CN104388448A - Maize PLA2 (Phospholipase A2) gene ZmsPLA2-1 and application thereof - Google Patents

Abstract

The invention discloses a maize PLA2 (Phospholipase A2) gene ZmsPLA2-1. A nucleotide sequence of the cDNA (complementary Deoxyribonucleic Acid) of the gene is shown as SEQ ID NO.1, and an encoded amino acid sequence is shown as SEQ ID NO.2. The invention further discloses the application of the gene to cultivation of plants with the changed resistance characteristic. The gene is cloned from maize and is recombined into a plane expression vector in a positive-sense form or a negative-sense form, the recombined gene is guided into plant cells by utilizing the transgenic technology, a transgenic seedling is obtained, and a drought-resistant and cold-resistant transgenic plant is cultivated.

Description

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

Technical field

The invention belongs to crop bioengineering breeding field, specifically, relate to a kind of corn phospholipase A_2 gene ZmsPLA2-1 and changing the application in plant stress-resistance proterties.

Background technology

Phosphatide, as the framework ingredient of cytolemma, stimulates also transmission of information to enter in the process of cell in cytolemma impression and plays very important effect.Lipid signaling approach and photosynthesis, hormone regulating and controlling etc. affect plant growth and development process and degeneration-resistant intracellular signaling is in close relations.Phospholipase A (phospholipase A, the PLA) family of hydrolytic phosphatide is made up of phospholipase A1 (PLA1) and Phospholipase A2 (PLA2) two class.The two hydrolysis of catalyze phospholipid on sn-1 and sn-2 position respectively.Wherein PLA2 catalyze phospholipid generates lysophospholipid (lysophospholipids) and free fatty acid (the free fatty acids of sn-1 bit strip acyl chain; FFA), be extensively present in bacterium, plant, mammiferous tissue, cell and secretory product.In animal, PLA2 plays an important role in many important vital movements are as processes such as lipid digestion, cytoplasmic metabolism, intracellular signaling, inflammatory mediator, the invasion of defence pathogen and injuries.In plant, PLA2 also participates in multiple vital movement, as aging, damage, stress response, pathogenic agent defence and inducing secondary metabolite accumulation etc.PLA2 can be divided into five types: secretor type PLA2 (sPLA2s), kytoplasm Ca ²⁺dependent form PLA2 (cPLA2s), Ca ²⁺not dependent form PLA2 (iPLA2s), PAF Acyl-hydrolase (PAF-AH) and lysosome PLA2s.Compared with animal, PLA2 seldom has research in plant, although having reported PLA2 at 1970s can be lysophospholipid and free fatty acids by phospholipid hydrolysis, being definitely applied to of the various enzymes of Phospholipase A2 family is not set forth clear so far completely yet.Kytoplasm Ca is not found in plant ²⁺dependent form cPLA2, its PLA2 be sPLA2 and the class patatin PLA2 (PAT-PLA2) with animal iPLA2 homology mainly.PAT-PLA2 is the lipid acyl hydrolase having PLA2 and PLA1 activity concurrently.The specific PLA2 of plant is sPLA2.

The sequence of sPLA2 gene has been cloned and has been obtained from several plants.1999, Kim etc. and deng respectively from the flower and paddy rice of carnation clone obtain the complete sequence (sequence number is respectively AF064732, AJ238116 and AJ238117) of possible sPLA2 gene.2003, Lee etc. and Bahn etc. search for Arabidopis thaliana database and find there are 4 with the isozyme of animal sPLA2 homology, are respectively AtPLA2-α (At2g06925), AtPLA2-β (At2g19690), AtPLA2-γ (At4g29460) and AtPLA2-δ (At4g29470); Do not find in Arabidopis thaliana, to there is kytoplasm Ca ²⁺dependent form PLA2 (cPLA2).2010, Liao and Burns cloned and obtains two sPLA2 gene C ssPLA2-α and CsPLA2-β from sweet orange, and finds that light regulates and controls the expression of these two genes.

Plant sPLA2 in the similarity lower (about 15%) of amino acid levels and animal sPLA2, the Ca between them ²⁺coupling collar and avtive spot element similarity are 50%.According to the sorting criterion of animal sPLA2s, the sPLA2 of plant can be divided into an independently type.Plant sPLA2 can be divided into again two hypotypes: the first hypotype has AtPLA2-α, the sPLA2 of elm, sPLA2-II and sPLA2-III of paddy rice, tomato and carnation sPLA2s, and the amino acid sequence similarity in hypotype is 47%; Second hypotype has the sPLA2-I of AtsPLA2-β ,-γ ,-δ and paddy rice.Amino acid sequence similarity in hypotype is 54%.Amino acid sequence similarity between hypotype is 29%.Plant sPLA2 comprises the characteristic domain PA2C of PLA2.This structural domain is by Ca ²⁺coupling collar (YGKYCGXXXXGC) and catalytic site element (DACCXXHDXC) composition.Catalytic site element has extremely conservative His/Asp bivalent.At the Ca of plant ²⁺coupling collar finds two conservative amino acid residues in animal sPLA2, Tyr and Gly, they participate in hydrogen bond and are formed.The same with the sPLA2 of animal, the sPLA2 of plant have also discovered the N-end signal peptide of mediating proteins secretion.The transient expression assay of onion epidermis cell finds that AtPLA2-β and AtPLA2-γ is secreted in intercellular substance really.All comprise 12 cysteine residues very guarded in the plant sPLA2s identified, the latter may form six pairs of disulfide linkage.The research of animal sPLA2s has shown that these cysteine residues are very important to the structure and activity of enzyme.After sPLA2 excises signal peptide in endoplasmic reticulum, be secreted into intercellular substance with mature form.The enzymic activity of the mature form of AtPLA2-β and AtPLA2-γ improves 2 times and 1.3 times than before processing respectively.This change is owing to the structural rearrangement of enzyme molecule in the course of processing.

The plasma membrane and microsomal membrane of plant different tissues all detect the activity of PLA2, comprises embryo, endosperm, culturing cell, cotyledon, plumular axis and root.Plant sPLA2 the earliest from elm seed endosperm purifying obtain.It is the low-molecular-weight water-soluble protein of a kind of about 15kD, and to heat, sour, organic solvent is extremely insensitive, but very responsive to disulfide bond reducing agent, and activity needs alkaline pH and Ca ²⁺.AtPLA2-α and AtPLA2-β still keeps the activity of 80-95% after 5 minutes in boiling water treating, and when reductive agent-5mMDTT exists, activity is only original 35-65%.In sweet orange, find the active light cycle influences of sPLA2, after the photoperiod starts 11 hours, sPLA2 activity is the highest, and under lasting dark condition, sPLA2 activity is in lower state always.Ca ²⁺the enzyme that concentration affects sPLA2s is lived, along with Ca ²⁺concentration is elevated to 10mM, and the specific activity of AtPLA2-α continues to raise, and AtPLA2-β, AtPLA2-γ, AtPLA2-δ is at the Ca of micro-concentration of rubbing ²⁺lower enzymic activity reaches plateau value.The sPLA2 enzymic activity of elm and Ca ²⁺relation similar to AtPLA2-α.The Ca that AtsPLA2s enzymic activity needs ²⁺concentration and pH value difference, imply that the activity of the sPLA2s when plant is subject to external stimulus may be subject to Ca ²⁺the adjustment of level and pH value change.

Stream signal people about PLA2 know little about it.Someone infers that sPLA2 may be subject to G-protein regulation, because heterotrimer G-protein activation thing---mastoparan can stimulating plant PLA2 active, but not yet have direct evidence to prove that sPLA2 and G-protein signal is coupled.For the research of PLA2 downstream target gene, more depend on and infer from the biochemistry of sPLA2 and Molecular injury, known (1) PLA2 may play a role in the intracellular signaling of intraor extracellular, and in plant, polyunsaturated fatty acid and lysophospholipid can activated protein kinases; (2) PLA2 regulates and controls stomatal opening, and blue light and ruddiness are by activating H ⁺-pump induction stomatal opening, and this effect can be suppressed by the inhibitor of sPLA2, illustrates that sPLA2 may participate in the light signal transduction of guard cell; (3) PLA2 may participate in the cell elongation of mediating growth element induction.AtsPLA2-β expression intensity in seedling and active growth tissue is higher, and by growth hormone induction, the Arabidopsis plant of AtsPLA2-β process LAN produces the petiole and inflorescence stem that extend, and causes cell elongation retardation to the RNAi of this gene; (4) effect of PLA2 in plant senescence, in plant, product lysophospholipid such as LPE and the LPI of PLA2 can special suppression PLD alpha actives, and known PLD α causes Cellular membraneous damage and promotes plant senescence; (5) can produce free linolenic and linoleic after PLA2 acts on film fat, they can be used as the substrate of lipoxygenase (lipoxygenase, LOX) in octadecenoic acid approach, produce lipid peroxide, and produce jasmonic further.And JA is the important regulon in plant defense response, the expression of various plants defensive raction genes involved can be activated as system stress signal molecule, promote the synthesis etc. of pisatin accumulation and pathogenesis-related protein.JA can independently or via systemin signal transduction and transmit the synthesis of inducible protein enzyme inhibitors, the defensive raction of involved in plant body, and transcribing of phenylpropyl alcohol ammonia lyase gene can be started, regulation and control chalcone synthase, vegetative storage protein, be rich in the expression of the relevant injury defensin gene such as cell wall protein of oxyproline.In addition, in animal, the product P tdOH of PLD can activate PLA2, illustrates to there is complicated interaction between PLA2 and PLD.

Applicant clones corn secretor type phospholipase A_2 gene ZmsPLA2-1, and the aminoacid sequence of its cDNA nucleotide sequence and coding is as shown in SEQ ID No.1 and SEQ ID No.2.Containing the typical PA2c structural domain of sPLA2 in the aminoacid sequence of coding, the highest with RicesPLA2-I similarity on amino acid levels, be 81%, belong to the second hypotype.

The long 575bp of ZmsPLA2-1cDNA sequence, infers that it comprises the ORF of the 5 ' Fei FanGTTYi district (5 ' UTR) of 14bp and the 3 ' non-translational region (3 ' UTR) of 138bp and a 426bp.This ORF encodes 141 amino acid, and its molecular weight and iso-electric point are respectively 153kDa and 8.60.Through retrieval, have not been reported about corn phospholipase A_2 gene ZmsPLA2-1 and the application in change plant stress-resistance proterties thereof.

Summary of the invention

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

Corn phospholipase A_2 gene ZmsPLA2-1 of the present invention, is characterized in that: the nucleotide sequence of described gene cDNA is as shown in SEQ ID No.1; The aminoacid sequence of its coding is as shown in SEQ ID No.2.

Corn phospholipase A_2 gene ZmsPLA2-1 of the present invention is cultivating the application in adverse-resistant characteristic change plant.

The invention provides the scheme that utilizes corn secretor type phospholipase A_2 gene ZmsPLA2-1, ZmsPLA2-1 is applied to plant transgene, produces the engineering plant that degeneration-resistant proterties changes, be applied to crop breeding for stress tolerance.

Corn ZmsPLA2-1 cloning and expressing is analyzed

First, from corn gene group DNA or cDNA, clone ZmsPLA2-1 gene, the latter expresses rear generation 141 amino acid whose albumen.Concrete steps are: carry out BLAST with the RicesPLA2-I (AJ238116) of paddy rice sPLA2 gene family in the EST storehouse of corn, obtain sequence C K827370.Sequential analysis shows that this est sequence comprises complete ORF.Adopt PCR method to clone complete cDNA sequence from corn cDNA library, SEQ ID No.1 and ZmsPLA2-1 is identified to obtain in order-checking.Sequential analysis shows, the long 575bp of ZmsPLA2-1cDNA sequence comprises the opening code-reading frame of 414bp, 141 amino acid of encoding.On amino acid levels ZmsPLA2-1 and other plant sPLA2 have higher similarity, the typical PA2c structural domain containing sPLA2.ZmsPLA2-1 and RicesPLA2-I similarity is the highest, is 81%.PA2c structural domain comprises a Ca ²⁺integrated structure (YGKYCGxxxxGC) and an avtive spot element (DACCxxHDxC).

5 ' of ZmsPLA2-1cDNA terminal sequence is carried out BLAST in NCBI, obtains the sequence of a series of different lengths.The sequence of getting the 2000bp of the initiator codon 5 ' upstream of gene is promotor, the online software of plantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) is utilized to analyze, find the promoter region at ZmsPLA2-1 gene, possible heat stress that DNA normal chain distributes is correlated with cis element 2, ABA is correlated with cis element 1, MeJA is correlated with cis element 2, and growth hormone is correlated with cis element 1, GA ₃relevant cis element 1; Drought stress that minus strand distributes is correlated with cis element 1, defends and coerces relevant cis element 2, and MeJA is correlated with cis element 2, and growth hormone is correlated with cis element 2.The expression of this prompting ZmsPLA2-1 gene may be subject to drought stress, heat stress, ABA, MeJA, GA ₃with the regulation and control of the signals such as growth hormone, corn growth and to Response to stress in have important effect.

Extract the mRNA of the root of the milpa grown under suitable condition, stem, leaf, stamen, gynoecium and seed, reverse transcription synthesis cDNA.According to 3 ' non-translational region or non-conservative district design primer, carry out real-time RT-PCR detection.Result shows: ZmsPLA2-1 has expression in each organ, and wherein in root, expression intensity is the highest, and be secondly seed, be stem again, in blade, female fringe and tassel, expression amount is lower.The corn seedling getting different abiotic stress process is material, and extracting mRNA reverse transcription becomes cDNA, take Ubiquitin as reference gene, detects ZmsPLA2-1 gene expression abundance, with 2 ^{-△ △ CT}method calculate the relative expression quantity of each gene.Show that, under osmotic stress treatment condition, ZmsPLA2-1 all significantly raises in leaf and root, during process 12h, expression amount is respectively 7 times and about 5 times before treatment.The expression of this gene is induced by osmotic stress, and namely they take part in the response of plant to osmotic stress.Under cold Stress treatment condition, this gene expression amount when 4 DEG C of process 6h is the highest.In leaf and root, expression amount reaches 2 times and about 3 times before treatment respectively, shows that this gene also plays an important role in plant is to cold stress response.

ZmsPLA2-1 plant expression vector construction

Go out by pcr amplification the ZmsPLA2-1 inverted defined gene that 5 ' end band has KpnI restriction enzyme site, 3 ' end band has the ZmsPLA2-1 of SacI restriction enzyme site justice gene or 5 ' end band has SacI restriction enzyme site, 3 ' end band has KpnI restriction enzyme site, after KpnI and SacI double digestion, reclaim this fragment.KpnI and SacI double digestion is carried out to plant expression carrier plasmid pCPE simultaneously, reclaim carrier segments.Carry out ligation after the two being mixed, obtain plasmid pCPE-ZmsPLA2-1 (±).1 BamHI restriction enzyme site is contained in goal gene district.Carry out enzyme with restriction enzyme BamHI and cut qualification, except carrier ribbon, plasmid pCPE-ZmsPLA2-1 (+) should have a 136bp electrophoretic band, and plasmid pCPE-ZmsPLA2-1 (-) should have a 440bp electrophoretic band.The plant expression vector of ZmsPLA2-1 gene justice or anti-sense versions insertion is obtained through qualification.In this plasmid, target gene ZmsPLA2-1 merges with its justice or anti-sense versions and corn Ubiquitin1 promotor or Arabidopis thaliana RD29A promotor, transfer-gen plant selective marker is weedicide grass fourth phosphine resistant gene bar, and the latter is started by the promotor of the 35SRNA of cauliflower mosaic virus.

Genetic Transformation in Higher Plants

Different transgenic methods is selected to obtain transgenic plant according to the characteristic of transgenic acceptor.The present invention adopts agriculture bacillus mediated heredity method and particle bombardment to carry out genetic transformation.Set forth for corn below and transform and transformed plant screening process.

In the genetic transformation being material with corn inbred line embryo callus, get 9-15 days corn inbred line fruit ears after pollination self, peel off bract, be placed in 70% alcohol and soak 5min, the rataria that under aseptic condition, picking is about 1.0-1.5mm size is inoculated on inducing culture, cultivate and obtain crisp, flaxen II type callus 4-6 week, then every 10-15 days succeeding transfer culture once.The II type callus obtained is as the acceptor material of genetic transformation.

Ordinary method is adopted to prepare particle gun bullet.Namely take the bronze of 1.0 μm of sizes, after 70% washing with alcohol, leave standstill 15 minutes, centrifugal segregation supernatant liquor; Thoroughly clean 3 times with sterilized water again, then 50% sterile glycerol (final concentration is the micro-bullet of 60mg/ml) is stored for subsequent use.During use, vortex breaks bronze aggegation in 5 minutes, adds 5 μ l plasmid DNA (1 μ g/ μ l), 50 μ l12.5M CaCl successively ₂, 20 μ l 0.1M spermidines, application of sample limit, limit vortex.Then, continue vortex 2 ~ 3 minutes, leave standstill 1 minute.Centrifugal abandon supernatant liquor after, add 70% ethanol leave standstill.Then centrifugally abandon supernatant liquor, then use dehydrated alcohol resuspended, sampling is added on micro-missile-borne body.Micro-bullet consumption is for often to play 0.5mg.

In the culture dish of diameter 9cm, pour the thick substratum of 0.4cm into, then callus high-density is put into the every ware bombardment of culture dish once.Bombardment parameters is got: the distance can splitting disk and carrier is 2.5cm, and the distance that carrier is netted with stop is 0.8cm, micro-bullet flying distance 6--9cm.Other parameter presses working instructions value.After bombardment, material renewal cultivation 3 days darkling, then proceeds to material in the new substratum of components unchanged and cultivates 3 weeks, the target gene proceeded to is given full expression to.Material is proceeded to the enterprising row filter of substratum being added with selective agent (0.08% weedicide grass fourth phosphine).Step sizing 3 generation, per 15 days generations.The tissue block of browning death is eliminated during subculture.Through the resistant calli of screening proceed to do not add selective agent substratum in illumination 16 hours/after renewal cultivation 1 generation of the world, proceed on division culture medium and break up seedling.The seedling that callus produces is taken root in root media, moves into flowerpot, plant large Tanaka, self-fertility when growing to about 10cm height after strong sprout.

At the genetic transformation utilizing agrobacterium-mediated transformation to carry out maize bud point and rye grass Multiple Buds, then for the plant weedicide aqueous solution, screening is sprayed to T0.For the non-transgenic plant of the sprinkling 1.5% weedicide grass fourth phosphine aqueous solution, the blade tip leaf margin place transforming seedling for 4 days afterwards starts jaundice of wilting, and after one week, whole blade becomes withered and yellow, and after 12 days, whole strain turns yellow withered dead gradually; And the blade of Partial Conversion seedling can keep green or yellow leaf degree is comparatively light, show obvious Herbicid resistant, and have 1-2 sheet young leaves to grow in two weeks after herbicide spray.The Herbicid resistant seedling filtered out is transplanted to land for growing field crops, and artificial autocopulation pollinates.Proceed herbicide screening to T1 for plant, resistant plant carries out PCR detection.

The qualification of transformed plant

Extract Herbicid resistant seedling leaf DNA and be used for PCR detection.According to the primers of riddled basins bar and goal gene ZmsPLA2-1, carry out PCR detection respectively.The object band of the object band of the 552bp of bar gene, the ZmsPLA2-1 justice 804bp object band of gene and the 891bp of inverted defined gene has been amplified respectively from transfer-gen plant.Primer sequence is that transgenosis is special, in transfer-gen plant, the size of pcr amplified fragment is consistent with the amplified band carrying these gene plasmids (positive control) respectively, and the object band of formed objects does not appear in not genetically modified adjoining tree (negative control).Proceed PCR to T2 for plant to detect, in some T2 are for strain, external source gene isolation ratio meets Mendelian inheritance ratio, is tentatively defined as the strain of transgenosis genetic stability.

The T2-T4 choosing the inheritance stability of the PCR positive is material for transfer-gen plant and non-transfer-gen plant, extracts blade RNA, is detected the expression intensity of target gene, with Ubiquitin gene for internal standard gene by Real time RT-PCR.Turn at corn in the different strains of ZmsPLA2-1 justice gene, the expression intensity of ZmsPLA2-1 gene has raising in various degree, is 1.31-4.34 times of contrast; Turning in ZmsPLA2-1 inverted defined gene strain, in corn, the expression intensity of ZmsPLA2-1 gene declines, and is 0.42-0.62 times of non-non-transgenic control lines.In the strain of rye grass transfer ZmsPLA2-1 gene, genetically modified gene expression abundance is variant because strain is different, and in the different generations of some strains, transgene expression is stablized.

The drought resistance of transfer-gen plant detects

For corn, under normal growing conditions, 3 leaf phase transfer-gen plants and non-non-transgenic control self-mating system have different in phenotype.Compared with contrast self-mating system, the root system turning ZmsPLA2-1 justice plant is comparatively flourishing, and the plant above ground portion of ZmsPLA2-1 antisense strain is more flourishing.Analyze the biomass of 3 leaf phase transfer-gen plants and adjoining tree under normal growing conditions, show that to turn the growth of ZmsPLA2-1 justice gene strain underground part very fast, the dry-matter of accumulation is significantly higher than and contrasts self-mating system.The transgenic corns and non-non-transgenic control seedling that grow to tri-leaf period are carried out arid lethal experiment, through the drought stress process of 6 days, non-non-transgenic control seedling, antisense ZmsPLA2-1 seedling and part turn the little seedling leaf of ZmsPLA2-1 process LAN close to withered, basal part of stem deliquescing, after recovery is watered, or most of plant is dead in strain, or complete stool system plant is dead.But the drought resistance turning ZmsPLA2-1 process LAN seedling is obvious difference because strain is different, and the drought resistance of most strain significantly improves, water if recover after drought stress, most strain can comparatively fast restore normal growth.

For assessment bloom before drought stress impact that transgenic corn plant is grown, start when plant grew to for 10 leaf phase to control soil relative water content at about 50%-55%, carry out drought stress process, 8 days afterwards recovery normally water, plant started to take out hero after one week.Observe the character mutation of transgenic corn plant and non-non-transgenic control lines in stress during drought stress, and the every physical signs of mensuration of drawing materials respectively for a week after watering in 0,2,4,6,8 day of drought stress process and recovery.Under normal growing conditions, the ground morphological specificity turning ZmsPLA2-1 justice gene strain with grow speed compared with non-non-transgenic control self-mating system without significant difference.After being subject to drought stress, non-transfer-gen plant and turn ZmsPLA2-1 inverted defined gene strain plant and comparatively early occur lack of water symptom early, at Continuous Drought after 5 days, blade turns yellow, withered, and the blade wilting mild degree of ZmsPLA2-1 justice plant, leaf look comparatively emerald green.Under normal growing conditions, the mda content of transgenic line and contrast self-mating system and cytolemma Ion leakage rate are without significant difference.Along with the prolongation of drought stress time, mda content and cytolemma Ion leakage rate obviously raise, and the elevation amplitude of its transfer ZmsPLA2-1 justice gene strain is all less.After coercing 8 days, mda content and the cytolemma Ion leakage rate of ZmsPLA2-1 justice strain are 19.64 ~ 20.24nmol/g FW and 34.7 ~ 37.6%, significantly lower than (the 21.86nmol/g FW and 41.7%) of contrast self-mating system; Mda content and the cytolemma Ion leakage rate of antisense strain be respectively 21.62 ~ 22.64nmol/g FW and 45.5 ~ 50.4%, higher than or be significantly higher than contrast self-mating system.After recovery is watered 7 days, compare with antisense strain with contrast self-mating system, the mda content and the Ion leakage rate level that turn ZmsPLA2-1 justice strain still keep lower level, and namely cell damage is light.

For understanding the forfeiture degree of drought stress conditions lower blade moisture, determine the relative water content (RWC) in different corn strain blade.Before drought stress, between each strain, relative water content does not have significant difference, and after drought stress, all strain leaf r elative water content all reduce, but after coercing 2 days, in different strain blade, RWC fall is different.Compare with ZmsPLA2-1 justice strain, the leaf water loss of non-non-transgenic control self-mating system and ZmsPLA2-1 antisense strain strain is more.At the 8th day of drought stress, two just strain blade RWC were reduced to 70.9%, 71.1%, and antisense strain blade RWC is reduced to about 64.0%, and compared with contrast self-mating system (67.8%), difference all reaches pole conspicuous level.After recovering normally to water, leaf r elative water content starts again to rise, but just strain is higher than contrast self-mating system, and namely the recovery of the water content of just strain blade is faster than contrast self-mating system.These results show that ZmsPLA2-1 justice gene strain blade has better water retention capacity under drought stress conditions, maintain normal cell turgor preferably, are conducive to the growth of plant.

Soluble sugar is a kind of as compatible solute, under drought stress, play provide protection to plant.Measure the soluble sugar content in transgenic line and non-non-transgenic control self-mating system blade under drought stress conditions, can find before drought stress process, blade Soluble adhesion molecule transgenic line and between contrasting difference not obvious.Cause soluble sugar content in all corn strain blades to rise after drought stress process, but ascensional range is different.To turn in ZmsPLA2-1 justice gene strain blade soluble sugar content apparently higher than non-non-transgenic control self-mating system, as when coercing the 8th day, the leaf soluble of two just strains is 82.5,78.7mg/g DW, and contrast self-mating system for 67.7mg/g DW, difference reaches conspicuous level, and antisense strain also have accumulated the soluble sugar content (72.6mg/g DW) more than contrast.After recovery is watered 7 days, in each strain blade, soluble sugar content reduces gradually, but in just strain still higher than contrast self-mating system.These results show that the blade cell of ZmsPLA2-1 justice strain can accumulate more soluble sugar solute, likely make the solute potential that cell remains lower, and then can keep moisture better, maintain cell turgor, from the injury of drought stress.

In order to study drought stress to the photosynthetic impact of milpa, determine transgenic corns strain and Net Photosynthetic Rate, the stomatal conductance of contrast self-mating system under normal growing conditions, drought stress conditions and rehydration condition.Normally watering under water condition, the Net Photosynthetic Rate of ZmPLA2-1 justice gene strain a little more than non-non-transgenic control self-mating system, after drought stress, with stress time the different corn strain of prolongation between difference strengthen gradually.In whole stress during drought stress, compared to contrast self-mating system, the Decrease in Net Photosynthetic Rate turning ZmPLA2-1 justice gene strain is comparatively slow.As when drought stress 6 days, the Net Photosynthetic Rate of just strain is reduced to 49.9% before Stress treatment, is significantly higher than (41.3%) of contrast self-mating system; And now ZmPLA2-1 inverted defined gene strain is 21.7%, significantly lower than contrast self-mating system.In stress during drought stress, the variation tendency of stomatal conductance is with Net Photosynthetic Rate, and the stomatal conductance that turns ZmPLA2-1 justice strain is significantly higher than contrast self-mating system, antisense strain.Stomatal conductance declines and causes CO ₂it may be cause the important factor that in stress during drought stress, photosynthesis declines that supply reduces.In addition, after recovery is watered 7 days, the Net Photosynthetic Rate of all strains and pore lead angle value all to be increased, turn ZmPLA2-1 justice gene strain still higher than contrast self-mating system.After photosynthetic capacity stronger under drought stress and rehydration, higher recovery rate shows that turning ZmsPLA2-1 justice gene strain improves plant drought-resistant ability.

In drought tests rain-proof shelter, milpa before blooming is carried out to the drought stress of continuous 6 weeks, then recover normally to water, observe transfer-gen plant during this period and contrast the character mutation of self-mating system plant under drought stress, statistics single plant yield and cell production after results.It is influenced less that result shows to turn growing of ZmsPLA2-1 justice gene strain plant, and grain yield is high.

Difference under comprehensive drought stress conditions in the physical signs of different strain plant and output draws, turn ZmsPLA2-1 justice gene strain cytosol content increasing degree when running into drought stress large, decrease the loss of cell moisture, better can keep the water content of cell, cell membrane damage is little, and photosynthetic capacity is comparatively strong, and growing of plant is influenced less, therefore grain yield is high, shows and can adapt to drought stress environment better compared with non-non-transgenic control lines.The drought resistance difference turning the strain of ZmsPLA2-1 inverted defined gene and adjoining tree is little.

Turn ZmsPLA2-1 gene plant resistance to cold to detect

Usual low temperature stress is divided into and damages to plants caused by sudden drop in temperature (0-15 DEG C) and freeze injury (being less than 0 DEG C) two kinds, the plant being distributed in different areas is different to the TL of low temperature, and the genetic expression in the resistance to cold of plant and plant materials, microbial film lipid forms and the accumulation of small-molecule substance has substantial connection.Low temperature mainly passes through to the injury that plant is caused the structure destroying film, photosynthesis is obstructed, produces the death that toxicant and active oxygen etc. finally cause cell and plant.Plant has the process of an adaptation and self-control to Chilling stress.Corn is more responsive to low temperature stress, under cryogenic vulnerable death.Low temperature easily makes corn seedling come to harm, and Wang etc., by quick-thawing again after Corn Root Tip Cells slow freezing, find growing of the root, stem and leaf recovering corn after under normal condition obviously delayed; The discoveries such as Fngels, maize root system is processed 3 days under cryogenic with under normal condition simultaneously, and cauline leaf fresh weight, leaf area and root/shoot ratio obviously decline.Subzero treatment (3 DEG C) is carried out, its seedling soluble sugar and sucrose content, plant leach liquor ions content significant difference in body compared with under normal condition in Course of Corn Seed Germination process.

The morphological specificity no significant difference of ZmPLA2-1 gene overexpression plant and non-non-transgenic control lines under normal growing conditions, but growth velocity is higher, and ZmPLA2-1 gene antisense expresses plant, and difference is not obvious compared with the control.Turn the analysis of ZmPLA2-1 gene plant resistance to cold can in the controlled environment chamber in carry out, plant to be planted grew to for 3 leaf phases, first 10 DEG C process 1 day, then drop to-2 DEG C (rye grasses) or 4 DEG C (corn) lasting process 3 days, then room temperature restoration ecosystem 3 days.Under cold condition, transfer-gen plant is all suppressed with the growth of non-transfer-gen plant, but turns the impaired comparatively light of ZmPLA2-1 gene overexpression strain, and growth difference compared with wild type control of antisense gene plant is significantly not clear.4 DEG C of process corn seedlings are after 3 days, non-transgenic reference plant occurs that blade wilting dehydration, limb are curling, antisense ZmPLA2-1 gene strain is than contrast plant leaf more wilting dehydration and color burn, and turn ZmPLA2-1 gene overexpression strain and show as that blade is unfolded, leaf color is light green, there is not obvious crimp in limb, shows and improve resistance to cold compared with non-non-transgenic control yet.

Detect and turn the gene expression abundance of ZmPLA2-1 gene in the different strain of corn, find that the gene expression abundance of process LAN strain before cold Stress treatment is about 1.6 times of adjoining tree, and the ZmPLA2-1 abundance of antisense expression strain is about the 0.4-0.6 of non-non-transgenic control lines doubly, difference all reaches extremely remarkable, i.e. transgenosis functionating in plant.After 4 DEG C of process 6h, the ZmPLA2-1 gene expression abundance of non-non-transgenic control strain about raises 2 times, process LAN strain about raises 5.5 times compared with before process, namely ZmPLA2-1 gene expression abundance is far above non-non-transgenic control lines, and the gene expression abundance change of this gene is little in antisense expression strain, show the raising of the ZmPLA2-1 gene expression abundance that transgene silencing mechanism effectively controls, reduce plant resistance to cold.This ZmPLA2-1 gene expression abundance determined from transcriptional level and plant resistance to cold closely related, cold Stress treatment inducing maize ZmPLA2-1 gene expression amount raises.

Under stress conditions, plant carrys out excess activity oxygen in scavenger cell by the activity improving superoxide-dismutase (SOD) and peroxidase (POD), reduce the injury of active oxygen or other peroxide radical cell membrane systems, thus maintain the stability of film and macromolecular structure.With before cold Stress treatment, in cold Stress treatment and the plant leaf recovering room temperature measure the activity of superoxide-dismutase (SOD) and peroxidase (POD) for material, draw: before process, SOD with POD activity activity difference compared with transgenic line of non-non-transgenic control is little, SOD and the POD activity of subzero treatment render transgenic strain and non-transgenic reference strain raises all to some extent, but the elevated-levels of different strain enzymic activity is different.In subzero treatment and recovery process, the SOD of ZmPLA2-1 gene overexpression strain is active in wild-type, and the SOD of antisense expression strain is active in low-temperature process significantly lower than non-non-transgenic control.

Under cold stress conditions, maize leaf cell dehydration, active o content rises, and causes blade cell film impaired, causes cell plasma percolation ratio to increase.This measuring is cold coerce before and after each strain blade cell Electrolytic leakage.Result shows before cold Stress treatment, and the leaves ions percolation ratio difference of different strain is not obvious, and the cell membrane damage degree of subzero treatment render transgenic strain and non-transgenic reference strain raises all to some extent, but different strain degree of injury is different.Turn the cell membrane damage of ZmPLA2-1 gene overexpression strain lower than 60% during 4 DEG C of process 3 days, and non-transgenic reference is about 65%, and higher than process LAN strain, and the cell membrane damage of antisense strain is more serious than non-transgenic reference.

Embodiment

Embodiment 1: turn ZmPLA2-1 gene and create drought-resistant maize self-mating system and application

1) foundation of receptor system with Inbred Lines used in China's agriculture production for material, as the selfed seed of Zheng 58, prosperous 7-2, DH4866 etc.Isolated culture induction stem apex produces grows thickly sprout tuber, carries out genetic transformation with the sprout tuber that grows thickly for acceptor.Used medium has:

Seed germination medium: KNO ₃1900mg/l, NH ₄nO ₃1650mg/l, CaCl ₂2H ₂o 440mg/l, MgSO ₄7H ₂o370mg/l, KH ₂pO ₄h ₂o 170mg/l, FeSO ₄7H ₂o 27.8mg/l, ZnSO ₄7H ₂o 10mg/l, MnSO ₄4H ₂o22.3mg/l, H ₃bO ₃10mg/l, KI 0.83mg/l, Na ₂moO ₄2H ₂o 0.5mg/l, CuSO ₄5H ₂o 0.025mg/l, CoCl ₂6H ₂o 0.025mg/l, vitamin 10.0mg/l, pyridoxine hydrochloride 1.0mg/l, nicotinic acid 1.0mg/l, glycine 2.0mg/l, inositol 100.0mg/l, vitamin H 0.05mg/l, casein hydrolysate 500mg/l, sucrose 30g/l, agar powder 7g/l, pH 5.8 ~ 6.0.For seed germination.Liquid nutrient medium then removes agar powder.

A substratum: seed germination medium adds 6-BA 4.5 ~ 9.0 μm of ol/l and 2,4-D, 1.0 ~ 3.0 μm of ol/l, produce Multiple Buds tissue block and Multiple Buds tissue block succeeding transfer culture for inducing isolated culture bud point.

B substratum: seed germination medium adds 6-BA 4.5 μm of ol/l and IBA (indolebutyric acid) 1.8 μm of ol/l, for Multiple Buds tissue block differentiation seedling.

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

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

Minimum medium and plant growth regulating substance pressure sterilizing; Microbiotic and weedicide isoreactivity composition filtration sterilization, add after medium sterilization.

Seed sterilization and sprouting: corn seed 70% alcohol immersion 10 minutes, then soak 10--15 minute with 0.1% mercury chloride, then with sterilized water washing 3--5 time.After sterilizing, seed is placed in aseptic triangular flask and sprouts, and puts into a small amount of (30---40 milliliter/250 milliliter triangular flask) sterilized water in bottle, is placed on 1--2 days under dark condition (23---30 DEG C) after sealing.Sprout (showing money or valuables one carries unintentionally) afterwards seed to be placed on minimum medium and to continue to sprout under dark condition.

Stem tip culture and the induction of Multiple Buds tissue block, subculture, differentiation: the plumule of germinating seed extend only 3---5 centimetre time, peel off coleoptile and spire, cut the epicotyl and stem apex that are about 5 millimeter, be inoculated into (24--27 DEG C) cultivation under dark on A substratum, and cut the plumular axis of elongation in time and peel off spire.Cultivate after 6--10 days, stem apex starts irregular growth of expanding, and several warty or digitation appear in the meristematic tissue expanded.After 20 days, start to form indefinite bud and embryoid on the surface of warty or digitation.Generally every 4 weeks succeeding transfer culture once.In succeeding transfer culture, if Multiple Buds tissue block is grown thickly, budlet is on the high side, and 2,4-D concentration get 3.0 μm of ol/l; If Multiple Buds tissue block callusization is heavier, though there is a large amount of Meristematic cell mass, seldom there is indefinite bud in surface, can by 2, and 4-D concentration reduces to 1.0 μm of ol/l, continues to cultivate then to regenerate a large amount of warty or digitation.Multiple Buds tissue block on A substratum, minority has the generation of adventive root.Exist the same with spire, what adventive root also affected tissue block expands growth and embryoid and the generation of budlet of growing thickly, and needs to remove early.Multiple Buds tissue block is being transferred on B substratum after 2--3 days, and color and luster turns yellow gradually, and quality is more pliable and tougher, and within 5--6 days, microspike appears in rear surface.Visible each phase embryoid and indefinite bud is observed under scanning electron microscope.Embryoid and indefinite bud are grown rapidly, form on tissue block surface the budlet that grows thickly.

2) with Multiple Buds tissue block be acceptor conversion and plant regeneration

By the agrobacterium tumefaciens (as AGL0 and LBA4404) with binary vector (Mini--Ti plasmid is with selective agent resistant gene and ZmPLA2-1 gene), at additional antibiotic LB substratum, (often liter of substratum contains: tryptone 10g, yeast extract 5g, NaCl 10g, pH 7.0, autoclaving) at 28 DEG C concussion cultivate, concussion speed is 110rpm (rev/min), makes bacterium be in logarithmic phase.Then at 3,000 rpm centrifugal 10 minutes, supernatant liquor is abandoned.The thalline liquid seeds germination medium (namely seed germination medium composition reduces by half, and removes agar powder) of 1/2 concentration washs, then collected by centrifugation.Suspended by the liquid inducing clumping bud substratum of thalline by 1/2 concentration of adding Syringylethanone (acetosyringone, As) 100 μm of ol/l, dilution 5--20 is doubly for transforming again.

The Multiple Buds tissue block of cultivating after getting subculture 13--20 days is transgene receptor.Transform with agrobacterium-mediated transformation, after transforming, material carries out renewal cultivation darkling.Grow thickly budlet or the Multiple Buds tissue block of agroinfection cultivate 7--12 days, bacteria growing inhibiting on the substratum being added with cephamycin (Cefotaxime) 250mg/l or Pyocianil (Carb) 500mg/l in dark.Growing thickly budlet or Multiple Buds tissue block step sizing 3-4 generation on the substratum being added with selective agent (as careless fourth phosphine 0.08%) after renewal cultivation or after micro-organisms, obtains transgenic cell and budlet.In screening and culturing, most Multiple Buds tissue block is dead gradually.The tissue block of survival is transferred to and removes 2 without selective agent, the A substratum of 4-D produces budlet after renewal cultivation.

Budlet is placed on irradiation on seedling substratum to cultivate, light intensity 2000-3000lx, illumination 14-15 hour/day.Seedling proceeds in root media when growing to 3-4 sheet leaf takes root.Cultivate about 15 days, the seedling of about 40% produces new root.For seedling of not taking root, its base portion of cut wound, transfers on new root media and cultivates, and after 10 days, most of plant produces root system.After seedling of taking root washes away substratum, be transplanted to vermiculite be cultivation medium flowerpot in grow.Plant grows under natural lighting, day temperature 22--28 DEG C, and night, temperature 15--21 DEG C, watered the inorganic salts ingredients of the seed germination medium of 1/2 concentration every other day.Grow about 2 weeks, nursery transplant produces flourishing root system, is then colonizated in field.

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

The blade getting transplant survival plant carries out Molecular Detection determination transfer-gen plant.Then by transfer-gen plant (T0) bagging self-fertility.T1 seed from different T0 plant is broadcast in greenhouse or the field with safeguards, gets blade after emerging and adopt PCR method to detect transgenosis, and smear weedicide (as 0.2% careless fourth phosphine) solution qualification resistance.By PCR, the positive and plantlet of transplant of Herbicid resistant is to field, bagging self-fertility after growing up.

In transfer-gen plant progeny selection, first get T2 of uniform size for transgenic seed and non-non-transgenic control self-mating system planting seed in vinyl disc of the same size, the homogeneous loam of equivalent is housed in dish, and often coil sowing 20, every strain at least plants 3 dishes.Normally water, treat that seedling grew to for 3 leaf phases, choose the consistent vinyl disc of growing way and carry out drought resistance viewing test.The method of drought stress process be disposable water sufficient moisture after stop watering, until the plant leaf of most strain is withered, basal part of stem deliquescing, recover again to water, observe each strain and recovering the upgrowth situation after watering, filter out the strain that drought resistance significantly improves than contrast self-mating system.Then carry out transfer-gen plant bloom before drought stress process.

Corn bloom before drought stress test carry out under field conditions (factors), avoid plant to drench with rain during Stress treatment.Choose T2 for transgenic seed and contrast self-mating system planting seed in large flowerpot of the same size, when plant grew to for 5 leaf phase, every basin stays seedling 2 strain, namely contrasts 1 strain, transgenic plant 1 strain.Plant grows to 10 leaves normally watering under water condition, then chooses the consistent milpa of growing way and is divided into two groups, one group be in drought condition under grow, soil absolute water content remains on 15-17%, continues process one week, then recovers normally to water.Another group remains under abundance waters water condition and grows, in contrast.Often group establishes 6 repetitions.In the pouring of control water in earlier stage, Osmotic treatment group plant by day 9 ~ 18 periods blades is wilted, and night, blade recovered to stretch; In the control water pouring later stage, blade continues to wilt.After recovery is normally watered one week, plant starts to take out hero.(blade the morning 8 time about start to wilt) is designated as drought stress process 0 day to control plant to water the 1st day, sampling after normally watering one week in 0,2,4,6,8 day that processes and recovery, measures RWC, cytolemma Ion leakage rate, mda content, soluble sugar content, proline content and photosynthesis index etc. respectively.Measure plant height and the green number of blade flowering period after Osmotic treatment, statistics is bloomed and weave silk the timed interval (Anthesis-silking interval, ASI).The mensuration of Pollen Activity adopts IKI staining.Getting mature anther is positioned on slide glass, adds 1 distilled water, is smashed to pieces by flower pesticide with tweezers, release pollen granule, then adds 1 ~ 2 I ₂-KI solution-dyed.Covered, in low power Microscopic observation, statistics Pollen Activity.

The milpa of experience 8 days drought stresses, recovery starts to take out hero after watering one week, and the phase is added up plant plant height and holds the green number of blade at this moment.Normally watering under water condition, growing with to contrast the phenotypic difference existed between self-mating system not obvious of transgenic corn plant, the measurement result of some indexs also demonstrate that observation conclusion.Bloom drought stress process in early stage after 8 days, and the average plant height of all strains all significantly reduces, but the amplitude reduced between different corn strain is different.Compared to the homology plant normally watered under water condition, the reduction amplitude turning ZmsPLA2-1 justice gene strain plant height is respectively 21.9% and 22.2%, and reduction amplitude is less than (28.7%) of non-non-transgenic control self-mating system; The plant height turning ZmsPLA2-1 inverted defined gene strain reduces difference 28.4% and 31.9%, reduces amplitude little with the difference contrasting self-mating system.

After experience drought stress, hold green number of blade significant difference between different strain, the greenery number turning ZmsPLA2-1 justice gene plant is apparently higher than non-non-transgenic control self-mating system and antisense gene plant.The female tassel of drought stress before blooming to milpa is grown and is created deep effect.After Osmotic treatment, the flowering time of all strains is postponed, and female tassel is grown inharmonious, and causes the normal pollen quantity of milpa to reduce and the rising of pollen sterile quantity.Drought stress is less than not genetically modified turning bloom-weave silk the timed interval impact of (Anthesis silking interval, ASI) of ZmsPLA2-1 justice gene plant.After drought stress, the pollen amount of non-non-transgenic control self-mating system is few, turns ZmsPLA2-1 antisense plant similar with non-non-transgenic control lines, and it is more to turn ZmsPLA2-1 justice gene plant pollen amount.The flower pesticide getting different strain adopts the dyeing of IKI method to carry out cytological observation, and find out in the pollen granule of non-non-transgenic control lines and only have smaller portions to be contaminated for blueness, major part presents light tan.The pollen being blueness by dye is the pollen of rich in starch, and its vitality is strong, presents filemot pollen development bad, and after pollination, Setting percentage is lower.And the normal loose powder of tassel energy of adopted gene plant of becoming a full member, most POLLEN MORPHOLOGY is normal, and major part is mazarine by IKI dyeing, and color is darker.This illustrates that the pollen abortion rate of non-non-transgenic control lines is high, and adopted gene plant of becoming a full member is grown normal at its pollen majority after drought stress process, and vitality is strong.

By transgenosis and non-non-transgenic control maize seed in arid experiment shed, plant to be planted grew to for 10 leaf phases, controlled irrigation amount in arid canopy, soil relative water content is made to remain on about 50%-55%, drought stress process is carried out to plant, continues 6 weeks, then water adequate water.In observation Osmotic treatment process, plant grows.After fruit ear maturation, observation statistics is carried out to Ear Characters.After drought stress, the blade turning ZmsPLA2-1 justice strain plant comparatively stretches, and leaf color jade green, affect little by drought stress, and non-non-transgenic control self-mating system plant leaf is seriously curling, indivedual Plant Leaf look shoals.After drought stress, different strain Ear Characters and output show, the average spike length of ZmsPLA2-1 justice strain is obviously greater than non-non-transgenic control self-mating system, and difference between inverted defined gene strain and non-non-transgenic control is not remarkable.From grain weight per panicle, the grain weight per panicle of ZmsPLA2-1 justice strain is obviously many and non-non-transgenic control self-mating system, and grain weight per panicle increases by 34.1%, 29.9% than (50.8 ± 9.0g) of contrast self-mating system respectively.From 100-grain weight, though each transgenic line with contrast between self-mating system variant, do not reach significant difference degree.These results show, turn ZmsPLA2-1 justice gene plant and have to the drought stress run into before blooming the resistance significantly improved, reproductive development is influenced less, thus make grain yield be significantly higher than contrast self-mating system.

Embodiment 2: turn ZmPLA2-1 gene and create Maize Cold Resistant self-mating system and application

1. corn aseptic seedling obtains

The seed of maize elite inbred line, by 70% alcohol immersion 8 minutes, then soaks 8-12 minute with 0.1% mercury chloride, then with sterilized water washing 3-5 time.Constantly seed is rocked, to ensure that surface sterilization is thorough during sterilizing.After sterilizing, seed is placed in aseptic triangular flask and sprouts, and puts into a small amount of sterilized water 1-2 days under dark condition (25-28 DEG C) in bottle.After Seed sprouting (showing money or valuables one carries unintentionally), place it in modified MS medium and sprout under dark condition.When plumule elongation stops 3-4 centimetre, peel off coleoptile and 2-3 sheet spire, emergent stem pointed tip growing tip.

2. Agrobacterium is cultivated and activation

Will with the concussion cultivation at 28 DEG C in additional antibiotic LB substratum of the agrobacterium tumefaciens (AGL1 or LBA4404) of binary vector (Mini-Ti plasmid is with herbicide resistance gene bar and ZmPLA2-1 fusion gene), concussion speed is 110r/min, makes bacterium be in logarithmic phase.Then under 3000r/min centrifugal 10 minutes, supernatant liquor is abandoned.Thalline 1/2MS liquid nutrient medium washing, collected by centrifugation.Again the 1/2MS liquid nutrient medium of thalline with interpolation 100 μm of ol/l Syringylethanones is suspended, be diluted to OD ₆₀₀0.2 ~ 0.4 for transforming.

3. corn aseptic seedling transforms

(1) bacterium liquid is poured in the culture dish of 4.5 cm diameters, inclination culture dish, makes the aseptic seedling exposing stem apex growing tip be immersed in bacterium liquid, 0.5 × 10 ⁵8-12 minute is processed under Pa normal atmosphere.

(2) the bud point aseptic filter paper after contaminating blots, and germinating seed is placed in modified MS medium and cultivates 2-3 days in dark, and culture temperature is 22-24 DEG C.Then aseptic seedling is put and cultivate 2 days under diffuse light.

(3) aseptic seedling after being cultivated by irradiation is transplanted in the flowerpot being covered with upper strata vermiculite lower floor loam, and covers plant top with vermiculite.Then allow plant grow under natural lighting, day temperature 22-28 DEG C, night, temperature 15-21 DEG C, watered 1/2 modified MS medium inorganic salt every other day.

4. transformed plant screening and field planting

After transformed plant grows 3 leaves, spraying herbicide (Hoechst Schering AgrEvo GmbH, containing weedicide glufosinate ammonium) aqueous solution, concentration is 9.6ml-10.8ml / L, falls drop with plant and is advisable.Unconverted adjoining tree stops growing after 4 days after spraying, starts death after 9 days.After spraying, some individual changes are similar with adjoining tree, and other individual continued propagation, change not obvious for transformed plant.When the plant that survives grows to 5 leaf, by its field planting to field, bagging selfing is set seeds.

5. transfer-gen plant progeny analysis

T1 grows to 3 leaf phase 10.8ml for plant the process of/L the aqueous solution, observes statistics resistance and sensitive individuals ratio; Adopt round pcr to detect foreign gene, and add up the segregation ratio of foreign gene in progeny plant.Survive plantlet of transplant to land for growing field crops, bagging selfing.T2 plant, except bagging selfing is set seeds, adopts round pcr to detect foreign gene, and carries out Southernblotting checking; RT-PCR technology is adopted to check transgene expression intensity.

6. the resistance to cold of transfer-gen plant offspring detects

Milpa is along with the prolongation of deepfreeze time, and leaf soluble has rising trend, and in 10 DEG C of process soluble sugar amount rising after 1 day, 4 DEG C of process turned ZmPLA2-1 gene overexpression strain soluble sugar content at 55-61mg g after 3 days ^-1between DW, apparently higher than non-non-transgenic control lines, and the soluble sugar content of antisense gene strain is less than non-non-transgenic control lines, is 45mg g ^-1about DW.Before cold Stress treatment, the total free aminoacids total amount of transgenic line does not have significant difference compared with non-non-transgenic control strain; After Stress treatment; the total amount of total free aminoacids increases to some extent compared with before process; except methionine(Met) (Met); other free amino acid concentrations has rising in various degree after being subject to cold coercing; wherein proline(Pro) (Pro) content obviously increases; proline(Pro) can accumulate when plant materials is forced in a large number as osmotic protection material, and the proline content increasing degree turning ZmPLA2-1 gene overexpression strain is significantly higher than non-non-transgenic control lines.In addition, Glu and the Val content that turns ZmPLA2-1 gene overexpression strain after deepfreeze is significantly higher than contrast, and Gly content is then starkly lower than contrast, the plant of antisense expression then with contrast close.These differences, demonstrate the regulation and control that ZmPLA2-1 gene participates in corn resistance to cold.

ZmsPLA2-1 process LAN improves the resistance to cold of milpa, along with cold under cold condition stress responsive gene ZmDREB1A, ZmMPK1, ZmMPK2, ZmMPK7, ZmHK3 at the gene expression abundance of ZmsPLA2-1 in process LAN strain higher than non-non-transgenic control lines.The series of genes that the regulation and control of DREB transcription factor are relevant to adverse circumstance is expressed, and comprises drought stress response gene.Their abduction deliverings in transgenic plant can activate the expression of the degeneration-resistant functional gene in a series of downstream, and improve the resistance of plant, wherein ZmDREB1A expression amount obviously raises the expression that can activate cold-resistant genes involved.Mitogen-activated protein kinase (mitogcn-activated protein kinase, MAPK) be the important component of signal transduction pathway MAPK cascade reaction in organism, by transmitting born of the same parents' internal/external signal, mediate multiple biology and abiotic stress reaction.Therefore, after corn is forced, ZmsPLA2-1 product may have activated the expression of these stress-related genes, thus enables plant adapt to stressful environmental better.

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

For the transgenic line that selected resistance to cold is significantly improved, measure the change of plant Net Photosynthetic Rate under different light intensity and temperature, measure 3-7 leaf phase seedling growth velocity and biomass, measure single plant yield, and with non-transfer-gen plant for contrast.Under field cultivating condition, carry out the observation of Yield Traits In Corn after selecting excellent transgenic line and compare, selecting cold-resistant high yield strain to enter corn breeding experiment.

Embodiment 3: turn ZmPLA2-1 gene and create rye grass Drought Resistance Germplasm and application

Rye grass is Gramineae lolium plant, one year raw rye grass and English ryegrass point.English ryegrass cries again perennial root rye grass, and annual ryegrass is Italian ryegrass again, and originate in southwestern Europe, north African and southwest, Asia, the 1950's is introduced into China as high quality forage.Rye grass tillers many, and output is high, and quality is soft, and the green phase is long, is good green feed; Its well developed root system in addition, suitable natural disposition is strong, and resistance to trample, has certain Salt And Alkali Tolerance potentiality, can increase the soil organism, improve Soil structure, prevent erosion, and is again good turfgrass.But drought tolerance is not high, be necessary to improve its drought tolerance.

1), the foundation of rye grass Multiple Buds system

The substratum setting up rye grass Multiple Buds system used has: inducing culture (MS+0.5mg/L 2,4-D+2.0mg/L6-BA), proliferated culture medium (MS+2.0mg/L 6-BA), root media (1/2MS+1.0mg/L NAA).

Ryegrass seed through 70% alcohol sterilizing 1 minute, 0.2% mercuric chloride sterilizing 5 minutes, aseptic washing 4 times.Sprout under MS is without dark condition on hormone culture-medium, temperature 25 ± 2 DEG C.After 4-5 days, when plumule is stretched to 2-3cm, cut the explant sprouted as induced bundle in shoot tip meristem position.

By the stem apex position (2-3mm is long) of the seed seedling of sterile culture cut, be inoculated on the inducing culture containing different concns 6-BA and 2,4-D.Culture temperature 25 ± 2 DEG C, illumination 12h/d, forms the sprout tuber that grows thickly that base portion expands for light intensity 800-1000Lx, 15-20 days afterwards.Within 20 days, add up the inductivity of Multiple Buds afterwards.Explant was cultivated after 2 days on the inducing culture of suitable concentration 6-BA and 2,4-D, and stem apex and epicotyl start to extend.After 7-8 days, epicotyl part is withered, and stem apex and outer leaf sheath thereof expand, and from the stem apex expanded, 2-3 budlet occur on the surface, within 10-15 days, forms the Multiple Buds of 8-10 intensive budlet composition afterwards.The kind of exogenous hormone and the form of concentration to rye grass Multiple Buds play a part very important.Substratum without 6-BA and 2,4-D does not have the generation of Multiple Buds; Only adding 6-BA and do not add the substratum of 2,4-D, though there is Multiple Buds to be formed, bud number is few, and little bud-leaf look dark green, and blade face macula lutea even appears in partial vitrification.On the substratum of 2,4-D concentration lower (0.1-0.2mg/L), the budlet bending blade of induction or distortion, bastem portion is comparatively thin, and inductivity is also lower.In 2,4-D concentration higher than on the inducing culture of 0.5mg/L, bastem portion callusization is comparatively serious, and have more new generation, sprout tuber blade is more, but budlet generation quantity is few.At MS+0.5mg/L 2,4-D+2.0mg/L 6-BA) on substratum, little bud-leaf look bud green, sprout tuber growing way is better, and the stem apex percentage producing Multiple Buds is high, average each sprout tuber budlet number also average at most (6.79).Therefore, in follow-up work, select this substratum to be inducing culture.Substratum needed for the inducing clumping bud of different varieties rye grass and Hormone Conditions difference little.

The sprout tuber that grows thickly is peelled off older leaf sheath and spire, transfers on different proliferated culture medium after being divided into simple bud respectively, every 20 days subcultures once.Light intensity 800-1000Lx, light application time 12h/d, temperature 25 ± 2 DEG C.The sprout tuber that grows thickly of expanding is cut into simple bud or little sprout tuber (about 2mm size) by multiplication culture after 20 days, forward succeeding transfer culture in the different proliferated culture medium of hormone combination respectively to.After 2 days, simple bud or budlet BOB(beginning of block) expand, and from the stem apex expanded, 2-3 budlet occur on the surface, and within 5 days, formed afterwards by the Multiple Buds formed of 8-10 intensive budlet, the kind of exogenous hormone and the propagation of concentration to rye grass Multiple Buds play a part very important.Do not producing Multiple Buds without bud point in vitro on hormone culture-medium, bud point extends into plantlet.Be added with on 1.0mg/L 6-BA substratum, the sprout tuber that grows thickly generally is made up of 2-3 budlet, and budlet proliferation rate is low.Succeeding transfer culture on the substratum of 3.0mg/L 6-BA, the bud-leaf look of growing thickly of induction is dark green, the vitrifying of part budlet, and there is macula lutea on blade face, has a small amount of albefaction bud to occur.Proliferated culture medium is the suitableeest with MS+2.0mg/L 6-BA, and average bud number (kind bay 9.96) (kind Tai Chuilaite 10.62) at most of each Multiple Buds, Multiple Buds growing way is also best., after 3 generations, budlet hyperplasia is very vigorous on this substratum every 20 days subculture Multiple Buds once, and simple bud or little sprout tuber can produce the budlet of about 50 after 20 days in cultivation.

The sprout tuber that grows thickly proliferated culture medium growing 20 days is separated into simple bud, and transfer on root media, illumination is increased to 1500-2000Lx.The rootlet starting adularescent for general 9th day occurs, and within 20 days, formed flourishing root system, rooting rate reaches 100%.After 20 days, open bottle cap, add a little tap water, hardening 3 days.Clean the substratum of root, be transplanted in the flowerpot of upper strata vermiculite, lower floor's loam.The seedling of the flourishing root system of tool is easy to transplant, survival rate 100%.

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

Cut blade after rye grass Multiple Buds (succeeding transfer culture 8-10 days) is shelled into simple bud, the meristematic tissue exposing pointed tip of sprouting carries out agroinfection.Get the bacterium liquid that 10ml grows to logarithmic phase, the centrifugal 10min of 4000rpm, collect thalline, be resuspended in by thalline in liquid nutrient medium, dilution makes final OD ₆₀₀for about 0.4-0.6, add the Syringylethanone (AS) of 0.1mmol/L dip-dye forward direction bacterial suspension.The bud point stripped is immersed in this suspension, under vacuum tightness is 0.05MPa pressure, contaminates 5min.Taking-up bud point, blots bacterium liquid with aseptic dry filter paper, proceeds to Dual culture and cultivates based on light culture 3-4 days at 25 ± 2 DEG C.Bud point after Dual culture forwards on the micro-organisms base that adds containing 100mg/L cephamycin and suppresses Agrobacterium to grow, and after 8 days, established Multiple Buds is separated into simple bud, be put into be added with selective agent substratum on screen.After each screening and culturing, select survival budlet and continue screening.As the succeeding transfer culture rye grass Multiple Buds of 8 days is separated and is cut into 2-3mm size, be inoculated into containing cultured continuously 3 generation in 0.08% careless fourth phosphine substratum, per 15 days generations.Budlet after screening was through the renewal cultivation of 8 days, and transfer on root media, illumination is increased to 1500-2000Lx.After 20 days, open bottle cap, add a little tap water, hardening 3d.Clean the substratum of root, be transplanted in the flowerpot of upper strata vermiculite, lower floor's loam.Water twice nutritive medium, intensive care, survival rate 100%.

Plant to be planted in flowerpot after two months, extracts plant leaf DNA, carry out pcr amplification and Southern hybridization check by CTAB method.According to the primers of riddled basins and goal gene ZmsPLA2-1, carry out PCR detection respectively.Part Herbicid resistant plant amplifies object fragment, and non-non-transgenic control lines is 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 positive is carried out counting generation amplification at subculture medium, obtains a large amount of budlets, then transfer in flowerpot.After seedling survives, January is carried out Stress treatment in general growth again.Carrying out in drought stress test, each strain is planted 6 basins, the strain of every basin 5.One basin is used for normally watering (satisfy the demand, but bottom flowerpot not flowing water), and a basin is used for arid lethal test, and all the other 4 basins are for controlling water (drought stress) test.When on-test, sufficient moisture filled with by flowerpot, sampling and measuring physical signs before treatment.Arid lethal test group material continues not water, until plant above ground portion dries up, takes a picture; Then recover to water (every day is enough), take a picture after 5-7 days, see difference between strain; Continued growth was taken a picture after 10 days, saw difference between strain, recorded each strain plant above ground portion and to dry up required number of days, recovered strain number and required time that young leaves occurs after watering.Control water material is drawn materials when drought stress (watering sufficient moisture content rear blade to start to occur being designated as control water 0 day when wilting) 1,6,10,14 day survey physical signs, and take a picture when the 2nd day the morning 10 ~ 11, at the end of drought stress and after recovering enough watering after controlling water, see difference between strain.Along with the prolongation of control water time, the damaged membrane evil degree of different strain increases day by day, but transgenic line is for non-non-transgenic control, anti-lack of water ability is strong, some material membrane good stabilities, mda content is obviously lower, and difference reaches significance degree, reflects them and has the drought tolerance significantly improved.In addition, in drought stress process, genetically modified material chlorophyll content compared with non-non-transgenic control reduces comparatively slow, is still significantly higher than contrast, demonstrates and can keep good green at drought condition lower blade to chlorophyll content when controlling water 14 days.

4), the field of drought-enduring transgenosis rye grass is selected and is utilized

Rye grass strain excellent for drought resistance is transplanted and to bloom pollination in isolated area, gather in the crops seed.Filial generation sowing is carried out the observation of tree characteristics and is selected further in isolated area.By Analysis of Comprehensive Traits, from the Tai Chuilaite strain turning ZmsPLA2-1 gene, select the strain of 3 drought tolerance excellences, from the bay strain turning ZmsPLA2-1 gene, select the strain of 2 drought tolerance excellences.To be bloomed pollination in isolated area by continuous 2 generations again, obtain the drought resisting new variety (being) of neat and consistent.

Claims (6)

1. a corn phospholipase A_2 gene ZmsPLA2-1, is characterized in that: the nucleotide sequence of described gene cDNA is as shown in SEQ ID No.1; The aminoacid sequence of its coding is as shown in SEQ ID No.2.

2. corn phospholipase A_2 gene ZmsPLA2-1 described in claim 1 is cultivating the application in adverse-resistant characteristic change plant.

3. apply as claimed in claim 2, it is characterized in that: described resistance refers to drought tolerance or resistance to cold; Described plant refers to herbaceous crops and the herbage of cultivation.

4. apply as claimed in claim 3, it is characterized in that: described plant refers to corn or rye grass.

5. apply as claimed in claim 2, it is characterized in that: the method for described application is from corn, clone ZmsPLA2-1 gene, with justice or anti-sense versions by this gene recombination in plant expression vector, utilize transgenic technology that fusion gene is imported plant; From transfer-gen plant offspring, filter out the transgenic line that resistance significantly improves or reduces, obtain the new germ plasm in breeding with using value.

6. apply as claimed in claim 5, it is characterized in that: described ZmsPLA2-1 gene has cDNA form or genomic gene form, its encoding sequence is built into fusion gene with anti-sense versions or just form.