CN1298213C - Technology of obtaining gene transferred plant without selecting mark through division process - Google Patents
Technology of obtaining gene transferred plant without selecting mark through division process Download PDFInfo
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- CN1298213C CN1298213C CNB031432891A CN03143289A CN1298213C CN 1298213 C CN1298213 C CN 1298213C CN B031432891 A CNB031432891 A CN B031432891A CN 03143289 A CN03143289 A CN 03143289A CN 1298213 C CN1298213 C CN 1298213C
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Abstract
The present invention relates to a technology for obtaining a transgenic plant without a selective mark through division processing, which has the technical scheme that callus formed after conversion is sliced into a tablet equal to or less than 1mm<3>, the sliced processing times are equal to or greater than 2 times, goal genes are traced by a method that genes can be directly detected, a homozygous and an exact homozygous conversion body can be separated by division processing in many times, and the transgenic plant without the selective mark is further obtained. The technology has the advantages that the possibly produced adverse effects of the selective mark in the transgenic plant are avoided, and the limitation and the defects existed in the existing various selective mark elimination technology are stepped over; furthermore, the technology also has the characteristics of easy operation and convenient generalization and application, and is suitable for all plants successful in tissue culture.
Description
Technical field
The present invention relates to a kind of method, belong to plant transgenic technology by dividing processing acquisition non selecting sign transgene plant.
Background technology
Genetically modified crops have become the high-tech product that the market temptation is arranged now most, but present transgenic technology mostly relies on selected marker.Selected marker in the genetically modified plants may have a negative impact from many aspects, and the transgenic technology of development marker-free is current active demand.
Since nineteen eighty-three obtained transgene tobacco first, plant transgene branch art was updated or is innovated, and developed some kinds of methods, as agrobacterium-mediated transformation, particle bombardment, microinjection, pollen tube passage method etc.What wherein, use the most successfully is agrobacterium-mediated transformation and particle bombardment.But because the efficient of all transgenic technologys is all lower at present, the marker gene that the importing of " genes of interest " is easy to discern on need contacting is usually selected.This marker gene is called " selected marker " or " selection markers " again.Most genetically modified plants all obtain by " selection markers ".These " marks " still also bring or a series of problem of having hidden though help people to realize the selection of transformant.Can be divided into five aspects substantially: 1, may influence expression of gene.2, limit a plurality of genetic transformation.3, may influence some proterties of crop.4, may influence Environmental security.5, may jeopardize personal safety.
For selected marker may bring many unfavorable, scientists has had understanding for a long time, and has begun the exploration of the method for transformation of relevant removal selected marker earlier.End is got up, and mainly contains the following aspects:
1, utilize the cotransformation method that genes of interest and selected marker are integrated in two T-DNA respectively, be implemented in it on carrier or be implemented in two carriers respectively, with such carrier or two carriers transformation receptor plant simultaneously, obtain to isolate the offspring of only containing genes of interest from the cotransformation body by hereditary separation principle behind the cotransformation body by screening.But utilize the cotransformation method to obtain not contain the transformation generation of selected marker, it is high that prerequisite is that cotransformation efficient is wanted, and two genes in its cotransformation body can separate, for many important crops, because the cotransformation rate is lower, the application of this method and the effect after the application have been limited greatly.
2, utilizing locus specificity recombinant technique locus specificity reorganization system is the dna sequence dna of a class uniqueness, generally it can be divided into two parts, and a part is the gene of coding recombinase, and another part is the specificity site that the gene that inserts can be excised automatically.What wherein study morely is the Cre/lox system.The principle of utilizing the Cre/lox system to remove selection markers is marker gene to be implemented between two 34bp distinguished sequences of lox system, i.e. the lox site, genes of interest is implemented in outside the lox site, after screening transformant, induce the Cre expression of enzymes, will be positioned at the selected marker excision in lox site.The specific site recombination system, though showed higher controllability, too complicated, also have a deadly defect in addition, promptly can't excise specific site.This is for be still unnecessary " rubbish " as selected marker by plant modification.
3, utilizing the method transposons swivel base of transposons swivel base is to realize reconfiguring of gene by the transposition of transposase in the transposons system.Transposons is made up of autonomous member and the autonomous member's two parts of non-, and as the Ac/Ds system of corn, its autonomous member Ac gene code transposase is used for the swivel base of self and non-autonomous member Ds gene.There are hundreds of nucleotide sequences at non-autonomous member's two ends, and the centre can change or insert bigger foreign gene into.The non-autonomous member who inserts foreign gene is still shifted by transposase together together with autonomous member.Genes of interest is placed the outside of whole transposons, selected marker is located at non-autonomous member inside, the plant after the conversion can transfer selected marker by transposition, isolates unmarked transgenic progeny at last.But, can utilize the crop of transposons mechanism also fewer at present, the activity of transposons is very little in many important crops, and the frequency that swivel base takes place is very low.
4, utilize the homologous recombination effect promptly to import one section and the identical sequence of selected marker both sides DNA again, will include the dna fragmentation displacement of selected marker by the reorganization of homology preface, thereby obtain the transfer-gen plant that selected marker weeds out.This technology is called gene targeting again, and that uses on microorganism and animal is very successful, but also is in the exploratory stage on plant, does not still have a convictive evidence.
5, utilizing additive method mainly is to utilize certain that plant and the useful gene of people are replaced selected marker, as color, drought resisting, anti-salt etc.But the gene of this respect is big, the complex structure of volume mostly, is selecting to be difficult to judgement on the effect.In addition, the gene that can be used as selected marker from this respect is to count seldom.
Summary of the invention
Technical problem to be solved by this invention provides a kind of method that obtains the non selecting sign transgene plant by dividing processing.
The technical solution adopted for the present invention to solve the technical problems:
The callus that transforms back formation is cut into as far as possible little fritter, the volume≤1mm of fritter
3Treat that the fritter back of growing up follows the trail of the cell or tissue that target gene is arranged with the method that can be directly gene be detected (evaluation), by dividing processing repeatedly, dividing processing number of times 〉=2 time, just separablely go out to isozygoty and accurate transformant of isozygotying, and then obtain the transfer-gen plant of marker-free; Its techniqueflow is as follows:
(1), the acceptor that transforms of conversion processing can be explant, also can be callus; Can be agrobacterium mediation converted, also can use the particle gun mediated transformation.
(2), the explant induction of tissue culture after with conversion processing become callus and by cultivating further amplification, with the callus after the conversion processing by cultivating further amplification.
(3), the callus that obtains after with conversion processing of dividing processing is divided into as far as possible little fritter, the volume≤1mm of fritter
3, and clocklike be arranged on the medium in the position of original callus relation by each fritter; After callus after to be split is grown up, select the callus that conversion is arranged, be divided into again≤1mm with the method that can be directly target gene be detected (evaluation)
3Fritter; After callus after treating to cut apart is for the second time grown up, find out the many zones of transformed calli earlier, again the callus in this zone is detected one by one, determine the concrete distribution that transforms callus, choose the callus differentiation plant that has transformed then from the central area of transformed calli compact district or further cut apart, evaluation and plant regeneration; The number of times of dividing processing 〉=2 time.
(4), Molecular Detection is selected the callus that conversion is arranged with the method that PCR etc. can be directly detects (evaluation) to target gene.
(5), the conversion that will select of plant regeneration callus be divided into regeneration plant.
(6), plant identifies regeneration plant identified one by one, keeps two all positive plant of blade of all topmosts.
After cutting apart for the first time, treat to adopt the exclusive method that reduces by half that it is identified after callus is grown up, the exclusive method that reduces by half is: the callus of growing up after will cutting apart is divided into two groups, being put in two little centrifuge tubes respectively identifies, eliminate reactionless group, callus in the positive group is two groups of samplings again, identifies, so repeatedly until finding the callus that conversion is arranged.For the influence that prevents to take a sample to callus, should reserve certain time interval that allows its recovery to callus when repeatedly taking a sample.
After cutting apart for the second time, treat to adopt the method for dividision into groups that it is detected after callus is grown up, the method of dividision into groups is: for the more processing of block number, divide into groups by the relation of arranging of callus on plate, same group put same little centrifuge tube, define the callus and the maximum group of transformed calli of conversion according to testing result, then all callus in this group are detected one by one, define the distribution of transformed calli, choose the most central one.
The invention has the beneficial effects as follows and avoided the issuable adverse effect of the selected marker in the genetically modified plants, and existing limitation of the technology of having crossed over existing various removal selected markers and shortcoming, it also has characteristics easy and simple to handle, easy to utilize, but the present invention is applicable to the plant of all tissue culture successes.
Description of drawings
Fig. 1 is the schematic diagram that carried out 64 fens.
Fig. 2 is the distribution situation of transformant on the initial callus (dark position is the part that has transformed).
Fig. 3 is transformed calli after 64 minutes for the first time (dark position is the part that has transformed).
Fig. 4 is transformed calli after 64 minutes for the second time (dark position is the part that has transformed).
Fig. 5 is genetically modified hair T-5 tomato.
Embodiment
Embodiment 1: utilize split plot design that gus gene is imported tobacco.
Transform with agrobacterium-mediated transformation, the acceptor tobacco is kind SR1, and donor is the pCAMBIA1301 plasmid that carries gus gene, and agrobacterium strains is LBA4404.Infect and be total to the medium of cultivating and be: B5+ glutamine 584mg/l+ betain 117.2mg/l+ proline 0.12mg/l+ inositol 400mg/l+30g/l sucrose, pH 5.5.The OD of Agrobacterium when infecting
600Value is 0.5, and additional 5mg/l acetosyringone infected 30 minutes.Cultivate on the medium of additional 10mg/l Agar altogether and carry out, the time is 3 days.The medium of cultured calli is B5+584mg/l glutamine+0.6mg/l BA+0.03mg/l IAA+250mg/lCarb.When the callus after the conversion processing grows to the diameter 0.5cm left and right sides, cut apart by the method (60 quartering) of Fig. 1 signal, be divided into 64 at every turn.For ease of testing result is mapped with transforming the position, the small cell cluster after dividing equally is placed on the medium in proper order by original adjacent relation.In order to analyze the effect of cutting apart, with " each block number * segmentation times " or " each dividing processing institute block count and " as cutting apart degree of going forward one by one.After cutting apart for the 1st time,, identify, select the callus that conversion is arranged, be divided into 64 again and proceed to cultivate with " exclusive method reduces by half " to the callus of growing up.After cutting apart for the 2nd time, to the callus of growing up, detect with " method of dividision into groups ", find the many zones of transformed calli earlier, again the callus in this zone is detected one by one, determine the concrete distribution of transformed calli, the callus that has transformed is chosen in the central area of transformed calli compact district then.Go forward one by one according to this, carried out dividing processing altogether 5 times.
Experimental result shows, the explant after the conversion processing, on the medium of no selection pressure, cultivate 12 days just the president to the nearly 0.5cm of diameter.Randomly draw the callus of some and carry out the X-gluc staining analysis, the original callus of 80-90% all has conversion, but the ratio of transformant number in the monoblock callus is very low, and transformation event mainly occurs in the wound part (see figure 2) of explant periphery.
Be divided into 64 with the callus that transforms back diameter 0.5cm is even, be cultured to original size again and need 11 days approximately.The degree of going forward one by one of cutting apart after once cutting apart is 64 * 1=64.The result that the callus of growing up after once cutting apart is detected shows, the cell that has transformed in the callus of conversion is arranged has shown certain enrichment (see figure 3).
The degree of going forward one by one of cutting apart after the secondary splitting is 64 * 2=128.Testing result shows, transformed calli after the secondary splitting has begun to occur in flakes, have the ratio of callus in total callus number of conversion obviously to improve, and the enrichment of transformant also obviously improve (see figure 4) in the callus that conversion is arranged of growing up after the secondary splitting.The result that the 80 strain regrowths that differentiated by the callus after the secondary splitting are detected shows that homozygote 45 strains, 3 strains of accurate homozygote, chimera 10 strains, unconverted 22 strains are wherein arranged.The above plant of accurate homozygote (homozygote+accurate homozygote) shared ratio (being effective conversion ratio) in total strain tree is: (45+3)/and 80=60.0%.
The degree of going forward one by one of cutting apart after cutting apart for three times is 64 * 3=192.Detect the sub-callus of cutting apart for three times, the distribution in flakes that has wherein had the callus of conversion to be rendered as to expand significantly, in total callus number, accounting for quite high ratio, and the most of callus in the transformed calli area has become the pure callus that has transformed.In the 80 strain regrowths that differentiate by it, homozygote 67 strains, 3 strains of accurate homozygote, chimera 6 strains, unconverted 4 strains, effectively conversion ratio is 87.5%.
The progress of cutting apart after cutting apart for four times is 64 * 4=256.Detecting the sub-callus of cutting apart for four times, almost all is to have transformed calli, the overwhelming majority to have the callus of conversion to become the pure callus that has transformed.80 strain regrowths by its regeneration are detected, homozygote 78 strains, 0 strain of accurate homozygote, chimera 2 strains, unconverted 0 strain, effectively conversion ratio has reached 97.5%.
Cutting apart progress after cutting apart for five times is 64 * 5=320.Detecting the sub-callus of cutting apart for five times, all is the pure callus that has transformed, and 80 strain regrowths all are the transformant of isozygotying.
Cut apart also with " 30 dichotomy " and can obtain transformed plant, compare, when cutting apart degree of going forward one by one when close, the effect of cutting apart also close (seeing attached list 1) with " 60 quartering ".
Embodiment 2: utilize split plot design that chitinase gene is imported tobacco
Acceptor is with embodiment 1, and donor becomes the pAHCGG1 plasmid that carries chitinase gene, and agrobacterium strains is LBA4404.Cut apart the processing that degree of passing is 64+32+32, spent 37 day time altogether, homozygote 3 strains are arranged, accurate homozygote 3 strains, chimera 2 strains, unconverted 2 strains in the 10 strain regrowths from being induced to regeneration plant.
Embodiment 3: utilize split plot design that " beta-1,3-glucanase gene " imported tobacco
Acceptor is with embodiment 1, and donor becomes the pAHGGG1 plasmid that carries the beta-1,3-glucanase gene, and agrobacterium strains is LBA4404.Cut apart the processing that degree of going forward one by one is 64+64, spent 34 day time altogether, homozygote 3 strains, accurate fit 3 strains, chimera 1 strain, unconverted 3 strains are arranged in the 20 strain regrowths from being induced to regeneration plant.Cut apart the processing that degree of going forward one by one is 64+32+32, spent 37 day time altogether, homozygote 2 strains, 4 strains of accurate homozygote, chimera 2 strains, unconverted 2 strains are arranged in the 20 strain regrowths from being induced to regeneration plant.
Embodiment 4: utilize split plot design that " beta-1,3-glucanase gene " changed over to the tomato (see figure 5)
Acceptor becomes tomato, and kind is hair T-5 (kind of extensively planting in the current production), and donor becomes the pAHGGG1 plasmid that carries the beta-1,3-glucanase gene, and agrobacterium strains is LBA4404.Hormone in medium during callus culture is adjusted into BA 1.2mg/l+IAA 0.2mg/l.Cut apart the processing that degree of going forward one by one is 32+32+32+32 (128), from the callus induction to the regeneration plant, spent 48 day time altogether.To from 20 at random the result that detects of 20 strain regrowths of callus be: homozygote 6 strains, 5 strains of accurate homozygote, chimera 4 strains, unconverted 5 strains, effectively conversion ratio is 55.0%.
Subordinate list 1: cut apart degree of going forward one by one action effect list
| Cut block count | The cutting number of times | Cut apart degree of going forward one by one | Need the time (d) | Chimera number (strain) | Accurate homozygote number (strain) | Homozygote number (strain) | Effective transformant number (strain) | Effective conversion ratio (%) |
| 32 | 2 | 64 | 29 | 8 | 5 | 13 | 18 | 22.5 |
| 3 | 96 | 36 | 16 | 9 | 18 | 27 | 33.8 | |
| 4 | 128 | 43 | 14 | 9 | 38 | 47 | 61.3 | |
| 5 | 160 | 50 | 12 | 10 | 42 | 52 | 65.0 | |
| 64 | 2 | 128 | 34 | 10 | 3 | 45 | 48 | 60.0 |
| 3 | 192 | 45 | 4 | 3 | 67 | 70 | 87.5 | |
| 4 | 256 | 56 | 2 | 0 | 78 | 78 | 97.5 | |
| 5 | 320 | 67 | 0 | 0 | 80 | 80 | 100.0 |
Claims (2)
1, a kind of method that obtains the non selecting sign transgene plant by dividing processing, comprise that conversion processing, tissue culture, Molecular Detection, plant regeneration, plant identify each step, it is characterized in that between tissue culture and Molecular Detection two steps, being provided with the dividing processing step, press following operation:
(1), the callus that obtains after the conversion processing is divided into volume≤1mm
3Fritter;
(2), after the callus after to be split grows up, directly target gene is detected, select the callus that conversion is arranged, be divided into again≤1mm
3Fritter;
(3), segmentation times 〉=2 time;
(4), cut apart, all regularly be arranged in medium on by concerning in the position of female callus originally small callus after cutting apart from for the second time at every turn;
(5), after the callus after treating to cut apart is for the second time grown up, find out the many zones of transformed calli earlier, again the callus in this zone is detected one by one, determine the concrete distribution of transformed calli, choose transformed calli from the central area of transformed calli compact district then and be used to break up plant, or further cut apart.
2, a kind of method that obtains the non selecting sign transgene plant by dividing processing according to claim 1, it is characterized in that after cutting apart for the second time, treat to adopt the method for dividision into groups that it is detected after callus is grown up, the method of dividision into groups is: for the more processing of block number, divide into groups by the relation of arranging of callus on plate, same group be put in same little centrifuge tube, determine the group and the maximum group of transformed calli of the callus of conversion according to testing result, then all callus in these groups are detected one by one, determine the distribution situation of the callus of conversion, choose its central authorities one.
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| CN1298213C true CN1298213C (en) | 2007-02-07 |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07147971A (en) * | 1993-07-23 | 1995-06-13 | Gist Brocades Nv | Recombinant strain free from selection marker gene, method for preparation thereof and use of said strain |
| CN1356389A (en) * | 2000-12-08 | 2002-07-03 | 大连理工大学 | Plant transgenic method without selective marker |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH07147971A (en) * | 1993-07-23 | 1995-06-13 | Gist Brocades Nv | Recombinant strain free from selection marker gene, method for preparation thereof and use of said strain |
| CN1356389A (en) * | 2000-12-08 | 2002-07-03 | 大连理工大学 | Plant transgenic method without selective marker |
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