WO2010127579A1 - Gène codant la bétaïne aldéhyde déshydrogénase de la mangrove et ses utilisations - Google Patents

Gène codant la bétaïne aldéhyde déshydrogénase de la mangrove et ses utilisations Download PDF

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WO2010127579A1
WO2010127579A1 PCT/CN2010/071536 CN2010071536W WO2010127579A1 WO 2010127579 A1 WO2010127579 A1 WO 2010127579A1 CN 2010071536 W CN2010071536 W CN 2010071536W WO 2010127579 A1 WO2010127579 A1 WO 2010127579A1
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mangrove
aldehyde dehydrogenase
badh
dehydrogenase gene
betaine aldehyde
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PCT/CN2010/071536
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English (en)
Chinese (zh)
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孙超
王君丹
陈文华
刘晓霞
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创世纪转基因技术有限公司
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Publication of WO2010127579A1 publication Critical patent/WO2010127579A1/fr

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    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0008—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • 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
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance

Definitions

  • the invention relates to the field of plant genetic engineering, in particular to a mangrove betaine aldehyde dehydrogenase gene and application thereof in the development of salt-tolerant, cold-tolerant and drought-tolerant transgenic plants.
  • osmotic regulation which actively maintains osmotic balance by actively accumulating a large number of small molecular organic osmolytes, such as polyols, sugars, amino acids and their derivatives, in cells. Moisture in the body, these substances are called osmotic regulators.
  • Betaine is recognized as the most important cell-compatible substance in the protection of bacteria, plants, animals and other organisms. It is a kind of quaternary ammonium compound. Studies have shown that betaine is almost no longer synthesized. It is further metabolized and belongs to permanent or semi-permanent osmotic regulators. It has been paid more and more attention in the research of plant salt tolerance, drought tolerance and cold tolerance.
  • Betaine in plants can be divided into 4 types according to their structure and synthetic route, namely Gly betaine. , Pro betaine, Hydroxyproline betaine and Alanine betaine ( ⁇ -2 ⁇ -betaine) .
  • Gly betaine Pro betaine
  • Hydroxyproline betaine Hydroxyproline betaine
  • Alanine betaine ⁇ -2 ⁇ -betaine
  • Many studies have shown that the osmotic protection of glycine betaine is usually the strongest, and the glycine betaine is present in a variety of algae and seed plants of at least 10 families. Therefore, the existing studies mostly use glycine betaine. the Lord.
  • BADH Betaine aldehyde dehydrogenase
  • Arakawa et al. isolated and purified BADH from spinach leaves and prepared antibodies.
  • Weretilnyk et al. first isolated the mRNA of BADH from spinach, and proved that it was regulated by salt on the basis of successful expression in vitro.
  • Weretilnyk et al. first cloned BADH from spinach.
  • the cDNA of the gene which is 1819 bp in length, includes a 67 bp 5' non-coding region, a 1491 bp open reading frame and a 239 bp 3' non-coding region.
  • the entire BADH gene sequence of spinach and Chisui Valley, and all exon and intron regions of rice except for the 5' and 3' flanking sequences have also been cloned.
  • the BADH gene of higher plants has been studied to some extent in terms of structure, expression characteristics and its relationship with plant system evolution.
  • the object of the present invention is to provide a mangrove betaine aldehyde dehydrogenase A gene that increases the tolerance of a transgenic plant to abiotic stress caused by salinity, drought, and low temperature.
  • a mangrove betaine aldehyde dehydrogenase gene was cloned from mangrove with the nucleotide sequence shown in SEQ ID NO: 1.
  • a second object of the present invention is to provide a prokaryotic expression vector and a eukaryotic expression vector comprising a mangrove betaine aldehyde dehydrogenase gene.
  • the eukaryotic expression vector is a plant expression vector.
  • a plant cell, tissue or plant transformed with the plant expression vector A plant cell, tissue or plant transformed with the plant expression vector.
  • a third object of the present invention is to provide a betaine aldehyde dehydrogenase gene for use in the cultivation of salt tolerant, drought tolerant and cold tolerant plant varieties.
  • a fourth object of the present invention is to provide a protein sequence encoded by a mangrove betaine aldehyde dehydrogenase gene having SEQ ID NO: 3 The amino acid sequence shown.
  • the expression of the protein in the recipient plant is the key to the cultivation of the salt-tolerant, drought-tolerant and cold-tolerant plant varieties of the present invention.
  • the present invention clones the mangrove betaine aldehyde dehydrogenase (BADH) gene from the mangrove, the gene sequence thereof and the salt-tolerant BADH reported in the literature.
  • the gene has the highest homology, and the encoded protein has a conserved amino acid sequence unique to the same protein: QLFIDGE.
  • a prokaryotic expression vector and a binary plant expression vector were constructed using the mangrove betaine aldehyde dehydrogenase gene.
  • Mangrove betaine aldehyde dehydrogenase gene can be highly expressed in E. coli, and in high salt environment, transfer to BADH The E. coli growth of the gene is better than that of the untransferred BADH Gene E. coli.
  • the results of transgenic tobacco showed that the cloned mangrove betaine aldehyde dehydrogenase gene could be highly expressed in tobacco, and the transgenic tobacco obtained had obvious improvement in tolerance to salt stress, osmotic stress and low temperature stress.
  • Figure 1 is a schematic diagram of RNA detection of mangrove RNA
  • Figure 2 is a schematic diagram showing the results of 5'Race identification of the mangrove BADH gene
  • Figure 3 is a schematic diagram showing the results of 3'Race identification of the mangrove BADH gene
  • Figure 4 is a flow chart showing the construction of the prokaryotic expression vector EPSPS-pET30a;
  • Figure 5 is a schematic diagram showing the results of expression analysis of mangrove BADH gene in E. coli
  • Figure 6 is a schematic diagram showing the growth state of control Escherichia coli in LB medium with different NaCl concentrations
  • Figure 7 is a schematic diagram showing the growth state of BADH engineering bacteria in LB medium with different NaCl concentration
  • Figures 8a and 8b are flow diagrams showing the construction of the binary plant expression vector pCAMBIA300BADH
  • Figure 9 is a schematic diagram of the results of the seed germination test.
  • the whole idea of the present invention includes the following points: the betaine aldehyde dehydrogenase gene (BADH gene) is cloned from the salt-tolerant plant mangrove, and the prokaryotic expression vector and the binary plant expression vector are constructed, respectively transformed into E. coli and tobacco, and transferred to BADH.
  • BADH gene betaine aldehyde dehydrogenase gene
  • the high-salt culture of Escherichia coli was carried out, and the stress of BADH gene-positive tobacco was tested to verify the function of BADH gene.
  • the method comprises the following steps: 1. extracting total RAN of mangrove, preparing cDNA by reverse transcription; 2. amplifying the expression sequence tag of BADH gene from mangrove cDNA by PCR; 3. cloning the full length sequence of BADH gene by RACE method. The following is described in further detail:
  • the above degenerate primers P1 and P2 were used for PCR amplification to obtain an expression sequence tag (EST) of the BADH gene, and the base sequence thereof is shown in SEQ ID NO: 2.
  • the EST sequence was highly homologous to the known BADH gene, up to 88%.
  • the cloned sequence was determined to be the EST sequence of the mangrove BADH gene.
  • GSP-RT 5'-GGGTCCGATACTTTGATG-3'
  • GSP1 5'-CCGATACTTTGATGTTCTCAGAC-3'
  • GSP2 5'-CTCCAAAAGTTGTGCTGCAATGCTCTC-3'
  • AAP 5'-GGCCACGCGTCGACTAGTACGGGIIGGGIIGGGIIG-3'
  • Cycle parameters 94 °C for 5 minutes; 94 °C for 30 seconds, 55 °C for 30 seconds, 72 °C 2 minutes; 30 cycles; 72 ° C for 7 minutes; 5 ° C;
  • Cycle parameters 94 °C for 30 seconds, 55 °C for 30 seconds, 72 °C for 2 minutes for 30 cycles; 72 °C 7 minutes; 5 °C.
  • the 5' end sequence of mangrove BADH gene was obtained by RACE method.
  • the results of PCR identification are shown in Fig. 2.
  • PCR amplification conditions were: 94 ° C 5 minutes; 94 ° C 30 seconds, 52 ° C 30 seconds, 72 ° C 2 minutes 30 cycles; 72 ° C 7 minutes; 5 ° C.
  • the results of 5'Race and 3'Race were ligated with DNAman software to obtain the full-length sequence of mangrove BADH gene.
  • the base sequence is SEQ ID. NO: 1 is shown.
  • the full-length sequence of the BADH gene can be obtained by RT-PCR.
  • the primer is BADH5': 5'-CAAAACCCACTGAAGAGTCC-3'
  • the amplification conditions were: 94 ° C for 5 minutes; 94 ° C for 30 seconds, 56 ° C for 45 seconds, 72 ° C for 1 minute. 30 cycles; 72 degrees for 5 minutes.
  • the mangrove BADH gene carrying the 5'UTR and 3'UTR sequences has the base sequence shown in SEQ ID NO:4.
  • the betaine aldehyde dehydrogenase encoded by the mangrove BADH gene of the present application has an amino acid sequence of SEQ ID NO: 3 is shown.
  • the prokaryotic expression vector EPSPS-pET30a of PET system was constructed. The specific construction process is shown in Figure 4.
  • the E. coli DH5 ⁇ was transformed with the prokaryotic expression vector EPSPS-pET30a.
  • the BADH gene of mangrove specifically expressed a protein band of 55KD in E. coli DH5 ⁇ .
  • the results of SDS-PAGE electrophoresis identification are shown in Fig. 5.
  • E. coli DH5 ⁇ itself contains a substrate required for BADH gene metabolism, the function of the BADH gene was initially verified directly using Escherichia coli DH5 ⁇ . After induced by IPTG for 3 hours, E. coli DH5 ⁇ was directly transferred into LB medium with different NaCl concentration (1%, 3%, 5%, 7%), and its OD value (bacterial concentration) was measured at different times. The growth conditions are shown in Tables 1 and 2.
  • Table 1 Cell concentration (OD value) of control cells grown at different NaCl concentrations at different times
  • the binary plant expression vector pCAMBIA300BADH which drives the drought-inducible promoter RD29A to drive BADH gene expression, is shown in Figures 8a and 8b.
  • Sph I was digested with the vector Rd29A-BADH-Tnos-PMD and then digested with EcoRI to obtain a plant expression cassette fragment in which the Ra29A promoter drives BADH gene expression.
  • the fragment was inserted into the vector pCAMBIA2300, and the final vector was named pCAMBIA2300BADH.
  • the tobacco seeds were soaked in 75% alcohol for 30 s, and then immersed in 0.1% liters of mercury for 8 min for surface disinfection.
  • the sterile tobacco seeds were placed on MS medium (30 g/L sucrose) and sterilely germinated to prepare sterile seedlings.
  • the leaves of the sterile seedlings were cut into 5 mm ⁇ 5 mm leaf discs, and the non-transgenic tobacco leaf discs were transferred into 1.5%, 2%, 2.5%, and 3%, respectively.
  • the differentiation medium of NaCl a differentiation medium to which no NaCl was added was used as a control.
  • the transgenic T1 seed and non-transgenic seeds were cultured on MS medium containing 5% NaCl. The results are shown in Figure 9.
  • the transgenic T1 seed is seeded, at least 90% of the transgenic T1 seeds can be germinated, B plate
  • the middle is a control non-transgenic seed with a germination rate of about 15%, indicating that the transgenic seeds have better tolerance to salt stress.
  • Use 5% The PEG MS medium and the 4 °C low temperature treatment germination test also confirmed that the transgenic seeds also had good tolerance to osmotic stress and low temperature stress.

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Abstract

Cette invention concerne le gène codant la bétaïne aldéhyde déshydrogénase (BADH) de la mangrove et ses utilisations dans les études sur l'alimentation de nouvelles espèces végétales présentant certaines tolérances en matière de teneur en sel, de sécheresse et de température basse. Le gène codant la bétaïne aldéhyde déshydrogénase de la mangrove comporte la séquence nucléotidique SEQ ID NO: 1. Ce gène est utilisé pour construire des vecteurs d'expression procaryotes et des vecteurs d'expression de plantes binaires; E. coli et le tabac sont transformés respectivement avec ces vecteurs; E. coli génétiquement modifié par BADH est cultivé dans des conditions de forte teneur en sel; et des expériences de stress abiotique sont conduites sur le tabac transgénique BADH positif. Le gène codant la bétaïne aldéhyde déshydrogénase de la mangrove s'exprime en grande quantité dans E. coli, et dans des conditions de forte teneur en sel, la croissance d'E. coli génétiquement modifié par BADH est meilleure que celle d'E. coli non génétiquement modifié. Les résultats d'identification du tabac transgénique montrent que le gène codant la bétaïne aldéhyde déshydrogénase de la mangrove clonée est capable de s'exprimer en grande quantité dans le tabac, et que le tabac transgénique obtenu présente une bonne capacité à tolérer une certaine quantité de stress abiotique.
PCT/CN2010/071536 2009-05-08 2010-04-02 Gène codant la bétaïne aldéhyde déshydrogénase de la mangrove et ses utilisations WO2010127579A1 (fr)

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CN107630026B (zh) * 2017-11-06 2020-10-30 中国科学院新疆生态与地理研究所 极端耐干齿肋赤藓醛脱氢酶基因及其编码蛋白

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CN115725602A (zh) * 2022-10-10 2023-03-03 山东省花生研究所 花生AhBADH1基因及其应用

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