CN103667265B - The application of tomato LoxD gene in regulating plant resistance - Google Patents
The application of tomato LoxD gene in regulating plant resistance Download PDFInfo
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Abstract
The invention discloses the application of tomato LoxD gene in regulating plant resistance.The invention provides a kind of DNA fragmentation, its nucleotides sequence to be classified as in sequence table sequence 2 from 5 ' end 106-570 position Nucleotide.Present invention also offers LoxD albumen and the application of encoding gene in regulating plant insect-resistance and/or disease resistance thereof, wherein the aminoacid sequence of LoxD albumen is the sequence 1 in sequence table; The nucleotides sequence of described LoxD protein coding gene is classified as the sequence 2 in sequence table.Experiment of the present invention proves, the present invention obtains transgenic Fructus Lycopersici esculenti by suppressing the expression of LoxD gene in wild-type tomatoes, its disease resistance and insect-resistance are all lower than wild-type tomatoes, thus prove, suppress the expression of plant LoxD gene to reduce plant resistance to insect and disease resistance, illustrate that this gene of LoxD is relevant to disease resistance of plant.
Description
Technical field
The present invention relates to biological technical field, particularly relate to the application of a kind of tomato LoxD gene in regulating plant resistance.
Background technology
LOX (lipoxygenase, lipoxidase) is prevalent in plant, animal and fungi, also there is this fermentoid in discovered in recent years bacterium.Different according to the position of LOX catalyzed oxidation two ethylene linkage, can be divided into: 9-lipoxygenase and 13-lipoxygenase; Whether be positioned in chloroplast(id) according to LOX albumen, LOX can be divided into the large class of Type1 and Type2 two.In plant, LOX not only take part in its growth and development process, chews in the Resistant reaction such as food and physical abuse also play an important role plant resistant insect.All have the Lox gene of multiple copy in the Plant Genome of tomato, Arabidopis thaliana and other species, and different Lox genes has different biological functions.In the biosynthetic process of JA, the Lox silence that LOX tobacco RNAi induces inhibits JA to synthesize, thus causes transfer-gen plant to lose resistance to insect.The afunction of Arabidopis thaliana LOX-2 destroys the JA synthesis of injured induction, but does not affect flower development.Separately have bibliographical information to show, Lox3, Lox4 in Arabidopis thaliana also take part in JA synthesis, and the two prominent male flower fertility of lox3lox4 is affected, and causes male sterile.The sex-determination factor TASSELSEED1 of corn is that the afunction of the JA synthetase 1 3-Lipoxygenase be positioned in plastid, TASSELSEED1 causes corn male inflorescence can not normal development.These results illustrate, the JA synthesis of LOX regulation and control has vital role in plant resistance to environment stress reaction and fertility.At present, the homologous gene obtaining 6 Lox has been separated in tomato, wherein, LOXD participates in JA synthesis, and the ALA catalysis that discharges from plasma membrane is formed 13-HPOT (13-Hydroperoxy-octadecatrienoic acid), and 13-HPOT is through AOS, AOC, the a series of enzymatic reaction such as OPDA and ACX1, generates JA, and then induces a series of signal to forward approach to.As far back as 1997, LoxD gene was cloned out, but still needed more detailed research to the gene function of LoxD.
There are some researches show, at the transgenic Fructus Lycopersici esculenti (35S:Prosystemin of overexpression systemin precursor-gene (Prosystemin), 35S:PS), the resistance protein composing type ground high expression level of jasmonic induction, thus improves greatly to the resistance of insect.
Summary of the invention
An object of the present invention is to provide a kind of RNA molecule.
RNA molecule provided by the invention is the RNA that the sequence 2 in sequence table is transcribed from the DNA molecular shown in 5 ' end 106-570 position Nucleotide.
LoxD albumen and the application of encoding gene in regulating plant insect-resistance and/or disease resistance thereof are the scope of protection of the invention; In above-mentioned application, the aminoacid sequence of described LoxD albumen is the sequence 1 in sequence table; The nucleotides sequence of described LoxD protein coding gene is classified as the sequence 2 in sequence table.
In above-mentioned application, described regulating plant insect-resistance and/or disease resistance are following 1) or 2): 1) reduce plant resistance to insect and/or disease resistance; 2) plant resistance to insect and/or disease resistance is improved.
In above-mentioned application, described disease is graw mold of tomato, and described worm is bollworm.
In above-mentioned application, the pathogenic bacteria of described graw mold of tomato is the pathogen of Botrytis cinerea (Botrytis cinerea).
In above-mentioned application, described plant is monocotyledons or dicotyledons, and described dicotyledons is specially tomato.
Another object of the present invention is to provide a kind of method of cultivating transgenic plant.
Method provided by the invention, in inactivation or suppression object plant, LoxD protein coding gene is expressed, obtains transgenic plant; Described transgenic plant have following 1) and 2) in the characteristic of at least one:
1) insect-resistance of described transgenic plant is lower than described object plant;
2) disease resistance of described transgenic plant is lower than described object plant.
Described disease is graw mold of tomato, and the pathogenic bacteria of described graw mold of tomato is specially the pathogen of Botrytis cinerea (Botrytiscinerea); Described worm is bollworm.
In aforesaid method, described object plant is monocotyledons or dicotyledons, and described dicotyledons is specially tomato.
In aforesaid method, the aminoacid sequence of described LoxD albumen is the sequence 1 in sequence table; The nucleotide sequence of described LoxD protein coding gene is specifically the sequence 2 in sequence table.
In above-mentioned application or aforesaid method, above-mentioned insect-resistance by after worm feeding plant, insect resistance protein expression amount and/or take food rear body weight and embody in plant; Above-mentioned insect resistance protein is specially PI-II;
Above-mentioned disease resistance, by after inoculation pathogenic bacteria, is expressed with disease-resistant related gene (PI-II, PR1b1 and/or Solyc07g006380) in plant leaf bacterial plaque diameter and/or plant leaf and is embodied.
Above-mentioned inactivation or suppress LoxD protein coding gene in object plant to express by inactivation will be used for or suppresses recombinant vectors that in object plant, LoxD protein coding gene is expressed to import in object plant realize; This recombinant vectors is DNA fragmentation is inserted the carrier obtained between the multiple clone site of pCAMBIA 1301; Wherein, this DNA fragmentation is made up of forward fragment, intron and reverse fragment; Described reverse fragment nucleotides sequence is classified as the reverse complementary sequence of forward fragment; The nucleotides sequence of described forward fragment is classified as sequence 2 in sequence table from 5 ' end 106-570 position Nucleotide; In an embodiment of the present invention, recombinant vectors is pCAMBIA 1301-LoxDRNAi, for above-mentioned DAN fragment being inserted the carrier obtained between the PstI restriction enzyme site of pCAMBIA1301.
Experiment of the present invention proves, the present invention obtains transgenic Fructus Lycopersici esculenti by suppressing the expression of LoxD gene in wild-type tomatoes, its disease resistance and insect-resistance are all lower than wild-type tomatoes, thus prove, suppress the expression of plant LoxD gene to reduce plant resistance to insect and disease resistance, illustrate that LoxD gene is relevant to disease resistance of plant.
Accompanying drawing explanation
Fig. 1 is for suppressing LoxD genetic expression reduction tomato opposing insect
Fig. 2 is for suppressing LoxD genetic expression in reduction tomato opposing Botrytis virus
Embodiment
The experimental technique used in following embodiment if no special instructions, is ordinary method.
Material used in following embodiment, reagent etc., if no special instructions, all can obtain from commercial channels.
Quantitative experiment in following embodiment, all in triplicate, results averaged.
In following embodiment, LoxD protein amino acid sequence is the sequence 1 in sequence table, and the nucleotides sequence of its encoding gene LoxD is classified as the sequence 2 in sequence table.
In following embodiment, tomato adopts tomato conventional variety CM (Castlemart, hereinafter also referred to wild-type tomatoes), purchased from American Tomato Germplasms center (TGRC, http://tgrc.ucdavis.edu/).
The recombinant vectors of embodiment 1, suppression LoxD genetic expression obtains
Extract the total serum IgE of tomato (Solanum lycopersicon) kind CM, the cDNA that reverse transcription obtains is template, carries out pcr amplification with primer 1 and primer 2.
Primer 1:5 '-GCG
cTCGAGaACCAGCAGTTTCAAGGAAGGAGG-3 ' (upstream primer, underscore number of base is XhoI recognition site)
Primer 2: 5 '-GCG
aCTAGTaACCGGACCACATGCAAACCCCT-3 ' (downstream primer, underscore number of base is SpeI recognition site)
Obtain 465bpPCR product, through order-checking, it, from 5 ' end 106-570 position Nucleotide, is LoxD Gene Partial fragment that the nucleotides sequence of this PCR primer is classified as sequence 2 in sequence table.
After above-mentioned PCR primer XhoI and SpeI double digestion, (Gan D is recorded with the PUCCRNAi carrier through same double digestion, Zhang J, Jiang H, Jiang T, Zhu S, Cheng B.Bacterially expresseddsRNA protects maize against SCMV infection.Plant Cell Rep.2010.29 (11): 1261-8., the public can obtain from Chinese Academy of Sciences's heredity with developmental biology institute) be connected, transformation of E. coli DH5 α, selects positive colony.The plasmid extracting positive colony checks order, this plasmid is between XhoI and the SpeI restriction enzyme site of PUCC RNAi carrier, insert sequence 2 in sequence table from 5 ' end 106-570 position Nucleotide, show that the recombinant vectors built is correct, be denoted as PUCC-LoxD RNAi Forward.
By above-mentioned PCR primer through XhoI and SpeI double digestion, be connected with the PUCC-LoxD RNAi Forward recombinant vectors through isocaudarner SalI and XbaI double digestion, transformation of E. coli DH5 α, selects positive colony.The plasmid extracting positive colony checks order, this plasmid is that sequence 2 in SalI and XbaI of PUCC-LoxD RNAi Forward carrier in insertion sequence table is from 5 ' end 106-570 position Nucleotide, show that the recombinant vectors built is correct, be denoted as PUCC-LoxD RNAi.
After the PUCC-LoxD RNAi carrier PstI enzyme built is cut, reclaim the fragment of 1175bp, (Cheng H is recorded with the pCAMBIA1301 carrier cut through same enzyme through this fragment, Song S, Xiao L, Soo HM, ChengZ, Xie D, Peng J.Gibberellin Acts through Jasmonate to Control the Expressionof MYB21, MYB24, and MYB57 to Promote Stamen Filament Growth in Arabidopsis.PLoS Genet.2009.5 (3): e1000440., the public can obtain from Chinese Academy of Sciences's heredity with developmental biology institute) be connected, transform DH5 α, picking mono-clonal carries out enlarged culturing, extract plasmid, utilize PstI digestion verification positive plasmid (1175bp's is the positive), called after pCAMBIA 1301-LoxD RNAi, for suppressing the recombinant vectors of LoxD genetic expression.
The acquisition of the transgenic Fructus Lycopersici esculenti of embodiment 2, LoxD RNAi and Function Identification
One, the transgenic Fructus Lycopersici esculenti of LoxD RNAi
1, the recombinant bacterium of LoxD genetic expression is suppressed to obtain
The recombinant vectors pCAMBIA 1301-LoxD RNAi transformation Agrobacterium AGLO(of the suppression LoxD genetic expression obtained by embodiment 1 is recorded Li C, Liu G, Xu C, Lee GI, Bauer P, Ling HQ, Ganal MW, HoweGA.The tomato suppressor of prosystemin-mediated responses2 gene encodes afatty acid desaturase required for the biosynthesis of jasmonic acid and theproduction of a systemic wound signal for defense gene expression.Plant Cell.2003.15 (7): 1646-1661., the public can obtain from Chinese Academy of Sciences's heredity with developmental biology institute), picking mono-clonal, in cultivating with 28 DEG C of overnight shakings in the antibiotic LB liquid nutrient medium of Rifampin containing that enzyme element of card, obtain transformant.
Transformant is carried out bacterium liquid PCR to identify, adopt primer 1 and primer 2, what obtain 465bp is positive recombinant bacterium, by positive recombinant bacterium called after AGL0/pCAMBIA1301-LoxD RNAi, and be stored in-70 DEG C for subsequent use.
2, the transgenic Fructus Lycopersici esculenti of LoxD RNAi
1), tomato conversion is correlated with the preparation of substratum
Liquid MS medium: 4.4g MS salt, 30g sucrose and water are mixed, be settled to 1L with water, adjusts between pH to 5.8 ~ 6.0 with 1mol/LKOH, autoclaving.
Seed growth substratum (1/2MS substratum): 2.2g MS salt, 30g sucrose and water are mixed, be settled to 1L with water, adjusts between pH to 5.8 ~ 6.0 with 1mol/L KOH, adds the agar of 0.8%, autoclaving.
(be total to) culture medium (D1) in advance: by 4.4g MS, 1.0mg zeatin (Zeat in) and 30g sucrose soluble in water, be settled to 1L with water, adjust between pH to 5.8 ~ 6.0 with 1mol/L KOH, add the agar of 0.8%, autoclaving.
Screening division culture medium (2Z): by soluble in water to 4.4g MS salt, 2.0mg zeatin, 50mg kantlex, 100mg inositol, 0.5mg folic acid and 20g sucrose, 1L is settled to water, adjust between pH to 5.8 ~ 6.0 with 1mol/L KOH, add the agar of 0.8%, autoclaving.
Root media: by soluble in water to 4.4g MS salt, 50mg kantlex, 0.5mg folic acid, 0.5mg indolebutyric acid and 30g sucrose, be settled to 1L with water, adjusts between pH to 5.8 ~ 6.0 with 1mol/L KOH, adds the agar of 0.8%, autoclaving.
2), the preparation of the transgenic Fructus Lycopersici esculenti of LoxD RNAi
(1) preparation of transformation explant
Get wild-type tomatoes (kind CM), select full, the large seed of grain, soak 20min with 40%NaCl, with aseptic water washing 5 times, be seeded on seed growth substratum, in 25 DEG C, illumination cultivation under 16h illumination/8h dark condition.Sprout after 8 days, aseptically cotyledon diced (action should soon) with sharp scissors, cotyledon dice is inoculated in precultivation medium, in 25 DEG C, cultivate under 16h illumination/8h dark condition, after 2d, can be used for tomato conversion.
(2) preparation of liquid is infected
The AGL0/pCAMB IA 1301-LoxD RNAi saved backup is inoculated in containing in corresponding antibiotic LB liquid nutrient medium, 28 DEG C, 200rpm incubated overnight.Next day is forwarded in new LB liquid nutrient medium with the ratio of 1:100,28 DEG C, 200rpm is cultured to OD600=0.7.Bacterium liquid, with the centrifugal 10min of 5000rpm, abandons supernatant liquor, collects thalline.With liquid MSO substratum Eddy diffusion thalline, after being diluted to OD600=0.4, add the Syringylethanone of 50uL 0.074mol/L, for subsequent use.
(3) explant conversion, screen and take root
What the cotyledon piece that step (1) obtains is immersed respectively (2) prepare infects liquid 10min, then D1 substratum (substratum puts filter paper) Dual culture is inoculated in 2 days, be transferred to screening and culturing in screening division culture medium (2Z), every 2 weeks subcultures once, produce resistant buds after cultivating 8 weeks.When Elongation of adventitious bud is to 3cm, with scalper, resistant buds is cut, and be forwarded on root media, carry out root culture, by the T of taking root
0routine Management in soil is moved into, results T for transfer-gen plant
1for the transgenic Fructus Lycopersici esculenti seed of LoxD RNAi.
The condition of above-mentioned Dual culture, screening and culturing, root culture is: temperature is 25 DEG C, 16h illumination/8h dark.
3), the qualification of the transgenic Fructus Lycopersici esculenti of LoxD RNAi
The tomato leaf mosquito forceps of growth two leaf one heart stages of 18 days is made physical abuse, and at the direction parallel clips twice of the vertical main lobe arteries and veins of each leaflet, after 1 hour, clip above-ground plant parts, uses liquid nitrogen flash freezer rapidly ,-80 DEG C of preservations.Extract T
1for the RNA of the transgenic Fructus Lycopersici esculenti blade of LoxD RNAi, reverse transcription obtains cDNA, and with upstream primers F: ACTCATCAGCACCGACATCG, downstream primer R:ACTCTCCAGAAAGAACTCCTGC is primer, take wild-type tomatoes as contrast, carries out RT-PCR.Take actin as internal reference, the upstream primer for the reference gene that increases is F:TTGCTGACCGTATGAGCAAG, and downstream primer is R:GGACAATGGATGGACCAGAC.
Result T
1be 0.0205 for the relative expression quantity of LoxD gene in the transgenic Fructus Lycopersici esculenti of LoxD RNAi;
In wild-type tomatoes, the relative expression quantity of LoxD gene is 0.0142;
Can find out, with wild-type tomatoes ratio, T
1expression for the LoxD gene in the transgenic Fructus Lycopersici esculenti of LoxDRNAi is suppressed, and is positive transgenic plant.
4) acquisition of homozygous transgenic plants
Positive T is accredited as by above-mentioned
1transgenic Tomato Plants sowing for LoxDRNAi obtains T
2for seed, (wild-type CM is growing containing on the substratum of Totomycin, and its main root and over-ground part all can be subject to serious suppression to broadcast screening on the 1/2MS substratum of 800 μ L/L Totomycin after sterilization; Transgenic line is substantially not suppressed owing to having hygromycin resistance growth), to Totomycin have resistance for T
2for the transgenic Fructus Lycopersici esculenti of LoxDRNAi, obtain homozygote.
Adopting uses the same method proceeds in wild-type tomatoes by empty carrier pCAMBIA 1301, obtains T
0in generation, turns empty carrier tomato, sowing, sowing, until obtain T
2in generation, turns empty carrier tomato.Adopting uses the same method detects its LoxD gene relative expression quantity, and result shows, T
2in generation, to turn in empty carrier tomato leaf LoxD expression amount compared with wild-type tomatoes, without significant difference.
Two, the transgenic Fructus Lycopersici esculenti of LoxD RNAi affects insect-resistant
1, insect feeding experiment
Bollworm (Helicoverpaarmigera) is documented in document " Wu K.; Y.Lu; H.Feng; Y.Jiang; and J.Zhao.2008.Suppression of cotton bollworm in multiple crops in Chinain areas with Bttoxin-containing cotton.Science.321 (5896): 1676-1678. " and is disclosed, and the public can obtain from Chinese Academy of Sciences's heredity with developmental biology institute.
Before insect is fed, be documented in by transgenic Fructus Lycopersici esculenti 35S:PS(in " Howe GA; Lightner J; Browse J; Ryan CA.1996.An octadecanoid pathway mutant (JL5) of tomato is compromisedin signaling for defense against insect attack.Plant Cell.1996.8 (11): 2067-77. ", the public can obtain from Chinese Academy of Sciences's heredity with developmental biology institute), wild-type tomatoes, T
2in generation, turns empty carrier tomato and T
2transgenic Fructus Lycopersici esculenti for LoxDRNAi is cultivated under normal conditions: illumination 16h, dark 8h, intensity of illumination 70microeinsteins m
-2s
-2, temperature 25 DEG C.When plant grows to 4 weeks, get growing way consistent 2 the length of time bollworm be carefully put on plant, let alone freely to take food.Above-mentioned each strain is got 20 strains and is tested, and every plant puts 3 bollworms.
2, protease-inhibitor Ⅱ detects
Proteinase inhibitor in plant materials is a kind of resistance protein resisting insect pest infestation.This small basic proteins molecule being rich in Serine, reaches pest-resistant object by suppressing the activity of the digested enzyme of insect gut.20 century 70 professors Ryan find, insect pest infestation and physical abuse can the synthesis of inducible protein enzyme inhibitors, the expression amount of proteinase inhibitor is as the important indicator of power weighing plants against insects resistance, be the downstream gene of JA signal transduction path, raise by JA, and LoxD is first key enzyme in catalysis JA route of synthesis, therefore, the reduction of LoxD gene expression amount, causes the reduction of JA content, finally causes the reduction of protease-inhibitor Ⅱ expression amount.
Until feed when the 7th day, Taking Pictures recording is carried out to plant and bollworm, and utilize immunodiffusion(ID) to measure protease-inhibitor Ⅱ content, specific as follows:
A. the preparation of protease-inhibitor Ⅱ dish
1) 10 × buffer (radioimmunodiffusion dish damping fluid) stock solution (500ml) is first prepared:
Drug weight final concentration
Tris Base 12.1g 200mM Tris
NaCl 45g 9％NaCl
Adjust pH to 8.5, be settled to 500 milliliters with distilled water, autoclaving, when for subsequent use after solution cooling.
2) in 250ml triangular flask, 100ml 1 × buffer(10ml 10 × buffer+90ml distilled water is added), add bar magnet simultaneously and stir on heating magnetic stirring apparatus.
3) add 2g agar (Noble Agar), heating is directly seethed with excitement, and agar particle is fully dissolved.
4) slowly stirring allows solution be cooled to 58 DEG C gradually, the protease-inhibitor Ⅱ goat-anti serum adding 1ml (obtains with protease-inhibitor Ⅱ antigen immune sheep, the nucleotides sequence of this protease-inhibitor Ⅱ encoding gene is classified as the sequence 3 in sequence table), this solution is 1% sero-fast immunocell agar stationary liquid.
5) this stationary liquid of 11ml is added the culture dish (Falcon351012Integrid) of a scale square, make it cover the bottom of culture dish completely, form uniform one deck.Closed with sealed membrane after cooling, for subsequent use under being kept at 4 DEG C of conditions.
B, protease-inhibitor Ⅱ content radioimmunodiffusion measure
1) the injured blade of tomato (bollworm take food after blade) respectively after results process, grinding extruding blade.
2) drawing 5 μ l tomato leaf juice is added in the aperture of the corresponding lattice central authorities of the protease-inhibitor Ⅱ dish prepared, and places 24 hours.
3) in protease-inhibitor Ⅱ dish, pour the acetic acid solution of 7% into, allow solution cover card, react after 5 minutes, outwell acetic acid solution, use distilled water flushing.
4) result viewing, the diameter (centimetre) of the immunodiffusion(ID) ring in Accurate Measurement protease-inhibitor Ⅱ dish, is accurate to millimeter.Be applied to the content that following formulae discovery goes out protease-inhibitor Ⅱ.Protease-inhibitor Ⅱ content formula: protease-inhibitor Ⅱ content (μ g/ml juice)=(ring diameter data × ring diameter data × 10000-625) × 0.016.
Result as shown in Figure 1,
1A is for observing 35S:PS, wild-type tomatoes and T
2for the phenotype of the transgenic Fructus Lycopersici esculenti of LoxD RNAi, can find out, T
2transgenic Fructus Lycopersici esculenti for LoxD RNAi more easily comes to harm in insect administering transgenic, and almost to be chewed food by bollworm clean for its plant leaf.And insect chew food after 35S:PS and wild-type tomatoes still grow normally.
1B is 35S:PS, wild-type tomatoes and T
2for protease-inhibitor Ⅱ content results in the transgenic Fructus Lycopersici esculenti of LoxD RNAi, as seen from the figure, before insect chews food, the proteinase inhibitor content of 35S:PS is 58.6mg/mL, wild-type and T
20 is for protease-inhibitor Ⅱ content in the transgenic Fructus Lycopersici esculenti of LoxDRNAi; After insect chews food, 35S:PS, wild-type and T
2436.7mg/mL is respectively, 454.2mg/mL and 18.6mg/mL for protease-inhibitor Ⅱ content in the transgenic Fructus Lycopersici esculenti blade of LoxDRNAi; Show, after insect chews food, the protease-inhibitor Ⅱ albumen in 35S:PS and wild-type plant blade is subject to induced strong accumulation, and T
2obvious induction is not subject to for the protease-inhibitor Ⅱ albumen in the transgenic Fructus Lycopersici esculenti blade of LoxD RNAi.
1C is 35S:PS, wild-type tomatoes and T
2for the insect growth situation on the transgenic Fructus Lycopersici esculenti of LoxD RNAi, can find out, feed 7 days time observe, T
2healthy for the insect growth on the transgenic Fructus Lycopersici esculenti of LoxD RNAi, body weight is comparatively large, and this is due to T
2considerably less for protease-inhibitor Ⅱ protein accumulation amount in the transgenic Fructus Lycopersici esculenti of LoxDRNAi, so insect growth is healthy, the speed that body weight increases along with feeding time is also fast compared with the insect on 35S:PS and WT lines.
1D is 35S:PS, wild-type tomatoes and T
2for the insect body weight on the transgenic Fructus Lycopersici esculenti of LoxD RNAi; Feed 7 days time, 35S:PS, wild-type tomatoes and T
2for mean body weight 26.7mg, 62.8mg and 241.8mg respectively of every insect on the transgenic Fructus Lycopersici esculenti of LoxDRNAi.
Wild-type tomatoes and T
2in generation, turns the result of empty carrier tomato without significant difference.
Show from the above results, the transgenic Fructus Lycopersici esculenti of LoxDRNAi be subject to insect chew food after the accumulation of insect resistance protein protease-inhibitor Ⅱ be even 0 on a small quantity, insect body weight simultaneously on the transgenic Fructus Lycopersici esculenti of LoxD RNAi increases, illustrate and to compare with wild-type with 35S:PS, the resistance of transgenic Fructus Lycopersici esculenti to insect of LoxD RNAi significantly reduces, show that LoxD gene has pest-resistant effect, suppress its expression that insect-resistance can be made to reduce.
Three, the transgenic Fructus Lycopersici esculenti of LoxD RNAi is on the impact of the pathogen infection of graw mold of tomato
1, the cultivation of Botrytis and spore are collected
The pathogenic bacteria of graw mold of tomato is that the pathogen of Botrytis cinerea (Botrytis cinerea) is documented in Abuqamar S, ChaiMF, Luo H, Song F, Mengiste T.Tomato protein kinase 1b mediates signaling ofplant responses to necrotrophic fungi and insect herbivory.Plant Cell.2008.20 (7): the 1964-83. public can obtain from Chinese Academy of Sciences's heredity with developmental biology institute.
Picking a small amount of graw mold of tomato pathogenic bacterium the pathogen of Botrytis cinerea (Botrytis cinerea) mycelia, is seeded on potatodextrose agar (PDA) substratum, when cultivating 20 days under being 12h/12h condition in 24 ° of C photoperiods, collects spore.With tweezers scraping mycelia on substratum, put into aqua sterilisa, fully vibrate, make spore depart from mycelia; Utilize 8 layers of filtered through gauze suspension to remove mycelia, by centrifugal for filtrate 1000rpm 8min; Carefully outwell supernatant liquor, with potato dextrose broth (PDB) resuspended spore, utilize blood counting chamber to carry out spore concentration calculating and be adjusted to experimental concentration 5 × 10
5individual, obtain the pathogen of Botrytis cinerea spore liquid.
2, excised leaf connects bacterium experiment
By 35S:PS, wild-type tomatoes, T
2in generation, turns empty carrier tomato and T
2transgenic Fructus Lycopersici esculenti for LoxDRNAi is cultivated under normal conditions: illumination 16 hours, dark 8 hours, intensity of illumination 70microeinsteins m
-2s
-2, temperature 25 DEG C.When plant grows to 4 weeks, choose the plant that growth is consistent, the tomato leaflet of clip the 3rd leaf position, be put on 0.8% agar plate, and petiole base is inserted in agar, in the middle part of every sheet leaf, drip the pathogen of Botrytis cinerea spore liquid that 5 μ L above-mentioned 1 obtain, be put in illumination box and cultivate 72h, culture condition is: illumination 16 hours, dark 8 hours, intensity of illumination 70microeinsteins m
-2s
-2, temperature 25 DEG C.Then statistics bacterial plaque diameter is measured.The strain of each strain 10, tests in triplicate, results averaged.
Phenotypic Observation result as shown in Figure 2 A, Isolated leaf inoculation the pathogen of Botrytis cinerea spore liquid 3 days, the bacterial plaque diameter comparatively T of 35S:PS and wild-type
2bacterial plaque for the transgenic Fructus Lycopersici esculenti of LoxDRNAi is little, but does not have significant difference therebetween, shows as susceptibility difference.
Isolated leaf inoculation the pathogen of Botrytis cinerea spore liquid adds up bacterial plaque diameter result for 3 days as shown in Figure 2 B, 35S:PS, wild-type tomatoes and T
2bacterial plaque diameter for the transgenic Fructus Lycopersici esculenti of LoxD RNAi is respectively 0.5cm, 0.5cm and 1.2cm; Visible T
2after infecting 3 days for the transgenic Fructus Lycopersici esculenti of LoxDRNAi with bacterium, its bacterial plaque diameter is significantly higher than 35S:PS and wild-type, is almost the two 2.4 times, shows as strong susceptibility.
Wild-type tomatoes and T
2in generation, turns empty carrier tomato result without significant difference.
3, Live leaf connects bacterium experiment
1) Live leaf connects bacterium
By 35S:PS, wild-type tomatoes, T
2in generation, turns empty carrier tomato and T
2transgenic Fructus Lycopersici esculenti for LoxD RNAi is cultivated under normal conditions: illumination 16 hours, dark 8 hours, intensity of illumination 70microeinsteins m
-2s
-2, temperature 25 DEG C.When plant grows to 20 days, choose the plant that growth is consistent, every sheet leaflet drips 5 μ L and states the 1 the pathogen of Botrytis cinerea spore liquid obtained, every sheet great Ye drips three leaflets, to ensure that every strain plant drips the spore of identical amount.There is the plant of spore liquid to be put in airtight synthetic glass case by dripping, guaranteeing the super-humid conditions that the pathogen of Botrytis cinerea infects.The strain of each strain 5, tests in triplicate, results averaged.
Respectively at inoculation 1 day, 2 days and 3 days time get tomato leaf, and with liquid nitrogen flash freezer, in-80 DEG C of preservations, to extract for RNA, and follow-up tolerant gene expression analysis.
2) disease resistence gene detection of expression
A, tomato leaf RNA extract
The extraction of total serum IgE is extracted by Trizol method:
1) by above-mentioned for each strain 1) after the tomato leaf got of different time clays into power in liquid nitrogen, get 100mg blade powder and proceed in the RNase-free centrifuge tube being added with 1mL Trizol extracting solution, concussion mixing.
2) lapping liquid room temperature (25 DEG C) is placed 5 minutes, and add 0.2mL chloroform, cover tightly centrifuge tube, acutely sway centrifuge tube 20 seconds, room temperature places 3 minutes.Centrifugal 15 minutes of 4 DEG C of 12000g.
3) get upper water and make an appointment 600 μ L in a new centrifuge tube, add 0.5mL Virahol, room temperature places 10 minutes, 4 DEG C, centrifugal 10 minutes of 12000g.
4) abandoning supernatant, adds 75% ethanol of 1mL, and vortex mixes, centrifugal 1 minute of 12000g at 4 DEG C.
5) repeating step 4), careful abandoning supernatant, then room temperature or vacuum-drying 10 minutes, be dissolved in the distilled water of DEPC process, obtain RNA.
The synthesis of B, strand cDNA
1) in the RNase-free centrifuge tube of 200 μ L, following reagent is added:
Template ribonucleic acid (total serum IgE) 2 μ g
Primer Oligo (dT) 18 (0.5 μ g/ μ L) 1 μ L
RNase-free ddH2O mends to 13 μ L
2) centrifugal 3 ~ 5s after mixing gently, 70 DEG C are reacted 5 minutes, are placed in rapidly cooled on ice 5 minutes, then add:
3) final volume is 25L.Slightly centrifugal after mixing gently, 42 DEG C are reacted 60 minutes, and 94 DEG C of reactions, after 5 minutes, are put and carried out subsequent experimental or-20 DEG C of freezen protective on ice, obtain cDNA.
C, fluorescence quantitative RT-RCR analysis
1) reaction system
SYBR Premix Ex Taq（2×）10μL
2) response procedures
95℃，30s；45cycles of（95℃，10s；60℃，20s；72℃，20s）
3) data analysis
The cycle number of Ct value for experiencing when fluorescent signal in PCR pipe reaches the thresholding of setting.
Δ Ct=Ct (Gene)-Ct (Actin), with 2
-Δ Ctvalue weigh the transcriptional level of gene.
Gene and the primer of the quantitative PCR analysis expression related to are as follows:
Reference gene is Actin, and the upstream primer for the reference gene that increases is F:TTGCTGACCGTATGAGCAAG, and downstream primer is R:GGACAATGGATGGACCAGAC;
Protease-inhibitor Ⅱ (PI-II, the nucleotide sequence of this gene is as sequence 3), the upstream primer for amplification protein enzyme inhibitors II is F:AATTATCCATCATGGCTGTTCAC, and downstream primer is R:CCTTTTTGGATCAGATTCTCCTT;
The nucleotides sequence of this gene of PR1b1(is classified as sequence 4), the upstream primer for the PR1b1 that increases is F:TGGTATTAGCCATATTTCACTC, and downstream primer is R:CACATTGGTTGGTAGCGTAG;
The nucleotides sequence of this gene of Solyc07g006380(is classified as sequence 5), the upstream primer for the Solyc07g006380 that increases is F:GTCTTGGCAATGATGCTCTTTGTTAC, and downstream primer is R:TGAGATTTTGTCAAATACACATGG.
The detected result that above-mentioned disease resistence gene is expressed is as shown in Fig. 2 C-E, specific as follows:
Fig. 2 C is the expression of results of PI-II gene, as seen from the figure, after inoculation pathogenic bacteria, compared with 35S:PS, wild-type tomatoes, and T
2significantly reduce for the relative expression quantity of PI-II gene in the transgenic Fructus Lycopersici esculenti of LoxD RNAi; And PI-II inoculation after 1 day expression amount the highest, along with the prolongation of time declines gradually.
Fig. 2 D is the expression of results of PR1b1 gene, as seen from the figure, after inoculation pathogenic bacteria, compared with 35S:PS, wild-type tomatoes, and T
2significantly reduce for the relative expression quantity of PR1b1 gene in the transgenic Fructus Lycopersici esculenti of LoxD RNAi.
Fig. 2 E is the expression of results of the Solyc07g006380 gene homologous gene of PDF1.2 (in the tomato), as seen from the figure, after inoculation pathogenic bacteria, compared with 35S:PS, wild-type tomatoes, and T
2significantly reduce for the relative expression quantity of Solyc07g006380 gene in the transgenic Fructus Lycopersici esculenti of LoxD RNAi; And Solyc07g006380 gene expression amount when inoculation 1 day is the highest, along with the prolongation of time declines gradually.
Wild-type tomatoes and T
2in generation, turns empty carrier tomato result without significant difference.
As can be seen from the above results, after the pathogenic bacteria the pathogen of Botrytis cinerea Botrytis cinerea of inoculating tomato gray mold, the blade bacterial plaque size of the transgenic Fructus Lycopersici esculenti of LoxDRNAi and disease resistence gene PI-II, PR1b1, Solyc07g006380 expression amount are all lower than 35S:PS, wild-type tomatoes, illustrate that LoxD gene has the effect of anti-graw mold of tomato worm, suppress its expression that anti-graw mold of tomato can be made to reduce.
Claims (1)
1. a RNA molecule is the RNA that the sequence 2 in sequence table is transcribed from the DNA molecular shown in 5 ' end 106-570 position Nucleotide.
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| CN111575254B (en) * | 2020-05-14 | 2022-03-15 | 安徽农业大学 | Lipoxygenase, encoding gene CsLOX3 and application thereof |
| CN112646819B (en) * | 2021-01-17 | 2023-04-18 | 浙江师范大学 | Use of gene to enhance resistance to tomato gray mold |
| CN115491380B (en) * | 2022-06-15 | 2024-05-17 | 山东大学 | Plant lipoxygenase gene LOX and application thereof in broad-spectrum disease resistance of plants |
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| CN1824778A (en) * | 2004-10-18 | 2006-08-30 | 中国科学院遗传与发育生物学研究所 | Tomato anti insect related gene, its coding protein and application |
| CN102146126A (en) * | 2011-01-07 | 2011-08-10 | 中国科学院遗传与发育生物学研究所 | Protein related to insect resistance and encoding gene and application thereof |
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| CN1824778A (en) * | 2004-10-18 | 2006-08-30 | 中国科学院遗传与发育生物学研究所 | Tomato anti insect related gene, its coding protein and application |
| CN102146126A (en) * | 2011-01-07 | 2011-08-10 | 中国科学院遗传与发育生物学研究所 | Protein related to insect resistance and encoding gene and application thereof |
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