CN102191253B - Plant disease-resistant regulation and control gene UEP and application thereof - Google Patents
Plant disease-resistant regulation and control gene UEP and application thereof Download PDFInfo
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- CN102191253B CN102191253B CN 201110083858 CN201110083858A CN102191253B CN 102191253 B CN102191253 B CN 102191253B CN 201110083858 CN201110083858 CN 201110083858 CN 201110083858 A CN201110083858 A CN 201110083858A CN 102191253 B CN102191253 B CN 102191253B
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Abstract
The invention provides a plant disease-resistant regulation and control gene. An open reading frame of the gene is 471 bp in length; and the gene encodes a ubiquitin extended protein consisting of 156 amino acids. The N end of the gene coded product is a ubiquitin molecule comprising 76 amino acids and the C end of the gene coded product is 40S ribosomal protein s27a comprising 80 amino acids. The gene is a high-quality disease-resistant gene resource. A disease-resistant material obtained by the gene has high resistance and a wide disease-resistant spectrum and can promote growth of plants. The gene widely joints in the regulation and control on resistance of various diseases caused by various pathogens by translating various kinds of targeted proteins and regulating modification after translation and joints in the regulation and control on growth and development of the plants. Due to overexpression of a tobacco UEP gene, the resistance of tobacco to various diseases such as Leptosphaeria maculans, wildfire, viral disease and the like is obviously improved. The plant disease-resistant regulation and control gene is applicable to creation and breeding of disease-resistant plant materials and varieties which have high resistance and wide disease-resistant objects and can promote growth of the plants.
Description
Technical field
The invention belongs to biological technical field, relate to a kind of plant disease-resistant regulatory gene (UEP) and uses thereof.
Background technology
1, Gene Clone in Plant
Methods of plant gene cloning is a lot, and along with the mensuration of various plants genome sequence is finished, the methods of plant gene cloning of based on database sequence obtains using more and more widely.The method operation steps mainly comprises the design of primers based on conserved sequence, and purpose plant tissue RNA extracts, ThermoScript II-polymerase chain reaction (RT-PCR), the PCR product is connected with carrier, and the bacterium that connects product transforms plasmid extraction, enzyme is cut check, sequencing analysis etc.
2, plant transgenic technology
Be used for foreign gene is imported the purpose plant, thereby obtain the transgenic plant of foreign gene-carrying.Comprise particle bombardment, agrobacterium-mediated transformation etc.Agrobacterium-mediated transformation comprises goal gene is cloned into plant expression vector, expression vector is to the conversion of Agrobacterium, carry the Agrobacterium of expression vector to the infection of purpose plant tissue, the steps such as detection analysis of gene transformation and expression in the regeneration of transfer-gen plant and the plant.
3, disease resistance of plant detects analytical technology
The phytopathy original comprises the broad varietys such as oomycetes, fungi, bacterium, virus and nematode.The inoculation method of dissimilar pathogens is different.For oomycetes, general first in culture dish on the moistening filter paper, cultivate 4 ~ 5d about 18 ℃ and make and produce a large amount of ascus that embrace.After will collect embrace in the distilled water that ascus is placed on sterilization (4000 ~ 6000/mL), 4 ℃ of lower 2 ~ 4h that cultivate make it to discharge zoospore, embrace a sub-suspension inoculation plant to move about.For fungi, can produce conidial, usually with conidial suspension spray inoculation plant.Do not produce conidial, then with the inoculation such as mycelia piece plant.For bacterium, often with the methods such as thallus suspension liquid leaf-cutting, acupuncture, injection inoculation plant.For virus, often with the virus particle of purifying or the outer transcription product friction of prosthesis, injection or by the communication medias such as insect inoculation plant.For nematode, normal second instar larvae with some amount contacts in plant root basal part of stem or the rhizosphere soil inoculates plant.In most cases, need the relative humidity that keeps higher, suitable temperature and illumination condition after the inoculation.By different time points Continuous Observation disease generation symptom, add up sickness rate after the inoculation, the morbidity severity calculates disease index.By with the comparative analysis of susceptible control plant incidence, the disease resistance of hard objectives plant.
4, Resistant breeding technology
Mainly can be divided into traditional breeding for disease resistance and pass through genetically engineered breeding for disease resistance two large classes.The tradition breeding for disease resistance can only be used the nearer disease-resistant resource of genetic affinity because there is natural hereditary isolation phenomenon to make disease-resistant resource usable range be subject to remarkable restriction, and need to repeatedly hybridize and backcross etc., so the seed selection cycle is long, and need a large amount of manpower and materials.And the genetic engineering breeding method is to import plant by the disease-resistant regulatory gene with external source by the agriculture bacillus mediated technology such as method, makes it obtain the disease resistance that does not originally have.Therefore, the genetic engineering breeding method has been broken the restriction of natural hereditary isolation phenomenon, has widened disease-resistant resource usable range, and has the advantages that operation is relatively simple and convenient, cultivation period short, need not a large amount of artificial material resources.In addition, can import a broad-spectrum disease resistance regulatory gene, perhaps can import a plurality of disease-resistant regulatory genes in the kind of plant, therefore have the suitable especially advantages such as wide spectrum, permanent disease-resistant kind of cultivating.
Summary of the invention
The purpose of this invention is to provide a kind of plant disease-resistant regulatory gene, called after UEP, its nucleotide sequence is shown in SEQ ID:1, the long 471bp of the open reading frame of this gene (ORF), a kind of ubiquitin extended proteins (Ubiquitin extension protein) of encoding, be comprised of 156 amino acid, its sequence is shown in SEQ ID:2.This gene encoding production N end is 76 amino acid whose ubiquitin molecules, and the C end then is 80 amino acid whose 40S ribosomal protein s27a.The Ben Shi cigarette (
Nicotiana benthamiana)
UEPThe overexpression of gene strengthen tobacco to balck shank (
Phytophthora parasiticaVar.
Nicotianae), wildfire (
Pseudomonas syringaePv.
Tabaci) and virus disease (
Tobacco rattle virus) etc. the resistance (specifically seeing the explanation among the embodiment 2) of multiple diseases.
Another object of the present invention provides described gene and obtains application in the broad-spectrum disease resistance material turning the UEP gene plant by initiative.Realize by following steps:
(1)
UEPThe structure of genetic expression structure and obtaining
Will
UEPGene ORF inserts a plant expression vector, makes it be subjected to the expression of ordering about of strong promoter.
(2) transform
UEPObtaining of the Agrobacterium of genetic expression structure
Will
UEPThe genetic expression structure transforms the agrobacterium strains that plant is had strong infection ability by methods such as electric shocks.
(3) turn
UEPThe initiative of gene plant and obtaining
Will by agrobacterium-mediated transformation
UEPGene imports the purpose plant, obtains to turn
UEPThe plant of gene.
(4) turn
UEPWhat gene plant isozygotied and is obtains
Respectively with antibiotics resistance and
UEPGenetic expression is observation index, detects transfer-gen plant offspring's proterties separation case, obtain that characters of progenies no longer separates, and can genetic stability contain turning that unit point integrates
UEPThe plant of gene is isozygotied and is.
(5) high resistance, lasting, broad-spectrum disease resistance turns the isozygoty Screening and Identification that is and obtaining of UEP gene plant
To turn
UEPIt is material that gene plant isozygotys, and detects the resistance of analyzing the disease that is caused by all kinds of pathogens such as oomycetes, fungi, bacterium, virus and nematodes, obtains turning of broad-spectrum disease resistance
UEPGene plant.
Advantage of the present invention: (1) is provided by the invention
UEPGene is High quality and diseases resistance regulatory gene resource, utilizes
UEPIt is strong that the disease-resistant material that gene obtains has resistance, and disease-resistant spectrum is wide, and the advantage such as Promoting plant growth simultaneously.Breeding for disease resistance often faces that the material and the variety resistance that obtain are strong not, and disease-resistant object is too single or narrow, follows the puzzlement of the problems such as growth and development of plants is suppressed when disease resistance strengthens.Provided by the invention
UEPGene is ubiquitin extended proteins (Ubiquitin extension protein) gene that is comprised of N end ubiquitin molecule and C end 40S ribosomal protein s27a.This gene is by the adjusting to the translation of all kinds of target proteinses and posttranslational modification, the regulation and control of the resistance of the multiple diseases that wide participation causes all kinds of pathogens, and also participation is to the regulation and control of growth and development of plants, tobacco
UEPThe overexpression of gene significantly strengthen tobacco to balck shank (
Phytophthora parasiticaVar.
Nicotianae), wildfire (
Pseudomonas syringaePv.
Tabaci) and virus disease (
Tobacco rattle virus) etc. the resistance of multiple diseases.And overexpression
UEPThe transfer-gen plant g and D contrast of gene is better.Therefore,
UEPGene is applicable to formulate the resistance level height, and disease-resistant object is wide, and the disease-resistant plants material of energy Promoting plant growth and initiative and the seed selection of kind.(2) obtain disease-resistant material cycle weak point.The method of obtaining disease-resistant plants material and kind mainly contains conventional traditional breeding way and the genetic engineering breeding method of utilizing disease-resistant regulatory gene.Traditional breeding way has available disease-resistant scope of resource and is subjected to natural heredity isolation restriction, and the seed selection cycle is long, needs the shortcomings such as a large amount of artificial material resources.The genetic engineering breeding method has then that available disease-resistant scope of resource is wide, operation is relatively simple and convenient, cultivation period short, need not a large amount of artificial material resources, the suitable especially advantages such as wide spectrum, permanent disease-resistant kind of cultivating.The present invention utilizes disease-resistant regulatory gene
UEP, adopt gene engineering method, the characteristics such as wide spectrum, permanent disease-resistant vegetable material are cultivated in initiative, and it is short to have a cycle, and seed selection is quick.
Description of drawings
Fig. 1 is
UEPTransgenic tobacco plant inoculation black shank bacterium (
Phytophthora parasiticaVar.
Nicotianae) 2 days symptom figure after the inoculated by hypha block.
Fig. 2 is
UEPTransgenic tobacco plant inoculation prairie fire germ (
Pseudomonas syringaePv.
Tabaci) thallus suspension liquid (OD
600Be 0.1) rear 2 days symptom figure.
Embodiment
The present invention is further described in conjunction with the accompanying drawings and embodiments.
Embodiment 1
The present invention has set up the High quality and diseases resistance regulatory gene that a cover utilizes the contriver to clone
UEP, adopting the genetic engineering technique initiative and obtain the resistance level height, disease-resistant object is wide, and the technical system of the disease-resistant plants material of simultaneously Promoting plant growth growth.Key step comprises:
1) the Ben Shi cigarette (
Nicotiana benthamiana)
UEPGene cloning and preservation
Ben Shi cigarette provided by the invention (
Nicotiana benthamiana)
UEPGene obtains by the following steps clone.First designed primer NbUbi-F(5 '-gc according to tobacco in the nucleic acid database and tomato ubiquitin extended proteins est sequence
GgatccAtg cag atc ttc gtg aaa acc-3 ', italicized item is
BamH I restriction enzyme site) (sequence is shown in SEQ ID:3), and NbRlp-R(5 '-gc
GtcgacTca atc ggc acc ggc ctt gtt g-3 ', italicized item is
SalThe I restriction enzyme site) (sequence is shown in SEQ ID:4).Adopt TRIZOL reagent to extract the total RNA of Ben Shi Tobacco Leaves, adopt the amplification of RT-PCR method to obtain
UEPCDNA reclaims the PCR product by rubber tapping purifying behind 1% agarose gel electrophoresis, is connected with the pGEM-T carrier subsequently, and thermal shock transforms bacillus coli DH 5 alpha, shakes the bacterium overnight incubation in the LB substratum, extracts plasmid, adopts
BamThe H I/
SalWhether the plasmid that the I enzyme cuts and utilize the PCR check of NbUbi-F/NbRlp-R to extract comprises
UEPGene send company's sequence verification at last, thereby successfully clones and obtain Ben Shi cigarette UEP gene cDNA full length sequence.
Transformed and carried the Ben Shi cigarette
UEPThe intestinal bacteria of the carrier of gene order are stored in-80 ℃ of refrigerators.Therefore, can extract plasmid at any time by the activation bacterial strain, cut by pcr amplification and enzyme, with the Ben Shi cigarette
UEPThe gene subclone is used for the researchs such as transgenosis to the purpose carrier.
2)
UEPThe structure of genetic expression structure and obtaining
To carrying the Ben Shi cigarette
UEPThe carrier of gene order carries out
BamThe H I/
SalThe I double digestion reclaims by electrophoresis and rubber tapping
UEPGene ORF sequence, subclone enters the strong promoter in the plant expression vector---and after cauliflower mosaic virus (CaMV) 35S promoter, obtaining can strong expression
UEPThe expression of plants structure of gene.
3) transform
UEPObtaining of the Agrobacterium of genetic expression structure
Will
UEPThe genetic expression structure transforms the agrobacterium strains that plant is had strong infection ability by methods such as electric shocks, obtains and carries
UEPThe Agrobacterium of genetic expression structure.
4) turn
UEPThe initiative of gene plant and obtaining
Will by agrobacterium-mediated transformation
UEPGene imports the purpose plant.First to carry
UEPThe agroinfection purpose plant tissue explant of genetic expression structure.By the screening of resistant buds, take root, turning of regeneration obtained in obtaining and the step such as evaluation of resistant plant again
UEPGene plant T
0Generation.
5) turn
UEPThe isozygoty Screening and Identification that is and obtaining of gene plant
Respectively with antibiotics resistance and
UEPGenetic expression is observation index, detects transfer-gen plant offspring's proterties separation case, obtains turning of unit point integration that characters of progenies no longer separates, also energy genetic stability
UEPGene plant isozygotys and is.
6) broad-spectrum disease resistance turns
UEPThe isozygoty Screening and Identification that is and obtaining of gene plant
To turn
UEPIt is material that gene plant isozygotys, and detects the resistance of analyzing the disease that is caused by all kinds of pathogens such as fungi, bacterium and viruses, obtains turning of broad-spectrum disease resistance
UEPGene plant.
Turning of embodiment 2 high levels, lasting, broad-spectrum disease resistance
UEPObtaining of genetic tobacco plant
Main operational steps comprises:
1)
UEPThe structure of genetic expression structure and obtaining
Carry the Ben Shi cigarette to what the contriver laboratory had
UEPThe carrier of gene order carries out
BamThe H I/
SalThe I double digestion reclaims by electrophoresis and rubber tapping
UEPGene ORF sequence, subclone enters the strong promoter among the plant expression vector pCHF3---and after cauliflower mosaic virus (CaMV) 35S promoter, obtaining can strong expression
UEPThe expression of plants structure pCHF3: of gene:
UEP
2) transform
UEPObtaining of the Agrobacterium of genetic expression structure
Draw 1 ~ 2 μ l
UEPGenetic expression structure pCHF3::
UEP, add in the 40 μ l EHA105 Agrobacterium competent cells, rapid mixing, with Bio-Rad Gene Pulser II electric exciter at voltage 12.5kV/cm, electric capacity 25 μ F and resistance 400
Electricity swashs about 9ms under the condition, adds rapidly 1ml YEP substratum, and 28 ℃ of lower 180rpm shake renewal cultivation 1h.Draw 100 μ l bacterium liquid and evenly be coated with the YEP substratum that contains 100mg/L Streptomycin sulphate and 100mg/L spectinomycin, obtain the possibility transformant, further verify expression vector and goal gene with PCR
UEPExistence.Obtain and carry
UEPThe EHA105 Agrobacterium of genetic expression structure.
3) turn
UEPThe initiative of genetic tobacco plant and obtaining
Carry
UEPGenetic expression structure pCHF3::
UEPThe EHA105 Agrobacterium to the Tissues of Tobacco explant infection---tobacco seed is sowed on the 1/2MS substratum that does not contain hormone after surface sterilization, the blade of the aseptic seedling behind the germinating growth is cut into 0.5 ~ 1 cm size, as aseptic explant.Place first the division culture medium preculture 2-3d that contains 1.0mg/L 6-BA, changing final concentration over to is OD again
6000.2 in ~ 0.5 the Agrobacterium bacterium liquid, soak 8 ~ 15min, after blotting explant changed over to again and cultivate altogether 2d in the division culture medium, clean and be placed on the transgenosis that contains 100mg/L kantlex and 500mg/L cephamycin and select to continue in the substratum to cultivate.
Turn
UEPThe regeneration of genetic tobacco plant---during the long 2 ~ 3cm of indefinite bud that treats to grow on the explant that transgenosis selects to cultivate in the substratum, indefinite bud is changed in the root media that contains 100mg/L kantlex and 250mg/L cephamycin, be cultured to grow main root after, with the aseptic seedling hardening of uncapping, move at last soil and normally cultivate, obtain seed.The antagonism plant carries out pcr amplification testing goal gene simultaneously
UEPExistence, and Southern and Northern analyze genetically modified copy number and expression.Obtain turning of regeneration
UEPGenetic tobacco T
0Generation.
4) turn
UEPThe isozygoty Screening and Identification that is and obtaining of genetic tobacco
With T
0Carry out surface sterilization for the seed of gathering in the crops on the transgenic tobacco plant, be seeded on the 1/2MS substratum that is added with the 200mg/L kantlex, filter out the resistance seedling, the antagonism seedling adopts PCR to detect simultaneously
UEPThe existence of gene and expression.Behind the results seed, be seeded on the same resistance culture base, select resistance to be the offspring who separates near the 3:1 ratio with non-resistance, this Dai Miao is screened equally and analyzes.Selection is obtained the offspring and is not produced transgenosis separation phenomenon, and turning of integrating of unit point that can genetic stability
UEPGenetic tobacco isozygotys and is.
5) high level, lasting, broad-spectrum disease resistance turns
UEPThe isozygoty Screening and Identification that is and obtaining of genetic tobacco
To turn
UEPIt is material that genetic tobacco isozygotys, and inoculates respectively all kinds of pathogens, detects to analyze to turn
UEPGenetic tobacco is to the resistance of these diseases.Detecting the pathogen of analyzing comprises: oomycetes---tobacco black shank bacterium (
Phytophthora parasiticaVar.
Nicotianae); Mycophyta---tobacco brown spot pathogen (
Alternaria alternata), the frog eye leaf spot of tobacco bacterium (
Cercospora nicotianae), the Colletotricum destructivum bacterium (
Colletotrichum destructum), the tobacco rhizoctonia solani (
Rhizoctonia solani), the tobacco da mping-off fungi (
Pythium spp.), tobacco sclerotium germ (
Sclerotinia sclerotiorum); Bacterium class---tobacco ralstonia solanacearum (
Ralstonia solanacearum), the wildfire bacterium (
Pseudomonas syringaePv.
Tabaci); Virus type---tobacco mosaic virus (
Tobacco mosaic virus,
Cucumber mosaic virus), Tobacco Vein Banding Caused by Potato Virus Y virus (
Potato virus Y), the sick virus of tobacco rattle (
Tobacco rattle virus), tobacco leaf curl virus (
Tobacco leaf curl virus); And threadworms---tobacco root-knot (
Meloidogyne incognita) etc.
Analyze caused the resistance of disease by all kinds of pathogens such as oomycetes, fungi, bacterium, virus and nematodes by detecting, obtain resistance level height, disease-resistant the turning of broad spectrum durable
UEPGenetic tobacco.
Relevant turning
UEPIt is performance to the detected result of the strong resistance of black shank and wildfire referring to Fig. 1 and Fig. 2 that genetic tobacco isozygotys.Fig. 1 is
UEPTransgenic tobacco plant inoculation black shank bacterium (
Phytophthora parasiticaVar.
Nicotianae) 2 days symptom figure after the inoculated by hypha block.A and B are the blade of UEP transgenic tobacco plant; C and D are for only turning the blade of the adjoining tree of empty carrier; The blade (C, D) of adjoining tree has showed very serious disease symptom, and more than half leaf is downright bad, and the blade (A, B) of UEP transgenic tobacco plant also only shows faint downright bad symptom.Show that the UEP transgenic tobacco plant has showed the strong resistance to balck shank.
Fig. 2 is
UEPTransgenic tobacco plant inoculation prairie fire germ (
Pseudomonas syringaePv.
Tabaci) thallus suspension liquid (OD
600Be 0.1) rear 2 days symptom figure.A and B are the blade of UEP transgenic tobacco plant; C and D are for only turning the blade of the adjoining tree of empty carrier; The blade (C, D) of adjoining tree has showed very serious disease symptom, and inoculation position is seriously downright bad, and the blade (A, B) of UEP transgenic tobacco plant does not show obvious downright bad symptom.Show that the UEP transgenic tobacco plant has showed the strong resistance to wildfire.
Turning of embodiment 3 high levels, lasting, broad-spectrum disease resistance
UEPObtaining of gene tomato plant
Main operational steps comprises:
1)
UEPThe structure of genetic expression structure and obtaining
Carry the Ben Shi cigarette to what the contriver laboratory had
UEPThe carrier of gene order carries out
BamThe H I/
SalThe I double digestion reclaims by electrophoresis and rubber tapping
UEPGene ORF sequence, subclone enters the strong promoter among the plant expression vector pCHF3---and after cauliflower mosaic virus (CaMV) 35S promoter, obtaining can strong expression
UEPThe expression of plants structure pCHF3: of gene:
UEP
2) transform
UEPObtaining of the Agrobacterium of genetic expression structure
Draw 1 ~ 2 μ l
UEPGenetic expression structure pCHF3::
UEP, add in the 40 μ l EHA105 Agrobacterium competent cells, rapid mixing, with Bio-Rad Gene Pulser II electric exciter at voltage 12.5kV/cm, electric capacity 25 μ F and resistance 400
Electricity swashs about 9ms under the condition, adds rapidly 1ml YEP substratum, and 28 ℃ of lower 180rpm shake renewal cultivation 1h.Draw 100 μ l bacterium liquid and evenly be coated with the YEP substratum that contains 100mg/L Streptomycin sulphate and 100mg/L spectinomycin, obtain the possibility transformant, further verify expression vector and goal gene with PCR
UEPExistence.Obtain and carry
UEPThe EHA105 Agrobacterium of genetic expression structure.
3) turn
UEPThe initiative of gene tomato plant and obtaining
Obtaining of aseptic tomato seedling and explant---get tomato seeds, first water washes 10 min and floats shrivelled seed, binds up with gauze, and 55 ℃ of water-bath 30 min add the effect that tween washes to increase sterilizing agent.75% alcohol disinfecting 30 s on Bechtop use 10% clorox (NaClO) surface sterilization 25 min again, ceaselessly rock therebetween, and with aseptic water washing four times, clean the residual disinfectancy agent of seed-coat.Blot on the aseptic filter paper, seed is sowed on the 1/2MS substratum, behind cultivation 3 d, tomato seeds breaks in the seed coat and shows money or valuables one carries unintentionally under 28 ℃ of dark conditions.Change into and cultivate aseptic seedling (25 ℃ are cultivated under 16/8 h, the 3000 lx illumination conditions) under the light, two cotyledons launch fully behind the 3-4 d, and true leaf not yet grows, and are the required aseptic seedling of experiment.Get its cotyledon as explant, used for agroinfection.
Carry
UEPGenetic expression structure pCHF3::
UEPThe EHA105 Agrobacterium tomato is organized the infection of explant---with cotyledon explant behind 3 d of preculture on the pre-culture medium, with agroinfection 5 ~ 10 min, blot bacterium liquid at sterilization filter paper, place upper 2 d of cultivation of common culture medium (not added with antibiotic), subsequently explant is changed over to evoking adventive bud on the selective differentiation substratum that contains 50 mg/L kantlex, per 2 all subcultures are once until the bud height reaches 0.5 cm.
Turn
UEPThe regeneration of gene tomato plant---indefinite bud is downcut from the callus base portion, be transferred on the stem elongation medium, cultivated for 2 weeks.Move to again root media, until tip height reaches 3 cm or root reaches 3 cm; The uncork hardening, and transplant to Nutrition Soil, normal management is obtained seed to yielding positive results.The antagonism plant carries out pcr amplification testing goal gene simultaneously
UEPExistence, and Southern and Northern analyze genetically modified copy number and expression.Obtain turning of regeneration
UEPGene tomato T
0Generation.
4) turn
UEPThe isozygoty Screening and Identification that is and obtaining of gene tomato
With T
0Carry out surface sterilization for the seed of gathering in the crops on the Transgenic Tomato Plants, be seeded on the 1/2MS substratum that is added with the 200mg/L kantlex, filter out the resistance seedling, the antagonism seedling adopts PCR to detect simultaneously
UEPThe existence of gene and expression.Behind the results seed, be seeded on the same resistance culture base, select resistance to be the offspring who separates near the 3:1 ratio with non-resistance, this Dai Miao is screened equally and analyzes.Selection is obtained the offspring and is not produced transgenosis separation phenomenon, and turning of integrating of unit point that can genetic stability
UEPThe gene tomato is isozygotied and is.
5) high level, lasting, broad-spectrum disease resistance turns
UEPThe isozygoty Screening and Identification that is and obtaining of gene tomato
To turn
UEPIt is material that the gene tomato is isozygotied, and inoculates respectively all kinds of pathogens, detects to analyze to turn
UEPThe gene tomato is to the resistance of these diseases.Detecting the pathogen of analyzing comprises: oomycetes---tomato late blight bacterium (
Phytophthorainfestans); Mycophyta---botrytis cinerea (
Botrytis cinerea), tomato early blight bacterium (
Alternaria solani), the tomato wilt bacterium (
Fusarium oxysporiumF.sp.
Lycopersici), tomato base rot disease bacterium (
Rhizoctonia solani), the sclerotinia of tomato bacterium (
Sclerotinia sclerotiorum); Bacterium class---bacterial wilt of tomato bacterium (
Ralstonia solanacearum); Virus type---tomato virus disease virus (
Tomato mosaic virus,
Tobacco mosaic virus,
Cucumber mosaic virus); And threadworms---tomato root-knot eelworm disease (
Meloidogyne incognita) etc.
Analyze caused the resistance of disease by all kinds of pathogens such as oomycetes, fungi, bacterium, virus and nematodes by detecting, obtain resistance level height, disease-resistant the turning of broad spectrum durable
UEPThe gene tomato.
Turning of embodiment 4 high levels, lasting, broad-spectrum disease resistance
UEPObtaining of Gene in Potato plant
Main operational steps comprises:
1)
UEPThe structure of genetic expression structure and obtaining
Carry the Ben Shi cigarette to what the contriver laboratory had
UEPThe carrier of gene order carries out
BamThe H I/
SalThe I double digestion reclaims by electrophoresis and rubber tapping
UEPGene ORF sequence, subclone enters the strong promoter among the plant expression vector pCHF3---and after cauliflower mosaic virus (CaMV) 35S promoter, obtaining can strong expression
UEPThe expression of plants structure pCHF3: of gene:
UEP
2) transform
UEPObtaining of the Agrobacterium of genetic expression structure
Draw 1 ~ 2 μ l
UEPGenetic expression structure pCHF3::
UEP, add in the 40 μ l EHA105 Agrobacterium competent cells, rapid mixing, with Bio-Rad Gene Pulser II electric exciter at voltage 12.5kV/cm, electric capacity 25 μ F and resistance 400
Electricity swashs about 9ms under the condition, adds rapidly 1ml YEP substratum, and 28 ℃ of lower 180rpm shake renewal cultivation 1h.Draw 100 μ l bacterium liquid and evenly be coated with the YEP substratum that contains 100mg/L Streptomycin sulphate and 100mg/L spectinomycin, obtain the possibility transformant, further verify expression vector and goal gene with PCR
UEPExistence.Obtain and carry
UEPThe EHA105 Agrobacterium of genetic expression structure.
3) turn
UEPThe initiative of Gene in Potato plant and obtaining
Obtaining of aseptic potato set and explant---to contain the aseptic potato test-tube plantlet of MS culture medium culturing of 3% sucrose.Choose the seedling age test-tube plantlet in 3 weeks, clip not with about 0.5cm stem section of axillalry bud as the acceptor material that transforms.
Carry
UEPGenetic expression structure pCHF3::
UEPThe EHA105 Agrobacterium to the infection of potato tissue explant---stem explants is incubated on the callus inducing medium.Medium's PH Value 5.8, culture temperature are 18-20 ℃, and illumination is 1500--20001ux, illumination every day 16 hours.Under 25 ℃ of dark, carry out preculture 2d after 3 weeks.Pre-incubated substratum is for containing 0.2 mg/L BA, 0.5 mg/L GA
3And the MS substratum of 2 mg/L zeatin.Explant is at OD
600Be about and soak 10 min in 0.5 the agrobacterium suspension, take out explant, blot surperficial bacterium liquid with aseptic filter paper, then change under dark, 28 ℃ of conditions and cultivate altogether 2 d, then explant is changed on the selective differentiation substratum that contains 50 mg/L kantlex and 300mg/L cephamycin over to evoking adventive bud differentiation under 25 ℃ of continuous illuminations.Per 2 ~ 3 all subcultures once, until the bud height reaches 1 ~ 1.5 cm.
Turn
UEPThe regeneration of Gene in Potato plant---indefinite bud is downcut from the callus base portion, be transferred to the root media that contains 50 mg/L kantlex and 250mg/L cephamycin, until tip height reaches 3 cm or root reaches 3 cm; The uncork hardening, and transplant to Nutrition Soil, normal management is obtained seed and potato seed to yielding positive results.The antagonism plant carries out pcr amplification testing goal gene simultaneously
UEPExistence, and Southern and Northern analyze genetically modified copy number and expression.Obtain turning of regeneration
UEPGene in Potato T
0Generation.
4) turn
UEPGene in Potato is clonal to obtain and breeds
Continue to cultivate through pcr amplification and Southern and Northern analysis
UEPThat obtains after the transgenosis turns
UEPThe Gene in Potato plant obtains potato seed.This potato seed can be used for vegetative propagation.
5) high level, lasting, broad-spectrum disease resistance turns
UEPThe Screening and Identification of Gene in Potato and obtaining
To turn
UEPThe Gene in Potato vegetative propagation is material, inoculates respectively all kinds of pathogens, detects to analyze to turn
UEPGene in Potato is to the resistance of these diseases.Detecting the pathogen of analyzing comprises: oomycetes---phytophthora infestans (
Phytophthorainfestans); Mycophyta---target bacterium (
Alternaria solani), the potato wilt bacterium (
Fusarium spp.), the potato cinerea bacterium (
Botrytis cinerea), the black scurf of potato bacterium (
Rhizoctonia solani); Bacterium class---Potato Ring Rot (
Corynebacterium sepedonicum); Virus type---rugose mosaic virus of potato virus (
Potato virus X,
Potato virus Y); And threadworms---potato pratylenchus disease (
Pratylenchus coffeae) etc.
Analyze caused the resistance of disease by all kinds of pathogens such as oomycetes, fungi, bacterium, virus and nematodes by detecting, obtain resistance level height, disease-resistant the turning of broad spectrum durable
UEPGene in Potato.
＜110〉Zhejiang University
＜120〉a kind of plant disease-resistant regulatory gene UEP and purposes
<160> 4
<210> 1
<211> 471
<212> DNA
＜213〉Ben Shi cigarette (Nicotiana benthamiana)
<220>
<221> CDS
<222> (1)…(471)
<400> 1
atgcagatct tcgtgaaaac cctgacgggt aagaccatca cccttgaggt cgaatcctcc 60
gacacaatcg acaacgtgaa ggcaaagatc caggataagg aaggcattcc accggaccag 120
cagcggctca ttttcgccgg aaaacagctt gaggatggac gaaccctagc tgattacaac 180
atccagaagg aatccactct ccatttggtg ctccgtctcc gtggtggtgc taagaagagg 240
aagaagaaga cttacaccaa gcctaaaaag attaagcaca agaagaagaa ggttaagctt 300
gctgtgttac agttttacaa ggtggatgat tctggaaaag tccagaggct tcgtaaggag 360
tgtcctaacg ctgagtgtgg tgctggtact ttcatggcaa accactttga ccgtcactac 420
tgtggtaagt gtggcctcac ttacgtttac aacaaggccg gtgccgattg a 471
<210> 2
<211> 156
<212> PRT
＜213〉Ben Shi cigarette (Nicotiana benthamiana)
<400> 2
Met Gln Ile Phe Val Lys Thr Leu Thr Gly Lys Thr Ile Thr Leu Glu
1 5 10 15
Val Glu Ser Ser Asp Thr Ile Asp Asn Val Lys Ala Lys Ile Gln Asp
20 25 30
Lys Glu Gly Ile Pro Pro Asp Gln Gln Arg Leu Ile Phe Ala Gly Lys
35 40 45
Gln Leu Glu Asp Gly Arg Thr Leu Ala Asp Tyr Asn Ile Gln Lys Glu
50 55 60
Ser Thr Leu His Leu Val Leu Arg Leu Arg Gly Gly Ala Lys Lys Arg
65 70 75 80
Lys Lys Lys Thr Tyr Thr Lys Pro Lys Lys Ile Lys His Lys Lys Lys
85 90 95
Lys Val Lys Leu Ala Val Leu Gln Phe Tyr Lys Val Asp Asp Ser Gly
100 105 110
Lys Val Gln Arg Leu Arg Lys Glu Cys Pro Asn Ala Glu Cys Gly Ala
115 120 125
Gly Thr Phe Met Ala Asn His Phe Asp Arg His Tyr Cys Gly Lys Cys
130 135 140
Gly Leu Thr Tyr Val Tyr Asn Lys Ala Gly Ala Asp
145 150 155
<210> 3
<211> 29
<212> DNA
＜213〉artificial sequence
<220>
＜223〉tobacco and the design of tomato ubiquitin extended proteins est sequence and the synthetic that provide according to the DFCI database are as primer
<400> 3
gc
ggatccatg cag atc ttc gtg aaa acc
<210> 4
<211> 30
<212> DNA
＜213〉artificial sequence
<220>
＜223〉tobacco and the design of tomato ubiquitin extended proteins est sequence and the synthetic that provide according to the DFCI database are as primer
<400> 4
gc
gtcgactca atc ggc acc ggc ctt gtt g
Claims (1)
1. plant disease-resistant regulatory gene, called after UEP, its nucleotide sequence is shown in SEQ ID:1, the long 471bp of the open reading frame of this gene, a kind of ubiquitin extended proteins of encoding is comprised of 156 amino acid, and its sequence is shown in SEQ ID:2, this gene encoding production N end is 76 amino acid whose ubiquitin molecules, and the C end is 80 amino acid whose 40S ribosomal protein s27a.
2. a kind of plant disease-resistant regulatory gene according to claim 1 obtains application in the broad-spectrum disease resistance material turning the UEP gene plant by initiative, it is characterized in that, be the application that obtains in the disease-resistant material of black shank and wildfire turning by initiative that the UEP genetic tobacco isozygotys.
A kind of plant disease-resistant regulatory gene according to claim 1 in preparation, detect and to turn isozygoty the application in the method that is of UEP gene tomato, potato.
4. according to claim 2 or 3 described application, it is characterized in that, realize by following steps:
(1)
UEPThe structure of genetic expression structure and obtaining
Will
UEPGene ORF inserts a plant expression vector, makes its expression of ordering about that is subjected to strong promoter,
(2) transform
UEPObtaining of the Agrobacterium of genetic expression structure
Will
UEPThe genetic expression structure transforms the agrobacterium strains that plant is had strong infection ability by electric-shocking method,
(3) turn
UEPThe initiative of gene plant and obtaining
Will by agrobacterium-mediated transformation
UEPGene imports the purpose plant, obtains to turn
UEPThe plant of gene,
(4) turn
UEPWhat gene plant isozygotied and is obtains
Respectively with antibiotics resistance and
UEPGenetic expression is observation index, detects transfer-gen plant offspring's proterties separation case, obtain that characters of progenies no longer separates, and can genetic stability contain turning that unit point integrates
UEPThe plant of gene is isozygotied and is,
(5) high resistance, lasting, broad-spectrum disease resistance turns the isozygoty Screening and Identification that is and obtaining of UEP gene plant
To turn
UEPIt is material that gene plant isozygotys, and detects the resistance of analyzing the disease that is caused by all kinds of pathogens such as oomycetes, fungi, bacterium, virus and nematodes, obtains turning of broad-spectrum disease resistance
UEPGene plant.
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| CN1757740A (en) * | 2005-08-22 | 2006-04-12 | 中国林业科学研究院林业研究所 | Plant bivalent anti-reverse gene bielement expression carrier |
| CN101250221A (en) * | 2008-04-15 | 2008-08-27 | 中国科学院遗传与发育生物学研究所 | Plant ABA signal transduction regulatory protein as well as coding gene and use thereof |
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| CN1757740A (en) * | 2005-08-22 | 2006-04-12 | 中国林业科学研究院林业研究所 | Plant bivalent anti-reverse gene bielement expression carrier |
| CN101250221A (en) * | 2008-04-15 | 2008-08-27 | 中国科学院遗传与发育生物学研究所 | Plant ABA signal transduction regulatory protein as well as coding gene and use thereof |
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| Chan A等.GenBank:XP_002525394.1.《GenBank》.2009, * |
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