CN105400792A - Application of corn kernel factor gene ZmNF-YA3 to changing plant resistance tolerance - Google Patents
Application of corn kernel factor gene ZmNF-YA3 to changing plant resistance tolerance Download PDFInfo
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- 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
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- 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
Abstract
The invention discloses application of a corn kernel factor gene ZmNF-YA3 to changing plant resistance tolerance. The ZmNF-YA3 gene is cloned from corns, the gene is reconstructed into a plant expressing carrier in a forward or reverse mode or in an RNAi structural mode, and a fusion gene is formed; transgenic plants are obtained through the transgenic technology; by detecting transgenic expression and measuring resistance tolerance of the plants, the transgenic plants with resistance tolerance obviously improved and offsprings of the transgenic plants with resistance tolerance obviously improved are screened from the transgenic plants, and new plant germplasm with application value is created. The cDNA sequence of the corn kernel factor gene ZmNF-YA3 is as shown in SEQ ID No.1, the coded amino acid sequence of the corn kernel factor gene ZmNF-YA3 is as shown in SEQ ID No.2; the plants refer to cultivated herbage crops or corn or cotton. The application of the corn kernel factor gene ZmNF-YA3 has great significance in cultivating high-yield resistance tolerance transgenic crops.
Description
Technical field
The invention belongs to the bioengineering breeding field of farm crop, specifically, relate to a kind of corn nf gene ZmNF-YA3 and changing the application in stress resistance of plant.
Background technology
NF-Y (nuclearfactor-y) is ubiquitous a kind of transcription factor complex in eukaryote, be made up of 3 different subunits (NF-YA/CBF-B/HAP2, NF-YB/CBF-A/HAP3 and NF-YC/CBF-C/HAP5), and regulate the expression of goal gene by the promotor acting on other regulatory factors.NF-YA is the specific subunit of DNA sequence dna, is attached on core pentamer Nucleotide CCAAT motif (motif) of gene promoter area.NF-YB and NF-YC is the subunit of structurally similar histone H2A and H2B respectively.The CCAAT motif of complete NF-Y transcription complex in target gene promoters is combined, transcribing of regulation and control target gene.NF-Y complex body can work as an activating transcription factor or repressor, and the combination of itself and DNA and transcriptional regulatory activity are also subject to other transcription factor and regulate, and the latter is worked by interacting with NF-Y subunit.Plant NF-Y can be divided into NF-YA, NF-YB and NF-YC tri-families, and there are 8 ~ 39 members in every family, has occurred the variation of structure and function, forms complicated gene family, and each family different members take part in different regulation and control.Existing plant NF-Y gene function analysis discloses, and the gene of three families all has member to take part in vine growth and development regulation and control or plant and microbial interaction, and the response to environment stress.Some subfamilies member defines the specific function participating in different development pathway and metabolic process during evolution, as fetal development regulation and control, root system development and flowering time control, er stress adjustment, drought stress response, infected by microbes and nitrogen-fixing root nodule formation etc.Some plant NF-Y family members also play an important role on drought resisting, salt-tolerance character, and process LAN AtNF-YB1 and ZmNF-YB2 can strengthen plant drought resistance.AtNF-YA5 be proved to be drought stress and response dormin (ABA) Arabidopis thaliana root and leaf in up-regulated.
Corn NF-Y family gene quantity is large, and retrieval maize genomic sequence, finds corn C CAAT-HAP2/NF-YA family member 36, CCAAT-HAP3/NF-YB member 28, CCAAT-HAP5/NF-YC member 25.The functional study of these NF-Y family genes is still at the initial stage, and only sees corn ZmNF-YB2 gene overexpression and improves the research report of milpa drought tolerance and the patent of Monsanto Company's application.About the application of corn nf gene ZmNF-YA3 in change stress resistance of plant has no report.
Summary of the invention
For current present Research, the problem to be solved in the present invention is to provide a kind of corn nf gene ZmNF-YA3 and is changing the application in stress resistance of plant.
Corn nf gene ZmNF-YA3 of the present invention is changing the application in stress resistance of plant.
Wherein: the cDNA sequence of described corn nf gene ZmNF-YA3 is as shown in SEQIDNo.1, and the aminoacid sequence of its coding is as shown in SEQIDNo.2; Described plant refers to the herbaceous crops of cultivation, or corn, or cotton; Described change stress resistance of plant refers to the drought resistance and/or thermotolerance that change plant.
In above-mentioned application: described corn nf gene ZmNF-YA3 gene has cDNA form or this ZmNF-YA3 gene with just form, anti-sense versions or its RNAi structure formation.
The application method of corn nf gene ZmNF-YA3 of the present invention in change stress resistance of plant is: from corn cDNA or genome, clone ZmNF-YA3 gene order, recombinate in plant expression vector with full length gene form or this gene with justice or anti-sense versions or RNAi structure formation, form fusion gene, then adopt transgenic technology that fusion gene is imported vegetable cell, obtain transfer-gen plant; Wherein, described ZmNF-YA3 gene merges with just form, anti-sense versions or its RNAi structure formation and promotor, and this promotor has stress induced and/or composing type.By detecting transgene expression intensity and carrying out macroscopical identification to plant, therefrom filter out transfer-gen plant and the offspring thereof of growth and the obvious change of resistance generation, create the plant new germ plasm in plant breeding with using value.Concrete:
In the present invention, first with the responsive self-mating system 65232 of drought-enduring self-mating system neat 319 and arid for material, adopt the process of chip hybridization Technical comparing osmotic stress in seedling stage (with 3 leaf phase corn seedlings of the husky training of 18%PEG solution pouring, respectively after treatment 12h, 24h, 48h and recovery water after 24h) on different genotype transcript profile impact, have selected the transcription factor that the variation tendency in different genotype of differential expression is different.Then, adopt quantitative RT-PCR technology to carry out expression pattern analysis to them, therefrom select ZmNF-YA3/GRMZM5G857944 gene and carry out transgenic research.Under normal growing conditions, the gene expression abundance of ZmNF-YA3/GRMZM5G857944 gene in Zea mays root is greater than in leaf; In root, the gene expression abundance of drought-enduring self-mating system neat 319 is 2.5 times of arid responsive self-mating system 65232; In leaf, the expression amount of neat 319 only has 65232 1/2.During osmotic stress process 24h, in the blade of drought-enduring self-mating system neat 319, gene expression abundance slightly rises, and significantly declines in root, is about before treatment 1/9; All significantly decline in the leaf and root of the responsive self-mating system 65232 of drought, fall is about 30%.At osmotic stress process 48h, in the leaf of neat 319, root, the change of ZmNF-YA3 gene expression abundance is little, and gene expression abundance has certain rising in 65232 and leaf.After rehydration 24h, in the root of neat 319, ZmNF-YA3 expresses and rises rapidly, but obviously declines in the root of 65232; And the gene expression abundance of ZmNF-YA3 returns to the level before close to Stress treatment in leaf.Namely this genetic expression is by the adjustment of osmotic stress, and it is variant to express change in different genotype, that osmotic stress suppresses in root, in leaf to osmotic stress reaction because genotype is different obvious difference, infer its root growth and development with corn and drought resistance relevant.
In the present invention, by ZmNF-YB3 genes encoding frame respectively with stress induced promotor as fusions such as Prd29B, Prd29A, restructuring, in plant expression vector, adopts agrobacterium-mediated transformation or particle bombardment that fusion gene is proceeded to plant, produces transgenic line.As in the generation of transgenic corns, cotton, construct carrier pCambia1300-Prd29B::ZmNF-YA3 (+)-PCaMV35S::bar and pCambia1300-Prd29B::ZmNF-YA3 (-)-PCaMV35S::bar, agrobacterium-mediated transformation or particle bombardment is adopted to proceed in Elite Maize Inbred Lines or cotton improved seeds by the T-DNA district of target plasmid, selfing after transformation seedlings transplant survival, results seed.Detect walking around progeny of plants by Herbicid resistant screening and molecular biology for detection (PCR, Southernblotting, RT-PCR), produce transgenic line.Then by selfing and the Molecular Identification in continuous 3 generations, transgenic homozygous strain is created.In addition, according to ZmNF-YA3 gene specific sequence, construct RNAi structure and recombinate in plant expression vector pFGC5941, producing transform plastids pFGC5941-Prd29B::ZmNF-YA3RNAi-PCaMV35S::bar and carry out maize genetic conversion, obtain and turn RNAi structure milpa.
In the present invention, obtain transgenic homozygous plant by continuous selfing subculture.First, under normal cultivation condition, determine whether transfer-gen plant grows normal, and then analyze transfer-gen plant and adjoining tree (transgene receptor genotype) Resistant Difference under stress conditions.Under normal cultivation condition, transfer-gen plant growth and morphology is grown normal, and due to the characteristic of promotor, transgenosis ZmNF-YA3 or its antisense construct or RNAi structure present stress induced expression.In transgenic corns, part process LAN strain ZmNF-YA3 expression intensity under arid or cold condition is significantly higher than non-transfer-gen plant, and the corn strain of part antisense gene or RNAi structure, the expression of Inner source ZmNF-YA3 is subject to obvious suppression, and namely ZmNF-YA3 expression level is significantly lower than non-transfer-gen plant.Basic step and the method for degeneration-resistant detection experiment in the present invention is sketched below for transgenic corns strain.
In drought resistance experiment, the seed of the homozygous lines turning ZmNF-YA3 gene or its RNAi structure is sowed respectively in vinyl disc, grows under being placed in suitable condition.Stop during the plant 4 leaf phase watering, Osmotic treatment 7-10 days (being determined by envrionment temperature and relative humidity), observes the difference of plant wither degree, when after most plant base portion deliquescing lodging, recovery is watered, and observation plant restoration ecosystem situation and surviving rate, determine the drought resistance of plant.Result shows, ZmNF-YA3 gene overexpression enhances the drought resistance of plant in seedling stage, and ZmNF-YA3 gene deregulation expresses the drought resistance of seedling reducing plant.
Simultaneously, by turn the corn seed of ZmNF-YA3 gene and its RNAi structure and the sowing same period of recipient genotypes seed flowerpot or/and land for growing field crops, drought stress process is carried out respectively in male and female Jointing stage (9 ~ 13 leaf phase), pollination period of blooming and filling stage, namely watered by control and avoid drenching with rain, keep soil relative water content at 55-65%, about 15 days time length, observe transfer-gen plant proterties and state of growing, detect relevant physical signs.General open pollination, results fruit ear also carries out species test.Result shows, the drought resistance of part ZmNF-YA3 process LAN strain is significantly higher than recipient genotypes, and single plant yield is better than recipient genotypes, and part reaches difference pole conspicuous level; And turn the plant damage symptoms weight of ZmNF-YA3 inverted defined gene and RNAi structure, it is slow to coerce restoration ecosystem after removing, and the economic characters such as a single plant's output are significantly lower than recipient genotypes.Therefrom select drought resistance and grain yield and the non-transgene gene type strain that there were significant differences.Yield compari@test under the latter enters suitable cultivation condition and under drought stress conditions, therefrom selects the transgenic line meeting breeding objective.Comprehensive many-sided test result, selects excellent transfer-gen plant bagging selfing and isozygotys, and carry out combining ability test, selects combining ability or the transgenosis self-mating system that be improved identical with recipient genotypes for Corn Single-Cross Stock seed selection.
In the present invention, the thermotolerance further defining the corn gene process LAN plant that stress induced promoter starts significantly improves than acceptor self-mating system plant, and the milpa thermotolerance turning RNAi structure declines than acceptor self-mating system plant.
Above-mentioned many-sided experimental result indication, the present invention carrys out coordinate plant growth by control ZmNF-YA3 genetic expression and grows and resistance, the significant and widespread use for cultivation high yield resistant transgenic farm crop.
Embodiment
Embodiment 1: turn corn ZmNF-YA3 gene and create drought-resistant maize self-mating system
1) structure of maize genetic conversion carrier
When building ZmNF-YA3 process LAN structure, the ZmNF-YA3 sequence of cloning from corn cDNA, be inserted in plant expression vector pCambia1300-Prd29B::MCS-PCaMV35S::bar with total length form, form fusion gene with Prd29B, produce plasmid pCambia1300-Prd29B::ZmNF-YA3-PCaMV35S::bar.After enzyme cuts qualification, plasmid is imported in agrobacterium tumefaciens AGL0 or LBA4404, for Genetic Transformation in Higher Plants.
When building ZmNF-YA3RNAi structure, first Multiple Sequence Alignment being carried out to ZmNF-YA gene family member, determining ZmNF-YA gene-specific region, select to build RNAi structure based on specific regions.For the ease of restructuring, add XhoI restriction enzyme site at 5 ' end of interference fragment, add NcoI restriction enzyme site at 3 ' end; Equally, add XbaI enzyme cutting site at 5 ' end of reverse interference fragment, 3 ' end adds BamHI restriction enzyme site.With the ZmNF-YA3 gene of having cloned for template, pcr amplification is adopted to go out interference fragment.Then the forward order fragment XhoI of carrier pFGC5941-Prd29B-PCaMV35S::bar and pcr amplification and NcoI is carried out enzyme to cut, reclaim carrier and forward fragment, connect with T4 ligase enzyme, obtain recombinant plasmid pFGC5941-Prd29B::ZmNF-YA3RNAi (-)-PCaMV35S::bar.The latter's enzyme cuts qualification, determines to carry out next step reaction correctly.Reverse object fragment XbaI and BamHI by recombinant plasmid and pcr amplification carries out enzyme and cuts, and reclaims carrier and reverse fragment, connects with T4 ligase enzyme, generation recombinant plasmid pFGC5941-Prd29B::ZmNF-YA3RNAi-PCaMV35S::bar.Cut qualification by enzyme, determine correct recombinant plasmid.The latter imports in agrobacterium tumefaciens AGL0 or LBA4404, for Genetic Transformation in Higher Plants.
2) foundation of receptor system
With Inbred Lines used in China's agriculture production if the selfed seed of Zheng 58, prosperous 7-2, DH4866 etc. is for parent material.Seed 70% alcohol immersion 10 minutes, then soak 10-15 minute with 0.1% mercury chloride, then with sterilized water washing 3-5 time.After sterilizing, seed is placed in aseptic triangular flask and sprouts, and puts into a small amount of (30-40 milliliter/250 milliliter triangular flask) sterilized water in bottle, is placed on 1-2 days under dark condition (23-30 DEG C) after sealing.Sprout (showing money or valuables one carries unintentionally) afterwards seed to be placed on minimum medium and to continue to sprout under dark condition.When plumule elongation stops 3-4 centimetre, peel off coleoptile and 2-3 sheet spire, emergent stem pointed tip growing tip is used for agroinfection.
3) conversion of acceptor and plant regeneration
By the agrobacterium tumefaciens (as AGL0 and LBA4404) with binary vector (Mini--Ti plasmid is with selective agent resistant gene and ZmNF-YA3), at additional antibiotic LB substratum, (often liter of substratum contains: tryptone 10g, yeast extract 5g, NaCl10g, pH7.0, pressure sterilizing) at 28 DEG C concussion cultivate, concussion speed is 110rpm (rev/min), makes bacterium be in logarithmic phase.Then at 3,000 rpm centrifugal 10 minutes, supernatant liquor is abandoned.The thalline MS liquid nutrient medium of 1/2 concentration washs, then collected by centrifugation.Again the MS liquid nutrient medium of thalline by 1/2 concentration of adding Syringylethanone (acetosyringone, As) 100 μm of ol/l is suspended, dilution OD
6000.25-0.65 for transforming.During conversion, first bacterium liquid is poured in the culture dish of 4.5 cm diameters, inclination culture dish, makes aseptic seedling stem apex growing tip be immersed in bacterium liquid, 0.5 × 10
58-12 minute is processed under Pa normal atmosphere.Then blotted by the bud point aseptic filter paper after dip-dye, be put on solidified MS media by seedling after transfection and cultivate 2-3 days in dark, culture temperature is 22-24 DEG C.Thereafter transfection seedling is put and cultivate 2 days under diffuse light, be transplanted in the flowerpot of upper strata vermiculite lower floor loam, and cover plant top with vermiculite.After plant grows vermiculite, the 3 leaf phases that grew under natural lighting spray selective agent grass fourth phosphine (0.12% concentration) solution, day temperature 22-28 DEG C, and night, temperature 15-21 DEG C, watered 1/2MS substratum inorganic salt every other day.
4) Resistance detecting of transfer-gen plant and Selection utilization
After 3 leaf phase unconverted plant spraying herbicide grass fourth phosphines, within about 4 days, stop growing, after 9 days, start death.After spraying, some individual changes are with unconverted adjoining tree, and other individual continued propagation, change not obvious for transformed plant.When the plant that survives grows to 5 leaf, by its field planting to field, bagging selfing is set seeds.T1 seed from different T0 plant is broadcast in greenhouse or the field with safeguards, 3 the leaf phase plant spray careless fourth phosphine (0.15% concentration) solution.After first quarter moon, non-transfer-gen plant is dead, the survival of transfer-gen plant majority, continued growth.The blade getting transplant survival plant carries out Molecular Detection determination transfer-gen plant.Then by transfer-gen plant (T1) bagging self-fertility.By continuous 2-3 for selfing, obtain transgenic homozygous strain.Detect and the qualification of tree characteristics through transgene expression level, filter out transfer-gen plant and offspring thereof that objective trait obviously changes, create in breeding the new germ plasm with application prospect.
In drought tolerance experiment, by turn ZmNF-YA3 gene and the corn of its RNAi structure and the seed of acceptor self-mating system broadcast flowerpot and land for growing field crops, drought stress process is carried out respectively in male and female Jointing stage (9 ~ 13 leaf phase), pollination period of blooming and filling stage, namely watered by control and avoid drenching with rain, keep soil relative water content at 55-65%, about 15 days time length, and carry out the detection of transfer-gen plant character observation and physical signs, open pollination, results fruit ear also carries out species test.Data analysis shows that ZmNF-YA3 process LAN strain drought resistance is obviously better than acceptor self-mating system and turns RNAi structure, not only plant damage symptoms is light, coerce restoration ecosystem after removing very fast, and the economic characters such as a single plant's output are significantly better than acceptor self-mating system and turn RNAi structure; And turn the plant drought resistance of most independent transformants of RNAi structure and grain yield lower than acceptor self-mating system.
Embodiment 2: turn corn ZmNF-YA3 gene and cultivate the heat-resisting self-mating system of corn
1) structure of maize genetic conversion carrier
With example 1, but replace Arabidopis thaliana RD29B protein promoter Prd29B with corn heat shock protein Hsp70 gene promoter Phsp, the target gene ZmNF-YA3 in conversion carrier exists with " Phsp ∷ ZmNF-YA3-Tnos " form.Tnos is 3 ' tail district of the rouge alkali synthetase gene that Ti-plasmids carries.Recombinant plasmid proceeds in agrobacterium tumefaciens AGL1 or LBA4404 for genetic transformation.
2) generation of corn gene plant
With example 1.
3) transfer-gen plant offspring analysis and selection
T1 grows to 3 leaf phase 10.8ml for plant
the aqueous solution sprays, and observes statistics resistance and sensitive individuals ratio; Adopt round pcr to detect foreign gene, and add up the segregation ratio of foreign gene in progeny plant.Survive plantlet of transplant to land for growing field crops, bagging selfing.T2 for plant, adopts round pcr to detect foreign gene and carries out Southernblotting checking, and adopt RT-PCR technology to check transgene expression intensity except bagging selfing is set seeds.Selected transgenic line is measured to the change of plant Net Photosynthetic Rate under different light intensity and temperature, and with non-transfer-gen plant for contrast, under field cultivating condition, carry out the observation of Yield Traits In Corn after selecting excellent transgenic line and compare, selecting specular removal high yield strain to enter biological safety test and corn breeding experiment.
4) thermotolerance turning ZmNF-YA3 corn detects
Thermotolerance experiment in seedling stage: will at 28 DEG C of (irradiations, 13h/d)/22 DEG C (dark, 2h is grown under the milpa turning ZmNF-YA3 gene of isozygotying of growth moves into 36 DEG C (irradiations) 11h/d), continuous heat treatment 4 days (irradiation 13h/d at 39 DEG C again, dark 11h/d), then restoration ecosystem at 28 DEG C.After heat treatment, only have in non-non-transgenic control and ZmNF-YA3 gene overexpression plant indivedual strain and acceptor self-mating system plant gap little, and the thermotolerance of most transgenic line is significantly better than acceptor self-mating system, wherein partial transgenic strain is injured not obvious.
Jointing stage plant thermotolerance experiment: milpa and the acceptor self-mating system seed of transgenic homozygous are broadcast in flowerpot, (temperature is not higher than 32 DEG C under optimal growth conditions for plant, be not less than 22 DEG C) grow to the 12-13 leaf phase, then 4h is grown under being moved into 36 DEG C (irradiations), at 40 DEG C, continue thermal treatment 5 days (irradiation 13h/d, dark 11h/d) again, move into restoration ecosystem under 32 DEG C of (light)/26 DEG C (secretly), irradiation 13h/d, dark 11h/d.After thermal treatment, non-non-transgenic control lines blade is almost all impaired, and lower blade is dead, and tassel dysplasia, without normal pollen, occurs without female fringe; To turn in ZmNF-YA3 strain most thermotolerance and be significantly better than acceptor self-mating system, wherein part strain blade is injured gently, only lower blade thirst and comes off, but tassel is grown and is still had a strong impact on, pollen abortion rate is up to 60%, female fringe is grown and is also significantly postponed, and the florescence is sterile, self-fertility difficulty.
Filling stage plant thermotolerance experiment: (temperature is not higher than 35 DEG C under optimal growth conditions to broadcast the milpa of the transgenic homozygous in flowerpot and acceptor self-mating system plant, be not less than 22 DEG C) grow to latter 10 days of pollination, 24h is grown under moving into 36 DEG C (irradiations), thermal treatment 5 days (irradiation 13h/d are continued again at 40 DEG C, dark 11h/d), move into restoration ecosystem under 32 DEG C of (light)/26 DEG C (secretly), irradiation 13h/d, dark 11h/d.After thermal treatment, acceptor self-mating system plant part is dead, almost blade is all impaired for survival plant, lower blade is dead, fruit ear diminishes, the bald 3/4-1/2 accounting for Ear-Length, and kernal number is less than not being subject to 1/2 of stressed plants, 100-grain weight is not less than being subject to 3/4 of stressed plants, and single plant yield significantly declines.Turning in ZmNF-YA3 gene strain only has the extent of injury of minority strain and acceptor self-mating system close, the thermotolerance of most strain is better than acceptor self-mating system, wherein the plant leaf of partial transgenic strain is injured gently, just lower blade thirst, but fruit ear is grown and is still had a strong impact on, baldly account for 1/3 of Ear-Length, kernal number is than significantly not reducing by stressed plants, 100-grain weight generally declines 30%, though single plant yield obviously declines, be significantly higher than acceptor self-mating system plant through processing equally.
According to the character observation under different growth conditions and Yield compari@, select the heat-resisting corn breeding material of process LAN ZmNF-YA3 gene, and then cultivate the heat-resisting self-mating system of corn.
Embodiment 3: turn corn ZmNF-YA3 gene and create Resistance Strain of Cotton against material
1. the structure of Cotton Transformation carrier
When building ZmNF-YA3 process LAN structure, the ZmNF-YA3 sequence of cloning from corn cDNA, be inserted in plant expression vector pCambia1300-Prd29B::MCS-PCaMV35S::als with total length form, form fusion gene with Prd29B, produce plasmid pCambia1300-Prd29B::ZmNF-YA3-PCaMV35S::als.After enzyme cuts qualification, plasmid is imported in agrobacterium tumefaciens AGL0 or LBA4404, for Genetic Transformation in Higher Plants.
2. cotton aseptic seedling obtains
Get the seed of cotton inbred lines or improved seeds, slough surperficial fine hair with the vitriol oil, by 70% alcohol immersion 2 minutes, then soak 10-12 minute with 0.1% mercury chloride, then with sterilized water washing 3-5 time.Constantly seed is rocked, to ensure that surface sterilization is thorough during sterilizing.After sterilizing, seed is placed in aseptic triangular flask and sprouts, and puts into a small amount of sterilized water (30-40 ml water/250 milliliter triangular flask) in bottle, is placed on 1-2 days under dark condition (23-30 DEG C) after sealing.After Seed sprouting (showing money or valuables one carries unintentionally), place it in modified MS medium that ammonium salt reduces by half and sprout under dark condition.When Embryo stem extension is to 3-5 centimetre, peel off a slice cotyledon, emergent stem end growing tip.
2. Agrobacterium is cultivated and activation
Will with agrobacterium tumefaciens (AGL1 or LBA4404) concussion cultivation at 28 DEG C in additional antibiotic YEP substratum of binary vector (Mini-Ti plasmid is with weedicide grass fourth phosphine resistant gene als), concussion speed is 110r/min, makes bacterium be in logarithmic phase.Then under 3000r/min centrifugal 10 minutes, supernatant liquor is abandoned.The 1/2 improvement MS liquid nutrient medium washing that thalline ammonium salt reduces by half, collected by centrifugation.Suspended by the 1/2MS liquid nutrient medium that the ammonium salt that thalline adds 100mg/l Syringylethanone reduces by half, dilution 10-25 is doubly for transforming again.
3. cotton aseptic seedling transforms
(1) bacterium liquid is poured in the culture dish of 4.5 cm diameters, inclination culture dish, makes the shoot apex exposing stem apex growing tip be immersed in 4-5 minute in bacterium liquid.
(2) the bud point aseptic filter paper after contaminating blots, and root is inserted and in dark, cultivates 2-3 days without in the modified MS medium of growth regulator, culture temperature is 24-26 DEG C.Then aseptic seedling is put and cultivate 2 days under diffuse light.
(3) aseptic seedling after being cultivated by irradiation is transplanted in the flowerpot of upper strata vermiculite lower floor loam, and vermiculite covers plant top.Then allow plant grow under natural lighting, day temperature 22-28 DEG C, night, temperature 18-23 DEG C, watered 1/2 modified MS medium inorganic salt every other day.
4. transformed plant screening and field planting
After transformed plant grows 3 leaves, spray 1.5mg/l chlorsulfuron (Shenyang Agricultural Chemicals Factory produces, the effective constituent 25%) aqueous solution, fall drop with plant and be advisable.Unconverted adjoining tree stops growing after spraying for 3 days, within about 12 days, starts dead.After spraying, some individual changes are similar with adjoining tree, and other individualities have strong resistance, continued propagation for transformed plant.When survival plant grew to for 5 leaf phase, by its field planting to field.
5. the molecular biology identification of antiweed plant
When antiweed plant grows to 7-8 leaf, get blade and extract DNA, adopt round pcr to detect foreign gene.PCR positive plant carries out Southernblotting detection.In the 186 strain antiweed PCR positive plants obtained, the plant of about more than 32% (60/186) is trans genie individual.
6. the qualification of transfer-gen plant and utilization
The selfing of transfer-gen plant Post flowering or sisters' knot are in fact.Planting seed, in land for growing field crops or greenhouse, is got blade and is extracted DNA when the plant 4-6 leaf phase, adopt round pcr whether to detect progeny plant with foreign gene, and add up foreign gene at filial generation segregation ratio.Result shows, with aseptic seedling shoot apical meristem for transgene receptor is organized, effectively can obtain transfer-gen plant.Further, in partial transgenic strain offspring, foreign gene mode of inheritance meets Mendelism, and stable in going down to posterity.Transgenic homozygous system, through field character determination and resistant determination, selects the transgenic line keeping acceptor kind fundamental characteristics and drought-enduring thermotolerance to be significantly improved, and improves for cotton variety.
Claims (5)
1. a corn nf gene ZmNF-YA3 is changing the application in stress resistance of plant.
2. apply as claimed in claim 1, it is characterized in that: the cDNA sequence of described corn nf gene ZmNF-YA3 is as shown in SEQIDNo.1, and the aminoacid sequence of its coding is as shown in SEQIDNo.2; Described plant refers to the herbaceous crops of cultivation, or corn, or cotton; Described change stress resistance of plant refers to the drought resistance and/or thermotolerance that change plant.
3. apply as claimed in claim 1, it is characterized in that: described corn nf gene ZmNF-YA3 gene has cDNA form or this ZmNF-YA3 gene with just form, anti-sense versions or its RNAi structure formation.
4. apply as claimed in claim 1, it is characterized in that, the application method of described corn nf gene ZmNF-YA3 in change stress resistance of plant is: from corn cDNA or genome, clone ZmNF-YA3 gene order, recombinate in plant expression vector with full length gene form or this gene with justice or anti-sense versions or RNAi structure formation, form fusion gene, then adopt transgenic technology that fusion gene is imported vegetable cell, obtain transfer-gen plant; By detecting transgene expression intensity and carrying out macroscopical identification to plant, therefrom filter out transfer-gen plant and the offspring thereof of growth and the obvious change of resistance generation, create the plant new germ plasm in plant breeding with using value.
5. apply as claimed in claim 4, it is characterized in that, described ZmNF-YA3 gene merges with just form, anti-sense versions or its RNAi structure formation and promotor, and wherein said promotor has stress induced and/or composing type.
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