CN104388448B - A kind of corn phospholipase A_2 gene ZmsPLA2-1 and its application - Google Patents

Abstract

The invention discloses a kind of corn phospholipase A_2 gene ZmsPLA2 1, wherein the nucleotide sequence of the gene cDNA is as shown in SEQ ID No.1；The amino acid sequence that it is encoded is as shown in SEQ ID No.2.The invention also discloses application of the gene in cultivating adverse-resistant characteristic and changing plant, it is to be cloned from corn, it will be in the genetic recombination to plant expression vector with justice or anti-sense versions, recombination is imported into plant cell using transgenic technology, transfer-gen plant is obtained, cultivates the cold-resistant genetically modified plants of drought resisting.

Description

A kind of corn phospholipase A_2 gene ZmsPLA2-1 and its application

Technical field

The invention belongs to crop bioengineering breeding fields, specifically, are related to a kind of corn phospholipase A_2 gene ZmsPLA2-1 And its application in plant stress-resistance character is changed.

Background technology

Framework ingredient of the phosphatide as cell membrane is experienced to stimulate and transmit during information enters cell and be sent out in cell membrane Wave highly important effect.Lipid signaling approach and photosynthesis, hormone regulating and controlling etc. influence plant growth and development process and degeneration-resistant Signal transduction is in close relations.Phospholipase A (phospholipase A, the PLA) family of hydrolytic phosphatide by phospholipase A1 (PLA1) and Two class of phospholipase A2 (PLA2) forms.The two is catalyzed the hydrolysis of phosphatide on sn-1 and sn-2 positions respectively.Wherein PLA2 is catalyzed phosphorus Fat generates sn-1 lysophosphatides (lysophospholipids) and free fatty acids (free fatty with acyl chain Acids, FFA), it is widely present in bacterium, plant, the tissue of mammal, cell and secretion.PLA2 is in many in animal Important vital movement such as lipid digestion, cytoplasmic metabolism, signal transduction, inflammatory mediator, the invasion of defence pathogen and injury etc. It plays an important role in the process.PLA2 also assists in a variety of vital movements in plant, such as aging, damage, stress response, pathogen Defence and inducing secondary metabolite accumulation etc..PLA2 can be divided into five types：Secreting type PLA2 (sPLA2s), cytoplasm Ca²⁺It relies on Type PLA2 (cPLA2s), Ca²⁺Type PLA2 (iPLA2s), PAF Acyl- hydrolases (PAF-AH) and lysosome PLA2s are not depended on.With Animal is compared, PLA2 few researchs in plant, although in 1970s it has been reported that phospholipid hydrolysis can be haemolysis by PLA2 Phosphatide and free fatty, but the definite of the various enzymes of phospholipase A2 family is applied to so far also without illustrating completely clearly Chu.Cytoplasm Ca is not found in plant²⁺Dependent form cPLA2, PLA2 are mainly sPLA2 and the class homologous with animal iPLA2 patatin PLA2(PAT-PLA2).PAT-PLA2 is the lipid acyl hydrolase for having PLA2 and PLA1 activity concurrently.Plant specificity PLA2 is sPLA2.

The sequence of sPLA2 genes is cloned from several plants and is obtained.1999, Kim etc. andDeng respectively from health It is that clone obtains the complete sequences of possible sPLA2 genes (sequence number is respectively AF064732, AJ238116 in fragrant flower and rice And AJ238117).2003, Lee etc. and the search arabidopsis database such as Bahn found that the isodynamic enzyme homologous with animal sPLA2 has 4, respectively AtPLA2- α (At2g06925), AtPLA2- β (At2g19690), AtPLA2- γ (At4g29460) and AtPLA2-δ(At4g29470)；Do not find that there are cytoplasm Ca in arabidopsis²⁺Dependent form PLA2 (cPLA2).2010, Liao and Burns is cloned from sweet orange has obtained two sPLA2 gene C ssPLA2- α and CsPLA2- β, and find that light regulates and controls the two bases The expression of cause.

Similitudes of the plant sPLA2 in amino acid levels and animal sPLA2 is relatively low (about 15%), the Ca between them²⁺Knot Cyclization and active site element similitude are 50%.According to the sorting criterion of animal sPLA2s, the sPLA2 of plant can be divided into One independent type.Plant sPLA2 can be divided into two hypotypes again：First hypotype has AtPLA2- α, the sPLA2 of elm, rice SPLA2-II and sPLA2-III, tomato and carnation sPLA2s, the amino acid sequence similarity in hypotype is 47%；Second Hypotype has the sPLA2-I of AtsPLA2- β ,-γ ,-δ and rice.Amino acid sequence similarity in hypotype is 54%.Between hypotype Amino acid sequence similarity be 29%.Plant sPLA2 includes the characteristic domain PA2C of PLA2.The structural domain is by Ca²⁺Knot Cyclization (YGKYCGXXXXGC) and catalytic site element (DACCXXHDXC) composition.Catalytic site element has extremely conservative His/ Asp bivalents.In the Ca of plant²⁺Two conservative amino acid residues, Tyr and Gly in animal sPLA2 are found on coupling collar, they Hydrogen bond is participated in be formed.As the sPLA2 of animal, the N- end signals of mediating proteins secretion are had also discovered on the sPLA2 of plant Peptide.The transient expression assay of onion epidermis cell finds that AtPLA2- β and AtPLA2- γ are secreted into space between cells really.It has reflected All comprising 12 extremely conservative cysteine residues in fixed plant sPLA2s, the latter is likely to form six pairs of disulfide bond.Animal The research of sPLA2s has shown that these cysteine residues are particularly important to the structure and activity of enzyme.SPLA2 is cut in endoplasmic reticulum After signal peptide, space between cells is secreted into mature form.The enzymatic activity difference of the mature form of AtPLA2- β and AtPLA2- γ Than improving 2 times and 1.3 times before processing.It is this to change the structural rearrangement for being attributed to enzyme molecule in process.

The activity of PLA2 is all detected on the plasma membrane and microsomal membrane of plant different tissues, it is thin including embryo, endosperm, culture Born of the same parents, cotyledon, plumular axis and root.Plant sPLA2 is purified from elm seed endosperm obtain earliest.It is a kind of 15kD or so low molecule The water-solubility protein of amount, extremely insensitive but extremely sensitive to disulfide bond reducing agent to heat, acid, organic solvent, activity needs alkaline pH And Ca²⁺.AtPLA2- α and AtPLA2- β still maintain the activity of 80-95% in boiling water treating after five minutes, and reducing agent- In the presence of 5mMDTT, activity is only original 35-65%.SPLA2 activity light cycle influences are found in sweet orange, in the photoperiod SPLA2 activity highests after starting 11 hours, under lasting dark condition, sPLA2 activity is constantly in compared with low state.Ca²⁺Concentration The enzyme activity of sPLA2s is influenced, with Ca²⁺Concentration is increased to 10mM, and the specific activity of AtPLA2- α persistently increases, and AtPLA2- β, AtPLA2- γ, AtPLA2- δ are in the Ca of micro- concentration of rubbing²⁺Lower enzymatic activity has reached plateau value.The sPLA2 enzymatic activitys and Ca of elm²⁺ Relationship it is similar to AtPLA2- α.The Ca that AtsPLA2s enzymatic activitys need²⁺Concentration and pH value are different, imply in plant by the external world The activity of sPLA2s may be by Ca during stimulation²⁺The adjusting of horizontal and pH value variation.

Stream signal people about PLA2 know little about it.Some have speculated that sPLA2 may be by G- protein regulations, because different three Aggressiveness G- protein activation objects --- mastoparan can stimulate plant PLA2 active, but there has been no positive evidences to prove SPLA2 and G- protein signals couple.Research for PLA2 downstream target genes is more depended on from the biochemical of sPLA2 and is divided Subcharacter analysis speculates, it is known that (1) PLA2 may play a role in outer signal transduction in the cell, in plant more insatiable hungers It can activated protein kinase with aliphatic acid and lysophosphatide；(2) PLA2 regulates and controls stomatal opening, and blue light and feux rouges can be by activating H⁺- Pump induction stomatal opening, and this effect can be inhibited by the inhibitor of sPLA2, illustrate that sPLA2 may participate in the light of guard cell Signal transduction；(3) PLA2 may participate in the cell elongation of mediating growth element induction.AtsPLA2- β are in seedling and active growth group It is higher to knit middle expression intensity, and by growth hormone induction, the Arabidopsis plant that AtsPLA2- β are overexpressed generates the petiole and flower of elongation Sequence stem, and cell elongation retardation is caused to the RNAi of the gene；(4) effects of the PLA2 in plant senescence, PLA2 in plant Product lysophosphatide such as LPE and lysophosphatidylinositol can specifically inhibit PLD alpha actives, it is known that PLD α cause Cellular membraneous damage and Promote plant senescence；(5) PLA2 can generate free linolenic and linoleic after acting on film fat, they can be used as octadecene The substrate of lipoxygenase (lipoxygenase, LOX) in sour approach generates lipid peroxide, and further generates jasmonic. And JA is the important regulator in plant defense response, and various plants defense reaction can be activated as system stress signal molecule The expression of related gene promotes pisatin accumulation and synthesis of pathogenesis-related protein etc..JA can be independently or via systemin Signal transduction and transmit inducible protein enzyme inhibitor synthesis, participate in the defense reaction of plant, and phenylpropyl alcohol ammonia can be started and split Solve the related wound such as the transcription of enzyme gene, regulation and control chalcone synthase, vegetative storage protein, cell wall protein rich in hydroxyproline The expression of evil defensin gene.In addition, the product PtdOH of PLD can activate PLA2 in animal, illustrate to exist between PLA2 and PLD multiple Miscellaneous interaction.

Applicant clones corn secreting type phospholipase A_2 gene ZmsPLA2-1, cDNA nucleotide sequence and coding Amino acid sequence is as shown in SEQ ID No.1 and SEQ ID No.2.It is typical containing sPLA2 in the amino acid sequence of coding PA2c structural domains with RicesPLA2-I similitude highests on amino acid levels, are 81%, belong to the second hypotype.

The long 575bp of ZmsPLA2-1cDNA sequences, thus it is speculated that it includes the 5 ' Fei FanGTTYi areas (5 ' UTR) of 14bp and 138bp 3 ' non-translational regions (3 ' UTR) and a 426bp ORF.The ORF encodes 141 amino acid, molecular weight and isoelectric point point It Wei not 153kDa and 8.60.Through retrieval, about corn phospholipase A_2 gene ZmsPLA2-1 and its in plant stress-resistance character is changed Application have not been reported.

Invention content

For current present Research, the object of the present invention is to provide a kind of corn phospholipase A_2 gene ZmsPLA2-1 and Its application in plant stress-resistance character is changed.

Corn phospholipase A_2 gene ZmsPLA2-1 of the present invention, it is characterised in that：The nucleotide of the gene cDNA Sequence is as shown in SEQ ID No.1；The amino acid sequence that it is encoded is as shown in SEQ ID No.2.

Applications of the corn phospholipase A_2 gene ZmsPLA2-1 of the present invention in cultivating adverse-resistant characteristic and changing plant.

The present invention provides a scheme using corn secreting type phospholipase A_2 gene ZmsPLA2-1, by ZmsPLA2-1 Applied to plant transgene, the engineering plant that degeneration-resistant character changes is generated, applied to crop breeding for stress tolerance.

Corn ZmsPLA2-1 is cloned and expression analysis

First, ZmsPLA2-1 genes are cloned from corn gene group DNA or cDNA, one 141 is generated after the latter's expression The albumen of amino acid.The specific steps are：With the RicesPLA2-I (AJ238116) of rice sPLA2 gene families corn EST Library carries out BLAST, obtains sequence C K827370.Sequence analysis shows that this est sequence includes complete ORF.Using PCR method Complete cDNA sequence is cloned from corn cDNA library, the i.e. ZmsPLA2-1 of SEQ ID No.1 are identified to obtain in sequencing.Sequence point Analysis shows the opening code-reading frame that the long 575bp of ZmsPLA2-1cDNA sequences includes 414bp, encodes 141 amino acid.In amino acid The sPLA2's of ZmsPLA2-1 and other plant has higher similitude in level, the typical PA2c structural domains containing sPLA2. ZmsPLA2-1 and RicesPLA2-I similitude highests are 81%.PA2c structural domains include a Ca²⁺Integrated structure (YGKYCGxxxxGC) and an active site element (DACCxxHDxC).

5 ' the terminal sequences of ZmsPLA2-1cDNA in NCBI are subjected to BLAST, obtain a series of sequence of different lengths Row.The sequence for taking the 2000bp of the 5 ' upstream of initiation codon of gene is promoter, utilizes plantCARE online softwares (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) it is analyzed, it finds The promoter region of ZmsPLA2-1 genes, possible heat stress correlation cis element 2 is distributed in DNA normal chains, and ABA is related Cis element 1, MeJA correlations cis element 2, auxin correlation cis element 1, GA₃Related cis element 1；Minus strand Upper distribution drought stress correlation cis element 1, defence cis element 2 related to stress, MeJA correlations cis element 2, Auxin correlation cis element 2.This prompting ZmsPLA2-1 genes expression may by drought stress, heat stress, ABA, MeJA、GA₃With the regulation and control of the signals such as auxin, there is important role in corn growth and to Response to stress.

The mRNA of the root of plant, stem, leaf, stamen, gynoecium and seed grown under extraction suitable condition, reverse transcription are closed Into cDNA.According to 3 ' non-translational regions or non-conservative area design primer, real-time RT-PCR detections are carried out.The result shows that： ZmsPLA2-1 has expression in each organ, wherein the expression intensity highest in root, is secondly seed, is again stem, blade, Expression quantity is relatively low in female fringe and tassel.The corn seedling for taking different abiotic stress processing is material, extraction mRNA and reverse transcription Into cDNA, using Ubiquitin as reference gene, ZmsPLA2-1 gene expression abundances are detected, with 2^{- △ △ CT}Method calculate it is each The relative expression quantity of gene.It obtains under osmotic stress treatment conditions, ZmsPLA2-1 is significantly raised in leaf and root, place Expression quantity is respectively 7 times and 5 times of before processing or so when managing 12h.The expression of the gene is induced by osmotic stress, i.e., they are participated in The response of plant pair osmotic stress.Under the conditions of cold Stress treatment, gene expression quantity highest when handling 6h for 4 DEG C.In leaf It reaches 2 times and 3 times or so of before processing respectively with expression quantity in root, shows that the gene also rises emphatically in the cold stress response of plant pair It acts on.

ZmsPLA2-1 plant expression vector constructions

5 ' ends are gone out with the ZmsPLA2-1 justice of KpnI restriction enzyme sites, 3 ' ends with SacI restriction enzyme sites by PCR amplification Gene or 5 ' ends with SacI restriction enzyme sites, 3 ' ZmsPLA2-1 antisense genes of the end with KpnI restriction enzyme sites, through KpnI and The segment is recycled after SacI double digestions.KpnI and SacI double digestions are carried out to plant expression carrier plasmid pCPE simultaneously, recycling carries Body segment.Coupled reaction is carried out after the two is mixed, obtains plasmid pCPE-ZmsPLA2-1 (±).Contain 1 in target gene area A BamHI restriction enzyme sites.Digestion identification is carried out with restriction enzyme BamHI, in addition to carrier ribbon, plasmid pCPE- ZmsPLA2-1 (+) should have a 136bp electrophoretic band, and plasmid pCPE-ZmsPLA2-1 (-) should have a 440bp electrophoretic band. The identified plant expression vector for obtaining ZmsPLA2-1 genes justice or anti-sense versions insertion.In the plasmid, target gene ZmsPLA2-1 is merged with its justice or anti-sense versions with corn Ubiquitin1 promoters or arabidopsis RD29A promoters, turns base Because plant selected marker be herbicide glufosinate-resistant Bar gene, the latter by cauliflower mosaic virus 35SRNA promoter Start.

Genetic Transformation in Higher Plants

Different transgenic methods is selected to obtain genetically modified plants according to the characteristic of transgenic acceptor.The present invention uses Agriculture bacillus mediated hereditaryization method and particle bombardment carry out genetic transformation.Conversion and conversion are illustrated by taking corn as an example below Plant screening process.

Using corn inbred line embryo callus as in the genetic transformation of material, take after pollination self 9-15 days corns from Friendship is fruit ear, peels off bract, is placed in 70% alcohol and impregnates 5min, the rataria of picking about 1.0-1.5mm sizes under aseptic condition It is inoculated on inducing culture, culture obtains crisp, flaxen II type callus in 4-6 weeks, then per 10-15 days after being commissioned to train It supports primary.Acceptor material of the obtained II types callus as genetic transformation.

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% ethyl alcohol 15 minutes, centrifugation removal 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 add 5 μ l Plasmid DNA (1 μ g/ μ l), 50 μ l12.5M CaCl₂, 20 μ l 0.1M spermidines, while sample-adding while vortex.Then, continue vortex 2~3 minutes, stand 1 minute.From After the heart abandons supernatant, 70% ethyl alcohol is added to stand.It is then centrifuged for abandoning supernatant, then be resuspended with absolute ethyl alcohol, sampling is added in micro- missile-borne On body.Micro- bullet dosage is often plays 0.5mg.

The culture medium of 0.4cm thickness is poured into the culture dish of diameter 9cm, callus high density is then put into culture dish In per ware, bombardment is primary.Bombardment parameters take：The distance that disk and carrier can be split is 2.5cm, and the distance of carrier and blocking net is 0.8cm, micro- bullet flying distance 6--9cm.Other parameters press operation instructions value.Material darkling renewal cultivation 3 after bombardment My god, then material is transferred in the new culture medium of components unchanged and cultivated 3 weeks, give full expression to the target gene being transferred to.By material It is transferred on the culture medium added with selective agent (0.08% herbicide glufosinate) and is screened.3 generation of step sizing, often for 15 days.After For when eliminate the tissue block of browning death.Resistant calli by screening is transferred on the culture medium for be not added with selective agent in illumination After 1 generation of 16 hours/world renewal cultivation, it is transferred on differential medium and breaks up seedling.The seedling that callus generates is in training of taking root It supports and takes root in base, flowerpot is moved into after strong sprout, it is long to be planted to during about 10cm high to big Tanaka, self-fertility.

Using agrobacterium-mediated transformation to maize bud point and rye grass Multiple Buds carry out genetic transformation, then to T0 for plant It is sprayed and screened with herbicide aqueous solution.For spraying the non-transgenic plant of 1.5% herbicide glufosinate aqueous solution, turn after 4 days Change and start jaundice of wilting at the blade tip leaf margin of seedling, entire blade becomes withered and yellow after a week, and whole strain flavescence is gradually withered dead after 12 days It dies；And the blade of Partial Conversion seedling can keep green or yellow leaf lesser extent, show apparent Herbicid resistant, And there is 1-2 piece young leaves to grow in the two weeks after herbicide spray.The Herbicid resistant seedling filtered out is transplanted to crop field, Artificial autocopulation pollinates.Herbicide screening is continued for plant to T1, resistant plant carries out PCR detections.

The identification of transformed plant

Herbicid resistant seedling leaf DNA is extracted to detect for PCR.According to riddled basins bar and target gene The primers of ZmsPLA2-1 carry out PCR detections respectively.Bar genes are amplified respectively from transfer-gen plant The purpose band of the 891bp of the purpose band of 552bp, the 804bp purpose bands of ZmsPLA2-1 justice genes and antisense gene. Primer sequence is that transgenosis is special, and the size of pcr amplified fragment is respectively with carrying these gene plasmid (sun in transfer-gen plant Property control) amplified band it is consistent, and the adjoining tree (negative control) of non-transgenosis does not occur the purpose item of same size Band.PCR detections are continued for plant to T2, external source Gene Isolation ratio meets Mendelian inheritance ratio in some T2 are for strain Example stablizes hereditary strain for transgenosis by primarily determining.

The T2-T4 of the inheritance stability of the selection PCR positives is material for transfer-gen plant and non-transfer-gen plant, extracts blade RNA detects the expression intensity of target gene by Real time RT-PCR, using Ubiquitin genes as internal standard gene.In jade Rice turns in the different strains of ZmsPLA2-1 justice genes, and the expression intensity of ZmsPLA2-1 genes has different degrees of raising, It is 1.31-4.34 times of control；In ZmsPLA2-1 antisense gene strains are turned, the expression intensity of ZmsPLA2-1 genes in corn Decline, be 0.42-0.62 times of non-non-transgenic control lines.In the strain of rye grass transfer ZmsPLA2-1 genes, transgenosis Gene expression abundance because strain difference due to it is variant, some strains different generations transgenic express stablize.

The drought resistance detection of transfer-gen plant

By taking corn as an example, 3 leaf phase transfer-gen plants and non-non-transgenic control self-mating system are in phenotype under normal growing conditions On have different.Compared with compareing self-mating system, the root system for turning ZmsPLA2-1 justice plant is more flourishing, and ZmsPLA2-1 is anti- The plant above ground portion of adopted strain is more flourishing.3 leaf phase transfer-gen plants and the biomass of adjoining tree under normal growing conditions are analyzed, It obtains and turns the growth of ZmsPLA2-1 justice gene strains under ground portion comparatively fast, the dry matter of accumulation is significantly higher than control self-mating system. The transgenic corns for growing to tri-leaf period and non-non-transgenic control seedling are subjected to the lethal experiment of arid, are coerced by the arid of 6 days Compel processing, non-non-transgenic control seedling, antisense ZmsPLA2-1 seedlings and part turn ZmsPLA2-1 and be overexpressed small seedling leaf Close to drying up, basal part of stem softens, and restores after watering or most of plant death or complete stool system plant are dead in strain.But turn The drought resistance of ZmsPLA2-1 overexpression seedlings difference due to strain difference is apparent, and the drought resistance of most strains significantly improves, if Restore watering after drought stress, most strains can comparatively fast restore normal growth.

The influence that drought stress grows transgenic corn plant before blooming for assessment, starts when plant was grown to 10 leaf phase Controlling soil relative water content, progress drought stress processing restores normal watering, plant one week in 50%-55% or so after 8 days After start to take out it is male.Observation stress during drought stress transgenic plant and the character mutation of non-non-transgenic control lines, and in 0,2,4,6,8 day and the measure items physical signs of drawing materials respectively in recovery watering latter week that drought stress is handled.In normal growth Under the conditions of, the ground morphological feature and growth and development rate that turn ZmsPLA2-1 justice gene strains are selfed with non-non-transgenic control System is compared to no significant difference.After by drought stress, non-transfer-gen plant and turn ZmsPLA2-1 antisense gene strain plant compared with Early there is water shortage symptom morning, after Continuous Drought 5 days, blade turns yellow, is withered, and the blade of ZmsPLA2-1 justice plant is wilted Mild degree, leaf color are more emerald green.Under normal growing conditions, the mda content and cell membrane of transgenic line and control self-mating system Ion leakage rate is without significant difference.With the extension of drought stress time, mda content and cell membrane Ion leakage rate are apparent Raising, the elevation amplitude of transfer ZmsPLA2-1 justice gene strains are smaller.Stress is after 8 days, ZmsPLA2-1 justice strains Mda content and cell membrane Ion leakage rate be 19.64~20.24nmol/g FW and 34.7~37.6%, substantially less than Compare (the 21.86nmol/g FW and 41.7%) of self-mating system；The mda content of antisense strain and cell membrane Ion leakage rate point Not Wei 21.62~22.64nmol/g FW and 45.5~50.4%, be higher than or be significantly higher than control self-mating system.Restoring to water After 7 days, compared with control self-mating system and antisense strain, turn the mda content of ZmsPLA2-1 justice strains and Ion leakage rate Level still keeps reduced levels, i.e. cell damage is light.

To understand the forfeiture degree of leaf water under drought stress conditions, determine opposite in different corn strain blades Water content (RWC).Before drought stress, relative water content is not significantly different between each strain, after drought stress, all strains Leaf r elative water content reduces, but after stress 2 days, RWC falls are different in different strain blades.Together ZmsPLA2-1 justice strains are compared, the leaf water loss of non-non-transgenic control self-mating system and ZmsPLA2-1 antisense strain strains It is more.At the 8th day of drought stress, two justice strain blade RWC were reduced to 70.9%, 71.1%, and antisense strain blade RWC is reduced to 64.0% or so, and compared with compareing self-mating system (67.8%), difference has reached the pole level of signifiance.Restore normal After watering, leaf r elative water content is begun to ramp up again, but just strain is higher than control self-mating system, i.e., just strain blade The recovery of water content is faster than control self-mating system.These the result shows that ZmsPLA2-1 justice gene strain blades in drought stress Under the conditions of have better water holding capacity, preferably maintain normal cell turgor, be conducive to the growth of plant.

Soluble sugar is a kind of as compatible solute, and protective effect is played to plant under drought stress.Measure the arid side of body Soluble sugar content under the conditions of compeling in transgenic line and non-non-transgenic control self-mating system blade, it is possible to find at drought stress Before reason, blade Soluble adhesion molecule difference unobvious between transgenic line and control.Cause after drought stress processing Soluble sugar content rises in all corn strain blades, but ascensional range is different.Turn ZmsPLA2-1 justice gene strain blades Middle soluble sugar content is apparently higher than non-non-transgenic control self-mating system, and such as when coercing the 8th day, the blade of two just strains can Dissolubility sugared content is 82.5,78.7mg/g DW, and it is 67.7mg/g DW to compare self-mating system, and difference reaches the level of signifiance, instead Adopted strain is also accumulated from than compareing more soluble sugar contents (72.6mg/g DW).After watering is restored 7 days, each strain blade Middle soluble sugar content gradually decreases, but being still higher than in just strain compares self-mating system.These the result shows that The blade cell of ZmsPLA2-1 justice strains can accumulate more soluble sugar solutes, it is possible to cell be made to remain relatively low Solute potential, and then moisture can be preferably kept, cell turgor is maintained, from the injury of drought stress.

In order to study drought stress photosynthetic influence on plant, transgenic corns strain and control are determined certainly It hands over and ties up to Net Photosynthetic Rate under the conditions of normal growing conditions, drought stress conditions and rehydration, stomatal conductance.Normally watering Under the conditions of, the Net Photosynthetic Rate of ZmPLA2-1 justice gene strains is slightly above non-non-transgenic control self-mating system, after drought stress, It is gradually increased with the difference between the different corn strains of extension of stress time.In entire stress during drought stress, compared to right According to self-mating system, the Decrease in Net Photosynthetic Rate for turning ZmPLA2-1 justice gene strains is more slow.Such as in drought stress 6 days, just The Net Photosynthetic Rate of adopted strain is reduced to 49.9% before Stress treatment, is significantly higher than (41.3%) of control self-mating system；And this When ZmPLA2-1 antisense genes strains be 21.7%, substantially less than compare self-mating system.In stress during drought stress, stomatal conductance Variation tendency with Net Photosynthetic Rate, the stomatal conductance for turning ZmPLA2-1 justice strains is significantly higher than control self-mating system, antisense Strain.Stomatal conductance decline leads to CO₂It may be cause photosynthesis decline in stress during drought stress one that supply, which is reduced, Key factor.In addition, after watering is restored 7 days, the Net Photosynthetic Rate and stomata of all strains, which lead angle value, all to be increased, and is turned Remaining above for ZmPLA2-1 justice gene strains compares self-mating system.Higher after stronger photosynthetic capacity and rehydration under drought stress Regeneration rate show that turning ZmsPLA2-1 justice gene strains improves plant drought-resistant ability.

In drought tests rain-proof shelter, the drought stress of continuous 6 weeks is carried out to plant before blooming, then restores normal Watering, observes transfer-gen plant with compareing character mutation of the self-mating system plant under drought stress, unites after harvest during this period Count single plant yield and cell production.The result shows that the growth and development for turning ZmsPLA2-1 justice gene strain plant is impacted smaller, Grain yield is high.

Difference under comprehensive drought stress conditions in the physical signs and yield of different strain plant obtains, turns ZmsPLA2- 1 just gene strain cytosol content increasing degree when encountering drought stress is big, reduces the loss of cell moisture, can The water content of cell is preferably kept, cell membrane damage is small, and photosynthetic capacity is stronger, and the growth and development of plant is impacted smaller, because This grain yield is high, shows that drought stress environment can be better adapted to compared with non-non-transgenic control lines.Turn The strain of ZmsPLA2-1 antisense genes and the drought resisting gender gap of adjoining tree are little.

Turn the detection of ZmsPLA2-1 gene plants cold resistance

Usual low temperature stress, which is divided into, damages to plants caused by sudden drop in temperature (0-15 DEG C) and two kinds of freeze injury (being less than 0 DEG C), is distributed in the plant of different regions It is different to the degree of tolerance of low temperature, gene expression, biomembrane lipid composition and small molecule in the cold resistance and plant of plant The accumulation of substance has substantial connection.It is mainly structure by destroying film that low temperature is injured caused by plant, makes photosynthesis It is obstructed, generates the death that toxicant and active oxygen etc. eventually lead to cell and plant.There are one adapt to Chilling stress for plant With the process of self-control.Corn is more sensitive to low temperature stress, under cryogenic vulnerable death.Low temperature easily makes corn Seedling comes to harm, and quick-thawing again after Corn Root Tip Cells slow freezing is found in the case where restoring normal condition corn afterwards by Wang etc. The growth and development of root, stem and leaf significantly lag；The discoveries such as Fngels, by maize root system under cryogenic with normal condition similarly hereinafter When handle 3 days, cauline leaf fresh weight, leaf area and root/shoot ratio are decreased obviously.Low-temperature treatment (3 is carried out during Course of Corn Seed Germination DEG C), seedling internal soluble sugar and cane sugar content, plant leachate ions content difference compared under normal condition are shown It writes.

Under normal growing conditions ZmPLA2-1 gene overexpressions plant with the morphological feature of non-non-transgenic control lines without bright Significant difference is different, but growth rate is higher, and ZmPLA2-1 gene antisenses express plant difference unobvious compared with the control.Turn ZmPLA2-1 The analysis of gene plant cold resistance can middle progress in the controlled environment chamber, plant to be planted length to 3 leaf phases, 10 DEG C first are handled 1 day, then are dropped It is persistently handled 3 days to -2 DEG C (rye grasses) or 4 DEG C (corns), then room temperature restoration ecosystem 3 days.Under cryogenic conditions, transgenosis is planted Strain and the growth of non-transfer-gen plant are suppressed, but turn the impaired relatively light of ZmPLA2-1 gene overexpression strains, turn anti- The growth of adopted gene plant difference compared with wild type control is unknown significantly.4 DEG C of processing corn seedlings are after 3 days, non-transgenic pair There is blade wilting dehydration, limb curling according to plant, antisense ZmPLA2-1 genes strain is more wilted than control plant leaf Dehydration and color burn, and turn ZmPLA2-1 gene overexpression strains show as blade unfold, leaf color it is light green, limb There is not apparent crimp, show to improve cold resistance compared with non-non-transgenic control.

Detection turns gene expression abundance of the ZmPLA2-1 genes in corn difference strain, finds to be overexpressed strain at cold stress Gene expression abundance before reason is about 1.6 times of adjoining tree, and the ZmPLA2-1 abundance of antisense expression strain is about non-transgenosis pair According to 0.4-0.6 times of plant, difference reaches extremely significantly, i.e., transgenosis functions in plant.After 4 DEG C handle 6h, not The ZmPLA2-1 gene expression abundances of non-transgenic control strain about raise 2 times, are overexpressed strain and 5.5 are about raised compared with before processing Times, i.e., ZmPLA2-1 gene expression abundances are far above non-non-transgenic control lines, and the expression of the gene is rich in antisense expression strain Degree variation is little, shows the raising of ZmPLA2-1 gene expression abundances that transgene silencing mechanism effectively controls, it is resistance to reduce plant Cold property.This ZmPLA2-1 gene expression abundance determined from transcriptional level and plant cold resistance are closely related, and cold Stress treatment lures Lead the up-regulation of corn ZmPLA2-1 gene expression amounts.

Under stress conditions, plant is by improving the activity of superoxide dismutase (SOD) and peroxidase (POD) come clear Except intracellular excess activity oxygen, reduce the injury of active oxygen or other peroxide radicals to cell membrane system, so as to maintain The stability of film and macromolecular structure.Using before cold Stress treatment, in cold Stress treatment and restore room temperature plant leaf as material The activity of superoxide dismutase (SOD) and peroxidase (POD) is measured, is obtained：The SOD of the non-non-transgenic control of before processing and POD activity activity difference compared with transgenic line is little, low-temperature treatment render transgenic strain and non-transgenic reference strain SOD and POD activity all increased, but the elevated-levels of different strain enzymatic activity are different.In low-temperature treatment and recovery process The SOD activity of middle ZmPLA2-1 gene overexpressions strain is apparently higher than wild type, and the SOD activity of antisense expression strain is low Non- non-transgenic control is substantially less than in warm processing procedure.

Under cold stress conditions, maize leaf cell dehydration, active o content rises, and blade cell film is caused to be damaged, is caused Cell plasma percolation ratio increases.Each strain blade cell Electrolytic leakage before and after the cold stress of this measuring.The result shows that Before cold Stress treatment, the leaves ions percolation ratio difference unobvious of different strains, low-temperature treatment render transgenic strain and non-turn The cell membrane damage degree of genetic contrast strain is all increased, but different strain degree of injury is different.4 DEG C processing 3 days when Turn the cell membrane damage of ZmPLA2-1 gene overexpression strains less than 60%, and non-transgenic reference is 65% or so, is higher than Strain is expressed, and the cell membrane damage of antisense strain is more serious than non-transgenic reference.

Specific embodiment

Embodiment 1：Turn ZmPLA2-1 genes and create drought-resistant maize self-mating system and application

1) foundation of receptor system is using Inbred Lines used in China's agricultural production as material, as Zheng 58, prosperous 7-2, The selfed seed of DH4866 etc..Cultured in vitro induction stem apex generates the sprout tuber that grows thickly, and to grow thickly, sprout tuber carries out genetic transformation as receptor. Used medium has：

Seed germination medium：KNO₃1900mg/l, NH₄NO₃1650mg/l, CaCl₂·2H₂O 440mg/l, MgSO₄· 7H₂O 370mg/l, KH₂PO₄·H₂O 170mg/l, FeSO₄·7H₂O 27.8mg/l, ZnSO₄·7H₂O 10mg/l, MnSO₄· 4H₂O 22.3mg/l, H₃BO₃10mg/l, KI 0.83mg/l, Na₂MoO₄·2H₂O 0.5mg/l, CuSO₄·5H₂O 0.025mg/ L, CoCl₂·6H₂O 0.025mg/l, thiamine hydrochloride 10.0mg/l, puridoxine hydrochloride 1.0mg/l, niacin 1.0mg/l, sweet ammonia Sour 2.0mg/l, inositol 100.0mg/l, biotin 0.05mg/l, casein hydrolysate 500mg/l, sucrose 30g/l, agar powder 7g/l, pH 5.8~6.0.It is sprouted for seed.Fluid nutrient medium then removes agar powder.

A culture mediums：Seed germination medium adds 1.0~3.0 μm of ol/l of 4.5~9.0 μm of ol/l of 6-BA and 2,4-D, For cultured in vitro bud point to be induced to generate Multiple Buds tissue block and Multiple Buds tissue block squamous subculture.

B culture mediums：Seed germination medium adds 6-BA 1.8 μm of ol/l of 4.5 μm of ol/l and IBA (indolebutyric acid), is used for Multiple Buds tissue block breaks up seedling.

Seedling culture medium：Seed germination medium adds 6-BA 3.6 μm of ol/l of 2.25 μm of ol/l and IBA, for growing thickly Budlet develops into seedling.

Root media：Seed germination medium adds 2.8~3.6 μm of ol/l of IBA, for the small seedling rooting of unrooted.

Minimal medium and plant growth regulating substance pressure sterilizing；Antibiotic and the filtering of herbicide isoreactivity ingredient are gone out Bacterium adds in after medium sterilization.

Seed sterilization and sprouting：Corn seed is impregnated 10 minutes with 70% ethyl alcohol, then impregnates 10--15 with 0.1% mercury chloride Minute, then with sterile water washing 3--5 times.Seed is placed in sterile triangular flask and sprouts after sterilizing, and a small amount of (30--- is put into bottle 40 milliliters/250 milliliters triangular flasks) sterile water, it is placed on after sealing under dark condition (23---30 DEG C) 1--2 days.It sprouts and (shows money or valuables one carries unintentionally) Seed is placed on minimal medium afterwards continues to sprout under dark condition.

Shoot Tip Culture and the induction of Multiple Buds tissue block, subculture, differentiation：When the plumule elongation of germination seed stops 3---5 centimetres, Plumule and spire are removed, cuts and is about 5 millimeters or so of epicotyl and stem apex, be inoculated on A culture mediums the (24-- under dark 27 DEG C) culture, and cut the plumular axis of elongation in time and peel off spire.After culture 6--10 days, stem apex starts irregularly to expand life It is long, occur several wartys or digitation on the separate living tissue expanded.After 20 days, start on the surface of warty or digitation Form adventitious bud and embryoid.General every 4 weeks squamous subcultures are primary.In squamous subculture, the budlet if Multiple Buds tissue block is grown thickly On the high side, 2,4-D concentration takes 3.0 μm of ol/l；If Multiple Buds tissue block callusization is heavier, though there is a large amount of Meristematic cell mass, But seldom there is adventitious bud in surface, can be by 2, and 4-D concentration is reduced to 1.0 μm of ol/l, continue culture then regenerate a large amount of wartys or Digitation.Multiple Buds tissue block on A culture mediums, minority have the generation of adventitious root.Existing with spire, adventitious root also shadow The generation for expanding growth and embryoid and the budlet that grows thickly of tissue block is rung, needs to remove early.Multiple Buds tissue block is being transferred to B On culture medium after 2--3 days, color and luster gradually turns yellow, and quality is more flexible, and microspike occurs in rear surface within 5--6 days.Under scanning electron microscope Observe visible each phase embryoid and adventitious bud.Embryoid and adventitious bud are developed rapidly, and the budlet that grows thickly is formed on tissue block surface.

2) conversion using Multiple Buds tissue block as receptor and plant regeneration

The root nodule agriculture of binary vector (Mini--Ti plasmids are with selective agent resistant gene and ZmPLA2-1 genes) will be carried In the LB culture mediums of additional antibiotic, (every liter of culture medium contains bacillus (such as AGL0 and LBA4404)：Tryptone 10g, yeast carry Take object 5g, NaCl 10g, pH 7.0, high pressure sterilization) at 28 DEG C shake culture, concussion rate be 110rpm (rev/min), make thin Bacterium is in exponential phase.Then it centrifuges 10 minutes at 3,000 rpm, abandons supernatant.Thalline is sprouted with the liquid seeds of 1/2 concentration Culture medium (i.e. seed germination medium ingredient halves, and removes agar powder) is sent out to wash, then be collected by centrifugation.Again by thalline addition second The liquid inducing clumping bud culture medium of 1/2 concentration of 100 μm of ol/l of acyl syringone (acetosyringone, As) suspends, dilution 5--20 times is used to convert.

It is transgene receptor to take the Multiple Buds tissue block cultivated 13--20 days after subculture.Turned with agrobacterium-mediated transformation Change, material darkling carries out renewal cultivation after conversion.Grow thickly budlet or the Multiple Buds tissue block of Agrobacterium infection are added with cephalo 7--12 is cultivated in dark on the culture medium of mycin (Cefotaxime) 250mg/l or carbenicillin (Carb) 500mg/l My god, inhibit bacteria growth.After renewal cultivation or after micro-organisms grow thickly budlet or Multiple Buds tissue block added with selective agent (such as Glufosinate 0.08%) culture medium on step sizing 3-4 generations, obtain transgenic cell and budlet.In screening and culturing absolutely mostly Number Multiple Buds tissue block is gradually dead.Remove 2 by what the tissue block of survival was transferred to no selective agent, it is extensive on the A culture mediums of 4-D Budlet is generated after multiple culture.

Budlet is placed on irradiation culture on seedling culture medium, light intensity 2000-3000lx, 14-15 hours/day of illumination.Seedling It is transferred in root media and takes root when growing to 3-4 piece leaves.Culture 15 days or so, about 40% seedling generate new root.For not giving birth to Offspring, its base portion of cut wound are transferred on new root media and cultivate, and most of plant generate root system after 10 days.Rooted seedling is washed After removing culture medium, be transplanted to using vermiculite as the flowerpot of cultivation medium in grow.Plant grows under natural lighting, day temperature 22--28 DEG C, 15--21 DEG C of night temperature pours the inorganic salts ingredients of the seed germination medium of 1/2 concentration every other day.Growth 2 weeks or so, transplanting Seedling generates flourishing root system, is then colonized in field.

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

The blade of transplant survival plant is taken to carry out Molecular Detection and determines transfer-gen plant.Then transfer-gen plant (T0) is covered Bag self-fertility.T1 seeds from different T0 plant are broadcast in greenhouse or there is the field of protective equipment, blade is taken after emergence Transgenosis is detected using PCR method, and smears herbicide (such as 0.2% glufosinate) solution identification resistance.By the PCR positives and weeding The plantlet of transplant of agent resistance is to field, bagging self-fertility after growing up.

In transfer-gen plant progeny selection, take first T2 of uniform size for transgenic seed and non-non-transgenic control from Friendship is that seed is seeded in vinyl disc of the same size, and the homogeneous loam of equivalent is housed in disk, and often disk sows 20, every plant 3 disks are at least planted by system.Normal watering treats that seedling length to 3 leaf phases, chooses the consistent vinyl disc of growing way and carries out drought resistance observation experiment. The method of drought stress processing is to stop watering after being disposably watered with moisture, until the plant leaf of most strains dries up, stem Base portion softens, then restores to water, and observes upgrowth situation of each strain after restoring to water, and filters out drought resistance than control self-mating system The strain significantly improved.Then drought stress before transfer-gen plant is bloomed is carried out to handle.

Drought stress experiment carries out under field conditions (factors) before corn is bloomed, and plant is avoided to drench with rain during Stress treatment.It chooses T2 is seeded in for transgenic seed and control self-mating system seed in big flowerpot of the same size, when plant was grown to 5 leaf phase, is stayed per basin 2 plants of seedling compares 1 plant, 1 plant of transgenic plant.Plant grows under the conditions of normal watering to 10 leaves, and it is consistent then to choose growing way Plant is divided into two groups, and one group grows under drought condition, and soil absolute water content is maintained at 15-17%, lasting to handle One week, then restore normal watering.Another group is maintained under the conditions of sufficient watering and grows, as control.Every group sets 6 repetitions. It is poured early period in control water, Osmotic treatment group plant is wilted in 9~18 period of daytime blade, and night blade restores stretching, extension；In control water The later stage is poured, blade is persistently wilted.Restore normal watering after a week, plant starts to take out male.Plant is controlled into the 1st day (leaf of watering Piece starts to wilt at the morning 8 or so) drought stress processing 0 day is denoted as, 0,2,4,6,8 in processing day and recovery are normal respectively Watering sample after a week, measure RWC, cell membrane Ion leakage rate, mda content, soluble sugar content, proline content with And photosynthesis index etc..Osmotic treatment measures plant height and greenery the piece number after florescence, and statistics is bloomed and time interval of spinning (Anthesis-silking interval, ASI).The measure of pollen activity uses iodine-potassium iodide decoration method.Take mature anther It is positioned on glass slide, adds 1 drop distilled water, smashed to pieces anther with tweezers, discharge pollen grain, then add 1~2 drop I₂- KI solution contaminates Color.Covered, in low power Microscopic observation, statistics pollen activity.

The plant of 8 days drought stresses is undergone, restores watering and starts to take out hero after a week, plant plant height is counted in this period With hold greenery the piece number.Under the conditions of normal watering, the growth and development of transgenic corn plant with compare between self-mating system existing for Phenotypic difference unobvious, the measurement result of some indexs also demonstrate observation conclusion.After drought stress early period of blooming is handled 8 days, The average plant height of all strains significantly reduces, but the amplitude reduced between different corn strains is different.Compared to normal watering Under the conditions of homology plant, the reduction amplitude for turning ZmsPLA2-1 justice gene strain plant heights is respectively 21.9% and 22.2%, drop Low amplitude is less than (28.7%) of non-non-transgenic control self-mating system；Turning the plant height of ZmsPLA2-1 antisense gene strains reduces difference 28.4% and 31.9%, it reduces amplitude and the difference for compareing self-mating system is little.

After undergoing drought stress, greenery the piece number significant difference is held between different strains, turns ZmsPLA2-1 justice gene plants Greenery number be apparently higher than non-non-transgenic control self-mating system and antisense gene plant.Drought stress before blooming is to corn The female tassel development of plant produces deep effect.After Osmotic treatment, the flowering time of all strains is postponed, female tassel development It is uncoordinated, and the normal pollen quantity of plant is caused to reduce and the raising of pollen sterile quantity.Drought stress is to turning ZmsPLA2-1 Bloom-the spin influence of time interval (Anthesis silking interval, ASI) of just gene plant is less than to not Transgenosis.After drought stress, the pollen amount of non-non-transgenic control self-mating system is few, turns ZmsPLA2-1 antisenses plant with not turning Genetic contrast plant is similar, and it is more to turn ZmsPLA2-1 justice gene plant pollen amounts.The anther of different strains is taken using iodo- Potassium Iodide Methods dyeing carries out cytological observation, it will be seen that only has smaller portions in the pollen grain of non-non-transgenic control lines and is contaminated and be Blue, it is most of that fawn is presented.It is rich amyloid pollen to be contaminated for the pollen of blue, and viability is strong, presents yellowish-brown The pollen development of color is bad, and Setting percentage is relatively low after pollination.And the normal loose powder of tassel energy for adopted gene plant of becoming a full member, most pollen shapes State is normal, and most of dyed by iodine-potassium iodide is navy blue, and color is deeper.This illustrates that the pollen of non-non-transgenic control lines loses Rate height is educated, and normally, viability is strong for the development of its pollen majority after being handled by drought stress for adopted gene plant of becoming a full member.

By transgenosis and non-non-transgenic control maize seed in arid experiment shed, plant to be planted length to 10 leaf phases, control arid Irrigation amount in canopy, makes soil relative water content be maintained at 50%-55% or so, carries out drought stress processing to plant, continues 6 Week, then pour adequate water.The growth and development of plant during observation Osmotic treatment.After fruit ear maturation, Ear Characters are carried out Observation statistics.After drought stress, the blade for turning ZmsPLA2-1 justice strain plant more stretches, leaf color jade green, by drought stress Influence small, and the curling of non-non-transgenic control self-mating system plant leaf is serious, and indivedual Plant Leaf discolorations are shallow.Not homophyletic after drought stress It is that Ear Characters and yield show that the average spike length of ZmsPLA2-1 justice strains is significantly greater than non-non-transgenic control self-mating system, And the difference between antisense gene strain and non-non-transgenic control is not notable.From the point of view of grain weight per panicle, ZmsPLA2-1 justice strains Grain weight per panicle significantly mostly with non-non-transgenic control self-mating system, grain weight per panicle respectively than compare self-mating system (50.8 ± 9.0g) Increase by 34.1%, 29.9%.From the point of view of 100-grain weight, though each transgenic line is variant between self-mating system with compareing, it is not achieved Significant difference degree.These results indicate that turn ZmsPLA2-1 justice gene plant to the drought stress encountered before blooming with bright The aobvious resistance improved, reproductive development is impacted smaller, so as to which grain yield be made to be significantly higher than control self-mating system.

Embodiment 2：Turn ZmPLA2-1 genes and create Maize Cold Resistant self-mating system and application

1. corn aseptic seedling obtains

The seed of maize elite inbred line is impregnated 8 minutes with 70% ethyl alcohol, then is impregnated 8-12 minutes with 0.1% mercury chloride, Then with sterile water washing 3-5 times.Sterilization period constantly shakes seed, to ensure that surface sterilizing is thorough.Seed is placed on after sterilizing It is sprouted in sterile triangular flask, a small amount of sterile water is put into 1-2 days under dark condition (25-28 DEG C) in bottle.Treat Seed sprouting (dew After in vain), place it in modified MS medium and sprouted under dark condition.When plumule elongation stops 3-4 centimetres, plumule is removed And 2-3 piece spires, emergent stem pointed tip growth cone.

2. Agrobacterium cultivates and activation

Binary vector will be carried (Mini-Ti plasmids carry herbicide resistance gene bar and ZmPLA2-1 fusion) Shake culture, concussion rate are agrobacterium tumefaciens (AGL1 or LBA4404) at 28 DEG C in the LB culture mediums of additional antibiotic 110r/min makes bacterium be in exponential phase.Then it is centrifuged 10 minutes under 3000r/min, abandons supernatant.Thalline is with 1/ 2MS fluid nutrient mediums wash, and are collected by centrifugation.Again by the thalline 1/2MS fluid nutrient mediums of 100 μm of ol/l acetosyringones of addition It suspends, is diluted to OD₆₀₀0.2~0.4 is used to convert.

3. corn aseptic seedling converts

(1) bacterium solution is poured in the culture dish of 4.5 cm diameters, tilts culture dish, make the sterile of exposing stem apex growth cone Seedling is immersed in bacterium solution, 0.5 × 10⁵It is handled 8-12 minutes under Pa atmospheric pressure.

(2) the bud point after disseminating is blotted with aseptic filter paper, and germination seed is placed in modified MS medium and is cultivated in dark 2-3 days, cultivation temperature was 22-24 DEG C.Then aseptic seedling is put and cultivated 2 days under diffuse light.

(3) aseptic seedling after irradiation culture is transplanted in the flowerpot for being covered with upper strata vermiculite lower floor loam, and is covered with vermiculite At the top of lid 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 pours 1/2 improvement every other day MS culture medium inorganic salts.

4. transformed plant screens and field planting

After transformed plant grows 3 leaves, herbicide is sprayed(Hoechst Schering AgrEvo GmbH, contain Have herbicide glufosinate ammonium) aqueous solution, a concentration of 9.6ml -10.8ml/ L falls drop with plant It is advisable.Unconverted adjoining tree stops growing after 4 days after spraying, starts after 9 days dead.Transformed plant after spraying, some Individual variation is similar with adjoining tree, other individual continued propagations change unobvious.When the plant that survives grows to 5 leaf, by it It is colonized to field, bagging selfing is set seeds.

5. transfer-gen plant progeny analysis

T1 grew to for 3 leaf phases for plant and uses 10.8mlThe processing of/L aqueous solutions, observation statistics resistance and sensitive individuals Ratio；Foreign gene is detected, and count segregation ratio of the foreign gene in progeny plant using round pcr.The plant that survives moves It plants to crop field, bagging selfing.T2 plant detect foreign gene, and carry out in addition to bagging selfing is set seeds, using round pcr Southern blotting are verified；Transgene expression intensity is checked using RT-PCR technology.

6. the cold resistance detection of transfer-gen plant offspring

For plant with the extension of cold treatment time, leaf soluble has raising trend, is handled 1 day at 10 DEG C Soluble sugar amount raising afterwards, 4 DEG C of processing turn ZmPLA2-1 gene overexpression strain soluble sugar contents in 55-61mg g after 3 days^{- 1}Between DW, hence it is evident that higher than non-non-transgenic control lines, and the soluble sugar content of antisense gene strain is than non-transgenosis pair It is small according to plant, it is 45mg g^-1DW or so.Before cold Stress treatment, free amino acid total amount and the non-transgenosis pair of transgenic line No significant difference is compared according to strain；After Stress treatment, the total amount of free amino acid increased compared with before processing, in addition to first Outside, other free amino acid concentrations have different degrees of raising, wherein proline to methyllanthionine (Met) after by cold stress (Pro) content significantly increases, and proline can be accumulated largely as osmotic protection substance when plant is forced, and turns The proline content increasing degree of ZmPLA2-1 gene overexpression strains is significantly higher than non-non-transgenic control lines.In addition, Glu the and Val contents for turning ZmPLA2-1 gene overexpression strains after cold treatment are significantly higher than control, and Gly contents are then apparent low In control, and the plant of antisense expression then with compare it is close.These differences show that ZmPLA2-1 genes participate in corn cold resistance Regulation and control.

ZmsPLA2-1 is overexpressed the cold resistance for improving plant, along with stress responsive gene cold under cryogenic conditions ZmDREB1A, ZmMPK1, ZmMPK2, ZmMPK7, ZmHK3 are higher than not in gene expression abundances of the ZmsPLA2-1 in strain is overexpressed Non-transgenic control lines.DREB transcription factors regulate and control to be expressed with the relevant series of genes of adverse circumstance, including drought stress response Gene.Their induced expressions in transgenic plants can activate a series of expression of the degeneration-resistant functional gene in downstreams, improve plant Resistance, wherein ZmDREB1A expression quantity significantly raises the expression that can activate cold-resistant related gene.Mitogen-activated protein Kinases (mitogcn-activated protein kinase, MAPK) is signal transduction pathway MAPK cascade reactions in organism Important component, by transmitting intracellular external signal, mediate a variety of biologies and abiotic stress reaction.Therefore, when corn is coerced After compeling, ZmsPLA2-1 products may have activated the expression of these stress-related genes, so as to which plant be enable to better adapt to coerce Compel environment.

7. the utilization of cold-resistant transfer-gen plant

For the transgenic line that selected cold resistance is significantly improved, measure plant net light at a temperature of in different light intensity Close the variation of rate, measure 3-7 leaf phases seedling growth rate and biomass, measure single plant yield, and using non-transfer-gen plant as Control.The observation of Yield Traits In Corn is carried out under field cultivating condition after selecting excellent transgenic line and is compared, is selected Cold-resistant high yield strain enters corn breeding experiment.

Embodiment 3：Turn ZmPLA2-1 genes and create rye grass Drought Resistance Germplasm and application

Rye grass is point of grass family Lolium plant, one year raw rye grass and English ryegrass.It is perennial black Wheat straw is called perennial root rye grass, and annual ryegrass is called Italian ryegrass, originates in southwestern Europe, north African and Asia southwest, The 1950s is introduced into China as high quality forage.Rye grass tiller is more, and yield is high, and soft texture, the green phase is long, is good Green feed；In addition its well developed root system, adaptability is strong, resistance to trample, has certain saline-alkali tolerant potentiality, it is organic to increase soil Matter improves soil texture, prevents erosion, and be good turfgrass.But drought tolerance is not high, it is necessary to it is drought-enduring to improve its Property.

1), the foundation of rye grass Multiple Buds system

Establishing the culture medium used in rye grass Multiple Buds system has：Inducing culture (MS+0.5mg/L 2,4-D+2.0mg/ L 6-BA), proliferated culture medium (MS+2.0mg/L 6-BA), root media (1/2MS+1.0mg/L NAA).

Ryegrass seed sterilizes 1 minute through 70% alcohol, and 0.2% mercuric chloride sterilizes 5 minutes, sterile to wash 4 times.In MS without sharp It is sprouted under dark condition on plain culture medium, 25 ± 2 DEG C of temperature.After 4-5 days, when plumule is extended to 2-3cm, shoot tip meristem is cut Explant of the position as induction Multiple Buds.

By the stem apex position (2-3mm long) of the seed seedling of the sterile culture cut, be inoculated into containing various concentration 6-BA and On the inducing culture of 2,4-D.25 ± 2 DEG C, illumination 12h/d, light intensity 800-1000Lx of cultivation temperature forms base after 15-20 days What portion was expanded grow thickly sprout tuber.The inductivity of Multiple Buds is counted after 20 days.Induction of the explant in suitable concentration 6-BA and 2,4-D is trained It supports after being cultivated 2 days on base, stem apex and epicotyl start to extend.Epicotyl part is withered after 7-8 days, and stem apex and its outer leaf sheath are swollen Greatly, 2-3 budlet occurs from the stem apex surface expanded, the Multiple Buds of 8-10 intensive budlet compositions are formed after 10-15 days.Outside The type and concentration of exogenous estrogen play the form of rye grass Multiple Buds particularly important effect.In no 6-BA and 2,4-D Culture medium on there is no the generation of Multiple Buds；Only 6-BA is being added to be not added with the culture medium of 2,4-D, though there are Multiple Buds to be formed, bud number Few, budlet leaf color is dark green, partial vitrification or even blade face macula lutea occurs.In the training of 2,4-D concentration relatively low (0.1-0.2mg/L) It supports on base, the bending of budlet blade or distortion of induction, bastem portion is thinner, and inductivity is relatively low.It is higher than 0.5mg/ in 2,4-D concentration On the inducing culture of L, bastem portion callusization is more serious, there is more new root, and sprout tuber blade is more, but budlet sends out Raw quantity is few.In MS+0.5mg/L 2,4-D+2.0mg/L 6-BA) on culture medium, budlet leaf color is bud green, and sprout tuber growing way is preferable, The stem apex percentage for generating Multiple Buds is high, and average each sprout tuber budlet number is also average most (6.79).Therefore, in subsequent work In, the culture medium is selected as inducing culture.Culture medium and Hormone Conditions needed for the inducing clumping bud of different cultivars rye grass Difference is little.

The sprout tuber that grows thickly is peelled off to older leaf sheath and spire, is divided into after simple bud and being transferred to respectively on different proliferated culture mediums, Every 20 days subcultures are primary.Light intensity 800-1000Lx, light application time 12h/d, 25 ± 2 DEG C of temperature.Multiplying culture will expand after 20 days The sprout tuber that grows thickly be cut into simple bud or small sprout tuber (about 2mm sizes), go to subculture in the different proliferated culture medium of hormone combination respectively Culture.After 2 days, simple bud or budlet BOB(beginning of block) expand, and 2-3 budlet occurs from the stem apex surface expanded, and are formed after 5 days by 8- The Multiple Buds of the composition of 10 intensive budlets, the type and concentration of exogenous hormone, which play the proliferation of rye grass Multiple Buds, extremely to be weighed The effect wanted.In vitro bud point does not generate Multiple Buds on no hormone culture-medium, and bud point is extended into plantlet.Added with 1.0mg/L On 6-BA culture mediums, the sprout tuber that grows thickly generally is made of 2-3 budlet, and budlet proliferation rate is low.In the culture medium of 3.0mg/L 6-BA Upper squamous subculture, the Multiple Buds leaf color of induction is dark green, and there is macula lutea on part budlet vitrifying, blade face, there is a small amount of albefaction bud. Proliferated culture medium is most suitable with MS+2.0mg/L 6-BA, and (kind is safe for the average bud number at most (kind bay 9.96) of each Multiple Buds Chui Laite 10.62), Multiple Buds growing way is preferably also.Primary every 20 days subculture Multiple Buds on the culture medium, budlet increases after 3 generations Life is very vigorous, and simple bud or small sprout tuber can generate the budlet of 50 or so after 20 days in culture.

The sprout tuber that grows thickly that 20 days are grown on proliferated culture medium is separated into simple bud, is transferred on root media, illumination increases It is added to 1500-2000Lx.The rootlet for beginning with white for general 9th day occurs, and has formed flourishing root system within 20 days, rooting rate reaches 100%.After 20 days, bottle cap is opened, adds in a little tap water, hardening 3 days.Clean root culture medium, be transplanted to upper strata vermiculite, In the flowerpot of lower floor's loam.The seedling of the flourishing root system of tool is easy to transplant, and survival rate is up to 100%.

2), agrobacterium-mediated transformation generates transfer-gen plant

Blade is cut after rye grass Multiple Buds (squamous subculture 8-10 days) are shelled into simple bud, exposes the mitogenetic of budding pointed tip Tissue carries out Agrobacterium infection.Taking 10ml long, 4000rpm centrifugation 10min collect thalline, by bacterium to the bacterium solution of exponential phase Weight is suspended from fluid nutrient medium, and dilution makes final OD₆₀₀For 0.4-0.6 or so, added in before dip dyeing to bacterial suspension The acetosyringone (AS) of 0.1mmol/L.The bud point stripped is immersed in the suspension, in the case where vacuum degree is 0.05MPa pressure Disseminate 5min.Bud point is taken out, bacterium solution is blotted with sterile dry filter paper, is transferred to co-cultivation culture based on light culture 3-4 at 25 ± 2 DEG C My god.Bud point after co-cultivation, which goes to add, inhibits Agrobacterium growth on the micro-organisms base containing 100mg/L cephalosporins, after 8 days, Established Multiple Buds are separated into simple bud, is put on the culture medium added with selective agent and screens.After each screening and culturing, select and deposit Budlet living continues to screen.Such as the squamous subculture rye grass Multiple Buds of 8 days are separated and are cut into 2-3mm sizes, be inoculated into containing Continuously cultivated for 3 generations in 0.08% glufosinate culture medium, per 15 days generations.Budlet after screening passes through the renewal cultivation of 8 days, is transferred to On root media, illumination increases to 1500-2000Lx.After 20 days, bottle cap is opened, adds in a little tap water, hardening 3d.It cleans The culture medium of root is transplanted in the flowerpot of upper strata vermiculite, lower floor's loam.Nutrient solution twice, intensive care are poured, survival rate reaches 100%.

Plant is planted into flowerpot after two months, is extracted plant leaf DNA with CTAB methods, is carried out PCR amplification and Southern Hybridization check.According to riddled basins and the primers of target gene ZmsPLA2-1, PCR detections are carried out respectively.Portion Herbicid resistant plant is divided to amplify target fragment, non-non-transgenic control lines are without specific band.

3), the Drought Stress Tolerance Analysis of A of transgenosis bud and transfer-gen plant

The Herbicid resistant budlet of the PCR positives is obtained into a large amount of budlet, then in subculture medium into line number generation amplification It is transferred in flowerpot.After seedling survives, general grow carries out Stress treatment January again.In drought stress experiment is carried out, each strain System's 6 basins of plant, per 5 plants of basin.For normally watering and (meeting needs, but flowerpot bottom not flowing water), a basin causes one basin for arid Dead experiment, remaining 4 basin are tested for controlling water (drought stress).In on-test, flowerpot is filled with enough moisture, and before processing is measured by sampling Physical signs.Arid lethal test group material continues not water, until plant above ground portion dries up, photograph；Then restore watering (enough daily), takes a picture after 5-7 days, sees difference between strain；Continued growth is taken a picture after 10 days, sees difference between strain, records each strain It is that plant above ground portion dries up required number of days, restores that the strain number and required time of young leaves occur after watering.Water material is controlled in control water Materials survey physical signs when handling and (be watered with when moisture content rear blade starts to wilt and be denoted as control water 0 day) 1,6,10,14 day, and It takes a picture during the 2nd day morning 10~11, at the end of drought stress and after restoring enough waterings after control water, sees difference between strain.With The extension of water time is controlled, the damaged membrane evil degree of different strains increasingly increases, but transgenic line is relative to non-transgenosis In contrast, anti-water shortage ability is strong, some material membrane stabilities are good, and mda content is significant lower, and difference reaches notable journey Degree, reflecting them has the drought tolerance significantly improved.In addition, material and non-transgenosis in drought stress process transgenic Control reduces slower compared to chlorophyll content, is still significantly higher than control to chlorophyll content during control water 14 days, shows in arid Under the conditions of blade can keep preferable green.

4), the field selection and utilization of drought-enduring transgenosis rye grass

The excellent rye grass strain transplanting of drought resistance is bloomed pollination in isolated area, harvest seed.Filial generation sowing is being isolated The observation and further selection of tree characteristics are carried out in area.By Analysis of Comprehensive Traits, hang down to coming from the Thailand for turning ZmsPLA2-1 genes 3 excellent strains of drought tolerance are selected in special strain, selected from the bay strain for turning ZmsPLA2-1 genes 2 it is drought-enduring The excellent strain of property.It is bloomed pollination in isolated area by continuous 2 generation again, obtains the drought resisting new varieties (being) of neat and consistent.

Claims (1)

1. a kind of corn phospholipase A_2 geneZmsPLA2-1Application in drought tolerance or cold resistance rye grass is cultivated, feature It is：The method of the application is cloned from cornZmsPLA2-1Gene, with justice or anti-sense versions by the genetic recombination Into plant expression vector, fusion is imported into rye grass using transgenic technology；It is filtered out from transfer-gen plant offspring The transgenic line that drought tolerance or cold resistance significantly improve obtains the rye grass of drought tolerance or cold resistance；Wherein, the geneZmsPLA2-1CDNA nucleotide sequence as shown in SEQ ID No.1, it is describedZmsPLA2-1Gene has cDNA forms or base Because of a group gene forms, coded sequence is built into fusion in the form of anti-sense versions or justice.