Cold-injury-resistance gene of lycopersicon esculentum mill and application

A tomato and gene technology, applied in the field of plant biology, to achieve the effects of improving adaptability, promoting stable and high yield, and expanding the planting range

Active Publication Date: 2017-05-31
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The invention discloses a cold-injury-resistance gene of lycopersicon esculentum mill and an application. On the basis of virus induced gene silencing (VIGS) technology, the novel chilling-injury-resistance related gene SISLD of the lycopersicon esculentum mill is discovered, and the gene, through silencing, can result in obvious decrease in low temperature tolerance ability of a tomato plant. Based upon analysis on an amino acid sequence encoded by the gene, protein encoded by the gene is [delta]8-sphingomyelin dehydrogenase, which is related to lipid metabolism of neurolemma, and the enzyme (the [delta]8-sphingomyelin dehydrogenase) plays an important role in a reaction that a plant resists to low-temperature stress. According to the research, a nucleotide sequence and an amino acid sequence of the gene are provided; and meanwhile, the invention also relates the application of the gene in cold-resistance breeding of tomato. The SISLD gene, when applied to anti-freezing breeding of the tomato, can improve the adaptability of tomato's high-quality varieties, extend a planting range of the tomato's high-quality varieties and promote stable production and high-yield production of the tomato's high-quality varieties.

Application Domain

OxidoreductasesFermentation +3

Technology Topic

Nucleic acid sequencingNucleic acid sequence +18


  • Cold-injury-resistance gene of lycopersicon esculentum mill and application


  • Experimental program(3)

Example Embodiment

[0012] Example 1: VIGS treatment of tomato
[0013] The formula of the LB solid medium used below is: 10g/L Tryptone, 5g/L Yeast extract, 10g/L Sodium Chloride (NaCl), 15-20g/L agar powder, use Adjust the pH to 7.4 with 10M NaOH, with the remainder being water; sterilize at 121°C for 20 minutes, and when the temperature drops to about 55°C (not hot), add the final concentration of 30mg/L Kanamycin (Kanamycin, Kan), or Add the final concentration of 30mg/L Kanamycin (Kanamycin, Kan), 50mg/L Rifampicin (Rifampicin, Rif), 50mg/L Gentamicin (Gentamicin, Gen), pour the plate and store for later use; LB liquid The medium formula is: 10g/L Tryptone, 5g/L Yeast extract, 10g/L Sodium Chloride (NaCl), adjust the pH to 7.4 with 10M NaOH, and the balance is water; Sterilize at 121°C for 20min. After the temperature drops to about 55°C (not hot) or after cooling, add the final concentration of 30mg/L Kanamycin (Kanamycin, Kan), or add the final concentration of 30mg/L Kanamycin (Kanamycin, Kan), 50 mg/L rifampicin (Rifampicin, Rif), 50 mg/L gentamicin (Gentamicin, Gen).
[0014] The specific steps of the embodiment are described below:
[0015] 1) Tomato RNA extraction and cDNA acquisition
[0016] For the tomato RNA extraction method, please refer to the instructions of Huayueyang Biotech Ultrafast Plant RNA Extraction Kit. The specific steps are as follows: Take 0.1g of tomato leaves ground with liquid nitrogen in a 1.5mL centrifuge tube, add 1mL of cell lysate, and mix thoroughly; add 300μL of protein-removing solution and 200μL of chloroform, cover the tube, and shake vigorously to mix Homogenize for 30s; centrifuge at 12,000×g at room temperature for 10 minutes, transfer the supernatant (not more than 700μL) to a 1.5mL RNase-free centrifuge tube; add an equal volume of rinsing solution, invert and mix thoroughly, and mix the mixture (not more than 700μL) Transfer to a centrifugal adsorption column, centrifuge at 12,000×g at room temperature for 1 min, discard the penetrating solution; add 500 μL of wash column, at room temperature 12,000×g, centrifuge for 1 min, discard the penetrating solution, and repeat. Centrifuge at 12,000×g for 1 min at room temperature to remove the remaining liquid; transfer the centrifugal adsorption column to an RNase-free centrifuge tube, add 50 μL of RNA eluate, and leave it at room temperature for 5 min; centrifuge at 12,000×g at room temperature for 1 min, which will contain tomato RNA The penetration liquid is stored in a refrigerator at -80°C.
[0017] Reverse transcription of tomato RNA into cDNA using PrimeScript from Takara TM RT reagent Kitwith gDNA Eraser (Perfect Real Time) kit. Take out -80°C frozen tomato RNA, after thawing, add DNase according to the instructions, digest the DNA at 42°C for 2min, then add reverse transcription reagent, 37°C for 15min, 85°C for 5sec; get reverse transcribed cDNA.
[0018] 2)△ 8 -Cloning of sphingomyelin dehydrogenase gene (SlSLD)
[0019] Using bioinformatics analysis method, search tomato in tomato genome database△ 8 -Sphingomyelin dehydrogenase gene (SlSLD), to get 1 candidate sequence. According to the sequence information, the primer sequence for amplifying the full-length sequence of the SlSLD gene was designed, the upstream primer SlSLDF: 5'-GGATCCCAGGGTAAGGTGTATGATGT-3' and the downstream primer SlSLDR: 5'-TCTAGAGCAATTCCCAGTAAGGAC-3'.
[0020] Refer to the instructions for the reaction system. For PCR amplification, use Takara's PrimeSTAR Max Premix (2×) 10 μL, 10 μM upstream primer SlSLDF and 10 μM downstream primer SlSLDR each 0.5 μL, reverse transcribed cDNA template 0.1 μL, add double distilled water to make up 20 μL. The PCR amplification program is 98°C 30sec, 1 cycle; 98°C 15sec, 60°C 15sec, 72°C 30sec, 35 cycles; 72°C 10min, 1 cycle. From this amplification, △ 8 -The full-length sequence of the sphingomyelin dehydrogenase gene (SlSLD1). The full-length sequence was digested with restriction enzymes BamH I and Xho I (37℃ digestion reaction for 2h), and then connected to the expression vector pTRV2 vector (37℃ Restriction enzymes BamH I and Xho I were digested for 2h) to obtain the recombinant plasmid pTRV2-SlSLD, which was sent to the company for sequencing. △ 8 -The nucleic acid sequence of sphingomyelin dehydrogenase gene (SlSLD) is shown in SEQ ID NO. 1, which encodes △ 8 -The amino acid sequence of the sphingomyelin dehydrogenase gene is shown in SEQ ID NO.2. After confirming that the sequence is correct, the recombinant plasmid pTRV2-SlSLD was transformed into E. coli DH5α by heat shock method (heat shock at 42°C for 90sec), and cultured at 37°C in LB solid containing 30mg/L Kanamycin (Kan) Cultured on the base for 1 day, pick the positive single clone in 1mL of 30mg/L Kanamycin (Kanamycin, Kan) LB liquid medium, incubate at 200 rpm for 1 day at 37℃, to OD 600 The value is about 1.0, stored in 15% glycerin, and placed in the refrigerator at -80°C for later use.
[0021] 3) Agrobacterium-mediated△ 8 -VIGS of sphingomyelin dehydrogenase gene (SlSLD)
[0022] Pick the strains stored in 2), place them in 5mL LB liquid medium (containing 30mg/L Kanamycin (Kanamycin, Kan)), incubate at 200 rpm at 37°C for 1 day, To OD 600 The value is about 1.2, and then the recombinant plasmid pTRV2-SlSLD is extracted with a small plasmid kit. Take 1 μL of pTRV1 plasmid and pTRV2-SlSLD plasmid and add 30 μL of GV3101 competent cells that have been thawed on ice. The competent cells are transferred to a 2mm motor cup and transformed by motor. The specific parameters are as follows: select A. tumefaciens under Bacterial under Pre-Set Protocols, voltage 2400V, capacitance 25μF, resistance 200Ω, electric shock cup Cuvette is 2mm . After the electric shock, add non-resistant LB liquid medium, mix upside down, transfer to a 1.5mL centrifuge tube, incubate at 28°C at 200 rpm for 2h, and then add the bacteria to 30mg/L kanamycin (Kanamycin, Kan), 50mg/L rifampicin (Rifampicin, Rif), 50mg/L gentamicin (Gentamicin, Gen) on LB solid medium for 2-3 days.
[0023] Pick a positive single clone in 1mL of LB liquid medium containing 30mg/L Kanamycin (Kanamycin, Kan), 50mg/L Rifampicin (Rifampicin, Rif), 50mg/L Gentamicin (Gen) Medium, cultured at 200 rpm for 1 day at 28°C, to OD 600 The value is around 1.0. Centrifuge at 4000×g for 15 minutes, collect the precipitate, add 30 mg/L kanamycin (Kanamycin, Kan), 50 mg/L rifampicin (Rifampicin, Rif), 50 mg/L gentamicin (Gentamicin, Gen ) LB liquid medium to make the OD of pTRV1/GV3101 bacterial solution 600 Value is 0.4, the OD of pTRV2-SlSLD/GV3101 bacterial solution 600 The value is 0.4. Then mix the two bacterial liquids at a ratio of 1:1, and let stand at room temperature for 3 to 6 hours. The tomato seedlings that had just grown the first true leaf were infected by the method of vacuum injection, and pTRV2/GV3101 was used as the control, and the infection was carried out at the same time. The infected seedlings were cultured for 30 days under the conditions of 21℃, 16:8 (day: night). Fluorescence quantitative PCR analysis was used to identify the degree of gene suppression. The specific results are shown in Table 1.
[0024] Table 1 Expression of SlSLD gene in tomato plants after VIGS
[0025] Plant SlSLD gene expression *
[0026] Note: *The expression level of the control gene is 1.

Example Embodiment

[0027] Example 2: Low temperature stress test of SlSLD gene silenced plants (treatment group) and control group
[0028] The plants of the SlSLD gene silenced (treatment group) and the control group were subjected to stress treatment at 4°C, and photographed to record the phenotypic changes of the plants in a low temperature environment. From figure 1 It can be seen that when the SlSLD gene silenced and control plants were treated at low temperature for 6h, the leaf edges appeared curled, and the changes in the treatment group were more obvious; at 24h, the control group still maintained the state at 6h, and the leaves appeared Slight curl, but the whole plants in the treatment group appeared wilting and could no longer grow normally.

Example Embodiment

[0029] Example 3: Determination of plant physiological indicators after low temperature stress test
[0030] The physiological indicators of tomato leaves treated with low temperature for 6 hours were measured, including electrical conductivity, malondialdehyde content, soluble polysaccharide content, superoxide dismutase activity, peroxidase activity and chlorophyll content. The specific operation is as follows:
[0031] To prepare the supernatant, weigh 0.1g tomato leaves, add 2.5mL of pre-cooled 50mM phosphate buffered saline solution (pH7.0), grind on ice, centrifuge the homogenate at 4℃, 4,000×g for 10min, and collect The supernatant is placed in a refrigerator at 4°C and used for the determination of indicators 2-5 below.
[0032] 1) Determination of relative electrolytic leakage (REL)
[0033] Take 0.05 g of tomato leaves from the control group and the treatment group after low-temperature treatment, and wash the electrolytes on the surface of the leaves with double distilled water. The leaves were placed in a centrifuge tube containing 10 mL of double-distilled water, soaked at room temperature for 22 hours, and then measured with a conductivity meter (Jingke DDS-307A), the value obtained was R1. Treat the centrifuge tube containing tomato leaves in boiling water for 30 minutes, and then wait for it to fall to room temperature, then re-measure with a conductivity meter, and the value obtained is R2. Calculation formula: conductivity (REL)=R/R2×100%.
[0034] 2) Determination of Malondialdehyde (MDA) content
[0035] The content of MDA in leaves was determined by thiobarbital method. Take 200 μL of the supernatant and place it in a 1.5 mL centrifuge tube, add an equal volume of 0.6% thiobarbituric acid solution, and place the mixture in a boiling water bath for 30 minutes. After cooling, the absorbance was measured at wavelengths of 532nm, 600nm and 450nm. Calculation formula: MDA concentration (μmol/L)=6.45×(OD 532 -OD 600 )-0.56×OD 450.
[0036] 3) Determination of soluble polysaccharide content
[0037] Anthrone colorimetry was used to determine the content of soluble polysaccharides in leaves. Take 100 μL of the supernatant and place it in a 1.5 mL centrifuge tube, and add 400 μL of a sulfuric acid solution containing 0.2% anthrone. The mixture was subjected to a boiling water bath for 5 min. After cooling, the absorbance was measured at a wavelength of 630nm, and a standard curve was drawn with glucose.
[0038] 4) Determination of superoxide dismutase (SOD) activity
[0039] The activity of SOD was measured using Biyuntian WST-8 kit, and the specific operation steps refer to the instructions provided by the kit.
[0040] 5) Determination of peroxidase (POD) activity
[0041] Guaiacol colorimetry was used to determine the activity of POD. Take 10μL supernatant and add 190μL containing 0.12% H 2 O 2 With 0.56% guaiacol, the absorbance is measured at a wavelength of 470nm, and the reading is performed every 1 minute until the reaction is terminated. The change in absorbance per minute is 1 unit of enzyme activity (U). The unit of POD activity is U/mg protein.
[0042] 6) Determination of chlorophyll content
[0043] Weigh 0.02 g tomato leaves, soak them in 2 mL 80% acetone overnight in the dark, centrifuge at 8,000×g for 10 min, and collect the supernatant. The absorbance was measured at wavelengths of 647nm and 665nm. Calculation formula: chlorophyll concentration (mg/g) = (17.90×OD 647 +8.08×OD 665 )×Soaking liquid volume (ml)/1000/fresh weight (g).
[0044] It can be seen from Table 2 that compared with the control plants, the ability of the SlSLD gene silenced plants was significantly weaker to resist cold. The conductivity of the treatment group was higher than that of the control group, indicating that the suppression of the gene expression would cause the tomato plants to be more stressed under low temperature conditions. The content of malondialdehyde was also higher in the treatment group than in the control group, while the activities of superoxide dismutase and peroxidase were indeed higher in the control group. In addition, the chlorophyll content of tomato leaves whose SlSLD gene was silenced was lower than that of the control group. Therefore, cold stress experiments show that the silencing of the SlSLD gene will weaken the ability of tomato to resist cold damage.
[0045] Table 2 Physiological indexes of tomato leaves after low temperature stress
[0046] Physiological index Control group Treatment group Conductivity (%) 45.64±5.55 *
[0047] Note: * indicates that there is a significant difference between the control group and the treatment group, p≤0.05.


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