CN1137995C - Method for creating plant gene label system - Google Patents

Abstract

The present invention provides a method for constructing a label system of plant genes, which comprises: a DNA fragment is constructed to cause the DNA fragment to comprise a transposase encoding gene controlled by an inducible system or a promoter for the specific expression of calluses, a nonautonomy insertion element depending on transposase and a marker gene for plant sieving; plant cells are converted by the constructed DNA fragment or a DNA granule containing the fragment; converted plant cells are sieved; the expression of the transposase is induced to cause the converted plant cells to regenerate complete plants. The method largely simplifies a large amount of field purification and a hybridization process which are required by a conventional method and shortens required time.

Description

A kind of method of setting up plant gene label system

Technical field

The present invention relates to a kind of method of setting up plant gene label system.

Background technology

Genetics is the science that natural population or mutagenesis populational variation rule are studied.By thereby the genetic analysis of variation law in the mapping population is confirmed to the gene (gene) that causes variation and at the relevant position on karyomit(e).This variation can be isolating, as most of point mutation; Also can be successive, as some complex characters (some important economical characters particularly, as: output and plant height), these proterties be mostly by controlled by multiple genes.

In recent years, the Plant Genome Research progress is rapid, and arabidopsis gene group sequence is all determined in the end of the year 2000, and the genome sequence of paddy rice is measured and also soon finished this year.Along with the rapid propelling of Plant Genome order-checking plan, accumulated the DNA of plants sequence of a large amount of Unknown Function among the Genebank, how to illustrate emphasis and focus that function that its biological significance promptly identifies the genome full gene has become Plant Genome Research.The mutant approach is the means of using the most widely of at present gene in the Plant Genome being carried out functional analysis, and has obtained very ten-strike in plant genetics and molecular biology fundamental research.Classical chemistry (EMS) or physics (plasma, gamma-rays) mutagenesis allow us to obtain whole genome gene saturation mutation with high frequency under relatively easy situation, be that each gene all takes place once to suddenly change, with the map based cloning program mutator gene cloned then.Owing to highly dense gene mapping and the yeast artificial chromosome (YAC) of a series of overlapping genes group DNA or the existence of bacterial artificial chromosome (BAC), this program can relatively comparatively fast be carried out in model plant such as Arabidopis thaliana.And along with finishing of genome sequence, the map based cloning strategy also will quicken greatly.But concerning the low most of plants of molecule marker saturation ratio, map based cloning will be a very very long process undoubtedly.

Can insert the DNA element of plant chromosome such as transposon or agrobatcerium T-DNA at random and also can be used as the generation of mutagenic compound inducing plant afunction mutant.Along with being decrypted gradually of Plant Genome sequence, methods such as T-DNA and transposon insertion sudden change are used more and more widely in gene functional research.Because the sequence of insertion element is known, can obtain to insert segmental flank (Souer et al., 1995 in a short period of time by Inverse PCR or Tail PCR method; Liu et al., 1995), and check order, therefore the gene order that is inserted into can clone out easily by the method for PCR again, and can set up T-DNA or Ds flanking sequence database.Therefore, the utilization of insertion sudden change method provides a kind of method of rapidly effective clonal mutation gene for us.

The agrobatcerium T-DNA label technique promptly is inserted into T-DNA in the Plant Genome at random by the Agrobacterium-mediated Transformation technology.The improvement of agriculture bacillus mediated plant transgenic technology and perfect makes the function of utilizing the T-DNA label that the full genome of plant is suddenlyd change and then studying all genes of genome become possibility.T-DNA is in case insertion is just almost no longer mobile, and therefore, institute's regenerated mutant quite stable is more easily preserved.But the T-DNA label is only applicable to use the plant of agrobacterium mediation converted.And, be based upon the saturated mutant library that each gene in the genome contains a sudden change label at least, the conversion mutant quantity that need obtain is very big.

Nineteen fifty-one Mclintock finds to exist one section movably dna sequence dna in the corn gene group, it can be by a series of processes such as cutting, reintegrate from a genomic position " jump " to another position, called after transposable element or transposon (transposon) afterwards.Afterwards, except that corn, people were Common Snapdragon, and petunia also finds to have the transposon existence in the plants such as paddy rice.

Transposon can be divided into two big classes substantially: with the transposon and the retrotransposon (retrotransposon) of DNA-DNA mode swivel base.First kind transposon can be by dna replication dna or is directly excised the dual mode acquisition and can move fragment, inserts in the genomic dna again.Autonomy according to swivel base, this class component can be divided into autonomous transposable element and non-autonomous transposable element again, the former itself can encode transposase and carry out swivel base, the latter then needs when autonomous element exists can swivel base, as in the Ac/Ds of corn system, Ac (Activator) belongs to autonomous element, and Ds (Dissociation) then is non-autonomous element, must be in the presence of the Ac element could swivel base (Girel and Saedler, 1992).The second class transposon is called again and returns first (retroposon) (Hirochika, 1997), it is newfound in recent years transposable element by RNA mediation swivel base, structure and duplicate with retrovirus (retrovirus) similar, just there is not the necessary env gene of virus infection, it is by transcribing synthetic mRNA, the synthetic new element of reverse transcription is incorporated into and finishes swivel base in the genome again, 1 copy number of revolution seat will increase by 1 part, thus it be at present known to the movable genetic constitution of a class of quantity maximum in the higher plant.3 types of retrotransposons have been found at present altogether: the Tyl-copia class, Ty3-gypsy class and LINE (long interspersed nuclear elements) class transposon, preceding two classes are to have long terminal repetition transposon, and LINE class transposon is not long terminal repetition.Retrotransposon in the higher plant mainly belongs to the Tyl-copia class, and it is very extensive to distribute, and has almost covered all higher plant kinds (Hirochika et al., 1996).About 156 kinds of the plant transposon that lands in gene pool at present, the most frequently used is the Ac/Ds system that derives from corn.

Some phenomenons that the last molecularity of biological heredity evolution causes have not only been explained in the discovery of transposon, also provide strong tool for genetically engineered and molecular biology research, make the people can be under the situation of the biochemical property of not understanding gene product and expression pattern, separating clone plant gene, i.e. transposon tagging (transposon tagging).Its principle is to utilize the insertion of transposon to cause transgenation, based on the transposon sequence, from the gene library of mutant strain, filter out the clone who has this transposon, it must contain the partial sequence of the mutator gene adjacent with the transposon sequence, utilize this part sequence from the wild type gene library, to obtain complete gene (Girel and Saedler, 1992) again.

1984, at first in corn, separated the bronze gene with transposon tagging, this genes encoding the key enzyme of popcorn pigment route of synthesis---the yellow 3-O-glucanotransferase (Fedoroff et al., 1984) of UDP-grape carbohydrate.People such as Baker have proved that at first the Ac/Ds transposable element of corn has effect in transgene tobacco, after this find again that Ac/Ds has activity (Baker et al., 1986 in as Arabidopis thaliana, tomato, petunia, flax, potato, soya bean and paddy rice in other many species; Hirochika et al., 1996; Hirochika, 1997).From petunia, successfully cloned an anthocyanin synthetic gene with the Ac element in 1993, and started the beginning (Chuck et al., 1993) with external source transposon clone gene in heterologous host.After this utilize the transposon tagging technical point from many plant genes.

The transposable element system that plant genetic engineering is commonly used is divided into natural and artificial reconstructed two big classes at present, and the former comprises autonomous element single-factor system and retrotransposition element system, and the latter mainly is engineered double factor system.

Autonomous transposable element single-factor system has been utilized the dTpH1 transposon of Mu transposon, Ac transposon and the petunia of higher autonomous transposon of transposition activity such as corn, cloned Arabidopis thaliana albinism gene (albino), male fertile gene, tomato genes such as disease-resistant gene Cf-9 (Knappet al., 1994a).This swivel base system has two big advantages: the one, in plant, insert the copy number height, and can reach more than 100 as the average copy number of each genome of Mu element, therefore can under the natural culture condition of land for growing field crops, obtain the mass mutation individuality; The 2nd, only need just all genes of energy mark of the more a spot of relatively plant of screening.Yet also there are some problems in this system: the autonomous high-frequency swivel base of transposable element might excise transposase and stay some sequences and cause permanent sudden change; Autonomous swivel base may cause gene function to recover automatically in somatocyte; Autonomous element excision stay some fragments make transposable element can not with mutant phenotype be divided into from, the difficulty that these have all increased screening and cloning has hindered the popularization (Lucas et al., 1995) of transposon tagging.

Though retrotransposon is done as a whole, a lot of or even the maximum element of the first species of copy number in the whole plants genome, but it has comprised many subgroups, by subgroup only form by one or several copy, these are easier to identification with the composition ratio that single copy or low copy mode exist, the swivel base activity that experimental results show that retrotransposon simultaneously can be activated in tissue culture, so they are the very potential transposon tagging systems of a class.People such as Hirchick in 1996 just utilize paddy rice retrotransposon Tos17 to set up paddy gene and knock out system (gene knock-out system), Tos17 can be activated in tissue culture procedures, insert in the rice genome, make gene knock-out (Hirochika, 1997).Sato in 1999 etc. utilize this system to separate 6 paddy rice kn1-type homeobox gene, have found the mutator gene OSH15 (Sato et al., 1999) that causes that rice plant is downgraded.

Recently Lucas etc. imports Arabidopis thaliana (Lucas et al., 1995) with the activated Ty1-copia class retrotransposon in the tobacco, finds that it can carry out swivel base in the latter, thereby new copy is inserted in the open reading-frame (ORF) of other gene.In succession its is imported in tomato and the paddy rice again afterwards, in new host, express, and host's endogenous retrotransposon do not influence the swivel base of new importing transposon, illustrate that retrotransposon is not subjected to the influence of floristics difference.Retrotransposon in the dicotyledons not only can be in the allos dicotyledons swivel base, also can in monocotyledons, express, this provides more wide prospect for retrotransposon is used for transposon tagging.

Double factor transposon system is the transposon tagging system once artificial reconstructed mistake.It is made up of the autonomous transposable element that a non-autonomous transposable element and transformation later self can not swivel bases, and latter's transposase of encoding causes the former swivel base.Make up the plant expression vector (Fig. 1) that contains two elements respectively, transform the strain system that plant cultivation contains nonautonomy transposon and transposase respectively respectively, offspring's purifying is again by transfer-gen plant hybridization, at the mutant of F2 generation with regard to obtaining to be caused by transposon in a large number.By allos transposon (mainly being corn transposon such as Ac/Ds, En/Spm, or Mu), people are at several plant (Knappet al., 1994b such as Arabidopis thaliana, petunia, Common Snapdragon, tomato, corns; Lucas et al., 1995; Sato et al., 1999; Izawa et al., 1997) in, obtain various mutant plant colony, Shimamoto etc. utilize the binary vector system, cultivate the rice strain that contains the Ds transposable element and contain Ac transposable element transposase (AcTPase) gene respectively, obtained the mutant (Izawa et al., 1997) of a large amount of dwarfings, florescence change by screening by hybridization.

Though the transposon tagging system of the double factor transposon Ac/Ds of system only needs a hundreds of initial independent Ds transformant, by obtaining new mutant body colony with the transfer-gen plant hybridization of expressing the Ac transposase.Significantly reduced the transformation amount.But the required purifying in later stage field, hybridization workload are very big.And transgenic plant must just can obtain pure lines through the several generations screening in the field.Filial generation need be expanded the numerous Ac of making transposase through several generations again and separate with the Ds insertion element, and mutant is stablized, and workload is very big.

Though the T-DNA label technique can be obtained unit point synergy preferably, and has advantages such as inserting stable, easy maintenance, the quite big conversion of its needs colony.Set up saturated mutant library, promptly genomic each gene has at least one to insert label, needs 12.5 ten thousand independent transfer-gen plants approximately even if the plant Arabidopis thaliana of genome minimum will obtain the insertion of 95% saturation ratio, and paddy rice is then up to more than 450,000.Transformation in early stage amount is very big.Therefore, traditional method all is difficult to carry out in the foundation in most of plant mutants storehouse.

Summary of the invention

The purpose of this invention is to provide a kind of method of setting up plant gene label system.

But we utilize the transposase gene of inducible system control Ac/Ds transposon system, and change plant over to Ds.Do not inducing under the situation, the Ac transposase gene is in silence state, and tool does not activate Ds swivel base ability.The Ac transposase of inducing by moment is expressed, and obtains mutant in the Plant Genome thereby activate the Ds swivel base and insert at random.Owing to the swivel base of Ds is to induce the Ac transposase to take place with inductor, does not need to realize, thereby save heavy field hybridization work by hybridizing with the plant that contains the Ac transposase.And any one insertion mutant all can be used as a sudden change source and produces new mutant.Like this, just can induce number of times by controlling moment, obtain plant saturation mutation colony at short notice from limited parent material.On the other hand, do not inducing under the situation because the existence of no Ac transposase, mutant can keep stable and heredity, thereby save in the traditional method and repeatedly expanded numerous transposase and Ds insertion element of separating by the field, so that insert stable step and the program of mutant, saved the plenty of time, human and material resources and financial resources.

Because by superinduce, insertion element Ds is swivel base again, so can utilize limited parent material to make the insertion state that reaches capacity fast by inductive method repeatedly.In Arabidopis thaliana, only need 100-300 strain left and right sides parent material.In paddy rice, also only need the initial transgenic line about 500-1000.This be 5,000 to 10,000 of traditional method workload/.Shorten the needed time of acquisition saturated mutant colony greatly, saved financial resources, material resources.Particularly importantly the foundation of this method makes those be difficult to transform or the foundation in the incomplete plant mutant of conversion system storehouse becomes possibility.

The invention provides a kind of method of setting up plant gene label system, may further comprise the steps:

(a) make up a kind of first dna fragmentation, make it comprise a kind of gene and a kind of plant screening mark gene of the coding transposase by the control of inducible system or callus specific expressing promoter, with a kind of second dna fragmentation, make it comprise a kind of nonautonomy insertion element and a kind of plant screening mark gene that depends on transposase; Perhaps, make up a kind of the 3rd dna fragmentation, make it comprise a kind of gene, a kind of nonautonomy insertion element and a kind of plant screening mark gene that depends on transposase of the coding transposase by the control of inducible system or callus specific expressing promoter;

(b) with the first constructed dna fragmentation and second dna fragmentation or contain the plasmid of these dna fragmentations, perhaps with the 3rd constructed dna fragmentation or contain the plasmid transformed plant cells of this dna fragmentation;

(c) filter out by the plant transformed cell;

(d) induce transposase to express;

(e) make by plant transformed cell regeneration and go out whole plant.

In the method for the invention, described dna fragmentation refers to contain the dna molecular by the transposase of inducible system (or callus specific expressing promoter) control; It can be the dna fragmentation of synthetic, also binary vector plasmid such as the pBin 19 that is used for agrobacterium tumefaciens or Agrobacterium rhizogenes conversion plant, pCambia, pBI 101 grades and other plasmids such as the pUC that can breed can be included in prokaryotic organism, pBluescript is in the PCR vector plasmid.These plasmids and use the construction process of these plasmids to be undertaken by method well known in the prior art.

Described transposase can be to derive from different biological (comprising plant or non-plant), and the transposon system that has autonomous or nonautonomy swivel base ability in plant is as the Ac/Ds in corn source, En/Spm, Mu, the Tam in the Common Snapdragon, dTph1 in the petunia, the Tos17 in the paddy rice etc.

Described transposase gene is coding autonomy transposase and nonautonomy transposase gene, as the Ac transposase gene in corn source or the transposase gene in other plant source.

The described nonautonomy swivel base insertion element that depends on transposase is as Ds element etc.Can be between its inverted terminal repeat sequence of nonautonomy swivel base insertion element (TIR) according to the purpose of setting up mutant library

(1) do not contain other dna fragmentations; Or

(2) contain selectable marker gene; Or

(3) comprise the no promotor or core promoter (minimal the promoter)-reporter gene fragment of only having, to be used for trapping of enhanser and promotor or gene trap; Or

(4) contain promotor and be used for gene activation etc.

Described plant screening mark gene can be the antibiotic-screening marker gene, as anti-kantlex selection markers gene neomycin phosphotransferase (NPTII), and moisture resistance mycin selection markers gene hygromix phosphotransferase (hpt); Or (Haldrup etal., 1998) such as non-antibiotic selection markers gene such as xylose isomerases.

But the inducible system of described control transposase can be alcohol inducible system (alc), but sugar sterol inducible system, but tsiklomitsin inducible system (tet), but lactose inducible system (lac), but cupric ion inducible system, and plant endogenous inducible system such as heat shock (heat shock), weedicide (safener), damage inducible system etc. and callus specific expressing promoter.

In the method for the present invention, described vegetable cell refers to derive from cell, tissue or the plant as all plants such as Arabidopis thaliana, tobacco, potato, paddy rice, wheat, corn, rape, cotton, soybean.The conversion of cell or plant refers to by protoplastis-chemical mediated method (Ca ²⁺, PEG), the particle gun mediated method, agrobacterium-mediated transformation, electrization, pollen tube imports, the combination of any method such as microinjection or several method.

In the method for the present invention, described transformant (or plant callus, plant) screening refers to utilize microbiotic or other selection markers genes, as contains the cell of neomycin phosphotransferase (NPTII) gene or callus can be screened for thing such as G 418 etc. for deriving by kantlex or its; The cell or the callus that contain hygromycin phosphotransferase gene can be screened by Totomycin etc.Obtaining can to adopt Southern or PCR method behind the resistant calli cell, dot blot equimolecular detection means detects it, contains the Ac transposase gene to determine it.

In the method for the present invention, the abduction delivering of transposase can be by physical method such as heat-inducible, damage is induced or at the chemical induction of different inducible systems, as alcohol inducible system alcohol, tsiklomitsin inducible system tsiklomitsin, the dexamethasone that sugar sterol inducible system is used, the lac inducible system also can be realized by its corresponding surrogate or derivative with realizations such as glucose.Abduction delivering can be in any period such as the callus stage of plant, and in the vegetation growth of plant stage, generative growth phase, seed take place and seed germination stage etc., but is best with the callus stage.But transposase also can be controlled it by the callus specific expressing promoter and express in order to replace inducible system at the plant callus phasic specificity.Screen the transfer-gen plant that when inducing, has Ac transposase high expression level and when not inducing, do not express with Northern or Western.

In the method for the present invention, make to be gone out whole plant by plant transformed cell regeneration and mainly refer to also comprise non-other stripped method regenerated plant that nourish and generate by exsomatizing by the transformant regenerated plant that includes above-mentioned DNA element.

Brief Description Of Drawings

Fig. 1 contains the double factor swivel base system plasmid figure of Ds element and Ac transposase.

A. the encode transposable element of transposase.The transposase of Ac element is controlled by cauliflower mosaic virus (CaMV) 35S promoter.Contain antibiotic-screening marker gene neomycin phosphotransferase (NPTII) on the carrier simultaneously, control by rouge alkali synthetase gene (NOS) promotor.

B. the part fragment that lacks the Ac element obtains nonautonomy transposon Ds element, contains antibiotic-screening marker gene hygromycin phosphotransferase gene (HPT) on the carrier simultaneously, is controlled by the CaMV 35S promoter.Tocs, octopine synthase gene (OCS) terminator.Tcamv, the cauliflower mosaic virus terminator.

The structure of Fig. 2 inducible promoters-Ac transposase plasmid and Ds plasmid.

PCC-Ac01, the Ac transposase plasmid under the evoked promoter control.Kalamycin resistance gene is by the Ubiquitin promoter regulation, and the Ubiquitin promotor includes its first intron, expresses to strengthen resistant gene.But the transcription regulaton factor alcR of alcohol inducible system is by paddy rice actin 1 promotor control, but the Ac transposase gene by alcohol inducible system alcA control, under the situation of no inductor existence, transposase gene is not expressed.Alcohol combines with transcription regulaton factor alcR and changes its conformation after adding inductor alcohol, combines with alcA then, and the Ac transposase gene is transcribed, thereby produces transposase.

PCC-Ds02, the nonautonomy Ds element plasmid of dependence transposase.Plasmid contains the hygromycin gene of cauliflower mosaic virus (CaMV) 35S promoter regulation and control and relies on the nonautonomy swivel base Ds element of transposase, includes the recognition sequence that two terminal repeats (TIR) are used for swivel base.Being added with the intestinal bacteria glucuronidase reporter gene (GUS) that contains incomplete intron between two terminal repeats (TIR) traps to be used for promotor.LB, the agrobatcerium T-DNA left margin; RB, the agrobatcerium T-DNA right margin.

The evaluation of Fig. 3 alc:gus transgenic rice plant.The M.DNA molecular weight standard; 1. positive control; 2. negative control plant; 3-15 is the independent transfer-gen plant of intending.

The evaluation of Fig. 4 alc:gus transgenic paddy rice induced activity.

Induce the alc:gus transgenic paddy rice with the alcohol of 100mL 1% in the mode of root pouring, induce 48h for the first time after superinduce once, carry out behind the 120h that GUS is active to be detected.1 is the contrast of non-transgenic paddy rice, and 2-9 is respectively transgenic line ta56,8a15,8a31, ta3, ta4, ta80,8a1,8a2.

Fig. 5 waters paddy rice to the active influence of alc system induction with different ethanol concns.

Alcohol with 100mL different concns (v/v) is induced transgenic paddy rice in root pouring mode, and superinduce once detects the GUS activity when inducing 48h for the first time during 120h.CK, the non-transgenic paddy rice.A, transgenic line 8a2; B, transgenic line ta4.

Fig. 6 alcohol dosage is to the active influence of alc system induction.

Alcohol with 100mL different concns (v/v) is induced transgenic paddy rice ta4 by aquicultural mode, collects blade behind the successive induction 96h and detects the GUS activity.CK, the non-transgenic paddy rice.

Fig. 7 alcohol induction time is to the effect of alc system.

Induce the alc transgenic paddy rice with the alcohol of 100mL in the mode of root pouring, induce behind the 48h superinduce once (twice induce shown in arrow among the figure) for the first time, induce the back to gather primary vane for the first time, be used for the GUS determination of activity every 24h.CK is the non-transgenic paddy rice.A, transgenic line ta4; B, transgenic line ta56.

Fig. 8 alcohol induction time is to the effect of alc system.

Alcohol with 100mL is induced alc transgenic line ta4 in aquicultural mode, induces the back to gather primary vane every 24h, is used for the GUS determination of activity.CK is the non-transgenic paddy rice.

Fig. 9 alc system is in the comparison of Different Organs induced activity.

Alcohol with 100mL 2% is induced transgenic line ta4 in the mode of root pouring, and superinduce is once gathered root, stem, leaf during 48h behind the 96h, detects its GUS activity.

The Histochemical localization of Figure 10 GUS in the transgenic paddy rice blade. induce transgenic line ta4 with the alcohol of 100mL 2% in the mode of root pouring, during 48h superinduce once, the 96h rear blade detects its activity through free-hand section with X-Gluc.A. before inducing, after B. induces 96 hours.

Embodiment

Induce the transposon tagging system to form by the autonomous transposable element that a non-autonomous transposable element and transformation later self can not swivel bases, latter's transposase of encoding causes the former swivel base, but this method is utilized alcohol inducible system (Jepson, et al 1998) control Ac transposase gene is expressed, do not inducing under the situation, the Ac transposase gene is in silence state, and tool does not activate Ds swivel base ability.Induce back Ac transposase to be expressed and activate the Ds swivel base, obtain mutant in the Plant Genome thereby insert at random.Therefore, this method can begin from limited parent material to induce number of times by controlling moment, obtains plant saturation mutation colony at short notice.On the other hand, do not inducing under the situation because no Ac transposase exists, it is stable and hereditary that mutant can keep.

The structure of embodiment 1 inducible promoters-Ac transposase plasmid and Ds plasmid

The transposon tagging system that induces that we adopt is divided into two parts: the Ac transposase plasmid (pCC-Ac01) under the evoked promoter control and contain the nonautonomy Ds element plasmid that relies on transposase.Two plasmids are all in can be used for the binary vector plasmid of Agrobacterium-mediated Transformation.The pCC-Ac01 plasmid contains following composition: kalamycin resistance gene is by the Ubiquitin promoter regulation, and the Ubiquitin promotor comprises its first intron, expresses (Cornejo et al., 1993) to strengthen resistant gene.But the transcription regulaton factor alcR of alcohol inducible system is by paddy rice actin 1 promotor control, but the Ac transposase gene by alcohol inducible system alcA control, under the situation of no inductor existence, the Ac transposase gene is not expressed.Alcohol combines with transcription regulaton factor alcR and changes its conformation after adding inductor alcohol, combines with alcA then, and the Ac transposase gene is transcribed, thereby produces transposase.

The pCC-Ds02 plasmid contains the hygromycin gene of cauliflower mosaic virus (CaMV) 35S promoter regulation and control and relies on the nonautonomy swivel base Ds element of transposase, includes the recognition sequence that two terminal repeats (TIR) are used for swivel base.The intestinal bacteria glucuronidase reporter gene (GUS) that is added with the incomplete intron that contains 5 ' end between two terminal repeats (TIR) is to be used for promotor trapping (see figure 2).

The conversion of embodiment 2 paddy rice

Sophisticated seed is removed clever shell, the alcohol sterilization with 70% 1-2 minute, 0.1% mercuric chloride soaked 10-15 minute, with twice of aseptic water washing, clorox with 10-15% soaked 30-45 minute again, used aseptic water washing 3-4 time, was seeded on the inducing culture.The dark cultivation after 7-10 days peeled off the seed embryo, receives to be cultured on the subculture medium to grow callus.

Method according to (1994) such as Hiei imports to binary vector pBin19alc:gus in the paddy rice by agrobacterium mediation method.The single colony inoculation of picking Agrobacterium in 20ml YEB liquid nutrient medium (containing kantlex 50mg/mL), 28 ℃ (150rpm) 2-3 days.In centrifugal 3 minutes of 4 ℃ of following 5000rpm, remove supernatant, be resuspended in the AAM substratum (containing the 0.1mmol/L Syringylethanone), 28 ℃ of lucifuges were shaken 1-2 hour, to OD ₆₀₀=0.6-0.9.Select that growth conditions is good, after the particulate state callus immerses and shake 20 minutes in the Agrobacterium nutrient solution, left standstill 30 minutes, take out and blot unnecessary bacterium liquid, callus is inoculated on the common substratum cultivates with aseptic filter paper.After 2-3 days, treat that Agrobacterium grows to the bacterial plaque under the visible callus but when not covering with callus, callus is put into wide-necked bottle,, shake several at every turn, in water, lose thread thalline with aseptic water washing 3-5 time; In the sterilized water that contains the 500mg/L Pyocianil, soaked 30-60 minute then, wash again at last one time, place airing on the aseptic filter paper, change screening culture medium (containing 150mg/L G418 and 500mg/L Pyocianil) screening over to.

Embodiment 3 intends the regeneration of transformed plant

After Agrobacterium is infected, rice callus is after cultivating 3-5 days on the common culture medium, change over to and contain G418 (gentamicin derivative, toxicity mechanism is identical with kantlex) screened for 3 weeks on the NB substratum of 100mg/L, containing 4 weeks of screening on the NB substratum of 150mg/L G418 again, resistant calli changes pre-differentiation substratum (G418 150mg/L) 3 weeks of illumination cultivation over to subsequently, on division culture medium (G418 150mg/L), break up then, regeneration plant is transplanted to soil after taking root on containing the strong seedling culture base of G418 150mg/L, further identifies.

Embodiment 4 Molecular Identification

Method according to (1983) such as Dellaporta is extracted the genomic dna of intending transformed plant, and the primer special with the gus gene coding region carries out pcr amplification, thereby filters out transfer-gen plant.According to the method for (1989) such as Sambrook transgenic paddy rice being carried out Southern analyzes.Carry out Southern hybridization with gus gene coding region specific probe, and in the negative non-transgenic adjoining tree without any band (Fig. 3), illustrate that exogenous origin gene integrator is in these plan transgenic plant genomes.The optimization 1. alcohol inducible systems of embodiment 5 alcohol inducible systems in transgenic paddy rice are in the preliminary evaluation of transfer-gen plant induced activity

But induce situation in order to understand the alcohol inducible system in paddy rice, we have made up the alcohol inducible system: intestinal bacteria glucuronidase (GUS) reporter gene carrier, and change paddy rice over to by above-mentioned method for transformation.Obtain 22 positive plants by Molecular Detection.We choose 8 plant, adopt the mode of root pouring to induce the gus expression of gene of transfer-gen plant with 1% alcohol, induce 48h for the first time after superinduce once, collect blade after 120 hours, carry out the analysis of GUS fluorescence activity.The result shows that the active background activity with the non-transgenic adjoining tree of the GUS of plant inducer leading is approaching, induces the GUS activity in the transgenic paddy rice of back can improve 25 to 126 times (Fig. 4).This illustrates under the non-induction state that the alc system can rigorous control expression of exogenous gene, and the height that the alcohol of proper concn can induction exogenous gene is expressed.Strain is that 8a2, ta4, ta56 are used for further experiment.2. the optimization A. ethanol concn of inductive condition is to the active influence of alc system induction

Alcohol with 0.1%, 0.5%, 1%, 2% and 5% adopts root pouring and aquicultural mode to induce transfer-gen plant respectively.

When adopting the mode of root pouring, induce 48h for the first time after superinduce once, gather blade behind the 120h, measure its GUS activity.The result shows that transgenic line 8a2 is in sample time, and 0.1% alcohol can be induced the expression of its gus gene; The GUS activity is induced at 0.5% and 1% alcohol can improve 14 to 21 times under the situation; 2% alcohol is induced most effective, and the GUS activity can improve about 40 times; And 5% alcohol is induced efficient decrease (Fig. 5).

Inducing strain with different concns alcohol in the water culture mode is ta4.Behind the successive induction 96h, gather blade and carry out GUS fluorescence activity mensuration.Found that 0.1% alcohol can induce that GUS is active to improve 16 times, 1% alcohol can make the expression activity of alc:gus improve 160 times.And 5% alcohol only makes active about 20 times (Fig. 6) of increasing of GUS.

Thereby adopt root pouring or the aquicultural mode of inducing, in certain ethanol concn scope, the induced activity of alc system depends on the concentration of alcohol.When root pouring mode was induced the alc system, 2% alcohol was induced best results; And when adopting the water culture mode to induce, 1% alcohol is the most effective.May influence gene transcription and translate because the alcohol of higher concentration can produce paddy rice coerce, thereby the alcohol of higher concentration induces efficient to reduce (as the alcohol of 2% and 5% concentration in the alcohol of 5% in the root pouring mode and the water culture mode) on the contrary.Therefore adopt 2% alcohol when root pouring mode is induced in further experiment as inductor, the water culture mode then with 1% alcohol as inductor.B. the alcohol induction time is to the effect of alc system

We adopt root pouring and water culture dual mode to induce transgenic paddy rice, have studied the effect of alcohol induction time to the alc system.

When adopting the mode of root pouring, induce two transgenic line ta4, ta56, induce mode as previously mentioned with 2% alcohol.Induce the back to gather primary vane for the first time, after inducing 120h, detect the GUS activity every 24h.The result show strain be ta4 when inducing 48h for the first time, GUS is active to improve 18 times; The GUS activity is increased to 66 times behind the 72h; The GUS activity is the strongest during 96h, and being approximately increases by 144 times; The GUS activity begins to reduce afterwards, is 118 times (Fig. 7) before inducing approximately during 120h.

When inducing transgenic line ta4 in the water culture mode, the spirituous solution successive induction 96h with 1% gathers primary vane every 24h, detects the GUS activity.The result shows that the GUS activity continues to increase, and induces for the first time that GUS is active behind the 48h improves about 7 times, and the GUS activity can increase by 160 times (Fig. 8) behind the 96h.

This presentation of results alcohol can rapid induction alc system.Because proteinic translation will lag behind transcribing of mRNA, can infer that in fact the ale system is faster to the response of alcohol.The C.alc system is in the comparison of Different Organs induced activity

Be the inducibility of research alc system in the transgenic paddy rice Different Organs, we induce strain with 2% alcohol by the mode of root pouring is ta4, induces mode as previously mentioned.Induce back 96h for the first time, gather its root, stem, leaf respectively, carry out the GUS fluorescence activity and measure.The result shows that the GUS activity is the strongest in leaf, is 160 times before inducing approximately, and in the stem root GUS active more weak relatively be 7 to 12 times (Fig. 9) before inducing approximately.Blade before and after inducing is done free-hand section, carry out histochemical stain then.The result shows that before inducing, GUS does not all have expression in the blade in all cells, and GUS all has very strong expression activity (Figure 10) after 96 hours in comprising mesophyll cell, vascular tissue but induce.

Embodiment 6 contains the acquisition of Ac transposase transgenic plant

Utilize Agrobacterium rice conversion as previously mentioned, screening method changes the pCC-Ac01 plasmid over to paddy rice, obtains resistant plant by the kantlex screening.With Ac transposase gene fragment is that probe carries out Molecular Detection.And utilize the sophisticated program of inducing that positive plant is induced.Detect the expression amount of inducing front and back Ac transposase mRNA by Northern, choose and induce preceding nothing to express, and induce the plant of back high expression level Ac transposase.

Embodiment 7 is that background material changes the Ds plasmid over to agrobacterium-mediated transformation with above-mentioned transfer-gen plant

Not expressing to have before above-mentioned the inducing, is that starting materials changes the nonautonomy transposon Ds plasmid that contains dependence Ac transposase over to by agrobacterium-mediated transformation and induce the transfer-gen plant of back high expression level Ac transposase.Contain Ds with Southern method (or PCR method) screening and insert fragment transgenic positive callus cell.

Embodiment 8 induces Ac transposase transient expression

Utilize the alcohol set up the system of inducing that the transgenic positive callus cell that not only contains the Ac transposase but also contain the nonautonomy swivel base insertion element Ds that relies on the Ac transposase is induced.

Embodiment 9 makes above-mentioned callus cell regeneration plant by tissue culture

Make above-mentioned callus cell regeneration plant by tissue culture.The Southern method detects the genetic behavior of Ds in offspring's individual plant.Each plant that contains Ds all can be regarded an independent mutation body as.

Reference

Baker，B.，Schell，J.，Lorz，H.，and?Fedoroff，N.V.(1986).Transposition?ofthe?maize?controlling?element?activitor?in?tobacco.Proc.Natl.Acad.Sci.USA83，4844-4848.

Bradford，M.M.(1976a).A?rapid?and?sensitive?method?for?the?quantitationof?microgram?quantities?of?protein?utilizing?the?principle?of?protein-dye?binding.Anal.Biochem.72，248-254.

Chuck，G.，Robbins，T.，and?Nijjar，C.(1993).Tagging?and?cloning?ofpetunia?flower?color?gene?with?the?maize?transposable?element?Activitor.PlantCell?5，371-378.

Cornejo，MJ.，Luth，D.，Blankenship，K.M.，Anderson，O.D.，and?Blechl，A.E.(1993).Activity?of?a?maize?ubiquitin?promoter?in?transgenic?rice.Plant?Mol.Biol.23，567-581.

Dellaporta，S.L.，Wood，J.，and?Hicks，J.B.(1983b).A?rapid?method?forDNA?extraction?from?plant?tissue.Plant?Mol.Biol.Rep.1，19-21.

Fedoroff，N.V.，Furtek，D.B.，and?Nelson，O.E.(1984).Cloning?of?thebronze?locus?in?maize?by?a?simple?and?generalizble?procedure?using?thetransposable?element?Activator(Ac).Proc.Natl.Acad.Sci.USA?81，3825-3829.

Girel，A.and?Saedler，H.(1992).Plant-transposable?elements?and?genetagging.Plant?Mol.Biol.19，39-49.

Haldmp，A.，Petersen，S.G.，and?Okkels，F.T.(1998).The?xylose?isomerasegene?from?Thermoanaerobacterium?thermosulfurogenes?allows?effectiveselection?of?transgenic?plant?cells?using?D-xylose?as?the?selection?agent.PlantMol.Biol.37，287-296.

Hiei，Y.，Ohta，S.，Komari，T.，and?Kumashiro，T.(1994c).Efficienttransformation?of?rice(Oryza?sativa?L.)mediated?by?Agrobacterium?andsequence?analysis?of?the?boundaries?of?T-DNA.Plant?J.6，271-282.

Hirochika，H.(1997).Retrotransposons?of?rice：their?regulation?and?use?forgenome?analysis.Plant?Mol?Biol?35，231-240.

Hirochika，H.，Otsuki，H.，Yoshikawa，M.，Otsuki，Y.，Sugimoto，K.，andTakeda，S.(1996).Autonomous?transposition?of?the?tobacco?retrotransposonTtol?in?rice.Plant?Cell?8，725-734.

Izawa，T.，Ohnishi，T.，Nakano，T.，Ishida，N.，Enoki，H.，Hashimoto，H.，Itoh，K.，Terada，R.，Wu，C.，Miyazaki，C.，Endo，T.，Iida，S.，and?Shimamoto，K.(1997).Transposon?tagging?in?rice.Plant?Mol?Biol?35，219-229.

Jefferson，R.D.，Kavanagh，T.A.，and?Bevan，M.W.(1987).GUS?fusions：-glucuronidase?as?a?sensitive?and?versatile?gene?fusion?marker?in?higherplants.EMBO?J.6，3901-3907.

Knapp，S.，Larondelle，Y.，RoBberg，M.，Furte，D.，and?Theres，K.(1994a).Transgenic?tomato?lines?containing?Ds?element?at?defined?genomic?positions?astools?for?targeted?transposon?tagging.Mol.Gen.Gen.243，666-673.

Knapp，S.，Larondelle，Y.，Rossberg，M.，Furtek，D.，and?Theres，K.(1994b).Transgenic?tomato?lines?containing?Ds?elements?at?defined?genomic?positionsas?tools?for?targeted?transposon?tagging.Mol?Gen.Genet.243，666-673.

Liu，Y.G.，Mitsukawa，N.，Oosumi，T.，and?Whittier，R.F.(1995).Efficientisolation?and?mapping?of?Arabidopsis?thaliana?T-DNA?insert?junctions?bythermal?asymmetric?interlaced?PCR.Plant?J?8，457-463.

Lucas，H.，Feuerbach，F.，Kunert，K.，Grandbastien，M.A.，and?Caboche，M.(1995).RNA-mediated?transposition?of?the?tobacco?retrotransposon?Tnt1?inArabidopsis?thaliana.EMBO?J?14，2364-2373.

Sambrook，J.，Fritsch，E.F.，and?Maniatis，T.(1989d).Molecular?Cloning：ALaboratory?Manual.(New?York：Cold?Spring?Harbor?Laboratory?Press).

Sato，Y.，Sentoku，N.，Miura，Y.，Hirochika，H.，Kitano，H.，and?Matsuoka，M.(1999).Loss-of-function?mutations?in?the?rice?homeobox?gene?OSH15?affectthe?architecture?of?internodes?resulting?in?dwarf?plants.EMBO?J?18，992-1002.

Souer，E.，Quattrocchio，F.，de?Vetten，N.，Mol，J.，and?Koes，R.(1995).Ageneral?method?to?isolate?genes?tagged?by?a?high?copy?number?transposableelement.Plant?J?7，677-685.

Claims (14)

1. method of setting up plant gene label system may further comprise the steps:

(a) make up a kind of first dna fragmentation, make it comprise a kind of gene and a kind of plant screening mark gene of the coding transposase by the control of inducible system or callus specific expressing promoter, with a kind of second dna fragmentation, make it comprise a kind of nonautonomy insertion element and a kind of plant screening mark gene that depends on transposase; Perhaps, make up a kind of the 3rd dna fragmentation, make it comprise a kind of gene, a kind of nonautonomy insertion element and a kind of plant screening mark gene that depends on transposase of the coding transposase by the control of inducible system or callus specific expressing promoter;

(b) with the first constructed dna fragmentation and second dna fragmentation or contain the plasmid of these dna fragmentations, perhaps with the 3rd constructed dna fragmentation or contain the plasmid transformed plant cells of this dna fragmentation;

(c) filter out by the plant transformed cell;

(d) induce transposase to express;

(e) make by plant transformed cell regeneration and go out whole plant.

2. in accordance with the method for claim 1, wherein, the described plasmid that contains these dna fragmentations is the binary vector plasmid that is used for agrobacterium tumefaciens or Agrobacterium rhizogenes conversion plant.

3. in accordance with the method for claim 2, wherein, described plasmid is pBin 19, pCambia, or pBI 101.

4. in accordance with the method for claim 1, wherein, the described plasmid that contains these dna fragmentations is the plasmid that can breed in prokaryotic organism.

5. in accordance with the method for claim 4, wherein, described plasmid is pUC, pBluescript, perhaps PCR vector plasmid.

6. in accordance with the method for claim 1, wherein, described transposase is to derive from the transposon system that has autonomous or nonautonomy swivel base ability in the plant.

7. in accordance with the method for claim 6, wherein, described transposase is the Ac/Ds in corn source, En/Spm, Mu, the Tam in the Common Snapdragon, the dTph1 in the petunia, the perhaps Tos17 in the paddy rice.

8. in accordance with the method for claim 1, wherein, the described nonautonomy swivel base insertion element that depends on transposase is the Ds element.

9. in accordance with the method for claim 1, wherein, described plant screening mark gene is an anti-kantlex selection markers gene neomycin phosphotransferase (NPTII), moisture resistance mycin selection markers gene hygromix phosphotransferase (hpt); Or xylose isomerase.

10. in accordance with the method for claim 1, wherein, but the inducible system of described control transposase is alcohol inducible system (alc), but sugar sterol inducible system, but tsiklomitsin inducible system (tet), but lactose inducible system (lac), but cupric ion inducible system, perhaps heat shock, weedicide, damage inducible system.

11. in accordance with the method for claim 1, wherein, described vegetable cell refers to derive from the cell of Arabidopis thaliana, tobacco, potato, paddy rice, wheat, corn, rape, cotton or soybean.

12. in accordance with the method for claim 1, wherein, transformed plant cells refers to by protoplastis-chemical mediated method (Ca ²⁺, PEG), the particle gun mediated method, agrobacterium-mediated transformation, electrization, pollen tube imports, and the combination of microinjection or these methods is carried out.

13. in accordance with the method for claim 1, wherein, induce transposase to express to be the callus stage plant, in the vegetation growth of plant stage, generative growth phase, seed take place or the seed germination stage carries out.

14. in accordance with the method for claim 1, wherein, transposase is expressed by carrying out under the control of callus specific expressing promoter.

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