CN102102108B - 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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- CN102102108B CN102102108B CN201010567337.2A CN201010567337A CN102102108B CN 102102108 B CN102102108 B CN 102102108B CN 201010567337 A CN201010567337 A CN 201010567337A CN 102102108 B CN102102108 B CN 102102108B
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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 selected-marker-free transgenic farm crop novel method
Background technology
Food problem is social harmony, stable important substance basis, is one of key problem of each country care.Solving Food Security is the prerequisite ensureing the efficient and Sustainable development of national economy.In recent years, along with the deterioration of environment, the minimizing of cultivated area, the growth of population, the food problem of China is faced with increasing challenge.The traditional breeding method of simple dependence can not solve the situation of more and more sternly completing, and therefore utilizes genetic engineering means to improve variety of crops, improves crop yield, cultivates excellent crop varieties and have great importance.
From nineteen eighty-three the first transgenic plant cultivate successfully, transgenic technology is widely applied.Present 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.But along with the quickening of genetically modified crops merchandized handling, 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 the methods such as agriculture bacillus mediated, particle gun to be transferred in plant materials by foreign gene, carry out assisting sifting by selected marker and obtain proceeding to goal gene moral transgenic positive plant.But, selectable marker gene is neither gene also non-goal gene in plant materials, this unnecessary existence may cause some potential hazards, be mainly manifested in: 1, current selectable marker gene used mostly is antibiotics resistance gene, such as kalamycin resistance gene, hygromycin gene etc., these marks are with after transgenic product commercialization, transfer in pathogenic micro-organism, fears are entertained that can affect the resistance strengthening pathogenic micro-organism, causes antibiotic inefficacy.2, some marker gene is herbicide resistance markers's gene, if hybridized with nearly edge ruderal with the genetically modified crops of this kind of resistant maker gene, this resistant maker gene is parallel to be transferred in weeds, likely can develop immunity to drugs superweed, brings great harm to agriculture production.3, people suspect that genetically modified marker gene and product thereof may produce certain harm to human or animal.4, marker gene floats in environment and can disturb ecological stability, and causes certain harm to environment.Therefore, the transgenosis transformation system setting up efficient high-throughput marker-free is one of the important goal and urgent task of transgenic plant genetic engineering now, is also the hot issue that global plant breeding scholar is concerned about.
At present, the method obtaining non selecting sign transgene plant mainly contains Site-specific recombinase, Transposon System and cotransformation system, and wherein cotransformation system is the method that research is many, Application comparison is ripe.Site-specific recombinase be utilize that recombinase catalysis two is short, restructuring between specific dna sequence, reject selectable marker gene, thus obtain the genetically modified crops of marker-free.Its principle is, selectable marker gene is inserted between two Species specific sequences, after conversion of plant, more specific expressed specially site recombinase gene, reject selective marker by Site-specific recombinase.Site-specific recombination system conventional in plant genetic engineering now mainly contains,: 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 the method to obtain the transgenic plant of marker-free, generally need twice transformation or sexual hybridization to obtain recombinase, make the procurement process of selected-marker-free transgenic crop too complicated, be unfavorable for the commercial development of genetically modified crops.The regulatable site specific system of latest developments simplifies the process of marker-free gene cultivation, but its mechanism of action still needs further research, and actually operating also has much room for improvement.Transposon System can utilize special seat enzyme from the position transfer of plant chromosome to another position, utilizes this restructuring characteristic can reject the selective marker of genetically modified crops.AcPDs and SpmPdSpm is two transposon families studying the most deep.But, because most of cut by the transposable element of modified (as inserting ipt gene) after can reinsert again other position in genome, the cell only having swivel base to slip up could form the normal genetically modified crops of phenotype.The method efficiency comparison of this rejecting selectable marker gene is low, is restricted in the commercialization of genetically modified crops.Initial cotransformation system refers to two independently plasmids, one of them contains selectable marker gene, another contains goal gene, transform object cell simultaneously, two plasmids can be integrated into vegetable cell simultaneously, cultivate the transformed plant offspring of this co-integration, through the genetic recombination of perfect stage, selectable marker gene is separated with goal gene, thus obtains the transformed plant only containing goal gene not with selectable marker gene.Cotransformation system requirements cotransformation frequency wants DNA that is high and cotransformation in recipient cell, be in not chain state..In actually operating, the common integrating frequency of transgenosis being arranged in different conversion carrier is often lower, and is often incorporated into the same site of acceptor gene group.In order to overcome the above problems, investigator constructs again the super binary vector containing two T-DNA, to be inserted into respectively in same plasmid in two separate T-DNA districts by selectable marker gene and goal gene.The problem that this super binary vector makes integration efficiency low obtains alleviation.But, double T-DNA transformed acceptor cell, the common integration transformation cell major part obtained is for being incorporated into the same site of acceptor gene group, being incorporated into during descendant inheritting is separated of this situation can not be separated the goal gene transformed plant obtaining marker-free, how to reject this integration by the easiest method is key one step improving cotransformation efficiency, is also the necessary ways that the transgenic technology of marker-free is introduced to the market.
Present invention improves over traditional double T-DNA carrier, a promotor and specific gene is added between double T-NDA region, called after double T-DNA+1 vectors, like this when double T-DNA co-integration is to chromosomal same position, this specific gene will be expressed under the startup of promotor, the characteristic of this specific gene can be utilized directly to reject this transfer-gen plant by easy method, the plant selecting goal gene and selectable marker gene to be incorporated into coloured differently body or same karyomit(e) different positions is altogether cultivated, the transfer-gen plant obtaining marker-free is separated by descendant inheritting, so greatly save manpower, material resources and financial resources, commercialization in order to non selecting sign transgene advances an important step.
Summary of the invention
An object of the present invention is the transgene carrier setting up a kind of efficient marker-free, called after double T-DNA+1 vectors, this carrier comprises two T-DNA functional domains, carry selectable marker gene and goal gene respectively, region between these two T-DNA structural domains, carries a promotor and specific gene.The marker gene of this carrier can be any marker gene that can be used for Plant Transformation, as: hygromycin phosphotransferase gene, kalamycin resistance gene, phosphine silk mycin transferase gene etc., goal gene comprises any gene having production application to be worth: gene of such as anti insect gene, drought resisting base, cold tolerance gene, antiweed etc.Promotor comprises the endogenous promotor of any plant materials, exogenous promoter, constitutive promoter, induction sexual type promotor, tissue-specific promoter, organ inducible promoter.Specific gene comprises any lethal gene and reporter gene such as red fluorescent protein gene, green fluorescence protein gene, blue florescence protein gene and gus gene etc.
Another object of the present invention is to provide a kind of genetically modified crops system utilizing this double T-DNA+1 vectors to obtain marker-free, and specific implementation process comprises:
1, structure carries the double T-DNA carrier of corresponding goal gene and proceeds to Agrobacterium.
2, utilize agriculture bacillus mediated gene transformation, the plant expression vector built is transformed target plant
3, for transgenic seedling, molecule and phenotype analytical are carried out to acquisition T0, rejects the common integration transgenosis offspring containing selectable marker gene and specific gene, select containing selectable marker gene and goal gene but do not cultivate containing the transgenic progeny of specific gene.
4, the T1 that plantation screening obtains, for transgenic line, selects the transfer-gen plant only containing goal gene by PCR.Obtain only containing the transgenic homozygous strain of goal gene further by hereditary segregation ratio.
Set forth the present invention further below in conjunction with embodiment, and do not form the restriction to the claims in the present invention scope.
Accompanying drawing explanation
Fig. 1 expression of plants double T-DNA+1 vectors KT350;
Fig. 2 expression of plants double T-DNA carrier KT349;
Fig. 3 KT350 transgenosis T1 for seed phenotypes analysis, wherein: for the purpose of A, gene and marker gene are incorporated into the T1 of karyomit(e) same position altogether for seed; For the purpose of B, 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 vectors KT350
KT350 structure is shown in accompanying drawing one, and be double T-DNA+AsRED gene, a double T-DNA is 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 the pKAT-1 in this laboratory for template, expand 35s-AsRed-Tnos region, and import BamHI site, be building up on T-easy carrier by ligation, further sub clone construction is to containing on the carrier KT349 (structure is shown in accompanying drawing two) of double T-DNA.Enzyme in building process is cut, connect, reclaim, purifying equimolecular technology, is all undertaken by the method in molecular cloning handbook.(molecular cloning: laboratory manual, Sambrook et al, New York:Cold Spring harbor laboratory 1989 press)
The acquisition of embodiment two: KT350 rice transformation seedling
The Mature seed of rice shelled first uses 70% alcohol immersion 1-2 minute, then 30 minutes are soaked with 0.1% mercuric chloride, carry out surface sterilization (preferably carrying out on shaking table), aseptic water washing 3-4 time, again seed to be placed on aseptic filter paper after suck dry moisture, be placed on mature embryo calli induction media, 26 DEG C of light culture.Select the callus Dual culture that color and luster is yellowish.
The Agrobacterium AGL0 containing KT350 carrier at the flat lining out of the YM containing 50mg/L Kanamycin, 28 DEG C of dark culturing 2-3 days, Agrobacterium thalline is collected with a metal spoon, be suspended in Dual culture CM liquid nutrient medium, adjustment cell concentration is 0.3-0.5 to OD600, add Syringylethanone, make its final concentration be 100mM, be the agrobacterium suspension of Dual culture rice transformation.
The callus selecting state better (succeeding transfer culture 5-7 days, color and luster yellowish) puts into the aseptic triangular flask of 100ml, adds appropriate agrobacterium suspension (ensuring enough bacterium liquid and material), transforms.Callus after Dual culture is placed in the screening culture medium containing 50mg/l Totomycin, 26 DEG C of light culture.After two-wheeled screening in the resistant calli that grows, the resistant calli selecting milk yellow densification goes on the division culture medium containing 50mg/L Totomycin and cultivates.When the bud of resistant calli differentiation grows to about 2cm, seedling is moved on on root media, cultivate about two weeks.Select the seedling of high about 10cm, well developed root system, wash away substratum with warm water, transplant in greenhouse and bury.
Embodiment three: KT350 transgenosis T0 is for the screening of seedling and acquisition
The transgenic paddy rice seedling of what embodiment two obtained have hygromycin resistance, has three kinds of situations, a kind ofly proceeds to rice seedlings for selectable marker gene list; The second is the transgenic paddy rice seedling that selectable marker gene and KY1 gene are incorporated into karyomit(e) same position altogether, in this case, AsRED is also incorporated on 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 karyomit(e) altogether, and this strain is that offspring can be separated the strain obtaining non selecting sign transgene seedling.Generally, the transgenic line of the second will account for about 70% of transgenic seedling, transformation system in the past rejects the seedling stage that this transformation system will arrive T1 generation, pass through round pcr, screening is not only containing target gene KY1 but containing the strain of selectable marker gene, such screening not only workload is large, and the cycle is long, is unfavorable for the commercialization of transgenic technology.In the present invention, can utilize the expression of AsRED, directly reject the seed that seed is red T1 generation, greatly reduce workload, the High efficiency Commercial of render transgenic changes into as possibility.
The embodiment work well afoot of the present invention in soybean, cotton, corn, Chinese sorghum.
Claims (8)
1. cultivate a method for efficient selected-marker-free transgenic crop, comprise the following steps:
A) structure goal gene and selectable marker gene 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, wherein said specific gene is all lethal genes or reporter gene;
B) double T-DNA+1 vectors built is transformed object farm crop, obtain transformed plant;
C) by expression characterization and the Molecular Detection of specific gene, for transgenic seedling, molecule and phenotype analytical are carried out to acquisition T0, reject the common integration transgenosis offspring containing selectable marker gene and 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) plantation screens the T1 that obtains for transgenic line, selects the transfer-gen plant only containing goal gene, obtain the genetically modified crops of marker-free further by hereditary segregation ratio by PCR.
2. in accordance with the method for claim 1, goal gene is any anti insect gene, anti-ageing gene, anti-herbicide gene, quality-improving gene.
3. in accordance with the method for claim 1, goal gene is any adversity gene.
4. in accordance with the method for claim 1, goal gene is any yield improvement gene.
5. in accordance with the method for claim 1, promotor between two T-DNA structural domains is any plant endogenous promotor and exogenous promoter, comprises constitutive promoter, induction sexual type promotor, tissue-specific promoter or the fusion fragment by control domain and promotor.
6. in accordance with the method for claim 1, reporter gene comprises red fluorescent protein gene, blue florescence protein gene, green fluorescence protein gene, gus gene.
7. in accordance with the method for claim 1, it is characterized in that object farm crop comprise all monocotyledonss and dicotyledons.
8. method according to claim 7, monocotyledons is paddy rice, corn, wheat, Chinese sorghum, millet, barley, oat, rye, and dicotyledons is cotton, soybean, tobacco, rape, tomato.
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| 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 |
| 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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| CN1654662A (en) * | 2004-02-09 | 2005-08-17 | 中国科学院遗传与发育生物学研究所 | Method for cultivating transgenic plant without selective marker and its special expression vector |
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| CN1654662A (en) * | 2004-02-09 | 2005-08-17 | 中国科学院遗传与发育生物学研究所 | Method for cultivating transgenic plant without selective marker and its special expression vector |
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| A new technique for activation tagging in Arabidopsis;Pogorelko GV et al;《Gene》;20080515;第414卷(第1－2期);67-75 * |
| 通过构建三段T-DNA载体高频率获得无标记转基因大豆植株的研究;叶兴国等;《生物工程学报》;20070131;第23卷(第1期);138-144 * |
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