WO2003097827A1

WO2003097827A1 - A method for breeding salt-tolerant tomato plants

Info

Publication number: WO2003097827A1
Application number: PCT/CN2003/000241
Authority: WO
Inventors: Yinxin Li; Zhiqing Zhu; Gengxiang Jia
Original assignee: Institute Of Botany
Priority date: 2002-04-05
Filing date: 2003-04-03
Publication date: 2003-11-27
Also published as: AU2003236099A1; CN1450165A; CN1233833C

Abstract

The present invention discloses a method for breeding salt-tolerant tomato plants, which comprises introducing the cDNA of betaine aldehyde dehydrogenase (BADH) gene or its fragment into tomato explant and growing the salt-tolerant tomato. The explant and the introduction method can be various.The present method is of important theoretical and practical value to increasing salt tolerance of cultivated tomato. The method will be widly used in breeding for improving quantity of tomato.

Description

TECHNICAL FIELD

The invention relates to a method for cultivating salt-tolerant tomatoes in the field of plant biotechnology.

Background technique

Plants grow in the natural environment and are often affected by adverse environmental conditions. Among them, salt damage is an important factor that restricts the geographical distribution of plants and affects their yield and quality to a certain extent (Boyer JS, 1982. Plant productivity and environment. Science 218: 443-448). During long-term evolution, plants have formed mechanisms to adapt and resist salt stress. The common one is the synthesis of small molecules such as amino acid derivatives (proline, betaine, etc.), sugars, alcohols, and so on. Betaine protects plant cells by balancing the osmotic pressure of cells and the surrounding environment, the quaternary structure of stable proteins, and the enzyme system within the cells to protect plant cells to reduce damage to plants under salt stress

(Bernard T, Ayache M, Rudulier DL, 1988. Restoration of growth and enzymic activities of Escherichia coli Lac- mutants by glycinebetaine. CR Acad Sci III 307: 99-104; Papageorgiou GC, Murata N, 1995. The unusually strong stabilizing effects of glycinebetaine on the structure and function in the oxygen-evolving photosystem II complex. Photosynthesis Research 44: 243-252).

Studies have shown that plants that can synthesize betaine have strong salt tolerance. In plant cells, 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). The gene encoding the synthetic BADH enzyme has been successfully cloned in many plants, such as weedynyk (EA, Hanson AD, 1990. Molecular cloning of a plant betaine aldehyde dehydrogenase, a enzyme implicated in adaption to salinity and drought. Proc Natl Acad Sci USA 87: 2745-2749) Mountain spinach (Xiao Gang, Zhang Gengyun, Liu Fenghua, Wang Jun, Chen Shouyi, Li Cong, Geng Huazhu, 1995. Study on betaine aldehyde dehydrogenase gene of mountain spinach. Science Bulletin, 40 (8): 741-745), sugar beet (McCue KF, Hanson AD, 1992. Effects of soil salinity on the expression of betaine aldehyde dehydrogenase in leaves: investigation of hydraulic, ionic and biochemical signals. Aust J Plant Physiol 19: 555- 564), sorghum (Wood AJ, Saneola H, Rhides D, Joly RJ, Goldbrough PB, 1996. Betaine aldehyde dehydrogenase in Sorghum Plant physiol 110: 1301-1308) and the like. The results show that many plants, such as Arabidopsis, tobacco and tomato, are salt-sensitive plants that cannot synthesize and accumulate betaine (Weretilnyk EA, Bednarek S, McCue KF, Rhodes D, 1989. Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons. Planta 178: 342-352; Rhodes D, Hanson AD, 1993. Quaternary ammonium and tertiary sulfonium compounds in higher plants. Annu Rev Plant Physiol 44: 357-384; Nuccio ML, Russel BL, Nolte KD, Rathinasabapathi B, Gage DA, Hanson AD, 1998. The endogenous choline supply limits glycinebetaine synthesis in transgenic tobacco expressing choline monooxygenase. Plant J 16: 487-496). This has aroused people's interest in introducing betaine to synthesize betaine to improve salt tolerance of salt-sensitive plants.

There have been some reports on improving plant salt tolerance by introducing betaine synthesis pathways (Rathinasabapathi B, McCue KF, Gage DA, Hanson AD, 1994. Metabolic engineering of glycinebetaine synthesis: plant betaine aldehyde dehydrogenases lacking typical, transit peptides are targeted to tobacco chloroplasts where they confer betaine aldehyde resistance. Planta 193: 155-162; Liu Fenghua, Guo Yan, Gu Dongmei, Xiao Gang, Chen Zhenghua, Chen Shouyi, 1997. Study on Salt Tolerance of Transgenic Plants with Betaine Aldehyde Dehydrogenase Genes. (1): 54-58; Guo Yan, Zhang Li, Xiao Gang, Cao Shouyun, Gu Dongmei, Tian Wenzhong, Chen Shouyi, 1997. Expression of Betaine Aldehyde Dehydrogenase Gene in Rice and Salt Tolerance of Transgenic Plants. China Science (C Series), 27 (2): 151-155; Trossat C, Rathinasabapathi B, Hanson AD, 1997. Transgenically expressed betaine aldehyde dehydrogenase efficiently catalyzes oxidation of dimethylsulfoniopropionaldehyde and ω-aminoaldehydes. Plant Physiol 113: 1457-1461; Li silver , Chang Fengqi, Du Liqun, Guo Beihai, Li Hongjie, Zhang Jinsong, Chen Shouyi, Zhu Zhiqing, 2000. Salt tolerance of transgenic watercress with betaine aldehyde dehydrogenase gene. Chinese Journal of Botany, 42 (5): 480-484), but with Vegetables are rarely studied.

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 . Studies have shown that the endogenous BADH synthesis pathway is lacking in tomato plants (McCue KF, Hanson AD, 1990. Drought and Salt tolerance: towards understanding and application. Tibtech 8: 358- 362; Weretilnyk EA, Bednarek S, McCue KF, Rhodes D, 1989. Comparative biochemical and immunological studies of the glycine betaine synthesis pathway in diverse families of dicotyledons. Planta 178: 342-352). Therefore, by introducing the BADH gene into tomatoes, it is expected to establish a tomato betaine synthesis pathway, thereby improving the cultivation of tomato varieties. Salt tolerance.

Invention Disclosure

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.

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.

When the Agrobacterium-mediated method is used, 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. After co-cultivation, the leaves of tomato true leaves were transferred to IM + kanamycin 50 mg / L + carbenicillin 500 mg / L medium for differentiation culture. During the differentiation culture, light was applied for 12 hours a day. After the differentiation culture, 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 α- ³² P-dCTP labeled probe. Transgenic plants should also be tested for BADH activity and salt tolerance.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the physical structure of BADH-containing cDNA or its coding region sequence binary expression vector pBin438.

Figure 2—A shows the death of uninfected leaves on Kana screening media

Figure 2—B is a transgenic callus

Figure 2—C is a transgenic plant

Figure 3—A is the PCR test result of transgenic tomato TG0

Figure 3—B is the Southern blot result of transgenic tomato TG0

Figure 3—C is Northern blot results of transgenic tomato TG0

Figure 4 shows the BADH activity of transgenic tomato plants TG0.

Figure 5: Changes in leaf electrical conductivity of transgenic tomato plants under salt stress

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.

The best way to implement the invention

Material

(1) 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.

(2) Taq enzymes and primers used in PCR reactions were purchased and synthesized by Shanghai Shengong Company. Restriction enzymes and Random Primer Labeling System Kit were purchased from Takara. Trizol was purchased by GIBC0. (3) As shown in FIG. 1, the binary expression vector pBin438 contains a double 35S promoter and a TMV Ω 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. ,

Example 1.Agrobacterium transformation and screening of transgenic tomatoes

(1) A binary expression vector pBin438 containing BADH cDNA was introduced into Agrobacterium according to a conventional method

(2) 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.

Observation results showed that leaves that were not infected by Agrobacterium (control group, as shown in Figure 2-A) turned yellow or white after 2 weeks on the screening medium, the leaves died, and no wounds grew on the wound. Agrobacterium-infected tomato leaves began to show green shoots at the leaf margins after 2-3 weeks (as shown in Figure 2-B); plantlets differentiated after 5-6 weeks. When these plantlets reached 2-3 cm in height, they were transferred to MS + kanamycin 500 mg / L + carbenicillin 500 mg / L medium to take root. The results are shown in Figure 2-C. These plantlets Can take root smoothly and grow normally.

(3) Naming of transgenic plants

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.

(4) Molecular detection of contemporary transgenic strain TG0

(a) Extraction of genomic DNA

Genomic DNA was extracted by CTAB method from young leaves of 0.1-0.2 g of differentiated plants.

(b) PCR reaction

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). The results are shown in Figure 3-A, which shows that the BADH gene has been integrated into the genomes of six strains. In the figure, WT represents the uninfected control plants; 1, 2, 3, 5, 8, and 9 represent the strains, respectively. TG0—1, TG0—2, TG0-3, TGO-5, TG0—8, TG0—9.

(c) Southern hybridization

Genomic DNA (20 μg) 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. Are 5'-AGAATGGCGTTCCCA ATTCCTGCTC-3, and 5, '-TTCAAGGAGACTTGTACCATCCCCA-3'. The results are shown in Figure 3-B, which 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

(d) Nothern hybridization

The 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. In the figure, 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.

(5) BADH activity test of transgenic plant TG0

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. Expression of betaine aldehyde dehydrogenase gene in rice and salt tolerance of transgenic plants. Science in China (Series C), 27 (2): 151-155). An enzyme unit is defined as a standard reaction system Consumption of 1 nmol of NAD per milligram of protein per minute.

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. In Figure 4, 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.

(6) Determination of relative electrical conductivity (REc) of transgenic plant TG0 leaves

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.

The results are shown in Figure 5, which shows that the electrical conductivity of tomato leaves gradually increases with the increase of salt stress, indicating that the damage to the cells is getting more and more serious. However, it can be seen that in the 5 strains that normally express BADH genes, the conductivity was reduced to varying degrees, indicating that transgenic tomatoes with BADH genes have improved salt tolerance to varying degrees. In the figure, WT represents the uninfected control strains; Linel, Line3, Line5, Line8, and Line9 represent the strains TG0-1, TGO-3, TGO-5, TG0-8, and TG0-9, respectively.

Example 2: Determination of TGI kana resistance separation ratio and molecular identification of BADH transgenic tomatoes

(1) 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.

In this experiment, 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.

Table 1: Kana resistance isolation ratio of TG1 generation of BADH transgenic tomatoes

Measured value Expected value

Species 1 to X ² significance

Resistance sensitive

TG1-1 147 53 150 50 0.624 0.25 <P <0.5

TG1-8 145 55 150 50 0.414 0.5 <P <0.75

(2) PCR amplification and Northern hybridization of TG1 generation of BADH transgenic tomato

The 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. In FIG. 7, 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.

Example 3 Detection of Salt Tolerance in BADH-Transgenic Tomato TG1 Plants

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.

The results are shown in Fig. 8, which 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. In Figures 8 and 9, 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

OmM 90m 140mM OmM 90mM 140mM OmM 90mM 140mM

WT 6.60 a 3.25 c 0.60 d 9.68 a 4.40 d 0.62 e 8.4 a 0.0 e 0.0 e

TGl-1 6.52 a 5.04 b 2.91 c 9.72 a 6.86 b 5.84 cd 8.3 a 3.7 b 1.6 d

TG1-8 6.65 a 4.64 b 3.10 c 9.83 a 6.12 be 6.06 be 8.5 a 4.2 b 2.8 c Note: The difference of the same letter after the number is not significant.

Industrial applications

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.

Claims

Claim

1. A method for cultivating a salt-tolerant tomato 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.

The method according to claim 1, characterized in that: the explant is a young tissue of a tomato plant or a callus generated from them.

The method according to claim 2, characterized in that: the young tissue is a cotyledon or true leaf leaf, stem, torus, embryo or hypocotyl.

4. The method according to claim 3, wherein the explant is a true leaf blade.

5. The method according to claim 1 or 2 or 3 or 4, characterized in that: the cDNA of the BADH or a fragment thereof is transferred into a tomato explant by Agrobacterium tumefaciens.

6. The method according to claim 5, wherein: said Agrobacterium is Agrobacterium

7. The method according to claim 5, characterized in that: said Agrobacterium-mediated method is to dilute Agrobacterium LBA4404 bacterial solution to 0D ₅₆ with a hormone-free MS liquid culture medium. When it is equal to 0.01 to 0.1, the tomato true leaf leaves are infected, and the tomato true leaf leaves soaked by Agrobacterium are placed at MS + IAAO. 2mg / L + BA0. 2mg / L + ZT0. Lmg / L. Co-culture for 24 to 72 hours.

8. The method according to claim 7, characterized in that: after the restoration of culture, the leaves of tomato true leaves are transferred to IM + kanamycin 50 _mg / L + carbenicillin 500 mg / L medium for differentiation and culture.

9. The method according to claim 8, characterized in that: after the differentiation culture, the plants obtained from the differentiation culture are transferred to MS + kanamycin 500 mg / L + carbenicillin 500 mg / L medium root.

10. The method according to claim 1, wherein: the cDNA of the BADH or a fragment thereof is introduced into a tomato explant by a gene gun method.

11. The method according to claim 1, characterized in that: the obtained transgenic plants should be tested for genomic DNA molecules.

12. The method according to claim 11, wherein: the molecular detection is synthesized using BADH cDNA as a template, and is performed with an a- ³² p-dCTP labeled probe.