WO2021093258A1 - VvDUF642基因引起植物种子败育的用途 - Google Patents

VvDUF642基因引起植物种子败育的用途 Download PDF

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WO2021093258A1
WO2021093258A1 PCT/CN2020/083508 CN2020083508W WO2021093258A1 WO 2021093258 A1 WO2021093258 A1 WO 2021093258A1 CN 2020083508 W CN2020083508 W CN 2020083508W WO 2021093258 A1 WO2021093258 A1 WO 2021093258A1
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vvduf642
protein
nucleic acid
gene
seedless
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French (fr)
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张颖
刘崇怀
刘锐涛
樊秀彩
李民
姜建福
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中国农业科学院郑州果树研究所
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    • 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
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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
    • C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
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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
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention relates to the technical field of genetic breeding, in particular to the use of VvDUF642.
  • Grapes belong to the grape family Vitis berries, and their fruits can be widely used in wine making, fresh food, juice making, and drying. Grape fruits are loved by people because of their sweet and sour taste, rich flavor, and rich nutrition. Among them, natural seedless grapes are popular because of their easy cultivation, easy processing and convenient eating. In developed countries, more than 50% of the fresh and dried grapes consumed are seedless varieties. The natural seedless formation pathway of grapes is divided into parthenocarpy (Parthenocarpy) and seed abortion type (Stenospermacarpy). Parthenocarpy is caused by poor pollination and fertilization. Seedless fruits are mainly found in the ears of seedless varieties.
  • Seed abortion is the most stable way for grapes to form seedless fruits, and it is also an important genetic characteristic that needs to be maintained in the cultivation of seedless varieties. Using modern molecular biology methods to study the molecular mechanism and genetic law of seedless grapes can improve the efficiency of cultivating new varieties of seedless grapes.
  • Grape is a close-flowered pollinating plant and has been fertilized when it blooms. Therefore, the study of grape seed development often starts before the flower.
  • Professor Michael Striem mapped the abortion process of grape seeds according to the development of grape seeds. With fertilization as the core, it is divided into ovule period (pre-flowering), fertilization period (flowering), and seed coat development period (5-10 days after flowering). Endosperm development period (11-30 days after flowering), embryo development period (31-40 days after flowering), after the completion of the entire development process, a normal seeded fruit will be formed.
  • the technical problem to be solved by the present invention is to provide the use of VvDUF642 in seed abortion.
  • the present invention provides the application of any one of the following I) to V) in inducing abortion or deformation of plant seeds;
  • nucleotides are substituted, deleted or added in the nucleotide sequence of the nucleic acid molecule of III) and can encode the same or similar functional protein;
  • V A substance capable of increasing the level or activity of at least one of I) to V).
  • VvDUF642 protein the substance that induces abortion or deformation of plant seeds.
  • amino acid sequence of the VvDUF642 protein is shown in SEQ ID NO:1;
  • sequence of the nucleic acid molecule encoding the protein shown in SEQ ID NO: 1 is shown in SEQ ID NO: 2.
  • plants that can cause seed deformation or abortion are from the grape family, cruciferous family or solanaceae.
  • the plant for experimental verification is grape, Arabidopsis or tomato.
  • the experiment of the present invention shows that the VvDUF642 gene is continuously highly expressed in seedless varieties, but there is no obvious change in the expression level in nucleated varieties.
  • the gene transforms Arabidopsis thaliana and causes Arabidopsis seed modification, and transforms tomato causes tomato seed abortion. .
  • the present invention also provides a preparation for causing plant seed abortion or deformation, including at least one of the following i) to v):
  • VvDUF642 protein or nucleic acid molecule encoding VvDUF642 protein i) VvDUF642 protein or nucleic acid molecule encoding VvDUF642 protein
  • VvDUF642 an inducer that promotes the expression of VvDUF642 gene.
  • the present invention also provides a method for inducing plant seed abortion, which includes increasing the level and/or activity of endogenous VvDUF642 protein in plants, or making plants that do not contain VvDUF642 express VvDUF642 protein.
  • the method for expressing VvDUF642 protein in a plant that does not contain VvDUF642 includes:
  • the Agrobacterium is used to infect plant seeds or explants.
  • nucleic acid sequence encoding the VvDUF642 protein is shown in SEQ ID NO: 2
  • the backbone vector of the expression vector is pCAMBIA1303, and the insertion sites of the nucleic acid sequence encoding the VvDUF642 protein are NcoI and BstEII.
  • the recombinant host is Agrobacterium, specifically Agrobacterium LBA4404.
  • VvDUF642 can also be used as a marker for seedless grape screening and breeding.
  • the present invention also provides the application of any one of the following a) to d) in the selection and breeding of seedless grapes;
  • VvDUF642 Detection reagents for proteins that have been substituted, deleted or added one or more amino acids in the amino acid sequence of VvDUF642 and have the same or similar functions as VvDUF642;
  • a detection reagent for nucleic acid molecules that are substituted, deleted or added to the nucleotide sequence of the nucleic acid molecule of c) and can encode the same or similar functional protein.
  • a preparation for screening seedless grape germplasm resources which comprises at least one of a) to d):
  • VvDUF642 Detection reagents for proteins that have been substituted, deleted or added one or more amino acids in the amino acid sequence of VvDUF642 and have the same or similar functions as VvDUF642;
  • a detection reagent for nucleic acid molecules that are substituted, deleted or added to the nucleotide sequence of the nucleic acid molecule of c) and can encode the same or similar functional protein.
  • the detection of the present invention includes the detection of expression amount or activity level.
  • the Western blot method is used to detect the expression level of VvDUF642 protein; the real-time PCR method is used to detect the transcription level of VvDUF642 gene.
  • a method for screening seedless grape germplasm resources which includes:
  • VvDUF642 gene transcription level of grape germplasm or detect the expression level or activity of VvDUF642 protein.
  • the tested sample is the young leaves of grapes.
  • the screening method of the present invention only by detecting the young leaves of the grapes, the prediction of whether the grapes have nuclei can be realized, and the breeding cycle can be shortened.
  • the seed embryos of nucleus and seedless varieties 5-7 days after flowering are separated and collected as core test materials, and transcriptome analysis and protein expression analysis are carried out. 60 specifically expressed proteins were screened. After further screening using Western blot technology, the protein VvDUF642, which is highly expressed in seedless grapes, was obtained. The transcriptome results showed that the VvDUF642 gene continued to be highly expressed in seedless varieties, while the expression level in nucleated varieties There is no obvious change.
  • the gene transforms Arabidopsis thaliana into Arabidopsis thaliana seeds, and transforms tomato into tomato that causes tomato seed abortion.
  • the gene or protein can be used for the construction or screening of seed aborted grape varieties.
  • Figure 1 shows the expression level of DUF642 gene transcription level, in which CS is Morita Seedless and GR is Red Earth;
  • Figure 2 shows the difference in the protein expression of DUF642 between Red Globe and Sentena Seedless
  • FIG. 3 shows the difference in protein expression of DUF642 between nucleated and non-nucleated varieties
  • Figure 4 shows that DUF642 gene transfer into Arabidopsis thaliana causes the shape of Arabidopsis seeds to become smaller
  • Figure 5 shows the transfer of DUF642 gene into Arabidopsis thaliana causes a reduction in the thousand-seed weight of Arabidopsis seeds
  • FIG. 6 shows the expression level of DUF642 gene in wild-type Arabidopsis and DUF642 transgenic Arabidopsis
  • Figure 7 shows that DUF642 gene causes tomato seed abortion in tomato, forming seedless fruit, and reducing fruit volume
  • Figure 8 shows that DUF642 gene transfer into tomato causes a decrease in the average fruit weight of tomato
  • Figure 9 shows the expression level of DUF642 gene in wild-type tomato and DUF642 transgenic tomato.
  • the present invention provides the use of VvDUF642, and those skilled in the art can learn from the content of this article and appropriately improve the process parameters to achieve it.
  • all similar replacements and modifications are obvious to those skilled in the art, and they are all deemed to be included in the present invention.
  • the method and application of the present invention have been described through the preferred embodiments. It is obvious that relevant personnel can modify or appropriately change and combine the methods and applications herein without departing from the content, spirit and scope of the present invention to realize and apply the present invention.
  • Invent technology is possible.
  • test materials used in the present invention are all common commercially available products, all of which can be purchased in the market.
  • the nucleic acid molecule encoding the VvDUF642 protein includes genomic DNA, cDNA, recombinant DNA or mRNA, or hnRNA encoding the VvDUF642 protein; or nucleic acid molecules that are reverse complementary to the aforementioned DNA, cDNA, recombinant DNA or mRNA.
  • nucleic acid molecules can be modified or optimized according to actual needs to make gene expression more efficient; for example, 1
  • the codons of the VvDUF642 gene of the present invention can be changed while maintaining the amino acid sequence of the VvDUF642 gene of the present invention according to the preferred codons of the recipient plant To meet the preference of the recipient plant. 2 Or modify the gene sequence adjacent to the initiating methionine to enable effective translation initiation; for example, use an effective sequence known in plants for modification.
  • 3It is connected to various plant-expressed promoters to facilitate its expression in plants; the promoters can include constitutive, inducible, timing regulation, developmental regulation, chemical regulation, tissue optimization and tissue-specific promoters; The choice of promoter will vary with expression time and space requirements, and also depends on the target species; 4Introduction of enhancer sequences, such as intron sequences (for example from Adhl and bronzel) and viral leader sequences (for example, from TMV , MCMV and AMV).
  • enhancer sequences such as intron sequences (for example from Adhl and bronzel) and viral leader sequences (for example, from TMV , MCMV and AMV).
  • the vector can be a plasmid, cosmid, phage or virus vector.
  • the host can be fungi, bacteria, algae or cells.
  • VvDUF642 For plants that do not contain VvDUF642, chemical methods, shotgun methods, microinjection, electroporation and other methods can be used to introduce VvDUF642 gene fragments into plant cells, or homologous recombination, zinc finger nuclease, TALEN, CRISPR and other methods The gene fragment of VvDUF642 is introduced into plant cells.
  • DUF642 gene in seedless varieties increased significantly than that in seedless varieties: the five stages of fruit development (ovule stage ( Before flowering), fertilization period (flowering), seed coat development period (5-10 days after flowering), endosperm development period (11-30 days after flowering), and embryo development period (31-40 days after flowering) for transcriptome Sequencing showed that the expression of this gene did not differ significantly between the ovule stage and the fertilization stage. From the seed coat development stage, the expression of the DUF642 gene continued to increase in seedless varieties, while the expression level in nucleated varieties did not change significantly ( figure 1).
  • the DUF642 gene sequence uses the primer found in Vector NTI 11 to design primers to clone the DUF642 gene sequence.
  • the primers are shown in Table 1.
  • the PCR amplification uses NEB PhusionTM ultra-fidelity enzyme amplification.
  • the reaction system is: HF buffer 10 ⁇ L, 2.5mmol/L dNTPs 2.5 ⁇ L, template 2 ⁇ L, upstream and downstream primers (10mmol/L) each 2.5 ⁇ L, Phusion ultra-fidelity Enzyme 0.5 ⁇ L, double distilled water to make up to 50 ⁇ L.
  • the PCR reaction program is: pre-denaturation at 98°C for 3min; denaturation at 98°C for 10s, annealing at 58°C for 10s, extension at 72°C for 30s, a total of 31 cycles; the final extension at 72°C for 10min, and storage at 4°C.
  • the PCR product was electrophoresed on a 2% agarose gel, and the amplified product was recovered using the Omega Gel extract recovery kit.
  • the purified product was purified using TaKaRa's EXTaqTM polymerase plus A. Reaction system: 10xbuffer 2 ⁇ L, 2.5mmol Mg2+2 ⁇ L, 2.5 2 ⁇ L of dNTPs in mmol/L and 14 ⁇ L of template.
  • Reaction procedure react at 72°C for 10 min.
  • the sequencing primers are T7 and T7 Terminal universal primers .
  • the cDNA sequence of the DUF642 gene is shown in SEQ ID NO: 2.
  • PCR amplification template uses sequence-verified DUF642-T plasmid; plant expression vector uses pCAMBIA1303; Agrobacterium strain is LBA4404; NEB NCO I and Bst E II endonuclease, Axygen small amount of plasmid extraction
  • the kit was purchased from Zhengzhou Bomei Company; 1/2MS, Hygromycin B, Kanamycin, etc.
  • Arabidopsis planting conditions are 22°C, 16h light, 8h dark, and water enough water two days before transformation.
  • the transformed Agrobacterium was shaken to an OD value of about 2.0, concentrated and centrifuged at 5000g room temperature for 5 minutes, discarded the supernatant and resuspended with 10% sucrose solution, centrifuged at 5000g room temperature for 5 minutes and resuspended with 10% sucrose solution to OD1.0, added Sillwet L-77 to a final concentration of 0.02%.
  • Select Arabidopsis thaliana flowers to be transformed when they are just exposed to white soak the inflorescences in the Agrobacterium solution for 1 min, and remove the excess bacterial solution on the stems with filter paper. Place it in the dark at 22°C for two days, pay attention to moisturizing, and transform again every 7 days. About three weeks after transformation, the seeds are collected when the fruit clips are mature.
  • VvDUF642 gene transfer into Arabidopsis causes the Arabidopsis seeds to become smaller.
  • the thousand-seed weight of wild-type Arabidopsis seeds and the thousand-seed weight of Arabidopsis seeds transformed with the VvDUF642 gene were measured and counted. The results are shown in Fig. 5, and the results show that the transformation of DUF642 gene into Arabidopsis causes the Arabidopsis seed to become smaller. Thousand-seed weight statistics show that the high expression of the DUF gene causes the Arabidopsis seed to become smaller, from 0.023 g of the wild type to 0.015 g.
  • Example 3 DUF642 gene causes tomato seed abortion
  • Tomato transgenic the constructed plant expression vector pDUF642 is transgenic for tomato
  • Seed sterilization Take a few grams of tomato seeds and place them in a sterile Erlenmeyer flask, first wash with sterile water for 2 minutes, 75% alcohol for 1 minute, then soak in 5% hypochlorous acid solution for 5-8 minutes, and wash with sterile water for 20 minutes , Wash it twice, and place it in a sterile filter board to dry.
  • Sowing Sow the sterilized seeds in a culture bottle with a plant spacing of 0.8 ⁇ 1.0cm. Seal the remaining sterilized seeds and place them in a dry place for the next use. Frequent ultraviolet light irradiation in the ultra-clean table will reduce the germination rate of the seeds.
  • the sown culture bottle is placed in a dark place for 2 to 3 days, and after the seeds germinate, they are placed in a light tissue culture box to grow for 4 to 5 days. Culture conditions: 23 ⁇ 2°C, 16h/d light and 8h/d dark.
  • Pre-cultivation of explants Pre-cultivation of tomato explants for 1 to 2 days to enlarge the edges of the explants is beneficial to tomato infection and transformation.
  • Agrobacterium infection solution Agrobacterium OD600 is 0.1 ⁇ 0.2, infection is 10 ⁇ 15min, MS suspension pH is 5.4. Dry in the sterile filter paperboard. The filter paperboard can be replaced multiple times to absorb excess Agrobacterium.
  • Co-cultivation Place the dried explants in a co-cultivation medium and culture for 2 days in the dark. Temperature: 23 ⁇ 2°C, the co-cultivation medium is the same as the pre-cultivation medium.
  • Delayed screening Wash the co-cultured explants with 1g/L cephalosporin water twice for 15min (or 1g/L cephalosporin water once and sterile water once, each for 15min). After drying in the filter paperboard, it is placed in the delayed screening medium. Cultivate under light for 3 to 5 days.
  • the average fruit weight of wild-type tomato fruits and the average fruit weight of tomato seeds transformed with the VvDUF642 gene were measured and counted. The results are shown in Figure 8. The results show that the transformation of DUF642 gene into tomato causes the tomato fruit to become smaller.

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Abstract

提供了VvDUF642基因，其在无核葡萄品种中持续高表达，在有核葡萄品种中表达量无明显变化，转化拟南芥以后可引起拟南芥种子变形，转化番茄后可引起番茄种子败育。还提供了VvDUF642基因或蛋白在种子败育葡萄品种的构建或筛选中的用途。

Description

VvDUF642基因引起植物种子败育的用途 技术领域
本发明涉及遗传育种技术领域，尤其涉及VvDUF642的用途。
背景技术
葡萄(Vitis spp.)属于葡萄科葡萄属浆果，其果实可以广泛的应用于酿酒、鲜食、制汁、制干。葡萄果实因其酸甜适口，风味浓郁，营养丰富深受人们喜爱，其中天然无核葡萄因其栽培简易，易加工且食用方便而广受欢迎。在发达国家，超过50％消费的鲜食和制干葡萄为无核品种。葡萄天然无核形成途径分为单性结实型(Parthenocarpy)和种子败育型(Stenospermacarpy)，其中单性结实为授粉受精不良造成，主要出现有核品种果穗中偶见的无核果粒，而种子败育是葡萄正常受精后种子发生败育形成天然无核果实。种子败育是葡萄形成无核果实最稳定的途径，也是培育无核品种中需要保持的重要遗传特性。通过现代分子生物学方法研究葡萄无核形成的分子机理和遗传规律，可以提高培育无核葡萄新品种的效率。
对于葡萄无核形成的遗传机制，科学家进行了广泛的研究，借助现代分子生物学的研究手段，获得了较好的进展。葡萄为闭花受粉植物，开花时就已经完成了受精，因此研究葡萄种子发育过程往往从花前开始。Michael Striem教授根据葡萄种子的发育绘制了葡萄种子的败育过程，以受精为核心，分为胚珠期(花前)，受精期(开花)，种皮发育期(花后5-10天)，胚乳发育期(花后11-30天)，胚发育期(花后31-40天)，完成整个发育过程后形成正常的有核果实，如果种子败育就会留下残核或者种痕，形成无核果实。申请人利用切片对种子发育过程持续观察，完整的记录了种子的败育过程，结果表明无核葡萄品种的种子能够正常受精，但是因为种皮不能正常发育而产生败育，确定了种皮发育期为败育的关键期。在葡萄栽培生产中，有些品种如‘阳光玫瑰’可以利用赤霉酸(GA)处理花后果穗的方法获得无核果实，而处理的时间是花后5-7天(种皮发育期)，说明GA干扰正常种皮的发育可以引起种子败育。
发明内容
有鉴于此，本发明要解决的技术问题在于提供VvDUF642在种子败育方面的用途。
本发明提供了如下I)～V)中的任一项在诱导植物种子败育或变形中的应用；
I)、VvDUF642蛋白；
II)、在VvDUF642蛋白的氨基酸序列中经取代、缺失或添加一个或多个氨基酸，且与VvDUF642具有相同或相似功能的蛋白；
III)、编码I)或II)所述蛋白的核酸分子；
IV)、在III)的所述核酸分子的核苷酸序列中经取代、缺失或添加一个或多个核苷酸，且能编码相同或相似功能蛋白的核酸分子；
V)、能够提高I)～V)中的至少一种的水平或活性的物质。
本发明中，诱导植物种子败育或变形的物质为VvDUF642蛋白：
所述VvDUF642蛋白的氨基酸序列如SEQ ID NO:1所示；
编码SEQ ID NO:1所示蛋白的核酸分子的序列如SEQ ID NO:2所示。
本发明中，能够引起种子变形或败育的植物来自葡萄科、十字花科或茄科。一些实施例中，进行实验验证的植物为葡萄、拟南芥或番茄。
本发明实验表明，VvDUF642基因在无核品种中持续高表达，而在有核品种中表达量无明显变化，该基因转化拟南芥以后引起拟南芥种子变型，转化番茄后引起番茄种子败育。
本发明中还提供了一种引起植物种子败育或变形的制剂，包括如下i)～v)中的至少一种：
i)、VvDUF642蛋白或编码VvDUF642蛋白的核酸分子；
ii)、包含编码VvDUF642蛋白的核酸的表达载体；
iii)、含有ii)的重组宿主；
iv)、增强VvDUF642基因表达的启动子或增强子；
v)、促进VvDUF642基因表达的诱导剂。
本发明还提供了一种引起植物种子败育的方法，包括提高植物内源 VvDUF642蛋白的水平和/或活性，或使不含有VvDUF642的植物表达VvDUF642蛋白。
在一些具体实施例中，所述使不含有VvDUF642的植物表达VvDUF642蛋白的方法包括：
构建包含编码VvDUF642蛋白的核酸的载体，转化入农杆菌；
以所述农杆菌感染植物种子或外植体。
本发明中，所述编码VvDUF642蛋白的核酸序列如SEQ ID NO:2所示
所述表达载体的骨架载体为pCAMBIA1303，编码VvDUF642蛋白的核酸序列的插入位点为NcoⅠ和BstEⅡ。
所述重组宿主为农杆菌，具体为农杆菌LBA4404。
VvDUF642还可以作为无核葡萄筛选育种的标志物。
因此，本发明还提供了如下a)～d)中的任一项在无核葡萄筛选育种中的应用；
a)、VvDUF642蛋白的检测试剂；
b)、在VvDUF642蛋白的氨基酸序列中经取代、缺失或添加一个或多个氨基酸，且与VvDUF642具有相同或相似功能的蛋白的检测试剂；
c)、编码a)或b)所述蛋白的核酸分子的检测试剂；
d)、在c)的所述核酸分子的核苷酸序列中经取代、缺失或添加一个或多个核苷酸，且能编码相同或相似功能蛋白的核酸分子的检测试剂。
一种筛选无核葡萄种质资源的制剂，其包括a)～d)中的至少一种：
a)、VvDUF642蛋白的检测试剂；
b)、在VvDUF642蛋白的氨基酸序列中经取代、缺失或添加一个或多个氨基酸，且与VvDUF642具有相同或相似功能的蛋白的检测试剂；
c)、编码a)或b)所述蛋白的核酸分子的检测试剂；
d)、在c)的所述核酸分子的核苷酸序列中经取代、缺失或添加一个或多个核苷酸，且能编码相同或相似功能蛋白的核酸分子的检测试剂。
本发明所述检测包括表达量或活性水平的检测。
本发明实施例中，对VvDUF642蛋白表达水平的检测采用Western blot的方法；对VvDUF642基因转录水平的检测采用real-time PCR的方式。
一种筛选无核葡萄种质资源的方法，其包括：
检测葡萄种质的VvDUF642基因转录水平或检测VvDUF642蛋白的表达水平或活性。
所述检测的样本为葡萄的幼嫩叶片。
利用本发明所述的筛选方法，仅对葡萄的幼嫩叶片进行检测就可实现葡萄果实是否有核的预测，缩短育种周期。
本发明围绕种子败育的关键期，分离采集有核无核品种花后5-7天的种胚为核心试材，开展了转录组分析和蛋白表达分析。筛选到特异表达的蛋白60个，利用Western杂交技术进一步筛选后获得在无核葡萄中高表达的蛋白VvDUF642，转录组结果表明VvDUF642基因在无核品种中持续高表达，而在有核品种中表达量无明显变化，该基因转化拟南芥以后引起拟南芥种子变形，转化番茄后引起番茄种子败育。该基因或蛋白可以用于种子败育葡萄品种的构建或筛选。
附图说明
图1示DUF642基因转录水平的表达量，其中，CS为森田尼无核，GR为红地球；
图2示红地球和森田尼无核中DUF642的蛋白表达差异；
图3示有核和无核品种中DUF642的蛋白表达差异；
图4示DUF642基因转入拟南芥中引起拟南芥种子形状变小；
图5示DUF642基因转入拟南芥中引起拟南芥种子千粒重降低；
图6示DUF642基因在野生型拟南芥和DUF642转基因拟南芥中的表达量；
图7示DUF642基因在番茄中引起番茄种子败育，形成无核果实，果实体积降低；
图8示DUF642基因转入番茄中引起番茄平均果重降低；
图9示DUF642基因在野生型番茄和DUF642转基因番茄中的表达量。
具体实施方式
本发明提供了VvDUF642的用途，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。
本发明采用的试材皆为普通市售品，皆可于市场购得。
所述编码VvDUF642蛋白的核酸分子包括编码VvDUF642蛋白的基因组DNA、cDNA、重组DNA或mRNA、hnRNA；或者与上述DNA、cDNA、重组DNA或mRNA呈反向互补的核酸分子。
上述核酸分子可根据实际需要进行修饰或优化，从而使基因表达更高效；例如，①可根据受体植物所偏爱的密码子，在保持本发明所述VvDUF642基因的氨基酸序列的同时改变其密码子以符合受体植物的偏爱性。②或修饰邻近起始甲硫氨酸的基因序列，以使翻译有效起始；例如，利用在植物中已知的有效的序列进行修饰。③与各种植物表达的启动子连接，以利于其在植物中的表达；所述启动子可包括组成型、诱导型、时序调节、发育调节、化学调节、组织优选和组织特异性启动子；启动子的选择将随着表达时间和空间需要而变化，而且也取决于靶物种；④引入增强子序列，如内含子序列(例如来源于Adhl和bronzel)和病毒前导序列(例如来源于TMV、MCMV和AMV)。
本发明中，所述载体可为质粒、黏粒、噬菌体或病毒载体。所述的宿主可为真菌、细菌、藻类或细胞。
对于不含有VvDUF642的植物，可采用化学法、鸟枪法、微注射，电穿孔等方法，将VvDUF642的基因片段导入植物细胞，也可通过同源重组、锌指核酸酶、TALEN、CRISPR等方法将VvDUF642的基因片段导入植物细胞。
下面结合实施例，进一步阐述本发明：
实施例1
1.DUF642基因在无核品种中表达量增加显著高于有核品种：通过转录组对无核品种(森田尼无核)和有核品种(红地球)果实发育的5个时期(胚 珠期(花前)，受精期(开花)，种皮发育期(花后5-10天)，胚乳发育期(花后11-30天)，胚发育期(花后31-40天))进行转录组测序，结果显示该基因的表达量在胚珠期和受精期没有显著差异，从种皮发育期开始，DUF642基因在无核品种中表达量持续增加，而在有核品种中表达量变化不显著(图1)。
2.果实发育过程中，DUF642蛋白表达量在无核品种中表达量增加显著高于有核品种：通过westernblot检测，DUF642蛋白的表达量在无核白中的表达量远高于玫瑰香(图2)。
3.大样本验证：选取8个品种和两个无核化处理的发育期果实作为样品进行Westernblot验证，果实发育过程中，DUF642蛋白在无核品种中高表达，而在有核品种中低表达，在醉金香和阳光玫瑰无核处理后表达量提高，证实了该基因表达量与葡萄无核性状的紧密连锁关系(图3)。
4.RNA提取
选取葡萄的幼嫩叶片，利用Bioteke植物RNA提取试剂盒提取植物总RNA。使用Thermo Scientific Nanodrop 1000微量紫外可见光分光光度计测量浓度并经琼脂糖凝胶电泳确定RNA的完整性。利用TakaRa公司的RR047反转录试剂盒得到cDNA的第一条链，作为PCR克隆的模板。
5.DUF642cDNA序列的获得：
DUF642基因序列使用Vector NTI 11中的primer find设计引物克隆DUF642基因序列，引物见表1。PCR扩增使用NEB的PhusionTM超保真酶扩增，反应体系为：HF buffer 10μL，2.5mmol/L的dNTPs 2.5μL，模板2μL，上下游引物(10mmol/L)各2.5μL，Phusion超保真酶0.5μL，双蒸水补至50μL。PCR反应程序为：98℃预变性3min；98℃变性10s，58℃退火10s，72℃延伸30s，共31个循环；最后72℃延伸10min，4℃保存。PCR产物经2％的琼脂糖胶电泳，使用Omega Gel extract回收试剂盒回收扩增产物，纯化后的产物使用TaKaRa的EXTaqTM聚合酶加A，反应体系：10xbuffer 2μL，2.5mmol的Mg2+2μL，2.5mmol/L的dNTPs 2μL，模板14μL。反应程序：72℃反应10min。参照pGEMT-easyTM载体构建说明书构建DUF642-T载体，转化E.coli DH5α大肠杆菌进行蓝白斑筛选，挑单克隆送生工生物工程(上海) 股份有限公司测序，测序引物为T7和T7 Terminal通用引物。
表1 DUF642基因克隆引物
Figure PCTCN2020083508-appb-000001
6.DUF642基因的cDNA序列如SEQ ID NO:2所示。
实施例2 DUF642基因引起拟南芥种子变型
1.构建植物表达载体：PCR扩增模板使用测序验证过的DUF642-T质粒；植物表达载体选用pCAMBIA1303；农杆菌菌株为LBA4404；NEB的NCO Ⅰ和Bst E Ⅱ内切酶，Axygen小量质粒提取试剂盒购于郑州博美公司；1/2MS、潮霉素B、卡那霉素等购于郑州宝赛生物公司；PEG4000、Cellulase R10、Mecerozym R10、mannitol、氯化钾、MES、BSA、0.45μm滤头等购于郑州超研生物公司，构建植物表达载体pDUF642。
2.拟南芥原生质体制备转化参考Maas C的方法(Maas et al,1995)，使用激光共聚焦显微镜观察转化培养好的拟南芥原生质体。核定位信号分析预测使用Nuc Pre(http://www.sbc.su.se/～maccallr/nucpred/cgi-bin/single.cgi)。
3.农杆菌LBA4404感受态制备：在含有50mg/L利福平的LB固体培养基上划板，挑取单胞在含有50mg/L利福平的LB液体培养基中摇菌48h至菌液浑浊，按1：100比例接种到50ml新鲜的含有50mg/L利福平的LB液体培养基上摇菌5h。将菌液置于冰上冰浴30min，4℃，5000g离心5min；弃上清加入1ml 0.1％的氯化钙悬浮，冰浴5min，4℃，5000g离心5min；弃上清加入800μL0.02％的氯化钙重悬浮，分装8管每管100μL冰浴备用。
4.制备好的农杆菌感受态中加入2μg需要转化的质粒，混匀，液氮速冻1min，37℃水浴5min，加入1ml LB液体培养基，28℃，200rpm，摇菌5h。离心浓缩涂到含有50mg/L利福平和50mg/L的卡那霉素的LB平板上，28℃培养48h，挑取单菌落验证。
5.拟南芥种植条件为22℃，16h光照，8h黑暗，转化前两天浇足水。转化的农杆菌摇菌至OD值为2.0左右，5000g室温浓缩离心5min，弃上清加 10％的蔗糖溶液重悬浮，5000g室温离心5min加10％的蔗糖溶液重悬浮至OD1.0，添加Sillwet L-77至终浓度0.02％。选择拟南芥花刚露白时转化，将花序浸泡到农杆菌菌液中1min，取出用滤纸沾吸茎上多余的菌液。22℃黑暗条件下放置两天，注意保湿，隔7天再转化一次。转化后三周左右，待果夹成熟收取种子。
6.干燥的种子放在2ml EP管中加入900μL含0.2％的吐温20的70％的乙醇摇9min。弃上清加入90％的乙醇洗3遍，最后用100％乙醇悬浮倒在灭菌的滤纸上干燥。配置1/2MS培养基，倒板前添加潮霉素B至终浓度25mg/L。将灭菌的拟南芥种子均匀的洒在1/2MS培养基上，密封放4℃冰箱中两天再转到22℃培养箱中培养，一周后观察筛选结果，可以正常生长为转基因型拟南芥阳性植株，连续筛选两代后，获得纯系T2代种子进行形态观察。
7.表型观察：
如图4所示，与野生型拟南芥的种子相比，VvDUF642基因转入拟南芥后引起拟南芥种子变小。
8.测量数据：
测量并统计野生型拟南芥种子的千粒重和转入VvDUF642基因的拟南芥种子的千粒重，结果如图5所示，结果表明DUF642基因转入拟南芥后引起拟南芥种子变小。千粒重统计显示，DUF基因的高表达导致拟南芥种子变小，由野生型0.023g变小到0.015g。
9.转基因拟南芥DUF642基因表达数据：
检测并统计野生型拟南芥种子以及转入VvDUF642基因的拟南芥种子的中DUF642基因的表达量，结果如图6，结果显示，VvDUF642基因在转基因拟南芥中高表达。
实施例3 DUF642基因引起番茄种子败育
1.番茄转基因：构建好的植物表达载体pDUF642进行番茄转基因
种子灭菌:取番茄种子数克置于无菌三角瓶中，先用无菌水清洗2min，75％酒精清洗1min，再用5％的次氯酸溶液浸泡5-8min，无菌水清洗20min，清洗2次，置于无菌滤纸板内晾干。
2.播种：将灭菌好的种子播种于培养瓶中，株距0.8～1.0cm。将剩下的灭菌的种子封好放于干燥处待下次取用，在超净台内频繁紫外光照射会降低种子发芽率。播种的培养瓶置于暗处2～3天，待种子露白发芽后置于光照组培箱内生长4～5d。培养条件：23±2℃，16h/d光照8h/d黑暗。
3.外植体的制备：番茄种子生长7～8d后，子叶完全展开，用手术刀取子叶，切除子叶柄及子叶尖，留中间部分切为2～3段作为外植体。
4.外植体预培养：番茄外植体预培养1～2d使外植体边缘膨大有利于番茄侵染转化。
5.农杆菌侵染液：农杆菌OD600为0.1～0.2，侵染10～15min，MS悬浮液PH 5.4。在无菌滤纸板内晾干，可多次置换滤纸板来吸取多余的农杆菌。
6.共培养：将晾干的外植体置于共培培养基中，在暗处培养2d。温度：23±2℃，共培培养基与预培养培养基相同。
7.延迟筛选：将共培后的外植体用1g/L的头孢水清洗15min两次(或者1g/L的头孢水清洗一次，无菌水清洗一次，各15min)。在滤纸板内晾干后，置于延迟筛选培养基中。时间3～5d，光照下培养。
8.愈伤的诱导及筛选：时间30-40d。愈伤分化及芽伸长(后期幼苗分化培养基)：时间30-40d。生根：待分化的幼苗生长至2-3cm左右，将其从愈伤上切除移至生根培养基中。时间：10-15d。
9.表型观察：
观察野生型番茄以及VvDUF642基因转入番茄后的表型，结果表明，VvDUF642基因转入番茄后引起番茄种子败育，形成无核果实(图7)。
10.测量数据：
测量并统计野生型番茄果实的平均果重、以及转入VvDUF642基因的番茄种子的平均果重，结果如图8所示，结果表明DUF642基因转入番茄后引起番茄果实变小。
11.转基因番茄DUF642基因表达数据：
检测并统计野生型番茄种子以及转入VvDUF642基因的番茄种子的中DUF642基因的表达量，结果如图9，结果显示，VvDUF642基因在转基因番茄中高表达。
以上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。

Claims (10)

  1. 如下I)～V)中的任一项在诱导植物种子败育或变形中的应用；
    I)、VvDUF642蛋白；
    II)、在VvDUF642蛋白的氨基酸序列中经取代、缺失或添加一个或多个氨基酸，且与VvDUF642具有相同或相似功能的蛋白；
    III)、编码I)或II)所述蛋白的核酸分子；
    IV)、在III)的所述核酸分子的核苷酸序列中经取代、缺失或添加一个或多个核苷酸，且能编码相同或相似功能蛋白的核酸分子；
    V)、能够提高I)～V)中的至少一种的水平或活性的物质。
  2. 根据权利要求1所述的应用，其特征在于，
    所述VvDUF642蛋白的氨基酸序列如SEQ ID NO:1所示；
    编码SEQ ID NO:1所示蛋白的核酸分子的序列如SEQ ID NO:2所示。
  3. 根据权利要求1所述的应用，其特征在于，所述植物来自葡萄科、十字花科或茄科。
  4. 一种引起植物种子败育或变形的制剂，其特征在于，包括如下i)～v)中的至少一种：
    i)、VvDUF642蛋白或编码VvDUF642蛋白的核酸分子；
    ii)、包含编码VvDUF642蛋白的核酸的表达载体；
    iii)、含有ii)的重组宿主；
    iv)、增强VvDUF642基因表达的启动子或增强子；
    v)、促进VvDUF642基因表达的诱导剂。
  5. 一种引起植物种子败育的方法，其特征在于，提高植物内源VvDUF642蛋白的水平和/或活性，或使不含有VvDUF642的植物表达VvDUF642蛋白。
  6. 根据权利要求5所述的方法，其特征在于，所述使不含有VvDUF642的植物表达VvDUF642蛋白的方法包括：
    构建包含编码VvDUF642蛋白的核酸的载体，转化入农杆菌；
    以所述农杆菌感染植物种子或外植体。
  7. 如下a)～d)中的任一项在无核葡萄筛选育种中的应用；
    a)、VvDUF642蛋白的检测试剂；
    b)、在VvDUF642蛋白的氨基酸序列中经取代、缺失或添加一个或多个 氨基酸，且与VvDUF642具有相同或相似功能的蛋白的检测试剂；
    c)、编码a)或b)所述蛋白的核酸分子的检测试剂；
    d)、在c)的所述核酸分子的核苷酸序列中经取代、缺失或添加一个或多个核苷酸，且能编码相同或相似功能蛋白的核酸分子的检测试剂。
  8. 一种筛选无核葡萄种质资源的制剂，其特征在于，包括a)～d)中的至少一种：
    a)、VvDUF642蛋白的检测试剂；
    b)、在VvDUF642蛋白的氨基酸序列中经取代、缺失或添加一个或多个氨基酸，且与VvDUF642具有相同或相似功能的蛋白的检测试剂；
    c)、编码a)或b)所述蛋白的核酸分子的检测试剂；
    d)、在c)的所述核酸分子的核苷酸序列中经取代、缺失或添加一个或多个核苷酸，且能编码相同或相似功能蛋白的核酸分子的检测试剂。
  9. 一种筛选无核葡萄种质资源的方法，其特征在于，包括：
    检测葡萄种质的VvDUF642基因转录水平或检测VvDUF642蛋白的表达水平或活性。
  10. 根据权利要求9所述的方法，其特征在于，所述检测的样本为葡萄的幼嫩叶片。
PCT/CN2020/083508 2019-11-14 2020-04-07 VvDUF642基因引起植物种子败育的用途 WO2021093258A1 (zh)

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