CN110272911A - Application of the AOX1a gene in terms of improving drought resistance in plants - Google Patents

Application of the AOX1a gene in terms of improving drought resistance in plants Download PDF

Info

Publication number
CN110272911A
CN110272911A CN201910603825.5A CN201910603825A CN110272911A CN 110272911 A CN110272911 A CN 110272911A CN 201910603825 A CN201910603825 A CN 201910603825A CN 110272911 A CN110272911 A CN 110272911A
Authority
CN
China
Prior art keywords
aox1a
gene
plant
plants
ala
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN201910603825.5A
Other languages
Chinese (zh)
Inventor
林宏辉
张大伟
乔康
邓星光
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Sichuan University
Original Assignee
Sichuan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Sichuan University filed Critical Sichuan University
Priority to CN201910603825.5A priority Critical patent/CN110272911A/en
Publication of CN110272911A publication Critical patent/CN110272911A/en
Pending legal-status Critical Current

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
    • C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0004—Oxidoreductases (1.)
    • C12N9/0055—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10)
    • C12N9/0057—Oxidoreductases (1.) acting on diphenols and related substances as donors (1.10) with oxygen as acceptor (1.10.3)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y110/00—Oxidoreductases acting on diphenols and related substances as donors (1.10)
    • C12Y110/03—Oxidoreductases acting on diphenols and related substances as donors (1.10) with an oxygen as acceptor (1.10.3)
    • C12Y110/03011—Ubiquinol oxidase (1.10.3.11)

Abstract

The present invention relates to gene engineering technology fields, disclose application of the AOX1a gene in terms of improving drought resistance in plants.The AOX1a gene is applied to improve the drought tolerance of plant, for the nucleotide sequence of the AOX1a gene as shown in SEQ ID NO:1, the plant is tomato.AOX1a gene participates in the drought tolerance of ethylene induction, and can be improved the drought tolerant of plant.The present invention provides a kind of Gene A OX1a of new raising drought resistance in plants, and transgenic engineering is had a good application prospect.

Description

Application of the AOX1a gene in terms of improving drought resistance in plants
Technical field
The present invention relates to gene engineering technology fields more particularly to a kind of AOX1a gene in terms of improving drought resistance in plants Application.
Background technique
Agricultural production is basis and the important component of social economy and substance production, and wherein plant production is raw in agricultural Occupy important fundamental position in production again.It may be subjected to various abiotic stress in growing process, to influence its yield.Now Global drying trend is serious, and drought stress, which becomes, leads to one of major stress of crop production reduction.Existing wild-type plant For showing lower tolerance under environment-stress, especially drought stress conditions.
Drought stress is to influence one of plant growth and development, main abiotic stress factor of yield and quality.However, planting Object drought resistance is the quantitative character by controlled by multiple genes, and monogenic conversion can not significantly improve the resistance of crop, this is also tight The development of the resistance crop of high yield is limited again.Drought stress can cause the variation of lots of genes product, to these genes of regulation Transcription factor and unlike signal approach crosstalk understand the improvement that will be helpful to crop Comprehensive Traits in depth.Change in crop During good, how to create that growth is normal, yield is unaffected, turns with stronger stress tolerance using existing achievement How the foreign gene being transferred to is preferably minimized the interference of crop self mechanism by gene plant, and activates and coerce in proper time How the expression for compeling induced gene makes stress inducible gene result from ideal cell tissue position and has suitable expression quantity Deng being all our problems in need of consideration when creating genetically modified plants.
Summary of the invention
The technical problem to be solved by the present invention is to improve the drought tolerance of plant from gene level, a kind of AOX1a gene is provided Application in terms of improving drought resistance in plants.
To achieve the goals above, the technical solution adopted by the present invention is that providing a kind of AOX1a gene to improve plant resistance to The AOX1a gene is applied to improve the drought tolerance of plant, the nucleotides sequence of the AOX1a gene by the application in terms of drought For column as shown in SEQ ID NO:1, the plant is tomato.
Further, the purposes includes cultivating the tomato plant with drought resistance enhancing purpose character.
The present invention also provides a kind of methods for improving drought resistance in plants, including by transgenic technology, AOX1a gene is existed It is overexpressed in plant, for the nucleotide sequence of the AOX1a gene as shown in SEQ ID NO:1, the plant is tomato.
Further, it the described method comprises the following steps: the building of AOX1a Overexpression vector；Kind of mediated by agriculture bacillus The genetic transformation of eggplant；Transgenic plant identification.
Further, the method also includes following steps: carrying out drought stress processing to the transgenic plant, sees Plant drought resisting phenotype is examined to be screened.
The beneficial effects of the present invention are improve drought resistance in plants by the way that AOX1a gene to be applied to, solve existing The problem of wild-type plant is for showing lower tolerance under environment-stress, especially drought stress conditions.After improvement Tomato plant strain growth is normal, yield is unaffected, has stronger stress tolerance.
Detailed description of the invention
Fig. 1 is the experimental technique route map of the embodiment of the present invention；
Fig. 2 is alignment's figure of each hypotype (AOX1a, AOX1b, AOX2) of AOX gene；
Fig. 3 is AOX1a Overexpression vector and AOX-RNAi silent carrier building schematic diagram；
Fig. 4 is that pMD19-T intermediate vector digestion identification and the connection of PBI121 expression vector are identified with digestion；
Fig. 5 is the AOX1a gene expression that plant is overexpressed using the method analysis wild-type tomatoes and AOX1a of qRT-PCR Amount；
Fig. 6 is the AOX1a gene expression amount using method the analysis wild-type tomatoes and RNAi plant of qRT-PCR；
Fig. 7 is the photo of the phenotype of the 14th day pretreated tomato plant of ACC of drought stress；
Fig. 8 is the relative water content measurement to wild type and Transgenic Tomato Plants；
Fig. 9 is the conductivity miss rate measurement to wild type and Transgenic Tomato Plants；
Figure 10 is the content of hydrogen peroxide measurement to wild type and Transgenic Tomato Plants；
Figure 11 is to carry out QT-PCR analysis to the expression variation of the marker gene P5CS1 of drought stress response pathway；
Figure 12 is that the variation to the marker gene NCED3 of drought stress response pathway carries out QT-PCR analysis；
Figure 13 is to carry out QT-PCR analysis to the expression variation of the marker gene RD29A of drought stress response pathway；
Figure 14 is that the embodiment of the present invention carries out the expression variation of the marker gene RD29B of drought stress response pathway QT-PCR analysis；
Figure 15 is fluorescence quantification PCR primer list.
Specific embodiment
To make the objectives, technical solutions, and advantages of the present invention clearer, below with reference to embodiment and attached drawing, to this Invention is described in further detail.
The embodiment of the present invention provides a kind of Gene A OX1a (Alternative oxidase1a) for improving drought resistance in plants, The nucleotides sequence of the gene is classified as shown in SEQ ID NO:1, and the nucleotides sequence is classified as CDS sequence, the protein of coding Amino acid sequence is as shown in SEQ ID NO:2.AOX1a gene is applied to cultivate and turns base with drought resistance enhancing purpose character Because of plant, tomato plant strain growth after improvement is normal, yield is unaffected, has stronger stress tolerance.
The experimental technique route of the embodiment of the present invention is as shown in Figure 1, comprising the following steps:
The clone of 1 target gene
The extraction of 1.1 tomato total serum IgEs
1. 1g tomato leaf is taken to be placed in sterilized mortar, suitable liquid nitrogen is added and quickly grinds；
2. the material after grinding is transferred in 1.5mL centrifuge tube, the mixed liquor of 500 μ L phenol, chloroform, isoamyl alcohol is added (phenol: chloroform: the volume ratio of isoamyl alcohol is 25:24:1), 500 μ L RNA extracting solutions [20mM Tris-HCl (pH 8.0), 1% (W/V) SDS, 200mM NaCl, 5mM EDTA] and 2v% beta -mercaptoethanol, the 200 anhydrous second of μ L are finally added into centrifuge tube again Alcohol；30s or so is mixed on vortex oscillator is denaturalized protein sufficiently；
3. 4 DEG C, 12000rpm, being centrifuged 10min；
4. taking supernatant, the 8M LiCl, -20 DEG C of precipitating 30min of 1/2 volume is added；
5. 4 DEG C, 12000rpm, being centrifuged 10min, precipitating is collected；
6. precipitating is cleaned 1~2 time with 70v% ethyl alcohol, it is dried in vacuo；
7. 50 μ L ddH are added2O is stored in -20 DEG C of refrigerators, spare.
1.2 reverse transcriptions synthesize cDNA
All RNA carried out digestions with Dnase I before reverse transcription and remove DNA.Then SuperScript is used IIIReverse Transcriptase (Invitrogen) carries out reverse transcription in 20 μ L systems, synthesizes the first chain cDNA.Tool Steps are as follows for body:
1. 4 μ L RNA are added into 0.5mL EP pipe, 1 μ L Oligdt, 1 μ L dNTP, to 14 μ L, of short duration centrifugation is mixed for moisturizing After even, 65 DEG C of 5~10min of water-bath；Cooled on ice is set in taking-up；
2. 4 μ L 5 × buffer, 1 μ L DTT, 0.5 μ L Rnaseout are added, mix, 37 DEG C of water-bath 2min；
3. 0.5 μ L M-MLV, 37 DEG C of water-bath 1h is added；
4. 70 DEG C of water-bath 15min terminate reaction.
The amplification of 1.3 AOX genetic fragments
In order to construct AOX1a overexpression vector, referring to AOX1a gene (accession number on NCBI AY034148 primers) expand its whole open reading frame (Open reading frame, ORF).Specific primer Sequence is as follows:
AOX1a gene magnification primer:
AOX1a-F:5'-TATTTGCCTTCTTCCTCAAGTTTC-3'
AOX1a-R:5'-AAAAAGGAACAAAATAGTGACGGAC-3'
The AOX1a gene magnification primer of the restriction enzyme site containing BamHI and ECoRI:
AOX1a-F:5'-CGCGGATCCTATTTGCCTTCTTCCTCAAGTTTC-3'(BamHI)
AOX1a-R:5'-CCGGAATTCAAAAAGGAACAAAATAGTGACGGAC-3'(ECoRI)
PCR amplification is carried out using TAKARA Primer STAR HS high fidelity enzyme.PCR amplification system: totally 50 μ L, wherein 5 μ L 10 × rTaq DNA buffer, 3 μ L MgCl2(2.5mmolL-1), template are 2 μ L, upstream and downstream primer each 2 μ L, 1 μ L DNTP (the 2.5mmolL of rTaq archaeal dna polymerase (2.5U/ μ L) and 2 μ L-1), then plus ddH2O to 50 μ L.
Response procedures are as follows: 95 DEG C of initial denaturation 3min, 30 circulations of following reaction (94 DEG C, 1min；54 DEG C, 30s；72 DEG C, 50s), last 72 DEG C of extensions 10min.
In order to construct AOX silent carrier, this experiment first to each hypotype of AOX gene (AOX1a, AOX1b and AOX2) sequence into Row compares, and takes homologous sequence design primer (Fig. 2), specific as follows:
Positive segment:
AOX-UF:5'-TGTATTTTTTCAGAGGAGATATGGT-3'
AOX-UR:5'-ATTATTAGTCGCTTAGTGATACCCA-3'
Reversed segment:
AOX-DF:5'-AGAGCAATGATGTTAGAGACAGTGG-3'
AOX-DR:5'-GCTTAGTGATACCCAAGTGGTGCTG-3'
Introduce the primer of restriction enzyme site:
Positive segment:
AOX-UF:5'-CCCCCGGGGGTGTATTTTTTCAGAGGAGATATGGT-3'(SmaI)
AOX-UR:5'-CGCGGATCCATTATTAGTCGCTTAGTGATACCCA-3'(BamHI)
Reversed segment:
AOX-DF:5'-CCCCCGGGGGAGAGCAATGATGTTAGAGACAGTGG-3'(SmaI)
AOX-DR:5'-CCGGAATTC GCTTAGTGATACCCAAGTGGTGCTG-3'(EcoRI)
For PCR amplification system with described previously, annealing temperature is respectively as follows: 53 DEG C of positive segment；Reversed 54 DEG C of segment.
2 vector constructions (as shown in Figure 3)
2.1 PCR product purification and recoveries
It is recycled using PCR product purification kit (Shanghai Sangon Biotech Company) and purifies amplified production.
Specific purification step is as follows:
1. the gel cut is weighed, isometric B2 sol solutions, 50 DEG C of water-bath 8min are added.It runs up and down during water-bath It mixes, until being completely dissolved glue.
2. lysate is transferred in purification column, 9 000rpm are centrifuged l min, discard and throw away liquid.
3. 500 μ L rinsing liquids are added, it is placed at room temperature for 3min, then 9000rpm is centrifuged l min, discards and throws away liquid.
4. being repeated once step 3..
5. 12000rpm is centrifuged 2min at room temperature, the residual liquid in column is dried.
6. 30~50 μ L ddH2O are added, it is placed at room temperature for 5min, then 12000rpm is centrifuged 2min, and collection throws away liquid.
2.2 being connect with pMD19-T carrier
It is carried out according to pMD19-T carrier specification.Following component is sequentially added in 0.5mL PCR pipe, then in 14 DEG C It is connected overnight in thermostat water bath.It is specific as follows:
DNA fragmentation (2 μ g/ μ L) 4.5 μ L of purifying
Solution I 5μL
pMD19-T vector 0.5μL
10 μ L of total volume.Connecting pMD19-T carrier is as intermediate vector, and subsequent be connected on expression vector carries out Operation.It is specific as follows:
By pMD19-T carrier BamHI/EcoRI double digestion, large fragment is recycled.While to the successful AOX1a's of identification After PCR fragment BamHI/EcoRI double digestion, it is attached and is reacted with the pMD19-T carrier large fragment of recycling with T4 ligase, As AOX1a intermediate vector.In addition, with BamHI/SmaI and SmaI/EcoRI respectively to the positive segment of AOX, reversed segment into It after row double digestion, is attached and is reacted with the pMD19-T carrier large fragment of recycling with T4 ligase, carried among as AOX-RNAi Body.
The digestion of the building of 2.3 plant expression vectors --- plasmid and connection (Fig. 4)
2.3.1 the double digestion of PBI121 plasmid
Double digestion is carried out to expression vector PBI121 with BamHI and ECoRI, recycles target fragment.Concrete operation method is such as Under: 5 μ L of plasmid, 1 BamHI μ L, 1 EcoRI μ L, 10 × Q cut buffer, 2 μ L, dd H211 μ L of O, reaction total volume are 20 μ L, 37 DEG C of water-baths are stayed overnight after mixing.Agarose gel electrophoresis detects digestion as a result, then using gel reclaims kit (GIANGEN, Beijing) recycles purpose segment, spare.
2.3.2 the building of AOX1a Overexpression vector
1. carrying out double digestion to pMD19-T plasmid with BamHI and ECoRI, target gene fragment is recycled.
It is reacted 2. the target gene segment of recycling is attached with PBI121 recycling segment with T4 ligase.In view of inserting The molar ratio difference entered between target gene and carrier DNA is larger, therefore template and the sample-adding amount of carrier will be according to recycling pieces Disconnected concentration adjusts.
10 μ L of target gene segment
PBI121 recycles 2 μ L of segment
T4DNA ligase 1μL
10×buffer 2μL
dd H2O 5μL
After the above sample blending, 14 DEG C of air bath connections are overnight.In case conversion is used.
2.3.3 the building of AOX-RNAi expression vector
1. carrying out double digestion to pMD19-T plasmid with BamHI/EcoRI, target gene fragment is recycled.
It is reacted 2. the target gene segment of recycling is attached with PBI121 recycling segment with T4 ligase.Concrete operations It is identical as AOX1a Overexpression vector connection.
After the above sample blending, 14 DEG C of air bath connections are overnight.In case conversion is used.
2.4 conversion Escherichia coli
1. 10 μ L connection products are added in 100 μ L competent cells, gently shake up, place 30min on ice；
2. heat shock 90s in 42 DEG C of water-baths, is immediately placed in 3~5min of cooled on ice after heat shock；
3. 200 μ L LB liquid mediums are added into pipe, after standing l min, 37 DEG C of culture l h of shaking table are put it into, are made Bacterium restore normal growth.
4. taking out 5000g is centrifuged l min, supernatant is abandoned；200 μ L LB liquid mediums are added.
It is screened 5. being coated on after above-mentioned bacterium solution is shaken up on the solid LB media containing 50mg/mL antibiotic, 37 DEG C of trainings It supports overnight.
The identification of 2.5 recombinant plasmids
Single colonie is chosen after bacterium colony is grown and carries out recombinant plasmid identification, including bacterium colony (bacterium solution) PCR selects positive strain, mentions It takes plasmid order-checking and carries out sequence alignment.Specific step is as follows:
1. choosing single colonie (5~10) to be inoculated into the LB liquid (3~5mL) containing antibiotic, 37 DEG C of shaking tables (200rpm) shake culture 10~12 hours, the bacterium solution after culture carry out bacterium solution PCR, and whether identification is successfully connected to load for segment On body；
2. taking 1 μ L bacterium solution as template, with target fragment cloning primer and section 2 × TsingKe Master Mix is held up (Red, TSE001) is that reactive component carries out bacterium solution PCR, and PCR response procedures are 95 DEG C of initial denaturation 3min, next reaction 30 Circulation (94 DEG C, 1min；54 DEG C, 30s；72 DEG C, 50s), last 72 DEG C of extensions 10min；
3. PCR product is taken to carry out agarose gel electrophoresis, choose with the consistent bacterial strain of positive control purpose band as sun Property clone；
4. positive colony bacterial strain bacterium solution a part is saved strain, another part extracts plasmid or is fed directly to sequencing company It is sequenced；
5. sequencing sequence is compared with objective gene sequence, retains the sun of exact matching after sequencing result return Property bacterial strain as constructing successful recombinant vector.
Also it can extract plasmid and carry out plasmid PCR, the extraction of plasmid is carried out by plasmid extraction kit (Shanghai Sangon Biotech Company), It is specific as follows:
1. the bacterium solution for taking 4mL to be incubated overnight, 12000rpm is centrifuged 1min, abandons supernatant to the greatest extent；
2. being precipitated with the buffer P1 suspended bacterial that 250 μ L have been added to RNase A, suspending should uniformly, should not there are small Fungus block.
3. 250 μ L buffer P2 are added, spin upside down mildly and fully mixing 4~6 times, cracks thallus sufficiently, Until forming bright solution.This step is no more than 5min；
4. 350 μ L buffer P3 are added, spin upside down mildly and fully mixing 6~8 times, 12000rpm centrifugation 10min；
5. aspiration step 4. in supernatant and be transferred in DNA adsorption column, 12000rpm be centrifuged 1min, abandon filtrate；
6. 500 μ L rinsing liquids are added into adsorption column, 12000rpm is centrifuged 30s, abandons filtrate；
7. repeating step 6.；
8. 12000rpm is centrifuged 2min to eliminate rinsing liquid；
9. 30 μ L ddH are added2O, is stored at room temperature 3min, and 12000rpm is centrifuged 1min, eluted dna.
If PCR result is correct, sequencing company is sent to be sequenced bacterium solution or plasmid, all sequencings of this experiment are by Shanghai Sheng Gong company completes.
2.6 Agrobacterium-mediated Transformation
Correct plasmid is sequenced and carries out Agrobacterium-mediated Transformation, the specific method is as follows: the sense of 50uL Agrobacterium is added in the plasmid of 1.5uL By state cell (GV3101), 30min, liquid nitrogen frozen 5min are placed on ice.37 DEG C of water-bath heat shock 5min, are added the LB liquid of 1mL Culture medium is put into shaking table, and shaken cultivation 3-4h under the conditions of 28 DEG C, 3000rpm are centrifuged 3min and abandon 800uL supernatant, and 200uL is mixed Coated plate is dried, is sealed, 1-2d are cultivated in 28 DEG C of inversions.
The genetic transformation of the tomato of 3 mediated by agriculture bacillus
The preparation of 3.1 explants
1. the disinfection of seed
By tomato seeds with after aqua sterilisa repeated flushing 3~5 times, 1min, aqua sterilisa rinsing 3~5 are impregnated with 75v% alcohol It is secondary；Then 8min is impregnated with 1% (W/V) sodium hypochlorite, then is rinsed 3~5 times with aqua sterilisa, sowed on MS culture medium.
It is cultivated 2. the seed after disinfection is placed in 26 DEG C (16h illumination)/18 DEG C of (8h is dark) illumination boxs.
3. after germination, rough leaf not yet eruption when, cut cotyledon for genetic transformation test.
3.2 preculture
1. cotyledon both ends are cut away with sterilized blade, leave and take center portion, it is of length no more than in 1cm.
2. explant face down is placed on pre-culture medium.
3. 26 DEG C, 1~2d is cultivated under dark condition (or low-light).
The preparation of 3.3 recombinational agrobacteriums
Identified recombinational agrobacterium is taken, 4mLLB fluid nutrient medium (Kana containing 50mg/L and 50mg/L Rif) is inoculated in In, 28 DEG C of concussion (200rpm) cultures.When OD600 reaches 0.1~0.5, takes out and collects thallus (3000rpm is centrifuged 5min), It is resuspended again with suitable induced medium, dilution thallus, makes OD600 value 0.1 or so, for transfecting.
3.4 Agrobacterium infections and co-cultivation
By the explant after preculture, it is dipped into the diluted Agrobacterium bacterium solution of induced medium, is taken out after 15min, the phase Between ceaselessly oscillation mix.Extra bacterium solution is sucked with sterilized filter paper, is then placed in and is co-cultured in base, sealing, dark item 2d is co-cultured under part.
3.5 regeneration cultures (induction Multiple Buds)
After dark culturing 2d, explant is gone on the screening and culturing medium containing corresponding antibiotic, is placed in 26 DEG C of (16h light According to) cultivate in/18 DEG C of (8h dark) illumination boxs, until growing Multiple Buds.One subculture of every 2~3 weeks of period replacement.
3.6 culture of rootage
The well-grown Multiple Buds for the green that antagonism screening obtains, cut budlet with sterilized blade, are transferred to Screening is cultivated in root media.It is equally placed in 26 DEG C (16h illumination)/18 DEG C of (8h is dark) illumination boxs and cultivates, until Young shoot, which is taken root, to grow tall.The positive transformants seedling of acquisition is identified for PCR.
4. transgenic plant is identified
The extraction of 4.1 transgenic plant total DNAs
Well-grown resistant transgenic plant is chosen, extracts total DNA, the specific method is as follows:
1. taking 1g tomato leaf, liquid feeding nitrogen is quickly pulverized, and is transferred in 2mL EP pipe, and 650 μ L CTAB are added and extract Liquid, 10 μ L beta -mercaptoethanols, mixing of turning upside down.
2. being placed in 65 DEG C of water-bath 45min, during which turn upside down (should not too acutely, slow jog) several times.
3. taking out, 5min on ice is put.
4. 650 μ L chloroforms are added: isoamyl alcohol (volume ratio 24:1) is mixed by inversion, and makes it at milkiness shape.
5. 12000rpm is centrifuged 10min, takes supernatant.
6. 500 μ L chloroforms are added: isoamyl alcohol (volume ratio 24:1) is mixed by inversion, 12000rpm, is centrifuged 10min, is taken Clearly.
7. the dehydrated alcohol of 2 times of volumes is added, -20 DEG C are placed 2 hours or are stayed overnight.
8. 12000rpm is centrifuged 10min, precipitating is collected, is cleaned twice with 70v% ethyl alcohol.
9. being dried in vacuo, add 100 μ L ddH2DNA precipitating is resuspended in O.Add 2 μ L RNase (10 μ g/mL), 37 DEG C of water-baths 1h.200 μ L chloroforms are added after taking-up: isoamyl alcohol (volume ratio 24:1) is mixed by inversion, 12000rpm, is centrifuged 10min, is taken Clearly.
10. the dehydrated alcohol of 2 times of volumes is added, -20 DEG C are placed 2 hours or are stayed overnight.12000rpm is centrifuged 10min, collects Precipitating, is cleaned twice with 70v% ethyl alcohol.Vacuum drying, adds the ddH of 30~50 μ L2DNA precipitating is resuspended in O, is stored in -20 DEG C of ice It is spare in case.
The Total RNAs extraction of 4.2 transgene tomatos
Method is the same as 1.1
The resistant gene PCR of 4.3 transgenic plants is detected
It is specific as follows with neomycin phosphotransferase (NPTII) gene design primer:
Upstream NPTII-F:5'-GAGAGGCTATTCGGCTATG-3'
Downstream NPTII-R:5'-CTCAGAAGAACTCGTCAAGA-3'
PCR reaction condition are as follows: 94 DEG C, 5min；It is following 35 circulation (94 DEG C, 50s；58 DEG C, 30s；72 DEG C, 50s)；Most 72 DEG C of extension 10min afterwards.
4.4 analyze transgene tomato AOX1a gene expression amount using the method for qRT-PCR.It filters out Gene A OX1a and crosses table Up to AOX-RNAi silencing strain.
The total serum IgE that wild-type tomatoes WT and AOX1a are overexpressed plant and RNAi plant is extracted, is analyzed using qRT-PCR The gene expression amount of AOX1a.Transgene tomato and screening and identification are constructed by Agrobacterium infection technology and tissue culture technique. As shown in Figure 5, Figure 6,6 overexpression strains and 4 silencing strains are obtained by screening one.Wherein, " Sl " is The abbreviation of Solanumlycopersicum (tomato), SlAOX1a-OE are that the AOX1a of tomato is overexpressed strain.It is tied by qPCR Shown in fruit, the AOX1a of 6 overexpression strains has significant up-regulation in transcriptional level, nothing if other members such as AOX1b and AOX2 Significant change, and it is respectively #2 and #5 (OE-2#, OE-5#) that AOX1a, which raises most significant 2 overexpressions strain, wherein OE-2# Middle AOX1a has raised about 5 times or so, and AOX1a has raised about 4 times or so in OE-5#.In silencing plant, AOX1a, AOX1b and AOX2 has a different degrees of downward, silencing efficiency most significantly two strains of 13# and 19#.
5 Drought Resistance Analysis
The tomato seedling grouping for growing to 3-4 weeks size is taken, one group is 3 disks, and a disk is 20 basins.ACC (1- amino-cyclopropane Carboxylic acid 1-aminocyclopropane-1-carboxylic acid) pretreatment uses pre-configured 1L solution, it includes kind Various a great number of elements and microelement needed for eggplant growth, solution is poured into pallet.Control is the nutrient solution without ACC. Plant is put into illumination box after having poured water, the environment for manufacturing arid is not rewatered, continues 14 days.
5.1 AOX1a can respond Ethylene Signal and drought stress
The phenotype of the 14th day pretreated transgenosis AOX tomato plant of ACC of drought stress is as shown in Figure 7.Observation shows The blade wilting degree of the pretreated AOX1a-RNAi19# of ACC will be significantly greater than WT and AOX1a-OE2#, AOX1a silent mutation Body surface reveals worse drought tolerance, shows that ACC and AOX1a can participate in regulation drought stress.
Physiological responses variance analysis under 5.2 stress conditions
Relative water content, conductivity miss rate, content of hydrogen peroxide are all physical signs important under environment stress, we Determine a series of physiological data analysis.
Under drought stress, the intracorporal variation for keeping moisture ability of plant, to WT and AOX1a correlated inheritance mutant from Body plant relative moisture content is determined.Method is as follows: the tomato leaf after taking control group and Stress treatment claims its fresh weight (FW).Then blade is immersed in distilled water, Leaf water absorption is made to reach saturation state.It takes out blade and sucks surface water with blotting paper Point, be placed in weighing bottle weighing immediately and obtain saturation water suction weight (TW), then claim after 105 DEG C of drying leaf tissue is dry Weight (DW) calculates the relative water content of blade: relative water content (%)=(FW-DW)/(TW-DW) with following formula.Test knot Fruit as shown in figure 8, the experimental results showed that, the relative water content in AOX1a-OE will be significantly larger than WT and AOX1a deletion mutation Body.
Plant cell membrane plays a part of to adjust control intraor extracellular mass exchange, its selection permeability is its most important function One of can.When plant is under stress conditions, cell membrane is increased by different degrees of destruction, the permeability of film, selects permeability funeral It loses, intracellular portion Electrolyte Leakage.Degree and the intensity of adverse circumstance that membrane structure is destroyed, duration, crop varieties it is anti- The factors such as property are related.Therefore, the measurement of membrane permeability often can be used as a physical signs of adverse circumstance injury, be widely used in plant In Resistance Physiology research.We have carried out conductance measurement to the related mutants of AOX1a, the method is as follows: weigh 0.5g tomato Blade is cut into the tissue dissection of 0.5-1.0cm, after then being cleaned with deionized water, is placed in test tube of the 25mL with plug, And about 25mL deionized water is added, it is evacuated 15 minutes with vacuum pump and (is put halfway with the air for extracting blade surface and space between cells out Gas 2-3 times).Then it is settled to 25mL again at room temperature, and stands 1 hour, measure conductivity with DDS-II type electric conductivity instrument (Ri).Sample cell is placed in boiling water bath 5 minutes to reach 100% exudation after having measured, be cooled to after room temperature measure again it is total Conductivity (Rt) indicates membrane permeability with relative electrolyte leakage R0 (%)=Ri/Rt.
Test results are shown in figure 9, the results showed that and the conductivity in material, which is overexpressed, in AOX1a is lower than control group, and RNAi silencing material electric conductivity is then higher than control group.
Plant makes H under adverse circumstance or when aging, since activity in vivo oxygen metabolism is reinforced2O2It accumulates.H2O2It can be straight It connects or oxidative cell nucleic acid, protein and other indirectly, and sustains damage cell membrane, to accelerate cell Aging and disintegration.We have carried out H to material2O2Assay, the method is as follows:
1. about 0.5g tomato leaf is taken to be placed in mortar, 0.1% (W/V) trichloroacetic acid (TCA) of 5mL is added, grinds rapidly It is milled to homogenate；
2. quickly homogenate is transferred in 2mL EP pipe, 12000rpm, 4 DEG C, it is centrifuged 15 minutes；
3. supernatant 0.5mL is taken to be transferred in new 2mL EP pipe, the kaliumphosphate buffer of 0.5mL 10mM is added (pH7.0), 1mL 1M KI is added, mix and is reacted at room temperature 20 minutes；
4. taking TCA solution as blank control using the light absorption value at microplate reader measurement 390nm；
5. according to H2O2OD390 standard curve, calculate the H of sample2O2Content (nmolg-1FW)。
Shown in test result Figure 10, H in the pretreated overexpression plant of ACC2O2Content is substantially less than control group, and ACC is pre- H in the silencing plant of processing2O2Then it is apparently higher than control group.The result shows when AOX function is suppressed, ethylene is lured The drought tolerance led substantially reduces, and when the enhancing of AOX function, the drought-resistance ability that ethylene is induced then has certain enhancing, explanation AOX1a takes part in the drought stress of ethylene induction, and is overexpressed AOX1a and can be improved the drought resistance of plant.
Gene expression difference under 5.3 analysis stress conditions
Each genotype seedling drought stress processing that 3-4 weeks is grown under long-day conditions, extracts total serum IgE, reverse transcription is Real-time fluorescence quantitative PCR analysis is carried out after cDNA.Realtime fluorescent quantitative PCR experiment used kit is Trans Start Reaction system is added according to product description in Green qPCR SuperMix (Transgen), dry using primer amplified Non-irrigated stress response gene；PCR reacts on fluorescence quantitative PCR instrument BIO-RAD CFX ConnectTM Real-Time System It carries out.House-keeping gene Actin is as reference gene, using △ △ CTMethod calculates the relative expression levels of testing gene, i.e. root It is analyzed according to the PCR cycle number (CT value) that fluorescence signal in each PCR reaction reaches given threshold, using Actin as internal reference, Calculate the △ C of each sampleT=(CT,interesting gene-CT, atcin), then calculate △ △ CT=(△ CT-△CT,wt(0h)), The relative expression quantity of testing gene=2- △ △ CT.With wild type WT with no treatment for 1.0, each mutant and each is calculated Handle the relative expression quantity relative to WT.It is independent to test in triplicate, take △ △ CTAverage value is calculated.
Drought stress can induce the synthesis of ABA (abscisic acid abscisic acid), and ABA passes through activation downstream responses base The variation of the controlling gene expression of response because of mediated plant to stress, these regulations ABA synthesis and ABA response can certain journey The variation of ABA signal and stress response signal is reacted on degree.Therefore, we have detected ABA in AOX1a related mutants plant and close At the expression of key gene and the ABA response marker gene for mediating stress response, these genes include P5CS1, NCED3, RD29 Deng.The fluorescence quantification PCR primer list of said gene is as shown in figure 15.As shown in Figure 11 to 14, the experimental results showed that, arid Stress can induce the expression of these genes, but its expression quantity will be significantly higher than WT, explanation in AOX1a2# and AOX1a5# AOX1a, which is overexpressed plant, all has stronger drought resistance.
It follows that AOX1a gene participates in the drought tolerance of ethylene induction, and it can be improved the drought tolerant of plant, it is right It is had a good application prospect in transgenic engineering.
Above-described embodiment is only preferred embodiment, the protection scope being not intended to limit the invention, in spirit of the invention With any modifications, equivalent replacements, and improvements made within principle etc., should all be included in the protection scope of the present invention.
Sequence table
<110>Sichuan University
<120>application of the AOX1a gene in terms of improving drought resistance in plants
<130> ZX19044
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1482
<212> DNA
<213>tomato (Solanum lycopersicum)
<400> 1
tatataaaat gagtgtattt cattaaagaa attattacca tcacatataa taaaatatat 60
atatataata ttattatttg ccttcttcct caagtttctt tccaaacttc cggcgaacat 120
tttttttgat tccagatttt gatcggaagt tatcaaacat gatgacccgt ggagcaacaa 180
ggatgacacg agttgtcatg ggtcatatgg gtccacgtta cttttcaact acagttctgc 240
gaaataatcc cgggaccgga gttgttgttg gagtcgctgc cggacttttg catggttttc 300
cggcgaatcc atcggagaaa gtggcagtaa cgtgggttag gcatttttcg gctatgggtt 360
cacggagcgc tagtactgcg gctttgaatg ataagcaaca agagaaggaa agtagtgaca 420
aaaaagtgga gaacaccgcc accgccaccg ccgctgtaaa cggtggtgtt ggtaaatctg 480
tggtgagtta ttggggggtt cctccttcaa aggctactaa accagatggt actgaatgga 540
aatggaattg ttttaggcca tgggagactt atgaagctga tatgtcgata gatttgacga 600
aacaccatgc gcctgtaacg tttttggata aatttgctta ttggactgtt aagatccttc 660
gtttccccac tgatgtattt tttcagagga gatatggttg cagagcaatg atgttagaga 720
cagtggcggc ggtgcctgga atggtgggag gtatgttgtt gcattgtaag tcattgaggc 780
gattcgaaca gagtggtgga tggatcaaag ctctgttaga agaagctgaa aacgagagga 840
tgcatttgat gactttcatg gaagttgcaa agccaaatgt atacgaacgt gctctggttt 900
tcgcagtgca aggcgtcttc ttcaacgctt actttgctgc ataccttatt tccccaaaat 960
tggctcatcg tatcgtcgga tatttggaag aagaggctgt acattcgtac accgagttcc 1020
tcaaggaatt ggacaatggt aacattgaga acgttcctgc tcccgctatt gctattgatt 1080
actggcgact gcctaaggat gccactctcc gcgatgttgt cttggttgtt cgggctgatg 1140
aggctcatca tcgcgatgtc aaccactatg catctgacat tcattaccaa ggacaacagc 1200
tgaaggactc accagcacca cttgggtatc actaagcgac taataataaa tacaaagata 1260
tgacttatca agctaaaagt ccgtcactat tttgttcctt tttttgaact ggtaatagag 1320
aatatgtagt aggtattact aatctcgaat gttgatgtac atatttttgt gtggatttgg 1380
ttcgttcttt gctgcctctt catcgataag aggtagagta ggatgacctc tgtatcctat 1440
atattcttga tgaaattata tcatcttttt gtctacttga at 1482
<210> 2
<211> 358
<212> PRT
<213>tomato (Solanum lycopersicum)
<400> 2
Met Met Thr Ala Gly Ala Thr Ala Met Thr Ala Val Val Met Gly His
1 5 10 15
Met Gly Pro Ala Thr Pro Ser Thr Thr Val Leu Ala Ala Ala Pro Gly
20 25 30
Thr Gly Val Val Val Gly Val Ala Ala Gly Leu Leu His Gly Pro Pro
35 40 45
Ala Ala Pro Ser Gly Leu Val Ala Val Thr Thr Val Ala His Pro Ser
50 55 60
Ala Met Gly Ser Ala Ser Ala Ser Thr Ala Ala Leu Ala Ala Leu Gly
65 70 75 80
Gly Gly Leu Gly Ser Ser Ala Leu Leu Val Gly Ala Thr Ala Thr Ala
85 90 95
Thr Ala Ala Val Ala Gly Gly Val Gly Leu Ser Val Val Ser Thr Thr
100 105 110
Gly Val Pro Pro Ser Leu Ala Thr Leu Pro Ala Gly Thr Gly Thr Leu
115 120 125
Thr Ala Cys Pro Ala Pro Thr Gly Thr Thr Gly Ala Ala Met Ser Ile
130 135 140
Ala Leu Thr Leu His His Ala Pro Val Thr Pro Leu Ala Leu Pro Ala
145 150 155 160
Thr Thr Thr Val Leu Ile Leu Ala Pro Pro Thr Ala Val Pro Pro Gly
165 170 175
Ala Ala Thr Gly Cys Ala Ala Met Met Leu Gly Thr Val Ala Ala Val
180 185 190
Pro Gly Met Val Gly Gly Met Leu Leu His Cys Leu Ser Leu Ala Ala
195 200 205
Pro Gly Gly Ser Gly Gly Thr Ile Leu Ala Leu Leu Gly Gly Ala Gly
210 215 220
Ala Gly Ala Met His Leu Met Thr Pro Met Gly Val Ala Leu Pro Ala
225 230 235 240
Val Thr Gly Ala Ala Leu Val Pro Ala Val Gly Gly Val Pro Pro Ala
245 250 255
Ala Thr Pro Ala Ala Thr Leu Ile Ser Pro Leu Leu Ala His Ala Ile
260 265 270
Val Gly Thr Leu Gly Gly Gly Ala Val His Ser Thr Thr Gly Pro Leu
275 280 285
Leu Gly Leu Ala Ala Gly Ala Ile Gly Ala Val Pro Ala Pro Ala Ile
290 295 300
Ala Ile Ala Thr Thr Ala Leu Pro Leu Ala Ala Thr Leu Ala Ala Val
305 310 315 320
Val Leu Val Val Ala Ala Ala Gly Ala His His Ala Ala Val Ala His
325 330 335
Thr Ala Ser Ala Ile His Thr Gly Gly Gly Gly Leu Leu Ala Ser Pro
340 345 350
Ala Pro Leu Gly Thr His
355

Claims (5)

  1. Application of the 1.AOX1a gene in terms of improving drought resistance in plants, which is characterized in that be applied to mention by the AOX1a gene The drought tolerance of high plant, for the nucleotide sequence of the AOX1a gene as shown in SEQ ID NO:1, the plant is tomato.
  2. 2. application of the AOX1a gene as described in claim 1 in terms of improving drought resistance in plants, which is characterized in that the use Way includes cultivating the tomato plant with drought resistance enhancing purpose character.
  3. 3. a kind of method for improving drought resistance in plants, which is characterized in that by transgenic technology, by AOX1a gene in plant It is inside overexpressed, for the nucleotide sequence of the AOX1a gene as shown in SEQ ID NO:1, the plant is tomato.
  4. 4. improving the method for drought resistance in plants as claimed in claim 3, which is characterized in that the described method comprises the following steps: The building of AOX1a Overexpression vector；The genetic transformation of the tomato of mediated by agriculture bacillus；Transgenic plant identification.
  5. 5. the method according to claim 4 for improving drought resistance in plants, which is characterized in that further comprising the steps of: to institute The transgenic plant stated carries out drought stress processing, and observation plant drought resisting phenotype is screened.
CN201910603825.5A 2019-07-05 2019-07-05 Application of the AOX1a gene in terms of improving drought resistance in plants Pending CN110272911A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201910603825.5A CN110272911A (en) 2019-07-05 2019-07-05 Application of the AOX1a gene in terms of improving drought resistance in plants

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201910603825.5A CN110272911A (en) 2019-07-05 2019-07-05 Application of the AOX1a gene in terms of improving drought resistance in plants

Publications (1)

Publication Number Publication Date
CN110272911A true CN110272911A (en) 2019-09-24

Family

ID=67962865

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201910603825.5A Pending CN110272911A (en) 2019-07-05 2019-07-05 Application of the AOX1a gene in terms of improving drought resistance in plants

Country Status (1)

Country Link
CN (1) CN110272911A (en)

Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU5137896A (en) * 1995-04-07 1996-10-23 Australian National University, The Plants with altered mitochondrial function
WO2000071668A2 (en) * 1999-05-20 2000-11-30 The Board Of Regents Of The University Of Nebraska Method for identifying components involved in signal transduction pathways in higher plants
WO2005047472A2 (en) * 2003-11-10 2005-05-26 Boyce Thompson Institute For Plant Research Increasing seed threonine content through alteration of threonine aldolase activity
CN101921774A (en) * 2010-05-24 2010-12-22 南京大学 Application of Dnaj-like protein and encoded gene thereof
WO2011074959A1 (en) * 2009-12-15 2011-06-23 Edwin Henricus Antonius Holman Transgenic ozone-resistant plants
WO2012151254A1 (en) * 2011-05-02 2012-11-08 Board Of Regents Of The University Of Nebraska Plants with useful traits and related methods
US20130219560A1 (en) * 2010-09-02 2013-08-22 Richard T. Sayre Reduction of post-harvest physiological deterioration
CN103305488A (en) * 2013-07-01 2013-09-18 中国农业科学院生物技术研究所 Plant drought resistance related protein as well as encoding gene and application thereof
US20140026254A1 (en) * 2012-07-17 2014-01-23 Industry Foundation Of Chonnam National University Gene Implicated in Drought Stress Tolerance and Transformed Plants with the Same
CN104988159A (en) * 2015-07-22 2015-10-21 江苏省农业科学院 Cotton drought resistant gene GhEXP1 and applications thereof
CN105018504A (en) * 2015-06-19 2015-11-04 镇江泰和益元生物科技有限公司 Transformed luciferase gene, recombinant vector containing transformed luciferase gene, engineering bacterial, construction method of recombinant vector, and fermentation method of engineering bacterial
CN105063063A (en) * 2010-06-24 2015-11-18 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and method for making the same
CN105543274A (en) * 2016-01-19 2016-05-04 浙江省农业科学院 Application of solanum lycopersicum SlWRKY81 gene
CN106636125A (en) * 2016-09-21 2017-05-10 华南农业大学 Method for improving basic stress resistance of plants and application of method
CN107557368A (en) * 2016-06-30 2018-01-09 未名生物农业集团有限公司 The plant and method that abiotic stress tolerance improves
CN109456394A (en) * 2018-11-19 2019-03-12 浙江大学 Tomato SlPIF4 gene, albumen and its application in raising plant frigostabile
CN109576280A (en) * 2018-11-20 2019-04-05 广州大学 New Zealand spinach TtASR gene and its coding albumen and application
CN109679971A (en) * 2019-01-14 2019-04-26 浙江大学 The PCR molecular labeling and application thereof of the important adversity gene AOX of watermelon

Patent Citations (18)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AU5137896A (en) * 1995-04-07 1996-10-23 Australian National University, The Plants with altered mitochondrial function
WO2000071668A2 (en) * 1999-05-20 2000-11-30 The Board Of Regents Of The University Of Nebraska Method for identifying components involved in signal transduction pathways in higher plants
WO2005047472A2 (en) * 2003-11-10 2005-05-26 Boyce Thompson Institute For Plant Research Increasing seed threonine content through alteration of threonine aldolase activity
WO2011074959A1 (en) * 2009-12-15 2011-06-23 Edwin Henricus Antonius Holman Transgenic ozone-resistant plants
CN101921774A (en) * 2010-05-24 2010-12-22 南京大学 Application of Dnaj-like protein and encoded gene thereof
CN105063063A (en) * 2010-06-24 2015-11-18 巴斯夫植物科学有限公司 Plants having enhanced yield-related traits and method for making the same
US20130219560A1 (en) * 2010-09-02 2013-08-22 Richard T. Sayre Reduction of post-harvest physiological deterioration
WO2012151254A1 (en) * 2011-05-02 2012-11-08 Board Of Regents Of The University Of Nebraska Plants with useful traits and related methods
US20140026254A1 (en) * 2012-07-17 2014-01-23 Industry Foundation Of Chonnam National University Gene Implicated in Drought Stress Tolerance and Transformed Plants with the Same
CN103305488A (en) * 2013-07-01 2013-09-18 中国农业科学院生物技术研究所 Plant drought resistance related protein as well as encoding gene and application thereof
CN105018504A (en) * 2015-06-19 2015-11-04 镇江泰和益元生物科技有限公司 Transformed luciferase gene, recombinant vector containing transformed luciferase gene, engineering bacterial, construction method of recombinant vector, and fermentation method of engineering bacterial
CN104988159A (en) * 2015-07-22 2015-10-21 江苏省农业科学院 Cotton drought resistant gene GhEXP1 and applications thereof
CN105543274A (en) * 2016-01-19 2016-05-04 浙江省农业科学院 Application of solanum lycopersicum SlWRKY81 gene
CN107557368A (en) * 2016-06-30 2018-01-09 未名生物农业集团有限公司 The plant and method that abiotic stress tolerance improves
CN106636125A (en) * 2016-09-21 2017-05-10 华南农业大学 Method for improving basic stress resistance of plants and application of method
CN109456394A (en) * 2018-11-19 2019-03-12 浙江大学 Tomato SlPIF4 gene, albumen and its application in raising plant frigostabile
CN109576280A (en) * 2018-11-20 2019-04-05 广州大学 New Zealand spinach TtASR gene and its coding albumen and application
CN109679971A (en) * 2019-01-14 2019-04-26 浙江大学 The PCR molecular labeling and application thereof of the important adversity gene AOX of watermelon

Non-Patent Citations (7)

* Cited by examiner, † Cited by third party
Title
AIGEN FU 等: "Alternative Oxidases (AOX1a and AOX2) Can Functionally Substitute for Plastid Terminal Oxidase in Arabidopsis Chloroplasts", 《THE PLANT CELL》 *
FEI XU 等: "The role of alternative oxidase in tomato fruit ripening and its methylation and chromatin patternin", 《JOURNAL OF EXPERIMENTAL BOTANY》 *
NCBI: "Solanum lycopersicum alternative oxidase 1a (AOX1a), mRNA", 《GENBANK DATABASE》 *
TONG ZHU 等: "Mitochondrial alternative oxidase-dependent autophagy involved in ethylene-mediated drought tolerance in Solanum lycopersicum", 《PLANT BIOTECHNOLOGY JOURNAL》 *
付丽军: "交替氧化酶在番茄防御病毒侵染中的作用及信号调控机制", 《中国优秀硕士学位论文全文数据库（电子期刊）》 *
刘杰: "在植物逆境耐受中调节机理的研究", 《中国优秀硕士学位论文全文数据库（电子期刊）》 *
廖杨文科: "线粒体呼吸电子传递在植物对烟草花叶病毒防御反应中的作用及其调控机制", 《中国优秀博士学位论文全文数据库》 *

Similar Documents

Publication Publication Date Title
CN109777810B (en) Application of PUB41 gene as negative regulatory factor in improving resistance to tomato gray mold and bacterial wilt
CN106868021B (en) Gene OsNAC1 for controlling rice seed size and application thereof
CN107937416A (en) Improve gene and its application of nitrogen fertilizer for paddy rice utilization ratio and yield
CN109021084A (en) Trifoliate orange Cold resistant genes PtrERF109 and its application in plant cold resistance genetic improvement
CN109111514A (en) And the breeding method and its relevant biological material of the transgenic wheat of anti-banded sclerotial blight and root rot
CN103194456B (en) Lilium regale antifungal gene Lr14-3-3 and application thereof
CN102978218B (en) Cloning of apple stress-resistant related gene MdSIMYB2 and application of cloning of apple stress-resistant related gene MdSIMYB2
CN110468150B (en) Application of RGS1 gene as negative regulatory factor in improving tomato bacterial leaf spot resistance in low-irradiation environment
CN108192920A (en) A kind of method that disease resistance of plant is improved using NDR1 genes
CN108588041B (en) Gossypium barbadense cytochrome P450 gene, and coding protein and application thereof
CN104531718A (en) Apple V-ATPase subunit gene MdVHA-B1S396A and stress-resistance application thereof
CN110358778B (en) Application of SlDALR2 gene in improving resistance to bacterial leaf spot of tomato
US20200216855A1 (en) Disease Resistant Plants Containing HIR3 Gene and Method for making the plants thereof
CN107663232B (en) Plant anti-adversity associated protein OsIAA18 and its encoding gene and application
CN102732553B (en) Improve the gene engineering method and material of plant products
CN101781654B (en) Novel cotton fungal disease-resistant gene GhMPK7 and application thereof
CN104988175B (en) Application of the tomato HsfA1a genes in plant autophagosome activity and drought resistance is improved
CN106892973A (en) Plant adversity resistance related protein GhMYB4 and encoding gene and application
CN110272911A (en) Application of the AOX1a gene in terms of improving drought resistance in plants
KR102000465B1 (en) Method of improving resistance of Bakanae disease
CN114032245A (en) Gene VLNHX3D in regulating plant cell Na+And/or K+Application in concentration
JP5164093B2 (en) Method for increasing resistance of rice to pathogens and pathogen-resistant rice transformants
CN110358774A (en) Control gene, protein, expression casette, expression vector, host cell, method and the application of Rice Flowering time
CN104558132B (en) Peanut DELLA gene families and its encoding gene and application
CN109053870A (en) Application of AtERF49 gene during plant responding high temperature stress

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20190924

RJ01 Rejection of invention patent application after publication