CN106282225A - A kind of negative regulation plant is to the gene of the resistance of Delphacidae insecticide and application thereof - Google Patents
A kind of negative regulation plant is to the gene of the resistance of Delphacidae insecticide and application thereof Download PDFInfo
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Abstract
The present invention relates to a kind of negative regulation plant to the gene of the resistance of Delphacidae insecticide and application thereof.Disclose TOM64 gene first closely related to the resistance of Delphacidae insecticide with plant, lower this gene and can give the plant resistance for Delphacidae insecticide.Therefore, TOM64 gene or the material of regulation TOM64 gene and method can be applicable to realize the improvement of plant variety.
Description
Technical field
The invention belongs to botany and genetic engineering field, more particularly it relates to an negative regulation
Plant is to the gene of the resistance of Delphacidae insecticide and application thereof.
Background technology
Plant suffer from the threat of various plant-feed insect during growth promoter, so planting
Thing needs a set of extremely complex defense system to resist the infringement of these insecticides.The autoimmune quilt of plant
During activation, innate immune response can be started, excite the expression of a series of biochemical reactions and gene,
Innate immune response includes that immunoreation (PTI) that the pattern recognition receptors of surface of cell membrane excites and plant are special
Resistance protein (R albumen) identification receptor of the property changed, the immunoreation (ETI) that secretion effect protein excites.Plant
Immunoreation be excited after, including jasmonic (JA), salicylic acid (SA), H2O2Compile Deng in plant
Knit a complicated defence signal network.In addition, plant also has composing type defense system, bag
Include the physical barriers such as the waxiness of plant surface, horny layer, lignin.And constitute the base of these defending against network
This unit is the gene in plant, process LAN or knock out certain gene may have influence on some of which prevent
Imperial path, thus demonstrate the enhancing to insect-resistant or weaken.And at the functional genome research of plant
In, mutant has important effect to the explaination of gene function, and equally, mutant library is also applied for grinding
Study carefully the function in terms of plants against insects resistance.
At present, plant such as grass to the research of Delphacidae insect-resistant molecular mechanism also in primary
In the stage, excavate the gene that Delphacidae insect-resistant is relevant, strengthen Delphacidae insect-resistant molecular mechanism
Understand, it will help field is for the preventing and treating of Delphacidae insecticide.
Summary of the invention
It is an object of the invention to provide a kind of negative regulation plant to the gene of the resistance of Delphacidae insecticide and
Application.
In a first aspect of the present invention, it is provided that a kind of raising plant resistance to insect, reduction plant plant height, increase
Plant tillering or the method improving plant products, described method includes: lower TOM64 polypeptide in plant
Expression or activity；The genome of described plant has TOM64 gene.
In a preference, the described expression lowering TOM64 polypeptide in plant or the method for activity
Including: knock out in the genome of plant or reticent TOM64 gene；Maybe downward TOM64 gene is turned
The lower adjustment of record, expression of polypeptides or polypeptide active proceeds in plant.
In another preference, described knock out or reticent TOM64 gene is to be inserted in by exogenous sequences
In 9th exon of TOM64 gene.
In another preference, described lower adjustment is that the interference of specificity interference TOM64 gene expression divides
Son.
In another preference, described disturbing molecule be TOM64 gene or its transcript be suppression or heavy
The silent dsRNA of target, antisensenucleic acids, siRNA, Microrna, maybe can express or be formed described
DsRNA, antisensenucleic acids, siRNA, the construction of Microrna.
In another preference, described raising plant resistance to insect is to improve plant for Delphacidae insecticide
Resistance.
In another preference, described plant includes: grass.
In another preference, described TOM64 polypeptide is selected from lower group:
The albumen of (a) such as SEQ ID NO:2 aminoacid sequence；
(b) by SEQ ID NO:2 aminoacid sequence through one or more (such as 1-30；Preferably 1-20
Individual；More preferably 1-10；More preferably 1-5) amino acid residue replacement, lack or add and formed,
And there is the albumen derivative by (a) of (a) protein function；Or
C protein sequence that () and (a) limit has 70% (preferably 80%；More preferably 90%；More preferably 95%；
More preferably 98%；More preferably 99%) above homology and there is the albumen derivative by (a) of (a) protein function.
In another preference, the encoding gene of described TOM64 comprises following sequence: SEQ ID NO:
1。
In another aspect of this invention, it is provided that the use of a kind of material reducing TOM64 gene expression or activity
On the way, for improving the insect resistace of plant, or it is used for reducing plant plant height, or is used for increasing plant tillering,
Or be used for improving plant products, or it is used for improving H in plant tissue2O2Content；The base of described plant
Because group has TOM64 gene.
In a preference, described reduction TOM64 gene expression or the material of activity are that specificity is done
Disturb the disturbing molecule of TOM64 gene expression；It is preferred that described disturbing molecule be TOM64 gene or
Its transcript is suppression or the reticent dsRNA of target, antisensenucleic acids, siRNA, Microrna,
Maybe can express or be formed the construction of described dsRNA, antisensenucleic acids, siRNA, Microrna.
The other side of the present invention, due to this disclosure, is aobvious to those skilled in the art
And be clear to.
Accompanying drawing explanation
Fig. 1, TAIL-PCR separate other adjacent sequence diagram.
Fig. 2, the insertion point of pest-resistant mutant T35 and anti-BPH individual plant are identified.
(A). the T-DNA insertion point of mutant T35；(B) the anti-BPH of .WT and T35 individual plant in seedling stage
Identify (n=8, figure is representative plant)；(C). the expression of mutant gene in fluorescence quantitative PCR detection T35,
Sheath in tillering stage taken from by material, and data are average ± SD (n=3), t inspection display significant difference (* *
P < 0.01, compared with wild type)；(D) the anti-BPH of .WT and T35 individual plant in tillering stage identifies that (n=8, figure is
Representative plant)；(E). Rice Resistance BPH individual plant in tillering stage identifies stem Harm (n=8, scale
=1.5cm).
The structure of Fig. 3, TOM64 polypeptide and associated protein phylogenetic analysis.
(A) structure of .TOM64 polypeptide；(B). part TOC64 and the evolution of TOM64 in different plants
Tree is analyzed.Os-Oryza sativa (Oryza sativa L.), ob-Oryza brachyantha (Oryza brachyantha),
Bd-Brachypodium distachyon (two fringe false bromegrasses), zm-Zea mays (Semen Maydis), si-Setaria
Italica (millet), at-Arabidopsis thaliana (arabidopsis), ps-Pisum sativum (Semen Pisi sativi),
Mt-Medicagotruncatula (Herba Medicaginis).
The complementary checking of Fig. 4, tom64 mutant.(A) .PCR identifies complementary transfer-gen plant.(B). mutually
After-culture strain brown planthopper resistant identifies (n=3).
Selectivity (A) and the egg laying amount (B) of brown paddy plant hopper are measured by Fig. 5, Oryza sativa L..Data are average (n=3),
And by t inspection significant difference, all there was no significant difference compared with wild type.
Fig. 6, Oryza sativa L. take food (A), growth (B) and survival rate (C) and measure BPH.Data are average
± SD (A, n=5；B and C, n=3), t inspection significant difference (* * P < 0.01 contrasts WT).
(C) in, mutant there was no significant difference in wild type at each time point.
The tolerance to insects of BPH is measured by Fig. 7, Oryza sativa L..Data are average ± SD (n=3), and t inspection shows
Write sex differernce (* * P < 0.01 contrasts WT).
The Subcellular Localization of Fig. 8, TOM64 polypeptide.
Fig. 9, rice tissue H2O2The mensuration of content.Data are average ± SD (n=3), and t inspection shows
Write sex differernce (* * P < 0.01 contrasts WT).
Figure 10, the evaluation of resistance of Rice Resistance BPH, data are that average ± SD (n=10), ANOVA are aobvious
Work sex differernce is analyzed, and different letters represents significant difference (P < 0.05).
The impact on rice yield correlated traits it is sheerly after Figure 11, TOM64 gene knockout.Data are average
Number ± SD (n=3), t inspection significant difference (* * P < 0.01 contrasts WT).(A) TOM64 is shown
Gene is knocked rear rice tillering in advance；(B) show that TOM64 gene causes Oryza sativa L. to become short after being knocked；(C)
Be pure lines Oryza sativa L. compared with wild type, ratio of productive tiller does not has notable difference；(D) TOM64 gene quilt is shown
After knocking out, the tillering quantity of meeting Oryza sativa L. significantly increases.
Detailed description of the invention
The present inventor through in-depth study, find mitochondrial outer membrane transport protein 64 (TOM64,
Translocon at the Outer envelope membrane of Mitochondria protein 64) and plant
Closely related to the resistance of Delphacidae insecticide, lower this gene and can give plant Delphacidae insecticide is resisted
Property.Therefore, TOM64 gene or the material of regulation TOM64 gene and method can be applicable to realize plant product
The improvement planted.
As used herein, described " plant " includes but not limited to: crucifer, grass family are planted
Thing.Such as, described " plant " includes but not limited to: Cruciferae Mus ear mustard belongs to such as arabidopsis, standing grain
Undergraduate course oryza plant such as Oryza sativa L., grass family Triticum plant such as Semen Tritici aestivi, grass family Zea plant such as Semen Maydis
Deng.
As used herein, described " Delphacidae insecticide " includes but not limited to: brown paddy plant hopper (Brown
Planthopper, BPH), white backed planthopper (white back planthopper, WBPH) and small brown rice planthopper (small
brown planthopper,SBPH)。
In the present invention, " TOM64 polypeptide (albumen) " refers to the polypeptide with SEQ ID NO:2 sequence,
Also include having and TOM64 polypeptide identical function, the variant form of SEQ ID NO:2 sequence.These
Variant form includes (but being not limited to): several (usually 1-50, preferably 1-30, more preferably
1-20, most preferably 1-10, the most more preferably as 1-8,1-5 individual) amino acid whose disappearance, insertion and/or
Replace, and C-terminal and/or N-terminal add or disappearance one or several (usually within 20,
Within preferably 10, within being more preferably 5) aminoacid.
Any Yu described TOM64 peptides homologous is high (such as with the sequence shown in SEQ ID NO:2
Homology is 70% or higher；Preferably, homology is 80% or higher；It is furthermore preferred that homology is
90% or higher, such as homology 95%, 98% or 99%) and there is TOM64 polypeptide identical function
Albumen is also included in the present invention.Derive from other species beyond Oryza sativa L. with shown in SEQ ID NO:2
The TOM64 polypeptide that the homology of sequence is higher is also included in the present invention.
The invention still further relates to code book invention TOM64 polypeptide or the polynucleotide sequence of its conservative variation's polypeptide
Row.Described polynucleotide can be DNA form or rna form.DNA form include cDNA,
Genomic DNA or the DNA of synthetic.DNA can be strand or double-strand.DNA is permissible
It is coding strand or noncoding strand.The coding region sequence of encoding mature polypeptide can with shown in SEQ ID NO:1
Coding region sequence is identical or the variant of degeneracy.As used herein, " variant of degeneracy " exists
The present invention refers to encode the protein with SEQ ID NO:2, but with the volume shown in SEQ ID NO:1
The code differentiated nucleotide sequence of region sequence.
The polynucleotide of the mature polypeptide of coding SEQ ID NO:2 include: the coding of an encoding mature polypeptide
Sequence；The coded sequence of mature polypeptide and various additional coding sequence；The coded sequence of mature polypeptide (with appoint
The additional coding sequence of choosing) and non-coding sequence.
Although should be understood that the TOM64 gene of the present invention is preferably obtained from grass such as Oryza sativa L., but obtain
From other plant with Oryza sativa L. TOM64 gene very high homology (such as have more than 70%, such as 80%, 85%,
90%, 95%, even 98% sequence thereto) other gene also within the scope of the present invention contemplates.
The Method and kit for of aligned sequences homogeny is also well known in the art, such as BLAST.
The TOM64 gene that the present invention relates to has important application valency in theoretical research and plant improvement
Value.This sequence can be applied to the insect resistace research of specified plant such as Oryza sativa L.；Further, its also with tune
The control tiller of plant, plant height, yield traits are correlated with.
The invention still further relates to a kind of method improveing plant, the method includes regulating TOM64 in described plant
The expression of polypeptide.
On the one hand, present invention also offers a kind of raising plant resistance to insect, reduction plant plant height, increase and plant
Thing tiller, the method improving plant products, described method includes: reduce TOM64 in described plant many
The expression (including making TOM64 polypeptide not express or low expression) of peptide or activity.
After the purposes knowing described TOM64 polypeptide, can use well known in the art multiple
Method regulates the expression of described TOM64 polypeptide.Such as can use well known in the art multiple
Method reduces the expression of TOM64 polypeptide or is allowed to loss of expression, such as by carrying antisense TOM64 base
The ceneme (such as expression vector or virus etc.) of cause is delivered on target spot so that cell or plant tissue are not
Express or reduce and express TOM64 polypeptide.
As one embodiment of the present invention, it is provided that a kind of reduce the expression of TOM64 polypeptide in plant
Method, described method includes:
(1) disturbing molecule of interference TOM64 gene expression is proceeded to plant cell, tissue, organ or kind
Son, it is thus achieved that be transformed into the plant cell of described disturbing molecule, tissue, organ or seed；
(2) plant cell of described disturbing molecule, tissue, organ or seed have been proceeded to by what step (1) obtained
Regeneration plant.
It is preferred that described method also includes:
(iii) select and proceeded to the plant cell of described carrier, tissue or organ；With
(iv) plant cell, tissue or the neomorph in step (iii) is become plant.
As another embodiment of the invention, it is provided that a kind of reduce the table of TOM64 polypeptide in plant
The method reached, described method includes: knock out in the genome of plant or reticent TOM64 gene.Example
As, described knock out or exogenous sequences can be inserted in TOM64 gene by reticent TOM64 gene
In exon.
The method that other suppression TOM64 gene or its homologous genes are expressed is well known in the art.
In an embodiment of the present invention, the present inventor is screened by the brown planthopper resistant of extensive mutant library,
It is finally obtained resistance strain, and then qualification has separated its mutant gene, and pest-resistant prominent to one of them
The function of variant and related gene thereof has carried out research checking.
Through field and indoor qualification, the inventors discovered that a strain paddy gene deletion mutant T35 height resists brown
Plant hopper.Find after analyzing further, this mutant code TOM64 gene.The silence of TOM64 gene is
Owing to T-DNA is inserted into the 9th exon position of this gene, causes tanscription termination, thus cause this
The silence of gene.Pest-resistant Analysis on Mechanism shows, the sudden change of this gene mainly improves Oryza sativa L. to brown paddy plant hopper
Toleration.After being replied by mutant gene, complement strain then loses and resists Delphacidae insecticide
Property, prove further, only can improve Oryza sativa L. after the expression of this gene is silenced to Delphacidae insecticide
Resistance.Subcellular Localization result shows, this gene mapping, in mitochondrion, improves in rice tissue
H2O2Content, thus improve the Oryza sativa L. resistance to Delphacidae insecticide.TOM64 mutant is to brown paddy plant hopper
Resistance can bring up to middle water resistant flat (3-5 level) from original high sense level (7-9 level).
Below in conjunction with specific embodiment, the present invention is expanded on further.Should be understood that these embodiments are only used for
The present invention is described rather than limits the scope of the present invention.The reality of unreceipted actual conditions in the following example
Proved recipe method, generally writes according to normal condition such as J. Pehanorm Brooker etc., Molecular Cloning: A Laboratory guide, the 3rd
Version, Science Press, the condition described in 2002, or according to the condition proposed by manufacturer.
I. materials and methods
1, the plantation of vegetable material is cultivated
Oryza sativa L. (Oryza sativa) pest-resistant cultivar RHT (Rathu Heenati) used by the pest-resistant screening of field high flux
Or IR56, sense worm kind TN1 (Taichung Native 1)；The wild type of T-DNA insertional mutagenesis library is
In spend 11 (ZH11), mutant is by Shanghai life science institute of Chinese Academy of Sciences plant molecular heredity state key
Laboratory provides.
Field Plants material is planted on village, often used in village names farm, Songjiang, Shanghai five, and all mutant materials are planted in specially
In enclosure wall for examination transgenic plant material.
2, plasmid used
PCAMBIA 2300: purchased from Cambia company, its bacterial strain resistance and plant resistance are Kmr,
For building complementation (reply) carrier of mutant.
PA7-GFP: purchased from Promega company, is used for building fusion vector, and the subcellular fraction of research gene is fixed
Position.
3, field and indoor insect resistance identification
Carrying and put abundant soil, field the last week in order, point ridge, every row spacing 1.2m, long 12m, between ridge
Away from 30cm.Wild type, pest-resistant comparison, sense worm comparison and mutant seeds are sub-packed in permeable pouch
In, after accelerating germination 2-3 days, it is seeded in uniformly and accomplishes fluently the ridge of line on the ground, each mutant sowing 10-15 grain,
Mutant side plants pest-resistant comparison (RHT or IR56) and sense worm comparison (TN1) respectively.Observe long after two weeks
Seedling situation, often row mutant is left to few 3 strain man power single stem rices, it is to avoid the plant of many seeds combines,
The growing way of Oryza sativa L. is paid close attention to every a few days.
Not spraying insecticide in field, appoints a brown paddy plant hopper to move into breeding, observes each dashing forward in each period of paddy growth
The victimization state of variant.If running into the brown paddy plant hopper less time, when Adult plant, by laboratory cultures
Brown paddy plant hopper is released to the most uniformly for examination field.Treat that TN1 is the most withered, mutant is carried out pest-resistant commenting
Valency, evaluation criterion is shown in Table 1.The resistance rank mutant less than ZH11 is pest-resistant mutant, higher than ZH11
Mutant for sense worm mutant.
Brown Planthopper Resistance is identified rating scale by table 1, Oryza sativa L. Adult plant
| Resistance rank | Symptoms (is investigated during TN1 death) | Resistance level |
| 0 | Plant strain growth is healthy, and on-bladed is injured | Immunity (Im) |
| 1 | The yellow of plant beginning | High anti-(HR) |
| 3 | Most plant turn yellow, the obvious yellow of plant part | Pest-resistant (R) |
| 5 | Most plant Huangs wither, and minority is withered | In anti-(MR) |
| 7 | Plant more than half are withered | Middle sense (MS) |
| 9 | All plant is withered | Sense worm (S) |
4, the extraction of oryza sativa genomic dna
Use CTAB method (2%CTAB, 1.4M NaCl, 20mM EDTA, 100mM/L Tris.HCl,
PH8.0)。
A) take in the centrifuge tube that rice leaf puts into 1.5mL, add after grinding with grinder after liquid nitrogen freezing
Enter the CTAB extract of 600 μ L.
B) 65 DEG C of heating 45min (softly mixing).
C) adding equal-volume chloroform, mixing, room temperature stands 10min.
D) 12000rpm, centrifugal 15min, take supernatant to new EP pipe.
E) isopyknic isopropanol is added, mixing ,-20 DEG C of precipitation 30min.
F) 12000rpm is centrifuged 10min, abandons supernatant, and 70% washing with alcohol precipitates twice.
G) centrifugal abandon supernatant after, be dried, with appropriate water dissolution.
H) integrity of 1% agarose gel electrophoresis detection genomic DNA.
5, the acquisition (TAIL-PCR) of the other adjacent sequence of mutant
With rice leaf genomic DNA as pcr template, carry out three-wheel PCR reaction.First round PCR:
With the genomic DNA that extracts as template, use the primer NTL1 on T-DNA and random primer.The
Two take turns PCR: with first round PCR primer as template, use the NTL2 primer on T-DNA and with power traction
Thing.Third round PCR: take turns PCR primer as template with second, use the NTL3 primer on T-DNA and
Random primer (Fig. 1).Third round PCR primer detects by 1% sepharose electrophoresis, has the sample of band to cut glue
Direct Sequencing after recovery, or be connected on cloning vehicle check order again.Sequencing result is first left with T-DNA
Border sequence comparison, after sequence excision correct for comparison, residue sequence is in NCBI or paddy gene networking
Stand and compare on (http://rice.plantbiology.msu.edu/index.shtml), determine insertion point.
Tail-PCR program is shown in Table 1.
6, T-DNA and phenotype be divided into from
Choose the progeny seed of the heterozygote of mutant, sow after accelerating germination, treat that rice seedlings length is to two leaves wholeheartedly
Time, the rice leaf that each clip about 2cm is long, extracts DNA.With insertion point both sides and T-DNA
On primer detect every young plant pure, miscellaneous or without the state inserted.Treat that rice seedlings recovers 3 days, cover is moulded
Material cover, carries out insect resistance identification according to the method that seedling stage, individual plant was identified.Interpretation of result according to insect resistance identification
Phenotype is divided into from situation with T-DNA's.
7, the complementary checking (back mutation) of mutant
Choose the own promoter sequence of genes of interest start codon ATG upstream 2kb, with gene
The coded sequence of cDNA inserts pCAMBIA2300 carrier together, and promoter is connected into the restriction enzyme site of selection
Being KpnI and BamHI, the connection restriction enzyme site of coded sequence is BamHI and XbaI.Pass through Agrobacterium
The callus of the method untransformed mutants of mediation, owing in mutant, T-DNA has hygromycin resistance,
Therefore select the transgene carrier blocking that resistance, and screen positive transgenic plant with kanamycin.Obtain
Positive transgenic plant is the complementary plant of mutant.Seedling stage, the method for individual plant insect resistance identification identified complementation
Can plant the phenotype of revertant.
8, the tolerance to insects of BPH is identified by Oryza sativa L.
To sow after strain to be measured (wild type and mutant) accelerating germination, the plantation two same strains of strain in each little square box
Rice seedlings, grow about 6 weeks, choose the consistent plant of growth, go secondary tiller, after recovering three days
Transparent plastic cover on cover, inside each cover (two young plants) inoculate 25 BPH one age nymph, comparison
Plant also covers transparent plastic cover, but does not connect worm.In time feeling worm kind (WT) and begin to change into yellowish-brown,
Take out and collect all BPH, after being dried 48h in 60 DEG C of baking ovens, weigh quality.Meanwhile, plant from
Remove in etui, clean root earth, after 75 DEG C of baking ovens are dried 60h, weigh quality.According to public affairs
The formula calculating coefficient of resistance to worm (TI): TI=[(Wc-Wt)/Wc]/BPH, wherein, WcFor not connecing the plant of worm
Dry weight, WtFor connecing the dry weight of the plant after worm, BPH is the dry weight of the BPH collected on every strain rice seedlings.
The coefficient of resistance to worm is the lowest, and Oryza sativa L. is the strongest to the tolerance to insects of BPH.Often group experiment sets three repetitions.
9, the Subcellular Localization of TOM64 polypeptide
(1) structure of Subcellular Localization carrier
TOM64 gene is removed the coding region sequence of termination codon by two enzyme action of XhoI and SpeI
Site is connected in PA7-GFP carrier before GFP, forms the fusion vector of PA7-TOM64-GFP.
(2) preparation of rice protoplast
A) seed disinfection and cultivation.ZH11 seed is 1min in 75% ethanol；Then 2.5% hypochlorous acid is used
Sodium washes 20min；At least clean with sterilized water 5 times.Sowing is on 1/2MS solid medium, at 12h/12h
Illumination/dark, under 26 DEG C of environment, germinates 3 days, and dark lower cultivation obtains the etiolated seedling of ZH11 for 7 days.
B) shears intercepts stem and the sheath of 40-60 rice seedling, holds into a branch of, is cut into sharp cutter
About 0.5mm strip.
C) the little bar sheared is transferred quickly in 0.6M mannitol (mannitol), dark under place 10min.
D) discard mannitol, little stick be immersed in enzymic digestion liquid, dark under, slightly shake
(60-80rpm)4-5hr。
Enzymolysis solution: 1.5%Cellulase R-10,0.75%Macerozyme R-10,0.6M mannitol,
10mM MES.KOH (pH 5.7), 10mM CaCl2, 0.1%BSA.
E) after enzymic digestion, add isopyknic W5 solution, firmly rock 10s with hands.
W5 solution: 154mM NaCl, 125mM CaCl2, 5mM KCl, 2mM MES.KOH at
pH 5.7。
F) by liquid mixed above by 40 μm nylon filter membrane filtrations, to a round bottom pipe；Clear with W5 solution
Oryza sativa L. little stick above filter wash film 3-5 time, is filtered through nylon membrane.
G) swinging bucket rotor is centrifuged 1500rpm, 3min, collects protobiont.
H) protoplast, centrifugal protoplast are cleaned with W5.
I) resuspended with MMG solution.Blood cell counting plate counting about 2 × 106Cell/mL.
MMG solution: 0.4M mannitol, 15mM MgCl2, 4mM MES.KOH (pH 5.7).
J) protoplast and vigor thereof are observed in FDA dyeing.
(3) conversion of rice protoplast
A) by 5-10 μ g plasmid and 100 μ L protobionts (about 2 × 105Individual cell) mixing.
B) the PEG solution that 110 μ l newly prepare is added.Mixed liquor room temperature, dark under place 10-20min.
PEG solution: 40% (W/V) PEG 4000,0.2M mannitol, 0.1M CaCl2。
C) 440 μ l W5 solution it are slowly added to gently.Upset pipe is allowed to mix gently.Centrifugal 1500rpm,
3min.Collect protoplast.
D) gently with the 1ml resuspended protoplast of WI solution.
WI solution: 0.5M mannitol, 20mM KCl, 4mM MES (pH 5.7).
E) protoplast is transferred to six orifice plates.6-16h is placed in room temperature under An.
(4) Mitotracker Red dyeing
Agents useful for same is MitoRed CMXRos (Invitrogen, M7512), dyeing is strict
Operate according to reagent description.
10, rice tissue H2O2The mensuration of content
Rice leaf sheath sample is ground to powder in liquid nitrogen, weighs 0.3g sample in 2ml centrifuge tube, adds 1ml
Deionized water fully mixes, and 13600g is centrifuged 10 minutes, and supernatant is testing sample.H2O2Content
Mensuration use Amplex Red Hydrogen Peroxide/PeroxidaseAssay kit (Invitrogen,
A22188), it is measured according to test kit description.
11, the TOM64 gene delection impact on rice yield traits
In order to analyze the impact on rice yield traits of the TOM64 gene delection, the present inventor is by hybridization
Offspring carries out separation screening, it is thus achieved that the pure lines seed of TOM64 gene delection, and is planted in indoor and field
Between, the character that its yield is relevant is observed and measured.
Embodiment 1, paddy gene deletion mutant T35 show the resistance stronger to brown paddy plant hopper (BPH)
The present inventor from field Preliminary screening to Rice Resistance brown paddy plant hopper mutant, select fractional mutant
Carry out indoor individual plant insect resistance identification, found that some mutant does not demonstrate preferable resistance to brown paddy plant hopper,
But there is the mutant (field test Brown Planthopper Resistance rank in 2012: 3 grades) of a strain code T 35, indoor single
Strain insect resistance identification shows, either seedling stage or tillering stage, relative to wild type ZH11, mutant T35
All show the strongest resistance (Fig. 2 B, D, E).
The analysis of other adjacent sequence shows, the T-DNA of this mutant is inserted in paddy gene
In 9th exon of LOC_Os02g51580 (this gene has 13 exons) (Fig. 2 A).
The analysis result of quantitative fluorescent PCR shows, this gene is not detected by expressing (Fig. 2 C) in mutant.
Thus proving, this mutant is gene lacks functionality mutant, and mutant shows brown paddy plant hopper the strongest
Resistance.That is, this mutant is the plant having knocked out TOM64 gene in genome.
The TOM64 polypeptide of embodiment 2, T35 mutant gene coding Oryza sativa L.
As it was previously stated, the gene of disappearance is made up of 13 exons and 12 introns in T35 mutant,
Total length 8864bp, a length of 2212bp of its full-length cDNA (KOME accession number: AK069965).This base
Because of one 614 amino acid whose albumen of coding.Its N end (aminoacid 20-38) has a membrane spaning domain,
Centre is a functional domain (aminoacid 75-462) with lactamase activity, and C end is that a tripeptides repeats sequence
The TPR domain (aminoacid 532-564) (Fig. 3 A) of row.This is plant distinctive plastid (chloroplast or line
Plastochondria) adventitia transport molecule TOC64 (chloroplast) or the structure of TOM64 (mitochondrion).Pass through
BLASTP (Altschul et al., 1990) program carries out further homology at NCBI albumen database and divides
Analysis, the present inventor is at Oryza brachyantha (Oryza brachyantha), millet (Setaria italica), Semen Maydis (Zea
Mays) arabidopsis (Arabidopsis thaliana) etc. and all have found the homologous protein of this albumen, with Oryza sativa L.
The homology of this albumen in source is respectively 89%, 76%, 74% and 53%, it was demonstrated that this gene is at unifacial leaf
In plant the most conservative, bigger with the homology difference of TOM64 in dicotyledon arabidopsis.To some
The phylogenetic analysis of the albumen with amidase domain shows that this albuminoid is chloroplast, mitochondrion and born of the same parents
Matter has distribution (Fig. 3 B).In T35 mutant, the albumen of disappearance is the mitochondrial outer membrane transport molecule of Oryza sativa L.
Albumen TOM64.
The cDNA sequence (SEQ ID NO:1) of TOM64 gene:
ATGGACTCCTCGGCGCGATCGGGCGGCGGAGGCACCGGCGGGTACACCAGCACTCG
CGTGTGGATGGTCGCGGGCGTCGCCATCGCCGGCGCCATCGTCTTCGTGGAGGCCGCGCG
GCGCAGGCGCCGGTGGCTGCGGGACAGGTCCGAGGTTCCCCCCGATTTCGGCGCCTTCTGC
TACCGCTTCGAGATCGCCCCGGCGCCGCAGCCTCCGCCTCCCGCCGCGCGCCAGCTACTCT
CGGGCCTCACATTCGCGGCCAGCGACAACTTCGAGATCGAGGGCTATGTGGCTGGGTTTG
GGAATCCGGACTGGAAGAGGACTCACAAGGCAGCCACGCGCACGGCGGTTCCTGTCACGA
TGCTGCAGAAGCAGGGGGGCACCTACGTTGGTAGCACAGTCATGGATGAGCTCGGATTTG
GTGTTTCTGGAGGAAATTTACACAATGGAACACCAATCAACCCAGCATCTCCATCACTCTT
CCCTGGTGGATCATGCAGTGGTTCTGCTGTGGCAGTTTCTGCACAACTTGTCGACTTTGCTC
TTGGTACTGATACAACTGGTGATGTAAGAATTCCAGCATGCTTCTGTGGTGTACTTTGTTTC
AAGTCCTCTCATGGGGTTGTATCTACTCTTGGGACCATTGCAAACTCTCAAAGTTTAGACA
CGATTGGATGGTTTGCACGAGATCCAAGTGTTCTGCATCGTGTTGGAGATGTTCTTTTACC
AGCTGCTACAGGTGGGCTTACGCAAACAAGGCAGTTATTTTTTGCTGACGATTGCTTTCAG
TTGCTGAAGGTCCCCAACGAGAAAACAGTAAATGTCATAGAAAATGCTATCCAGACATTA
CCAGGATATCAGCCACCTAAGCACATCAATATTGGTGAGTATATCAGTTCACATGTACCTA
GCCTGAAGGATTTTTGTGAACCCACTGTGGAGATGCTCGAAGGAATGTCAGCCTTGAAAG
CACTCTCAACAGTTATGCTGTTATTACAGAGATATGAATTCAAGACAAACCACGAGGATTG
GGTTAATACTGTAAAACCCAAGCTTGGGCTTGATACCTCTACTCGTGTGCTACAAGCTGTC
AATTCGAAAAGTGATAACATCAAATCTTTATACATTGTACGAAATGAATTGCGAGCTGCAC
TCAAGAATCTTTTAAAGGATACTGGAATTTTAGTTCTGCCAACCACAGCTGGATATCCTTT
AAAGAGGAATGCAAGGCAAAGACTTTCACCTGGGTTTGAAGATAGAATGAGTGCATTTGT
AGGCATTGCTACACTTTCAGGCTGCTGTCAGGCTGTGATTCCCTTAGGAAGTCACAATGAT
CATCCTATATCTCTTTCATTGCTAGCAGCACATGGATCAGACAAATTCCTTCTTCGCAATGT
CTTGTATATGTTTTCTTCCATCAAAGAACAAGTCGTTTTGGCGTCCAAGTTGGTAACTGCAC
CAGTAATCAATAGAGATGCTGATTTTGGTGCAGCAGAGTTGTTGAAAGAGAAGGGAAATA
GTGCTTTCAAAGGACGAAAATGGAGCAAGGCGGTTGAATTTTATTCTGATGCTATCAAATT
GAATGGCACAAATGCAACATATTACAGCAATAGAGCAGCTGCCTATCTGGAACTTGGCCG
TTATAAGCAAGCTGAAGCAGATTGTGAGCAGGCCTTACTCTTGGATAAAAAGAATGTTAA
AGCATATCTACGACGAGGGATCGCAAGAGAAGCTGTTCTGAATCACCAAGAAGCTCTTCA
AGATATCAGGCATGCTTTAGCTTTGGAGCCACAGAACAAAGCAGGCCTTTTGGCAGAGAG
AAGGCTGCAGAAAAAACTAAGGTGA
The aminoacid sequence (SEQ ID NO:2) of TOM64 polypeptide:
MDSSARSGGGGTGGYTSTRVWMVAGVAIAGAIVFVEAARRRRRWLRDRSEVPPDFGAF
CYRFEIAPAPQPPPPAARQLLSGLTFAASDNFEIEGYVAGFGNPDWKRTHKAATRTAVPVTML
QKQGGTYVGSTVMDELGFGVSGGNLHNGTPINPASPSLFPGGSCSGSAVAVSAQLVDFALGTD
TTGDVRIPACFCGVLCFKSSHGVVSTLGTIANSQSLDTIGWFARDPSVLHRVGDVLLPAATGGL
TQTRQLFFADDCFQLLKVPNEKTVNVIENAIQTLPGYQPPKHINIGEYISSHVPSLKDFCEPTVE
MLEGMSALKALSTVMLLLQRYEFKTNHEDWVNTVKPKLGLDTSTRVLQAVNSKSDNIKSLYI
VRNELRAALKNLLKDTGILVLPTTAGYPLKRNARQRLSPGFEDRMSAFVGIATLSGCCQAVIPL
GSHNDHPISLSLLAAHGSDKFLLRNVLYMFSSIKEQVVLASKLVTAPVINRDADFGAAELLKEK
GNSAFKGRKWSKAVEFYSDAIKLNGTNATYYSNRAAAYLELGRYKQAEADCEQALLLDKKN
VKAYLRRGIAREAVLNHQEALQDIRHALALEPQNKAGLLAERRLQKKLR
Pest-resistant phenotype after embodiment 3, TOM64 gene pairs mutant genetics complementation
The pest-resistant phenotype of mutant is further verified by the present inventor by genetic complementation experiment.
The promoter of TOM64 gene is connected into pCAMBIA 2300 carrier together with its coding region, passes through Agrobacterium
Mediated transformation tom64 mutant.
By the detection of RNA, the present inventor has successfully obtained three independent complementary transgenic paddy rice strains
(R1, R2 and R3) (Fig. 4 A).
Further brown planthopper resistant is identified and is shown that the resistance of the complementary transfer-gen plant proceeding to TOM64 gene is bright
Aobvious less than tom64 mutant, with wild type quite (Fig. 4 B), it was demonstrated that the phenotype of the anti-BPH of this mutant
It is owing to the disappearance of TOM64 gene causes.
Embodiment 4, the pest-resistant Mechanism Study of tom64 mutant
The insect resistace of plant can be realized by three aspects: non-preferendum (non preference), antibiosis and resistance to
Worm property (tolerance to insects).Selectivity test result between mutant tom64 and wild type ZH11 shows,
In the time of release brown paddy plant hopper 1 to 96h, brown paddy plant hopper number on wild type and mutant is close, and
(Fig. 5 A) is there was no significant difference at each time point.It addition, the test result display brown paddy plant hopper of egg laying amount
Egg laying amount on mutant slightly increases in wild type than it, but there was no significant difference (Fig. 5 B).This
Illustrate what the resistance of brown paddy plant hopper was realized by mutant tom64 not by non-preferendum.
The excretion of honeydew (brown paddy plant hopper Excreta) can point out the food ingestion of brown paddy plant hopper.By measuring brown paddy plant hopper
Honeydew excretion, the inventors discovered that, the brown paddy plant hopper taken food in wild rice, its honeydew amount is bright
The aobvious honeydew amount (Fig. 6 A) taking food mutant generation more than brown paddy plant hopper.It addition, compare wild type, mutant
Also significantly suppress the growth rate (Fig. 6 B) of brown paddy plant hopper.But, dead to brown paddy plant hopper in different plants
The mensuration of rate is but not significantly different from (Fig. 6 C).
These results explanation mutant tom64 can suppress taking food and growing of brown paddy plant hopper, but TOM64
Brown paddy plant hopper is not enough to lethal by disappearance, thus illustrates, mutant brown planthopper resistant passes through to a certain extent
Antibiosis realizes.The resistance of brown paddy plant hopper is had by phenotypic evaluation display mutant before relative to wild type
The raising of highly significant.
Further tolerance to insects experimental analysis shows, the coefficient of resistance to worm of tom64 mutant is substantially less than wild type
The coefficient of resistance to worm (Fig. 7) of ZH11, and the coefficient of resistance to worm is the lowest, tolerance to insects is the strongest, it was demonstrated that mutant flies brown
The tolerance to insects of louse has had obvious raising.
The analysis of the most pest-resistant mechanism, it was demonstrated that mutant tom64 is to pass through antibiosis to the resistance of brown paddy plant hopper
Play a role with tolerance to insects two aspect simultaneously.
Embodiment 5, the Subcellular Localization of TOM64 gene
Subcellular location has important guiding effect to research protein function.Arabidopsis TOM64 is found
Special it is positioned in mitochondrion.The present inventor utilizes the instantaneous table of Oryza sativa L. PA7-TOM64-GFP fusion protein
Take things philosophically and examine its Subcellular Localization.By control vector PA7-GFP and fusion vector rice transformation protoplasm respectively
Body, confocal microscope observation finds PA7-GFP ubiquitous expression in cell membrane and kytoplasm, and
Fusion protein is random distribution (Fig. 8 A) in kytoplasm.Further with mitochondrial specific dye Mitotracker
Dye, finds that the fluorescence of the two can well position (Fig. 8 B) altogether, it was demonstrated that the TOM64 polypeptide of Oryza sativa L. is also altogether
It is the most special being positioned in mitochondrion, consistent with prediction.
Embodiment 6, the disappearance of TOM64 gene improve rice tissue H2O2Content
TOM64 is the transport molecule of mitochondrial outer membrane, and its disappearance can affect the transhipment of a series of mitochondrial protein
Enter mitochondrion.And mitochondrion carries out aoxidizing Repiration, it it is the main cell device of plant generation active oxygen
One of.The present inventor, before and after brown paddy plant hopper takes food, determines H in rice leaf sheath respectively2O2Content, knot
Fruit finds, H in wild rice ZH112O2Content take food brown paddy plant hopper and front and back there is no significant change,
And H in mutant2O2Content can be by notable induction (Fig. 9) brown paddy plant hopper takes food 6h when.More important
, no matter before and after brown paddy plant hopper takes food, H in mutant tom642O2Content will be significantly higher than open country
Content (Fig. 9) in raw type.And H2O2It it is an important molecule in Oryza sativa L. resistance defense reaction.This explanation
The disappearance of TOM64 significantly improves H in rice tissue2O2Content, prompting which thereby enhance Oryza sativa L. to brown
The resistance of plant hopper.
The evaluation to Brown Planthopper Resistance of embodiment 7, mutant and wild type
In order to verify the phenotype of Rice Resistance brown paddy plant hopper further, the present inventor is respectively to wild type (wt), sudden change
Body (mutant), Heterozygous mutants offspring (Co-M, Co-WT, Co-H) and three complementary transgenic line
System (R1, R2, R3) has carried out the brown planthopper resistant level evaluation of indoor in seedling stage, when sense worm comparison TN1 is whole
Being evaluated time dead, evaluation criterion is: 0 grade the most killed for rice shoot；1 grade, the first leaf part jaundice；
3 grades, the first leaf and the jaundice of the second leaf part；5 grades, significantly turn to be yellow, some dwarfing of plant；7 grades, plant
Strain atrophy or seriously downgrade；9 grades, plant is dead.Result shows, wild type ZH11,3 complementary
Transgenic line and be divided into from heterozygote and wild type, their resistance rank is all 7-9, mutant
Tom64 and be divided into from the resistance rank of pure and mild mutant be 3-5, the resistance rank of pest-resistant comparison IR56
For 1-3 (Figure 10).
These results fully prove, the disappearance of TOM64 gene plays pass in raising Oryza sativa L. is to BPH resistance
The effect of key.
Embodiment 8, the impact on rice yield traits of the TOM64 gene delection
In order to further determine that the impact on rice yield traits of the TOM64 gene delection, the present inventor passes through
The mode of mutant tom64 selfing is obtained the pure lines of TOM64 gene delection (i.e. gene is knocked),
It is planted in indoor and field, and the character that its yield is relevant is observed and measured.
It was found that TOM64 gene is knocked rear rice tillering in advance (Figure 11 A), rice plants is obvious
Become short (Figure 11 B).TOM64 gene is knocked plant compared with wild rice, the ratio of productive tiller of the two
It is not significantly different from (Figure 11 C).Owing to the whole tiller number of the pure lines of TOM64 gene delection significantly increases,
Therefore, TOM64 gene is knocked the effective tillering quantity of rear Oryza sativa L. and significantly increases.And, the present inventor
Relatively finding, the mass of 1000 kernel of pure lines Oryza sativa L. and the grain number of every fringe of TOM64 gene delection are not significantly different from,
And the increase of tiller number promotes the increase of fringe of man power single stem rice, therefore, knock out this gene pairs and improve Oryza sativa L.
Yield has effect.
Sum up
1, present invention demonstrates that tom64 mutant improves Oryza sativa L. and resists Delphacidae insecticide (including brown paddy plant hopper)
Property.
2, pest-resistant study mechanism shows that this gene mutation inhibits the food ingestion of Delphacidae insecticide, thus improves
The Oryza sativa L. tolerance to insects to Delphacidae insecticide.
3, TOM64 gene delection significantly improves H in rice tissue2O2Content, therefore improve water
The rice resistance to Delphacidae insecticide.
4, the resistance of Delphacidae insecticide can be carried by tom64 mutant from original high sense level (7-9 level)
High to medium resistance level (3-5 level).
The all documents mentioned in the present invention are incorporated as reference the most in this application, just as each literary composition
Offer and be individually recited as with reference to like that.In addition, it is to be understood that reading the above-mentioned teachings of the present invention
Afterwards, the present invention can be made various changes or modifications by those skilled in the art, and these equivalent form of values are same
Fall within the application appended claims limited range.
Claims (10)
1. one kind is improved plant resistance to insect, reduction plant plant height, increase plant tillering or improves plant products
Method, it is characterised in that described method includes: lower expression or the work of TOM64 polypeptide in plant
Property；The genome of described plant has TOM64 gene.
2. the method for claim 1, it is characterised in that TOM64 in described downward plant
The expression of polypeptide or the method for activity include: knock out in the genome of plant or reticent TOM64 gene；
Or
The lower adjustment lowering TOM64 genetic transcription, expression of polypeptides or polypeptide active is proceeded in plant.
3. method as claimed in claim 2, it is characterised in that described lower adjustment is specificity interference
The disturbing molecule of TOM64 gene expression.
4. method as claimed in claim 3, it is characterised in that described disturbing molecule is TOM64
Gene or its transcript are suppression or the reticent dsRNA of target, antisensenucleic acids, siRNA, small
RNA, maybe can express or be formed described dsRNA, antisensenucleic acids, siRNA, Microrna
Construction.
5. the method for claim 1, it is characterised in that described raising plant resistance to insect is to carry
High plant is for the resistance of Delphacidae insecticide.
6. the method for claim 1, it is characterised in that described plant includes: grass family is planted
Thing.
7. the method for claim 1, it is characterised in that described TOM64 polypeptide is selected from down
Group:
The albumen of (a) such as SEQ ID NO:2 aminoacid sequence；
(b) by SEQ ID NO:2 aminoacid sequence through one or more amino acid residues replacement, lack
Lose or add and formed, and there is the albumen derivative by (a) of (a) protein function；Or
C protein sequence that () and (a) limit has more than 70% homology and has being spread out by (a) of (a) protein function
Raw albumen.
8. method as claimed in claim 7, it is characterised in that the encoding gene of described TOM64
Comprise following sequence: SEQ ID NO:1.
9. reduce a purposes for the material of TOM64 gene expression or activity, for improving the pest-resistant of plant
Property, or it is used for reducing plant plant height, or it is used for increasing plant tillering, or it is used for improving plant products, or
For improving H in plant tissue2O2Content；The genome of described plant has TOM64 gene.
10. purposes as claimed in claim 9, it is characterised in that described reduction TOM64 gene table
Reach or the material of activity is the disturbing molecule that specificity disturbs TOM64 gene expression；It is preferred that it is described
Disturbing molecule be TOM64 gene or its transcript be suppression or the reticent dsRNA of target, antisensenucleic acids,
SiRNA, Microrna, maybe can express or be formed described dsRNA, antisensenucleic acids, little interference
RNA, the construction of Microrna.
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| PCT/CN2016/084928 WO2016197887A1 (en) | 2015-06-10 | 2016-06-06 | Gene for negatively regulating resistance of plant against delphacidae insects and use thereof |
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| JP2021029248A (en) * | 2019-08-21 | 2021-03-01 | 広東省農業科学院植物保護研究所 | Paddy rice pest resistance regulatory protein, gene encoding the same, and uses thereof |
| CN112522297A (en) * | 2019-09-19 | 2021-03-19 | 中国科学院分子植物科学卓越创新中心 | Novel gene for regulating and controlling insect-resistant character of plant and application thereof |
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| CN110951748B (en) * | 2019-12-16 | 2021-10-22 | 武汉大学 | Rice brown planthopper resistant gene Bph37, protein, vector, host cell, molecular marker, method and application |
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| CN101356188A (en) * | 2005-11-08 | 2009-01-28 | 克罗普迪塞恩股份有限公司 | Plants having improved growth characteristics and a method for making the same |
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| CN101356188A (en) * | 2005-11-08 | 2009-01-28 | 克罗普迪塞恩股份有限公司 | Plants having improved growth characteristics and a method for making the same |
Non-Patent Citations (2)
| Title |
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| GENBANK: "BAD17240", 《GENBANK》 * |
| 胡远辉等: "甘油型油菜Toc33基因编码区的分离及蛋白序列的比较", 《西北植物学报》 * |
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| JP2021029248A (en) * | 2019-08-21 | 2021-03-01 | 広東省農業科学院植物保護研究所 | Paddy rice pest resistance regulatory protein, gene encoding the same, and uses thereof |
| CN112522297A (en) * | 2019-09-19 | 2021-03-19 | 中国科学院分子植物科学卓越创新中心 | Novel gene for regulating and controlling insect-resistant character of plant and application thereof |
| CN112522297B (en) * | 2019-09-19 | 2023-01-31 | 中国科学院分子植物科学卓越创新中心 | Gene for regulating and controlling insect-resistant character of plant and application thereof |
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