CN101643745B - Thellungiella V-pyrophosphatase gene (TsVPI) promoter sequence and application of deletion mutant thereof - Google Patents
Thellungiella V-pyrophosphatase gene (TsVPI) promoter sequence and application of deletion mutant thereof Download PDFInfo
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Abstract
The invention discloses a thellungiella V-pyrophosphatase gene (TsVPI) promoter sequence and the application of a deletion mutant thereof. A clone carrying a TsVPI promoter is screened from a genomic library of halophyte thellungiella, a 2200 nucleotide sequence positioned on the upstream of a TsVPI coding frame 5' is truncated as an overall-length promoter, promoter snippets with different lengths are obtained through PCR amplification, and then the promoter snippets are respectively blended with gus genes to be recombined into a plant expression vector to be converted into Arabidopsis; the TsVPI promoter and a part of the deletion mutant thereof are determined as salt-stress inducible type promoters by detecting the GUS enzymatic activity of transgenic plants, wherein a T5 promoter not only has a short sequence (667bp) but also has root specificity, and the TsVPI promoter and the T5 promoter are respectively connected with betA genes from colibacillus to be transmitted into tobaccos and corns so as to determine that the TsVPI promoter and the T5 promoter can normally exert functions in the transgenic tobaccos and the transgenic corns, are salt-stress inducible type strong promoters and have important application value in the industrialization development of plant gene engineering.
Description
Technical field
The invention belongs to the biotechnology breeding field of farm crop and forest, specifically, relate to clone, transformation and the application of a kind of plant gene promoter sequence (sequence of salt mustard coding vacuole skin pyrophosphatase gene TsVP1 promotor).
Background technology
Salt stress is one of main abiotic stress of occurring in nature, soil middle and high concentration Na
+G and D to many plants causes very big injury.Salting of soil and secondary salinization problem have become the restraining factors of world's Irrigation farming Sustainable development.Along with the scarcity of Increase of population and water resources, raising water resource utilization efficiency, abundant development and utilization saltings have become the important topic that involves the interests of the state and the people.In recent years, along with to Arabidopis thaliana, the further investigation of paddy rice isotype biology and developing rapidly of biotechnology, provide new approaches, novel method and novel material for cultivating height salt tolerable crop new variety, utilized molecular biology method and means to find that new genetic resources is the important step of cultivating the drought-enduring new variety of height salt tolerant.
Along with developing rapidly of Protocols in Molecular Biology and plant genetic engineering, utilize transgenic technology to change the proterties of some crops, thereby improve its to external world the tolerance of the stress conditions output that increases crop become a kind of important techniques.Foreign gene low expression level and non-specific expression in transgenic plant are important factors of restriction plant genetic engineering development, and major cause is to lack the ideal promotor.
Common promotor can be divided into constitutive promoter, organizing specific promotor and abduction delivering promotor.The genetic expression intensity that different promoters starts has very big difference, often has tangible species specificity.In some cases, one type promotor often has the characteristic of other types promotor concurrently.The organizing specific promotor is because the space specificity of its expression, can be with foreign gene localization and expression in transgenic plant, this not only can reduce the plant burden, alleviate the influence to the crop economical character, and can improve the concentration of exogenous genes products at privileged site, increase genetically modified effect.At present tissue-specific promoter is mainly used in following several respects: 1. improve freeze proof, drought resisting, the saline-alkaline tolerance of crop and prevent that fruit from rotting, 2. improve the color of flower characteristic, control ornamental flower, 3. improve disease resistance, insect-resistance and the antiweed ability of plant, 4. develop bio-reactor and be used for bio-pharmaceuticals, 5. create male sterile line and recover system, 6. (Wang Rui Pei, 2008) such as differentiation and development researchs that are used for plant.But the inducible promoter render transgenic is expressed under given conditions, the anxious open applications for the treatment of in the plant stress-resistance genetically engineered.The inducible promoter of having reported has stress-inducing promotor and chemical substance evoked promoter two big classes, last class comprises heat shock promoter, cold induced promoter, drought-induced promotor, damage evoked promoter etc., and back one class has hormone induction promotor, alcohol induced promotor, sterol evoked promoter etc.Although have the organ specific promoters of some amount and the promotor of abduction delivering at present; but in majority work; used promotor still is a constitutive promoter; as cauliflower mosaic virus 35SRNA gene promoter (CaMV35S), corn ubiquitin gene promotor (Ubi1); rice actin gene promoter (Act1) etc., its major cause be available inducible promoter and tissue-specific promoter be subjected to patent protection or the startup ability of promotor a little less than.
In plant genetic engineering, widely apply constitutive promoter, use the composing type strong promoter can improve expression of exogenous gene intensity, expression to goal gene has very strong promoter action to a certain extent, but some problems have also been brought thereupon, make gene product not only cause the waste of resource in the host as constitutive promoter, and often cause the change of some proterties of transgenic line at host's whole life and all histoorgan continuous expressions.Influence transgenic line and grow normally, make it hypoevolutism occur, phenomenons such as plant dwarfing.As constitutive expression GhDREB1 gene in cotton such as Shan,, compare obvious postponement its flowering period with adjoining tree though improved the resistance of plant.Nakashima etc. with the ubiquitin gene promotor constitutive expression OsNAC6 gene of corn, cause the plant strain growth phase to prolong in paddy rice, and output reduces.In addition, though some genes of constitutive expression can increase the resistance of crop, but the security to genetically modified crops also affects, can prevent disease and pest to a certain extent such as the constitutive expression anti insect gene, but, often cause certain difficulty for the release of transgenic line owing to also have this albumen in fruit or the seed.
In recent years, increasing scientific research person is willing to adopt inducible promoter or tissue-specific promoter to make up plant expression vector.Compare with composition type expression promoter, the transgenosis that they start only just has high strength to express under particular environment or in specific tissue, therefore can control the spatial and temporal expression of goal gene according to the experiment needs.Liu etc. utilize constitutive promoter CaMV35S and stress induced promoter RD29A to start downstream gene AtbZIP17 carrier construction and arabidopsis thaliana transformation respectively, the result shows that these two kinds of combinations all can improve the resistance of Arabidopis thaliana, but under the normal growth condition, this gene of constitutive expression causes the transgenic line hypoevolutism, but this gene of inducible expression does not then have this situation.Equally, people such as Kazuo cross expression OsNAC6 gene in paddy rice, utilize constitutive promoter to cause paddy growth to postpone output and reduce, but utilize the promotor of itself just can address this problem.Aryadee etc. utilize itself promoter expression gene Rab16A in tobacco, only under stress conditions this gene could be in leaf great expression, thereby improve its resistance, and transfer-gen plant its morphological development and output all are not affected compared with the control.
Though comparing with constitutive promoter, inducible promoter has lot of advantages, but present known inducible promoter seldom, application mainly still is relatively more classical promotors such as RD29A, and that China has a stress induced promoter of independent intellectual property right is few especially, and therefore the clone identifies that new stress induced promoter sequence and the central role element of finding out wherein have great importance.
The salt mustard is a kind of Arabidopis thaliana close relative species that grow in the saltings, also is the important model plant of research plant stress-resistance mechanism.The cDNA sequence similarity of salt mustard and Arabidopis thaliana is very high, but the salt mustard will be higher than Arabidopis thaliana far away for the resistance of abiotic stress, and the applicant has cloned the gene TsVP1 of coding vacuole skin Pyrophosphate phosphohydrolase in the salt mustard, and it has been carried out functional analysis.The homologous gene of this gene in Arabidopis thaliana is AVP1.Change these two genes over to yeast salt sensitive mutant respectively, find that they all can obviously improve the zymic salt resistance; In tobacco, cross respectively and express the salt tolerance that these two genes also can significantly improve tobacco, show that the function of these two genes has very high similarity.Detect the expression pattern of these two genes in salt mustard and Arabidopis thaliana respectively and find that under condition of salt stress, the TsVP1 expression of gene is subjected to tangible inducing action, and the expression of AVP1 does not have to change substantially.There is very big difference in this gene expression regulation mechanism that the two is described, clones the promoter sequence of TsVP1 gene and it is carried out functional analysis, and the keying action element of seeking wherein has great importance.
The higher plant promotor generally is made up of two portions: a part is to form ubiquity to transcribe structure needed, is commonly referred to the core promoter region, comprises transcripting start point and contiguous TATA frame; Another part is decision genetic transcription specificity and active zone, is made up of a plurality of conserved sequences, and these conserved sequences exist than big-difference at position, kind and the copy number of different promoters.Two portions all participate in the regulation and control of genetic transcription, but a back part plays main control action kou, seek keying action element in the promotor for the transformation of promotor with utilize significant.
Summary of the invention
At present present Research, the purpose of this invention is to provide a kind of plant gene promoter sequence (sequence of salt mustard coding vacuole skin pyrophosphatase gene TsVP1 promotor) and transformation and application.
The application method of salt mustard V-pyrophosphatase gene promotor of the present invention and deletion mutant thereof is from halophytes salt mustard clone's V-pyrophosphatase gene promoter sequence and carries out bioinformatic analysis.
General steps is to filter out the clone who carries the TsVP1 promotor from the genomic library of halophytes salt mustard, 2200 nucleotide sequences of intercepting TsVP1 opening code-reading frame 5 ' upstream are as the total length promoter sequence, obtain the promoter fragment T2～T7 of different lengths by pcr amplification as template, change Arabidopis thaliana over to after respectively itself and reporter gene being merged, determined that TsVP1 promotor and its part series deletion mutant are Salt Stress-induced type promotors, wherein the T5 promotor has stronger root-specific; TsVP1 promotor and T5 promotor change tobacco and corn over to linking to each other from colibacillary betA gene respectively, have determined that they can normally exercise the startup function in transgene tobacco and corn, are the strong promoters of Salt Stress-induced type.
Wherein, the described V-pyrophosphatase gene promotor full length sequence sequence (its sequence is shown in sequence table SEQ ID NO.3) that is V-pyrophosphatase gene (TsVP1) upstream 2200bp.Utilize PLANTCARE on-line analysis software analysis to find, there are a plurality of controlling elements in this sequence, comprise anaerobic induction response element ARE, drought-induced response element MBS, ABA response element ABRE, thermal induction response element HSE, Whitfield's ointment response element TCA element, jasmonic response element CGTCA motif, photoinduction response element SP1, ACE, AE-box, G-box etc. (seeing accompanying drawing 1).
Wherein, the structure of deletion mutant is according to the bioinformatic analysis result, utilize the interval region design upstream and downstream primer of these prediction elements, obtain the promoter fragment of different lengths by pcr amplification, produce complete deletion mutant (accompanying drawing 2), and they are connected the multiple clone site that enters among the plant expression vector pCAMBIA1391z respectively, and the downstream of multiple clone site is a gus reporter gene, the promoter fragment and the gus gene that promptly insert multiple clone site form fusion gene.
Wherein, the described promotor that is used for gene fusion construct can be the full length sequence of promotor, also can be partial sequence, also can be the deoxy polynucleotide according to the partial sequence synthetic.
Described application method is function and a utility value of determining promoter sequence by the expression that detects fusion gene in transfer-gen plant.
Wherein, described application method can also be by changeing transfer-gen plant and the offspring that the fusion gene acquisition improves or reduces stress resistance.
The salt thaliana promoter subsequence that the present invention cloned is used to make up the expression of plants structure, changes Agrobacterium GV3101 over to behind the insertion plant expression vector, by inflorescence infestation method arabidopsis thaliana transformation.The seed that transformed plant produces screens being added with on the antibiotic nutrient agar flat board, and the seedling of sprouting is transplanted in the nutrition soil, survives laggard performing PCR and detects, the screening transfer-gen plant.The continuous selfing of transfer-gen plant 2 generations generation is isozygotied and is.The latter is used for the transgene expression strength analysis.The promoter sequence of Arabidopis thaliana Avp1 gene with the clone links to each other with reporter gene simultaneously, the plant expression vector of gene fusion construct Avp1-gus is by difference of their promoter functions of variance analysis on two homologous gene expression patterns in the transfer-gen plant and utility value separately.
The expression pattern analysis of the said promotor control of the present invention, comprise staining analysis to TsVP1-T1 (the total length promotor connects gus gene) transfer-gen plant, determine the expressive site of this promotor promotor gene, comprise Arabidopis thaliana root, stem, leaf, flower and pod, find that the GUS expression intensity will be higher than other organs in root and leaf.Root dyeing result's histological observation shows, expresses mainly being distributed in the axis position at root GUS, and the expression amount in epiblem and the cortical tissue is low.And at the lateral root happening part, the GUS expression intensity will be apparently higher than other position of main root.
The said stress resistance of plant of the present invention is meant anti-(resist) drought, anti-(resisting) salt, resistance to cold, winter resistance, resistance to overhead flooding injury, thermotolerance that plant shows, resists characteristic and combinations thereof such as (anti-) radiativity, disease resistance, insect-resistance, antiweed on whole strain, organ and/or cell levels.This anti-(anti-) property can show some etap or the time of infertility on the plant level, can weigh or estimate by multiple index.
The said plant of the present invention mainly refers to higher plant, comprises all kinds of crops and economic plants, ornamental plant, ecological vegetable, weeds etc.; Comprise angiosperm, gymnosperm; Comprise draft and xylophyta etc.
The clone and the analysis of V-pyrophosphatase gene coding region 5 ' upstream region
Use available from the λ BlueSTARTM Xho I Half-site Arms KIT of Merck company and
KIT makes up salt mustard small segment genomic library.Extract genomic dna from salt mustard blade, MboI partly digests with restriction enzyme.To the part end-filling of part enzymolysis DNA, carry out electrophoresis at 0.6% low melting-point agarose gel then, downcut the gel piece that contains 10-23 Kb dna fragmentation with scalpel after electrophoresis is finished and reclaim DNA, be used for ligation behind the recovery product purification.Ligation is independently carried out in the centrifuge tube of 3 aseptic 0.5mL respectively.Every pipe lambda phage carrier 1 μ L (500ng/ μ L), genomic DNA fragment is respectively 2,4,6 μ L, and 1 μ L T4 dna ligase adds sterilized water to volume 10 μ L, connects 16-24h in 4 ℃.To connect then in the careful packaging extract that is added to 50 μ L thawing of product, (50mM Tris-Cl, pH 7.3,100mM NaCl, 10mM MgCl to add 500 μ L SM buffer
2, 0.01%Glutin), the vortex mixing adds 25 μ L chloroforms gently, be divided into behind the vortex mixing gently 120 inferior storehouses be kept at 4 ℃ standby.
Sequence information according to salt mustard TsVP1 gene, at its 5 ' end design PCR primer PTsVP-P1 (5 ' GTGGCGTCGGCGTTTCTTC 3 ') and PTsVP-P2 (5 ' CTTGGCGACGACACTCTGC 3 '), adopt PCR method sizing screening salt mustard genomic library.The inferior storehouse of PCR male is divided into secondary storehouse screens once more, carries out the screening of plaque blotting through after 3 grades of screenings plate being paved in the inferior storehouse of the positive.During the filter membrane trace, the about 0.5-0.8mm of plaque diameter, good dispersion does not link to each other mutually.Filter membrane should be put into flat board 4 ℃ of 1h at least before inhaling seal in advance, and then nylon membrane being covered length has on the flat board of an amount of plaque, positions with 3 asymmetric apertures.Film all soaks the back and continue to place 1min on flat board.Take out nylon membrane, trace faces up, and room temperature is placed 10min and dried.
Get the TsVP1cDNA that 1 μ g reclaims, adopt the DIG-High Prime KIT of Roche Holding Ag (Roche) to carry out probe mark, the method for mark is seen the test kit specification sheets.The DIG Nucleic AcidDetection KIT of Roche company is used in hybridization, colour developing, and operating process and method are seen the test kit specification sheets.
Method with in situ hybridization is separated positive plaque, during hybridization, nylon membrane is successively placed on by 5min on the saturated filter paper of sex change liquid (0.5MNaOH, 1.5 M NaCl), neutralizer (0.5M TrisCl, pH 7.4; 1.5 M NaCl) 5min on the saturated filter paper repeats once; Then film is washed 5min in 2 * SSC solution.After nylon membrane disposed, room temperature was placed 30min and is dried, and placed 80 ℃ of oven dry 2h then, wrapped with preservative film, deposit in 4 ℃ standby.The picking positive plaque is converted into λ BlueSTAR plasmid at random.After obtaining λ BlueSTAR plasmid, check order, obtain the sequence of gene coding region upstream 5452bp inserting fragment.
By salt mustard TsVP1 gene promoter sequence is carried out the Blast compare of analysis, its nearly 5 ' end, 850 nucleotide sequences and Arabidopis thaliana AT1G15670 have very high similarity (87%).Nucleotide sequence to the 4475bp of the 4587bp of salt mustard TsVP1 gene and upstream gene transcribed spacer thereof and Arabidopis thaliana AVP1 gene and upstream gene transcribed spacer thereof compares, and finds the similarity very low (＜10%) of the promoter sequence of the promoter sequence of salt mustard TsVP1 gene and Arabidopis thaliana H+-PPase gene A VP1.By the cis-acting elements in NSITE-PL (http://www.softberry.com) the programanalysis promoter sequence, salt mustard TsVP1 gene promoter sequence contains 18 different cis-regulating element, comprises 27 (can different regulatory factors interact with 25) cis-acting elements in the Arabidopis thaliana AVP1 promoter sequence.Wherein have 5 cis-acting elements (RSP00079, RSP00377, RSP00420, RSP00635 is that two promoter sequences are common RSP00653).And in salt mustard TsVP1 gene promoter sequence, found a plurality ofly to coerce relevant cis-acting elements (as the ABRE cis-acting elements: RSP00049).The number and the kind of these two cis-acting elements that gene promoter sequence comprised exist greatest differences, and be corresponding with their different expression patterns.
The essential characteristic of V-pyrophosphatase gene promotor
According to checking order and utilizing information biology tool analysis result, the nucleotide sequence (accompanying drawing 1) of 2200bp of getting nearly TsVP1 gene opening code-reading frame upstream is as the total length promotor, utilizing PLANTCARE on-line analysis software that it is carried out bioinformatic analysis finds, the controlling element that exists in this promoter sequence has: anaerobic induction response element ARE, drought-induced response element MBS, ABA response element ABRE, thermal induction response element HSE, Whitfield's ointment response element TCAelement, methyl jasmonate response element CGTCA motif, photoinduction response element SP1, ACE, AE-box, G-box etc. infer that the genetic expression of this promotor control is subjected to inducing of salt or drought stress etc.
The complete deletion mutant of V-pyrophosphatase gene promotor make up and with the reorganization of reporter gene
According to V-pyrophosphatase gene TsVP1 promoter sequence, interval region design upstream and downstream primer at the controlling element of predicting, adopt conventional molecule clone technology to obtain the promoter fragment of different lengths by pcr amplification, be connected respectively to plant expression vector, as pCam is the multiple clone site place of the reporter gene gus upstream of pCAMBIA1391z and pROK2, constructs complete deletion mutant.
With the recombinant plasmid transformed bacillus coli DH 5 alpha that builds, the screening transformed clone extracts recombinant plasmid and checks order, and selects the correct plasmid of reorganization to adopt freeze-thaw method to transform Agrobacterium GV3101 then.Clone's the promoter sequence of Arabidopis thaliana Avp1 gene is also linked to each other with reporter gene, make up the Avp1-gus expression vector, the Agrobacterium of adopting same program acquisition to carry the Avp1-gus expression vector then.With the Agrobacterium is that intermediary will change Arabidopis thaliana respectively over to by the gus gene that different promoters (fragment) starts, and detects the expression pattern of different promoters (fragment) promotor gene.Arabidopis thaliana transforms and adopts the flower dip method.
The pattern analysis of V-pyrophosphatase gene total length promoter expression
Observe by GUS dyeing, find that the GUS expressive site that this promotor starts comprises root, stem, leaf, flower and angle fruit the transgenic arabidopsis plant that changes T1 (TsVP1 total length promotor connects the gus gene structure) structure.Wherein the expression intensity in root and leaf will be higher than other organs.At root, the GUS expressive site that this promotor starts mainly is the middle shaft position of root, and the GUS expression amount is low in the root cortical tissue, and promptly dyeing is very shallow.In main root, lateral root happening part GUS expression amount will be apparently higher than other positions of main root.By GUS enzyme assay, confirmed that further the expression intensity of gus gene in plant root and leaf that this promotor starts will be higher than (accompanying drawing 3) of other organs to the Different Organs of changeing T1 structure plant.
The Arabidopis thaliana plant that changes the T1-gus structure is handled with 200mM NaCl, handling the measurement GUS enzymic activity of drawing materials respectively in 3,6,12,16 and 24 hours, the observation of dyeing simultaneously.The result draws by enzyme assay, and GUS is expressed under the condition of salt stress and is significantly improved, and reaches maximum value 16 hours activity, and this moment, the GUS activity of above-ground plant parts was 4 times of plant of being untreated approximately, is be untreated plant 6 times (accompanying drawings 4) approximately in the root.Dyeing is observed and is found that under condition of salt stress, in the root, especially the GUS of tip of a root place staining power increases substantially.Because be not subjected to salt stress to handle the blade of plant, the expression amount of GUS is very high, be difficult to find out the considerable change of salt stress processing back GUS staining power by dyeing.
The expression pattern analysis of V-pyrophosphatase gene promoter deletion mutant
With a series of deletion mutant respectively with the gus gene fusion, change Arabidopis thaliana over to.Transfer-gen plant is carried out staining analysis, find that the expression intensity of gus genetic expression in leaf that T2 fragment (1517bp) starts will be starkly lower than T1, and the gene that starts respectively in T2 fragment, T3 fragment (905bp), T4 fragment (788bp) and T5 fragment (667bp) difference is not obvious each other aspect expression specificity and the intensity.T6 fragment (537bp) is compared with the segmental startup activity of T5, and the GUS expression amount is obviously low in the root.The gene that T6 fragment and T7 fragment (328bp) start, not significantly difference aspect expression intensity and specificity.These results show, compare with complete TsVP1 promotor, exist in the sequence that the T2 fragment is lacked to promote the reporter gene controlling element that high strength is expressed in leaf; And compare with the T5 fragment, there is the element that promotes that reporter gene efficiently expresses in root in the sequence that the T6 fragment lacks.
Transfer-gen plant is carried out staining analysis, find that gus gene its expression intensity under condition of salt stress that T2, T3, T4, T5 start respectively is significantly increased the enhancement highly significant at tip of a root place.And analyze discovery to changeing the T6-gus gene plant, this salt inducing action disappears.
Relatively T3 fragment and T4 fragments sequence find that the part that the T3 fragment has more exists and the relevant functional element of ground organizing specific expression.The bioinformatic analysis that this section sequence is carried out is found, there are 10 light response elements, 1 ABA response element, 1 arid response element, 1 heat responsive element and 1 diel rhythm controlling element, also have and to grow relevant element with leaf portion palisade tissue 2,3 of the elements of unknown function are in addition arranged.
In the diversity sequence of T5 and T6, there be the element relevant with the root specifically expressing, there is the element of response salt stress, the latter can make reporter gene significantly raise in the expression at plant tip of a root position under the condition of salt stress.This section diversity sequence is carried out bioinformatic analysis, the functional element that find to have 4 predictions is respectively photoinduction response element G-box, methyl jasmonate response element CGCTA-box, with plant defense and coerce and respond relevant elements T C-richrepeats and protein binding site Box III.The promoter fragment of AVP1 in this section sequence (130bp) and the Arabidopis thaliana is carried out sequence alignment, find that these two sections promoter fragments have higher similarity, but exist sequence difference.
The genetic transformation of economic plants
Adopt agrobacterium-mediated transformation to obtain transgenic plant.Method for transformation commonly used has: infect method, the sprout tuber conversion method of growing thickly, aseptic seedling vegetative point conversion method etc. by inflorescence infestation method arabidopsis thaliana transformation, plant of Solanaceae leaf dish genetic transformation method, cereal crop callus conversion method and rataria.Usually adopt PCR method and molecular hybridization method to detect transformed plant, obtain positive transformed plant and carry out subsequent analysis.Below be that example is illustrated with tobacco leaf disc genetic transformation and corn inbred line aseptic seedling stem apex vegetative point conversion method.
A. the analysis of tobacco leaf disc genetic transformation and transfer-gen plant
Getting the tobacco aseptic seedling is material, utilizes Ye Panfa to transform.Agrobacterium strains is LBA4404; carrying the Mini-Ti plasmid or the plant stress-resistance genes involved betA that contain gus gene and plant screening mark gene neomycin phosphotransferase gene nptII (encodes from colibacillary choline dehydrogenase; catalysis is by the synthetic glycinebetaine of choline) and herbicide resistance gene, gus gene and anti contravariance related gene are started by TsVP1 promotor or its deletion mutant fragment respectively.Adopt conventional method for transformation to transform, the transgenosis budlet is screened on the inducing culture that contains selective agent kantlex (Kan) 150 μ g/ml, takes root on root media.Adopt PCR and Southern hybridization analysis method to determine transfer-gen plant.Detecting the positive plant that contains gus gene or salt-resistant related gene respectively by PCR method, is contrast with the empty carrier transformed plant that does not contain goal gene.Northern hybridization shows that transgenosis has very high expression level in tobacco, and hybridization signal does not appear in transfer-gen plant and the empty carrier transformed plant that do not contain goal gene.
The analysis of transgenic tobacco plant
In order to determine genetically modified expression specificity and expression intensity,, detect the GUS enzymic activity of root, blade and petiole for the tobacco seedling that changes the gus gene.Under the normal growth condition, the GUS enzymic activity of transgenic tobacco root difference between commentaries on classics TsVP1 promotor and the segmental plant of T5 is not remarkable, and the GUS enzymic activity of blade and petiole then is to change being significantly higher than of TsVP1 promotor plant to change T5 fragment plant.With 250mM NaCl solution soaking transgene tobacco seedling root system 24 hours, the GUS enzymic activity of transfer-gen plant root system, blade and petiole all raise significantly.Compare with the plant that changes the TsVP1 promotor, change the root system GUS enzymic activity no significant difference of T5 fragment plant, and the GUS enzymic activity rising amplitude of blade and petiole is relative less.Promptly TsVP1 promotor and T5 promotor all have the salt inducing properties in transgene tobacco, but the T5 promotor has stronger root-specific.
The blade that will change betA gene and adjoining tree (empty carrier transformation of tobacco and not transfer-gen plant) is cut into 0.5cm
2The leaf dish, respectively contain 1.0%, 1.5%, 2.0% and the substratum of 2.5%NaCl on cultivate, and measure its allometry amount.Containing on the substratum of 1.0%NaCl, various leaf dish growths are vigorous, produce callus differentiation and bud formation then, and allometry amount does not each other have marked difference.Be added with on the substratum of 1.5%NaCl, the tobacco leaf disc that changes TsVP1 promotor and T5 promotor is green, and the edge has callus to generate.Be added with on the substratum of 2.0%NaCl, the upgrowth situation obvious difference between various leaf dishes, from the about half death of the leaf dish of adjoining tree, all the other growths are relatively poor; The leaf dish that changes the T5 promotor is still survived, and growth better; The leaf dish allometry amount of changeing the TsVP1 promotor is apparently higher than coming rotation T5 promotor, far above the allometry amount of adjoining tree leaf dish.Promptly find by detecting the salt tolerance of changeing betA genetic tobacco leaf dish, changeing the tobacco of TsVP1 promotor compares with adjoining tree, its salt tolerance and drought tolerance obviously improve, and the tobacco that changes the T5 promotor is compared salt tolerance and drought tolerance increase rate not as changeing the T5 promotor with adjoining tree.Do not influence the physical signs of regeneration plant with the empty carrier transformation of tobacco that does not contain goal gene, so the change of transgene tobacco salt tolerance and drought tolerance is because the expression of transgenosis betA is facilitated, the less betA expression intensity in blade with its startup of tobacco leaf disc salt tolerance increase rate that changes the T5 promotor is low corresponding.
For understanding the drought-enduring sex difference of betA gene pairs transgenic tobacco plant that starts by different promoters, placing the MS inorganic salt solution that contains 10% (W/V) PEG 6000 to carry out osmotic stress the transgene tobacco seedling handles, detects the variation of blade relative water content.In the osmotic stress process, the tobacco leaf relative water content reduces gradually, commentaries on classics TsVP1 promotor plant relative water content changing down significantly is lower than adjoining tree, 94%-99% before 18h rear blade relative water content is handled from osmotic stress is reduced to 62%-66%, the blade relative water content that changes the T5 promotor is reduced to 64%-65% from 96%-98%, and the relative water content of transgenosis adjoining tree blade is not reduced to 54%-60% from 94%-98%.The expression intensity of betA gene in tobacco leaf that this explanation different promoters starts is variant, but the difference between the promotor does not reach significance degree.
B. the analysis of corn inbred line genetic transformation and transfer-gen plant
Bagging obtains key self-mating system of corn or cross-fertilize seed seed, with 70% alcohol immersion 10 minutes, with 0.1% mercury chloride immersion 10-12 minute, washs 3-5 time with sterilized water then again.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of sterilized water (30-40mL water/250mL triangular flask) in the bottle, is placed under the dark condition (23-30 ℃) 1-2 days after sealing.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the modified MS medium, under dark condition, continue to sprout.When treating that plumule is stretched to 3-5 centimetre, peel off coleoptile and 2-3 sheet spire, expose the stem apex growing tip.
To have binary vector (Mini-Ti plasmid or have TsVP1 promotor-betA and weedicide chlorsulfuron resistant gene als or T5 promotor-betA and als; the als gene is from the modification of going forward side by side of the Arabidopis thaliana plant of anti-chlorsulfuron; Li Guosheng etc.; 2000; " Science Bulletin "; agrobacterium tumefaciens 45:2181-2184) (AGL1 or LBA4404) 28 ℃ of concussions in additional antibiotic YEP substratum are cultivated, and make bacterium be in logarithmic phase.Under 3000r/min centrifugal 10 minutes then, abandon supernatant liquor.Thalline washs centrifugal collection with 1/2 improvement MS liquid nutrient medium.Thalline is suspended with the 1/2MS improvement liquid nutrient medium that adds the 100mg/l Syringylethanone, dilution 5-20 doubly is used for transforming again.At first bacterium liquid is poured in the culture dish during conversion, the inclination culture dish, the corn aseptic seedling top decompression that will expose the stem apex growing tip is immersed in 4-8 minute (AGL16 minute, LBA44048 minute) in the bacterium liquid.Bud point after will contaminating then blots with aseptic filter paper, root inserted in the modified MS medium in dark cultivated 2-3 days, culture temperature is 22-24 ℃, then aseptic seedling is placed under the scattered light and cultivated 2 days, aseptic seedling after again irradiation being cultivated is transplanted in the flowerpot that is covered with upper strata vermiculite and lower floor's loam, and vermiculite covers the plant top.Allow plant under natural lighting, grow then, warm 22-28 ℃ of day, at temperature 18-23 ℃ of night, water 1/2 modified MS medium inorganic salt every other day.
After transformed plant grows 3 leaves, spraying 20-30mg/l chlorsulfuron (consumption changes because of the self-mating system resistance, and Shenyang Agricultural Chemicals Factory produces, the effective constituent 25%) aqueous solution, is contrast with unconverted plant.The sprinkling amount is fallen drop with plant and is advisable.Adjoining tree stopped growing in sprinkling in back 3 days, and beginning is dead about 12 days.Plant after the conversion processing, the variation of some individualities is similar to adjoining tree, and other individualities then continue growth, no considerable change.The survival plant is long to 5 leaf phases, and the field is arrived in its field planting.The antiweed plant strain growth is got blade and is extracted DNA during to the 7-8 leaf, adopts round pcr to detect foreign gene, hands over solid to bagging selfing of PCR positive plant or sisters.
The planting seed that transformed plant produces is in the land for growing field crops or the greenhouse, plant is long gets blade extraction DNA to the 4-6 leaf during phase, adopt round pcr to detect and whether have foreign gene, the PCR positive plant is carried out Southern blotting detection and RT-PCR detection, therefrom select transfer-gen plant.Simultaneously transfer-gen plant is carried out the bagging selfing and reserve seed for planting, analyze the ratio of transgenosis individuality among the next generation, selecting that transgenosis isozygotys is to carry out the transgene expression analysis and the plant resistance detects.
The analysis of transgenic corn plant and utilization
Adopt to understanding genetically modified expression specificity and expression intensity, adopt the real-time quantitative RT-PCR method to detect betA expression of gene intensity in little shoot root of corn gene and the blade organ respectively.Because it is big from the different transgenic line betA expression of gene strength differences of same transferring structure, therefore, from offspring's strain system of 10 independent transformant, reject 2 higher and 2 measured values that expression amount strain on the low side is of expression amount, get the mean number of the measured value of other 6 strains systems and represent transgene expression intensity.Under the normal growth condition, the betA gene transcription abundance of transgenic corns root system difference between the strain of changeing TsVP1 promotor and T5 promotor is is not remarkable; And in blade, change being significantly higher than of TsVP1 promotor and change the T5 promotor.With 250mM NaCl solution soaking transgenic corns seedling root system 24 hours, the betA gene transcription abundance of transfer-gen plant root system and blade all raise significantly.Compare with the plant that changes the TsVP1 promotor, change the root system betA gene transcription abundance of T5 promotor plant and do not have considerable change, and blade betA gene transcription abundance rising amplitude is also less relatively.Promptly TsVP1 promotor and T5 promotor all show the Salt Stress-induced characteristic in transgenic corns, but the T5 promotor has stronger root-specific.
The planting seed that changes the betA gene is in the sand basin, and the pouring 0.8%NaCl aqueous solution is contrast with transgenosis selfing not.The not homophyletic of transfer-gen plant ties up to the salt solution pouring and shows different salt tolerances down, most strain systems that change the TsVP1 promotor show comparison according to the salt tolerance that significantly improves, the 5 leaf phase surviving rates that strains more than half are are more than 70%, and the contrast self-mating system emerge after very fast death, 5 leaf phase surviving rates are less than 20%, and plant seriously is injured.The strain system that changes the T5 promotor also shows the comparison illumination and shows the salt tolerance that improves, and salt tolerance is not obvious with the difference of changeing the TsVP1 promotor.The transfer-gen plant bagging selfing of selecting the salt tolerance excellence is isozygotied, and carries out combining ability test, and it is identical with the donor self-mating system or have the transgenosis self-mating system of raising to be used for the haloduric corn culturing cenospecies to pick out combining ability.
The corn seed and the not genetically modified contrast self-mating system seed that change the betA gene are broadcast in flowerpot, carry out drought stress in seedling stage (3-7 leaf phase), female tassel growth period (9-13 leaf phase), the pollination phase of blooming respectively and handle, detect arid and handle the difference of influence, damaged membrane degree, single plant yield and other economic characters and the physical signs of corn growth etc.Comprehensive many-sided test result, the corn of commentaries on classics TsVP1 promotor and T5 promotor is compared respectively according to plant and is shown the drought tolerance of obvious raising, and notable difference is not arranged because of promotor is different.The transfer-gen plant bagging selfing of selecting the drought tolerance excellence is isozygotied, and carries out combining ability test, and selection combining ability is identical with the donor self-mating system or have the transgenosis self-mating system of raising to be used for the seed selection of the corn seed of single cross.
Beneficial effect of the present invention
The invention discloses the functional element of salt mustard coding vacuole skin pyrophosphatase gene TsVP1 promoter sequence and its different sections, made up serial deletion mutant, by detecting the expression of gus gene in Arabidopis thaliana that starts respectively by them, tentatively determined their using value.And then in transgene tobacco and milpa, verify,, determined the normal functionating of TsVP1 promotor and T5 promotor energy in transgene tobacco and corn, be the strong promoter of Salt Stress-induced type.The value of this invention is:
1) provides Salt Stress-induced type strong promoter TsVP1 and T5 promotor for the plant stress-resistance breeding.In the plant transgene breeding for stress tolerance, lack the stress induced promotor of this class at present.
2) genetic expression that starts of T5 promotor intensity height not only, and expression intensity is different different because of organ, high strength is expressed in the root, and this is significant for starting the plant stress-resistance Expression of Related Genes.In addition, because sequence is short, is convenient to gene recombination and plant polygenic inheritance and transforms.
3) identify a series of transcriptional regulatory elements at the TsVP1 promoter region, and the analysis of structure and transfer-gen plant by complete deletion mutant, tentatively determined some elements in promotor gene is expressed about, for TsVP1 promotor and T5 promoter engineering lay the foundation.
Description of drawings
Fig. 1: TSVP1 promoter sequence and cis element analysis;
Fig. 2: different TsVP1 gene promoter fragments and reporter gene connection diagram
(+1 represents this gene transcription initiation site);
Fig. 3: the expression intensity of T1-gus gene in transgenic arabidopsis plant root and leaf will be higher than other organs;
Fig. 4: the variation in each tissue of the GUS enzymic activity Arabidopis thaliana that the TsVP1 promotor starts under 0.2M NaCl treatment condition.
Embodiment
Embodiment 1: change ZmPIS gene creation drought-enduring self-mating system of corn and application that the TsVP1 promotor starts
1) fusion gene of structure TsVP1 promotor and ZmPIS
Adopt conventional gene recombination method the TsVP1 promotor to be linked to each other the T-DNA district of the plant expression vector pCUA plasmid of after sequence verification, recombinating with ZmPIS.The pCUA plasmid carries plant selectable marker gene als for this laboratory makes up.Adopt freeze-thaw method to import among Agrobacterium LBA4404 or the AGL0 then.
2) produce to go up the key self-mating system of used corn with China be material in the foundation of receptor system, as Zheng 58, tuck in 478, tuck in 515 etc., take from and hand over seed sterilization, isolated culture to induce stem apex to produce the sprout tuber that grows thickly, and be that acceptor carries out genetic transformation with the sprout tuber that grows thickly.Used substratum has:
Seed germination substratum: KNO
31900mg/l, NH4NO
31650mg/l, CaCl
22H
2O 440mg/l, MgSO
47H
2O 370mg/l, KH
2PO
4H
2O 170mg/l, FeSO
47H
2O 27.8mg/l, ZnSO
47H
2O10mg/l, MnSO
44H
2O 22.3mg/l, H
3BO
310mg/l, KI 0.83mg/l, Na
2MoO
42H
2O0.5mg/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.Be used for seed germination.Liquid nutrient medium then removes agar powder.
The A substratum: the seed germination substratum adds 6-BA 4.5-9.0 μ mol/l and 2, and 4-D 1.0-3.0 μ mol/l is used to induce isolated culture bud point to produce grow thickly the bud tissue block and the bud tissue block succeeding transfer culture of growing thickly.
B substratum: additional 6-BA 4.5 μ mol/l of seed germination substratum and IBA (indolebutyric acid) 1.8 μ mol/l, the bud tissue block that is used to grow thickly differentiation seedling.
Become the seedling substratum: additional 6-BA 2.25 μ mol/l of seed germination substratum and IBA 3.6 μ mol/l, the budlet that is used to grow thickly develops into seedling.
Root media: the seed germination substratum adds IBA 2.8-3.6 μ mol/l, is used for the little seedling rooting of unrooted.
Minimum medium and plant growth regulating substance pressure sterilizing; Microbiotic and weedicide isoreactivity composition filtration sterilization add behind medium sterilization.
Seed sterilization and sprouting: corn seed with 0.1% mercury chloride immersion 10-15 minute, washs 3-5 time with sterilized water more then with 70% alcohol immersion 10 minutes.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of (30-40mL/250mL triangular flask) sterilized water in the bottle, is placed under the dark condition (23-30 ℃) 1--2 days after sealing.(showing money or valuables one carries unintentionally) back seed of sprouting is placed on the minimum medium and continues to sprout down in dark condition.
Stem tip culture and grow thickly that the bud tissue block is induced, subculture, differentiation: when the plumule elongation of germinating seed ends 3-5 centimetre, peel off coleoptile and spire, cut the epicotyl and the stem apex that are about 5 millimeter, be inoculated into (24-27 ℃) cultivation under dark on the A substratum, and in time cut the plumular axis of elongation and peel off spire.Cultivate after 6-10 days, stem apex begins the irregular growth of expanding, and occurs several wartys or digitation on the meristematic tissue that expands.After 20 days, begin to form indefinite bud and embryoid on the surface of warty or digitation.General per 4 all succeeding transfer culture once.In succeeding transfer culture, budlet is on the high side if the bud tissue block of growing thickly is grown thickly, and 2,4-D concentration is got 3.0 μ mol/l; Bud tissue block callusization is heavier if grow thickly, though a large amount of meristematic cell groups is arranged, indefinite bud seldom appears in the surface, can be with 2, and 4-D concentration is reduced to 1.0 μ mol/l, continues to cultivate then to produce a large amount of wartys or digitation again.The bud tissue block of growing thickly on the A substratum, minority has the generation of adventive root.Exist equally with spire, adventive root also influences the generation of expanding growth and embryoid and the budlet that grows thickly of tissue block, need remove early.The bud tissue block of growing thickly was transferred on the B substratum after 2-3 days, and color and luster is flavescence gradually, and quality is more pliable and tougher, and microspike appearred in the rear surface in 5-6 days.Observe visible each phase embryoid and indefinite bud under the scanning electron microscope.Embryoid and indefinite bud are grown rapidly, form the budlet that grows thickly on the tissue block surface.
3) with the bud tissue block of growing thickly be the conversion and the plant regeneration of acceptor
(every liter of substratum contains: tryptone 10g at additional antibiotic LB substratum will to have the agrobacterium tumefaciens (as AGL0 and LBA4404) of binary vector (the Mini-Ti plasmid has selective agent resistant gene and TsVP1 promotor-ZmPIS gene), yeast extract 5g, NaCl 10g, pH 7.0, pressure sterilizing) 28 ℃ of concussion cultivations down in, concussion speed be 110rpm (rev/min), make bacterium be in logarithmic phase.Under 3000rpm centrifugal 10 minutes then, abandon supernatant liquor.Thalline washs centrifugal again collection with the liquid seeds germination medium (be that the seed germination medium component reduces by half, remove agar powder) of 1/2 concentration.(acetosyringone, As) the liquid inducing clumping bud substratum of 1/2 concentration of 100 μ mol/l suspends, and dilutes 5-20 and doubly is used for transforming with adding Syringylethanone with thalline again.
Getting the bud tissue block of growing thickly of cultivating 13-20 days behind the subculture is transgene receptor.Transform with agrobacterium-mediated transformation, transform the back material and recover darkling to cultivate.Grow thickly budlet or the bud tissue block of growing thickly of agroinfection were cultivated 7-12 days bacteria growing inhibiting in dark on the substratum that is added with cephamycin (Cefotaxime) 250mg/l or Pyocianil (Carb) 500mg/l.Grow thickly budlet or the bud tissue block of growing thickly of recover cultivating after back or the antibacterial cultivation are being added with step sizing 3-4 generation on the substratum of selective agent, obtain transgenic cell and budlet.The exhausted big bud tissue block of growing thickly of counting is dead gradually in screening and culturing.Remove 2 with what the tissue block of survival was transferred to no selective agent, recover to cultivate generation budlet in back on the A substratum of 4-D.
Budlet is placed on into irradiation growth down on the seedling substratum, light intensity 2000-3000 lx, illumination 14-15 hour/day.Seedling length changes in the root media during to 3-4 sheet leaf takes root.Cultivated about 15 days, about 40% seedling produces new root.For the seedling of not taking root, its base portion of cut wound is transferred on the new root media and is cultivated, and most of plant produce root system after 10 days.Take root behind the seedling flush away substratum, being transplanted to the vermiculite is to grow in the flowerpot of cultivation medium.Plant grows under natural lighting, day warm 22-28 ℃, at temperature 15-21 ℃ of night, waters the inorganic salt composition of the seed germination substratum of 1/2 concentration every other day.Grow about 2 weeks, nursery transplant produces flourishing root system, is colonizated in the field then.
4) resistance of transfer-gen plant detects and selects and utilizes
The blade of getting the transplant survival plant carries out molecular Biological Detection and determines transfer-gen plant.With transfer-gen plant bagging self-fertility.Get planting seed in the sand basin, carried out continuous pouring 30 days with the 0.7%NaCl aqueous solution, the seedling of will surviving is transplanted to the land for growing field crops, treats the plant back bagging self-fertility of growing up.Its next generation is proceeded molecular biology identification and salt tolerance detect, and the bagging selfing isozygotys, obtained the salt tolerant self-mating system.
Embodiment 2: change the T5-ZmPIS gene and create the good drought resisting breeding material of wheat
1) fusion gene of structure T5 promotor and ZmPIS
Adopt conventional gene recombination method the T5 promotor to be linked to each other the T-DNA district of the plant expression vector pCUA-bar plasmid of after sequence verification, recombinating with ZmPIS.The pCUA-bar plasmid carries plant selectable marker gene bar for this laboratory makes up, and adopts freeze-thaw method to import among Agrobacterium LBA4404 or the AGL0 then.
2) the wheat aseptic seedling obtains
The seed of wheat improved seeds with 70% alcohol immersion 1-3 minute, soaked 8-12 minute with 0.1% mercury chloride again, then with sterilized water washing 3-5 time.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of sterilized water in the bottle in dark condition (20-25 ℃) 1-2 days down.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the modified MS medium and sprout down in dark condition.When treating that the plumule elongation ends 3-4 centimetre, peel off coleoptile and 2-3 sheet spire, emergent stem pointed tip growing tip.
3) Agrobacterium is cultivated and activation
To have agrobacterium tumefaciens LBA4404 28 ℃ of concussion cultivations down in additional antibiotic LB substratum of binary vector (the Mini-Ti plasmid has herbicide resistance gene bar and ZmPIS gene), concussion speed is 110r/min, makes bacterium be in logarithmic phase.Under 3000r/min centrifugal 10 minutes then, abandon supernatant liquor.Thalline washs with the 1/2MS liquid nutrient medium, centrifugal collection.Thalline is suspended with the 1/2MS liquid nutrient medium that adds the 100mg/l Syringylethanone, dilution 5-20 doubly is used for transforming again.
4) the wheat aseptic seedling transforms
(1) bacterium liquid is poured in the culture dish of 4.5 cm diameters, the inclination culture dish is immersed in the bacterium liquid, 0.5 * 10 the aseptic seedling of exposing the stem apex growing tip
5The Pa normal atmosphere was handled 3-6 minute down.
(2) the bud point after the dip-dye blots with aseptic filter paper, and germinating seed is placed on the modified MS medium of adding the 200mg/l glutamine and cultivated 2-3 days in dark, and culture temperature is 22-24 ℃.Then aseptic seedling is placed under the scattered light and cultivated 2 days.
(3) aseptic seedling after the irradiation cultivation is transplanted in the flowerpot that is covered with upper strata vermiculite lower floor loam, and covers the plant top with vermiculite.Allow plant under natural lighting, grow then, warm 22-28 ℃ of day, at temperature 15-21 ℃ of night, water 1/2 modified MS medium inorganic salt every other day.
5) transformed plant screening and field planting
After transformed plant grows 3 leaves, spraying herbicide
(Hoechst Schering AgrEvo GmbH contains weedicide glufosinate ammonium) aqueous solution, concentration is 9.6ml-10.8ml
, fall drop with plant and be advisable.Unconverted adjoining tree stops growing after back 5 days in sprinkling, and is dead about 15 days.Transformed plant is after sprinkling, and some individual variations are similar with adjoining tree, and other individual continuing grow, and change not obvious.When the plant of waiting to survive length arrives the 7-8 leaf, the field is arrived in its field planting, and promoted to tiller.
6) Molecular Detection of transformed plant
The antiweed plant is being stood up-jointing stage gets blade and extract DNA, adopts round pcr to detect transformed plant after vernalization treatment.The PCR positive plant carries out RT-PCR and detects, and in the antiweed plant that obtains, about about 15% is the transgenosis individuality.
6. transfer-gen plant progeny analysis
Plant by vernalization stage (the subzero treatment condition is different because of kind) was stood up under the long day, jointing, blossom and bear fruit.The seed that transfer-gen plant produces is put after immersion is sprouted in the refrigerator (0～2 ℃) and was carried out vernalization treatment 30-65 days.Seed 10.8ml after the part vernalization treatment
The aqueous solution is handled, and observes the individual ratio of statistics resistance and susceptibility; Planting seed after another part vernalization treatment carries out drought stress processing and high-salt stress respectively and handles at land for growing field crops and sand basin, screens drought-enduring and/or salt tolerant transfer-gen plant.Extract the leaf DNA of degeneration-resistant plant, adopt round pcr to detect foreign gene, and the segregation ratio of statistics foreign gene in progeny plant, obtain to be used for wheat breeding or directly to enter safety experiment and regional experiment after the pure lines.
The betA gene that embodiment 3, commentaries on classics T5 promotor start is created the good breeding for stress tolerance material of cotton
1) T5 promotor and betA gene Fusion gene and plasmid construction and Agrobacterium are cultivated
Adopt conventional gene recombination method the T5 promotor to be linked to each other the T-DNA district of the plant expression vector pCUA plasmid of after sequence verification, recombinating with the betA gene.The pCUA plasmid carries plant selectable marker gene als, adopts freeze-thaw method to import among Agrobacterium LBA4404 or the AGL0 then.Agrobacterium is cultivated and activates with example 2.
2) the cotton aseptic seedling obtains
Get the seed of cotton self-mating system or improved seeds, slough surperficial fine hair,, soaked 10-12 minute with 0.1% mercury chloride again, then with sterilized water washing 3-5 time with 70% alcohol immersion 1 minute with the vitriol oil.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of sterilized water (30-40 ml water/250 milliliter triangular flask) in the bottle, is placed under the dark condition (23-30 ℃) 1-2 days after sealing.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the modified MS medium that ammonium salt reduces by half and sprout down in dark condition.When treating that plumular axis is stretched to 3～5 centimetres, peel off a slice cotyledon, emergent stem end growing tip.
3) the cotton aseptic seedling transforms
(1) bacterium liquid is coated onto with on the stem apex growing tip of separating the plane pricking wound, and covers thereon with Agrobacterium wetted cotton ball, cultivated in the dark then 2-3 days, culture temperature is 24-26 ℃.Again aseptic seedling is placed under the scattered light and cultivated 2 days.
(2) aseptic seedling after the irradiation cultivation is transplanted in the flowerpot of upper strata vermiculite lower floor loam, allows plant under natural lighting, grow, warm 22-28 ℃ of day, at temperature 18-23 ℃ of night, water 1/2 modified MS medium inorganic salt every other day.
4) transformed plant screening and field planting
After transformed plant grows 3 leaves, spray 8mg/l chlorsulfuron (Shenyang Agricultural Chemicals Factory produces, the effective constituent 25%) aqueous solution, fall drop with plant and be advisable.Unconverted adjoining tree stopped growing in sprinkling in back 3 days, and beginning is dead about 12 days.Transformed plant is after sprinkling, and some individual variations are similar with adjoining tree, and other individualities have very strong resistance, continue growth.The survival plant is long during the phase, to arrive field with its field planting to 5 leaves.
5) molecular biology identification of antiweed plant
When antiweed plant length arrives the 7-8 leaf, get blade and extract DNA, adopt round pcr to detect foreign gene.The PCR positive plant carries out RT-PCR and detects.In the antiweed plant that obtains, the plant more than 10% is the transgenosis individuality.
6) molecular biology identification of transfer-gen plant filial generation
Selfing or sisters handed over solid after transfer-gen plant was bloomed.Planting seed is in the land for growing field crops or the greenhouse, get blade in the plant 4-6 leaf phase and extract DNA, adopt round pcr to detect progeny plant and whether have foreign gene, and the statistics foreign gene is at the filial generation segregation ratio, the result shows, in the transgenic line about 20%, the segregation ratio of foreign gene in progeny plant meets Mendelism.
7) resistance of transfer-gen plant is identified and is utilized
Get the seed of the transfer-gen plant that isozygotys, sowing is in flowerpot, and plant 2 leaves carry out subzero treatment during the phase, and by survival rate statistics and physiological index determining, filtering out cold-resistant strain is to be used for producing.Carry out in the sand basin, carried out continuous pouring 30 days with the 0.7%NaCl aqueous solution, the seedling of will surviving is transplanted to the land for growing field crops, treats the plant back bagging self-fertility of growing up.Its next generation is proceeded molecular biology identification and salt tolerance detect, and the bagging selfing isozygotys, obtained the salt tolerant self-mating system.This self-mating system can be used for preparing corn salt tolerant cross-fertilize seed.
The betA gene that embodiment 4, commentaries on classics T5 promotor start is created the drought-enduring breeding material of soybean
1) T5 promotor and betA gene Fusion gene and plasmid construction and Agrobacterium are cultivated
With example 3.But the Agrobacterium thalline of centrifugal collection suspends with the 1/2 improvement B5 liquid nutrient medium that adds the 100mg/l Syringylethanone, and dilution 5-10 doubly is used for transforming.
2) the soybean aseptic seedling obtains and genetic transformation
Get the seed of improved seeds,, soaked 10-12 minute, then with sterilized water washing 3-5 time with 0.1% mercury chloride with 70% alcohol immersion 2-3 minute.Constantly rock seed during the sterilization, thorough to guarantee surface sterilization.Sterilization back seed is placed in the aseptic triangular flask to be sprouted, and puts into a small amount of sterilized water (50-60mL water/250mL triangular flask) in the bottle, is placed under the dark condition (23-30 ℃) 2-3 days after sealing.Treat that seed sprouts after (showing money or valuables one carries unintentionally), place it on the improvement B5 medium and sprout down in dark condition.When treating that plumular axis grows to 3-5 centimetre, peel off a slice cotyledon and emergent stem end growing tip.
Step of converting is:
(1) bacterium liquid is poured in the culture dish of 4.5 cm diameters, the inclination culture dish is immersed in the bacterium liquid aseptic seedling top of exposing the stem apex growing tip, handles 3-6 minute under 0.5 * 105Pa normal atmosphere.
(2) the very useful aseptic filter paper of seedling after the dip-dye blots, and the seedling butt is inserted on the improvement B5 medium cultivated 3-4 days in dark, and culture temperature is 22-24 ℃.
(3) aseptic seedling is transplanted in the flowerpot that is covered with upper strata vermiculite lower floor loam, the top covers vermiculite, and natural lighting is growth down, warm 23-28 ℃ of day, at temperature 20-25 ℃ of night, waters 1/2 improvement B5 medium inorganic salt every other day.
3) transformed plant screening and field planting
Transformed plant is in long day (13-14 hour/day) growth down, after growing 3 leaves, (Shenyang Agricultural Chemicals Factory produces chlorsulfuron to spray 1.5-2.0mg/l (because of the genotype difference), effective constituent 25%) aqueous solution, falling drop with plant is advisable, unconverted adjoining tree stopped growing in sprinkling in back 4 days, and beginning is dead about 15 days.Transformed plant is after sprinkling, and the variation of some individualities is similar to adjoining tree, and other individual continuing grow, and change not obvious.The survival plant is long to 5 leaf phases, and the field is arrived in its field planting.
The antiweed plant strain growth is got blade and is extracted DNA during to the 7-8 leaf, adopts round pcr to detect foreign gene.The PCR positive plant carries out RT-PCR and detects.In the antiweed PCR positive plant that obtains, about 20% plant is the transgenosis individuality.
4) transfer-gen plant offspring's molecular biology identification and resistance are measured and are utilized
With the seed that transfer-gen plant produces, sow in the land for growing field crops or the greenhouse.Plant (T1 generation) 3-4 leaf is got blade and is extracted DNA during the phase, adopt round pcr to detect foreign gene, and carry out Southern blotting checking, adds up the segregation ratio of foreign gene in progeny plant simultaneously.Obtain transgenosis and isozygoty and carry out resistance after being and measure, screen degeneration-resistant strain system.
Sequence table
＜110〉Shandong University
＜120〉application of salt mustard V-pyrophosphatase gene promoter sequence and its deletion mutant
<141>2009-9-1
<160>3
<210>1
<211>19
<212>DNA
<213>PTsVP-P1
<400>1
gtggcgtcgg?cgtttcttc 19
<210>2
<211>19
<212>DNA
<213>PTsVP-P2
<400>2
cttggcgacg?acactctgc 19
<210>3
<211>2203
<212>DNA
＜213〉TSVP1 promoter sequence
<400>3
aagatttgct?acctcccatt?acgccaaaaa?taattttaaa?aaatcatcta?gatattatac 60
gttttctaga?taccaataaa?gtatctgaca?tgaataaaag?aataaataaa?ggaccacatg 120
ttcttaaaaa?tataactcac?atcaataaat?caaataattc?aacattttac?ctttaaacat 180
agaaatgaac?catccgtgac?ttttatcaaa?ataaatatat?ttctatgaag?aacaatagtt 240
cgtccatata?tttcaagttt?ctacgaatct?tattaagata?gattgtactg?gtcaccgttt 300
tatggcattt?tgtcaatata?taaaagatta?taaacataga?ggtatcacca?aattttggtg 360
aggctttgat?tgccttaatt?agaaaacaat?ataattacga?cagcgacgat?catctaatca 420
tgtcacggaa?agtcggaatt?gttaccgtgt?aaaatcatcc?gcttctttct?taattcaatt 480
attgttactt?taattagttg?gattggtttg?aattctacgt?ttacacctct?gcatttggat 540
aacctatgcc?atatagtatt?gattatataa?ctgataacta?agggaactaa?aatactaaat 600
cttgactgaa?agataataag?agaagtctct?agtccctaat?gtcagccaca?tacctaggaa 660
agatttttta?gttttcatta?cgtggtaaat?tttccagatt?gattagcaac?ctgcaaaaaa 720
gggaacaaga?gggaggaaga?taaagcatga?gtggtcacta?ctcactcact?gatactacaa 780
gaaattaatt?aatgctaaca?aaaatccaca?atataaattt?ttaattaaga?tattttttca 840
aaagagtatt?tatccgattg?gagggttgca?caccattcaa?tacacattaa?tacgtcacag 900
ccgctcgaaa?aggcctccgc?tatatatcca?tctttttgct?aaagaatttt?ttttttcgaa 960
gaatcatcaa?tttactttat?tcttggtctg?aggaaatatc?tatatacgtg?aagtgtgcac 1020
cttatcttgt?acagggcttc?cttcggaaaa?aaaaaaaatc?ttgtacaggg?cttccctcgt 1080
atgaagaaga?gataaactcg?taaaaaggtt?ttggaatcca?acttaatgga?acaaatgcca 1140
aaataattta?gtacaacgtc?gaatcaagtt?gcgaaagtac?tgtagaatcg?attcctatac 1200
agagacgaaa?cgagttctta?ttgcacttta?taatattgat?taaaaactga?aacttttttt 1260
gtttctccga?atattgttat?ctttatttca?cagctagata?tcaatccgca?catgcatata 1320
attagtttat?cttttcacaa?aaaagtatat?ccccaaataa?aatcccacgt?aaatgaaaac 1380
acagttttaa?tttttttttc?atagtgttta?cgataataca?catggtttgt?tgtaattaaa 1440
aagatagata?cggttttttt?tcaactgaaa?atttgacttt?gtatttatcc?taatgatatc 1500
aaatgattta?accatatcca?tatctgatag?tatgcacaca?cattgaatcc?ctcattgaaa 1560
attatgacga?gctgagattc?aatgatatta?gttagtccat?atacactgga?tttggtaaga 1620
ataccattac?tgtataaaca?ctcgaatata?ccatggataa?gcataagatt?cacctaatta 1680
attaatttcc?accagtgaga?aaaaaaggaa?aactaattaa?tttctacacg?tagacaattg 1740
actacgaatc?aaaaagagcg?ctaatcgtgt?acctgacagt?cggacggaca?caagcgcatg 1800
gatgcacacg?tagttggtgt?ctcttaccct?aaaatcgacg?gtgaagatgg?acacgagcgt 1860
tacttgtccc?tcgtgtctga?taactatcgg?tagacgaaac?gagattatac?cttccgacaa 1920
tttggagcgt?cggctgcacg?cgctttctaa?tcaaaaaaaa?gaaaaaaaaa?gaaacgaggg 1980
gtgtagcgta?aagaacggtg?taactaaccg?taccagattc?caactttcgt?tgtggatgtg 2040
agcatccaag?aggaggagag?tcagtgttat?aaaacgacac?gatatcctca?ccgagtttac 2100
gccttcattt?catcatctcg?tcgaaaacac?ttcccttcct?ttctctctac?tctctctctc 2160
tcgttatctt?cggtttctgc?tttctctatt?cggaggagag?atg 2203
Claims (6)
1. salt mustard V-pyrophosphatase gene promotor or its deletion mutant application in the preparation transgenic plant, wherein, the sequence of described V-pyrophosphatase gene promotor is shown in SEQ ID NO.3, and described deletion mutant is-667 to+1 of sequence of V-pyrophosphatase gene promotor.
2. application as claimed in claim 1, wherein, the clone obtains V-pyrophosphatase gene promotor from the genome of salt mustard, with this promotor is that template amplification obtains its deletion mutant, V-pyrophosphatase gene promotor or its deletion mutant are connected with adversity gene as promotor, thereby gene fusion construct, and change the fusion gene that makes up over to plant, detect the expression of this fusion gene in transgenic plant.
3. application as claimed in claim 1, wherein said V-pyrophosphatase gene promotor or described deletion mutant are the promotors of Salt Stress-induced type.
4. application as claimed in claim 3, wherein said deletion mutant has root-specific.
5. application as claimed in claim 1, the wherein transfer-gen plant that stress resistance is improved or reduces by the fusion gene acquisition that changes over to.
6. application as claimed in claim 5, described transgenic plant are transgene tobacco, transgenic corns, transgenic wheat, transgene cotton or genetically engineered soybean.
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| CN102002485A (en) * | 2010-10-26 | 2011-04-06 | 复旦大学 | Pyrophosphatase of ammopiptanthus mongolicus, and coding gene and application thereof |
| CN102757967B (en) * | 2011-06-10 | 2013-12-18 | 中国科学院遗传与发育生物学研究所 | Promoter and application thereof |
| CN102250900B (en) * | 2011-06-29 | 2013-01-30 | 北京师范大学 | Thellungiella salsuginea COR15a gene promoter and application thereof |
| CN102321662A (en) * | 2011-07-14 | 2012-01-18 | 中国科学院植物研究所 | Method for transforming stem tips of plants and special tool thereof |
| CN103484495A (en) * | 2012-12-05 | 2014-01-01 | 潍坊学院 | Application of thellungiella halophila DREB2A gene to culture high-salinity-tolerant drought-tolerant corn |
| CN103981187B (en) * | 2014-06-10 | 2016-02-10 | 山东大学 | The deletion mutant of corn phosphatidylinositols synthase gene promoter P-ZmPIS and application thereof |
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| CN1399512A (en) * | 1999-11-10 | 2003-02-26 | 康涅狄格州大学 | Stress-resistant oversized transgenic plant capable of growing in salinized soil |
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| Teruaki Taji et al."Large-scale collection and annotation of full-length enriched cDNAs from a model halophyte, Thellungiella halophila".《BMC Plant Biology》.2008,(第8期),全文. * |
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