CN107987141A

CN107987141A - A kind of applications of Maize kernel factor gene ZmNF-YA1 in stress resistance of plant transformation

Info

Publication number: CN107987141A
Application number: CN201810090650.8A
Authority: CN
Inventors: 张举仁; 王保海; 王洁敏; 李朝霞
Original assignee: Shandong University
Current assignee: Shandong University
Priority date: 2018-01-30
Filing date: 2018-01-30
Publication date: 2018-05-04
Anticipated expiration: 2038-01-30
Also published as: CN107987141B

Abstract

The invention discloses a kind of applications of Maize kernel factor gene ZmNF YA1 in stress resistance of plant transformation, it is that ZmNF YA1 genes are cloned from corn, with justice or anti-sense versions or RNAi structure types by the genetic recombination into plant expression vector, using transgenic technology by ZmNF YA1 channel genes plant；By detecting transgene expression and carrying out resistance measure to plant, the Transgenic plants and progenies for improving or reducing to stress resistance are filtered out, create the new germ plasm in plant breeding with application value.The present invention is adjusted using ZmNF YA1 genes by gene expression regulation to participate in stress resistance of plant, is of great significance for the transgenic crop for cultivating high yield.

Description

A kind of applications of Maize kernel factor gene ZmNF-YA1 in stress resistance of plant transformation

Technical field

The invention belongs to the bioengineering breeding field of crops, specifically, is related to a kind of Maize kernel factor gene ZmNF- Applications of the YA1 in stress resistance of plant transformation；I.e. by building, transgenosis is overexpressed structure and transgenic approach changes corn and resists The scheme and purposes of inverse property.

Background technology

NF-Y (nuclear factor-y) is a kind of transcription factor complex of generally existing in eucaryote, by 3 Different subunit (NF-YA/CBF-B, NF-YB/CBF-A and NF-YC/CBF-C) compositions, and by acting on other regulatory factors Promoter adjust the expression of target gene.Complete NF-Y transcription complexes and the CCAAT motifs in target gene promoters With reference to regulating and controlling the transcription of target gene.Wherein NF-YA is the specific subunit of DNA sequence dna, is attached to the core of gene promoter area On pentamer nucleotide CCAAT motifs (motif).NF-Y complexs can be used as an activating transcription factor or repressor to rise Effect, itself and the combination of DNA and transcriptional regulatory activity are also subject to other transcription factors to adjust, and the latter passes through mutual with NF-Y subunits Effect is worked.

In yeast and mammal, every kind of NF-Y subunits are encoded by a single-gene, these genes have a variety of montages Mode, have a variety of posttranslational modifications.In mammal, the physiological function and its participation adjusting approach of NF-Y complexs are including more Kind molecule biochemical process, such as endoplasmic reticulum stress, DNA damage and reparation, cell cycle regulating.But in plant, per middle NF-Y Subunit is encoded by a gene family, and therefore, NF-Y complexs have the diversity being made of different subunits.In arabidopsis In, NF-YA subunits are by 10 gene codes, and NF-YB subunits are by 13 gene codes, and NF-YC subunits are by 13 gene codes.Water Rice at least 11 NF-YA genes, 12 NF-YB genes, 8 NF-YC genes.In corn, 14 NF-YA have been had now been found that Gene, 18 NF-YB genes, 18 NF-YC genes.GUS expression analysis, hair are carried out to 36 NF-Y subunit genes of arabidopsis Now each subfamily member has complicated and diversified expression pattern, implies the work(of the different NF-Y complexs of these family members composition The variation of energy, adjusts the expression of several genes.

Work on hand shows that tri- subunit families members of NF-Y have both participated in the regulation and control of growth and development of plants.In arabidopsis Middle LEAFY COTYLEDON1 inductions vegetative cell develops into embryo, and two NF-Y subunit genes (AtLEC1/AtNF-YB9) are adjusted Embryo occurs and seed maturity.AtNF-YB9 and LEC1-LIKE (L1L/NF-YB6) is formed by inducing embryo and cell differentiation The expression of related gene regulates and controls arabidopsis embryonic development.Multiple NF-YA subunit genes are expressed in embryo in arabidopsis, such as NF- YA1, YA2, A3, A4, A6, A7, A8, and A^].It is super to ABA quick when seed is sprouted to be overexpressed NF-YA1, A5, A6, or A9 strain Sense, Exflagellation, embry ogenesis, seed morphology and sprouting are affected.These overexpression NF-Y materials can be directly from nutrition Orga- nogenesis somatic embryo.NF-YA3 and-A8 have highest expression quantity when globular embryo is to torpedo embryo period.nf-ya3nf- Ya8 double-mutant embryonic deaths, but nf-ya3 and nf-ya8 single mutants do not show obvious character mutation, thus it is speculated that the two Gene functional redundancy in arabidopsis body early embryo forming process.

Except influencing, plant embryos are formed and seed maturity, NF-Y transcription factors also adjust the nutrient growth or/and life of plant Reproductive growth.AtNF-YB2 is overexpressed by stimulating cell division and cell elongation to promote primary root to extend.Planted in beans pattern In thing Medicago truncatula, MtHAP2-1 is expressed in root nodule meristematic zone, and the differentiation to nodule cell plays an important role.It is overexpressed Kidney bean PvNF-YC1 and by RNAi disturb the research of the gene expression disclose NF-Y played in nodule organs' forming process it is important Effect.In picea wilsonii, Yu etc. has found that a NF-YC albumen adjusts the growth side of pollen tube by interacting with PwFKBP12 To.

The report NF-Y that worked adjusts drought resistance in plants.Be overexpressed respectively in arabidopsis and corn AtNF-YB1 and its Corn homologous gene ZmNF-YB2, in drought condition Transfer-gen plant growing way and survival rate better than nontransgenic plants.Core Piece testing result shows that AtNF-YB1 expression changes do not influence dehydration-response element associated proteins (dehydration- Responsive element binding proteins) or ABA- dependences drought resisting path, imply AtNF-YB1 may lead to Cross one and do not depend on ABA signal Drought Resistance Approaches and work.Multinomial work shows NF-Y and bZIP interactions, and bZIP albumen participates in ABA signal paths.

AtNF-YA5 genes are expressed by arid, osmotic stress and salt stress inducible up regulation.The transcriptional control of AtNF-YA5 by ABA mechanism regulatings are relied on, miR169 participates in its posttranscriptional modification.Under drought condition, miR169 lowers expression, which relies on ABA.35S::MiR169 and nf-ya5 mutant plants are more sensitive to drought stress.AtNF-YA5 regulation and control guard cell's stomatas Size, AtNF-YA5 also by activating the expression of stress response gene in other cells, as oxidative stress respond related gene, with Improve the drought tolerance of plant.In addition to AtNF-YA5, being overexpressed other AtNF-YA genes such as YA2, YA3, YA7, YA10 can also improve The tolerance of plant pair arid^[148].11 wheat NF-Y genes expression and expression under normal condition under drought condition Level is compared to having significant difference, wherein 8 are lowered expression, 3 up-regulated expressions by arid.One NF-YA gene of rice (OsHAP2E) be miR169 target, by salt stress induced expression, thereby increases and it is possible to by rely on ABA path regulate and control.OsNF-YA7 by Drought stress induced expression, but its expression is not responding to ABA.The drought tolerance for being overexpressed OsNF-YA7 rice plants is improved.Point Analysis OsNF-YA7 promoters find there are 3 DRE/CRT elements, which is adjusted transcription factor by the arid for not depending on ABA OsDREB2 regulates and controls.Therefore speculate that OsNF-YA7 mainly adjusts the drought tolerance of plant by not depending on the approach of ABA.Drought stress The transcriptional expression level of OsNF-YA2 is reduced, the drought tolerance of osnf-ya2 insertion and deletion mutant improves, and shows that this gene can It can be negative regulatory factor arid in rice.Wheat NF-YA genes TaNF-YA10-1 is overexpressed in arabidopsis can improve plant Drought tolerance, promote the growth of root system and plant, but to be overexpressed plant pair salt stress more sensitive by TaNF-YA10, shows the gene Regulatory function in response drought stress and salt stress is different.

The response that existing multinomial work report NF-Y transcription factors regulation and control plant coerces hot and cold.Table is crossed in arabidopsis The cold stress resistance of plant can be improved up to AtNF-YA2 or AtNF-YC1.Transcriptome analysis in AtNF-YA2 it turns out that be overexpressed Some abiotic stress responsive genes lower expression in plant, but the downward of these genes can improve AtNF-YA2 and be overexpressed plant Cold resistance mechanism it is not clear.AtNF-YC1 may be by relying on the way of C-REPEAT BINDING FACTORs (CBFs) Footpath adjusts freeze proof stress.

Influence getting worse of the heat stress to crop yield, this is the research topic that plant science is paid close attention at present.Sato etc. It was found that DREB2A can with DNA POLYMERASE II SUBUNIT B3-1 (DPB3-1/NF-YC10) interaction, especially in heat stress Under the conditions of.DPB3-1/NF-YC10 can form tripolymer with NF-YA2, NF-YB3, adjust related to the heat stress of DREB2A mediations The expression of gene.It is overexpressed DPB3-1/NF-YC10 and improves heat stress responsive genes HEAT SHOCK TRANSCRIPTION The expression of FACTOR A2 (HSFA2), and the expression of HSFA2 is lowered in nf-yc10 mutant^[158].It is overexpressed NF-YC10 does not influence the growth of Arabidopsis plant, and it is heat-resisting can to improve rice plant by overexpression arabidopsis AtNF-YC10 in rice Property without influence plant growth and development.NF-YC10 belongs to monophyletic group, highly conserved in land plant, therefore can conduct Create the candidate gene of heat stress resistance crop.

NF-Y transcription factors also participate in endoplasmic reticulum (ER) response.In arabidopsis, NF-YC2, NF-YA4 and NF-YB3 A transcription complex is formed with bZIP28, raises the expression of er stress related gene, prevents false folding and unfolded Albumen is in the accumulation of endoplasmic reticulum, the decline of alleviation protein synthesis.Although existing bioinformatic analysis result and chip data Understanding, the expression of NF-Y gene family members is regulated and controled by various abiotic stress, but for the specific degeneration-resistant Function and its mechanisms of plant Research is very few.

In many reports, the expression pattern of plant NF-YA gene families has Space-time speciality.Arabidopsis NF-YA man Expression analysis of the family member in different tissues finds there is common organ expression pattern, such as AtNF-YA4 between some members It is similar with the expression pattern in spending in leaf with AtNF-YA7, imply their functional redundancy.Zhang etc. is to 50 ZmNF-Y genes The identification of carry out system, and its expression pattern of chip data analysis is utilized, draw some ZmNF-Y family members in nutrition device Present specific expressed in official and reproductive organs, and there is also difference for the response between different members to biotic and abiotic stress It is different.By being inoculated with pathogen fusarium moniliforme, head smut bacterium and lawn anthrax-bacilus respectively, ZmNF-Y is to biotic for analysis Response, finds ZmNF-YA3, ZmNF-YA8 and ZmNF-YA12 up-regulated expression, and ZmNF-YA1 lowers expression.Luan Mingda etc. is to 7 A ZmNF-YA by ripe miR169 regulation and control carries out Subcellular Localization, it is found that these ZmNF-YA are positioned at nucleus In, but lack the ability of transcriptional activation.The expression pattern of ZmNF-YA and miR169 response environment stresses is analyzed, is found In maize leaf, expression response PEG, ABA and salt stress of ZmNF-YA and miR169, but be not expected between the two Negative correlativing relation.In corn root, short-term stress causes the expression of miR169 to be lowered (when 0-48 is small), and coerces (15 for a long time My god) expression of up-regulation miR169, and most of ZmNF-YA shows opposite expression change in short-term stress with miR169 Change.When responding environment stress, relation that the elongation of miR169 expressions and corn root is proportionate, and ZmNF-YA14 with Negative correlativing relation is presented in the elongation of root, i.e. miR169/ZmNF-YA14 regulation and control module may take part in corn root response environment stress Regulation process.In addition, in the expression quantity of overexpression ZmNF-YA14 up-regulation peroxidase related genes, positive scavenger-cell The accumulation of active oxygen, resistance of the enhancing corn to salt stress.

Corn NF-Y family gene quantity is big, retrieves maize genomic sequence, it is found that corn NF-YA families may member 36 A, NF-YB member 28, NF-YC member 25.But the functional study of these NF-Y family genes is still at the initial stage, retrieval Be sees only corn ZmNF-YB2 gene overexpressions improve plant drought tolerance research report and Monsanto Company application it is special Profit.Concrete application in relation to Maize kernel factor gene ZmNF-YA1 in stress resistance of plant transformation is rarely reported.

The content of the invention

For current present Research, the present invention provides a kind of Maize kernel factor gene ZmNF-YA1 in stress resistance of plant Application in transformation.Adjusted using ZmNF-YA1 genes by gene expression regulation to participate in stress resistance of plant, it is high for cultivating The transgenic crop of production is of great significance.

Applications of the Maize kernel factor gene ZmNF-YA1 of the present invention in stress resistance of plant transformation, is reflected from corn Not and ZmNF-YA1 sequences are cloned, with total length or partial sequence gene fusion construct, the promoter of the latter is connected to target base Before encoder block (in the form of just) or RNAi structures, then fusion is inserted into plant expression vector, using turn Recombination is imported plant cell by gene technology, obtains transfer-gen plant；By detecting transgene expression and plant being carried out Characters Identification, therefrom filters out transfer-gen plant and its offspring that objective trait is substantially change, creates and have in plant breeding There are the new germ plasm and new varieties of application prospect.Concrete scheme is as follows：

A kind of applications of Maize kernel factor gene ZmNF-YA1 in stress resistance of plant transformation, it is characterized in that：From corn ZmNF-YA1 genes are cloned, with justice or anti-sense versions or RNAi structure types by the genetic recombination to plant expression vector In, form fusion；Recombination is imported into plant using transgenic technology；By detect transgene expression and to plant into Row resistance measures, and filters out the Transgenic plants and progenies for improving or reducing to stress resistance, creates in plant breeding New germ plasm with application value；Wherein, the nucleotide sequence of the ZmNF-YA1 gene cDNAs is as shown in SEQ ID No.1, The amino acid sequence that it is encoded is as shown in SEQ ID No.2.

In applications of the above-mentioned Maize kernel factor gene ZmNF-YA1 in stress resistance of plant transformation：The ZmNF-YA1 genes There are cDNA forms or a genomic gene form, its coded sequence is in the form of just or anti-sense versions or the insertion of RNAi structure types are planted Thing expression vector, is built into fusion；Promoter is stress induced promoter or constitutive promoter.

Wherein：The stress resistance of plant includes drought tolerance, salt tolerance or heat resistance；The plant is corn, wheat or morning Ripe standing grain.

The expression analysis of ZmNF-YA1 genes

In Maize mechanism study, it is material with drought-enduring self-mating system Q319 and the sensitive self-mating system 65232 of arid, uses The processing of chip hybridization Technical comparing Drought at seedling stage (is poured 3 leaf phase corn seedlings of sand culture with 18%PEG solution, is being handled respectively 24h after 12h, 24h, 48h and recovery are watered afterwards) different genotype transcript profile is influenced, it have selected a collection of in different genotype The different transcription factor of variation tendency.Then, the expression intensity of these genes is verified using quantitative RT-PCR technology.From In select ZmNF-YA1/GRMZM2G000686 genes and studied.ZmNF-YA1 is expressed rich in root under optimal growth conditions Degree is significantly higher than blade.In root, the expression quantity of drought-enduring self-mating system Q319 is 2.5 times of arid sensitive self-mating system 65232； In leaf, the expression quantity of Q319 and 65232 is close, and 1/4 in about 65232.Osmotic stress handle 24h when, it is drought-enduring from Friendship is in the root of Q319, and gene expression abundance declines, about the 70% of before processing, and expression quantity significantly rises in 65232 root, and Higher than Q319's；In leaf, the change of Q319 and 65232 is in opposite trend, i.e. Q319 substantially rises, and 65232 drop below 1/10 in before processing root is horizontal.Osmotic stress handle 48h, in root the gene expression abundance of ZmNF-YA1 in Q319 with 24h when Compared to being increased slightly, and 65232 are remarkably decreased, slightly above the level of before processing；In leaf, the expression of the ZmNF-YA4 of Q319 Abundance increases considerably compared with during 24h, about 4 times of before processing, and 65232 are presented slightly rising.Rehydration 24h, ZmNF- Transcript abundances of the YA1 in 65232 and Q319 roots is almost equal, the expression of slightly above untreated first 65232；In leaf In, the gene expression abundance of Q319 drops below before processing level, and 65232 gene expression abundance slightly rises, and abundance is higher than Q319's.I.e. the expression of ZmNF-YA1 is that osmotic stress suppresses in Q319 roots, is osmotic stress induction in Q319 leaves；And It is that osmotic stress induces in 65232, is that osmotic stress suppresses in 65232 leaf.I.e. they are anti-to osmotic stress There should be notable difference, may be associated with its drought resistance.

The generation of transgenic corns

Promoter Prd29A of the ZmNF-YA1 gene codes frame respectively with desiccation stress induction is merged, plant table is arrived in restructuring Up in carrier, carrier pCambia1300-Prd29A is constructed::ZmNF-YA4-PCaMV35S::Bar, use are agriculture bacillus mediated The T-DNA areas of the plasmid are transferred to Elite Maize Inbred Lines by method or particle bombardment, and bagging is selfed after transformation seedlings transplant survival, Harvest seed.Detected by herbicide screening and molecular biology for detection (PCR, Southern blotting, RT-PCR) Walking around progeny of plants, obtains transfer-gen plant.Meanwhile according to ZmNF-YA1 gene specific sequences, construct RNAi structures And recombinate into plant expression vector, maize genetic conversion is carried out, obtains transgenic corn plant.Pass through the selfing in continuous 3 generation And Molecular Identification, generate transgenic homozygous strain.Transgenic corn plant form and growth and development under normal cultivation condition Normally, strain ZmNF-YA1 expression intensities are overexpressed and are significantly higher than non-transfer-gen plant, and turn the ZmNF- of RNAi structure strains YA1 expression intensities are substantially less than non-transfer-gen plant.

According to particle bombardment, corn (Zea mays L.) self-mating system plant 9-15 days childrens after pollination self are taken Embryo (1.0-1.5mm sizes), is inoculated on inducing culture, and culture obtains crisp, flaxen II type callus in 4-6 weeks, Then subculture medium per 10-15 days squamous subcultures once.Acceptor material of the obtained II types callus as genetic transformation Material.

Particle gun bullet is prepared using conventional method.The bronze of 1.0 μm of sizes is weighed, is stood after the washing of 70% ethanol 15 minutes, centrifugation removed supernatant；Thoroughly clean 3 times with sterile water again, then 50% sterile glycerol (final concentration of 60mg/ml Micro- bullet) storage it is spare.It is vortexed 5 minutes during use and breaks bronze aggegation, sequentially adds 5 μ l plasmids T-DNA (1 μ g/ μ l), 50 μ l 12.5M CaCl₂, 20 μ l 0.1M spermidines, while sample-adding while vortex.Then, continue vortex 2~3 minutes, stand 1 minute.Centrifugation After abandoning supernatant, 70% ethanol is added to stand.It is then centrifuged for abandoning supernatant, then is resuspended with absolute ethyl alcohol, sampling is added in micro- missile-borne body On.Micro- bullet dosage is often to play 0.5mg.The culture medium of 0.4cm thickness is poured into the culture dish of diameter 9cm, then by callus High density is put into culture dish bombards once per ware.Bombardment parameters take：The distance that disk and carrier can be split is 2.5cm, carrier with The distance for stopping net is 0.8cm, micro- bullet flying distance 6--9cm.Other parameters press operation instructions value.Material exists after bombardment Material, is then transferred in the new culture medium of components unchanged and cultivates 3 weeks, the target gene for making to be transferred to fills by renewal cultivation 3 days in the dark Divide expression.Material is transferred on the culture medium added with selective agent (such as 0.1% herbicide glufosinate) and is screened.Step sizing three Generation, often for 15 days.The tissue block for the death that becomes sticky is eliminated during subculture.Resistant calli by screening, which is transferred to, is not added with selective agent On culture medium when illumination 16 is small/1 generation of world renewal cultivation after, be transferred on differential medium and break up seedling.Callus produces Seedling take root in root media, move into flowerpot after strong sprout, it is long to be planted to during about 10cm high to big Tanaka, self-fertility.

The character detection and utilization of transgenic corn plant

To being selfed the obtained transgenic homozygous plant of subculture by continuous bag sleeving, it is first determined under normal cultivation condition Transfer-gen plant growth and development is normal, and then analyzes the Resistant Difference of transfer-gen plant and adjoining tree under stress conditions.

Heat resistance is tested：The jade for the transgenic homozygous that will be grown under 28 DEG C (irradiation, 13h/d)/22 DEG C (dark, 11h/d) Rice plant, which is moved under 36 DEG C (irradiations), grows 2h, then continuous heat 4 days (the irradiation 13h/d, dark 11h/d) at 39 DEG C, then The restoration ecosystem at 28 DEG C.After heat treatment, non-non-transgenic control (wt) almost all is dead.And pass through heat stress processing and turn The English grass of RNAi structure strains heat resistance compared with non-transfer-gen plant significantly improves, nontransgenic plants almost all Death, transgenosis suppress the expression aggrieved unobvious of strain, i.e. heat resistance is significantly better than control.

Cold resistance is tested：The jade for the transgenic homozygous that will be grown under 28 DEG C (irradiation, 13h/d)/22 DEG C (dark, 11h/d) Rice plant grows 1 day successively at 16 DEG C (irradiation 13h/d), 10 DEG C under (irradiation 13h/d), the then continuous cold treatment 3 at 4 DEG C My god (irradiation 13h/d), the then restoration ecosystem at 28 DEG C.In cold treatment, non-non-transgenic control (wt) growth is slow, bright after processing Show less than transgenic line；Handled 1 day at 4 DEG C, a small number of transgenosis are overexpressed plant and are damaged with non-non-transgenic control blade, leaf Point is often wilted, and turns RNAi structures plant to damaging to plants caused by sudden drop in temperature sensitivity, is damaged heavier；Restoration ecosystem 1 day after 4 DEG C of processing 3 days, turns For gene overexpression strain extent of injury significantly lower than control, only there is blade tip wilting in which part strain, and turns RNAi structures Plant is damaged weight, and seedling almost all is dead.

Drought tolerance is tested：Take and turn the pure of ZmNF-YA1 genes and its RNAi structures or anti-sense versions after abundant drying respectively The mature seed for closing strain is sowed in native disk, is put and is grown under suitable condition.Stop watering, Osmotic treatment one during the 4 leaf phase of plant In week, then recover watering, observes plant growth condition and survival rate, determines the drought resistance of plant.The result shows that no matter in seedling stage Or jointing stage reduction ZmNF-YA1 gene expressions reduce plant drought resistance, and ZmNF-YA1 gene overexpressions significantly improve plant The drought resistance of strain.

It will turn ZmNF-YA1 genes and its RNAi structure or the corn of anti-sense versions and the control self-mating system kind of non-transgenosis Son broadcast flowerpot and crop field, done respectively in seedling stage male and female Ear development (9~13 leaf phase), pollination period of blooming and pustulation period Non-irrigated Stress treatment, i.e., water by controlling and avoid drenching with rain, and keeps soil relative water content in 55-60% or so, duration 15 days, and the detection of transfer-gen plant character observation and physical signs is carried out, open pollination, the simultaneously species test of harvest fruit ear.It was found that turn ZmNF-YA1 is overexpressed strain drought resistance and is significantly higher than non-non-transgenic control and turns RNAi structures or anti-sense versions, not only plants Strain damage symptoms are light, and restoration ecosystem is fast after stress releases, and the economic characters such as a single plant's output are significantly better than non-transgenosis Compare and turn RNAi structures；And the plant drought resistance and grain yield for turning RNAi structures or anti-sense versions are significantly worse than and do not turn Genetic contrast.Embodiment 3 and embodiment 4 are shown in transgenosis English grass plant Resistance detecting and plant screening.

Tested by Resistance detecting, stress induced promoter start transgenosis be overexpressed the drought-enduring of plant, heat resistance and Cold resistance is significantly improved than non-non-transgenic control.On this basis, comprehensive many test results, select excellent transgenosis The selfing of plant bagging is homozygous, and carries out combining ability test, selects the transgenosis that coordinate force is identical with donor self-mating system or is improved Self-mating system is used for Corn Single-Cross Stock selection and breeding.

Embodiment

Embodiment 1：Turn ZmNF-YA1 genes and create Maize salt-tolerant inbred lines

1) foundation of receptor system is using Inbred Lines used in China's agricultural production as material, as Zheng 58, prosperous 7-2, DH4866 etc..Seed is soaked 8 minutes with 70% ethanol, then is soaked 8-12 minutes with 0.1% mercury chloride, then with sterile water washing 3-5 times.Sterilization period constantly rocks seed, to ensure that surface sterilizing is thorough.Seed is placed in sterile triangular flask and sprouts after sterilizing, A small amount of sterile water is put into 1-2 days under dark condition (25-28 DEG C) in bottle.After Seed sprouting (showing money or valuables one carries unintentionally), improvement is placed it in Sprouted on MS culture mediums under dark condition.When plumule elongation stops 3-4 centimetres, plumule and 2-3 piece spires, emergent stem are peeled off Pointed tip growth cone.

2) conversion of corn stem apex and plant regeneration structure carry the fusion base of Maize kernel factor gene ZmNF-YA1 complete sequences Because of (just form), transgenosis ZmNF-YA1 stress induced promoter RD29A/B or CaMV35RNA promoter or corn Ubiquitin promoters start.Fusion is recombinated to T-DNA zone to the root nodule for having plant herbicide resistant gene bar again Agrobacterium is answered in Mini--Ti plasmids, obtains genetic transformation carrier.

The root nodule agriculture of binary vector (Mini--Ti plasmids are with selective agent resistant gene and ZmNF-YA1 genes) will be carried In the LB culture mediums of additional antibiotic, (every liter of culture medium contains bacillus (such as LBA4404)：Tryptone 10g, yeast extract 5g, NaCl 10g, pH 7.0, pressure sterilizing) in shake culture at 28 DEG C, concussion speed is 110rpm (rev/min), is made at bacterium In exponential phase.Then centrifuge 10 minutes at 3,000 rpm, abandon supernatant.Thalline sprouts training with the liquid seeds of 1/2 concentration Base (i.e. seed germination medium component halves, and removes agar powder) washing is supported, then is collected by centrifugation.Again by thalline addition acetyl fourth The liquid modified MS medium of 1/2 concentration of 100 μm of ol/l of ketone musk (acetosyringone, As) suspends, and 5--20 times of dilution is used In conversion.Bacterium solution is poured in the culture dish of 4.5 cm diameters during conversion, culture dish is tilted, makes the nothing for exposing stem apex growth cone Vaccine is immersed in bacterium solution, is handled 8-12 minutes under 0.5 × 105Pa atmospheric pressure.Again by the bud point aseptic filter paper after dip dyeing Blot, germination seed is placed in modified MS medium to be cultivated 2-3 days in dark, and cultivation temperature is 22-24 DEG C.Then will be sterile Seedling is put to be cultivated 2 days under diffuse light.Aseptic seedling after irradiation culture is transplanted to and is covered with the flowerpot of upper strata vermiculite lower floor loam, And with the top of vermiculite covering plant.Then plant is allowed to be grown under natural lighting, 22-28 DEG C of day temperature, 15-21 DEG C of night temperature, every other day Pour 1/2 modified MS medium inorganic salts.

3) Resistance detecting and Selection utilization of transfer-gen plant

After transformed plant grows 3 leaves, 0.12% herbicide glufosinate aqueous solution is sprayed, falling drop with plant is advisable.Not Conversion adjoining tree stops growing after 4 days after spraying, starts after 9 days dead.After spraying, some individuals change transformed plant Similar with adjoining tree, other individual continued propagations, change unobvious.When the plant that survives grows to 5 leaf, it is colonized to field Between, bagging selfing is set seeds.Take the blade of transplant survival plant to carry out molecular Biological Detection and determine transfer-gen plant.It will then turn Gene plant (T0) bagging self-fertility.T1 seeds from different T0 plant are broadcast in greenhouse or there is the field of protective equipment Between, sprinkling 0.18% herbicide glufosinate aqueous solution of sprinkling, observes plant resistance.The antiweed plant that T1 generations filter out continues Bagging is selfed, and continues molecular biology identification and Resistance detecting to its filial generation.Pass through number generation selfing homozygosis and Resistance detecting And selection, it is final to obtain transgenic corns homozygous line.In Identification of Drought and selection, transgenic line is planted and flower respectively Basin, greenhouse and crop field, in 3 leaf phases, jointing stage and the early period of blooming, take out male and loose powder phase, pustulation period progress drought stress processing, survey Determine change and the grain yield of physiological parameter, filter out strong drought resistance, grain yield is dramatically increased than non-non-transgenic control material, And the grain yield with acceptor self-mating system under the conditions of suitable cultivation slightly has the strain improved.The strain can be used for preparation corn to resist Non-irrigated antiweed cenospecies.

The seed of homozygous transgenic line is sowed in the flowerpot of sand is filled, pours 0.5% or 0.7%NaCl's Aqueous solution, pours the 1/3MS culture medium inorganic salt solutions added with 0.5% or 0.7%NaCl, statistics emergence rate, leaf after emergence Piece degree of necrosis, 5 leaf phase survival rate of plant, therefrom select the excellent transgenic line of salt tolerance and are used for inbred line breeding and new product Kind is cultivated.

Embodiment 2：Turn ZmNF-YA1 genes and create the heat-resisting self-mating system of corn

1. the acquisition of corn gene plant

The seed of maize elite inbred line is sprouted, plasmid construction and genetic transformation are the same as embodiment 1.Transformed plant grows 3 Ye Hou, sprays herbicide(Hoechst Schering AgrEvo GmbH, contain herbicide glufosinate Ammonium) aqueous solution, concentration are 9.6ml -10.8mlFalling drop with plant is advisable.Unconverted adjoining tree Stop growing, start after 9 days dead after 4 days after spraying.After spraying, some individual changes are the same as adjoining tree phase for transformed plant Seemingly, other individual continued propagations, change unobvious.When the plant that survives grows to 5 leaf, it is colonized to field, bagging selfing Set seeds.

2. transfer-gen plant homozygosis and offspring analysis

T1 grew to for 3 leaf phases for plant and uses 10.8mlAqueous solution processing, observation statistics resistance and sensitiveness Body ratio；Foreign gene is detected using round pcr, and counts segregation ratio of the foreign gene in progeny plant.Survive plant It is transplanted to crop field, bagging selfing.T2 in addition to bagging selfing is set seeds, is carried out for plant using round pcr detection foreign gene Southernblotting is verified, and checks transgene expression intensity using RT-PCR technology.For selected transgenic line The change of plant Net Photosynthetic Rate under different temperatures (28~39 DEG C) and light intensity is measured, measures single plant yield and biomass, and Using non-transfer-gen plant as control.Yield Traits In Corn is carried out under field cultivating condition after selecting excellent transgenic line Observe and compare, select high yield specular removal strain to enter Heat tolerance identification experiment and corn breeding experiment.

3. transgenic line Heat tolerance identification is tested

Seedling stage heat-resistance test：The transgenic homozygous that will be grown under 28 DEG C (irradiation, 13h/d)/22 DEG C (dark, 11h/d) Plant move into 36 DEG C (irradiations) under grow 2h, then continuous heat 4 days (the irradiation 13h/d, dark 11h/d) at 39 DEG C, Then the restoration ecosystem at 28 DEG C.After heat treatment, non-non-transgenic control (wt) almost all is dead, and in transgenic line only Have that indivedual strains and non-non-transgenic control gap are little, and the heat resistance of most strains is significantly better than control, its middle part Divide the aggrieved unobvious of transgenic line, the as tentatively selected heat-resisting strain of transgenosis.

Pollination and Early filling stage heat resistant test：The seed of the tentatively selected heat-resisting strain of transgenosis is sowed in greenhouse, Pollination period is grown under optimal growth conditions, greenhouse temperature is then adjusted to 36 DEG C (irradiation, 13h/d)/30 DEG C of (dark, 11h/ D) grow 7 days under, then be adjusted to unite after growing 7 days, 20 days under 38 DEG C (irradiation, 13h/d)/30 DEG C (dark, 11h/d) by temperature Count plant greenery number and fruit ear number.The parameters such as fruit ear development condition, Setting percentage, 100-grain weight are observed after harvest, filter out excellent heat resistance Transgenic line.

Embodiment 3：Turn ZmNF-YA1 genes and create drought-enduring salt tolerant annual bluegrass

With the different cultivars of English grass (Poa pratensis L), such as prize, new song come, rugby-A, the noon The seed seedling stem apex at night etc. is test material, induces its base portion callus to expand, cuts and expand tissue, is placed on subculture and Cheng Congpei Support induction on base and Multiple Buds occur, the latter is in subculture and into persistent hyperplastic on clump culture medium, there is provided the acceptor material of transgenosis.Adopt With agrobacterium-mediated transformation by Studies of Transfer of Alien Genes Into Receptors cell, chosen acquisition transformed cells and plant.Agriculture bacillus mediated In genetic transformation, handled using surfactant Silwet L-77 and reduced pressure treatment effectively increases transformation frequency, it is established that one A efficient genotype restricts small annual bluegrass transgenic technology system.Concrete operations are as follows.

Prepare various culture mediums

Minimal medium：For modified MS medium (MS culture mediums inorganic salts, thiamine hydrochloride 10.0mg L^-1, hydrochloric acid pyrrole trembles Alcohol 1.0mg L^-1, nicotinic acid 1.0mg L^-1, glycine 2.0mg L^-1, inositol 100.0mg L^-1, biotin 0.05mg L^-1, junket egg White hydrolysate 500mg L^-1), sucrose 30g L^-1, agar powder 6.5g L^-1, pH 5.8--6.0.Sprouted for seed.Liquid Culture Base then removes agar powder.

Inducing culture：The minimal medium of additional different plant growth regulating substance (hormone) combinations.For most early For ripe standing grain genotype, 2,4-D concentration are 0.01-1.0mg.L^-1, 6- benzyl purines (6-BA) or kinetin or zeatin concentration 0-5mg.L^-1.Using solid medium.Most suitable hormone concentration becomes within this range because the genotype of culture materials is different It is dynamic.

Subculture and into clump culture medium：Solid medium, the Plant growth regulators of various combination are added in minimal medium Matter, 2,4-D concentration are generally 0-0.2mg L^-1, 6-BA or kinetin or zeatin concentration are 0-3mg L^-1, because of annual bluegrass base Changed because of type.

Root media：Minimal medium adds 0.002-2.0mg L^-1 methyl α-naphthyl acetate (NAA) or indolebutyric acid (IBA) or Yin Indolylbutyric acid (IAA), using solid medium, for the small seedling rooting of unrooted or strong seedling culture.Most suitable hormone concentration is because cultivating material The genotype of material is different and changes within this range.

Seed sterilization and sprout tuber induction of sprouting and grow thickly

Seed is sterilized 4-5 minutes with 70% alcohol, 0.2% mercuric chloride 10-15 minutes, aseptic water washing 3-5 times.The sterilizing phase Between constantly rock seed, to ensure that surface sterilizing is thorough.Seed sowing is on the aseptic filter paper of moistening after sterilizing, culture dish sealing After be placed under dark or low light condition in 20-28 DEG C of sprouting.Stem apex is cut between 10-15 days and is placed on inducing culture (addition 2,4-D 0.2mg L^-1, 6-BA 2mg L^-1) on cultivate, induction base portion expands, and expands rate for 100%.20 are cultivated on inducing culture After it, cut stem apex base portion and expand tissue (diameter 1-3mm) and be transferred to subculture and (take 2,4-D 0.1mg L into clump culture medium^-1, 6- BA 2mg L^-1) on induce Multiple Buds, after 15-20 days, majority expands base portion and differentiates Multiple Buds, left for 75% into clump rate The right side, is mostly 15-20 per clump bud number.Subculture is transferred to after the sprout tuber that will grow thickly segmentation and on clump culture medium, sprout tuber cell of growing thickly is fast Fast hyperplasia, general 15 days squamous subcultures are once.To obtain larger seedling, remove subculture and into removing 2,4-D in clump culture medium, Retain 2mg L-16-BA.Culture of ex vivo stem apex and stem apex base portion expand under 24 ± 2 DEG C, 500-1000Lx illumination (14h/d) Thing and the renewable intact plant of the sprout tuber that grows thickly.

English grass genetic transformation

ZmNF-YA1 gene clonings and Fusion gene construction and conversion plasmid restructuring will carry binary vector with example 1. The agrobacterium tumefaciens (such as AGL0 and LBA4404) of (Mini-Ti plasmids carry Plant selection marker gene) are additional antibiotic (every liter of culture medium contains LB culture mediums：Tryptone 10g, yeast extract 5g, NaCl 10g, pH 7.0, pressure sterilizing) in Shake culture at 28 DEG C, concussion speed are 170-180rpm (rev/min), bacterium is in exponential phase (OD₆₀₀=0.4- 0.6).Then centrifuge 10 minutes at 3,000 rpm, abandon supernatant.Thalline is washed with the liquid minimal medium of 1/2 concentration, then It is collected by centrifugation.The thalline liquid minimal medium of 1/2 concentration is suspended again, 5~20 times is diluted, adds acetosyringone (acetosyringone,As)100μmol L^-1It is used to turn afterwards with surfactant Silwet L-77 (concentration 0.1-0.3%) Change.

Annual bluegrass Multiple Buds are switched to the sharp growing point of exposure budding, is put into and 0.95-0.10 is disseminated and be accompanied by above-mentioned bacterium solution A atmospheric pressure (0.95 × 10⁵Pa--1×10⁴Pa) handle, disseminate 4-8 minutes.Then bacterium solution is sucked with aseptic filter paper, will grown thickly Sprout tuber is transferred to subculture and into co-culturing 2-3 days on clump culture medium, returns again to added with antibiotic cephalosporin (Cefotaxime) 250mgL^-1Or carbenicillin (Carb) 500mgL^-1Culture medium on cultivated in dark, inhibit bacteria growth.In antibacterial training The gradual restoration ecosystem of sprout tuber of growing thickly on base is supported, is transferred on the screening and culturing medium containing selective agent and screens again after 10-15 days.Continuously Screened for 3 generations, often for 15-20 days.Selective agent resistant gene bar or antibiotics resistance gene hpt (damp enzyme element phosphotransferase bases Cause).Then resistant tissues block is transferred to subculture and into breaking up seedling on clump culture medium.The latter is taken root length on root media Greatly.Seedling of taking root moves into flowerpot, and the every 5 days inorganic salt solutions for pouring 1/2 minimal medium, water once every other day.Transplanting The seedling survived takes blade to be used for molecular Biological Detection.Through PCR and Southern Northern blot analysis, (generation turns base to conversion ratio Because of the sprout tuber number * 100 of sprout tuber number/Agrobacterium infection of plant) it is 2~5% or so.Induction base portion expands more sensitive to light. Low 2,4-D concentration (<0.1mg.L-1), intensity of illumination>Easily differentiation is taken root during 1000Lx, is not easy to be divided into clump, when illumination is weak (500Lx), which expands, to be easier to, and is not easy to take root.In appropriate 2,4-D concentration, well-grown (expands group during light intensity about 1000Lx It is woven to pistac), than being more easy into clump at 500Lx (expanding tissue deviation white), and it is also more per clump bud number.Highly concentrated During degree 2,4-D, illumination power influences it not being very big, expands larger, is not easy to take root, but low into clump rate.This stage should be Suitable 2,4-D concentration illumination is more lower to cultivate in favor of later Cheng Cong.At first 5-8 days of sprout tuber squamous subculture of growing thickly, if light According to intensity>During 1000Lx, tissue is easily taken root, and is unfavorable for the differentiation of bud, and once being formed after Multiple Buds, easily promote when illumination is strong Differentiation.So first 5-8 days after squamous subculture, the sprout tuber that grows thickly is cultivated under dim light (about 500Lx), when growing several budlets After be put in intensity of illumination>Cultivated under 1000Lx, per clump bud number up to 15-25.

Screen determining for agent concentration

After herbicide aqueous solution filtration sterilization, add and remove casein hydrolysate (when culture medium temperature is less than 50 DEG C) In inducing culture.Glufosinate concentration is respectively 0.1%, 0.15%, 0.2%, 0.25%, i.e. 4 gradient concentrations.It will induce Multiple Buds be cut into after simple bud to be put on the culture medium containing various concentrations chlorsulfuron and cultivate, be inoculated with every kind of Selective agar medium small More than 100 plants of seedling.Every 15 days squamous subcultures once, co-continuous screening three generations.According to the survival rate after screening, different cultivars is determined The herbicide of seedling suitably screens dosage.Glufosinate selection concentration such as kind prize is 0.2%, step sizing at this concentration 3 generations, small shoot survival percent>10%.Survive seedling transgenosis PCR detections and be generally the positive.

Small transplantation of seedlings and the screening of transfer-gen plant

Seedling root induction on suitable root media.As root long 2-3cm, seedling is placed under natural light and is trained Support 1-2 days, then remove sealed membrane hardening 1-2 days, it (is loam under flowerpot, top is to be transplanted into flowerpot in early morning or dusk Vermiculite) in, a 1/2 minimal medium inorganic salt solution is poured within 5 days, watering in 3 days is once.Transplanted seedling growth temperature is 15-25 ℃.When seedling grows to 4-5 tiller, moisture is filled with enough, more than January, plant to be planted blade is all wilted to be recovered Continuous Drought after a week Watering, recovers faster plant progress clonal propagation after selecting rehydration.Part clone's transplantation of seedlings is treated into the flowerpot equipped with loam 0.3%~0.5%NaCl aqueous solutions are excessively poured when plant grows to 5 or so tillers, continuous pouring 3 days, makes soil salt content Reach setting concentration (being not higher than 0.5%NaCl), then maintain the 1-3 months, select survival plant and carry out clonal propagation.Meanwhile Seedling is cloned by the constant herbicide glufosinate aqueous solution repeated screening transfer-gen plant of spray concentration.It is excellent to drought-and salt-tolerance The strain of anti-glufosinate carries out space isolation or bagging, it is set seeds under the conditions of isolation.

The transfer-gen plant seed of harvest is seeded in flowerpot respectively, sprays removing for 0.25% concentration in the 4-5 leaf phases Careless agent glufosinate, survival plant plant carry out salt tolerant, drought tolerance detection, therefrom filter out the drought-enduring transgenosis of excellent salt tolerant again Stable strain, environmental greening is can be used to after the latter's breeding.

Embodiment 4：Turn ZmNF-YA1RNAi structures or anti-sense versions create heat-resisting English grass

The promoter Prd29A of ZmNF-YA1 anti-sense versions or RNAi structures respectively with desiccation stress induction is merged, then Recombinate and English grass is converted in plant expression vector, obtain transfer-gen plant.In natural conditions after the latter's transplant survival Under grow into 5-6 tillering, spray 0.25% glufosinate solution, survival plant is bred by asexual clonal, and Carry out transgenic molecules detection.The transgenosis annual bluegrass plant of acquisition is homozygous by the Molecular Identification and single plant in continuous 2 generation, produces Transgenic homozygous strain.Using transfer-gen plant as material, hot, cold Stress treatment has been carried out respectively, specify that transfer-gen plant Had differences with adjoining tree in resistance.Heat treatment method is as follows.

Transgenosis annual bluegrass plantlet of transplant produces a large amount of tiller formations after 2 months.Select the precocity of the transgenosis of neat and consistent To flowerpot and dixie cup, plant to be planted is cradled standing grain plantlet of transplant after growing 2 months, and when young leaves length to 10cm or so carries out heat resistance survey It is fixed.Material is put into growth cabinet during processing, controls photoperiod 12h/d, relative humidity 70%.During 40 DEG C of heat stress processing, Temperature is by 30 DEG C of (2h) → 32 DEG C (2h) → 34 DEG C (2h) → 36 DEG C (2h) → 38 DEG C (2h) → 40 DEG C (3d) → 23 DEG C (3d) ladders Degree change.During the processing of 46 DEG C of heat stresses, by 30 DEG C of (2h) → 32 DEG C (2h) → 34 DEG C (2h) → 36 DEG C (2h) → 38 DEG C (2h) → 40 DEG C of (3h) → 46 DEG C (5h) → 23 DEG C (7d) gradeds.The upgrowth situation of observation material during processing, including wilting degree, Survival rate, leaf color change, difference etc. between strain.Meanwhile respectively before treatment, 40 DEG C processing 2h, 12h, 3d and recover 3d and Related physiological parameters, including chlorophyll content, membrane damage, chlorophyll fluorescence, physiology are measured during 46 DEG C of processing 5h and recovery 10d Learn index measure according to《Modern plants Physiology Experiment guide》(soup Zhang Chengzhu volumes) carries out.Fv/Fm values are small by dark adaptation half When after measure.

During 46 DEG C of short term thermal Stress treatments, recover to train at different annual bluegrass plant are placed on 23 DEG C after 46 DEG C of processing 5h Support, observe the growing state of different disposal period plant, measure relative physiologic index, such as chlorophyll content, membrane damage, chlorophyll Change in fluorescence etc..After 46 DEG C of processing, reduce ZmNF-YA1 gene expression strains and be better than control strain, grow 10 at 23 DEG C Though whole plant survivals, growing way are widely different after it.The long potential difference of nontransgenic plants, heat stress are damaged seriously, recover slow；Drop Low ZmNF-YA1 gene expressions plant recovers fast, and tiller is more, and growing way is strong, and Newborn Leaves length of a film, shows them in heat stress It is impaired light, there is good heat resistance.In heat treatment, blade Fv/Fm is on a declining curve, is gradually increasing in recovery period.No Rise with annual bluegrass strain in the initial 2 days mda contents for being heat-treated and recovering, non-non-transgenic control is apparently higher than reduction ZmNF-YA1 gene expression plant, show that latter cell Lipid peroxidation metabolism degree is low.During heat stress processing and recovering, Non- transfer-gen plant hydronium exudation rate, which is significantly higher than it, reduces ZmNF-YA1 gene expression strains, the membrane damage when recovering 2 days Maximum is presented, the latter has the Membrane stability apparently higher than control in heat stress processing and convalescence, shows to reduce ZmNF- YA1 gene expressions significantly improve the heat resistance of plant.

In 40 DEG C of Long Time Thermal Stress treatments, handle 1 day, plant has slight wilting, and difference is little to each other；Processing 3 My god, there is notable difference in the plant wilting degree of different strains, and non-transfer-gen plant wilting is heavier, reduces ZmNF-YA1 gene tables There is the heat resistance significantly improved up to strain, blade is straight and upright, and dehydration is less.When recovering to handle, non-non-transgenic control is all dead Die, reduction ZmNF-YA1 gene expression strain heat resistanceheat resistants are good, and the survival rate of part strain is higher than 70%.40 DEG C of Stress treatments 3 days are right Caused by plant injury much larger than 46 DEG C of Stress treatments 5 it is small when, i.e., long period heat stress handle annual bluegrass cell can be made Into the damage for being difficult reparation.

The strain clonal propagation that heat resistance is significantly improved compared with the control, and 0.2% sprinkling herbicide glufosinate is molten Liquid (dosage is 3 times of crop field weeding), eliminates resistance plant on the weak side.The isolation pollination of remaining plant, is mixed by strain and receives seed, used In breeding of new variety.

Different transgenic lines from same kind tie up to temperature capability and also show notable difference, this is probably due to outer Source gene integration site and expression intensity difference in transfer-gen plant is related.By plant regeneration rate, survival after Stress treatment The measured value analysis of physical signs judges in rate and Stress treatment and recovery process, in 46 DEG C of short term thermal Stress treatments to first selection Expect relatively suitable, the cycle is short, and screening effect is preferable.40 DEG C of heat stresses handle 3 days and more than, can Effective selection to go out heat resistance excellent Different transgenic line, the latter can be directly used for heat-resisting annual bluegrass breeding of new variety.

Sequence table

<110>Shandong University

<120>A kind of applications of Maize kernel factor gene ZmNF-YA1 in stress resistance of plant transformation

<141> 2018-1-29

<160> 2

<170> PatentIn Version 3.5

<210> 1

<211> 1270

<212> cDNA

<213>Artificial sequence

<220>

<221>Corn

<222>（1）…（1270）

<223>The nucleotide sequence of ZmNF-YA1 gene cDNAs

<400> 1

agagatagga aagggcccaa cagctcaaca gaaaagccaa gcaaaggctg ctgcatactg 60

gaaggccctc tgtctgtgtg cgagcgcaag agaaagggag tcagagagag agagagaggg 120

aggagacctt gcagaggagc gaagcaagca aggtgggaaa gaggcagcag caagggcggc 180

gggctgccgg aaggggaaca tgctccctcc tcatctcaca gagaatggcg cggtgatgat 240

tcagtttggc catcagatgc ctgattacga ctccccggct acccagtcaa ccagtgagac 300

gagccatcaa gaagcgtctg gaatgagcga agggagcctc aacgagcata ataatgacca 360

ttcaggcaac cttgatgggt actcgaagag tgacgaaaac aagatgatgt cagcgttatc 420

cctgggcaat ccggaaacag cttacgcaca taatccgaag cctgaccgta ctcagtcctt 480

cgccatatca tacccatatg ccgatccata ctacggtggc gcggtggcag cagcttatgg 540

cccgcatgct atcatgcacc ctcagctggt tggcatggtt ccgtcctctc gagtgccact 600

gccgatcgag ccagccgctg aagagcccat ctatgtcaac gcgaagcagt accacgctat 660

tctccggagg agacagctcc gtgcaaagct agaggcggaa aacaagctcg tgaaaagccg 720

caagccgtac ctccacgagt ctcggcacct gcacgcgatg aagagagctc ggggaacagg 780

cgggcggttc ctgaacacga agcagcagcc ggagtccccc ggcagcggcg gctcctcgga 840

cgcgcaacgc gtgcccgcga ccgcgagcgg cggcctgttc acgaagcatg agcacagcct 900

gccgcccggc ggtcgccacc actatcacgc gagagggggc ggtgagtagg gagccccgac 960

actggcaact catccttggc ttatcagcga ttcgactcgg ctctccctcg tctgaaactg 1020

aactctctgc aactactgta actgtaacta aactgggtgt gcccggattg gcggtcgttc 1080

tgttctacta ctagtacctg ctacgcgtcg ttgggttggg tctggactag agagcgtgct 1140

ggttctttga tgaacttggc tggacttgag ggtgttgact agcgcgaagc tgagttccat 1200

gtaaaacttt tgcttcaaga ccgatgactg gcggcataat aagtagcagt aataaccatt 1260

cttctgtgtc 1270

<210> 2

<211> 249

<212> PRT

<213>Artificial sequence

<220>

<221>Corn

<222>（1）…（249）

<223>The amino acid sequence of ZmNF-YA1 gene codes

<400> 2

MLPPHLTENG AVMIQFGHQM PDYDSPATQS TSETSHQEAS GMSEGSLNEH NNDHSGNLDG 60

YSKSDENKMM SALSLGNPET AYAHNPKPDR TQSFAISYPY ADPYYGGAVA AAYGPHAIMH 120

PQLVGMVPSS RVPLPIEPAA EEPIYVNAKQ YHAILRRRQL RAKLEAENKL VKSRKPYLHE 180

SRHLHAMKRA RGTGGRFLNT KQQPESPGSG GSSDAQRVPA TASGGLFTKH EHSLPPGGRH 240

HYHARGGGE 249

Claims

1. a kind of applications of Maize kernel factor gene ZmNF-YA1 in stress resistance of plant transformation, it is characterized in that：From corn gram It is grand go out ZmNF-YA1 genes, with justice or anti-sense versions or RNAi structure types by the genetic recombination into plant expression vector, Form fusion；Recombination is imported into plant using transgenic technology；By detecting transgene expression and plant being carried out Resistance measures, and filters out the Transgenic plants and progenies for improving or reducing to stress resistance, creates and have in plant breeding There is the new germ plasm of application value；Wherein, the nucleotide sequence of the ZmNF-YA1 gene cDNAs is as shown in SEQ ID No.1, its The amino acid sequence of coding is as shown in SEQ ID No.2.

2. applications of the Maize kernel factor gene ZmNF-YA1 as claimed in claim 1 in stress resistance of plant transformation, it is characterized in that： The ZmNF-YA1 genes have cDNA forms or a genomic gene form, its coded sequence in the form of just or anti-sense versions or RNAi structure types are inserted into plant expression vector, are built into fusion；Promoter is stress induced promoter or composing type Promoter.

3. applications of the Maize kernel factor gene ZmNF-YA1 as claimed in claim 1 in stress resistance of plant transformation, it is characterized in that： The stress resistance of plant includes drought tolerance, salt tolerance or heat resistance.

4. applications of the Maize kernel factor gene ZmNF-YA1 as claimed in claim 1 in stress resistance of plant transformation, it is characterized in that： The plant is corn, wheat or annual bluegrass.