CN106754769B

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

Info

Publication number: CN106754769B
Application number: CN201611085754.7A
Authority: CN
Inventors: 周瀛; 杨子银; 曾兰亭; 蒋跃明; 段学武
Original assignee: South China Botanical Garden of CAS
Current assignee: South China Botanical Garden of CAS
Priority date: 2016-12-01
Filing date: 2016-12-01
Publication date: 2019-08-23
Anticipated expiration: 2036-12-01
Also published as: CN106754769A

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 Inappropriate ADH syndrome gene and application

Technical field

The invention belongs to plant biotechnology fields, specifically, being related to a kind of tomato Inappropriate ADH syndrome gene and application.

Background technique

Neurolemma lipid (Sphingolipids) is that a kind of complicated lipid containing fatty acid and long-chain sphingol skeleton is total Claim.It is urged on the net by serine palmityl coenzyme A transferase (Serine palmitoyltransferase, SPT) in endoplasm Change serine and palmitoyl coenzyme A and de novo formation.The lipoids is plant plasma membrane serves, the weight of tonoplast and endomembrane system Component part is wanted, 40% of lipid on film is accounted for about, is primarily present in the siphonal lobe of cell membrane.The siphonal lobe of cell membrane will affect the complete of film The permeability of whole property and ion, and the physiological status of cell membrane under cryogenic is the important indicator that Genes For Plant Tolerance damages to plants caused by sudden drop in temperature ability. In addition, neurolemma lipid, which can be reduced, generates a kind of 18 carbon amino alcohol containing unsaturated alkyl chain, i.e. sphingol (Sphinganine).It is a kind of long-chain base (Long chain base, LCB), can be by sphingomyelins dehydrogenase (Sphingolipid desaturase, SLD) catalysis generates unsaturated sphingomyelins.Largely studies have shown that unsaturated sheath phosphorus Rouge largely exists in hardy type plant, because the number of the content of material affects the height of plant cold-resistant ability.In quasi- south In mustard, the freezing tolerance of plant is can be enhanced in the rising of glucosylceramide (d18:1c24:0) content.Also it tests into one Step shows that the Arabidopsis plant of AtSLD gene mutation will appear chlorosis at low ambient temperatures, and plant is unable to normal growth.Separately Outside, the study found that heteroside ceramide has a quite high content on cell membrane and tonoplast, and the hydroxylated grape of height Glycosides ceramide is extremely important to the integrality for maintaining cell membrane and tonoplast.△⁸Sphingomyelins dehydrogenase is catalysis sphingolipid long-chain C8 dehydrogenations of base generate △⁸The key enzyme of unsaturated sphingomyelins.The sheath of acyl containing heteroside in soybean leaves can be promoted by damaging to plants caused by sudden drop in temperature processing The △ of ammonia alcohol⁸Unsaturated sphingomyelin content increases, it is therefore contemplated that △⁸Unsaturated sphingomyelins and △⁸Sphingomyelins dehydrogenase It is closely related with the winter resistance of soybean.It can be found that neurolemma lipid and its unsaturated sphingomyelins dehydrogenation from the research of forefathers Enzyme may affect the Inappropriate ADH syndrome ability of plant.However, it is still less about the dehydrogenase and the research of cold-resistant relationship, especially exist In terms of industrial crops, the connection of the two need further to illustrate.

Tomato (Lycopersicon esculentum Mill) is Solanaceae tomato platymiscium, is a kind of important economic work Object, it is closely bound up with national " vegetable basket project ".It is cold responsive type plant, chilling injury can be caused to the production of tomato compared with Big loss is to influence one of tomato whole year production amount and the principal element of market supply.Most tomato varieties are lower than 10 in temperature DEG C when growth and development be easy to be obstructed, when temperature be lower than 6 DEG C when show significantly to damage to plants caused by sudden drop in temperature symptom, so about tomato to low temperature ring The adaptability in border receives the extensive concern of scholars.In order to improve tomato variety, tomato is improved to the resistance of low temperature, is reduced The economic loss that tomato is damaged to plants caused by sudden drop in temperature, researchers are in membrane structure, inoxidizability enzyme, cell wall substance metabolism etc. at present Various aspects explore tomato Inappropriate ADH syndrome mechanism.But also there is not researcher to explore △⁸Sphingomyelins dehydrogenase kind Effect in eggplant low temperature adaptability.Therefore, the present invention not only from neurolemma lipid-metabolism, go to excavate tomato by this completely new angle The mechanism of Inappropriate ADH syndrome, while certain theoretical foundation also is provided to cultivate low temperature resistant tomato variety, it is fitted to tomato planting is improved Ying Xing, expansion planted area, increase yield etc. have great importance.

Summary of the invention

The purpose of the present invention is to provide a kind of tomato Inappropriate ADH syndrome gene SlSLD, the silencing of the gene can lead to tomato Low temperature tolerance ability reduces.The gene can be applied to the freeze proof breeding of tomato, can effectively improve the frost resistance of tomato variety.

It is another object of the present invention to provide a kind of tomato Inappropriate ADH syndrome genes to resist cold the application in breeding in tomato.It is logical SlSLD gene expression amount is crossed in raising plant to carry out plant frigostabile breeding, the suitable planting range of plant is made with this Expand.

A kind of tomato △⁸Sphingomyelins dehydrogenase gene, nucleotide sequence is as shown in SEQ ID NO.1.

Encode above-mentioned tomato △⁸The tomato △ of sphingomyelins dehydrogenase gene⁸Sphingomyelins dehydrogenase, amino acid sequence is such as Shown in SEQ ID NO.2.

By the expression for adjusting SlSLD gene described in the invention, thus it is possible to vary plant is to cold sensibility.

Compared with prior art, the invention has the following beneficial effects: the present invention to pass through virus induced gene silencing A kind of new tomato Inappropriate ADH syndrome related gene SlSLD of (Virus induced gene silencing, VIGS) scientific discovery, The silencing of the gene can cause the low temperature tolerance ability of tomato plant to be substantially reduced.To the amino acid sequence analysis of gene coding The albumen for proving the coded by said gene is a kind of △⁸Sphingomyelins dehydrogenase, related to neurolemma lipid-metabolism, the enzyme is in plant It resists and plays an important role in the reaction of low temperature stress.This research provides the nucleic acid sequence and its amino acid sequence of this gene Column also relate 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, The adaptability for improving tomato excellent variety, expands its planting range, promotes its stable yields and high yield.

Detailed description of the invention

Fig. 1 is that low temperature stress handles the phenotypic map after tomato plant.

Specific embodiment

The following examples are further illustrations of the invention, rather than limiting the invention.

Embodiment 1: the VIGS processing of tomato

The formula of LB solid medium used below are as follows: 10g/L tryptone (Tryptone), 5g/L yeast extract (Yeast extract), 10g/L sodium chloride (NaCl), 15~20g/L agar powder are remaining with 10M NaOH adjustment pH value to 7.4 Amount is water；121 DEG C, sterilize 20min, and when temperature is down to 55 DEG C or so (non-scald on hand), final concentration of 30mg/L kanamycins is added (Kanamycin, Kan), or final concentration of 30mg/L kanamycins (Kanamycin, Kan), 50mg/L rifampin is added (Rifampicin, Rif), 50mg/L gentamicin (Gentamicin, Gen), inverted plate save backup；LB liquid medium Formula are as follows: 10g/L tryptone (Tryptone), 5g/L yeast extract (Yeast extract), 10g/L sodium chloride (NaCl), with 10M NaOH adjustment pH value to 7.4, surplus is water；121 DEG C, sterilize 20min, and temperature is down to 55 DEG C or so and (is not scalded Hand) or it is cooling after, final concentration of 30mg/L kanamycins (Kanamycin, Kan) is added, or final concentration of 30mg/L is added Kanamycins (Kanamycin, Kan), 50mg/L rifampin (Rifampicin, Rif), 50mg/L gentamicin (Gentamicin,Gen)。

The specific steps of embodiment are illustrated below:

1) extraction of tomato RNA and the acquisition of cDNA

Tomato RNA extracting method refers to the ultrafast type plant RNA extraction kit operation instruction of Hua Yue ocean biotech firm.Specifically Steps are as follows: it takes 0.1g to be placed in 1.5mL centrifuge tube with the tomato leaf that liquid nitrogen is ground, 1mL cell pyrolysis liquid is added, it is sufficiently mixed It is even；300 μ L protein liquid removals and 200 μ L chloroforms are added, covers pipe lid, shakes vigorously and mix well 30s；Room temperature 12,000 × g, centrifugation Supernatant (not more than 700 μ L) is transferred in 1.5mL RNase-free centrifuge tube by 10min；Isometric rinsing liquid is added, sufficiently It is mixed by inversion, mixture (not more than 700 μ L) is transferred in centrifugal adsorbing column, room temperature 12,000 × g, be centrifuged 1min, abandoning is worn Transparent liquid；500 μ L are added and wash column liquid, room temperature 12,000 × g is centrifuged 1min, and abandoning penetrates liquid, is repeated once.Room temperature 12,000 × g, It is centrifuged 1min, to remove residual liquid；Centrifugal adsorbing column is transferred in RNase-free centrifuge tube, 50 μ L RNA are added Eluent is placed at room temperature for 5min；Room temperature 12,000 × g are centrifuged 1min, the liquid that penetrates containing tomato RNA are stored in -80 DEG C of ice Case.

By tomato RNA reverse transcription at cDNA, using the PrimeScript of Takara company^TM RT reagent Kit With gDNA Eraser (Perfect Real Time) kit.It takes out -80 DEG C and freezes tomato RNA, after melting, according to After DNA enzymatic is added in specification, 42 DEG C of digestion DNA 2min add Reverse Transcription, 37 DEG C of 15min, 85 DEG C of 5sec；It obtains inverse The cDNA of transcription.

2)△⁸The clone of sphingomyelins dehydrogenase gene (SlSLD)

Using bioinformatic analysis method, tomato △ is searched in tomato dna group database⁸Sphingomyelins dehydrogenase base Because of (SlSLD), 1 candidate sequence is obtained.According to the sequence information, designed for expanding the primer of SlSLD full length gene sequence Sequence, upstream primer SlSLDF:5 '-GGATCCCAGGGTAAGGTGTATGATGT-3 ' and downstream primer SlSLDR:5 '- TCTAGAGCAATTCCCAGTAAGGAC-3’。

Reaction system is referring to specification, PrimeSTAR Max Premix (2 ×) 10 the μ L, 10 μ of PCR amplification Takara M upstream primer SlSLDF and each 0.5 μ L of 10 μM of downstream primer SlSLDR, the 0.1 μ L of cDNA template of reverse transcription, adds distilled water to supply 20μL.PCR amplification program is 98 DEG C of 30sec, 1 circulation；98 DEG C of 15sec, 60 DEG C of 15sec, 72 DEG C of 30sec, 35 circulations；72 DEG C 10min, 1 circulation.Thus amplification obtains △⁸The full length sequence of sphingomyelins dehydrogenase gene (SlSLD1), full length sequence warp After restriction enzyme BamH I and Xho I digestion (37 DEG C of endonuclease reaction 2h), it is connected to (37 DEG C of expression vector pTRV2 carrier Restriction enzyme BamH I and Xho I digestion 2h) in, recombinant plasmid pTRV2-SlSLD is obtained, company is sent to be sequenced.△⁸Sheath The nucleic acid sequence of phosphatide dehydrogenase gene (SlSLD) is as shown in SEQ ID NO.1, the △ of coding⁸Sphingomyelins dehydrogenase gene Amino acid sequence as shown in SEQ ID NO.2.It is using heat shock method (42 DEG C of heat shock 90sec) that this is heavy after confirming that sequence is correct Group plasmid pTRV2-SlSLD is transferred in bacillus coli DH 5 alpha, in 37 DEG C in kanamycins containing 30mg/L (Kanamycin, Kan) LB It is cultivated 1 day on solid medium, picking positive monoclonal is in kanamycins containing 30mg/L (Kanamycin, Kan) LB liquid of 1mL In body culture medium, under the conditions of 37 DEG C, cultivated 1 day with 200 revs/min, until OD₆₀₀Value be 1.0 or so, be stored in 15% it is sweet It is spare to be put in -80 DEG C of refrigerators for oil.

3) △ of mediated by agriculture bacillus⁸The VIGS of sphingomyelins dehydrogenase gene (SlSLD)

Picking 2) in the strain deposited, be placed in LB liquid medium containing 5mL (kanamycins containing 30mg/L (Kanamycin, Kan it in)), under the conditions of 37 DEG C, is cultivated 1 day with 200 revs/min, until OD₆₀₀Value is 1.2 or so, then with small upgrading grain reagent Box extracts the plasmid pTRV2-SlSLD of recombination.1 μ L pTRV1 plasmid and pTRV2-SlSLD plasmid are taken, is separately added into set and exists The 30 μ L GV3101 competent cells to thaw on ice.Competent cell is transferred to the motor cup of 2mm type, passes through motor mode Converted, design parameter such as: selection Pre-Set Protocols under Bacterial under A.tumefaciens, voltage 2400V, capacitor 25 μ F, 200 Ω of resistance, electric shock cup Cuvette are 2mm.After electric shock, the LB liquid medium of non-resistant is added, It is mixed by inversion, is transferred to 1.5mL centrifuge tube, under the conditions of 28 DEG C, with 200 revs/min of culture 2h, then by the bacterium in 30mg/L card That mycin (Kanamycin, Kan), 50mg/L rifampin (Rifampicin, Rif), 50mg/L gentamicin (Gentamicin, Gen it is cultivated 2-3 days on LB solid medium).

Picking positive monoclonal is in the kanamycins containing 30mg/L (Kanamycin, Kan) of 1mL, 50mg/L rifampin (Rifampicin, Rif), in 50mg/L gentamicin (Gentamicin, Gen) LB liquid medium, under the conditions of 28 DEG C, with 200 revs/min are cultivated 1 day, until OD₆₀₀Value is 1.0 or so.At 4000 × g, it is centrifuged 15min, collects precipitating, addition contains 30mg/L kanamycins (Kanamycin, Kan), 50mg/L rifampin (Rifampicin, Rif), 50mg/L gentamicin (Gentamicin, Gen) LB liquid medium, so that the OD of pTRV1/GV3101 bacterium solution₆₀₀Value is 0.4, pTRV2-SlSLD/ The OD of GV3101 bacterium solution₆₀₀Value is 0.4.The two bacterium solution is mixed with the ratio of 1:1 again, and in being stored at room temperature 3~6h.By true The method of sky injection infects the tomato seedling for just growing rough leaf, is control with pTRV2/GV3101, while being infected. Seedling after infecting is placed in 21 DEG C, under the conditions of 16:8 (daytime: night), cultivates 30d.With quantitative fluorescent PCR analyze method come Identify the repressed degree of gene, concrete outcome is as shown in table 1.

After table 1VIGS in tomato plant SlSLD gene expression quantity

Plant	The expression quantity of SlSLD gene^*
		Plant 1	0.0064
Plant 2	0.0036
		Plant 3	0.0048
Plant 4	0.0032
		Plant 5	0.0031

Note: * is with the expression quantity of control group gene for 1.

The plant (processing group) of embodiment 2:SlSLD gene silencing and the low temperature stress of control group are tested

By the plant of (the processing group) of SlSLD gene silencing and control group under the conditions of 4 DEG C Stress treatment, and photograph to record Character mutation of the plant in low temperature environment.It will be seen from figure 1 that SlSLD gene silencing and control group plant is in low temperature When handling 6h, all there is curling in blade edge, and wherein the variation of processing group becomes apparent；When for 24 hours, control group still keeps 6h When state, there is slight curling in blade, and the plant of processing group is integrally all wilted, and normal growth has been unable to.

Embodiment 3: the physiological index determining of plant after low temperature stress test

The physical signs through low-temperature treatment 6h tomato leaf is measured, it is mainly soluble more including conductivity, mda content Sugared content, superoxide dismutase activity, peroxidase activity and chlorophyll content.Concrete operations are as follows:

Supernatant preparation is prepared, 0.1g tomato leaf is weighed, the 50mM phosphate buffered saline solution (pH of 2.5mL pre-cooling is added 7.0) it, is ground on ice, homogenate is centrifuged 10min under the conditions of 4 DEG C, 4,000 × g, collects supernatant, is placed in 4 DEG C of refrigerators, uses In the measurement of following 2-5 indexs.

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

The blade for taking 0.05g control group and processing group tomato after low-temperature treatment, with the electricity on distilled water cleaning blade surface Xie Zhi.Blade is placed in the centrifuge tube of the distilled water containing 10mL, in soaking at room temperature 22h, then uses conductivity meter (Jingke DDS-307A it) measures, resulting value R1.Centrifuge tube containing tomato leaf is handled into 30min in boiling water, then equal its drops to room Wen Hou is redeterminated with conductivity meter, resulting value R2.Calculation formula: conductivity (REL)=R/R2 × 100%.

2) measurement of malonaldehyde (Malondialdehyde, MDA) content

Using the content of thio barbital method measurement MDA in leaves.It takes the supernatant of 200 μ L to be placed in 1.5mL centrifuge tube, adds Enter 0.6% isometric thiobarbituricacidα- solution, mixture is subjected to boiling water bath 30min.After cooling, Yu Bochang 532nm, Light absorption value is surveyed under 600nm and 450nm.Calculation formula: MDA concentration (μm ol/L)=6.45 × (OD₅₃₂-OD₆₀₀)-0.56× OD₄₅₀。

3) measurement of soluble polysaccharide content

Using the content of soluble polysaccharide in anthrone colorimetry measurement blade.The supernatant of 100 μ L is taken to be placed in 1.5mL centrifugation The sulfuric acid solution that 400 μ L contain 0.2% anthrone is added in pipe.Mixture is subjected to boiling water bath 5min.After cooling, under Yu Bochang 630nm Light absorption value is surveyed, standard curve is drawn with glucose.

4) superoxide dismutase (Superoxide dismutase, SOD) active measurement

Using the activity of green skies WST-8 kit measurement SOD, concrete operation step explanation referring to provided by kit Book.

5) peroxidase (POD) active measurement

Using the activity of guaiacol colorimetric method measurement POD.10 μ L supernatants are taken, 190 μ L are added and contain 0.12%H₂O₂With The mixed liquor of 0.56% guaiacol surveys light absorption value under Yu Bochang 470nm, primary every 1min reading, until reaction terminating, with Light absorption value variation 1 per minute is 1 unit of enzyme activity (U).The active unit of POD is U/mg albumen.

6) measurement of chlorophyll content

Weigh 0.02g tomato leaf, with the acetone of 2mL 80% in the immersion overnight of dark place, under the conditions of 8,000 × g from Heart 10min collects supernatant.Light absorption value is surveyed under wavelength 647nm and 665nm.Calculation formula: chlorophyll concentration (mg/g)= (17.90×OD₆₄₇+8.08×OD₆₆₅) × soak volume (ml)/1000/ fresh weight (g).

From table 2 it can be seen that the plant chilling resistance of SlSLD gene silencing obviously weakens compared to the plant of control group. The conductivity of processing group is higher than control group, illustrates that the inhibition of the gene expression amount will lead to tomato plant and be coerced under cryogenic Urgent degree aggravates.The content of malonaldehyde is also that processing group is higher than control group, and superoxide dismutase and peroxidase Really control group is higher for activity.In addition, the chlorophyll content of tomato leaf is the plant of SlSLD gene silencing lower than control group. Therefore, show that the silencing of SlSLD gene can weaken the Inappropriate ADH syndrome ability of tomato by cold stress experiment.

The physical signs of tomato leaf after 2 low temperature stress of table

Physical signs	Control group	Processing group
			Conductivity (%)	45.64±5.55^*	71.47±4.09
Concentration of malondialdehyde (μm ol/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
Superoxide dismutase activity (U/mg albumen)	0.30±0.06^*	0.20±0.06
			Peroxidase activity (U/mg albumen)	2.51±0.41^*	1.12±0.28
Chlorophyll (mg/g fresh weight)	3.18±0.20^*	2.24±0.31

Note: * indicates that control group and processing group have the difference of conspicuousness, p≤0.05.

SEQUENCE LISTING

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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

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

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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

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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

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

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

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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

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

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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 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

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

435 440 445

Claims

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.