WO2003097827A1 - Procede de culture de plans de tomates halophiles - Google Patents

Procede de culture de plans de tomates halophiles Download PDF

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Publication number
WO2003097827A1
WO2003097827A1 PCT/CN2003/000241 CN0300241W WO03097827A1 WO 2003097827 A1 WO2003097827 A1 WO 2003097827A1 CN 0300241 W CN0300241 W CN 0300241W WO 03097827 A1 WO03097827 A1 WO 03097827A1
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WIPO (PCT)
Prior art keywords
tomato
badh
agrobacterium
salt
explant
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PCT/CN2003/000241
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English (en)
Chinese (zh)
Inventor
Yinxin Li
Zhiqing Zhu
Gengxiang Jia
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Institute Of Botany
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Priority to AU2003236099A priority Critical patent/AU2003236099A1/en
Publication of WO2003097827A1 publication Critical patent/WO2003097827A1/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 a method for cultivating salt-tolerant tomatoes in the field of plant biotechnology.
  • betaine is synthesized in a chloroplast by a two-step reaction.
  • the first step is to catalyze choline to form betaine aldehyde by choline monooxidase (CM0);
  • the second step is to oxidize betaine aldehyde to betaine under the action of betaine aldehyde dehydrogenase (BADH).
  • BADH betaine aldehyde dehydrogenase
  • the gene encoding the synthetic BADH enzyme has been successfully cloned in many plants, such as weedynyk (EA, Hanson AD, 1990.
  • Tomato is widely distributed in the world. It is a vegetable with high economic value and great market potential. Cultivated tomato varieties are generally sensitive to salt. Salinization of soil and irrigation water has greatly reduced tomato production and caused great economic losses (Foolad MR, 1999. Genetics of salt and cold tolerance in tomato: Quantitative analysis and QTL mapping. Plant Biotech 16: 55-64; Cuartero et al., 1999. Tomato and salinity. Sci Hortic 78: 83-125). As the available arable land area is decreasing and soil quality is deteriorating, improving the salt tolerance of cultivated tomatoes and enhancing the ability of tomatoes to adapt to poor soil environments can not only make full use of limited land resources, but also have great economic and social benefits .
  • the object of the present invention is to provide a method for growing salt-tolerant tomatoes.
  • the method for cultivating a salt-tolerant tomato provided by the present invention is to introduce a cDNA of a betaine aldehyde dehydrogenase gene (BADH) or a fragment thereof into a tomato explant to obtain a salt-tolerant tomato.
  • BADH betaine aldehyde dehydrogenase gene
  • the explants can be cotyledons or true leaf leaves, stems, toruses, embryos, hypocotyls and other young tissues or their callus; among them, true leaf leaves are preferred.
  • the BADH cDNA or a fragment thereof can be transferred into tomato explants through Agrobacterium tumefaciens (the Agrobacterium is preferably Agrobacterium LBA4404), or can be introduced into tomato explants by gene gun method, or can be mediated by viruses. Transduction is performed by conventional methods such as inductive method, PEG-mediated method, electroporation method, microinjection method, pollen tube channel method, and ultrasonic method.
  • the hormone-free MS liquid medium is used to dilute the Agrobacterium LBA4404 bacterial solution to 0D ⁇ equal to 0.01 to 0.1, and the tomato true leaf leaves are infected and will be soaked by Agrobacterium
  • the leaves of tomato real leaves were co-cultured in an IM medium consisting of MS + IAA 0.2 mg / L + BA 0.2 mg / L + ZT 0.1 mg / L for 24-72 hours.
  • the leaves of tomato true leaves were transferred to IM + kanamycin 50 mg / L + carbenicillin 500 mg / L medium for differentiation culture.
  • light was applied for 12 hours a day.
  • the plants obtained from the differentiation culture are transferred to MS + kanamycin 500 mg / L + carbenicillin rooted on 500 mg / L medium.
  • the resulting transgenic plants should be tested for genomic DNA molecules.
  • the molecular detection was synthesized using BADH cDNA as a template and was performed with an ⁇ - 32 P-dCTP labeled probe.
  • Transgenic plants should also be tested for BADH activity and salt tolerance.
  • Figure 1 shows the physical structure of BADH-containing cDNA or its coding region sequence binary expression vector pBin438.
  • Figure 2 shows the death of uninfected leaves on Kana screening media
  • FIG. 2 is a transgenic callus
  • Figure 2—C is a transgenic plant
  • FIG. 3 A is the PCR test result of transgenic tomato TG0
  • FIG. 3 is the Southern blot result of transgenic tomato TG0
  • FIG. 3 is Northern blot results of transgenic tomato TG0
  • Figure 4 shows the BADH activity of transgenic tomato plants TG0.
  • Figure 6 shows the results of PCR detection of transgenic tomato TG1 plants.
  • Figure 7 shows orthern blot results of transgenic tomato TG1 plants
  • Figure 8 shows the effect of salt stress on the germination rate of BADH transgenic TG1 seeds.
  • Figure 9 shows the change in electrical conductivity of BADH transgenic TG1 plants.
  • Seeds of Lycopersicon esculentum Mill were purchased from the Chinese Academy of Agricultural Sciences. The seeds were surface disinfected with 70% alcohol and 0.1% liter of mercury for 1 minute and 15 minutes, respectively, and then washed three times with sterile distilled water. Seeded in MS minimal medium without any hormones (Murashige T, Skoog F, 1962. A revised medium for rapid growth and bioassays in tobacco tissue cultures. Physiol Plant 15: 473-493). The temperature of the incubator is maintained at 25 ⁇ 2 ° C, the humidity is 70% -80%, and the light is illuminated for 12 hours every day.
  • the binary expression vector pBin438 contains a double 35S promoter and a TMV ⁇ fragment translation enhancer.
  • the cDNA of BADH or a fragment thereof is inserted into the BaraHI and Kpnl sites of pBin438 according to a conventional method.
  • a binary expression vector pBin438 containing BADH cDNA was introduced into Agrobacterium according to a conventional method
  • Agrobacterium LBA4404 containing the expression vector was cultured to OD 56 with liquid YEB + kanamycin 50 mg / L medium. After 0.5, diluted 10-fold with hormone-free MS liquid medium. Infection of sterile true leaves of Lycopersicon esculentum Mill (Lycopersicon esculentum Mill) for 5 minutes. Subsequently, the leaves were removed, and the surface bacterial solution was blotted with sterile filter paper, and placed in IM medium (MS + IAA 0.2 mg / L + BA 2.0 mg / L + ZT 0.1 mg / L) and co-cultured in the dark for 48 hours. hour. Then transfer to IM + kanamycin 50 mg / L + carbenicillin 500 mg / L medium for differentiation and culture, light for 12 hours a day.
  • IM medium MS + IAA 0.2 mg / L + BA 2.0 mg / L + ZT 0.1 mg / L
  • the contemporary transgenic lines are TG0 plus their respective line numbers, such as TG0-1, TG0-2.
  • the strict offspring of each line is TG1 plus its own line number, such as TG1-1, TG1-2.
  • Genomic DNA was extracted by CTAB method from young leaves of 0.1-0.2 g of differentiated plants.
  • the PCR reaction system follows standard methods.
  • the primers used were: 5'-AGAATGGCGTTCCCAATTCCTGCTC-3 'and 5'-TTCAAGGAGACTTGTACCATCCC CA-3'.
  • the reaction procedure was: 95 ° denaturation for 1 minute, 55 ° C annealing for 1 minute, 72 ° extension for 1.5 minutes, a total of 35 cycles (Xiao -Gang, Zhang Gengyun, Liu Fenghua, Wang Jun, Chen Shouyi, Li Cong, Geng Huazhu, 1995. Study on the gene of betaine aldehyde dehydrogenase from Spinach. Science Bulletin, 40 (8): 741-745).
  • FIG. 3-A shows that the BADH gene has been integrated into the genomes of six strains.
  • WT represents the uninfected control plants; 1, 2, 3, 5, 8, and 9 represent the strains, respectively.
  • Genomic DNA (20 ⁇ g) was digested with Hindlll and EcoRI, electrophoresed on a 1.0% agarose gel at 50V for 5 hours, and the DNA was transferred to a Hybond-nylon membrane.
  • the hybridization was performed according to the method of Sambrook J ( Sambrook J, Fritsch EF, Maniatis T, 1989. Molecular Cloning: a laboratory manual. Cold Spring Harbor Press, New York, pp362-491).
  • the probe was a 1.5 kb BanRI-Kpnl BAMc fragment in the cloning plasmid vector ( Figure 1), and BAJM cmp was used as a template to synthesize with a- 32 P-dCTP, Randon Primer Labeling System (Takara) kit, and primer sequences.
  • a- 32 P-dCTP Randon Primer Labeling System (Takara) kit, and primer sequences.
  • Takara Randon Primer Labeling System
  • Figure 3-B shows that the BADH gene has been integrated into the genomes of the six strains. However, it can be seen from the figure that the integration site and the copy number of the BADH gene are different in different strains.
  • WT represents uninfected control plants; 1, 2, 3, 5, 8, 9 represent lines TG0-1, TGO-2, TG0-3, TG0-5, TGO-8, TG0-9c
  • RNA was extracted by Trizol-step method, and electrophoresed with 1.2% formaldehyde denaturing gel.
  • the membrane, hybridization method and probe used were hybridized with Southern.
  • the results are shown in Figure 3—C.
  • the figure shows that one line TG0-2 could not detect the expression signal completely.
  • WT indicates the uninfected control plants; 1, 2, 3, 5, 8, 9 Respective strains are TG0-1, TGO-2, TG0-3, TG0-5, TG0-8, TG0-9.
  • Plants of different transgenic lines were transplanted into flowerpots filled with soil and vermiculite (1: 1), and grown under greenhouse conditions (25 ⁇ 2 ° C, humidity 60% -80%, 12 hours of light per day). Irrigation with MS nutrient solution. After 40 days, each transgenic line plant was divided into two groups. One group was irrigated with MS nutrient solution; the other group was irrigated with MS + NaCl until the final NaCl concentration reached 180raM. BADH activity was measured according to Guo Yan et al. (Guo Yan, Zhang Li, Xiao Gang, Cao Shouyun, Gu Dongmei, Tian Wenzhong, Chen Shouyi, 1997.
  • An enzyme unit is defined as a standard reaction system Consumption of 1 nmol of NAD per milligram of protein per minute.
  • Fig. 4 The results of BADH activity measurement are shown in Fig. 4, which shows that significant activity can be detected in the plants of 5 lines regardless of the presence or absence of NaCl stress. However, the BADH activity of transgenic plants under salt stress was significantly higher than that under no salt stress.
  • WT represents the uninfected control strains
  • L1, L3, L5, L8, and L9 represent the strains TG0-1, TGO-3, TGO-5, TG0-8, and TG0-9, respectively.
  • the relative conductivity was measured according to the method of Leopold et al. (Leopold AC, Toenniessen RPW, 1984. GH (eds) Salinity tolerance in plants, Wiley: New York).
  • the control and each transgenic line were treated with 0 mM, 90, 180 mM and 270 mM NaCl, respectively.
  • Example 2 Determination of TGI kana resistance separation ratio and molecular identification of BADH transgenic tomatoes
  • the transgenic strain TG0 whose BADH gene has indeed been integrated into the genome and can be correctly expressed by molecular identification, is self-fruiting.
  • the seeds were sown on filter paper impregnated with MS + kanamycin 100mg / L solution, and the solution was kept in a petri dish without filter paper, and sealed with Parafilm to reduce the effect of water evaporation on the concentration of antibiotics. After 20 days, the percentage of seedlings that were sensitive and insensitive to kanamycin was counted, and an X 2 test was performed.
  • kana resistance was identified for seeds obtained from two lines TG0-1 and TG0-8 that can normally express the BADH gene.
  • the X 2 test results showed that the separation of the two offspring populations was a 3 : 1 separation, which was in line with the Mendelian separation ratio.
  • the results are shown in Table 1. It can be inferred that the insertion site of the BADH gene in the two transgenic lines (TG0-1, TG0-8) is one or on the same chromosome and can be inherited to the offspring.
  • PCR amplification and Northern hybridization of TG1 generation of BADH transgenic tomato were the same as TG0 generation.
  • the PCR results in Figure 6 also prove that the insertion site of the BADH gene in the two transgenic lines (TG0_1, TG0-8) is one or on the same chromosome, and can be inherited to the offspring; A in the figure is TG1— 1, B is TGl-8.
  • the results of Northern hybridization in Fig. 7 show that all plants with BADH gene in their genome can detect the expression signal.
  • 1 and 2 are PCR-positive plants of TGI-1; 3 is a PCR-negative plant of TGI-1; 4 is a PCR-negative plant of TG1-8; and 5, 6 are PCR-positive plants of TG1-8.
  • the sterile TG1 seeds were placed on the MS medium in a test tube, and the number of germinated seeds, seedling height, root length and number of roots were recorded daily. SPSS statistical software was used for analysis of variance. At the same time, the leaf electrical conductivity of 60-day-old seedlings under 0niM and 180mM NaCl stress was measured.
  • Fig. 8 indicates that the seed germination rate of strain TG1-1 and strain TG1-8 is significantly higher than that of the control under the stress of 90mM and 140mM NaCl.
  • Table 2 shows that the seedling height, main root length, and number of fibrous roots of the seedlings are also greatly improved compared with the control.
  • Figure 9 shows that the electrical conductivity of the leaves of the transgenic plants was significantly lower than that of the control under NaCl stress.
  • WT represents the uninfected control strain.
  • Table 2 Ducan analysis of T1 plant growth indicators Seedling height (cm) Root length (cm) Number of roots (strips) Population
  • the present invention cleverly introduces the cDNA of a betaine aldehyde dehydrogenase gene (BADH) or a fragment thereof into a tomato explant, and successfully obtains a salt-tolerant tomato, which has important theory and practice for improving the salt-tolerant quality of cultivated tomatoes Significance, will be widely used in tomato quality improvement breeding.
  • BADH betaine aldehyde dehydrogenase gene

Abstract

La présente invention se rapporte à un procédé de culture de plans de tomates halophiles, qui consiste à introduire l'ADNc d'un gène de bétaine aldéhyde déshydrogénase (BADH) ou un fragment de ce gène dans un explant de tomates et à cultiver la tomate halophile. L'explant et le procédé d'introduction peuvent être de types différents. Le procédé de la présente invention présente une importance théorique et il s'avère intéressant d'un point de vue pratique pour accroître la résistance au sel d'une tomate cultivée. Ce procédé peut être largement mis en oeuvre dans la culture de tomates pour améliorer la qualité des tomates.
PCT/CN2003/000241 2002-04-05 2003-04-03 Procede de culture de plans de tomates halophiles WO2003097827A1 (fr)

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AU2003236099A AU2003236099A1 (en) 2002-04-05 2003-04-03 A method for breeding salt-tolerant tomato plants

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CN02105490.8 2002-04-05
CN 02105490 CN1233833C (zh) 2002-04-05 2002-04-05 一种利用山菠菜badh基因转化培育耐盐番茄的方法

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CN101831497A (zh) * 2010-05-11 2010-09-15 新疆农业科学院园艺作物研究所 一种鉴别番茄耐盐性状的分子标记方法
US8044190B2 (en) 2004-09-08 2011-10-25 Carmel-Haifa University Economic Corp. Ltd. Stress tolerant organisms expressing a map kinase homologue

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CN1307312C (zh) * 2004-05-25 2007-03-28 山东省林业科学研究院 四倍体刺槐转基因及组培快繁方法
CN1871896B (zh) * 2005-06-01 2010-06-09 天津农学院 高效番茄基因转化方法
CN100465276C (zh) * 2006-12-11 2009-03-04 中国科学院植物研究所 一种提高植物抗逆性的方法
CN101880678B (zh) * 2009-05-08 2011-09-21 创世纪转基因技术有限公司 一种红树甜菜碱醛脱氢酶基因及其应用
CN101787372A (zh) * 2010-03-29 2010-07-28 天津农学院 一种油葵的基因转化方法
CN103070004B (zh) * 2013-02-17 2014-12-03 中国科学院植物研究所 一种盐水灌溉转基因番茄的方法

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JPH08266179A (ja) * 1995-02-01 1996-10-15 Toyota Motor Corp 環境ストレス耐性植物及びその作出方法

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JPH08266179A (ja) * 1995-02-01 1996-10-15 Toyota Motor Corp 環境ストレス耐性植物及びその作出方法

Non-Patent Citations (1)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8044190B2 (en) 2004-09-08 2011-10-25 Carmel-Haifa University Economic Corp. Ltd. Stress tolerant organisms expressing a map kinase homologue
CN101831497A (zh) * 2010-05-11 2010-09-15 新疆农业科学院园艺作物研究所 一种鉴别番茄耐盐性状的分子标记方法

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AU2003236099A1 (en) 2003-12-02
CN1450165A (zh) 2003-10-22

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