CN102286522A - Method for cultivating transgenic rice without foreign gene in white rice through molecular deletion strategy - Google Patents
Method for cultivating transgenic rice without foreign gene in white rice through molecular deletion strategy Download PDFInfo
- Publication number
- CN102286522A CN102286522A CN2011101974356A CN201110197435A CN102286522A CN 102286522 A CN102286522 A CN 102286522A CN 2011101974356 A CN2011101974356 A CN 2011101974356A CN 201110197435 A CN201110197435 A CN 201110197435A CN 102286522 A CN102286522 A CN 102286522A
- Authority
- CN
- China
- Prior art keywords
- rice
- gene
- transgenic
- endosperm
- plant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Landscapes
- Breeding Of Plants And Reproduction By Means Of Culturing (AREA)
Abstract
The invention provides a method for cultivating transgenic rice without foreign gene in white rice through a molecular deletion strategy, which comprises the following steps of: constructing a rice transformation carrier of a site-specific recombination system based on endosperm specificity expression control; transforming rice calluses by the rice transformation carrier and carrying out sieving and differentiation for generating transgenic rice plants; and planting the transgenic rice plants and obtaining a transgenic rice plant strain, of which the foreign gene structure in the endosperm is deleted, but the foreign gene structure in other tissues can be stably integrated and expressed and can be stably inherited. The method adopts recombinase identification sequences, the deletion efficiency is effectively improved, and the highest 100-percent deletion effect can be reached. In the method, the specific promoter control technology of the rice endosperm is adopted, the method of self site-specific recombinase system deletion is combined, and the efficient and specific deletion of all foreign genes at endosperm parts of rice seeds can be realized.
Description
Technical field
The present invention relates to plant genetic engineering field, relate more specifically to a kind of method of cultivating transgenic paddy rice.
Background technology
Agricultural with human clothing, food, live, row etc. is movable closely bound up, agriculture production provides necessary basic substance for human survival and procreation.Since several thousand, the agrotechnique that the human use has grasped constantly improves crop yield and improves quality of agricultural product, satisfies society to ever-increasing demands of existence requisite such as grains.Entered since 21 century, worldwide, the population sharp increase, but farming land resources reduce rapidly, and ecotope goes from bad to worse.How to produce more food and support more people, and reduce destruction as far as possible, a difficult problem that has become a lot of national especially developing countries in the whole world to face ecotope.Through secular development, traditional breeding technology has reached a high level, but also reaches the bottleneck stage of a development simultaneously.It is limited to utilize the traditional agriculture biotechnology further to improve the space of grain yield again, and this will be difficult to satisfy the requirement that agriculture production grew continuously and fast in 21 century.
The plant transgenic technology that grows up the beginning of the eighties in last century, at present technical ripe, its application is also increasingly extensive.This technology has become increases grain yield in the world, reduce one of most important means of environmental pollution.According to the international Agricultural biotechnologies application service of ISAAA(tissue) demonstration of 2010 annual reports, through short 15 years commercialization, genetically modified crops accumulative total cultivated area had exceeded 1,000,000,000 hectares in 2010.In the period of 1996 to 2010, the Area Growth of genetically modified crops plantations 87 times, be the fastest crop technology that adopts on the modern agriculture history.Generally, the development of transgenic plant is a kind of trend.Paddy rice is as topmost food crop, and transgenic technology and the final commercialization trend that also is inevitable is adopted in mass-producing.So far, the research of transgenic paddy rice has also obtained significant progress.
For many years, transgenic plant also are faced with some disputes always in fast development.Although up to now studies show that genetically modified crops and product thereof do not bring security hidden danger, some people still worries the problem that the foreign gene structure in the transgenic plant can be brought influences environment and human health security.
Generally the consideration for the transgenic plant safety aspect has two aspects: the one, and whether environmental safety influences the variation of its surrounding enviroment as transgenic plant, and whether foreign gene may escape by modes such as pollen diffusions, and ecotope is caused danger or the like; Be edible safety on the other hand, maybe can cause allergic reaction or the like as whether genetically modified product is toxic.
Consideration based on the security aspect, people have carried out strict safety evaluation to transgenic plant, on the other hand in the process of research and development transgenic plant product, people are also in the technical system that develops new cultivation transgenic plant, in the hope of fundamentally solving the problem of security concern.Based on the molecule deletion strategy of site-specific recombinase and institute's recognition site specific sequence thereof is exactly people's one of method of cultivating transgenic plant that grows up in recent years, and one of them typical example is exactly the deletion of marker gene in the transgenic plant.At present, the cultivation of non selecting sign transgene plant has solved the worry of people to plant transgene system use selectable marker gene.
The locus specificity recombination system is that a class can make its DNA that reorganization takes place reorganization system by specific dna sequence is cut and reconnects.At present in plant effectively the locus specificity recombination system mainly comprise the Cre/ of coliphage p1
LoxpThe R/ of system, zygosaccharomyces
RsThe Flp/ of system, yeast saccharomyces cerevisiae
FrtThe Xis/ of system and escherichia coli
AttpSystem etc.Wherein, Cre/
LoxpAnd FLP/
FrtIt is the locus specificity recombination system of in plant, studying the most deeply and widely.This type systematic has become a kind of important tool of genetic manipulation at present, at aspects such as fixed point integration of foreign gene, gene knockout, gene mutagenesis and gene clones important effect is arranged all.People such as Rusell (1992. Mol Gen Genetic. 234:49 ~ 59) utilize Cre/
LoxPSystem, with coding sulfonylurea (sulfonylurea) resistance (acetolactate synthase,
Als) gene constructed
LoxPBetween the sequence, in transgene tobacco,, deleted by expressing the Cre enzyme
LoxPBetween the sequence
AlsGene.Similarly the strategy of locus specificity recombination system mediation has been widely used in cultivating the non selecting sign transgene plant.
The efficient of locus specificity recombination system mediated dna reorganization is to use the key that this strategy carries out gene elmination.In view of the efficient of conventional locus specificity recombination system has certain limitation, people such as Luo (2007. Plant Biotechnol J. 5:263-274) will
LoxPWith
FrtTwo specific recognition sites merge, and are built into a new chimeric specific recognition site
LoxP/frtThe experimental result of people such as Luo in tobacco shows, new chimeric specific recognition site
LoxP/frtUnder the mediation of recombinase protein Cre or FLP, its recombination efficiency is significantly higher than primary type
LoxPOr
FrtThe efficient of reorganization.In this new chimeric specific recognition site
LoxP/frtThe basis on, people such as Luo have created a gene knock out system.The expression of the recombinase by utilizing pollen or seed specific expression promotor, it can delete the transgenic structure in pollen in the transgene tobacco or the seed, and top efficiency can reach 100%(Luo K, et al. 2007. Plant Biotechnol J. 5:263 ~ 274).Generally speaking, the establishment of these locus specificity recombination systems makes people can delete the selectable marker gene in the transgenic plant, or the transgenic structure in the specific tissue, thereby reduces the worry of people to transgenic plant safety.
On paddy rice, the gene elmination technology also has been applied in the security control of transgenic paddy rice, as utilizes (Sreekala C. et al. 2005, Plant Cell Rep such as antibiotics resistance gene in these technology deletion transgenic paddy rices, 24, (2): 86-94).But because paddy rice is the topmost grains of people, so people are especially strict to the requirement of its security.In addition, the molecule deletion system efficiency that is applied at present in the paddy rice is lower, still is further improved.
For these reasons, the present invention aims to provide in a kind of high-level efficiency deletion transgenic paddy rice novel method of external source transgenic structure in the edible position of rice.The present invention is based on two bases: the one, paddy rice is a kind of extremely special food crop, its seed comprises endosperm and two parts of embryo.Wherein endosperm is main nutrition part, also is the edible part of rice; And embryo is as the carrier part of heredity, and it is removed clean substantially in the course of processing of rice and can not be eaten by people.The 2nd, tissue-specific promoter's technology and efficiently the locus specificity recombination system provide possibility for all foreign genes of deleting fully in the transgenic paddy rice.
Summary of the invention
In view of above situation,, realize obtaining the purpose of no foreign gene constructed products by transgenic plant in order to cultivate the transgenic paddy rice that the endosperm position does not have transgenic structure.The invention provides a kind of molecule deletion strategy and cultivate the method for the transgenic paddy rice of no foreign gene in the polished rice.
The method of the transgenic paddy rice of external source transgenic structure is removed in cultivation of the present invention in edible polished rice (endosperm) position, comprise following step:
A. make up rice conversion carrier based on the locus specificity recombination system of endosperm specificity expression control.It is characterized in that described rice conversion carrier contains following element: the recombinase recognition sequence; External source destination gene expression box; The recombinase gene expression cassette of endosperm specificity expression promoter control and the antibiotics resistance expression cassette that is used for the rice conversion screening.
B. utilize this conversion carrier rice transformation callus, screening, differentiation produce transgenic rice plant.
C. plant and plant transgenic rice plant, detect external source goal gene different sites in transfer-gen plant, comprise the expression level in root, stem, leaf, embryo and the endosperm, obtain the external source goal gene and in endosperm, deleted, and all stable integration and the transgenic rice plant strain that efficiently expresses are in other tissue by specificity.
D. plant and plant transgenic paddy rice strain of future generation system, the further integration and the expression of Molecular Detection foreign gene, acquisition external source transgenic structure in endosperm is deleted, and the external source transgenic structure can stable integration and the transgenic paddy rice strain system of expressing and can genetic stability in other tissue.
Described recombinase recognition sequence is meant and can be comprised loxp, frt, fusion loxp-frt, RS, attP, attB, attL, attR by site-specific recombinase specific recognition and the sequence of shearing.
Described external source goal gene is meant a kind of in adversity gene, anti insect gene, disease-resistant gene, anti-herbicide gene, anti-ageing gene, the quality-improving gene or fusion or the multivalent genetic be made up of said gene.
Described endosperm specificity expression promoter be meant in endosperm specific expressed and at other position expression promoter not, derive from the wild-type promotor of plant species or modify after promotor, comprise Gt1, Gluc.
Described recombinase gene is that segmental any wild-type recombinase gene between sequence also be sheared, be deleted to expression product can two site-specific sequences of specific recognition, recombinase gene behind series jump type recombinase gene or the sequence modification comprises CRE, FLP, R, PhiC31.
Described antibiotics resistance expression cassette comprises hygromycin gene, neomycin resistance gene, kalamycin resistance gene, herbicide resistance gene.
Described rice callus tissue is from long-grained nonglutinous rice or japonica rice.
The present invention also provides a kind of transgenic paddy rice strain system, be the transgenic paddy rice strain system that utilizes above-mentioned rice conversion carrier to cultivate to obtain and be the rice material that the parent is obtained by the sexual hybridization transformation.
Remarkable advantage of the present invention:
The present invention utilizes the recombinase recognition sequence, has improved the efficient of deletion effectively, is up to the effect of 100% deletion.The present invention adopts the promotor control techniques of rice endosperm specific, and the method for binding site specificity recombinase system oneself deletion can be at efficient special all foreign genes of deletion in the endosperm position of rice paddy seed.In paddy rice, the main edible of rice partly is exactly the endosperm part of rice paddy seed, substantially only contains the endosperm part of rice through the refining rice of processing, and does not contain other parts.So the refining rice of the transgenosis after the special deletion in endosperm position is just the same substantially with the main component of general rice, has no longer contained any other genetically modified composition.Helping solving people like this pays close attention to and worry the food safety of transgenic rice.Simultaneously, because the embryo of rice paddy seed partly is the carrier of heredity, the specificity deletion does not take place in it, and the transgenic structure that it has also is convenient to the breeding demand and the descendant inheritting of rice paddy seed, and this also helps keeping the fine quality and the characteristic of transgenic paddy rice itself.
Description of drawings
Fig. 1 is a schema of the present invention;
Fig. 2 is the structural representation of loxPFRT-MCS-loxPFRT, and LF is the two recognition sites of loxp and FRT among the figure, the multiple clone site that MCS adds for convenient follow-up clone;
Fig. 3 is a pJ4-GCS plasmid structural representation, Fig. 3 .a is the ring texture synoptic diagram, Fig. 3 .b is the linear structure synoptic diagram of important recombinase expression cassette, Gt1 represents endosperm specificity promoter, Crein represents to contain the recombinase of intron, and Tnos represents octopine synthase gene 3 ' end non-translational region;
Fig. 4 is a pJ4-AGS plasmid structural representation, Fig. 4 .a is the ring texture synoptic diagram, Fig. 4 .b is the linear structure synoptic diagram of important GUS expression cassette, Actin represents the promotor of Actin muscle, GUSA represents reporter gene glycuronidase gene first exon, and Tnos represents octopine synthase gene 3 ' end non-translational region;
Fig. 5 is a p1300-LF-GCSAGS plasmid structural representation, Fig. 5 .a is the ring texture synoptic diagram, Fig. 5 .b is the important recombinase and the linear structure synoptic diagram of GUS expression cassette, LB, RB are the border, the left and right sides of T-DNA, LF-L, LF-R represent the site, the left and right sides of two recognition sites, Gt1 represents endosperm specificity promoter, and GUSA represents reporter gene glycuronidase gene first exon, and Tnos represents octopine synthase gene 3 ' end non-translational region;
Fig. 6 is a p1300-LF-AGCSGS plasmid structural representation, Fig. 6 .a is the ring texture synoptic diagram, Fig. 6 .b is the important recombinase and the linear structure synoptic diagram of GUS expression cassette, LB, RB are the border, the left and right sides of T-DNA, LF-L, LF-R represent the site, the left and right sides of two recognition sites, Gt1 represents endosperm specificity promoter, and GUSA represents reporter gene glycuronidase gene first exon, and Tnos represents octopine synthase gene 3 ' end non-translational region;
Fig. 7 is Totomycin blade soaking and screening result, and the left side is the resistance blade among the figure, the right be responsive blade, represent the negative plant of transgenic positive plant and transgenosis respectively;
Fig. 8 is the electrophoresis result of the PCR detection of hptII gene, and the 1-5 swimming lane is respectively, sample 1, sample 2, positive control, negative control, DNA Marker;
Fig. 9 is for changeing root, stem, leaf, kind shell and the flower pesticide GUS histochemical stain result from left to right of p1300-LF-GCSAGS plasmid transgenic rice plant;
Figure 10 is for changeing root, stem, leaf, the kind shell GUS histochemical stain result from left to right of p1300-LF-AGCSGS plasmid transgenic rice plant;
Figure 11 is the result of GUS histochemical stain after changeing the seed of p1300-LF-GCSAGS plasmid transgenic paddy rice and contrasting the seed rip cutting, and Figure 11 .a is the fine seed of non-transgenic Japan; Figure 11 .b is for changeing the seed of p1300-LF-GCSAGS plasmid transgenic paddy rice;
Figure 12 is the result of GUS histochemical stain after changeing the seed of p1300-LF-AGCSGS plasmid transgenic paddy rice and contrasting the seed rip cutting, and Figure 12 .a is the fine seed of non-transgenic Japan; Figure 12 .b is for changeing the seed of p1300-LF-AGCSGS plasmid transgenic paddy rice.
Embodiment
Below be concrete case study on implementation of the present invention, further describe the present invention, but the invention is not restricted to this.
The used experiment material of the present invention is commercially available purchase product if no special instructions.
The structure of [embodiment 1] plant expression vector
The segmental clone of LoxPFRT-MCS-LoxPFRT
In order to obtain the loxPFRT-MCS-loxPFRT fragment (referring to patent: be used for the gene knock out system of transgenic plant safety control and contain its plant expression vector, Roc is bright etc., number of patent application 200510112579.1), design is synthesized and is had two loxP-FRT fusion recognition site sequences in the same way, between two sites, contain a multiple clone site sequence (MCS), simultaneously two restriction enzyme site SalI and NcoI have also respectively been designed, to be convenient to follow-up clone operations at segmental two ends.The rich Deco skill Development Co., Ltd that steps in this segmental synthetic trust Beijing finishes, and the synthetic fragment is implemented in pMD18-T(TAKARA, Dalian) on, called after pMD18T-LF after sequence verification.
The structure of loxPFRT-MCS-loxPFRT as shown in Figure 2.
1. Gt1-Cre-Tnos(GCS) expression casette and Actin-GUS-Tnos(AGS) acquisition of expression casette
PJ4-GCS and pJ4-AGS are this laboratory (Fujian Province Agriculture Science Academy, Institute of Biotechnology subordinate Fujian Province agricultural genetic engineering key lab) and preserve, and this plasmid is transformed in DH5 α host bacterium by heat shock, 30% glycerol stock ,-80 ℃ of cryopreservation.Its plasmid structural representation is referring to shown in Fig. 3,4.
2. the structure of plant expression vector
2.1 the structure of p1300-LF-GCSAGS carrier
PvuII/BglII double digestion plasmid pJ4-GCS reclaims the GCS fragment, and EcoRV/BglII double digestion plasmid pMD18T-LF is built into pMD18T-LF-GCS with the insertion of GCS fragment simultaneously; PJ4-AGS is behind the XhoI/ClaI double digestion, and the AGS fragment is inserted into the XhoI/ClaI site of pBluescriptKS, is built into pBlueKS-AGS; PBlueKS-AGS AGS fragment behind the XhoI/SmaI double digestion is inserted into the XhoI/StuI site of pMD18T-LF-GCS, is built into pMD18T-LF-GCSAGS; The preservation of agricultural genetic engineering key lab of pCAMBIA1300(Fujian Academy of Agricultural Sciences) this plasmid is transformed in DH5 α host bacterium 30% glycerol stock ,-80 ℃ of cryopreservation by heat shock.Behind the EcoRI/BamHI double digestion, klenow mends flat, from connecting, is built into p1300 ' then; PMD18T-LF-GCSAGS reclaims big fragment after the SalI enzyme is cut, insert the SalI site of p1300 ', is built into final plant expression vector p1300-LF-GCSAGS.
The structural representation of plasmid p1300-LF-GCSAGS is seen shown in Figure 5.
2.2 the structure of p1300-LF-AGCSGS carrier
PBlueKS-AGS AGS fragment behind the XhoI/XbaI double digestion is inserted into the SalI/XbaI site of p1300 ', is built into p1300-AGS; PMD18T-LF-GCS reclaims the LF-GCS fragment after the NcoI enzyme is cut, be inserted into the NcoI site of p1300-AGS, is built into final plant expression vector p1300-LF-AGCSGS.
The structural representation of plasmid p1300-LF-AGCSGS is seen shown in Figure 6.
The acquisition of [embodiment 2] transgenic paddy rice
1, NB culture medium prescription (minimum medium)
N6 is a large amount of: saltpetre KNO
32830 mg.L
-1, sulfate of ammoniac (NH
4)
2SO
4463 mg.L
-1, potassium primary phosphate KH
2PO
4400 mg.L
-1, sal epsom MgSO
47H
2O 185 mg.L
-1, calcium chloride CaCl22H2O 166 mg.L
-1
B5 trace: boric acid H
3BO
43 mg.L
-1, Liu Suan Manganese MnSO
4H
2O 7.58 mg.L
-1, zinc sulfate ZnSO
47H
2O 2 mg.L
-1, potassiumiodide KI 0.75 mg.L
-1, Sodium orthomolybdate Na2MoO
42H
2O 0.25 mg.L
-1, copper sulfate CuSO
45H
2O 0.025 mg.L
-1, cobalt chloride CoCl
26H
2O 0.025 mg.L
-1
Molysite: ferrous sulfate FeSO
47H
2O 27.8 mg.L
-1, disodium ethylene diamine tetraacetate Na
2EDTA 37.3 mg.L
-1
Inositol: inositol Myo-inositol 100 mg.L
-1
Organic composition: vitamin ThiamineHCl 10 mg.L
-1, pyridoxine hydrochloride PyridoxineHCl 1 mg.L
-1, nicotinic acid Niacin 1 mg.L
-1, caseinhydrolysate Casamino acids 300 mg.L
-1, glutamine Glutamine 250 mg.L
-1, proline(Pro) Proline 500 mg.L
-1, glycine Glycine 2 mg.L
-1, sucrose Sucrose 30000 mg.L
-1, agar Phytagel 2400 mg.L
-1
PH value 5.8-5.9.
Attention: organic composition can not autoclaving, must use the filter suction filtration ,-20 ℃ of preservations after the packing
2, the cultivation of callus
Young tender seed behind the water intaking rice children tassel blossom 12-15 d is peelled off clever shell, in 75% ethanol sterilization 0.5-1 min, receives solution sterilization 15-30 min with 10-30% hypochlorous acid again, in the super clean bench aseptic water washing 3-4 time, places on the aseptic filter paper and dries.Take out rataria with tweezers and dissecting needle, place inducing culture (minimum medium+2,4-D, 2 mg l
-1PH 5.8-5.9) on, 27 ° of C secretly cultivate.The bud scale etc. of excision rataria behind the 10-15 d only stays terminal cell and expands body, transfers in new inducing culture (minimum medium+2,4-D, 2 mg l
-1PH 5.8-5.9) in, per thereafter 20 d succeeding transfer culture 1 time on inducing culture, the embryo callus that obtains to grow after 3 times is rapidly made Agrobacterium-mediated Transformation.
3, agrobacterium mediation converted:
With reference to the specification sheets of BIO-RAD company electric exciter, p1300-LF-AGCSGS is transformed in the agrobacterium tumefaciens lba4404 by electrization with plant expression vector.
Get eugonic embryo callus in the sterilization culture dish, add respective concentration (OD
600Value 1-2) after agrobacterium liquid (agrobacterium tumefaciens lba4404 after above-mentioned carrier transforms) soaks 3-5 min the callus taking-up is dried, transfer then in the common substratum that is covered with an aseptic filter paper (minimum medium+2,4-D, 2 mg l
-1+ AS 100 μ M; PH 5.2) on, 28 ° of C lucifuges are cultivated 2-3 d altogether.After cultivating altogether, callus is transferred in the sterile petri dish, rinsed with sterile water is 3 times in the super clean bench, again with containing Pyocianil 250 mg l
-1Aseptic washing 1 time remove Agrobacterium to kill, blot with aseptic filter paper after taking out callus, transfer in containing Pyocianil 250 mg l
-1With hygromycin 50 mg l
-1Or PPT 15 mg l
-1Screening culture medium (minimum medium+2,4-D, 2 mg l
-1+ Pyocianil 250 mg l
-1+ hygromycin 50 mg l
-1(or PPT 15 mg l
-1, pH 5.8-5.9) and last 28 ° of C lucifuges cultivation screening resistant calli.The callus that infected is after cultivating 20-30 d on the screening culture medium, the resistant calli that the edge is grown is transferred and continue screening 1-2 time on new screening culture medium.
Transfer in division culture medium (minimum medium+KT 10 mg l at the resistant calli that the screening culture medium screening obtains
-1+ NAA 0.4 mg l
-1PH 5.8-5.9) on, 28 ° of C secretly cultivate 7-10 d, move to (14 h/d) cultivation under the illumination then, grow green point subsequently gradually, are divided into seedling at last.After seedling breaks up fully, transferring in root media, (1/2 MS inorganic salt (referring to that inorganic salt content reduces by half)+MS organic composition (are meant the organic composition in the NB culture medium prescription (minimum medium) of [embodiment 2], comprise vitamin ThiamineHCl 10 mg.L
-1, pyridoxine hydrochloride PyridoxineHCl 1 mg.L
-1, nicotinic acid Niacin 1 mg.L
-1, caseinhydrolysate Casamino acids 300 mg.L
-1, glutamine Glutamine 250 mg.L
-1, proline(Pro) Proline 500 mg.L
-1, glycine Glycine 2 mg.L
-1, sucrose Sucrose 30000 mg.L
-1, agar Phytagel 2400 mg.L
-1)+hygromycin 30 mg l
-1(or PPT 10 mg l
-1); PH 5.8-5.9) 28 ° of C, illumination 14 h/d cultivation 14-21 d is taken root and is screened.Behind the resistance seedling flush away root substratum that obtains after taking root, move in 1/10 MS nutritive medium water planting 3-5 d and can grow new root, transplant in the greenhouse then or the paddy field, solarium.
4, Totomycin detects
Get seedling stage greater than one month green blade, the about 5-10cm of blade length, otch is downward, be soaked in the Totomycin aqueous solution of 50mg/L, the overcoat freshness protection package prevents excessive vaporization, kept 3-4 days in room temperature. take out and observe the performance that blade is submerged the position, the blade of transgenic positive shows hygromycin resistance because of having hygromycin gene, is embodied in blade no change or a small amount of jaundice; The blade of the negative strain of transgenosis does not have hygromycin resistance, and tangible rust staining or edge mildew appear in blade.The result is referring to shown in Figure 7.
5, PCR detects
Extract plant genome DNA, adopt the CTAB method, process is as follows:
Get the rice leaf that the step 3 of about 0.1g grows and in liquid nitrogen, clay into power, an amount of powdered material is transferred in the 1.5mlEP pipe.1.5 * CTAB the damping fluid that adds 600 μ l preheatings (95 ℃), 65 ℃ of temperature are bathed 45min then.Take out the EP pipe, add 600 μ l chloroforms after being cooled to room temperature: primary isoamyl alcohol (24:1) mixes, and the centrifugal 10min of 10000rpm takes out the back and draws supernatant liquor in another new EP pipe then.The Virahol that adds 2/3 volume mixes, and ice bath to DNA filament occurs.Centrifugation DNA outwells supernatant liquor a little.Add 600 μ l75% washing with alcohol, place 30min.Outwell supernatant liquor, dry up DNA under the room temperature.Add 100 μ l ddH
2The O dissolving DNA.The total dna content of electrophoresis detection plant.PCR detects hyg resistant gene in the regeneration plant, synthetic a pair of primer, and upstream primer is: 5 '-TACACAGCCATCGGTCCAGA-3 ', downstream primer is: 5'-TAGGAGGGCGTGGATATGTC-3', the PCR reaction system sees Table 1.
Table 1 PCR reaction system
The PCR response procedures: 94 ℃, 5 minutes; 94 ℃, 1 minute, 56 ℃, 1 minute, 72 ℃, 1 minute, 35 circulations; 72 ℃ were extended 10 minutes.
The result that transgenosis PCR detects is referring to shown in Figure 8, and the sample that can amplify the 845bp band is the transgenic paddy rice positive plant, and the result shows that sample 1 and 2 is the transgenic positive plant.
Expression of exogenous gene analysis in [embodiment 3] transfer-gen plant
1. change the expression analysis of gus gene in the p1300-LF-GCSAGS plasmid transgenic paddy rice
GUS detects:
With transgenic plant material (blade, root, stem etc., embodiment 2 step 3) are thinly sliced, put into freshly prepared X-Gluc staining fluid (Promega, USA) in, 37 ℃ the insulation 1-12 hour.After treating fully dyeing, use 75%(v/v) alcohol decolouring 2-3 time, white to control material (wild-type plant) one-tenth.Pollen and seed since bag by stratum corneum with plant skin, the X-Gluc staining fluid is difficult to soak into, for abundant dyeing, generally add the X-Gluc dye liquor after, vacuumized earlier 5-10 minute, carry out the GUS tissue staining by above program again.
Gt1 is an endosperm specificity expression promoter, and in changeing p1300-LF-GCSAGS plasmid transgenic paddy rice, the recombinase of Gt1 control is specifically expressing in endosperm only in theory, thus the deletion foreign gene.Therefore, before the transgenic plant endosperm development all should be able to detect the great expression of gus reporter gene (control of actin promotor) in a organized way.Choosing respectively changes T0 in the p1300-LF-GCSAGS plasmid transgenic paddy rice and carries out the GUS tissue staining for root, stem, leaf and the materials such as clever shell and flower pesticide of plant, as shown in Figure 9, above each organ of transfer-gen plant with organize equal GUS stained positive, show and expressed gus gene.
2. change the expression analysis of gus gene in the p1300-LF-AGCSGS plasmid transgenic paddy rice
In theory in changeing p1300-LF-AGCSGS plasmid transgenic paddy rice, because the insertion deactivation of recombinase expression cassette before the endosperm specificity expression of Gt1 control starts, the deletion effect does not take place.Therefore, before the transgenic plant endosperm development should all can not detect the expression of gus reporter gene (control of actin promotor) in a organized way.Choosing respectively changes T0 in the p1300-LF-AGCSGS plasmid transgenic rice plant and carries out the GUS tissue staining for root, stem, leaf and the materials such as clever shell and flower pesticide of plant, as shown in figure 10, above each organ of transfer-gen plant with organize equal GUS dyeing negative, show and do not express gus gene.
The deletion analysis of foreign gene in [embodiment 4] transfer-gen plant endosperm
1, changes the deletion analysis of gus gene in the p1300-LF-GCSAGS plasmid transgenic paddy rice
After endosperm development, cre enzyme under the endosperm specificity promoter Gt1 control begins to express, thereby start the locus specificity reorganization deletion system of cre enzyme mediation, delete the nucleotide sequence between the two recognition sequences of two loxPFRT, thereby deleted the gus reporter gene expression cassette.Therefore, in the commentaries on classics p1300-LF-GCSAGS plasmid transgenic rice plant that special deletion takes place, the endosperm of seed part can not be expressed gus gene, and the GUS coloration result blueness can not occur; And the embryo of seed, because do not delete, so still can express gus reporter gene, the GUS coloration result shows as blueness.As shown in figure 11, the seed that non-transgenic Japan is fine, the all not painted intensification of its embryo and endosperm, change the seed of p1300-LF-GCSAGS plasmid transgenic paddy rice, its painted intensification in embryo position, and the not painted yet intensification in endosperm position, its result shows that efficient special deletion effect has taken place at the seed endosperm position of changeing p1300-LF-GCSAGS plasmid transgenic paddy rice.
2, change the deletion analysis of gus gene in the p1300-LF-AGCSGS plasmid transgenic paddy rice
After endosperm development, cre enzyme under the endosperm specificity promoter Gt1 control begins to express, thereby start the locus specificity reorganization deletion system of cre enzyme mediation, delete the nucleotide sequence between the two recognition sequences of two loxPFRT, thereby deleted cre recombinase gene expression cassette, removed the gene disruption effect.Therefore, in the commentaries on classics p1300-LF-AGCSGS plasmid transgenic rice plant that special deletion takes place, the endosperm of seed part energy normal expression gus gene, the GUS coloration result occurs blue; And the embryo of seed, because do not delete, so still can not express gus reporter gene, the GUS coloration result shows as does not have blue the appearance.As shown in figure 12, the seed that non-transgenic Japan is fine, the seed of p1300-LF-AGCSGS plasmid transgenic paddy rice is changeed in all not painted intensification of its embryo and endosperm, the not painted intensification in its embryo position, the then painted intensification in endosperm position, its result shows that efficient special deletion effect has taken place at the seed endosperm position of changeing p1300-LF-AGCSGS plasmid transgenic paddy rice.
The genetic stability analysis of the specificity of foreign gene deletion in [embodiment 5] transfer-gen plant endosperm
Select the seed that the endosperm of some amount has been grown in the different generations of the transfer-gen plant that obtains, the analysis of GUS tissue staining is carried out in rip cutting then, and method is the same.T0 for the time choose the positive strain system that the deletion effect takes place the offspring continue the sowing plantation, T1 for the time choose the strain system that the full positive or positive ratio meet 3:1 approximately and continue the sowing plantation, T2 for the time can screen the positive strain system of the generation endosperm-specific deletion of isozygotying.Table 2 is that transgenic line genetic stability from generation to generation detects and identifies, the result shows and obtained the transgenic line that the deletion of endosperm generation specificity and this proterties can stably entail the offspring.
Table 2 transgenic line genetic stability from generation to generation detects to be identified
Above detailed description of the present invention does not limit the present invention, and those skilled in the art can make various changes and distortion according to the present invention, and these changes and distortion all should belong to the scope of claims of the present invention.
Claims (8)
1. a molecule deletion strategy is cultivated the method for the transgenic paddy rice of no foreign gene in the polished rice, comprises following step:
1) structure is based on the rice conversion carrier of the locus specificity recombination system of endosperm specificity expression control; Described rice conversion carrier contains following element: the recombinase recognition sequence; External source destination gene expression box; The recombinase gene expression cassette of endosperm specificity expression promoter control and the antibiotics resistance expression cassette that is used for the rice conversion screening;
2) utilize this rice conversion carrier rice transformation callus, screening, differentiation produce transgenic rice plant;
3) plantation transgenic rice plant, detect external source goal gene different sites in transfer-gen plant, comprise the expression level in root, stem, leaf, embryo and the endosperm, obtain the external source goal gene and in endosperm, deleted by specificity, and in other tissue equal stable integration and the transgenic rice plant that efficiently expresses;
4) plantation transgenic paddy rice strain of future generation system, the further integration and the expression of Molecular Detection foreign gene, acquisition external source transgenic structure in endosperm is deleted, and the external source transgenic structure can stable integration and the transgenic paddy rice strain system of expressing and can genetic stability in other tissue.
2. molecule deletion strategy according to claim 1 is cultivated the method for the transgenic paddy rice of no foreign gene in the polished rice, it is characterized in that: described recombinase recognition sequence is meant can be by site-specific recombinase specific recognition and the sequence of shearing.
3. molecule deletion strategy according to claim 1 is cultivated the method for the transgenic paddy rice of no foreign gene in the polished rice, it is characterized in that: described external source goal gene is meant a kind of in adversity gene, anti insect gene, disease-resistant gene, anti-herbicide gene, anti-ageing gene, the quality-improving gene or fusion or the multivalent genetic be made up of said gene.
4. molecule deletion strategy according to claim 1 is cultivated the method for the transgenic paddy rice of no foreign gene in the polished rice, it is characterized in that: described endosperm specificity expression promoter be meant in endosperm specific expressed and at other position expression promoter not.
5. molecule deletion strategy according to claim 4 is cultivated the method for the transgenic paddy rice of no foreign gene in the polished rice, it is characterized in that: described endosperm specificity expression promoter derives from the wild-type promotor of plant species or the promotor after the modification.
6. molecule deletion strategy according to claim 1 is cultivated the method for the transgenic paddy rice of no foreign gene in the polished rice, it is characterized in that: described recombinase gene is that segmental any wild-type recombinase gene between sequence, the recombinase gene behind series jump type recombinase gene or the sequence modification also be sheared, be deleted to expression product can two site-specific sequences of specific recognition.
7. molecule deletion strategy according to claim 1 is cultivated the method for the transgenic paddy rice of no foreign gene in the polished rice, and it is characterized in that: described rice callus tissue is from long-grained nonglutinous rice or japonica rice.
8. transgenic paddy rice strain system, be the transgenic paddy rice strain system that utilizes the described rice conversion carrier of claim 1 to cultivate to obtain and be the rice material that the parent is obtained by the sexual hybridization transformation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101974356A CN102286522A (en) | 2011-07-15 | 2011-07-15 | Method for cultivating transgenic rice without foreign gene in white rice through molecular deletion strategy |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2011101974356A CN102286522A (en) | 2011-07-15 | 2011-07-15 | Method for cultivating transgenic rice without foreign gene in white rice through molecular deletion strategy |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN102286522A true CN102286522A (en) | 2011-12-21 |
Family
ID=45333279
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN2011101974356A Pending CN102286522A (en) | 2011-07-15 | 2011-07-15 | Method for cultivating transgenic rice without foreign gene in white rice through molecular deletion strategy |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN102286522A (en) |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107099531A (en) * | 2017-05-23 | 2017-08-29 | 华南农业大学 | A kind of anther specific expression promoter PV4 and its application |
| CN107475286A (en) * | 2017-08-04 | 2017-12-15 | 福建省农业科学院生物技术研究所 | A kind of green safe marker-free transgenic rice cultivating method |
| CN111655027A (en) * | 2017-12-22 | 2020-09-11 | 科沃施种子欧洲股份两合公司 | Regeneration of plants in the presence of histone deacetylase inhibitors |
| CN114480478A (en) * | 2022-03-07 | 2022-05-13 | 武汉轻工大学 | Recombinant vector for specifically rejecting exogenous genes in endosperm and application of recombinant vector in construction of transgenic corn without exogenous genes |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1292416A (en) * | 2000-08-25 | 2001-04-25 | 林忠平 | Molcular method for removing specific tissue of transgenic plant or target gene in organ by utilizing location recombination system |
| CN1603408A (en) * | 2003-09-30 | 2005-04-06 | 中国农业大学 | Method for time delete of specific exogenous gene using induction site-specific recombination system |
| CN1769446A (en) * | 2005-10-11 | 2006-05-10 | 西南大学 | Gene deleting system for transgenic plant safety control and plant expression vector containing same |
-
2011
- 2011-07-15 CN CN2011101974356A patent/CN102286522A/en active Pending
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1292416A (en) * | 2000-08-25 | 2001-04-25 | 林忠平 | Molcular method for removing specific tissue of transgenic plant or target gene in organ by utilizing location recombination system |
| CN1603408A (en) * | 2003-09-30 | 2005-04-06 | 中国农业大学 | Method for time delete of specific exogenous gene using induction site-specific recombination system |
| CN1769446A (en) * | 2005-10-11 | 2006-05-10 | 西南大学 | Gene deleting system for transgenic plant safety control and plant expression vector containing same |
Non-Patent Citations (4)
| Title |
|---|
| 《Plant Biotechnology Journal》 20070331 Keming Luo 等 'GM-gene-deletor'：fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants 第5卷, 第2期 * |
| 《中国优秀硕士学位论文全文数据库 农业科技辑》 20081215 邱淑萍 利用Cre/loxp系统定点删除转基因水稻胚乳中外源基因的初步研究 , 第2008/12期 * |
| KEMING LUO 等: "‘GM-gene-deletor’：fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants", 《PLANT BIOTECHNOLOGY JOURNAL》, vol. 5, no. 2, 31 March 2007 (2007-03-31) * |
| 邱淑萍: "利用Cre/loxp系统定点删除转基因水稻胚乳中外源基因的初步研究", 《中国优秀硕士学位论文全文数据库 农业科技辑》, no. 200812, 15 December 2008 (2008-12-15) * |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107099531A (en) * | 2017-05-23 | 2017-08-29 | 华南农业大学 | A kind of anther specific expression promoter PV4 and its application |
| CN107099531B (en) * | 2017-05-23 | 2020-04-17 | 华南农业大学 | Anther specific expression promoter PV4 and application thereof |
| CN107475286A (en) * | 2017-08-04 | 2017-12-15 | 福建省农业科学院生物技术研究所 | A kind of green safe marker-free transgenic rice cultivating method |
| CN111655027A (en) * | 2017-12-22 | 2020-09-11 | 科沃施种子欧洲股份两合公司 | Regeneration of plants in the presence of histone deacetylase inhibitors |
| US11700805B2 (en) | 2017-12-22 | 2023-07-18 | KWS SAAT SE & Co. KGaA | Regeneration of plants in the presence of histone deacetylase inhibitors |
| CN114480478A (en) * | 2022-03-07 | 2022-05-13 | 武汉轻工大学 | Recombinant vector for specifically rejecting exogenous genes in endosperm and application of recombinant vector in construction of transgenic corn without exogenous genes |
| CN114480478B (en) * | 2022-03-07 | 2024-04-19 | 武汉轻工大学 | Recombinant vector for specifically rejecting exogenous genes in endosperm and application of recombinant vector in construction of exogenous gene-free transgenic corn |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| EP3252162B1 (en) | Method for conducting site-specific modification on entire plant via gene transient expression | |
| ES2750530T3 (en) | Means and procedures to produce yield in plants | |
| US11674146B2 (en) | Gene combination and use thereof | |
| CN102174568B (en) | Method for in-situ transgenosis of corn mature embryo | |
| CN102286522A (en) | Method for cultivating transgenic rice without foreign gene in white rice through molecular deletion strategy | |
| CN103060369B (en) | Hybrid crop transgenic safety control method and gene deletion system for implementing same | |
| CN107881172B (en) | Stress inducible promoter, stress inducible promoter plant expression vector and method for inducing expression of target gene | |
| CN105200080B (en) | A kind of efficient fast and stable gene transformation method of tomato | |
| CN106916818B (en) | drought-induced promoter, preparation method thereof, recombinant expression vector and transformant | |
| JP2014527406A (en) | A method for osmotic interference processes in male meiotic cells | |
| CN107058317A (en) | A kind of pollen specific promoter and its application | |
| CN106834345A (en) | A kind of method that polygenes superposition cotransformation improves rape synthesis resistance | |
| CN102177242A (en) | Change in plant architecture | |
| Okeyo-Ikawa et al. | In planta seed transformation of Kenyan cowpeas (Vigna unguiculata) with P5CS gene via Agrobacterium tumefaciens. | |
| CN102174571B (en) | Method for culturing anti-greensickness cotton by using artificially synthesized antimicrobial peptide gene | |
| Mogali et al. | High efficiency transformation protocol for two Indian cotton (Gossypium hirsutum) varieties via pollen tube pathway | |
| CN103343139B (en) | Novel method for enhancing drought resistance of plants | |
| CN101979550B (en) | Cloning and application of maize phosphoenolpyruvate carboxykinase gene promoter | |
| CN104630237B (en) | A kind of appropriateness delays plant senescence and the fusion for improving stress resistance and its application | |
| CN104098661B (en) | The albumen relevant to rice stress-tolerance and chlorophyll content of rice and encoding gene thereof and application | |
| Yesmin et al. | Agrobacterium-mediated transformation of eggplant (Solanum melongena L.) using cotyledon explants | |
| US20240052002A1 (en) | Tomato-derived sijul gene regulating phloem development and use thereof | |
| CN116855528A (en) | LoZAT12 gene for regulating and controlling lily flower senescence and application thereof | |
| Almerei | Agrobacterium-mediated transformation of Syrian maize with anti-stress genes | |
| Clapham et al. | Transformation of Norway spruce (Picea abies) by particle bombardment |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| C12 | Rejection of a patent application after its publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20111221 |