CN106337059A - Method for improving citrus huanglongbing resistance - Google Patents
Method for improving citrus huanglongbing resistance Download PDFInfo
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- CN106337059A CN106337059A CN201610816705.XA CN201610816705A CN106337059A CN 106337059 A CN106337059 A CN 106337059A CN 201610816705 A CN201610816705 A CN 201610816705A CN 106337059 A CN106337059 A CN 106337059A
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Abstract
The invention belongs to the technical field of molecular biology, in particular relates to a method for improving citrus huanglongbing resistance, and aims to solve the technical problem of providing a new choice for improving the citrus huanglongbing resistance. The technical scheme of the invention is the method for improving the citrus huanglongbing resistance. The method is particularly as follows: integrating an antibacterial peptide gene into a citrus genome under the regulation and control of a phloem specific promoter, wherein the antibacterial peptide gene is an antibacterial peptide gene PR1aCB, CB or PR1aCBer; the CB has the nucleotide sequence as shown in SEQ ID No.1; the PR1aCB has the nucleotide sequence as shown in SEQ ID No.2; the PR1aCBer has the nucleotide sequence as shown in SEQ ID No.3. The phloem specific promoter guides resistance genes, such as the antibacterial peptide and the like, to be efficiently expressed in the phloem, so that the citrus huanglongbing resistance level is enhanced.
Description
Technical field
The invention belongs to technical field of molecular biology is and in particular to a kind of method of improvement citrus yellow shoot disease resistance.
Background technology
Citrus yellow shoot disease is one of most destructive disease on world's mandarin orange produces.All of mandarin orange product almost can be endangered
Kind.According to statistics, citrus yellow shoot disease spreads in China 19 provinces and regions, and injured area accounts for more than the 80% of the total cultivated area of mandarin orange, produces
Amount accounts for the 85% about of total output.It is blue that citrus Huanglongbing pathogen belongs to (cadidatus liberobacter) leather for Candidatus liberibacter
Family name's negative bacteria, plants (cadidatus including Asia kind (cadidatus liberobacterasiatium), Africa
) and South America kind (cadidatus liberobacteramericanus) liberobacteafricanum[2].Wood louse is yellow
Dragon disease pathogen enter plant host unique insect vector, in addition, pass through grafting, pathogen also can between different plants long away from
From propagation.Yellow twig pathogen lacks the typical and pathogenic excretory system of type, and this is probably that this antibacterial specificity resides in
In plant cell the reason growth[4].It is difficult to isolated culture yet with this pathogen, therefore the pathogenic machine with regard to yellow twig antibacterial
Reason is still known little about it.Mandarin orange produces and there is no the technical measures fundamentally solving citrus yellow shoot disease evil, can only take destruction
A series of destructiveness measure such as disease plant or morbidity orchard, thoroughly to remove citrus yellow shoot disease.For this reason, for orchard worker, warp
Very serious the developing rapidly of modern biotechnology of Ji loss provides new approach for Citrus Disease Resistance breeding, and it can obtain often
Rule breeding is difficult to the new type obtaining, and creates new germ plasm[5,6].However, due to mandarin orange genetic background high complexity, at present not yet
Separate and obtain effective endogenous resistance gene, Citrus Disease Resistance genetic engineering with the functional gene importing other biological source is still
Main, as the antibacterial peptide gene in the sources such as insecticide[6].
With the continuous development of plant transgenic technology, the genetic transfoumation of many citrus varieties is successfully established,
Simultaneously many functional genes are successfully imported in mandarin orange genome, obtain the transgenic line of many character improvements:
Cardoso et al.[11]Convert Fructus Citri sinensiss with antibacterial peptide gene attacin a and obtain the strain of anti-citrus ulcer.Also grind
Study carefully personnel with cecropin b, shiva a, Antibacterial Peptide d gene transformation mandarin orange, obtain the strain of multiple Canker-Resistance diseases
System, field resistance evaluation for many years shows that the resistances against diseases of transfer-gen plant can stable heredity[12].
Zhang Qingjie etc.[13]In vitro bacteriostatic experiment find the antibacterial peptide of Antherea pernyi Guerin-Meneville Immunity hemolymph and its purification to mandarin orange HUANGLONG
Sick pathogenicbacteria separation thing has killing action.Its result of study implies, expression antibacterial peptide gene may strengthen mandarin orange to HUANGLONG
The resistance of disease.However, so far, antibacterial peptide gene is unclear to the resistance mechanism and power of yellow twig in plant body,
There is not the report obtaining the new strain of anti-citrus yellow shoot disease yet[5,6,11,14].Therefore, it is necessary to carry out antibacterial peptide gene further plant
The research of the potential ability of anti-yellow twig and resistance mechanism in object is effectively utilizes antibacterial peptide gene to improve the disease-resistant of mandarin orange
Property theoretical and material base is provided.
Content of the invention
The technical problem to be solved in the present invention is for improving the resistance to yellow twig for the mandarin orange, providing a kind of new selection.
The technical scheme is that a kind of method of improvement citrus yellow shoot disease resistance, specific as follows: phloem is special
Antibacterial peptide gene under promoter regulation is incorporated in mandarin orange genome；Described antibacterial peptide gene is antibacterial peptide gene
Pr1acb, cb, or pr1acber, cb has the nucleotide sequence as shown in seq id no.1, and pr1acb has as seq id
Nucleotide sequence shown in no.2, pr1acber has the nucleotide sequence as shown in seq id no.3.
Specifically, the antibacterial peptide gene Expression element under phloem-specific promoter regulation and control is building up to plant expression vector
In, convert mandarin orange.
Specifically, above-mentioned phloem-specific promoter is mandarin orange phloem-specific promoter.
Preferably, mandarin orange phloem-specific promoter is Semen vignae sinensiss glycin-rich protein matter gene grp promoter.
Specifically, described grp promoter has the nucleotide sequence as shown in seq id no.4.
Specifically, the method for described conversion mandarin orange is agrobacterium tumefaciens-mediated transformation or particle bombardment.
At present, also not with regard to controlling antibacterial peptide gene to improve the research report of mandarin orange resistance using phloem-specific promoter
Road.Possible the reason, is: one, the intensity of most of phloem-specific promoters opens by force well below composing types such as camv 35s
Mover；2nd, as external source small-molecular peptides, in plant cell, easily by intracellular protein enzymatic degradation, activity drops antibacterial peptide significantly
Low.Therefore, phloem-specific promoter instructs the expression of antibacterial peptide to be often difficult to reach bactericidal effect it is impossible to effectively improve
The resistance level of transfer-gen plant.
Phloem-specific promoter of the present invention instructs the resistant genes such as antibacterial peptide in phloem high efficient expression, and then strengthens Citrus chachiensis Hort.
The resistance level of Fructus Citri tangerinae yellow twig.Drive the genes amplification mandarin orange such as antibacterial peptide eventually through phloem-specific promoter to yellow twig disease
The inductivity epidemic prevention reaction of pathogenic microorganism infringement, has highly important theoretical and economical for raising Citrus Disease Resistance and yield
It is worth.
Brief description
The structure flow chart of the antibacterial peptide gene expression vector under the regulation and control of Fig. 1, grp phloem-specific promoter
Kanamycin, kalamycin resistance gene；Amp, ampicillin resistance gene；Nptii, neomycin phosphoric acid turns
Move enzyme gene；Gus, β-gluconic acid glycoside enzyme gene；Camv 35s (35s), from the plant composition of cauliflower mosaic viruses
Property promoter；Nos, Opines synthase gene terminator.Skeleton carrier for building plant expression vector is
(transformation process is shown in document: secreting type cecropin b antibacterial peptide gene conversion to the pc carrier of transformation on the basis of pcambia1305.1
Blood orange improves its Canker-Resistance level；Gardening journal 2014,41 (3): 417 428), have under camv 35s promoter regulation
Gus gene, be easy to during Genetic Transformation in Higher Plants, transformant is carried out with the screening of gus dyeing；Mcb gene be cb,
Pr1acb or pr1acber gene.
Pcr the and southern blot checking in transgenic mandarin orange of Fig. 2, exogenous gene
A, the pcr checking of transfer-gen plant, primer g-f/c-r, g-f/a-r and g-f/e-r is respectively intended to verify
Grp::cb (gc), grp::prlacb (ga) and grp::prlacber (ge) carrier；Target fragment is respectively 755,1009
And 1025bp, subsequently passes through sequencing and also demonstrates amplification；m,dna marker；Nt, wild type；1～10, transgenic is planted
Strain；3 electrophoresis result are followed successively by the knot after conversion carrier grp::cb, grp::prlacb and grp::prlacber from top to bottom
Really；
B-d, the southern blot checking of partial transgenic transfer-gen plant, nt, wild type；B, grp::cb transgenic
Plant；C, grp::prlacber transfer-gen plant；D, grp::prlacb transfer-gen plant.
Fig. 3, real-time quantitative pcr detect
Cecropin b gene expression in a, a (conversion grp::prlacb carrier) transfer-gen plant；B, c (conversion
Grp::cb carrier) cecropin b gene expression in transfer-gen plant；C, e (conversion grp::prlacber carrier) turn base
Because of cecropin b gene expression in plant；Pcr detection using the rna extracting in vein and chooses the endogenous actin of Citrus
Gene is detected as reference gene；Abscissa is transfer-gen plant, and vertical coordinate is relative expression levels.
Fig. 4, the resistances against diseases assessment of greenhouse transfer-gen plant
A-e, the HUANGLONG disease symptoms of rear transfer-gen plant and nontransgenic plants are infected in grafting；A, transgenic line a6 (turn
Change grp::prlacb carrier), c8 (conversion grp::cb carrier) and non-transgenic strain infect 6 months after HUANGLONG disease symptoms
Show；The close-ups of b, the bud-leaf of health and blade；The close-ups of c, the bud-leaf caught an illness and blade；D and e, turn base
Because strain a6 and non-transgenic strain infect the HUANGLONG disease symptoms behind 18 and 24 months and show；F, infect after 18 and 24 months turn
The qpcr detection of pathogen quantity in gene plant (transgenic line a6 and c8) and nontransgenic plants middle arteries；clas cells
μg-1Of total dna (the pathogen amount of counting accurately in the total dna of every microgram), bar diagram top letter representation transfer-gen plant and non-turn
Significance of difference tukey between gene plant ' s detection and analysis result；
Fig. 5, grafting infect the microscopic findings of latter 12 months transgenic and nontransgenic plants middle arteries
Middle arteries phloem, xylem and parenchyma cell under om observation；Compared with nontransgenic plants, transgenic is planted
The bast zone of strain middle arteries is thinner.Pa, parenchyma cell；Ph, phloem, xy, xylem, f, phloem fiber.Scale=50 μm；Upper row
Figure is 10 times, and figure below is 60 times of amplifications；Nt is wild type, and a6 is transfer-gen plant, and infected is processed for counteracting toxic substances；no-
Infected is control treatment.
Fig. 6, hbl standard curve
Fig. 7, plant dna standard curve
Specific embodiment
Reagent chemicals in present example does not do that being of illustrating is commonly commercially available, and MATERIALS METHODS illustrates
All with reference to " Molecular Cloning:A Laboratory guide " (sambrook and russell, 2001).
Embodiment one method introduction
The extraction of 1.dna
Choose mandarin orange vein 0.1-1g, using aidlab company test kit (cat.no.dn15) extract genome dna, -20
DEG C save backup.
The extraction of 2.rna and the synthesis of cdna
Choose mandarin orange leaf vein 0.1g easyspin plant rna rapid extraction test kit (aidlab, cat.no.rn09)
Extract the total rna of blade, with the quality of non denatured agarose gel electrophoresiies and ultraviolet spectrophotometer Scanning Detction rna.Cdna
The synthesis of one chain is according to bio-rad iscripttmCdna synthesis kit (bio-rad, cat.no.170-8891) says
Bright book is carried out.The cdna of synthesis saves backup in -20.
3. the pcr amplification of genome sequence
10 ex pcr buffer(mg2+Free) 2.5 μ l, 2.5mmol/l dntps 2 μ l, 25mmol/l mgcl22μ
L, primer 1 (5 μm of ol/l) 1 μ l, primer 2 (5 μm of ol/l) 1 μ l, ex taq dna polymerase 1u, genome dna about 60ng, plus
ddh2O to 25 μ l.Amplification program is: 94 DEG C, 5min；94 DEG C, 30sec, 56 DEG C, 30sec, 72 DEG C, 1.5min, 35 circulations；
72 DEG C of extension 10min.
4.dna fragment reclaims, and connects, and converts escherichia coli dh5 α
Under uviol lamp, cut the agarose gel block containing purpose fragment with clean blade.Recovery method is with reference to test kit
The operation instructions of (roche company) are carried out.Reclaim fragment electrophoresis on agarose gel quantitative.
The foundation of enzyme action system and reaction condition all enter with reference to the description of roche company restriction enzyme enzyme reagent kit
OK.Enzyme action reclaim fragment, or amplification obtain recovery fragment be cloned into pgem-t/ by ligase kit specification
On pgem-t easy (promega) carrier.Coupled reaction system is as follows: 10 × t4 dna connects buffer 1 μ l, carrier dna piece
Section 1 μ l (50ng), external source connection product dna fragment 1 μ l, t4 dna ligase 1 μ l, supply the connector of 10 μ l with distilled water
System, carrier dna fragment and external source connection product dna fragment mol ratio=1 3.
16 DEG C of connection 2～8h.Afterwards connection product is converted escherichia coli dh5a competent cell, 37 DEG C of cultures.
Embodiment two vector construction
1. the acquisition of specific promoter
According to Semen vignae sinensiss phloem-specific promoter grp (genbank accession number: af250148.1), design primer (grp-f
And grp-r, be shown in Table 5), from Semen vignae sinensiss genome pcr amplification obtain a 600bp about fragment.Dna fragment clone will be expanded
Sequencing analysis are shown to be the grp specific promoter of Semen vignae sinensiss afterwards, see seq id no.4.
The primer sequence used in table 5 embodiment
2. the synthesis of antibacterial peptide gene spcb
According to cecropin antimicrobial peptides gene cecropin b sequence, there is the synthetic secretion of Shanghai biological engineering company limited
Type antibacterial peptide gene cb, pr1acb and pr1acber.Containing from Nicotiana tabacum L. pr1a base after 5 ' the end atg of pr1acb and pr1acber
Because of signal peptide sequence pr1a, and add plant translational enhancement sequences aaggagauauaaca in upstream, with the expression of enhancing gene.
And endoplasmic reticulum framing signal kdel (endoplasmic reticulum localization signal peptide) is contained at the 3 ' ends of pr1acber.After conversion Citrus, cb, pr1acb
To be respectively positioned in iuntercellular, Cytoplasm and endoplasmic reticulum with pr1acber albumen.
3. the structure of specifically expressing antibacterial peptide gene carrier
Vector construction flow process is shown in Fig. 1.All restricted enzyme are purchased from roche company, according to operation instructions operation.
Concrete operations are as follows: cb, pr1acb and pr1acber are cloned on pmd19, with bamh and sal enzyme action
Pmd-mcb and pc skeleton carrier, cb, pr1acb, pr1acber fragment reclaiming is connected with pc skeleton carrier, obtains pc-
35s-mcb；Bamh and hind enzyme action pc-35s-mcb carries, pmd19-grp, connects, obtains pc-grp-mcb, i.e. grp::
Cb, grp::prlacb and grp::prlacber carrier.
Embodiment three genetic transformation
1. with electrization, the plant expression carrier plasmid building is imported Agrobacterium eha105.
With reference to bio-rad micropulser instruction manual book, by above-mentioned carrier grp::cb, grp::prlacb and
Grp::prlacber is directed respectively into Agrobacterium eha105 by Electroporation conversion.
2. the vector integration of specifically expressing antibacterial peptide gene is to mandarin orange genome
Carry out the genetic transformation of mandarin orange by Agrobacterium tumefaciens mediated method, used culture medium is shown in Table 1.
The Agrobacterium tumefaciens mediated mandarin orange genetic transformation culture medium of table 1
Ms:murashige＆skoog culture medium (murashige＆skoog, 1962)；Gelrite: plant gel
(sigma, article No.: g1910)；Ba:6- benayl aminopurine；iaa；3-indolyl acetic acid；2,4-d:2,4- dichlorphenoxyacetic acid；cef:
Cephalo penicillin；Kan: kalamycin.
Above-mentioned expression vector imports brocade orange by agriculture bacillus mediated epicotylar method.Concrete grammar is as follows:
1st, the epicotylar acquisition of brocade orange seedling
Take fresh brocade orange to clean, with 70% ethanol surface sterilization, take out seed under sterile conditions, peel kind of a skin off, connect
Plant and sprout on ms solid medium, light culture 2 weeks at 28 DEG C, then cultivate 1 week under the photoperiod of 16h/d.Take sprouting children
Seedling epicotyl be cut into 1cm about stem section, for Agrobacterium-mediated genetic transformation.
2nd, convert the preparation of Agrobacterium
Agrobacterium bacterium solution for transfection adds 30% sterile glycerol to be stored in -70 DEG C of ultra cold storage freezers.Before transfection,
Containing 50mg/l kanamycin lb solid medium (0.5% sucrose (w/v), 0.1% bacto yeast extract (w/v),
1% antibacterial tryptone (w/v), 0.05%mgso4.7h2O (w/v), 1.5% agar powder (w/v) ph7.0) upper line
Culture.Choose Agrobacterium single bacterium colony, be inoculated in the lb fluid medium that 5ml contains identical antibiotic, 28 DEG C, 200r/min vibration training
Support overnight.After growing single bacterium colony, picking single bacterium colony in the lb fluid medium of 50mg/ml kanamycin, train by 28 DEG C of vibrations
Support overnight；Treat the od of bacterium solution600When reaching more than 1, it is diluted to 0.2 about with the lb fluid medium not containing kanamycin, continues
Persistent oscillation culture 3h about；When bacterium solution reaches exponential phase (od is 0.5 about), it is centrifuged 10min in 5000r/min, abandons
Supernatant, settling flux Agrobacterium is suspended in the ms fluid medium that ph is 5.4 and is used for transfecting.
3rd, the conversion of brocade orange epicotyl stem section
By be cut into 1cm about brocade orange epicotyl stem section soak 12min in Agrobacterium, then will be upper with aseptic filter paper
The bacterium solution that plumular axis shows blots；Explant is transferred in co-cultivation base, 26 DEG C of light culture 2d.
4th, the screening of transformant
After the completion of co-cultivation, epicotyl is transferred in screening culture medium, 28 DEG C of light culture 7d.Then, explant is existed
28 DEG C, cultivate under 16h illumination, subculture is once every two weeks.
5th, the seedling culture of transformant
When plumelet grows to more than 1cm, by its vitro grafting to aseptic brocade orange seedling, carry out in seedling culture medium
Culture；Whne seedling grow to 5cm about when be grafted onto on Trifoliate seedling, cultivated in greenhouse.The transgenic Citrus chachiensis Hort. obtaining
Fructus Citri tangerinae is not clearly distinguished from compareing of wild type in phenotype and growth promoter.
The pcr detection of example IV exogenous origin gene integrator and checking
Take Citrus leaf 100mg, extract genome dna using aidlab company test kit (cat.no.dn15), pcr examines
Survey the integration of antibacterial peptide gene, result is shown in Fig. 2 a (only illustrating the result of some positive plant in 2a).Reaction volume 25 μ
L, reaction condition: 94 DEG C of 3min；94 DEG C of 30s, 58 DEG C of 30s, 72 DEG C of 30s, 30 circulations；72℃10min.Detection primer g-f/c-
R is used for detecting grp::cb and grp::prlacb transfer-gen plant, and expanding fragment length is respectively 755bp and 1009bp.g-f/
E-r primer is used for detecting grp::prlacber transfer-gen plant, and expanding fragment length is 1,025bp.
The positive transgenic plant verified through pcr is extracted genome dna, then adopts hind enzyme action, electrophoresis, adopt
Do probe hybridization with the gus gene of digoxigenin labeled.Result is shown in Fig. 2 b, 2c and 2d, shows that exogenous gene has successfully been integrated into Citrus chachiensis Hort.
In Fructus Citri tangerinae genome, transgene copy number is between 1～5.
The realtime-pcr analysis of embodiment five antibacterial peptide gene expression
Take Citrus leaf, then carried with easyspin plant rna rapid extraction test kit (aidlab, cat.no.rn09)
Take the total rna of vein.The synthesis of cdna first chain is according to bio-rad iscripttmcdna synthesis kit(bio-rad,
Cat.no.170-8891) description is carried out.With real-time pcr test kit (bio-rad iqtmsybr green
Supermix, cat.no.170-8882ap) quantitative analyses antibacterial peptide gene expression.The detection primer of genes of interest is qcb-f
And qcb-r；The detection primer of reference gene actin is ctact-f and ctact-r.
Reaction volume 20 μ l.Reaction condition: 95 DEG C of 3min, 94 DEG C of 10s；56 DEG C of 10s, 72 DEG C of 10s, 40 circulations；72℃
10min.Test is repeated 3 times, and data carries out statistical analysiss with bio-rad cfx manager 2.0 software.Using 2-δδctMethod meter
Calculate the relative expression quantity of antibacterial peptide gene in transfer-gen plant: the sample defining water process is with reference to the factor, i.e. its antibacterial peptidyl
The expression position 1 of cause, then calculate yellow twig pathogen inoculation same sample with respect to reference to factor gene expression times
Number is 2-δδct, relative expression levels by a definite date.Testing result is shown in Fig. 3.Result shows, antibacterial peptide gene is high in transfer-gen plant
Horizontal expression, and it is not detected by the expression of antibacterial peptide gene in nontransgenic plants.
The foundation to yellow twig evaluation of resistance system for embodiment six transfer-gen plant
The foundation of standard curve, obtains yellow twig disease according to yellow twig pathogenic bacteria genome (genbank no.:cp001677)
The non-conserved sequences of opportunistic pathogen 16s gene, to this primers cla16s-f/cla16s-f, take Citrus vein 100mg, make
Extract genome dna with aidlab company test kit (cat.no.dn15), be pcr and expand, reaction volume 25 μ l, reaction condition:
94℃3min；94 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 35 circulations；72℃10min.Reclaim fragment, connect t carrier, import big
Enterobacteria, shakes bacterium, extracts plasmid, sequencing, obtains the plasmid vector containing yellow twig 16s, ultraviolet spectrophotometer is measured plasmid and carried
Bulk concentration, by formula copies=c/ (mw × na) (c: plasmid vector concentration, mw: plasmid size and relative molecular mass
Product, na: Avogadro's number), it is calculated corresponding copy number, do 10 times of gradient dilutions, be qpcr, obtain respective concentration
Yellow twig pathogen (hlb) standard curve, is shown in Fig. 6.Concentration is surveyed to vein dna, does gradient dilution, using qcla16s-f/
Qcla16s-r and ct18s-f/ct18s-f is qpcr and obtains standard curve to pathogen 16s and mandarin orange 18s gene respectively, sees
Fig. 7.Every ng Plant Genome dna cause of disease containing yellow twig bacterium amount=[(- 0.2718 × ct is obtained according to above formula16s+
10.624)/(-0.2749×ct18s+4.0531)]×103(12.7<ct16s<31.3；8.4<ct18s<26.5).
Embodiment seven transfer-gen plant is to yellow twig evaluation of resistance
1st, the detection of Citrus grafting twig yellow twig cause of disease is tested with passing poison
To malicious source branch Vein extraction dna, do conventional pcr poison source identification using cla16s-f/cla16s-r primer, then
Grafting is done using the method for artificial grafting to transfer-gen plant and passes poison experiment.Grafting part is the transgenic shoot from stock 10cm
On, the malicious bud of each transfer-gen plant average grafting 2-3.
2nd, evaluation of resistance
Start to do evaluation of resistance after grafting passes poison 3 months.First every plant of malicious source is extracted with dna be conventional pcr (primer is
Cla16s-f/cla16s-r primer) detect the material poison source band poison it is ensured that for test；Then transgenic Citrus material is taken to do
Evaluation of resistance, method particularly includes: extract the dna of identical Maturity young sprout vein, with qcla16s-f/qcla16s-r and ct18s-
F/ct18s-f is qpcr to pathogen 16s and mandarin orange 18s gene respectively, further according to above-mentioned standard curve and computing formula, calculates
Obtain the amount of yellow twig pathogen containing with respect to every nanogram Plant Genome, by spss7.0 software analysis, filter out disease
Opportunistic pathogen content is substantially less than the transfer-gen plant (ducan ' s test, p < 0.05) of non-transgenic wild type control (wt), monthly
Detect once, continuous detecting 1 year.Filter out to enhanced turn of yellow twig resistance finally according to this result and with reference to Phenotypic Observation
Gene plant.
Carried out the disease resistance detection of totally 69 independent transformation in greenhouse.In order to detect transfer-gen plant to phytopathy
The resistance of opportunistic pathogen (or direct yellow twig), non-transgenic (comparison) and transfer-gen plant offspring with grafting to leaf bud through row
Calas pathogen inoculates.First, each strain is chosen three plants of plant and is inoculated pathogen with grafting.After 3 months, with qpcr's
Method calculates the quantity of pathogen in rotaring gene plant blade tissue.In this experiment, fluorescence threshold thinks more than 31.3
Pathogen is not had to be detected in plant.In form 3, there is the pathogen quantity of 22 strains obvious in 69 transgenic lines
Less than non-transgenic reference.The pathogen quantity of this 22 strains after 3 months, is detected further with qpcr.Wherein there are 8 strains
The pathogen quantity of system (a5, a6, a12, a21, c8, c16, e3and e6) is still significantly lower than non-transgenic reference (table 2
With 4).In the observation of whole a year, the pathogen quantity of this 8 transgenic lines is all significantly lower than non-transgenic
Comparison, and the pathogen quantity of this two transgenic lines of a6 and c8 is substantially less than other transgenic lines (table 4).
Table 2 qpcr detects transfer-gen plant middle arteries calas pathogen quantity
In table 2, ct value is lower shows that concentration is higher.After infecting 3,6,9 and each strain after 12 months in 16s gene average
Fluorescence threshold period (ct).Mai represents the moon number after infecting.Repeat experimental calculation by three times and obtain standard deviation.
Table 3 qpcr detects transfer-gen plant middle arteries calas pathogen quantity after inoculating 3 months
In table 3, Superscript letters a, c, d, e, f represent the resistance significance difference opposite sex level of transfer-gen plant compared with comparison nt,
Tukey ' s detects (p < 0.05).
Table 4 qpcr detects transfer-gen plant middle arteries calas pathogen quantity
In table 4, after infecting 3,6,9 and each strain after 12 months in pathogen quantity (in the total dna of every microgram, pathogen is thin
Quantity).Mai represents the moon number after infecting.Repeat experimental calculation by three times and obtain standard deviation.Superscript letters a, c, b represent and turn base
Significance of difference tukey because between plant and nontransgenic plants ' s detection (p < 0.05) analysis result.
After inoculation infects 6 months, most of plant include nontransgenic plants and all start to fall ill, and after infecting 1 year, non-turn base
Because plant shows serious disease.But, transfer-gen plant does not but show obvious Visual symptoms (Fig. 4).A6 and
This two transfer-gen plants of c8 still do not show obvious symptom through the observation of 2 years, and contained by it, pathogen quantity is also obvious
Less than non-transgenic reference.Microscopic findings show and infect latter 12 months, and the middle arteries of transgenic and non-transgenic strain occur
Difference.The cell number of plies with the transfer-gen plant phloem of resistance is far less than nontransgenic plants, its phloem cell wall
Thickness also thinner compared with nontransgenic plants (Fig. 5).Result above shows, overexpression cecropin b can substantially suppress to invade
The paraplasm of plant phloem cell after dye.
It is concluded that phloem-specific expression antibacterial peptide gene can significantly increase the ability of the anti-yellow twig of Citrus, wherein
The resistance of pr1acb gene is the strongest, is secondly cb and pr1acber.
Above detailed description of the present invention is not intended to limit the present invention, and those skilled in the art can do according to the present invention
Go out various modifications and change, without departing from the spirit of the present invention, model defined in claims of the present invention all should be belonged to
Enclose.
Claims (6)
1. a kind of improvement citrus yellow shoot disease resistance method it is characterised in that: specific as follows: phloem-specific promoter is regulated and controled
Under antibacterial peptide gene be incorporated in mandarin orange genome；Described antibacterial peptide gene is antibacterial peptide gene pr1acb, cb, or
Pr1acber, cb have the nucleotide sequence as shown in seq id no.1, and pr1acb has the core as shown in seq id no.2
Nucleotide sequence, pr1acber has the nucleotide sequence as shown in seq id no.3.
2. as claimed in claim 1 improvement citrus yellow shoot disease resistance method it is characterised in that: by phloem-specific promoter
Antibacterial peptide gene Expression element under regulation and control is building up in plant expression vector, converts mandarin orange.
3. improvement citrus yellow shoot disease resistance as claimed in claim 1 or 2 method it is characterised in that: above-mentioned phloem is special
Promoter is mandarin orange phloem-specific promoter.
4. as claimed in claim 3 improvement citrus yellow shoot disease resistance method it is characterised in that: mandarin orange phloem specifically starts
Son is Semen vignae sinensiss glycin-rich protein matter gene grp promoter.
5. as claimed in claim 4 improvement citrus yellow shoot disease resistance method it is characterised in that: described grp promoter has
Nucleotide sequence as shown in seq id no.4.
6. improvement citrus yellow shoot disease resistance as claimed in claim 2 method it is characterised in that: the side of described conversion mandarin orange
Method is agrobacterium tumefaciens-mediated transformation or particle bombardment.
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| CN114774415A (en) * | 2022-04-06 | 2022-07-22 | 西南大学 | Plant promoter induced by huanglongbing and abscisic acid and application thereof |
| CN116410988A (en) * | 2023-04-28 | 2023-07-11 | 西南大学 | Method for improving citrus yellow dragon disease resistance by utilizing citrus RUB2 to regulate citrus ubiquitination pathway |
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| CN111246875A (en) * | 2017-10-23 | 2020-06-05 | 加利福尼亚大学董事会 | Compositions and methods for treating diseases of the genus bastnaobacter and other bacterial diseases |
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| CN116715733A (en) * | 2022-10-18 | 2023-09-08 | 深圳润康生态环境股份有限公司 | Antibacterial peptide STJ-2 for effectively inhibiting proliferation of citrus yellow dragon disease pathogenic bacteria and application thereof |
| CN116410988A (en) * | 2023-04-28 | 2023-07-11 | 西南大学 | Method for improving citrus yellow dragon disease resistance by utilizing citrus RUB2 to regulate citrus ubiquitination pathway |
| CN116410988B (en) * | 2023-04-28 | 2023-12-22 | 西南大学 | Method for improving citrus yellow dragon disease resistance by utilizing citrus RUB2 to regulate citrus ubiquitination pathway |
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