WO2020108031A1 - Gène gmgrf5-1 lié à la photosynthèse du soja, sa protéine codée et son utilisation - Google Patents

Gène gmgrf5-1 lié à la photosynthèse du soja, sa protéine codée et son utilisation Download PDF

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WO2020108031A1
WO2020108031A1 PCT/CN2019/106583 CN2019106583W WO2020108031A1 WO 2020108031 A1 WO2020108031 A1 WO 2020108031A1 CN 2019106583 W CN2019106583 W CN 2019106583W WO 2020108031 A1 WO2020108031 A1 WO 2020108031A1
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gmgrf5
soybean
protein
gene
plant
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Chinese (zh)
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傅永福
张晓玫
陈福禄
徐坤
禹国龙
陆明洋
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Insititute Of Crop Science Chinese Academy Of Agricultural Sciences
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8269—Photosynthesis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/827—Flower development or morphology, e.g. flowering promoting factor [FPF]

Definitions

  • the invention relates to the field of genetic engineering, in particular to soybean photosynthesis related genes, their encoded proteins and their application in plant flowering time and photosynthesis regulation.
  • Soybean is one of my country's five major crops, but the net imports of soybeans have significantly exceeded domestic soybean production in the past decade, and the annual imports have shown a rapid growth trend.
  • my country's soybean production safety is facing severe challenges.
  • the growth of the world’s population has led to a doubling of soybean production by 2050 to meet overall demand, but according to the current increase in output, only half of the demand will be reached by then (Tilman D et al. 2011 ; Ray et al., 2013). It is neither realistic nor possible to increase production by expanding planting area, because factors such as ecological security and desertification of land have forced the conversion of farmland to forests. Therefore, increasing soybean yield is the only way to go, and it has important strategic significance for the safe production of soybean in my country.
  • High-yield crops are nothing more than cultivating high-yielding varieties, applying organic fertilizers and yield-increasing agents, and improving cultivation methods.
  • the means of cultivating high-yielding varieties include traditional cross breeding and modern molecular breeding (including genetically modified breeding).
  • Transgenic breeding is carried out on the premise of a clear understanding of the function and mechanism of the target gene, so the cycle is shorter and the efficiency of yield increase is higher. There are also many reports on soybean production and resistance through genetic modification.
  • soybeans overexpressing Ncl genes have higher salt tolerance, and the yield in high-salt areas is 3.6 to 5.5 times higher than that of wild type (Do et al., 2016); LOS5/ABA3 ( Genes related to abscisic acid aldehyde dehydrogenase activity)
  • the transgenic soybean reduced the size of stomata opening and transpiration rate under drought conditions, drought resistance was significantly enhanced, and the yield under drought conditions was increased by about 21% compared with the wild type (Li et al., 2013);
  • Overexpression of sunflower Hahb-4 ethylene signal transduction pathway gene
  • Monsanto reported that overexpression of Arabidopsis BBX32 flowering genes in soybean increased yield by 5-7% (Preuss et al., 2012);
  • GRF Growth regulators
  • the purpose of the present invention is to provide soybean photosynthesis related gene GmGRF5-1, its encoded protein and its application in plant flowering time, photosynthesis and single plant yield regulation.
  • the soybean GmGRF5-1 protein provided by the present invention has an amino acid sequence comprising: 1) the amino acid sequence shown in SEQ ID No. 1; or 2) substituted, deleted or added in the amino acid sequence shown in SEQ ID No. 1
  • soybean GmGRF5-1 protein of the present invention also includes the amino acid sequence shown in SEQ ID No. 1 by substitution, substitution, and/or addition of one or several amino acids.
  • the soybean GmGRF5-1 protein has the same activity as the soybean GmGRF5-1 protein. Derived protein.
  • Another object of the present invention is to provide a GmGRF5-1 gene encoding the aforementioned soybean GmGRF5-1 protein.
  • the GmGRF5-1 (full name GROWTH-REGULATING FACTOR) gene has the nucleotide sequence shown in SEQ ID No. 2 or a nucleoside having 95% or more homology with the nucleotide sequence shown in SEQ ID No. 2 Acid sequence.
  • the soybean GmGRF5-1 gene of the present invention was cloned from soybean Tianlong 1 by RT-PCR.
  • the gene of the present invention includes the nucleic acid sequence encoding the protein.
  • codons and the preference of codons of different species, those skilled in the art may use codons suitable for expression of specific species as needed.
  • Another object of the present invention is to provide biological material carrying the GmGRF5-1 gene, the biological material being an expression cassette, an expression vector, a host cell or a host bacterium.
  • the plant expression vector of the present invention is pSoy1.
  • the GmGRF5-1 gene is constructed on the expression vector pSoy1 and expanded in E. coli DH5 ⁇ .
  • the Agrobacterium-mediated transformation method was used to transfer the GmGRF5-1 gene carried by pSoy1 to soybean Tianlong No. 1 to obtain a transgenic soybean overexpressing GmGRF5-1.
  • the results show that GmGRF5-1 has the functions of improving plant photosynthesis, increasing plant yield and delaying plant flowering.
  • the present invention also provides cloning vectors or various expression vectors containing GmGRF5-1 nucleotide sequence or fragments thereof, host cells containing the vector, transformed plant cells and transgenic plants containing the nucleotide sequence or specific fragments thereof .
  • Another object of the present invention is to provide the application of GmGRF5-1 gene and its encoded protein GmGRF5-1 in plant flowering time and photosynthesis regulation.
  • the invention provides application of soybean GmGRF5-1 protein or its coding gene or biological material containing the gene in delaying plant flowering time.
  • the invention provides the application of soybean GmGRF5-1 protein or its coding gene or biological material containing the gene in regulating plant photosynthesis and/or increasing plant yield.
  • the invention provides the application of soybean GmGRF5-1 protein or its coding gene or biological material containing the gene in the improvement of plant germplasm resources or cross breeding.
  • the use of GmGRF5-1 protein to delay the flowering of plants can solve the problem of flowering in hybrid breeding.
  • the invention provides the application of soybean GmGRF5-1 protein or its coding gene or biological material containing the gene in preparing transgenic plants.
  • the transgenic plant is a transgenic plant that has a delayed flowering time, increased chlorophyll content, increased lutein content, increased photosynthesis rate, and/or increased yield relative to a wild-type plant.
  • the chlorophyll is chlorophyll a and/or chlorophyll b.
  • the present invention also provides a method for preparing a transgenic plant with delayed flowering time, increased chlorophyll content, increased lutein content, increased photosynthesis rate and/or increased yield, which is introduced or overexpressed in the plant genome
  • the soybean GmGRF5-1 gene is introduced or overexpressed in the plant genome The soybean GmGRF5-1 gene.
  • the GmGRF5-1 gene and its encoded protein can regulate the flowering period, and can be used to solve the problem that the flowering period is not encountered in cross breeding, promote plant photosynthesis, and increase yield.
  • FIG. 2 is a schematic structural diagram of a cloning intermediate vector pGWCm according to Example 2 of the present invention.
  • FIG. 3 is a schematic structural diagram of the plant expression vector pSoy1 of Example 3 of the present invention.
  • Fig. 4 shows the subcellular localization of the protein encoded by the soybean photosynthesis-related gene GmGRF5-1 of the present invention in tobacco. From left to right, the location of GmGRF5-1-GFP in the nucleus; the location of the nuclear marker gene AHL22-RFP; the chloroplast; the bright field; the overlay of the first four images are shown in order from left to right.
  • FIG. 5 shows that soybean photosynthesis-related gene GmGRF5-1 transforms soybeans, resulting in delayed flowering.
  • the left picture shows the photos of wild-type (podded) and GmGRF5-1 transgenic soybeans (flowering stage), and the right picture shows the statistical results of the flowering time.
  • FIG. 6 shows that soybean photosynthesis-related gene GmGRF5-1 transforms soybeans, resulting in deeper soybean leaf color (A), increased chlorophyll content (B) and developed chloroplast matrix sheet (C).
  • FIG. 7 shows that soybean photosynthesis-related gene GmGRF5-1 transforms soybean, resulting in faster soybean photosynthesis rate (A) and increased yield per plant (B).
  • the forward primer 5'-ATGATGAGTGCAAGTGCAAGAA-3' and the reverse primer 5'-TCATTCATCGGTTTGGATTCTG-3' were cloned from soybean Tianlong 1 (Glycine max.L. Tianlong 1) and sequenced to obtain the GmGRF5-1 gene. As shown in SE QID NO.2; the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.1.
  • the PCR reaction program is: pre-denaturation at 95°C for 5 minutes, 94°C for 30 seconds, 55°C for 35 seconds, 72°C for 1 minute and 30 seconds, 25 cycles, and 72°C for 10 minutes extension.
  • the PCR product amplified from Example 1 was directly cloned into the pGWCm vector shown in FIG. 2 according to the TA cloning method.
  • the pGWCm vector was first hydrolyzed with Ahd I endonuclease, and then the digested product was recovered with a gel recovery kit to obtain T vector.
  • the PCR product and T vector were then ligated at 16°C, the ligated product was transformed into E. coli DH5 ⁇ , amplified therein, and positive clones were screened and sequenced.
  • the cloning vector of the soybean flowering gene GmGRF5-1 obtained in Example 2 and the plant expression vector pSoy1 shown in FIG. 3 were mixed in equal proportions and subjected to LR reaction (50 ng each of plasmids, 1 ⁇ l of LR enzyme, supplemented with H 2 O to the end (Volume 5 ⁇ l, mixed at 25°C for 6 hours or more), constructed GmGRF5-1 on pSoy1, used to overexpress soybean photosynthesis related gene GmGRF5-1 in plants, and study its function.
  • the method of plant transformation is carried out by Agrobacterium-mediated method.
  • the screening marker in the plant is Bar.
  • the flowering time statistics showed that the average number of days from sowing to first flowering of wild-type Tianlong 1 was about 26 days, and the average number of days from sowing to first flowering of the two transgenic soybean lines was 36 days.
  • Overexpression of GmGRF5-1 caused a delay in flowering period About 10 days.
  • the flowering period of transgenic soybean and wild-type soybean is significantly different.
  • Chlorophyll a(Chl)a (11.24A662-2.04A645) ⁇ V/W
  • Chlorophyll b(Chl)b (20.13A645-4.19A662) ⁇ V/W
  • Lutein (Car) ((1000A470-1.90 Chl a-63.14 Chl b)/214) ⁇ V/W
  • V in the formula refers to the volume of the acetone solution; W refers to the fresh weight of the sample.
  • Table 1 The test results are shown in Table 1. The data in the table are the average data of 10 leaves of OX-GmGRF5-1 (#2 and #3) and Tianlong No. 1, and the chlorophyll of 10 leaves of the two lines of transgenic soybean The difference in xanthophyll data is not significant. The difference in the above data for 10 leaves of wild-type soybean (Tianlong No. 1) is not significant.
  • the present invention After harvesting the above transgenic plants and control materials in the artificial climate room, the present invention also weighed the yield of each plant, and the measurement results are shown in Table 3.
  • the data in Table 3 is the average yield per plant of OX-GmGRF5-1 (#2 and #3) and Tianlong No. 1 (WT).
  • the difference in yield data per plant between the 10 transgenic soybeans of the two strains was not significant, and the yield data per plant between the 10 wild-type soybeans (Tianlong No. 1) was not significant. But the yield of single plant of transgenic soybean is significantly higher than that of wild type.
  • GmGRF5-1 As shown in Figure 7, overexpression of GmGRF5-1 will increase the net photosynthetic rate of the plant, which in turn will significantly increase plant yield per plant.
  • the invention provides a soybean GmGRF5-1 protein.
  • the soybean GmGRF5-1 protein provided by the present invention is: 1) a protein composed of the amino acid sequence shown in SEQ ID NO.1, or 2) substituted, deleted or added in the amino acid sequence shown in SEQ ID NO.1 A protein derived from 1) with one or several amino acids and equivalent activity.
  • the present invention also provides the gene GmGRF5-1 encoding the above protein, the nucleotide sequence of which is shown in SEQ ID NO. 2. Overexpression of the gene can delay plant flowering, promote photosynthesis, increase plant yield, and have good economic value and application prospects in agricultural production.

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Abstract

La présente invention concerne une protéine GmGRF5-1 de soja, qui est : 1) une protéine constituée de la séquence d'acides aminés telle que représentée dans SEQ ID NO. 1, ou 2) une protéine dérivée de 1) qui est obtenue par substitution, délétion ou addition d'un ou plusieurs acides aminés dans la séquence d'acides aminés telle que représentée dans SEQ ID NO. 1 et présente des activités équivalentes. La présente invention concerne également un gène GmGRF5-1 codant pour ladite protéine comprenant une séquence nucléotidique telle que représentée dans SEQ ID NO. 2. La surexpression du gène peut retarder la floraison de la plante, favoriser la photosynthèse, et augmenter le rendement de la plante, présentant une valeur d'application potentielle en production agricole.
PCT/CN2019/106583 2018-11-28 2019-09-19 Gène gmgrf5-1 lié à la photosynthèse du soja, sa protéine codée et son utilisation Ceased WO2020108031A1 (fr)

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Cited By (3)

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CN114195871A (zh) * 2020-09-02 2022-03-18 中国科学院遗传与发育生物学研究所 与油脂代谢调控相关的GmGRF5蛋白及其编码基因与应用
CN114989277A (zh) * 2022-04-27 2022-09-02 中国农业科学院作物科学研究所 GmGRF5-1及其编码蛋白在提高大豆种子蛋白质含量中的应用
CN121046446A (zh) * 2025-11-03 2025-12-02 中国农业科学院作物科学研究所 菜豆aurk及其互作蛋白基因在植物耐盐调控中的应用

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CN109627302B (zh) * 2018-11-28 2020-05-05 中国农业科学院作物科学研究所 大豆光合作用相关基因GmGRF5-1及其编码蛋白与应用
CN111454965B (zh) * 2020-04-10 2021-10-08 中国科学院东北地理与农业生态研究所 GmLMM2基因在调控植物叶绿素合成及PCD中的应用
CN114525299B (zh) * 2020-11-05 2023-06-02 中国农业科学院油料作物研究所 GmMYB14蛋白及其相关生物材料在调控植物株型和产量中的应用
CN112359050B (zh) * 2020-11-30 2021-12-31 中国科学院东北地理与农业生态研究所 菜豆金属蛋白酶PvFtsH2基因及其编码蛋白和应用
CN112707957B (zh) * 2021-02-10 2022-07-12 中国农业科学院作物科学研究所 大豆分生组织基因GmWUS2及其在根瘤发育中的应用
CN113337482B (zh) * 2021-05-18 2022-07-08 中国农业科学院作物科学研究所 大豆油菜素内酯合成酶基因GmDET2-1和GmDET2-2及其编码蛋白与应用
CN114262712B (zh) * 2022-01-25 2024-02-09 河南科技大学 一种普通小麦基因TaGRF5及其应用
CN114989278B (zh) * 2022-04-27 2025-09-02 中国农业科学院作物科学研究所 大豆光合作用相关基因GmGRF5-2及其编码蛋白与应用

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Cited By (3)

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Publication number Priority date Publication date Assignee Title
CN114195871A (zh) * 2020-09-02 2022-03-18 中国科学院遗传与发育生物学研究所 与油脂代谢调控相关的GmGRF5蛋白及其编码基因与应用
CN114989277A (zh) * 2022-04-27 2022-09-02 中国农业科学院作物科学研究所 GmGRF5-1及其编码蛋白在提高大豆种子蛋白质含量中的应用
CN121046446A (zh) * 2025-11-03 2025-12-02 中国农业科学院作物科学研究所 菜豆aurk及其互作蛋白基因在植物耐盐调控中的应用

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