents of the invention
有鉴于此，本发明提供了5’UTR片段和生物材料及在缩短葫芦科植物主茎长度中的应用、以及在葫芦科植物或其相关产品中的应用。通过对该5’UTR片段的删除，达到缩短植物(尤其是葫芦科植物)主茎长度的目的。In view of this, the present invention provides 5' UTR fragments and biological materials and their application in shortening the main stem length of Cucurbitaceae plants, and their application in Cucurbitaceae plants or related products. By deleting this 5’UTR fragment, the purpose of shortening the main stem length of plants (especially Cucurbitaceae plants) is achieved.
为了实现上述发明目的，本发明提供以下技术方案：In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions:
本发明一方面提供了一种5’UTR片段(YABBY1基因5’UTR的B-region序列)，该5’UTR片段包含如下核苷酸序列中的至少一种：One aspect of the present invention provides a 5'UTR fragment (B-region sequence of YABBY1 gene 5'UTR), which 5'UTR fragment includes at least one of the following nucleotide sequences:
a)核苷酸序列结构为：B1-X-B2 a) The nucleotide sequence structure is: B 1 -XB 2
其中，B1为SEQ ID NO：1所示序列，X为任意碱基或无碱基，B2为SEQ ID NO：2所示序列。Among them, B 1 is the sequence shown in SEQ ID NO: 1, X is any base or no base, and B 2 is the sequence shown in SEQ ID NO: 2.
B1序列为NNNAAGGCAGATCCAAGGA，其中，N为G或A；B 1 sequence is NNNNAAGGCAGATCCAAGGA, where N is G or A;
B2序列为GGTGGTTAATCTGTCAATCCCATCAATCAGTC。The B 2 sequence is GGTGGTTAATCTGTCAATCCCATCAATCAGTC.
b)上述a)中所示序列的互补序列、简并序列或同源序列，其中同源序列为与a)所示序列具有80％或以上同一性的核苷酸序列；b) The complementary sequence, degenerate sequence or homologous sequence of the sequence shown in a) above, wherein the homologous sequence is a nucleotide sequence having 80% or more identity with the sequence shown in a);
c)在严紧条件下与a)所示序列杂交的核苷酸序列或其互补序列；c) A nucleotide sequence that hybridizes to the sequence shown in a) under stringent conditions or its complementary sequence;
d)序列a)-c)中任一序列的cDNA序列。d) The cDNA sequence of any of the sequences a)-c).
在本发明提供的实施例中，碱基选自腺嘌呤(A)、鸟嘌呤(G)、胞嘧啶(C)、胸腺嘧啶(T)、尿嘧啶(U)。在本发明提供的具体实施例中，碱基为腺嘌呤(A)。In the embodiment provided by the present invention, the base is selected from adenine (A), guanine (G), cytosine (C), thymine (T), and uracil (U). In the specific embodiment provided by the present invention, the base is adenine (A).
在拟南芥等模式植物中，YABBY1基因具有负调控植株茎尖分生组织的功能。如拟南芥yabby1突变体中的植物茎尖分生组织大于对照野生型的茎尖分生组织；而在拟南芥中，35S启动子超量表达YABBY1基因可以抑制茎尖分生组织，使得拟南芥植株变得矮小。In model plants such as Arabidopsis thaliana, the YABBY1 gene has the function of negatively regulating the shoot apical meristem. For example, the plant shoot apical meristem in the Arabidopsis yabby1 mutant is larger than the control wild-type shoot apical meristem; in Arabidopsis, overexpression of the YABBY1 gene from the 35S promoter can inhibit the shoot apical meristem, making Arabidopsis plants become dwarfed.
本发明通过正向遗传学，克隆了中国南瓜矮化基因Bu，该基因编码一个YABBY转录因子(YABBY1)，在该基因的5’UTR里矮化南瓜植株相对于野生型南瓜植株缺失了76bp，导致了目的基因的蛋白翻译效率的大幅度提高。进一步分析发现缺失的76bp序列中存在一个葫芦科保守的顺式调控元件(命名为B-region)。本发明实施例中利用CRISPR-Cas9基因编辑工具，将南瓜、黄瓜、西瓜和甜瓜的YABBY1基因5’UTR的B-region序列删除或部分删除，在这些葫芦科作物中均表现为：各种B-region序列 的缺失在不同程度上提高了YABBY1的翻译效率，并且以剂量依赖性方式成比例地抑制植株的主茎长度。The present invention clones the Chinese pumpkin dwarf gene Bu through forward genetics. This gene encodes a YABBY transcription factor (YABBY1). In the 5'UTR of this gene, dwarf pumpkin plants are missing 76 bp compared to wild-type pumpkin plants. This leads to a substantial improvement in the protein translation efficiency of the target gene. Further analysis found that there was a conserved cis-regulatory element (named B-region) in the Cucurbitaceae family in the missing 76 bp sequence. In the embodiments of the present invention, the CRISPR-Cas9 gene editing tool is used to delete or partially delete the B-region sequence of the 5'UTR of the YABBY1 gene of pumpkin, cucumber, watermelon and melon. In these cucurbit crops, various B-region sequences are displayed. -region sequence The deletion improved the translation efficiency of YABBY1 to varying degrees and proportionally inhibited the main stem length of plants in a dose-dependent manner.
进一步的，同源序列为与原始核苷酸序列约80％或以上、81％或以上、82％或以上、83％或以上、84％或以上、85％或以上、86％或以上、87％或以上、88％或以上、89％或以上、90％或以上、91％或以上、92％或以上、93％或以上、94％或以上、95％或以上、96％或以上、97％或以上、98％或以上、99％或以上同一性的核苷酸序列，或其相对应的cDNA分子。Further, the homologous sequence is about 80% or more, 81% or more, 82% or more, 83% or more, 84% or more, 85% or more, 86% or more, 87% with the original nucleotide sequence. % or above, 88% or above, 89% or above, 90% or above, 91% or above, 92% or above, 93% or above, 94% or above, 95% or above, 96% or above, 97 % or above, 98% or above, 99% or above identity of the nucleotide sequence, or its corresponding cDNA molecule.
示例性地，所述“严格条件”是指探针将与其靶序列杂交至可探测程度超过与其它序列杂交(如至少2倍于背景)的条件。严格条件具有序列依赖性，且因环境的不同而不同。通过控制杂交和/或洗涤条件的严格性，可以鉴定与探针至少80％互补的靶序列。可选择地，可以调节严格条件以允许一些序列错配，使得探测到较低程度的相似性。Illustratively, the "stringent conditions" refer to conditions under which a probe will hybridize to its target sequence to a detectable extent that exceeds hybridization to other sequences (eg, at least 2 times the background). Stringent conditions are sequence dependent and vary depending on the environment. By controlling the stringency of hybridization and/or wash conditions, target sequences that are at least 80% complementary to the probe can be identified. Alternatively, stringent conditions can be adjusted to allow for some sequence mismatches such that lower degrees of similarity are detected.
在本发明提供的具体实施例中，a)中核苷酸序列包含SEQ ID NO：3～SEQ ID NO：5所示序列中的至少一种。In the specific embodiments provided by the present invention, the nucleotide sequence in a) includes at least one of the sequences shown in SEQ ID NO: 3 to SEQ ID NO: 5.
SEQ ID NO：3序列为：SEQ ID NO:3 sequence is:
GGGAAGGCAGATCCAAGGAGGTGGTTAATCTGTCAATCCCATCAATCAGTC，SEQ ID NO：3序列存在于南瓜中。GGGAAGGCAGATCCAAGGAGGTGGTTAATCTGTCAATCCCATCAATCAGTC, SEQ ID NO: 3 sequence exists in pumpkin.
SEQ ID NO：4序列为：SEQ ID NO:4 sequence is:
GAAAAGGCAGATCCAAGGAAGGTGGTTAATCTGTCAATCCCATCAATCAGTC，SEQ ID NO：4序列存在于黄瓜、甜瓜、西瓜、葫芦中。GAAAAGGCAGATCCAAGGAAGGTGGTTAATCTGTCAATCCCATCAATCAGTC, SEQ ID NO: 4 sequence exists in cucumber, melon, watermelon, and gourd.
SEQ ID NO：5序列为：SEQ ID NO:5 sequence is:
AAAAAGGCAGATCCAAGGAAGGTGGTTAATCTGTCAATCCCATCAATCAGTC，SEQ ID NO：5序列存在于冬瓜中。AAAAAGGCAGATCCAAGGAAGGTGGTTAATCTGTCAATCCCATCAATCAGTC, SEQ ID NO: 5 sequence exists in winter melon.
在本发明提供的另一实施例中，5’UTR片段还包含5’端侧翼序列A1和/或3’端侧翼序列A2，A1或A2为任意长度、任意碱基组合的序列。In another embodiment provided by the present invention, the 5'UTR fragment also includes a 5'-end flanking sequence A 1 and/or a 3'-end flanking sequence A 2 , where A 1 or A 2 is a sequence of any length and any combination of bases. .
在本发明提供的具体实施例中，5’端侧翼序列A1序列为AN(2)CN(4)CAN(7)N(8)AN(10)N(11)AN(13)N(14)N(15)N(16)N(17)N(18)N(19)N(20)N(21)N(22)N(23)N(24)N(25)AAN(28)N(29)N(30)AAAAN(35)，其中，第2、28-30位N为G或A，第4位N为G或T，第7、8、10位N为A或T，第11 位N为A或C，第13位N为A、T或无任何碱基，第14位N为C或无任何碱基，第15-17、19、21-23位N为A或无任何碱基，第18、20、24、25、35位N为A、G或无任何碱基。In the specific embodiment provided by the present invention, the 5' end flanking sequence A1 sequence is AN(2)CN(4)CAN(7)N(8)AN(10)N(11)AN(13)N(14 )N(15)N(16)N(17)N(18)N(19)N(20)N(21)N(22)N(23)N(24)N(25)AAN(28)N (29)N(30)AAAAN(35), where the 2nd and 28th-30th N is G or A, the 4th N is G or T, the 7th, 8th and 10th N is A or T, and the 7th, 8th and 10th N is A or T. 11 N at position 13 is A or C, N at position 13 is A, T or without any base, N at position 14 is C or without any base, N at positions 15-17, 19, 21-23 is A or without any base Bases, N at positions 18, 20, 24, 25 and 35 are A, G or no base.
本发明另一方面提供了一种生物材料，该生物材料为下述1)至5)中的任一种：Another aspect of the present invention provides a biological material, which is any one of the following 1) to 5):
1)删除上述5’UTR片段或其部分片段的敲除盒；1) A knockout cassette that deletes the above 5’UTR fragment or part of it;
2)删除上述5’UTR片段或其部分片段的敲除载体；2) A knockout vector that deletes the above 5’UTR fragment or part of it;
3)删除上述5’UTR片段或其部分片段的重组微生物；3) Recombinant microorganisms in which the above-mentioned 5’UTR fragment or partial fragments thereof are deleted;
4)删除上述5’UTR片段或其部分片段的植物细胞系；4) Plant cell lines in which the above-mentioned 5’UTR fragment or partial fragments thereof are deleted;
5)导入1)-3)中任意一种的植物原生质体、细胞或愈伤组织。5) Introduce the plant protoplasts, cells or callus of any one of 1)-3).
本发明另一方面提供了上述5’UTR片段或生物材料在缩短植物主茎长度中的应用。Another aspect of the present invention provides the application of the above-mentioned 5'UTR fragment or biological material in shortening the length of plant main stems.
在本发明提供的实施例中，植物为葫芦科植物。In the embodiment provided by the present invention, the plant is a Cucurbitaceae plant.
在本发明提供的具体实施例中，葫芦科植物包括但不限于南瓜(Cucurbita moschata)、黄瓜(Cucumis sativus)、西瓜(Citrullus lanatus)、甜瓜(Cucumis melo)、葫芦(Lagenaria siceraria)、冬瓜(Benincasa hispida)、丝瓜(Luffa cylindrica)、苦瓜(Momordica charantia)、佛手瓜(Sechium edule)、绞股蓝(Gynostemma pentaphyllum)、雪胆(Hemsleya chinensis)、栝楼(Trichosanthes kirilowii)、木鳖(Momordica cochinchinensis)、罗汉果(Siraitia grosvenorii)等。其它没有列举出来的葫芦科植物也在本发明的保护范围之内。In the specific embodiments provided by the present invention, Cucurbitaceae plants include but are not limited to pumpkin (Cucurbita moschata), cucumber (Cucumis sativus), watermelon (Citrullus lanatus), melon (Cucumis melo), gourd (Lagenaria siceraria), winter melon (Benincasa) hispida), Luffa cylindrica, Momordica charantia, Sechium edule, Gynostemma pentaphyllum, Hemsleya chinensis, Trichosanthes kirilowii, Momordica cochinchinensis, Luo Han Guo (Siraitia grosvenorii) etc. Other Cucurbitaceae plants not listed are also within the protection scope of the present invention.
本发明另一方面提供了一种葫芦科植物材料的培育方法，在受体植物中删除上述5’UTR片段或其部分片段，得到目的植物；与受体植物相比，目的植物的主茎长度缩短。On the other hand, the present invention provides a method for cultivating Cucurbitaceae plant materials. The above-mentioned 5'UTR fragment or partial fragment thereof is deleted in the recipient plant to obtain the target plant; compared with the recipient plant, the main stem length of the target plant is shorten.
本发明实施例中利用基因编辑工具将葫芦科作物中的YABBY1基因的5’UTR中保守的B-region序列删除，使所述基因的蛋白翻译效率提高，达到缩短葫芦科植物主茎长度的目的。In the embodiments of the present invention, gene editing tools are used to delete the conserved B-region sequence in the 5'UTR of the YABBY1 gene in cucurbit crops, so as to improve the protein translation efficiency of the gene and achieve the purpose of shortening the main stem length of cucurbit plants. .
本发明具体实施例中，利用基因编辑CRISPR-Cas9技术删除目的序列。将导入了CRISPR-Cas9系统的表达盒，或重组载体，或重组微生物应用于南瓜、黄瓜、西瓜和甜瓜等葫芦科植物的基因编辑中，可以对B-region序列进行删除，进而缩短这些葫芦科植物的主茎长度。 In specific embodiments of the present invention, gene editing CRISPR-Cas9 technology is used to delete the target sequence. The expression cassette, or recombinant vector, or recombinant microorganism introduced into the CRISPR-Cas9 system is applied to gene editing of cucurbit plants such as pumpkins, cucumbers, watermelons, and melons. The B-region sequence can be deleted, thereby shortening these cucurbits. The length of the main stem of the plant.
对于载体的具体种类不作限定，能够满足成功构建重组的表达载体，且该表达载体能够通过工程菌介导进入南瓜、黄瓜、西瓜和甜瓜中正常发挥CRISPR-Cas9系统的功能即可。具体的，载体包括但不限于PKSE401、PKSE402、pGREB32、pHR04、pCXUN-BE3，本发明具体实施例中选择PKSE402作为载体。There is no limit to the specific type of vector, as long as it can successfully construct a recombinant expression vector, and the expression vector can be mediated by engineering bacteria into pumpkin, cucumber, watermelon and melon to function normally of the CRISPR-Cas9 system. Specifically, vectors include but are not limited to PKSE401, PKSE402, pGREB32, pHRO4, and pCXUN-BE3. In specific embodiments of the present invention, PKSE402 is selected as the vector.
本发明具体实施例中，利用导入了上述表达盒和/或载体的微生物或植物细胞删除目的序列。In specific embodiments of the present invention, the target sequence is deleted using microorganisms or plant cells introduced with the above-mentioned expression cassette and/or vector.
本发明实施例中所述的表达盒和/或载体可以被引入到微生物或植物细胞中。此处，“微生物”是指引入所述CRISPR-Cas9系统的表达盒和/或载体，并且还包括携带所述表达盒和/载体的这种微生物的子代。微生物可以是任何原核或真核细胞生物。The expression cassettes and/or vectors described in the embodiments of the present invention can be introduced into microorganisms or plant cells. Here, "microorganism" refers to the expression cassette and/or vector introduced into the CRISPR-Cas9 system, and also includes the progeny of such microorganism carrying the expression cassette and/or vector. Microorganisms can be any prokaryotic or eukaryotic cell organism.
进一步，所述微生物包括但不限于农杆菌、枯草杆菌、枯草芽孢杆菌、大肠杆菌、乳酸菌、昆虫细胞，或本领域技术人员常用的其它合适作为宿主细胞的微生物。Further, the microorganisms include, but are not limited to, Agrobacterium, Bacillus subtilis, Bacillus subtilis, Escherichia coli, lactic acid bacteria, insect cells, or other microorganisms commonly used by those skilled in the art as suitable host cells.
进一步，微生物优选农杆菌。农杆菌的菌种包括但不限于：农杆菌EHA105、农杆菌LBA440、农杆菌EHA101、农杆菌GV3101。Furthermore, the microorganism is preferably Agrobacterium. Agrobacterium strains include but are not limited to: Agrobacterium EHA105, Agrobacterium LBA440, Agrobacterium EHA101, and Agrobacterium GV3101.
在本发明的一种实施方式中，包含CRISPR-Cas9系统的表达盒和/或载体的试剂和/或试剂盒用于培育葫芦科植物主茎缩短的材料。In one embodiment of the present invention, reagents and/or kits containing expression cassettes and/or vectors of the CRISPR-Cas9 system are used to cultivate materials with shortened main stems of Cucurbitaceae plants.
本发明另一方面提供了一种葫芦科植物材料或其相关产品，其为上述的植物细胞系、植物原生质体、细胞或愈伤组织生长形成的植物或其相关产品；或者采用上述葫芦科植物材料的培育方法获得的植物或其相关产品；Another aspect of the present invention provides a Cucurbitaceae plant material or its related products, which is a plant grown from the above-mentioned plant cell line, plant protoplast, cell or callus tissue or its related products; or the above-mentioned Cucurbitaceae plant is used Plants or their related products obtained by cultivation methods of materials;
相关产品为其植物部分、植物细胞、花粉或种子。The relevant products are plant parts, plant cells, pollen or seeds.
在本发明提供的实施例中，植物材料包括种质、株系、品系、商品种。In the embodiments provided by the present invention, plant materials include germplasm, strains, lines, and commercial varieties.
本发明另一方面提供了一种葫芦科植物材料的育种方法，包括将上述葫芦科植物材料自交，或与其它品种杂交，获得葫芦科植物材料。Another aspect of the present invention provides a breeding method for Cucurbitaceae plant materials, which includes self-crossing the above-mentioned Cucurbitaceae plant materials or crossing them with other varieties to obtain Cucurbitaceae plant materials.
在本发明的一种实施方式中，上述葫芦科植物的育种方法为主茎缩短的二倍体材料进行自交或与其他二倍体材料进行杂交，获得主茎缩短的材料后代。In one embodiment of the present invention, the above-mentioned breeding method for Cucurbitaceae plants involves selfing diploid materials with shortened main stems or hybridizing with other diploid materials to obtain offspring of materials with shortened main stems.
在本发明的一种实施方式中，上述葫芦科植物的育种方法为主茎缩短的二倍体材料加倍变成四倍体后与其他四倍体材料进行杂交后，再降倍变成二倍体材料后代。In one embodiment of the present invention, the breeding method of the above-mentioned Cucurbitaceae plants is to double the diploid material with shortened main stem into tetraploid, cross it with other tetraploid materials, and then reduce the doubling into tetraploid. Body material descendants.
在本发明的一种实施方式中，上述葫芦科植物的育种方法为主茎缩短的二倍体材料加倍变成四倍体后与其他四倍体材料进行杂交后，获得四倍体材料。In one embodiment of the present invention, the above-mentioned breeding method for Cucurbitaceae plants involves doubling diploid material with shortened main stems into tetraploid and then hybridizing with other tetraploid materials to obtain tetraploid material.
在本发明提供的实施例中，植物材料包括种质、株系、品系、商品种。 In the embodiments provided by the present invention, plant materials include germplasm, strains, lines, and commercial varieties.
本发明另一方面提供了一种葫芦科植物材料或其相关产品，其为上述葫芦科植物材料自交，或与其它品种杂交所形成的后代，以及后代生长形成的植物材料或其相关产品；Another aspect of the present invention provides a Cucurbitaceae plant material or its related products, which is the progeny formed by the above-mentioned Cucurbitaceae plant material being self-crossed or hybridized with other varieties, as well as the plant material or its related products formed by the growth of the progeny;
相关产品为其植物部分、植物细胞、花粉或种子。The relevant products are plant parts, plant cells, pollen or seeds.
本发明另一方面提供了一种由上述葫芦科植物材料或其相关产品制成的商业植物产品，商业植物产品包括食品、药品、化妆品/护肤品、饲料或其它产品。Another aspect of the present invention provides a commercial plant product made from the above-mentioned Cucurbitaceae plant material or its related products. Commercial plant products include food, medicine, cosmetics/skin care products, feed or other products.
在本发明提供的具体实施例中，食品包括但不限于：新鲜的果实、干燥的果实、冷冻的果实、腌制的果实、蜜制的果实、油炸的果实或其各种加工形式的果实；各种加工形式的果实包括但不限于果实条、果实块、果实片、果实颗粒、果实粉、果实酱、果实汁液、发酵产品。In the specific embodiments provided by the present invention, foods include but are not limited to: fresh fruits, dried fruits, frozen fruits, pickled fruits, honeyed fruits, fried fruits or fruits in various processed forms thereof. ; Fruits in various processed forms include but are not limited to fruit strips, fruit pieces, fruit slices, fruit granules, fruit powder, fruit paste, fruit juice, and fermented products.
在本发明提供的具体实施例中，药品包括：植物药用部分制成的各种剂型；植物药用部分可为根、茎、叶、花、果实、种子、瓜蒂等；剂型可为本领域中认可的任意剂型。In the specific embodiments provided by the present invention, medicines include: various dosage forms made from medicinal parts of plants; the medicinal parts of plants can be roots, stems, leaves, flowers, fruits, seeds, melon stems, etc.; the dosage forms can be this Any dosage form recognized in the field.
在本发明提供的具体实施例中，化妆品/护肤品包括但不限于：洗面奶、爽肤水、乳液、面霜、精华、面膜、眼霜、眼膜、隔离乳、防晒乳、粉底液、BB霜。In the specific embodiment provided by the present invention, cosmetics/skin care products include but are not limited to: facial cleanser, toner, lotion, cream, essence, facial mask, eye cream, eye mask, isolation lotion, sunscreen lotion, liquid foundation, and BB cream.
在本发明提供的具体实施例中，饲料包括但不限于：液态饲料、固态饲料、半固态饲料或饲料原料。In the specific embodiments provided by the present invention, feed includes but is not limited to: liquid feed, solid feed, semi-solid feed or feed raw materials.
本发明另一方面提供了一种制造商业植物产品的方法，包括获得上述葫芦科植物材料或其相关产品，制造商业植物产品。Another aspect of the present invention provides a method for manufacturing commercial plant products, including obtaining the above-mentioned Cucurbitaceae plant materials or related products and manufacturing commercial plant products.
与现有技术相比，本发明具有的有益效果为：Compared with the prior art, the beneficial effects of the present invention are:
本发明通过利用基因编辑工具，将葫芦科植物(南瓜、黄瓜、西瓜和甜瓜等)的YABBY1基因5’UTR的保守的B-region序列删除或部分删除，使得YABBY1的翻译效率提高，从而缩短了葫芦科植物主茎长度，达到葫芦科植物株型矮化和密植高产的目的，同时可以大大节约劳动力。The present invention uses gene editing tools to delete or partially delete the conserved B-region sequence of the 5'UTR of the YABBY1 gene of Cucurbitaceae plants (pumpkins, cucumbers, watermelons, melons, etc.), thereby improving the translation efficiency of YABBY1, thereby shortening The length of the main stem of Cucurbitaceae plants can achieve the purpose of dwarfing Cucurbitaceae plants and enabling dense planting and high yields, while also greatly saving labor.
附图说明Description of the drawings
图1示中国南瓜矮化基因Bu的图位克隆；Figure 1 shows the map-based cloning of Chinese pumpkin dwarfing gene Bu;
图注：a，F2分离群体中矮化池和蔓生池的混池测序分析，信号位点位于中国南瓜15号染色体；b，矮化基因Bu的精细定位；c，精细定位区间内存在8个候选基因； d，Bu候选基因的基因结构；e，Bu基因的进化树分析；Legend: a, mixed pool sequencing analysis of dwarf pool and creeping pool in F2 segregating population, the signal site is located on Chinese pumpkin chromosome 15; b, fine mapping of dwarf gene Bu; c, there are 8 genes in the fine mapping interval candidate genes; d, gene structure of Bu candidate gene; e, evolutionary tree analysis of Bu gene;
图2示葫芦科植物的YABBY1基因的5’UTR中存在一个保守的序列区域“B-region”；Figure 2 shows that there is a conserved sequence region “B-region” in the 5’UTR of the YABBY1 gene of Cucurbitaceae plants;
图注：葫芦科作物和模式植物YABBY1基因的5’UTR的序列比对，植物种类包括拟南芥(Arabidopsis thaliana)，番茄(Solanum lycopersicum)，6种葫芦科植物即中国南瓜(Cucurbita moschata)，黄瓜(Cucumis sativus)，甜瓜(Cucumis melo)，西瓜(Citrullus lanatus)，冬瓜(Benincasa hispida)和葫芦(Lagenaria siceraria)；下划线对应的区域，即葫芦科植物的YABBY1基因的5’UTR中存在的保守“B-region”；Legend: Sequence alignment of the 5'UTR of the YABBY1 gene of cucurbit crops and model plants. Plant species include Arabidopsis thaliana, tomato (Solanum lycopersicum), and six cucurbit species, namely Chinese pumpkin (Cucurbita moschata). Cucumber (Cucumis sativus), melon (Cucumis melo), watermelon (Citrullus lanatus), winter melon (Benincasa hispida) and gourd (Lagenaria siceraria); the underlined region is the conserved region present in the 5'UTR of the YABBY1 gene of Cucurbitaceae plants "B-region";
图3示中国南瓜CmoYABBY1基因5’UTR的B-region删除导致南瓜植株的主茎变短；Figure 3 shows that B-region deletion of the 5’UTR of the Chinese pumpkin CmoYABBY1 gene results in shorter main stems of pumpkin plants;
图注：a，中国南瓜CmoYABBY1基因5’UTR的B-region的CRISPR双靶位点设计，以及获得的编辑序列结果；b，中国南瓜T1代编辑纯合的植株表现为主茎缩短；c，将T1代编辑纯合的植株叶片去掉后更清晰的展示缩短的节间和主茎；Legend: a, CRISPR dual target site design of the B-region of the 5'UTR of the Chinese pumpkin CmoYABBY1 gene, and the obtained editing sequence results; b, Chinese pumpkin T1 homozygous edited plants show shortening of the main stem; c, After removing the leaves from plants homozygous for the T1 generation editor, the shortened internode and main stem can be more clearly displayed;
图4示黄瓜CsYABBY1基因5’UTR的B-region删除导致黄瓜植株的主茎变短；Figure 4 shows that B-region deletion of the 5’UTR of the cucumber CsYABBY1 gene causes the main stem of the cucumber plant to become shorter;
图注：a，黄瓜CsYABBY1基因5’UTR的B-region的CRISPR双靶位点设计，以及获得的编辑序列结果；b，黄瓜T1代编辑纯合的植株表现为主茎缩短；c，黄瓜T1代编辑纯合的植株主茎长度的统计；Legend: a, CRISPR dual target site design of the B-region of the 5'UTR of the cucumber CsYABBY1 gene, and the obtained editing sequence results; b, cucumber T1 generation homozygous edited plants show shortening of the main stem; c, cucumber T1 Statistics on the main stem length of plants homozygous for the editor;
图5示西瓜ClYABBY1基因5’UTR的B-region删除导致西瓜植株的主茎变短；Figure 5 shows that B-region deletion of the 5’UTR of the watermelon ClYABBY1 gene causes the main stem of the watermelon plant to become shorter;
图注：a，西瓜ClYABBY1基因5’UTR的B-region的CRISPR双靶位点设计，以及获得的编辑序列结果；b，西瓜T1代编辑纯合的植株表现为主茎缩短；c，西瓜T1代编辑纯合的植株主茎长度的统计；Legend: a, CRISPR dual target site design of the B-region of the 5'UTR of the watermelon ClYABBY1 gene, and the obtained editing sequence results; b, Watermelon T1 homozygous edited plants show shortening of the main stem; c, Watermelon T1 Statistics on the main stem length of plants homozygous for the editor;
图6示甜瓜CmYABBY1基因5’UTR的B-region删除导致甜瓜植株的主茎变短；Figure 6 shows that B-region deletion of the 5’UTR of the melon CmYABBY1 gene causes the main stem of the melon plant to become shorter;
图注：a，甜瓜CmYABBY1基因5’UTR的B-region的CRISPR双靶位点设计，以及获得的编辑序列结果；b，甜瓜T1代编辑纯合的植株表现为主茎缩短；c，甜瓜T1代编辑纯合的植株主茎长度的统计。Legend: a, CRISPR dual target site design of the B-region of the 5'UTR of the muskmelon CmYABBY1 gene, and the obtained editing sequence results; b, plants homozygous for editing in the muskmelon T1 generation show shortening of the main stem; c, muskmelon T1 Statistics on main stem length of plants homozygous for editing.
具体实施方式Detailed ways
本发明公开了5’UTR片段和生物材料及其在调控植物主茎长度中的应用、葫芦科 植物或其相关产品，本领域技术人员可以借鉴本文内容，适当改进工艺参数实现。特别需要指出的是，所有类似的替换和改动对本领域技术人员来说是显而易见的，它们都被视为包括在本发明。本发明的方法及应用已经通过较佳实施例进行了描述，相关人员明显能在不脱离本发明内容、精神和范围内对本文所述的方法和应用进行改动或适当变更与组合，来实现和应用本发明技术。The invention discloses 5'UTR fragments and biological materials and their application in regulating the length of plant main stems. Cucurbitaceae For plants or related products, those skilled in the art can learn from the content of this article and appropriately improve the process parameters. It should be noted that all similar substitutions and modifications are obvious to those skilled in the art, and they are deemed to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant persons can obviously make changes or appropriate changes and combinations to the methods and applications described herein without departing from the content, spirit and scope of the present invention to achieve and Apply the technology of this invention.
术语解释：Terminology explanation:
UTR(Untranslated Region)，即非翻译区。在分子遗传学中，是指任意一个位于mRNA链编码序列两端的片段。如果其位于5′端，则称为5′非翻译区(5'-untranslated region，5'-UTR)(或"前导序列，leader"),反之若位于3′端，则称为3′非翻译区(3'-untranslated region，3'-UTR)(或"尾随序列，trailer")。尽管它们被称为"非翻译区"，并且不是构成该基因的蛋白质编码区，但在5′非翻译区内的上游可读框可以被翻译成多肽；UTR (Untranslated Region), which is the untranslated region. In molecular genetics, it refers to any fragment located at both ends of the coding sequence of the mRNA chain. If it is located at the 5' end, it is called the 5'-untranslated region (5'-UTR) (or "leader sequence, leader"). Otherwise, if it is located at the 3' end, it is called the 3' untranslated region. Translated region (3'-untranslated region, 3'-UTR) (or "trailing sequence, trailer"). Although they are called "untranslated regions" and are not the protein-coding regions that make up the gene, the upstream open reading frame within the 5' untranslated region can be translated into polypeptides;
cDNA：全称complementary DNA，是一种互补脱氧核糖核酸；cDNA: The full name is complementary DNA, which is a kind of complementary deoxyribonucleic acid;
CRISPR/Cas9系统：CRISPR/Cas9是细菌和古细菌在长期演化过程中形成的一种适应性免疫防御，可用来对抗入侵的病毒及外源DNA。CRISPR/Cas9系统通过将入侵噬菌体和质粒DNA的片段整合到CRISPR中，并利用相应的CRISPR RNAs(crRNAs)来指导同源序列的降解，从而提供免疫性。此系统的工作原理是crRNA(CRISPR-derived RNA)通过碱基配对与tracrRNA(trans-activating RNA)结合形成tracrRNA/crRNA复合物，此复合物引导核酸酶Cas9蛋白再与crRNA配对的序列靶位点剪切双链DNA。而通过人工设计这两种RNA，可以改造形成具有引导作用的sgRNA(single-guide RNA)，足以引导Cas9对DNA的定点切割；CRISPR/Cas9 system: CRISPR/Cas9 is an adaptive immune defense formed by bacteria and archaea during the long-term evolution process. It can be used to fight against invading viruses and foreign DNA. The CRISPR/Cas9 system provides immunity by integrating fragments of invading phage and plasmid DNA into CRISPR and using corresponding CRISPR RNAs (crRNAs) to direct the degradation of homologous sequences. The working principle of this system is that crRNA (CRISPR-derived RNA) combines with tracrRNA (trans-activating RNA) through base pairing to form a tracrRNA/crRNA complex. This complex guides the nuclease Cas9 protein to pair with the sequence target site of crRNA. Cut double-stranded DNA. By artificially designing these two types of RNA, sgRNA (single-guide RNA) can be transformed to form a guiding function, which is enough to guide Cas9 to cut DNA at a specific location;
sgRNA(small guide RNA)：是向导RNA，在RNA编辑的过程中引导尿苷残基插入或缺失到动质体中，属于一种小型非编码RNA，可与pre-mRNA配对。gRNA编辑RNA分子，长度大约60-80个核苷酸，由单独的基因转录；sgRNA (small guide RNA): It is a guide RNA that guides the insertion or deletion of uridine residues into kinetoplasts during the RNA editing process. It is a small non-coding RNA that can be paired with pre-mRNA. gRNA edits RNA molecules, approximately 60-80 nucleotides in length, transcribed by individual genes;
杂交：两个基因型不同的个体相交。也指不同品种间的交配。植物可指不同品种间的异花传粉；Hybridization: The crossing of two individuals with different genotypes. Also refers to mating between different breeds. Plants can refer to cross-pollination between different species;
自交：两个基因型相同的个体相交。植物指自花传粉；Selfing: the intersection of two individuals with the same genotype. Plant refers to self-pollinating;
F2：是杂交或自交的子二代；F2: It is the second generation of hybrid or selfing;
染色体倍性(chromosome ploidy)：是指细胞中包含的染色体组数或基因组数。 正常的配子细胞中所包含的染色体数或半数的体细胞染色体数称为一套单倍染色体，用符号n表示。某种生物的完整的一套单倍染色体称为该种生物的基因组或染色体组。具有一个染色体组的细胞和由这样的细胞组成的个体称为单倍体(n)，具有两个染色体组的细胞或个体称为二倍体(2n)，具有两个以上整套染色体组的细胞或个体则称为多倍体，包括三倍体(3n)、四倍体(4n)等。由相同来源染色体组形成的多倍体称为同源多倍体，由不同来源不同染色体组形成的多倍体称为异源多倍体；Chromosome ploidy: refers to the number of chromosome sets or genomes contained in a cell. The number of chromosomes contained in normal gametocytes or half of the number of chromosomes in somatic cells is called a set of haploid chromosomes, represented by the symbol n. The complete set of haploid chromosomes of an organism is called the genome or chromosome set of that organism. Cells with one set of chromosomes and individuals composed of such cells are called haploids (n), cells or individuals with two sets of chromosomes are called diploids (2n), and cells with more than two complete sets of chromosomes are called diploids (2n). Or individuals are called polyploids, including triploid (3n), tetraploid (4n), etc. Polyploidy formed from chromosome sets from the same source is called autopolyploidy, and polyploidy formed from different chromosome sets from different sources is called allopolyploidy;
Sanger测序：Sanger法是根据核苷酸在某一固定的点开始，随机在某一个特定的碱基处终止，并且在每个碱基后面进行荧光标记，产生以A、T、C、G结束的四组不同长度的一系列核苷酸，然后在尿素变性的PAGE胶上电泳进行检测，从而获得可见DNA碱基序列的一种方法；Sanger sequencing: The Sanger method is based on the nucleotide starting at a fixed point, randomly ending at a specific base, and fluorescently labeling behind each base, resulting in A, T, C, G. A method of obtaining a visible DNA base sequence by electrophoresis on a urea-denatured PAGE gel using a series of four sets of nucleotides of different lengths;
InDel(insertion-deletion)插入缺失标记，指的是两种亲本中在全基因组中的差异，相对另一个亲本而言，其中一个亲本的基因组中有一定数量的核苷酸插入或缺失。根据基因组中插入缺失位点，设计一些扩增这些插入缺失位点的PCR引物，这就是InDel标记；InDel (insertion-deletion) insertion and deletion markers refer to the differences in the whole genome between two parents. Compared with the other parent, a certain number of nucleotides are inserted or deleted in the genome of one parent. According to the indel sites in the genome, design some PCR primers that amplify these indel sites. This is the InDel marker;
单核苷酸多态性(single nucleotide polymorphism，SNP)：主要是指在基因组水平上由单个核苷酸的变异所引起的DNA序列多态性。它是人类可遗传的变异中最常见的一种。占所有已知多态性的90％以上；Single nucleotide polymorphism (SNP): mainly refers to the DNA sequence polymorphism caused by the variation of a single nucleotide at the genome level. It is one of the most common heritable variations in humans. Accounts for more than 90% of all known polymorphisms;
基因组结构性变异(Structure Variantions，简称SVs)通常指基因组上大长度的序列变化和位置关系变化。基因组结构性变异类型很多，包括长度在50bp以上的长片段序列插入或者删除(Big Indel)、串联重复(Tandem repeate)、染色体倒位(Inversion)、染色体内部或染色体之间的序列易位(Translocation)、拷贝数变异(CNV)以及形式更为复杂的嵌合性变异。Genome structural variations (SVs) usually refer to large-length sequence changes and positional relationship changes on the genome. There are many types of genome structural variations, including insertion or deletion of long fragments of more than 50 bp (Big Indel), tandem repeats (Tandem repeats), chromosomal inversions (Inversion), and sequence translocations within or between chromosomes (Translocation). ), copy number variations (CNV), and more complex forms of mosaic variation.
核苷酸表：
Nucleotide table:
南瓜中的YABBY1基因，表示为CmoYABBY1，对应南瓜的基因号是CmoCh15G012090；黄瓜中的YABBY1基因，表示为CsYABBY1，对应黄瓜的基因号是CsaV3_5G003950；西瓜中的YABBY1基因，表示为ClYABBY1，对应西瓜的基因号是Cla97C01G003950；甜瓜中的YABBY1基因，表示为CmYABBY1，对应甜瓜的基因号是MELO3C000087.2。在葫芦科植物YABBY1基因的5’UTR中，存在一个保守的B-region区域。The YABBY1 gene in pumpkin is expressed as CmoYABBY1, and the gene number corresponding to pumpkin is CmoCh15G012090; the YABBY1 gene in cucumber is expressed as CsYABBY1, and the gene number corresponding to cucumber is CsaV3_5G003950; the YABBY1 gene in watermelon is expressed as ClYABBY1, and the gene number corresponding to watermelon The number is Cla97C01G003950; the YABBY1 gene in melon is expressed as CmYABBY1, and the gene number corresponding to melon is MELO3C000087.2. There is a conserved B-region in the 5’UTR of the YABBY1 gene in cucurbits.
本发明中所用试剂、仪器、菌种或生物材料等均可由市场购得。The reagents, instruments, bacterial strains or biological materials used in the present invention can all be purchased from the market.
下面结合实施例，进一步阐述本发明：The present invention will be further described below in conjunction with the examples:
实施例1中国南瓜矮化基因Bu的图位克隆Example 1 Map-based cloning of Chinese pumpkin dwarfing gene Bu
中国南瓜杂合商品种“无蔓4号”自交后，在后代群体里发生矮化性状和蔓生性状的分离，遗传学分析表明该矮化性状是由一个完全显性单基因Bu控制。通过矮化单株池和蔓生单株池的测序比较分析，在中国南瓜的15号染色体7.8-8.8Mb区间，鉴定到一个显著的信号峰(图1a)，即Bu基因初步定位于定位于该区间。通过对3200单株的F2分离群体筛选重组单株进行精细定位，结合重组单株表型和分子标记基因型分析，最后Bu基因定位在一个55kb的物理区间内(图1b)，通过序列的注释分析发现该区间内有8个候选基因(图1c)。通过对矮化单株池和蔓生单株池测序数据的变异分析，只发现了一个InDel(矮化南瓜相对于长蔓南瓜缺失了76bp序列)变异，位于基因号为CmoCh15G012090的5’UTR(图1d)，除此外，该55kb物理区间内没有其他任何变异(包括SNP、InDel和SV)。通过进化树分析(图1e)，发现CmoCh15G012090基因是一个YABBY家族的转录因子(即CmoYABBY1)。同时，针对Bu位点的76bp变异，我们设计了分子标记并检测了550份中国长蔓南瓜种质，发现这些长蔓南瓜种质均存在该76bp序列。综合上述的实验数据，我们认为CmoCh15G012090的5’UTR中76bp序列的缺失很可能是矮化表型发生的原因。After the Chinese pumpkin hybrid commercial variety "Wuman No. 4" was selfed, the dwarfing traits and vine traits were separated in the offspring population. Genetic analysis showed that the dwarfing traits were controlled by a completely dominant single gene Bu. Through comparative sequencing analysis of the dwarf single plant pool and the vine single plant pool, a significant signal peak was identified in the 7.8-8.8Mb interval of chromosome 15 of Chinese pumpkin (Figure 1a), that is, the Bu gene was initially located in this region. interval. By screening the F2 isolate population of 3200 individual plants for fine mapping of recombinant individuals, combined with phenotypic and molecular marker genotype analysis of the recombinant individual plants, the Bu gene was finally located within a 55kb physical interval (Figure 1b), and through sequence annotation The analysis found that there were 8 candidate genes in this interval (Fig. 1c). Through mutation analysis of the sequencing data of the dwarf single plant pool and the vine single plant pool, only one InDel (dwarf pumpkin deleted 76 bp sequence compared to the long vine pumpkin) mutation was found, which is located in the 5'UTR of the gene number CmoCh15G012090 (Figure 1d), except that there are no other variants (including SNP, InDel and SV) in this 55kb physical interval. Through evolutionary tree analysis (Fig. 1e), it was found that the CmoCh15G012090 gene is a transcription factor of the YABBY family (i.e., CmoYABBY1). At the same time, for the 76 bp variation in the Bu site, we designed molecular markers and tested 550 Chinese long-vine pumpkin germplasms, and found that the 76-bp sequence existed in these long-vine pumpkin germplasms. Based on the above experimental data, we believe that the deletion of 76 bp sequence in the 5’UTR of CmoCh15G012090 is likely to be the cause of the dwarf phenotype.
为了便于后续的阐述和分析，我们将CmoCh15G012090的5’UTR中缺失的76bp序列分为2部分，即25bp的A-region和51bp的B-region(图1d)。将葫芦科植物的YABBY1基因的5’UTR序列进行比对，结果显示B-region在葫芦科中较为保守(图2)，预示着在葫芦科作物里B-region作为一个保守的功能元件可能具有一定的生物学功 能。In order to facilitate subsequent elaboration and analysis, we divided the 76 bp sequence deleted in the 5'UTR of CmoCh15G012090 into 2 parts, namely the 25 bp A-region and the 51 bp B-region (Figure 1d). The 5'UTR sequence of the YABBY1 gene of Cucurbitaceae was compared, and the results showed that B-region is relatively conserved in Cucurbitaceae (Figure 2), indicating that B-region may serve as a conserved functional element in Cucurbitaceae crops. certain biological skills able.
南瓜YABBY1的B-region序列(SEQ ID NO：3)：
B-region sequence of pumpkin YABBY1 (SEQ ID NO: 3):
黄瓜YABBY1的B-region序列(SEQ ID NO：4)：
B-region sequence of cucumber YABBY1 (SEQ ID NO: 4):
甜瓜YABBY1的B-region序列(SEQ ID NO：4)：
B-region sequence of melon YABBY1 (SEQ ID NO: 4):
西瓜YABBY1的B-region序列(SEQ ID NO：4)：
B-region sequence of watermelon YABBY1 (SEQ ID NO: 4):
葫芦YABBY1的B-region序列(SEQ ID NO：4)：
B-region sequence of Cucurbita YABBY1 (SEQ ID NO: 4):
冬瓜YABBY1的B-region序列(SEQ ID NO：5)：
B-region sequence of winter melon YABBY1 (SEQ ID NO: 5):
实施例2 CmoYABBY1基因5’UTR的B-region片段或部分片段删除导致了南瓜植株的主茎变短Example 2 Deletion of the B-region fragment or part of the 5’UTR of the CmoYABBY1 gene causes the main stem of pumpkin plants to become shorter
利用CRISPR/Cas9工具，对南瓜CmoYABBY1(CmoCh15G012090)基因的B-region设计双靶位进行基因编辑，获得了一系列的缺失B-region南瓜突变体。具体步骤如下：Using the CRISPR/Cas9 tool, we designed dual target sites in the B-region of the pumpkin CmoYABBY1 (CmoCh15G012090) gene for gene editing, and obtained a series of pumpkin mutants lacking the B-region. Specific steps are as follows:
1.sgRNA序列的设计和CRISPR/Cas9载体的构建1. Design of sgRNA sequence and construction of CRISPR/Cas9 vector
在CmoYABBY1基因的B-region上设计双靶位点序列，长度均为19bp。双靶位点设计的位置如图3a，靶位点1的核苷酸序列为AAAAGGGAAGGCAGATCCA(5’-3’)(SEQ ID NO：6)，靶位点2的核苷酸序列为TGTTTGGTTGACTGATTGA(5’-3’)(SEQ ID NO：7)。Dual target site sequences were designed on the B-region of the CmoYABBY1 gene, both with a length of 19 bp. The position of the dual target site design is shown in Figure 3a. The nucleotide sequence of target site 1 is AAAAGGGAAGGCAGATCCA (5'-3') (SEQ ID NO: 6), and the nucleotide sequence of target site 2 is TGTTTGGTTGACTGATTGA (5 '-3') (SEQ ID NO: 7).
通过试剂盒In-Fusion HD Cloning Plus(Clontech,#638909)，利用In-Fusion克隆技 术将含有上述2个靶位点的序列构建入PKSE402载体中，PKSE402的酶切位点为Bsa I。Using In-Fusion cloning technology through the kit In-Fusion HD Cloning Plus (Clontech, #638909) The sequence containing the above two target sites was constructed into the PKSE402 vector, and the restriction site of PKSE402 was Bsa I.
将上步连接好的PKSE402-CRISPR/Cas9载体转化至大肠杆菌感受态DH5α，经过基因测序确定正确的载体质粒。Transform the PKSE402-CRISPR/Cas9 vector connected in the previous step into E. coli competent DH5α, and determine the correct vector plasmid through gene sequencing.
2.南瓜遗传转化和阳性转基因植株的获得2. Genetic transformation of pumpkin and acquisition of positive transgenic plants
将上述获得的PKSE402-CRISPR/Cas9载体通过热激转化转至农杆菌感受态细胞EHA105，得到重组菌EHA105/PKSE402-CRISPR/Cas9。The PKSE402-CRISPR/Cas9 vector obtained above was transferred into Agrobacterium competent cells EHA105 through heat shock transformation to obtain the recombinant strain EHA105/PKSE402-CRISPR/Cas9.
将重组菌EHA105/PKSE402-CRISPR/Cas9采用农杆菌侵染子叶的转化方法。将发芽的南瓜种子去除胚芽，并将子叶远轴端切割去除1/3，剩余的子叶作为外植体。将这些外植体转移到含有20mL农杆菌EHA105悬浮液的三角形瓶中，并对其进行超声处理，然后将南瓜外植体在农杆菌悬浮液中进行真空渗透。将外植体与农杆菌在潮湿的滤纸上在黑暗中共培养3天，然后转移至芽诱导培养基。培养3-4周后，由于PKSE402载体中携带标签绿色荧光蛋白，选择具有绿色荧光的植株即为T0代转基因南瓜植株。The recombinant strain EHA105/PKSE402-CRISPR/Cas9 was transformed using Agrobacterium to infect cotyledons. Remove the embryo from the germinated pumpkin seeds, cut the distal end of the cotyledons and remove 1/3, and use the remaining cotyledons as explants. These explants were transferred to triangular flasks containing 20 mL of Agrobacterium EHA105 suspension and sonicated, and then the pumpkin explants were vacuum infiltrated in the Agrobacterium suspension. Explants were co-cultured with Agrobacterium on moist filter paper in the dark for 3 days and then transferred to shoot induction medium. After 3-4 weeks of culture, since the PKSE402 vector carries the tagged green fluorescent protein, the plants with green fluorescence are selected as T0 generation transgenic pumpkin plants.
3.B-region发生突变的南瓜阳性植株鉴定和表型观察3. Identification and phenotypic observation of pumpkin-positive plants with mutated B-region
提取T0代转基因南瓜植株的基因组DNA作为模板，PCR扩增B-region区域，得到不同株系的PCR扩增产物。将不同株系的PCR扩增产物进行Sanger测序，根据测序结果与野生型南瓜的B-region区域进行比对分析。The genomic DNA of T0 generation transgenic pumpkin plants was extracted as a template, and the B-region region was amplified by PCR to obtain PCR amplification products of different strains. The PCR amplification products of different strains were subjected to Sanger sequencing, and the sequencing results were compared with the B-region of wild-type pumpkin.
测序比对结果表明，采用CRISPR/Cas9系统成功的实现了对南瓜CmoYABBY1的5’UTR中B-region片段或部分片段的删除编辑，造成了4种删除模式(图3a)。Sequencing comparison results showed that the CRISPR/Cas9 system was used to successfully delete and edit the B-region fragment or part of the 5'UTR of pumpkin CmoYABBY1, resulting in four deletion patterns (Figure 3a).
将获得的T0代南瓜基因编辑植株继续自交获得种子，T1代继续种植后观察表型。结果显示(图3b-3c)，与野生型对照的南瓜植株相比，CmoM-1、CmoM-2、CmoM-3和CmoM-4突变南瓜植株的主茎长度显著变短。The obtained T0 generation pumpkin gene-edited plants will continue to be selfed to obtain seeds, and the T1 generation will continue to be planted and the phenotypes will be observed. The results showed (Figure 3b-3c) that compared with wild-type control pumpkin plants, the main stem length of CmoM-1, CmoM-2, CmoM-3 and CmoM-4 mutant pumpkin plants was significantly shorter.
实施例3 CsYABBY1基因5’UTR的B-region片段或部分片段删除导致了黄瓜植株的主茎变短Example 3 Deletion of the B-region fragment or part of the 5’ UTR of the CsYABBY1 gene causes the main stem of cucumber plants to become shorter
利用CRISPR/Cas9工具，对黄瓜CsYABBY1(CsaV3_5G003950)基因的B-region设计双靶位进行基因编辑。The CRISPR/Cas9 tool was used to design dual target sites in the B-region of the cucumber CsYABBY1 (CsaV3_5G003950) gene for gene editing.
在CsYABBY1基因的B-region上设计双靶位点序列，长度均为19bp。双靶位点设 计的位置如图4a，靶位点1的核苷酸序列为AAAAGAAAAGGCAGATCCA(5’-3’)(SEQ ID NO：8)，靶位点2的核苷酸序列为ATTGGTAGTGACTGATTGA(5’-3’)(SEQ ID NO：9)。Dual target site sequences were designed on the B-region of the CsYABBY1 gene, both with a length of 19 bp. Dual target site design The calculated positions are shown in Figure 4a. The nucleotide sequence of target site 1 is AAAAGAAAAGGCAGATCCA (5'-3') (SEQ ID NO: 8), and the nucleotide sequence of target site 2 is ATTGGTAGTGACTGATTGA (5'-3'). ) (SEQ ID NO: 9).
载体质粒的构建和农杆菌侵染外植体过程同实施例2。The construction of vector plasmid and the process of infecting explants with Agrobacterium were the same as in Example 2.
转基因黄瓜植株的获得和B-region发生突变的黄瓜阳性植株鉴定过程同实施例2。The procedures for obtaining transgenic cucumber plants and identifying cucumber-positive plants with mutated B-region are the same as in Example 2.
测序比对结果表明，采用CRISPR/Cas9系统成功的实现了对黄瓜CsYABBY1的5’UTR中B-region片段或部分片段的删除编辑，造成了3种删除模式(图4a)。Sequencing comparison results showed that the CRISPR/Cas9 system was used to successfully delete and edit the B-region fragment or part of the 5'UTR of cucumber CsYABBY1, resulting in three deletion patterns (Figure 4a).
在上述编辑植株的T1代观察表型，结果显示(图4b-4c)，与野生型对照的黄瓜植株相比，CsM-1、CsM-2和CsM-3突变黄瓜植株的主茎长度显著变短。The phenotypes were observed in the T1 generation of the above edited plants. The results showed (Figure 4b-4c) that compared with the wild-type control cucumber plant, the main stem length of CsM-1, CsM-2 and CsM-3 mutant cucumber plants changed significantly. short.
实施例4 ClYABBY1基因5’UTR的B-region删除导致西瓜植株的主茎变短Example 4 B-region deletion of the 5’UTR of the ClYABBY1 gene results in shorter main stems of watermelon plants
利用CRISPR/Cas9工具，对西瓜ClYABBY1(Cla97C01G003950)基因的B-region设计双靶位进行基因编辑。The CRISPR/Cas9 tool was used to design dual target sites in the B-region of the watermelon ClYABBY1 (Cla97C01G003950) gene for gene editing.
在ClYABBY1基因的B-region上设计双靶位点序列，长度均为19bp。双靶位点设计的位置如图5a，靶位点1的核苷酸序列为AAAAGAAAAGGCAGATCCA(5’-3’)(SEQ ID NO：10)，靶位点2的核苷酸序列为TTGGGGTTTGACTGATTGA(5’-3’)(SEQ ID NO：11)。Double target site sequences were designed on the B-region of the ClYABBY1 gene, both with a length of 19 bp. The position of the dual target site design is shown in Figure 5a. The nucleotide sequence of target site 1 is AAAAGAAAAGGCAGATCCA (5'-3') (SEQ ID NO: 10), and the nucleotide sequence of target site 2 is TTGGGGTTTGACTGATTGA (5 '-3') (SEQ ID NO: 11).
载体质粒的构建同实施例2。The construction of vector plasmid is the same as in Example 2.
西瓜遗传转化过程如下：将西瓜种子萌发出芽，然后去除胚，将没有胚的子叶切成1.5×1.5-mm的小块。将含有PKSE402-CRISPR/Cas9的农杆菌侵染外植体。将侵染后的子叶外植体在黑暗中共培养4天，然后转移到选择性诱导培养基上，出现再生的不定芽后，将不定芽切下并转移到选择性延伸培养基上。培养数周后，选择具有绿色荧光的植株即为T0代转基因西瓜植株。The process of genetic transformation of watermelon is as follows: watermelon seeds are germinated, then the embryo is removed, and the cotyledons without embryos are cut into 1.5 × 1.5-mm pieces. The explants were infected with Agrobacterium containing PKSE402-CRISPR/Cas9. The infected cotyledon explants were cultured in the dark for 4 days, and then transferred to the selective induction medium. After regenerated adventitious buds appeared, the adventitious buds were cut out and transferred to the selective extension medium. After cultivating for several weeks, the plants with green fluorescence are selected as T0 generation transgenic watermelon plants.
B-region发生突变的西瓜阳性植株鉴定过程同实施例2。The identification process of watermelon-positive plants with mutated B-region is the same as in Example 2.
测序比对结果表明，采用CRISPR/Cas9系统成功的实现了对西瓜ClYABBY1的5’UTR中B-region区域的删除编辑，造成了3种删除模式(图5a)。Sequencing comparison results showed that the CRISPR/Cas9 system was used to successfully delete and edit the B-region region in the 5’UTR of watermelon ClYABBY1, resulting in three deletion patterns (Figure 5a).
在上述编辑植株的T1代观察表型，结果显示(图5b-5c)，与野生型对照的西瓜植株相比，ClM-1、ClM-2和ClM-3突变西瓜植株的主茎长度显著变短。 The phenotypes were observed in the T1 generation of the above edited plants. The results showed (Figure 5b-5c) that compared with the wild-type control watermelon plant, the main stem length of ClM-1, ClM-2 and ClM-3 mutant watermelon plants changed significantly. short.
实施例5 CmYABBY1基因5’UTR的B-region删除导致甜瓜植株的主茎变短Example 5 B-region deletion of the 5’UTR of the CmYABBY1 gene causes the main stem of the melon plant to become shorter
利用CRISPR/Cas9基因编辑工具，对甜瓜基因CmYABBY1(MELO3C000087.2)的B-region设计双靶位进行基因编辑。The CRISPR/Cas9 gene editing tool was used to design dual target sites in the B-region of the melon gene CmYABBY1 (MELO3C000087.2).
在CmYABBY1基因的B-region上设计双靶位点序列，长度均为19bp。双靶位点设计的位置如图6a，靶位点1的核苷酸序列为AAAAGAAAAGGCAGATCCA(5’-3’)(SEQ ID NO：12)，靶位点2的核苷酸序列为ATTGGTAGTGACTGATTGA(5’-3’)(SEQ ID NO：13)。Design dual target site sequences on the B-region of the CmYABBY1 gene, both with a length of 19 bp. The position of the dual target site design is shown in Figure 6a. The nucleotide sequence of target site 1 is AAAAGAAAAGGCAGATCCA (5'-3') (SEQ ID NO: 12), and the nucleotide sequence of target site 2 is ATTGGTAGTGACTGATTGA (5 '-3') (SEQ ID NO: 13).
载体质粒的构建和农杆菌侵染外植体过程同实施例2。The construction of vector plasmid and the process of infecting explants with Agrobacterium were the same as in Example 2.
转基因甜瓜植株的获得和B-region发生突变的甜瓜阳性植株鉴定过程同实施例2。The procedures for obtaining transgenic melon plants and identifying melon-positive plants with mutated B-region are the same as in Example 2.
测序比对结果表明，采用CRISPR/Cas9系统成功的实现了对甜瓜CmYABBY1的5’UTR中B-region片段或部分片段的删除编辑，造成了2种删除模式(图6a)。Sequencing comparison results showed that the CRISPR/Cas9 system was used to successfully delete and edit the B-region fragment or part of the 5'UTR of melon CmYABBY1, resulting in two deletion patterns (Figure 6a).
在上述编辑植株的T1代观察表型，结果显示(图6b-6c)，与野生型对照的甜瓜植株相比，CmM-1和CmM-2突变甜瓜植株的主茎长度显著变短。The phenotypes were observed in the T1 generation of the above edited plants, and the results showed (Figure 6b-6c) that compared with the wild-type control melon plant, the main stem length of CmM-1 and CmM-2 mutant melon plants was significantly shorter.
以上所述仅是本发明的优选实施方式，应当指出，对于本技术领域的普通技术人员来说，在不脱离本发明原理的前提下，还可以做出若干改进和润饰，这些改进和润饰也应视为本发明的保护范围。 The above are only preferred embodiments of the present invention. It should be noted that those skilled in the art can make several improvements and modifications without departing from the principles of the present invention. These improvements and modifications can also be made. should be regarded as the protection scope of the present invention.

Claims (13)

  1. 一种5’UTR片段，其特征在于，所述5’UTR片段包含如下核苷酸序列中的至少一种：A 5'UTR fragment, characterized in that the 5'UTR fragment contains at least one of the following nucleotide sequences:
    a)核苷酸序列结构为：B1-X-B2 a) The nucleotide sequence structure is: B 1 -XB 2
    其中，B1为SEQ ID NO：1所示序列，X为任意碱基或无碱基，B2为SEQ ID NO：2所示序列；Among them, B 1 is the sequence shown in SEQ ID NO: 1, X is any base or no base, and B 2 is the sequence shown in SEQ ID NO: 2;
    b)所述a)中所示序列的互补序列、简并序列或同源序列，其中所述同源序列为与a)所示序列具有80％或以上同一性的核苷酸序列；b) The complementary sequence, degenerate sequence or homologous sequence of the sequence shown in a), wherein the homologous sequence is a nucleotide sequence having 80% or more identity with the sequence shown in a);
    c)在严紧条件下与a)所示序列杂交的核苷酸序列或其互补序列；c) A nucleotide sequence that hybridizes to the sequence shown in a) under stringent conditions or its complementary sequence;
    d)所述序列a)-c)中任一序列的cDNA序列。d) The cDNA sequence of any of the sequences a)-c).
  2. 根据权利要求1所述的5’UTR片段，其特征在于，所述a)中核苷酸序列包含SEQ ID NO：3～SEQ ID NO：5所示序列中的至少一种。The 5' UTR fragment according to claim 1, wherein the nucleotide sequence in a) includes at least one of the sequences shown in SEQ ID NO: 3 ~ SEQ ID NO: 5.
  3. 一种生物材料，其特征在于，所述生物材料为下述1)至5)中的任一种：A biological material, characterized in that the biological material is any one of the following 1) to 5):
    1)删除权利要求1或2所述5’UTR片段或其部分片段的敲除盒；1) A knockout cassette that deletes the 5’ UTR fragment or partial fragment thereof according to claim 1 or 2;
    2)删除权利要求1或2所述5’UTR片段或其部分片段的敲除载体；2) A knockout vector that deletes the 5’ UTR fragment or partial fragment thereof according to claim 1 or 2;
    3)删除权利要求1或2所述5’UTR片段或其部分片段的重组微生物；3) A recombinant microorganism that deletes the 5’ UTR fragment or partial fragment thereof according to claim 1 or 2;
    4)删除权利要求1或2所述5’UTR片段或其部分片段的植物细胞系；4) A plant cell line in which the 5' UTR fragment or partial fragment thereof according to claim 1 or 2 is deleted;
    5)导入1)-3)中任意一种的植物原生质体、细胞或愈伤组织。5) Introduce the plant protoplasts, cells or callus of any one of 1)-3).
  4. 权利要求1-2中任一项所述5’UTR片段或权利要求3所述生物材料在缩短植物主茎长度中的应用。Application of the 5'UTR fragment of any one of claims 1-2 or the biological material of claim 3 in shortening the length of plant main stems.
  5. 根据权利要求4所述的应用，其特征在于，所述植物为葫芦科植物；The application according to claim 4, characterized in that the plant is a Cucurbitaceae plant;
    优选地，所述葫芦科植物包括南瓜、黄瓜、西瓜、甜瓜、葫芦、冬瓜、丝瓜、苦瓜、佛手瓜、绞股蓝、雪胆、栝楼、木鳖或罗汉果。Preferably, the Cucurbitaceae plants include pumpkin, cucumber, watermelon, melon, gourd, wax gourd, luffa, balsam pear, chayote, Gynostemma pentaphyllum, snow gallbladder, Trichosanthes aeruginosa, wood turtle or Luo Han Guo.
  6. 一种葫芦科植物材料的培育方法，其特征在于，在受体植物中将权利要求1或2所述5’UTR片段或其部分片段进行删除，得到目的植物；与受体植物相比，所述目的植物的主茎长度缩短。A method for cultivating Cucurbitaceae plant materials, which is characterized by deleting the 5'UTR fragment or partial fragments thereof according to claim 1 or 2 in the recipient plant to obtain the target plant; compared with the recipient plant, the The length of the main stem of the above-mentioned plants is shortened.
  7. 一种葫芦科植物材料或其相关产品，其特征在于，其为权利要求3所述的植物细胞系、植物原生质体、细胞或愈伤组织生长形成的植物材料或其相关产品；或者采 用权利要求6所述培育方法获得的植物材料或其相关产品；所述相关产品为其植物部分、植物细胞、花粉或种子。A Cucurbitaceae plant material or its related products, characterized in that it is a plant material formed by the growth of the plant cell line, plant protoplast, cell or callus described in claim 3, or its related products; or Plant materials or related products obtained by the cultivation method of claim 6; the related products are plant parts, plant cells, pollen or seeds.
  8. 根据权利要求7所述的葫芦科植物材料或其相关产品，其特征在于，所述植物材料包括种质、株系、品系或商品种。The Cucurbitaceae plant material or related products according to claim 7, wherein the plant material includes germplasm, strain, line or commercial variety.
  9. 一种葫芦科植物材料的育种方法，其特征在于，包括将权利要求7或8所述的葫芦科植物材料自交，或与其它品种杂交，获得葫芦科植物材料。A breeding method for Cucurbitaceae plant materials, which is characterized by comprising self-crossing the Cucurbitaceae plant materials described in claim 7 or 8, or crossing them with other varieties to obtain Cucurbitaceae plant materials.
  10. 一种葫芦科植物材料或其相关产品，其特征在于，其为权利要求7或8所述葫芦科植物材料自交，或与其它品种杂交所形成的后代，以及所述后代生长形成的植物材料或其相关产品；所述相关产品为其植物部分、植物细胞、花粉或种子。A Cucurbitaceae plant material or its related products, characterized in that it is the progeny formed by self-crossing the Cucurbitaceae plant material of Claim 7 or 8, or crossing with other varieties, and the plant material formed by the growth of the progeny or its related products; the related products are plant parts, plant cells, pollen or seeds.
  11. 一种由权利要求7或10所述的葫芦科植物材料或其相关产品制成的商业植物产品，其特征在于，所述商业植物产品包括食品、药品、化妆品/护肤品、饲料或其它产品。A commercial plant product made from the Cucurbitaceae plant material or related products according to claim 7 or 10, characterized in that the commercial plant product includes food, medicine, cosmetics/skin care products, feed or other products.
  12. 根据权利要求11所述的商业植物产品，其特征在于，所述食品包括：新鲜的果实、干燥的果实、冷冻的果实、腌制的果实、蜜制的果实、油炸的果实或其各种加工形式的果实；各种加工形式的果实包括果实条、果实块、果实片、果实颗粒、果实粉、果实酱、果实汁液、发酵产品；The commercial plant product according to claim 11, characterized in that the food includes: fresh fruits, dried fruits, frozen fruits, pickled fruits, honeyed fruits, fried fruits or various kinds thereof. Fruits in processed forms; fruits in various processed forms include fruit strips, fruit pieces, fruit slices, fruit granules, fruit powder, fruit paste, fruit juice, and fermented products;
    所述药品包括：植物药用部分制成的各种剂型；The medicines include: various dosage forms made from medicinal parts of plants;
    所述化妆品/护肤品包括：洗面奶、爽肤水、乳液、面霜、精华、面膜、眼霜、眼膜、隔离乳、防晒乳、粉底液、BB霜；The cosmetics/skin care products include: facial cleanser, toner, lotion, cream, essence, facial mask, eye cream, eye mask, isolation milk, sunscreen, liquid foundation, and BB cream;
    所述饲料包括：液态饲料、固态饲料、半固态饲料或饲料原料。The feed includes: liquid feed, solid feed, semi-solid feed or feed raw materials.
  13. 一种制造商业植物产品的方法，其特征在于，包括获得权利要求7或10所述的葫芦科植物材料或其相关产品，制造所述商业植物产品。 A method for manufacturing commercial plant products, characterized by comprising obtaining the Cucurbitaceae plant material or related products thereof according to claim 7 or 10, and manufacturing the commercial plant product.
PCT/CN2023/091275 2022-05-07 2023-04-27 Method for shortening length of main stem of cucurbitaceous plant and related product WO2023216907A1 (en)

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