CN106754769B - A kind of tomato Inappropriate ADH syndrome gene and application - Google Patents

Abstract

The invention discloses a kind of tomato Inappropriate ADH syndrome gene and applications.The present invention passes through virus induced gene silencing (Virus induced gene silencing, VIGS) scientific discovery a kind of new tomato Inappropriate ADH syndrome related gene SlSLD, the silencing of the gene can cause the low temperature tolerance ability of tomato plant to be substantially reduced.The albumen of the coded by said gene, which is a kind of △ 8- sphingomyelins dehydrogenase, to be proved to the amino acid sequence analysis of gene coding, related to neurolemma lipid-metabolism, which plays an important role in the reaction of plant resistant low temperature stress.This research provides the nucleic acid sequence and its amino acid sequence of this gene, also relates to the purposes that the gene is resisted cold in tomato in breeding.SlSLD gene can be applied to the freeze proof breeding of tomato, improves the adaptability of tomato excellent variety, expands its planting range, promote its stable yields and high yield.

Description

A kind of tomato chilling damage resistance gene and its application

technical field

The invention belongs to the field of plant biotechnology, and in particular relates to a tomato chilling damage resistance gene and its application.

Background technique

Sphingolipids is a general term for a class of complex lipids containing fatty acids and long-chain sphingosine skeletons. It is de novo synthesized on the endoplasmic reticulum by catalyzing serine and palmitoyl-CoA by serine palmitoyl-CoA transferase (SPT). This type of lipid is an important part of the plant cell plasma membrane, tonoplast membrane and inner membrane system, accounting for about 40% of the lipids on the membrane, and mainly exists in the outer leaflet of the cell membrane. The outer leaves of the cell membrane affect the integrity of the membrane and the permeability of ions, and the physiological state of the cell membrane under low temperature conditions is an important indicator of the plant's resistance to chilling damage. In addition, sphingolipids can be reduced to generate an octadecyl amino alcohol containing an unsaturated hydrocarbon chain, namely sphinganine. It is a type of long chain base (Long chain base, LCB), which can be catalyzed by sphingolipid desaturase (SLD) to generate unsaturated sphingomyelin. A large number of studies have shown that unsaturated sphingomyelin exists in large quantities in cold-resistant plants, because the content of this substance affects the level of plant cold resistance. In Arabidopsis, the increase of glucosylceramide (d18:1c24:0) content can enhance the frost resistance of plants. There are also experiments that further show that Arabidopsis plants with mutations in the AtSLD gene will develop chlorosis in low temperature environments, and the plants cannot grow normally. In addition, studies have found that glucosylceramide has a relatively high content in cell membranes and tonoplast membranes, and highly hydroxylated glucosylceramide is very important for maintaining the integrity of cell membranes and tonoplast membranes. △ ⁸ -sphingomyelin dehydrogenase is the key enzyme that catalyzes the dehydrogenation of C8 position of sphingolipid long chain base to generate △ ⁸ -unsaturated sphingomyelin. Chilling injury treatment can increase the content of △ ⁸ -unsaturated sphingomyelin containing glucosylceramide in soybean leaves. Therefore, it can be considered that △ ⁸ -unsaturated sphingomyelin and △ ⁸ -sphingomyelin dehydrogenase are related to cold resistance of soybean Sex is closely related. It can be found from previous studies that sphingolipids and their unsaturated sphingomyelin dehydrogenase may affect the ability of plants to resist chilling damage. However, there are still few studies on the relationship between the dehydrogenase and cold resistance, especially in economic crops, and the relationship between the two needs to be further elucidated.

Tomato (Lycopersicon esculentum Mill) is a plant of the genus Tomato in the family Solanaceae. It is an important economic crop and is closely related to the national "vegetable basket project". It is a cold-sensitive plant, and low-temperature chilling damage will cause great losses to tomato production, which is one of the main factors affecting tomato annual production and market supply. The growth and development of most tomato varieties are easily hindered when the temperature is lower than 10°C, and they show obvious symptoms of chilling injury when the temperature is lower than 6°C, so the adaptability of tomato to low temperature environments has attracted widespread attention from scholars. In order to improve tomato varieties, increase tomato resistance to low temperature, and reduce tomato economic losses from chilling damage, researchers have explored the mechanism of tomato resistance to chilling damage in terms of cell membrane structure, antioxidant enzymes, and cell wall substance metabolism. However, no researchers have explored the role of △ ⁸ -sphingomyelin dehydrogenase in tomato cold adaptation. Therefore, the present invention not only excavates the mechanism of tomato anti-chilling injury from the new angle of sphingolipid metabolism, but also provides a certain theoretical basis for cultivating low-temperature-resistant tomato varieties, which can improve the adaptability of tomato planting, expand the planting area, increase Yield and so on are of great significance.

Contents of the invention

The object of the present invention is to provide a tomato chilling damage resistance gene S1SLD, the silencing of the gene can lead to the reduction of tomato low temperature tolerance. The gene can be applied to tomato frost resistance breeding, and can effectively improve the frost resistance of tomato varieties.

Another object of the present invention is to provide an application of a tomato chilling damage resistance gene in tomato cold-resistant breeding. By increasing the expression level of the SlSLD gene in the plant, the low temperature tolerance breeding of the plant is carried out, so as to expand the suitable planting range of the plant.

A tomato Δ ⁸ -sphingomyelin dehydrogenase gene, the nucleotide sequence of which is shown in SEQ ID NO.1.

The amino acid sequence of tomato Δ ⁸ -sphingomyelin dehydrogenase encoding the above tomato Δ ⁸ -sphingomyelin dehydrogenase gene is shown in SEQ ID NO.2.

By modulating the expression of the SlSLD gene described in the present invention, the sensitivity of plants to cold can be altered.

Compared with the prior art, the present invention has the following beneficial effects: the present invention discovers a new tomato chilling resistance-related gene SlSLD through virus-induced gene silencing (VIGS) technology, and the silencing of this gene can lead to The low temperature tolerance of tomato plants was significantly reduced. Analysis of the amino acid sequence encoded by the gene proves that the protein encoded by the gene is a △ ⁸ -sphingomyelin dehydrogenase, which is related to the metabolism of nerve sphingolipids, and the enzyme plays an important role in the response of plants to resist low temperature stress. This study provides the nucleic acid sequence and amino acid sequence of this gene, and also involves the use of this gene in tomato cold-resistant breeding. The SlSLD gene can be used in tomato breeding for frost resistance, improve the adaptability of tomato elite varieties, expand its planting range, and promote its stable and high yield.

Description of drawings

Figure 1 is a phenotype diagram of tomato plants treated with low temperature stress.

Detailed ways

The following examples are to further illustrate the present invention, rather than limit the present invention.

Example 1: VIGS processing of tomato

The formula of LB solid medium used below is: 10g/L tryptone (Tryptone), 5g/L yeast extract (Yeast extract), 10g/L sodium chloride (NaCl), 15-20g/L agar powder, use Adjust the pH value to 7.4 with 10M NaOH, and the balance is water; sterilize at 121°C for 20 minutes, and when the temperature drops to about 55°C (not hot), add Kanamycin (Kanamycin, Kan) at a final concentration of 30mg/L, or Add final concentration of 30mg/L Kanamycin (Kanamycin, Kan), 50mg/L Rifampicin (Rifampicin, Rif), 50mg/L Gentamicin (Gentamicin, Gen), pour plate, save for later use; LB liquid The formula of the medium is: 10g/L tryptone (Tryptone), 5g/L yeast extract (Yeast extract), 10g/L sodium chloride (NaCl), adjust the pH value to 7.4 with 10M NaOH, and the balance is water; Sterilize at 121°C for 20 minutes, and after the temperature drops to about 55°C (not hot to the hands) or after cooling, add Kanamycin (Kanamycin, Kan) at a final concentration of 30mg/L, or add Kanamycin at a final concentration of 30mg/L (Kanamycin, Kan), 50mg/L rifampicin (Rifampicin, Rif), 50mg/L gentamicin (Gentamicin, Gen).

The concrete steps of embodiment are described below:

1) Extraction of tomato RNA and acquisition of cDNA

For the extraction method of tomato RNA, please refer to the instructions of Huayueyang Biological Company Ultra-fast Plant RNA Extraction Kit. The specific steps are as follows: Take 0.1 g of tomato leaves ground with liquid nitrogen and place in a 1.5 mL centrifuge tube, add 1 mL of cell lysate, and mix well; Homogenize for 30 seconds; centrifuge at 12,000×g at room temperature for 10 minutes, transfer the supernatant (not more than 700 μL) to a 1.5 mL RNase-free centrifuge tube; Transfer to a centrifugal adsorption column, centrifuge at 12,000×g at room temperature for 1 min, discard the passthrough; add 500 μL of column wash solution, centrifuge at 12,000×g at room temperature for 1 min, discard the passthrough, and repeat once. Centrifuge at 12,000×g at room temperature for 1 min to remove residual liquid; transfer the centrifugal adsorption column to an RNase-free centrifuge tube, add 50 μL RNA eluent, and place at room temperature for 5 min; centrifuge at 12,000×g at room temperature for 1 min, and the The breakthrough solution was stored in a -80°C freezer.

Tomato RNA was reverse-transcribed into cDNA using the PrimeScript ^TM RT reagent Kit with gDNA Eraser (Perfect Real Time) kit from Takara. Remove the frozen tomato RNA at -80°C, after thawing, add DNase according to the instructions, digest the DNA at 42°C for 2 minutes, then add reverse transcription reagent, 37°C for 15 minutes, 85°C for 5 sec; reverse transcribed cDNA was obtained.

2) Cloning of △ ⁸ -sphingomyelin dehydrogenase gene (SlSLD)

The tomato △ ⁸ -sphingomyelin dehydrogenase gene (SlSLD) was searched in the tomato genome database by bioinformatics analysis method, and a candidate sequence was obtained. According to the sequence information, primer sequences for amplifying the full-length sequence of the SlSLD gene were designed, the upstream primer SlSLDF: 5'-GGATCCCAGGGTAAGGTGTATGATGT-3' and the downstream primer SlSLDR: 5'-TCTAGAGCAATTCCCAGTAAGGAC-3'.

Refer to the instruction manual for the reaction system, use 10 μL of Takara’s PrimeSTAR Max Premix (2×) for PCR amplification, 0.5 μL each of 10 μM upstream primer SlSLDF and 10 μM downstream primer S1SLDR, 0.1 μL of reverse-transcribed cDNA template, and add double distilled water to make up 20 μL. The PCR amplification program was 98°C for 30 sec, 1 cycle; 98°C for 15 sec, 60°C for 15 sec, 72°C for 30 sec, 35 cycles; 72°C for 10 min, 1 cycle. The full-length sequence of the △ ⁸ -sphingomyelin dehydrogenase gene (SlSLD1) was thus amplified. After the full-length sequence was digested with restriction endonucleases BamH I and Xho I (37°C digestion reaction for 2 h), it was connected to The recombinant plasmid pTRV2-SlSLD was obtained from the expression vector pTRV2 vector (digested with restriction enzymes BamH I and Xho I for 2 h at 37°C) and sent to the company for sequencing. The nucleic acid sequence of the Δ ⁸ -sphingomyelin dehydrogenase gene (SlSLD) is shown in SEQ ID NO.1, and the amino acid sequence of the encoded Δ ⁸ -sphingomyelin dehydrogenase gene is shown in SEQ ID NO.2. After confirming that the sequence is correct, transfer the recombinant plasmid pTRV2-SlSLD into Escherichia coli DH5α by heat shock method (heat shock at 42°C for 90 sec), and culture it on solid LB containing 30 mg/L Kanamycin (Kanamycin, Kan) at 37°C Cultured on medium for 1 day, picked positive single clones in 1 mL of LB liquid medium containing 30 mg/L Kanamycin (Kanamycin, Kan), and cultured for 1 day at 37°C at 200 rpm until OD The ₆₀₀ value is about 1.0, stored in 15% glycerol, and stored in a -80°C refrigerator for later use.

3) VIGS of Agrobacterium-mediated Δ ⁸ -sphingomyelin dehydrogenase gene (SlSLD)

Pick the strains stored in 2), place them in 5 mL of LB liquid medium (containing 30 mg/L Kanamycin (Kanamycin, Kan)), and culture them at 37°C at 200 rpm for 1 day, When the OD ₆₀₀ value was about 1.2, the recombinant plasmid pTRV2-SlSLD was extracted with a small extraction plasmid kit. Take 1 μL of pTRV1 plasmid and pTRV2-SlSLD plasmid, and add them to 30 μL of GV3101 competent cells that have been thawed on ice. Transfer the competent cells to a 2mm type motor cup, and transform 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 electric shock, add non-resistant LB liquid medium, mix it upside down, transfer it to a 1.5mL centrifuge tube, and culture it at 200 rpm for 2 hours at 28°C, then add the bacteria in 30mg/L kanamycin (Kanamycin, Kan), 50mg/L rifampicin (Rifampicin, Rif), and 50mg/L gentamicin (Gentamicin, Gen) were cultured on LB solid medium for 2-3 days.

Pick positive single clones in 1 mL of LB liquid medium containing 30 mg/L Kanamycin (Kanamycin, Kan), 50 mg/L Rifampicin (Rifampicin, Rif), 50 mg/L Gentamicin (Gentamicin, Gen) , cultured at 200 rpm for 1 day at 28°C until the OD ₆₀₀ value was about 1.0. Centrifuge at 4000×g for 15 min, 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, so that the OD ₆₀₀ value of the pTRV1/GV3101 bacterial solution is 0.4, and the OD ₆₀₀ value of the pTRV2-SlSLD/GV3101 bacterial solution is 0.4. Then mix the two bacterial solutions at a ratio of 1:1, and let stand at room temperature for 3-6 hours. The tomato seedlings that had just grown the first true leaf were infected by vacuum injection, and the infection was carried out simultaneously with pTRV2/GV3101 as a control. The infected seedlings were placed at 21° C. under the condition of 16:8 (day:night) and cultured for 30 days. The degree of gene suppression was identified by the method of fluorescent quantitative PCR analysis, and the specific results are shown in Table 1.

Expression of SlSLD gene in tomato plants after table 1 VIGS

plant Expression of SlSLD gene^* plant 1 0.0064 plant 2 0.0036 plant 3 0.0048 plant 4 0.0032 plant 5 0.0031

Note: *The expression level of the gene in the control group was taken as 1.

Embodiment 2: the low temperature stress test of the plants (treatment group) and control group of SlSLD gene silencing

The plants of the SlSLD gene silenced (treatment group) and the control group were subjected to stress treatment at 4°C, and the phenotypic changes of the plants in the low temperature environment were recorded by taking pictures. As can be seen from Figure 1, when the plants of the SlSLD gene silencer and the control group were treated at low temperature for 6 hours, curling appeared at the edge of the leaves, and the change in the treatment group was more obvious; at 24 hours, the control group still maintained the state at 6 hours , the leaves were slightly curled, while the whole plants of the treatment group were wilting and could not grow normally.

Embodiment 3: Determination of physiological indexes of plants after low temperature stress test

The physiological indexes of tomato leaves treated with low temperature for 6 hours mainly included electrical conductivity, malondialdehyde content, soluble polysaccharide content, superoxide dismutase activity, peroxidase activity and chlorophyll content. The specific operation is as follows:

Preparation of supernatant: Weigh 0.1 g of tomato leaves, add 2.5 mL of pre-cooled 50 mM phosphate buffered saline (pH 7.0), grind on ice, centrifuge the homogenate at 4°C, 4,000×g for 10 min, collect The supernatant was placed in a 4°C refrigerator for the determination of the following items 2-5.

1) Determination of conductivity (Relative electrolytic leakage, REL)

Take 0.05 g of low-temperature-treated tomato leaves of the control group and the treatment group, and wash the electrolyte 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 (JingkeDDS-307A), and the obtained value was R1. Treat the centrifuge tube containing the tomato leaves in boiling water for 30 minutes, then wait for it to drop to room temperature, and measure it again with a conductivity meter, and the obtained value is R2. Calculation formula: conductivity (REL) = R/R2 × 100%.

2) Determination of malondialdehyde (Malondialdehyde, MDA) content

The content of MDA in the leaves was determined by the 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 min. After cooling, measure the absorbance at wavelengths of 532nm, 600nm and 450nm. Calculation formula: MDA concentration (μmol/L)=6.45×(OD ₅₃₂ −OD ₆₀₀ )−0.56×OD ₄₅₀ .

3) Determination of soluble polysaccharide content

The content of soluble polysaccharides in leaves was determined by anthrone colorimetry. Take 100 μL of the supernatant and place it in a 1.5 mL centrifuge tube, add 400 μL of sulfuric acid solution containing 0.2% anthrone. The mixture was placed in a boiling water bath for 5min. After cooling, measure the absorbance at a wavelength of 630nm, and draw a standard curve with glucose.

4) Determination of superoxide dismutase (Superoxide dismutase, SOD) activity

Biyuntian WST-8 kit was used to measure the activity of SOD, and the specific operation steps refer to the instructions provided by the kit.

5) Determination of peroxidase (POD) activity

The activity of POD was determined by guaiacol colorimetric method. Take 10 μL of supernatant, add 190 μL of a mixture containing 0.12% H ₂ O ₂ and 0.56% guaiacol, measure the absorbance at a wavelength of 470 nm, and read once every 1 min until the reaction is terminated, and the change in absorbance per minute 1 is 1 enzyme activity unit (U). The unit of POD activity is U/mg protein.

6) Determination of chlorophyll content

Weigh 0.02 g of tomato leaves, soak them in 2 mL of 80% acetone overnight in the dark, centrifuge at 8,000×g for 10 min, and collect the supernatant. Absorbance was measured at wavelengths of 647nm and 665nm. Calculation formula: chlorophyll concentration (mg/g)=(17.90×OD ₆₄₇ +8.08×OD ₆₆₅ )×volume of soaking solution (ml)/1000/fresh weight (g).

It can be seen from Table 2 that compared with the plants of the control group, the cold resistance ability of the plants silenced by the SlSLD gene was significantly weakened. The electrical conductivity of the treatment group was higher than that of the control group, indicating that the suppression of the expression of this gene would lead to aggravation of the stress of tomato plants 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 was lower than that of the control group in the SlSLD gene silenced plants. Therefore, cold stress experiments showed that the silencing of SlSLD gene would weaken the ability of tomato to resist cold stress.

Table 2 Physiological indicators of tomato leaves after low temperature stress

Physiological indicators control group treatment group Conductivity (%) 45.64±5.55^* 71.47±4.09 MDA concentration (μmol/g fresh weight) 6.23±0.31^* 7.41±0.33 Soluble polysaccharide content (mg/0.1g fresh weight) 3.24±0.28^* 2.28±0.26 SOD activity (U/mg protein) 0.30±0.06^* 0.20±0.06 Peroxidase activity (U/mg protein) 2.51±0.41^* 1.12±0.28 Chlorophyll (mg/g fresh weight) 3.18±0.20^* 2.24±0.31

Note: * indicates that there is a significant difference between the control group and the treatment group, p≤0.05.

SEQUENCE LISTING

<110> South China Botanical Garden, Chinese Academy of Sciences

<120> A Tomato Chilling Damage Resistance Gene and Its Application

<130>

<160> 2

<170> PatentIn version 3.5

<210> 1

<211> 1344

<212> DNA

<213> Tomato (Solanum lycopersicum cv. Micro-Tom)

<400> 1

atggcagatt caagaaagta catttctagt gaggaactga agaatcacaa caaaccaggg 60

gatctgtgga tatccattca gggtaaggtg tatgatgtat cagattgggt gaaggaacat 120

cctggtggtg atttcccact gttaaatctt gctggacagg atgttactga tgcatttgtt 180

gcatttcatc ctgctactgc ttggaagtat cttgacaagt tctttaaggg gttttacctc 240

aaggattatt ctgtttctga ggtatctacg gattatagaa ggcttgtgtc tgagttcact 300

aaaatggggt tgtttgaaaa gaaaggccat gtttgtttat tcaccatgtt cttaatgaca 360

atgttgtttt ccttaagtgt ttatggaatc ttgtattgtc atggtgtgtt ggcacatttg 420

ataagtggtg ccttgatggg gtgtctttgg attcagagtg ggtggattgg tcatgattca 480

gggcattatc aggtgatgag cactcgcgga ttcaacagat ttgctcaagt ccttactggg 540

aattgccttg ctggaatcag cattgcttgg tggaagtgga accacaatgc tcaccacatt 600

gcctgcaaca gtcttgaata tgaccctgat cttcaacaca tgccattctt tgtggtatct 660

tccaagtttt tcgactcact cacttcttat ttttacgata ggaagatgaa ttttgattct 720

tttactagat tcttggttag tcatcaacat tggacatttt atcctgttat gtgttttgct 780

agaatcaatt tgtttgctca gtcattcata ttgttgttat ccaacaaaaa tgtgccccat 840

cgagttcagg agcttttggg ggtggtttct ttctggattt ggtatccgtt gcttgtttct 900

ttcttgccaa actggggcga aagaattata tttgttcttg ctagtttcac agtgactgga 960

attcagcatg ttcaattctg tttaaaccat ttctcatctg aaatttatgt tgcaccacct 1020

aaaggaaatg attggtttga gaagcaaact aatggctctc tcgacatatc atgccctagt 1080

tggatggatt ggtttcatgg tggattgcag tttcagattg agcatcattt gtttcctaga 1140

ttaccaagat gccaactgag gaaagtctct ccctttgtga aagacctctg taaaaagcat 1200

ggtttgcctt acagttgtgt ctccttctgg aagtctaatg ttttgaccat cagcaccctc 1260

agagctgcag ctttgcaggc tcgggatta actaagcctg ttccaaaaaa tctagtttgg 1320

gaagccgtca acactcacgg ttga 1344

<210> 2

<211> 447

<212> PRT

<213> Tomato (Solanum lycopersicum cv. Micro-Tom)

<400> 2

Met Ala Asp Ser Arg Lys Tyr Ile Ser Ser Glu Glu Leu Lys Asn His

1 5 10 15

Asn Lys Pro Gly Asp Leu Trp Ile Ser Ile Gln Gly Lys Val Tyr Asp

20 25 30

Val Ser Asp Trp Val Lys Glu His Pro Gly Gly Asp Phe Pro Leu Leu

35 40 45

Asn Leu Ala Gly Gln Asp Val Thr Asp Ala Phe Val Ala Phe His Pro

50 55 60

Ala Thr Ala Trp Lys Tyr Leu Asp Lys Phe Phe Lys Gly Phe Tyr Leu

65 70 75 80

Lys Asp Tyr Ser Val Ser Glu Val Ser Thr Asp Tyr Arg Arg Leu Val

85 90 95

Ser Glu Phe Thr Lys Met Gly Leu Phe Glu Lys Lys Gly His Val Cys

100 105 110

Leu Phe Thr Met Phe Leu Met Thr Met Leu Phe Ser Leu Ser Val Tyr

115 120 125

Gly Ile Leu Tyr Cys His Gly Val Leu Ala His Leu Ile Ser Gly Ala

130 135 140

Leu Met Gly Cys Leu Trp Ile Gln Ser Gly Trp Ile Gly His Asp Ser

145 150 155 160

Gly His Tyr Gln Val Met Ser Thr Arg Gly Phe Asn Arg Phe Ala Gln

165 170 175

Val Leu Thr Gly Asn Cys Leu Ala Gly Ile Ser Ile Ala Trp Trp Lys

180 185 190

Trp Asn His Asn Ala His His Ile Ala Cys Asn Ser Leu Glu Tyr Asp

195 200 205

Pro Asp Leu Gln His Met Pro Phe Phe Val Val Ser Ser Lys Phe Phe

210 215 220

Asp Ser Leu Thr Ser Tyr Phe Tyr Asp Arg Lys Met Asn Phe Asp Ser

225 230 235 240

Phe Thr Arg Phe Leu Val Ser His Gln His Trp Thr Phe Tyr Pro Val

245 250 255

Met Cys Phe Ala Arg Ile Asn Leu Phe Ala Gln Ser Phe Ile Leu Leu

260 265 270

Leu Ser Asn Lys Asn Val Pro His Arg Val Gln Glu Leu Leu Gly Val

275 280 285

Val Ser Phe Trp Ile Trp Tyr Pro Leu Leu Val Ser Phe Leu Pro Asn

290 295 300

Trp Gly Glu Arg Ile Ile Phe Val Leu Ala Ser Phe Thr Val Thr Gly

305 310 315 320

Ile Gln His Val Gln Phe Cys Leu Asn His Phe Ser Ser Glu Ile Tyr

325 330 335

Val Ala Pro Pro Lys Gly Asn Asp Trp Phe Glu Lys Gln Thr Asn Gly

340 345 350

Ser Leu Asp Ile Ser Cys Pro Ser Trp Met Asp Trp Phe His Gly Gly

355 360 365

Leu Gln Phe Gln Ile Glu His His Leu Phe Pro Arg Leu Pro Arg Cys

370 375 380

Gln Leu Arg Lys Val Ser Pro Phe Val Lys Asp Leu Cys Lys Lys Lys His

385 390 395 400

Gly Leu Pro Tyr Ser Cys Val Ser Phe Trp Lys Ser Asn Val Leu Thr

405 410 415

Ile Ser Thr Leu Arg Ala Ala Ala Leu Gln Ala Arg Asp Leu Thr Lys

420 425 430

Pro Val Pro Lys Asn Leu Val Trp Glu Ala Val Asn Thr His Gly

435 440 445

Claims (1)

1. tomato △⁸Application of the sphingomyelins dehydrogenase gene in cold-resistant tomato plant breeding, the tomato △⁸Sphingomyelins is de- The nucleotide sequence of hydrogenase gene is as shown in SEQ ID NO.1.