CN102102108A - Method for cultivating efficient selected-marker-free transgenic crop by using double T-DNA+1 vectors - Google Patents
Method for cultivating efficient selected-marker-free transgenic crop by using double T-DNA+1 vectors Download PDFInfo
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Abstract
The invention provides a method for cultivating an efficient selected-marker-free transgenic crop by using double T-DNA+1 vectors. The method comprises the following steps of: constructing double T-DNA+1 plant expression vectors carrying a promoter and a specific gene between two T-DNA structural domains in which a target gens and a selected marker gene are positioned respectively; transforming the constructed double T-DNA+1 vectors into a target crop to obtain a transformed plant; screening a transformed strain in which the target gene and the selected marker gene are integrated in different chromosomes of the same plant or at different positions of the same chromosome according to the expression property of a specific gene and molecular detection; and obtaining the selected-marker-free transgenic crop according to a genetic separation ratio.
Description
Technical field
The present invention designs a kind of foundation of efficient non selecting sign transgene farm crop novel method
Background technology
Food problem is social harmony, stable important substance basis, is one of key problem of being concerned about of each country.Solve the grain security problem and be the prerequisite that ensures the efficient and Sustainable development of national economy.In recent years, along with deterioration, the minimizing of cultivated area, the Increase of population of environment, the food problem of China was faced with increasing challenge.Rely on traditional breeding method can not solve the situation of more and more sternly completing merely, therefore utilize genetic engineering means improvement variety of crops, raising crop yield, the good crop varieties of cultivation to have great importance.
From nineteen eighty-three the first transgenic plant cultivate successfully, transgenic technology has obtained widespread use.Transgenic plant have expanded to 35 genus, more than 120 species that comprise cash crop, food crop, vegetables, flowers, medicinal plant, fruit, trees and herbage etc. now.But along with the quickening that the genetically modified crops commercialization is produced, some potential problems are progressively appeared in one's mind out, selected marker to the safety issue of environment and food by the problem of extensive concern and discussion.Utilize methods such as agriculture bacillus mediated, particle gun that foreign gene is transferred in the plant materials, carry out assisting sifting by selected marker and obtain changing over to goal gene moral transgenic positive plant.But, selectable marker gene is neither the also non-goal gene of the intravital gene of plant, this unnecessary existence may cause some potential hazards, mainly show: 1, present used selectable marker gene mostly is antibiotics resistance gene, such as kalamycin resistance gene, hygromycin gene etc., these marks with the transgenic product commercialization after, transfer in the pathogenic micro-organism, fears are entertained that can influence the resistance that strengthens pathogenic micro-organism, causes antibiotic inefficacy.2, some marker gene is the Herbicid resistant marker gene, hybridizes with nearly edge ruderal if having the genetically modified crops of this kind resistant maker gene, and this resistant maker gene is parallel to be transferred in the weeds, the superweed that might develop immunity to drugs brings great harm to agriculture production.3, people suspect that genetically modified marker gene and product thereof may produce certain harm to the human or animal.4, marker gene floats in the environment and can disturb ecological stability, and environment is caused certain harm.Therefore, the transgenosis transformation system of setting up efficient high-throughput marker-free is one of the important goal of transgenic plant genetic engineering now and urgent task, also is the hot issue that global plant breeding scholar is concerned about.
At present, the method that obtains the non selecting sign transgene plant mainly contains locus specificity reorganization, transposon system and cotransformation system, and wherein the cotransformation system is that research is many, the application mature methods.Locus specificity reorganization is to utilize reorganization two weak points of recombinase catalysis, between specific dna sequence, rejects selectable marker gene, thereby obtains the genetically modified crops of marker-free.Its principle is, selectable marker gene is inserted between two specificity tumor-necrosis factor glycoproteinss, transform plant after, specific expressed more specially site recombinase gene is rejected selective marker by the locus specificity reorganization.Locus specificity recombination system commonly used mainly contains in plant genetic engineering now,: yeast FLPPFRTs site-specific recombination system, the RPRS system of Zygosaccharomyces rouxii, the CreP lox system of phage P1, and the Gin recombinase system of phage Mu.But, utilize this method to obtain the transgenic plant of marker-free, generally need twice transformation or sexual hybridization to obtain recombinase, make the procurement process of non selecting sign transgene crop too complicated, be unfavorable for the commercial development of genetically modified crops.The regulatable site specific system of latest developments has been simplified the process that marker-free gene is cultivated, but its mechanism of action still needs further research, and actually operating also has much room for improvement.The transposon system can utilize the special seat enzyme from a position transfer on the plant chromosome to another position, utilize this reorganization characteristic can reject the selective marker of genetically modified crops.AcPDs and SpmPdSpm are two transposon families of studying the most deeply.But, because most of other position that can be inserted again again after cut by the transposable element of modified (as having inserted the ipt gene) in the genome has only the cell of swivel base error could form the normal genetically modified crops of phenotype.The method efficiency ratio of this rejecting selectable marker gene is lower, is restricted in the commercialization process of genetically modified crops.Initial cotransformation system is meant with two plasmids independently, one of them contains selectable marker gene, another contains goal gene, transform the purpose cell simultaneously, two plasmids can be integrated into vegetable cell simultaneously, cultivate the transformed plant offspring of this common integration, through the genetic recombination of perfect stage, selectable marker gene separates with goal gene, thus only contain goal gene and not with the transformed plant of selectable marker gene.Cotransformation system requirements cotransformation frequency wants the DNA of height and cotransformation to be in not chain state in recipient cell..In the actually operating, the common integrating frequency of transgenosis that is arranged in different conversion carriers is often lower, and often is incorporated into the same site of acceptor gene group.In order to overcome the above problems, the investigator has made up the super binary vector that contains two T-DNA again, is about to selectable marker gene and goal gene and is inserted into respectively in the same plasmid in two separate T-DNA districts.This super binary vector makes the low problem of common integration efficiency obtain alleviation.But, the double T-DNA transformed acceptor cell, the common integration transformant that obtains is most of for being incorporated into the same site of acceptor gene group, being incorporated in the descendant inheritting separation of this situation can not separate the goal gene transformed plant that obtains marker-free, how to reject this integration with the easiest method is one step of key of improving cotransformation efficient, also is the necessary ways that the transgenic technology of marker-free is introduced to the market.
The present invention has improved traditional double T-DNA carrier, between double T-NDA zone, add a promotor and specific gene, called after double T-DNA+1 carrier, like this when double T-DNA is incorporated into chromosomal same position jointly, this specific gene will be expressed under the startup of promotor, can utilize the characteristic of this specific gene directly to reject this transfer-gen plant by easy method, the plant of selecting goal gene and selectable marker gene to be incorporated into coloured differently body or same karyomit(e) different positions is altogether cultivated, separate the transfer-gen plant that obtains marker-free by descendant inheritting, manpower, material resources and financial resources have been saved so greatly, for the commercialization of non selecting sign transgene has advanced an important step.
Summary of the invention
An object of the present invention is to set up a kind of transgene carrier of efficient marker-free, called after double T-DNA+1 carrier, this carrier comprises two T-DNA functional domains, carry selectable marker gene and goal gene respectively, a promotor and specific gene are carried in zone between these two T-DNA structural domains.The marker gene of this carrier can be any marker gene that can be used for Plant Transformation, as: Totomycin transferase gene, kalamycin resistance gene, phosphine silk mycin transferase gene etc., goal gene comprise any gene that has production application to be worth: such as gene of anti insect gene, drought resisting base, cold-resistant gene, antiweed or the like.Promotor comprises the endogenous promotor of any plant materials, exogenous promoter, constitutive promoter, induces sexual type promotor, tissue-specific promoter, organ inducible promoter.Specific gene comprises that any lethal gene and reporter gene are such as red fluorescent protein gene, green fluorescence protein gene, blue fluorescent protein gene and gus gene or the like.
Another object of the present invention provides a kind of genetically modified crops system of utilizing this double T-DNA+1 carrier to obtain marker-free, and specific implementation process comprises:
1, make up and carry the double T-DNA carrier of corresponding goal gene and change Agrobacterium over to.
2, utilize agriculture bacillus mediated gene transformation method, the plant expression vector that builds is transformed target plant
3, carry out molecule and phenotype analytical to obtaining T0 for transgenic seedling, reject the common integration transgenosis offspring of containing selectable marker gene and specific gene, select to contain selectable marker gene and goal gene but the transgenic progeny that do not contain specific gene is cultivated.
4, the T1 that the plantation screening obtains selects only to contain the transfer-gen plant of goal gene for transgenic line by PCR.Further separate than the transgenosis homozygous lines that obtains only to contain goal gene by heredity.
Further set forth the present invention below in conjunction with embodiment, and do not constitute restriction claim scope of the present invention.
Description of drawings
Fig. 1 expression of plants double T-DNA+1 carrier KT350
Fig. 2 expression of plants double T-DNA carrier KT349
T1 is for the seed phenotypes analysis, wherein for Fig. 3 KT350 transgenosis: A is that goal gene and marker gene are incorporated into the T1 of karyomit(e) same position altogether for seed; B is that goal gene and marker gene are incorporated into the T1 of karyomit(e) different positions altogether for seed.
Embodiment 1: the structure of double T-DNA+1 carrier KT350
The KT350 structure is seen accompanying drawing one, is double T-DNA+AsRED gene, and a double T-DNA is a selectable marker gene hptII hygromycin gene, and another double T-DNA is salt resistant gene KY1, and specific gene is red fluorescent protein gene A sRED.With this breadboard pKAT-1 is template, expands the 35s-AsRed-Tnos zone, and imports the BamHI site, is building up on the T-easy carrier by ligation, and further subclone is building up on the carrier KT349 (structure is seen accompanying drawing two) that contains double T-DNA.Enzyme in the building process is cut, is connected, recovery, purifying equimolecular technology, is all undertaken by the method in the molecular cloning handbook.(molecular cloning: laboratory manual, Sambrook et al, New York:Cold Spring harbor laboratory 1989press)
Embodiment two: the acquisition of KT350 rice transformation seedling
The paddy rice mature seed that shells is earlier with 70% alcohol immersion 1-2 minute, soaked 30 minutes with 0.1% mercuric chloride then, carry out surface sterilization (be preferably on the shaking table and carry out), aseptic water washing 3-4 time, again seed is placed on the aseptic filter paper behind the suck dry moisture, be placed on the mature embryo callus of induce substratum 26 ℃ of dark cultivations.Selecting the yellowish callus of color and luster cultivates altogether.
The Agrobacterium AGL0 that contains the KT350 carrier is rule containing on the YM flat board of 50mg/L Kanamycin, 28 ℃ dark culturing 2-3 days, collect the Agrobacterium thalline with a metal spoon, it is suspended in the common cultivation CM liquid nutrient medium, adjust cell concentration to OD600 be 0.3-0.5, add Syringylethanone, making its final concentration is 100mM, is the agrobacterium suspension that common cultivation rice transformation is used.
Select state better the callus of (succeeding transfer culture 5-7 days, color and luster yellowish) put into the aseptic triangular flask of 100ml, add an amount of agrobacterium suspension (assurance have enough bacterium liquid to contact get final product) with material, transform.Callus after cultivating altogether is placed on the screening culture medium that contains the 50mg/l Totomycin 26 ℃ of dark cultivations.In the resistant calli that grows after the two-wheeled screening, the resistant calli of selecting the milk yellow densification goes on the division culture medium that contains the 50mg/L Totomycin and cultivates.When the bud of resistant calli differentiation grows to about 2cm, seedling is moved on on the root media, cultivate about two weeks.Select the seedling of high about 10cm, well developed root system,, in the greenhouse, transplant and bury with warm water flush away substratum.
Embodiment three: KT350 transgenosis T0 is for the screening and the acquisition of seedling
The transgenic paddy rice seedling with hygromycin resistance that embodiment two obtains has three kinds of situations, a kind ofly changes the paddy rice seedling over to for the selectable marker gene list; Second kind of transgenic paddy rice seedling that is incorporated into the karyomit(e) same position for selectable marker gene and KY1 gene altogether, in this case, AsRED also is incorporated on the karyomit(e), the seed of paddy rice presents redness and sees accompanying drawing three, the third is the transgenic paddy rice seedling that selectable marker gene and KY1 gene are incorporated into different positions on the karyomit(e) altogether, and this strain is to be that the offspring can separate the strain system that obtains the non selecting sign transgene seedling.Generally speaking, second kind transgenic line will account for about 70% of transgenic seedling, transformation system is in the past rejected this transformation system will arrive the T1 seedling stage in generation, pass through round pcr, screen the strain system that only contains target gene KY1 but do not contain selectable marker gene, such screening not only workload is big, and the cycle is long, is unfavorable for the commercialization of transgenic technology.Among the present invention, can utilize the expression of AsRED, directly reject seed for the red T1 seed in generation, significantly reduce workload, the efficient commercialization of render transgenic becomes possibility.
The embodiment work well afoot of the present invention aspect soybean, cotton, corn, Chinese sorghum.
Claims (6)
1. method of cultivating efficient non selecting sign transgene crop may further comprise the steps:
A) make up goal gene and selectable marker gene and lay respectively at two independent T-DNA structural domains, between two T-DNA structural domains, take double T-DNA+1 plant expression vector of a promotor and specific gene.
B) double T-DNA that builds+1 carrier is transformed the purpose farm crop, obtain transformed plant.
C) by the expression characterization and the Molecular Detection of specific gene, screening goal gene and selectable marker gene are incorporated into the transgenic line of same plant coloured differently body and same karyomit(e) different positions.
D) separate than the genetically modified crops that obtain marker-free by heredity.
2. require describedly according to right 1, goal gene is any anti insect gene, adversity gene, anti-ageing gene, anti-herbicide gene, yield improvement gene, quality-improving gene or by the fusion gene or the multivalent genetic of said gene combination.
3. require described according to right 1, promotor between two T-DNA structural domains is any plant endogenous promotor and exogenous promoter, comprises constitutive promoter, induces the sexual type promotor, tissue-specific promoter, organ inducible promoter, adjustment type promotor or by the fusion fragment of adjusted and controlled territory and promotor.
4. require describedly according to right 1, the specific gene between two T-DNA structural domains is all lethal gene or reporter genes, and these reporter genes comprise red fluorescent protein gene, blue fluorescent protein gene, green fluorescence protein gene, gus gene etc.
5. require describedly according to right 1, it is characterized in that the purpose farm crop comprise all monocotyledonss and dicotyledons.
6. require describedly according to right 5, monocotyledons is paddy rice, corn, wheat, Chinese sorghum, millet, barley, oat, rye, and dicotyledons is cotton, soybean, tobacco, rape, tomato.
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Cited By (4)
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| CN103468792A (en) * | 2013-07-11 | 2013-12-25 | 江西省农业科学院水稻研究所 | Method used for detecting rice double T-DNA transgenic non-linked integration by molecular marker |
| CN103952426A (en) * | 2014-04-28 | 2014-07-30 | 四川农业大学 | Double reporter gene contained binary T-DNA (transferred deoxyribonucleic acid) carrier as well as construction method and application of double reporter gene contained binary T-DNA carrier |
| CN104830897A (en) * | 2015-05-04 | 2015-08-12 | 安徽师范大学 | Novel efficient selectable marker-free corn transgenic vector system construction method and application thereof |
| CN108588114A (en) * | 2018-05-03 | 2018-09-28 | 华中农业大学 | A kind of selection markers autonomous control rejecting transgene carrier and its application in corn marker-free transgenic breeding |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103468792A (en) * | 2013-07-11 | 2013-12-25 | 江西省农业科学院水稻研究所 | Method used for detecting rice double T-DNA transgenic non-linked integration by molecular marker |
| CN103468792B (en) * | 2013-07-11 | 2015-11-25 | 江西省农业科学院水稻研究所 | The molecular mark detection method of the non-chain integration of rice double T-DNA transgenic |
| CN103952426A (en) * | 2014-04-28 | 2014-07-30 | 四川农业大学 | Double reporter gene contained binary T-DNA (transferred deoxyribonucleic acid) carrier as well as construction method and application of double reporter gene contained binary T-DNA carrier |
| CN103952426B (en) * | 2014-04-28 | 2016-06-08 | 四川农业大学 | A kind of double T-DNA carrier containing double; two reporter genes and construction method thereof and application |
| CN104830897A (en) * | 2015-05-04 | 2015-08-12 | 安徽师范大学 | Novel efficient selectable marker-free corn transgenic vector system construction method and application thereof |
| CN108588114A (en) * | 2018-05-03 | 2018-09-28 | 华中农业大学 | A kind of selection markers autonomous control rejecting transgene carrier and its application in corn marker-free transgenic breeding |
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