WO2014040352A1 - 杂交作物转基因安全控制的方法和实现该方法的基因删除系统 - Google Patents

杂交作物转基因安全控制的方法和实现该方法的基因删除系统 Download PDF

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WO2014040352A1
WO2014040352A1 PCT/CN2012/086327 CN2012086327W WO2014040352A1 WO 2014040352 A1 WO2014040352 A1 WO 2014040352A1 CN 2012086327 W CN2012086327 W CN 2012086327W WO 2014040352 A1 WO2014040352 A1 WO 2014040352A1
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gene
promoter
plant
recombinase
specific
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French (fr)
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裴炎
邹修平
刘若尘
宋水清
侯磊
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西南大学
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    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8216Methods for controlling, regulating or enhancing expression of transgenes in plant cells
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology
    • C12N15/63Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/82Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
    • C12N15/8241Phenotypically and genetically modified plants via recombinant DNA technology
    • C12N15/8261Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
    • C12N15/8262Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield involving plant development
    • C12N15/8265Transgene containment, e.g. gene dispersal

Definitions

  • the present invention relates to the field of plant genetic engineering, and in particular to a method for safe control of transgenic hybrid crops and a system for carrying out the method. Background technique
  • Plant genetically modified technology has set off a new green revolution in agricultural production. It is estimated that by 2015, the number of farmers growing GM crops worldwide will reach more than 20 million in 40 countries, and the planting area will reach 200 million hectares (James, 2011). On the other hand, genes such as insect resistance, disease resistance, herbicide resistance and antibiotics introduced in GM crops have caused public concern, and people are concerned that the existence of these genes may cause potential harm to the ecological environment and human health. Even with catastrophic consequences. This concern and concern has intensified the public's hesitation in purchasing GM products. When told, the public is more willing to choose non-GM products.
  • Keenan (2002;) et al. proposed a technical idea for producing non-GM foods from transgenic plants: placing all foreign genes between recombinase recognition sites, and controlling recombinases with chemically-induced or tissue-specific promoters. Gene expression.
  • the applicant uses a transcriptional activation system to control the expression of the recombinant enzyme system, and constructs a set of biosafety control for the exogenous gene of sexually reproducing plants.
  • the automatic deletion of the binary system referred to as "GAEBS”
  • GAEBS achieves the stable inheritance of exogenous functional genes in the recombinase-mediated gene deletion system in sexually sexual reproduction; meanwhile, when it is necessary to delete the foreign gene, it can be sexually Hybridization, the recombinase genes originally isolated from different plants and the transcriptional activator genes controlling their expression are closed, and the transcription activator initiates the expression of the recombinase gene, and the recombinase will be derived from all foreign genes of the parents (including the recombinase gene).
  • hybrid corn In order to establish gene deletion technology in transgenic hybrid crops, gene deletion in hybrid seeds must be avoided to ensure that the foreign genes in the hybrid plants stably function and function (such as insect resistance, disease resistance, herbicide resistance, etc.); At the same time, the foreign genes in the pollen and seeds produced by the hybrid plants are completely deleted, so that the "gene escape" pathway through the pollen and seed pathways can be effectively blocked.
  • An object of the present invention is to provide a method for safe control of hybrid crop transgenes, which comprises introducing a plant flower primordial cell-specific promoter into a gene deletion system, and controlling the transcription activation system in the gene deletion system by the promoter, thereby realizing hybrid crops in vitro and abroad
  • the source gene is stably present in the hybrid seed and in the non-deleted tissues such as roots, stems and leaves of the F1 generation, and is not deleted, but is completely deleted in the pollen and seeds produced by the hybrid plant, thereby achieving the purpose of safe control of the hybrid crop transgene.
  • Another object of the present invention is to provide a plant flower primordial-specific promoter for use in the preparation of a safe transgenic plant.
  • the present invention also provides an automatic gene deletion binary system for safe control of hybrid crop transgenes, comprising a transcriptional activation system controlled by a plant flower primordial cell-specific promoter, controlled by said transcriptional activation system Recombinase system as well as exogenous gene expression control system.
  • the present invention also provides a method for preparing a transgenic plant by using the above-described gene automatic deletion binary system.
  • the key technology of the present invention is: constructing a transgenic plant automatic deletion binary system including a recombinase system, a transcription activation system and a foreign gene expression control system, and screening a suitable plant tissue-specific promoter in the deletion system to determine a promoter The tissue specificity and activity time window, the promoter controls the transcriptional activation system, the transcriptional activation system controls the initiation of the recombinase system, and the initiation of the recombinase system deletes all introduced foreign genes located between the recombinase-specific recognition sites.
  • the present application utilizes a flower primordial-specific promoter to control the transcriptional activation system, and recombinase-mediated gene deletion controlled by the transcriptional activation system is opened at a specific site and time of the plant, Even if the foreign genes in the transgenic hybrid seeds are retained, the vegetative tissues and organs (such as roots, stems, leaves, etc.) of the transgenic hybrid F1 plants exert the function of the exogenous gene (such as insect resistance, disease resistance, and herbicide resistance). Agents, etc., and make all foreign genes (including the recombinase itself) produce pollen and seeds in F1 plants.
  • the present invention in order to achieve the purpose of safe control of transgenes in a hybrid crop, the need to produce non-transgenic pollen and seeds by transgenic hybrid crops is satisfied, and the present invention is based on the invention patent application No. 201110179613.2. A glimpse of the results of the research. To achieve that the foreign gene is not deleted in the hybrid seed and is completely deleted in the pollen and seeds produced by the hybrid plant, the key depends on the tissue specificity of the tissue-specific promoter controlling the transcriptional activation system, and the initiation time window. And strength. Promoters suitable for use in the present invention should have the following characteristics: 1.
  • the promoter controlling the transcriptional activation system should be a promoter of germline cells, and must have both female cells and male cell specificity (eg, flower Specific promoters such as basal, pistil and stamen primordia; 2 promoter activity
  • the "time window" should be in diploid cells before meiosis and stop working after pollination to avoid exogenous genes being deleted in advance when hybridizing, and to prevent the loss of exogenous gene deletion efficiency of the plant ( If the promoter activity occurs in meiosis, it will result in the binary system being separated by meiosis).
  • a suitable flower primordium-specific promoter is finally obtained, and the transcriptional activation system is controlled by the flower primordium-specific promoter, and the transcriptional activation system controls the recombinase system to realize the foreign gene. 1
  • the hybrids are stable and are only deleted automatically in the pollen and seeds of the plants, thus meeting the needs of safe control of hybrid crop transgenes.
  • the tissue-specific promoter for controlling a transcriptional activator gene in the present invention includes a natural promoter derived from a plant, an animal, a microorganism, or a promoter artificially modified or synthesized; as a conceptual aspect of the present invention
  • the promoter described is the tobacco flower development C gene ntAG (tobacco AGAMOUSwwo/og ⁇ promoter ntAGIP1, the nucleotide sequence of which is shown in SEQ ID NO.
  • the plant flower primordium-specific promoter is applied to an automatic gene deletion binary system (also referred to as a gene deletion system) for the purpose of safety control of hybrid crop transgenes
  • the gene deletion system comprises a recombinase system, a transcriptional activation system and a foreign gene expression control system located in a first plant expression vector and a second plant expression vector
  • the recombinase system comprises a recombinase and a specific recognition site of the recombinase
  • the transcriptional activation system includes a transcriptional activator gene, a target promoter controlled by the transcriptional activator, and a plant flower primordial cell-specific promoter that controls the transcriptional activator gene
  • the exogenous gene expression control system includes controlling the foreign gene The expressed promoter and the introduced foreign gene.
  • the two plant expression vectors are constructed as: a first plant expression vector (also referred to as a Trigger plant expression vector, wherein each control element included is referred to as a Tngger element) comprising two homologous recombinase-specific recognition sites and a gene or nucleotide between the recombinase-specific recognition sites: a promoter that controls expression of a foreign gene; a multiple cloning site for introducing a foreign gene; a transcriptional activator gene; and a plant flower primordium A cell-specific promoter that controls the initiation of a transcriptional activator gene.
  • a second plant expression vector also referred to as a DETERTER plant expression vector, wherein each control element is referred to as a Deleter element
  • the expression vector comprising two homologous recombinase specific recognition sites and specific for the two recombinases a gene or nucleotide between the recognition sites: a promoter that controls expression of a foreign gene; a multiple cloning site for introducing a foreign gene; a recombinase gene; and a target promoter, the target promoter Controlled by the transcriptional activator gene, when activated, initiates recombinase gene expression.
  • the preferred recombinase-specific recognition site is selected from the group consisting of ⁇ , 1 ⁇ 2272, 1 ⁇ 5171 and the FRT recognition site
  • the recombinase gene is selected from the FLP, Cre and Cre mt recombinase genes, and the target promoter and
  • the transcriptional activation system composed of the transcriptional activator gene is a pOp/LhG4 transcriptional activation system.
  • the present invention also provides a method for preparing a safe transgenic plant, wherein plants carrying a Deleter element and a Tngger element, respectively, are hybridized as a parent to obtain a hybrid having a target trait, and the field production reflects the target trait.
  • the biological function in tissues such as roots, stems, leaves, etc., and the deletion function of the system is efficiently activated only in the flower primordial cells before meiosis occurs, and the deletion of all exogenous genes including the recombinase gene is achieved.
  • a safe transgenic plant can be prepared for the purpose of controlling the safety of the foreign gene of the hybrid crop.
  • the invention aims at the shortcoming of the "GAEBS” gene automatic deletion binary system which can not maintain the stable inheritance of the foreign gene in the hybrid crop breeding, and according to the characteristics of the hybrid crop and the technical requirements of the three-line, two-line hybrid seed production, creatively It is proposed to select the plant flower primordial cell-specific promoter to control the expression of the "GAEBS” system transcriptional activator gene, and then realize that the "GAEBS” system can be efficiently opened only before the generation of plant meiosis occurs, and all foreign genes are extracted from the flower primordial cells. Completely removed, producing pollen and safe edible seeds without any foreign genes.
  • the invention is applicable to hybrid crops prepared by three or two lines of corn, rice, rape and vegetables, and the first plant expression vector and the second plant expression vector are respectively introduced into the maintainer/sterile line and the restorer line of the crop.
  • the transgenic sterile line and the restorer line material are selected and purified by passage and used for hybrid seed production. Since the area required for hybrid seed production is much smaller than the planting area of hybrid plants, it can be carried out by the seed company in a closed environment, which can greatly reduce the ecological hazards caused by the exogenous gene "floating".
  • the vegetative organs of large-scale hybrid plants continue to play the role of transgenic (insect, disease resistance, herbicide resistance, etc.) due to the presence of foreign genes, but the foreign genes in pollen and seeds are all deleted.
  • the "gene escape” pathway through the pollen and seed pathways can be effectively blocked. More importantly, because there are no foreign genes and their encoded products in the seeds or fruits of crops (especially crops such as rice, corn, etc.), there are no food safety hazards in GMOs. Can be eliminated, the public's worries and opposition to the safety of genetically modified foods will also be eliminated.
  • the results of the transgenic tobacco experiment showed that the first transgenic plant and the second transgenic plant were sexually heterozygous.
  • hybrid seeding can not only obtain hybrids efficiently, but also effectively ensure the exogenous functional genes in roots, stems, leaves and other target tissues in field production. Effective expression, to achieve its biological function; At the same time, 100% of all foreign genes derived from the parents can be completely removed from the flower primordial cells of the hybrid progeny, producing pollen and edible seeds without any foreign genes.
  • Figure 1 Schematic representation of the structure of the Trigger plant expression vector for the expression of LhG4 ATO by the ntAGIP1 promoter.
  • kanamycin resistance gene
  • 2 X 35S a plant-constitutive promoter derived from cauliflower mosaic virus
  • GUS NPTII, ⁇ -glucosidase gene (GUS) and neomycin phosphotransferase gene ( ⁇ ) fusion gene for screening and identification of transgenic plants
  • nos opine synthase gene transcription termination sequence
  • LB T-DNA left border
  • RB T-DNA right border
  • loxp cre/loxp recombinase system Sequence of recognition sites.
  • the skeleton vector pL35SLhG4 for constructing a plant expression vector is described in the application No. 201110179613.2.
  • Figure 2 PCR analysis of non-specific deletion of foreign genes in leaves of F1 plants.
  • the target gene detected was m ⁇ and the gene. Approximately 1.2 kb of cre'" f and a 1.4 kb 2G ro specific band should be detected simultaneously in the hybrid plants.
  • M lane DNA molecule marker
  • H lane water as template
  • WT lane wild type tobacco DNA as template
  • 1, 2, 4-11, 13-17 lanes Total DNA of hybrid roots, stems and leaves as template
  • Lane 3 Trigger parental DNA as template
  • FIG. 1 Expression analysis of GUS and GFP in the hybridization pathway.
  • GUS GUS histochemical staining assay
  • GFP green fluorescence microscopy
  • Fl Deleter and Trigger hybrid F1 plants
  • F2 F1 generation selfed progeny
  • 1 seedling
  • 11 stem crosscut ; in: leaf; ⁇ : 2-3 period flower slitting; V: mature pollen.
  • the recombinase deletion system is split into two, producing two components: Deleter and Trigger.
  • the Deleter element includes a "recombinase gene (Cre) and a trait gene controlled by "Target promoter” ( ⁇ ); the Trigger element includes a germline cell-specific promoter (germlme P, such as a flower basal tissue-specific promoter).
  • the hybrid transcription factor (LhG4) gene as well as the foreign gene. Plant expression vectors were constructed by placing all of the two elements between the recombinase recognition sites (L). Introduce Trigger and Deleter into the plant genome, respectively.
  • the foreign gene is not deleted due to the absence of the recombinase gene; in the DETERTER transgenic plants, the ⁇ promoter is closed due to the absence of a hybrid transcription factor gene, and the controlled recombinase gene is not expressed. Genes will not be deleted. This ensures that the foreign gene is stably inherited in the sexual generation of the plant (: I ) o When producing hybrid seeds, Trigger and Deleter are recombined in the zygote by sexual hybridization.
  • Trigger Under the control of the germlme P promoter, Trigger remains silent in zygote, hybrid seed and F1 vegetative roots, stems and leaves, and gene deletion continues to be closed, ensuring target trait genes (such as insect-resistant and herbicide-tolerant genes) in hybrid generation. Nutrient roots, stems, leaves, etc. achieve their biological functions (11).
  • the germline P promoter initiates the Trigger, LhG4 gene expression in the flower primordial cells, specifically activates the ⁇ promoter, opens the recombinase gene expression, and then deletes all foreign genes, and subsequently, no exogenous
  • the flower primordial differentiation of the gene produces pollen and seeds that no longer have any foreign genes, controlling the potential biosafety hazards associated with pollen and seeds in field production (111).
  • Fig. 6 is a schematic view showing the structure of a Deleter plant expression vector constructed according to the method described in Application No. 201110179613.2. detailed description
  • Re-use phenol (Ph8.0): chloroform: isoamyl alcohol (25:24: 1) and chloroform: isoamyl alcohol (24: 1) for each extraction (10,000 r / min, centrifugation for 10 min), take Clear, 2.5 times volume of absolute ethanol was precipitated at -20 ° C for more than 30 min. At 13,000 r/min, the precipitate was collected by centrifugation for 10 min, the supernatant was discarded, and the precipitate was rinsed with 75% ethanol. The mixture was centrifuged under reduced pressure, and the precipitate was finally dissolved in 50-200 TE and stored at -20 °C until use.
  • the amplification procedure was: 94 ° C, 5 min; 94 ° C, 30 sec, 56 ° C, 30 sec, 72 ° C, l ⁇ 4 min, 35 cycles; 72 ° C extension for 10 min.
  • DNA fragments are recovered, ligated, and transformed into E. coli.
  • the establishment of the restriction enzyme system and the reaction conditions were carried out in accordance with the instructions of the Roche restriction endonuclease kit.
  • the recovered fragment or the amplified fragment obtained by amplification was cloned into pUCm-T (Shanghai Sangon) or GEM-T/pGEM-T Easy (Promega) vector according to the ligase kit instructions.
  • the ligation reaction system is as follows:
  • the molar ratio of the vector DNA fragment to the exogenous ligation product DNA fragment was 1:1.
  • API The API-specific promoter of Arabidopsis thaliana is named API.
  • Primer (SEQ ID N0.12, 13) was designed based on the sequence of the Arabidopsis spore mother cell-specific promoter SPL (GenBank accession number: AT4G27330), and the SPL promoter was amplified by PCR using Arabidopsis genomic DNA as a template. A fragment of about 2.7 kb in length was cloned, and the amplified DNA fragment was cloned and sequenced to indicate that it was a SPL-specific promoter of Arabidopsis thaliana, and was named SPL.
  • EMS1 specific promoter of Arabidopsis thaliana was named EMS1.
  • AJ583670.1 designed primers (SEQ ID NO. 16, 17), amplified the Lefsml promoter with tomato genomic DNA as a template, and obtained a fragment of about 1.0 kb in length. The amplified DNA fragment was cloned and sequenced and analyzed.
  • the Lefsml specific promoter of tomato named Lefsml. 2.
  • Flower primordial cell-specific promoter controls transcriptional activator gene construction of Tngger plant expression vector. Briefly, the pL35SLhG4 vector is ligated by restriction enzyme ligation (see application number
  • ntAGIPl Trigger
  • ntAGIP2 Trigger
  • AGIP Trigger
  • API Trigger
  • SPL Trigger
  • EMSl Trigger
  • Lefsml Trigger plant expression vector.
  • the structural diagram of the ntAGIPl::Tri gg er plant expression vector is shown in Figure 1, and the structure of the remaining Trigger plant expression vector is similar. All restriction enzymes were purchased from Roche and operated according to the instructions for use.
  • the constructed plant expression vector plasmid was introduced into Agrobacterium EHA105 by electroporation.
  • the above vector was introduced into Agrobacterium EHA105 by electroporation using the Bio-RAD MicroPulser User's Manual.
  • Table 1 The medium used for the genetic transformation of tobacco by Agrobacterium tumefaciens-mediated methods is shown in Table 1.
  • Table 1 Agrobacterium tumefaciens-mediated medium for tobacco genetic transformation
  • MSB MS inorganic salt + B5 organic
  • Tobacco seeds were sterilized with 1% sodium hypochlorite and germinated on solid medium MSB Q under conditions of 25 ° C, 16 hr light / 8 hr dark photoperiod.
  • a sterile sterile seedling that grows about one month later can be used as a transformed explant.
  • the cut leaf disc was immersed in the Agrobacterium liquid resuspended in MSB liquid basic medium, and the infusion was 10-20 mm at rest.
  • the bacterial liquid was decanted, and the excess bacterial liquid on the surface of the leaf disc was aspirated by a sterile absorbent paper, and the leaf disc was placed on the co-culture medium MSBi, and cultured at 24 ° C for 2 d.
  • the leaf discs were subjected to differentiation culture for 2 weeks in the screening medium MSB 2 under the conditions of 25 ° C, 16 hr light / 8 hr dark photoperiod. After the regenerating green callus appeared, transfer to the bud medium MSB 3 to promote bud production.
  • the resistant seedlings grow to 3-4 cm in length, they are cut into rooting medium MSB 4 to induce rooting.
  • the roots of the resistant seedlings grow to 2-3 cm long, the medium is washed, and the hydroponic seedlings are 2-3 days, transplanted into nutrient mash, and grown in a greenhouse.
  • Example 3 the Trigger plant expression vector constructed in Example 2 was transformed into tobacco by Agrobacterium-mediated transformation, GUS histochemical staining and PCR screening were used to identify transgenic plants, and GUS histochemical staining techniques were used to analyze and screen a single copy of Tngger transgene.
  • the progeny of the plant is homozygous and used for further studies.
  • the second plant expression vector Deleter (Fig. 6) referred to in the present invention was constructed by the method described in the invention patent application No. 201110179613.2, and a Deleter transgenic plant was obtained.
  • the flower primordial-specific promoter opening system deletes the foreign genes in the germ cells, and produces seeds and pollen without foreign genes.
  • Frequency is the key to assessing the efficiency of deletion of the GAEBS system controlled by the flower-based cell-specific promoter. To this end, the deletion efficiency of the system for the Deleter and Tngger was firstly calculated based on the phenotype of the F 2 generation seedling neutralizing genes produced by self-crossing seed germination.
  • F 2 plants should have four phenotypes: GFP+/GUS+, GFP+/GUS-, GFP7GUS+, and GFP7GUS—with a separation ratio of 9:3:3:1.
  • the ntAGIP1 promoter guidance system can remove the Trigger and Delete foreign genes up to 100%, and the average efficiency of the system is 68.6%, which is significantly higher than the deletion efficiency of other promoter-controlled systems. Therefore, the ntAGIP1 promoter from tobacco was selected for further study.
  • Trigger transgenic plants were selected as the male parent to hybridize with D198 Deleter to obtain hybrids (F1 generation), F1 plants were planted, and the enzyme activities of GFP and GUS in F1 self-fertilized seeds were analyzed. All Trigger transgenic plants are single copy homozygous plants.
  • a Delete Deleter efficiency (4 X GFF seedlings - total number of seedlings analyzed) /3 X analysis of total number of seedlings X 100;
  • e average deletion efficiency (%) (delete Deleter efficiency + delete Trigger efficiency) /2.
  • the e100% deletion efficiency means that all foreign genes (including Trigger and Deleter) from both parents are 100% removed from the F1 generation of selfed seeds.
  • Trigger lines analysis of GFP+/GFF seedlings a Deleted Effect effect GUS+/GUS-Number of seedlings b Deleted Trigger effect e Average deletion efficiency Total seedling number rate (% ⁇ ) Rate (% ⁇ ) (%) ntAGIPl-6 150 14/136 87 3/147 97 92 ntAGIPl-3 200 150/50 0 198/2 0 0 ntAGIPl-2 200 60/140 60 12/188 92 76 ntAGIPl-10 150 25/125 78 43/107 62 70 ntAGIPl-4 200 26 /174 83 0/200 100 91 ntAGIPl-17 100 38/63 50 43/57 43 46 ntAGIPl-11 225 45/180 73 9/216 95 84 ntAGIPl-8 150 99/51 12 86/64 24 18 ntAGIPl-5 120 19/101 79 22/98 76 77 ntAGIPl-9 2000 27/1973 98 20/1980
  • e average deletion efficiency (% ⁇ ) (delete Deleter efficiency + delete Trigger efficiency) /2.
  • F1 pollen is the main way to bring hidden dangers to genetically modified organisms.
  • the expression activity of GUS and GFP in mature pollen of F1 generation was detected, and the results showed that ntAGIP-85,-126,-137 Three strains of Triggers and D198 Deleter were not detected in the pollen of Fl generation, and the expression activity of GUS and GFP was not detected (evaluate the number of pollen 20,000 or more) (Fig. 3).
  • ntAGIPl -2 347 347/0 347/0 N ntAGIPl -3 496 496/0 496/0 N ntAGIPl -4 377 377/0 377/0 N ntAGIPl -5 371 371/0 371/0 N ntAGIPl -6 406 406/ 0 406/0 N ntAGIPl -8 366 366/0 366/0 N ntAGIPl -9 399 399/110 399/45 Y ntAGIPl-10 390 390/0 390/0 N ntAGIPl-11 325 325/0 325/0 N ntAGIPl -17 331 331/34 331/65 Y ntAGIPl-85 251 251/0 251/0 N ntAGIPl-89 205 205/0 205/0 N ntAGIPl-93 274 274/0 274/0 N ntAGIPl-95 246 246
  • ntAGIPl-109 381 381/0 381/0 N ntAGIPl-112 303 303/0 303/0 N ntAGIPl-113 334 334/0 334/0 N ntAGIPl-122 236 236/0 236/0 N ntAGIPl-125 338 338/ 0 338/0 N ntAGIPl-126 328 328/0 328/0 N ntAGIPl-132 238 238/0 238/0 N ntAGIPl-134 208 208/0 208/0 N ntAGIPl-137 310 310/0 310/0 N ntAGIPl -138 128 128/0 128/0 N
  • ntAGIPl-directed GAEBS system maintains the analysis of the effective expression of exogenous functional genes in F1 generation non-deleted tissues
  • the stability of the system in the generation of vegetative tissues is the key to the biological function of exogenous trait genes (such as insect resistance, disease resistance, etc.) in these tissues.
  • the stability of the system in the F1 plants was first detected by PCR, and the amplified target gene was cr e '" n 7J2G ro gene.
  • the genomic DNA of the plant leaves was extracted using a primer pair (SEQ ID No. 18, 19 and SEQ ID No. 20, 21) respectively, the stability of cre'"f] 2 ( ⁇ gene in the genome of these tissues was detected.
  • the PCR results showed that all F1 plants were detected at the same time with a length of about 1.2 kb.
  • the presence of cre'" f and the 1.3 kb 7J?G ro gene indicates that these plants contain both the Deleter and Trigger transgenes, and the foreign genes were not deleted in these tissues (Fig. 2).
  • GFP fluorescence detection and GUS histochemical staining were used to analyze the expression of reporter genes in the roots, stems, leaves and other tissues. The results showed that high expression of GFP and GUS genes was detected in non-deleted tissues such as roots, stems, leaves and flowers of F1 plants (Fig. 3-B).
  • the GAEBS system can effectively maintain the stability of exogenous functional genes (GFP and GUS) in F1 non-deleted tissues (roots, stems, leaves and flowers) after the hybrid progeny is closed.
  • F1 non-deleted tissues roots, stems, leaves and flowers
  • the ntAGIP1 promoter began to initiate the target gene at the center of the tobacco flower primordia (including pistil primordia and stamen primordia) until the flowering stage 3 ⁇ 4n ge t al., 2010; Therefore, in theory, after the system is closed in the generation cells, the ntAGIP1 promoter will remove the foreign gene from the center of the flower primordium from the early initiation of the ⁇ -generation flower primordial, and accordingly, use GFP green fluorescence detection. Techniques track the time window of exogenous gene deletion in floral organs.
  • Figure 4 shows that GFP green fluorescence disappeared from the center of the flower primordia before the hybrid progeny-7 period, and no green fluorescence signal was detected in the stamen and pistil populations of the subsequent -6 -1 period, but only red was detected. Fluorescent signal ( Figure 4A, B, C). In parental D198, the fluorescence signal was strongly present in the flowers of each period (Fig. 4D, E). These results indicate that the ntAGIP1 promoter initiates the automatic deletion of the binary system to efficiently delete foreign genes in the early flower primordium center before the F1 generation-7 period.
  • the present invention utilizes a flower-origin-specific promoter such as ntAGIP1 to control the spatiotemporal deletion of the GAEBS system, and successfully constructs an automatic deletion binary system GAEBS for controlling the safety of the exogenous gene of hybrid crops. 5.
  • the target trait gene is introduced into the hybrid crop through the system, thereby achieving: (1) maintaining the stable inheritance of the target trait gene in the sexual generation of the parent, facilitating the screening of the excellent transgenic line; (2) maintaining the target trait gene in the hybridization Stable transfer in the species, hybrid seed production can obtain transgenic hybrid seeds for production; (3) in field production, can effectively ensure that the target trait genes achieve their functions in non-deleted tissues (such as roots, stems and leaves) (such as Insects, disease resistance and herbicide resistance, etc.; (4) Gene automatic deletion of the binary system initiates 100% deletion of all exogenous genes including the recombinase system in the sexual cells before the generation of plant meiosis Pollen and seeds that do not contain any foreign genes.
  • the method of the invention is simple and convenient, and when the foreign gene needs to be deleted, all the foreign genes can be deleted 100%, the effect is remarkable, and the application prospect is good.
  • Keenan RJ Stemmer WPC (2002) Nontransgenic crops from transgenic plants. Nature
  • JRSM 101

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Abstract

本发明提供了一种杂交作物转基因安全控制的方法,其中构建包括重组酶系统、转录激活系统以及外源基因表达控制系统的转基因植物自动删除双元系统,通过将植物花原基细胞特异启动子作为所述转基因植物自动删除双元系统中的启动子控制转录激活系统,所述转录激活系统控制重组酶系统的启动,使导入到植物中的外源基因在F1代植株杂交种中以及根、茎、叶等非删除组织中稳定存在,但在F1代植株的花粉和种子中所述外源基因被删除,以实现杂交作物转基因安全控制。

Description

杂交作物转基因安全控制的方法和实现该方法的基因删除系统 技术领域
本发明涉及植物基因工程领域,具体涉及用于转基因杂交作物安全控制的方 法和实现该方法的系统。 背景技术
植物转基因技术已经在农业生产中掀起一场新的绿色革命。据估计,到 2015 年, 全球种植转基因作物的农民数量在 40个国家内将达到 2000万以上, 种植面 积将达到 2亿公顷 (James, 2011)。 另一方面, 在转基因作物中导入的抗虫、 抗病、 抗除草剂及抗生素等基因已引起了公众的担忧,人们担忧这些基因的存在可能会 对生态环境和人类健康带来潜在的危害, 甚至带来灾难性的后果。这种担心和顾 虑越来越加剧公众在购买转基因产品时的犹豫, 当被告之时, 公众更愿意选择非 转基因产品。人们对转基因作物生物安全性的担忧主要集中在两个方面: 一方面 是转基因食品可能对人类健康带来不利影响 (Key et al, 2008);另一个方面是转基 因植物通过花粉和种子扩散等方式, 可能对生态环境造成危害 (Ramessar et al., 2007) 这些担忧不断干扰着转基因产品的进一歩商业化发展。
许多分子生物技术已被成功用于控制外源基因生物安全的研究中 (Tut ja et al., 2012)。其中, 基于位点特异性重组酶系统 Cre/loxP和 FLP/FRT的基因删除技 术因具有操作简单易行, 删除效率高等优点, 近年来已被广泛应用到转基因植物 生物安全控制中。 2002 年, Keenan (2002;)等提出了一种从转基因植物生产非转 基因食品的技术思路: 即将所有外源基因置于重组酶识别位点之间, 用化学诱导 或组织特异启动子控制重组酶基因的表达。根据该思路, 本申请人与美国康涅狄 格大学合作, 成功构建了 "基因删除系统"("GM-gene-deletor") , 该技术能将所 有外源基因从 ^代烟草种子和花粉中彻底删除 (Luo et al, 2007)。 但该技术不能 直接用于水稻、 玉米、 油菜等有性繁殖植物中。 这是因为外源基因一旦从转基因 植物 TQ代的花粉和种子中去除, 无法通过有性繁殖的途径传给下一代, 在杂种 后代中不能实现外源基因的功能。针对这一问题,在本申请人的另一专利申请(申 请号 201110179613.2) 中, 本申请人利用转录激活系统控制重组酶系统的表达, 构建了一套用于有性繁殖植物外源基因生物安全控制的基因自动删除双元系统, 简称 "GAEBS", 实现了重组酶介导基因删除系统中外源功能基因在有性繁殖世 代中的稳定遗传; 同时, 当需要删除外源基因时, 可通过有性杂交, 使原本分别 于不同植株的重组酶基因和控制其表达的转录激活子基因合拢,转录激活子启动 重组酶基因的表达, 进而重组酶将来源于双亲的所有外源基因(包括重组酶基因 本身)从杂交后代中彻底删除。然而, 由于转录激活子置于组成型启动子(CaMV 35S)控制之下, 杂交发生的同时基因删除也就立即开始, 所以产生的 F1代杂交 种子是不含有外源基因的, 即非转基因种子, 尚不能在杂交作物的生产中应用。 在此,将申请号为 201110179613.2的专利申请所公开的内容全文引入本申请中作 为参考。
然而, 在现代农业中, 杂交优势利用已经成为增加产量、 改进品质的重要途 径。 如在中国和美国的玉米生产中, 几乎全部为杂交玉米; 而杂交水稻已占我国 水稻栽种面积的 50%和产量的 60%以上。要在转基因杂交作物中建立基因删除技 术, 必须避免杂交种子中的基因删除, 以保证杂交 代植株中的外源基因稳定 发挥其功能和作用 (如抗虫、 抗病、 抗除草剂等); 同时又要使杂交 植株产 生的花粉和种子中的外源基因被彻底删除,使通过花粉和种子途径的 "基因逃逸" 途径可以得到有效阻断。 更重要的是, 可以使作物(特别是像水稻、 玉米等这类 作为主食的作物) 的种子或果实 (Fmits)中没有任何外源基因及其编码产物的存 在,可以消除转基因生物存在的食品安全隐患以及公众对转基因食物安全性的忧 虑和反对。 但目前还没有解决上述问题的有效方案。 发明内容
本发明的一个目的在于提供杂交作物转基因安全控制的方法,通过将植物花 原基细胞特异启动子引入基因删除系统,通过该启动子控制基因删除系统中的转 录激活系统, 而实现了杂交作物中外源基因在杂交种子中以及 F1代的根、 茎、 叶等非删除组织中稳定存在, 不被删除、 而在杂交 代植株产生的花粉和种子 中被彻底删除, 达到杂交作物转基因安全控制的目的。
本发明的另一个目的在于提供植物花原基细胞特异启动子在制备安全的转 基因植物中的应用。
本发明还提供了一种用于杂交作物转基因安全控制的基因自动删除双元系 统, 该双元删除系统包含由植物花原基细胞特异启动子控制的转录激活系统、 由 所述转录激活系统控制的重组酶系统以及外源基因表达控制系统。
本发明还提供一种利用上述基因自动删除双元系统制备转基因植物的方法。 本发明的技术关键是: 构建包括重组酶系统、 转录激活系统以及外源基因 表达控制系统的转基因植物自动删除双元系统, 在此删除系统中筛选获得适宜 的植物组织特异启动子, 确定启动子的组织特异性和活性时间窗, 该启动子控 制转录激活系统, 转录激活系统控制重组酶系统的启动, 重组酶系统的启动删 除位于重组酶特异识别位点之间的所有导入的外源基因。 作为该删除系统启动 关键的植物特异性启动子, 本申请利用花原基特异启动子控制转录激活系统, 受转录激活系统控制的重组酶介导的基因删除在植物特定的部位和时间打开, 既使转基因杂交种子中的外源基因得以保留, 转基因杂交 F1代植株的营养组织 与器官中 (如根、 茎、 叶等) 发挥外源目的基因的功能 (如抗虫、 抗病、 抗除 草剂等), 又使所有外源基因 (包括重组酶本身) 在 F1 植株产生的花粉和种子
(或果实) 中被彻底删除。
根据本发明的一方面, 为了实现杂交作物中转基因安全控制的目的, 满足用 转基因杂交作物产生非转基因花粉和种子的需要, 本发明是在申请号为 201110179613.2的发明专利申请的基础上进行了进一歩的研究的成果。若要实现 外源基因在杂交种子中不被删除、 而在杂交 代植株产生的花粉和种子中被彻 底删除, 其关键取决于控制转录激活系统的组织特异启动子的组织特异性、启动 时间窗和强度。适用于本发明的启动子应具有下述特点: ①控制转录激活系统的 启动子应为性细胞 (: germline cells)特异启动子, 而且必须同时具有雌性细胞和雄 性细胞特异性 (如: 花原基、 雌蕊和雄蕊原基等特异启动子); ②启动子活性的
"时间窗口"应在减数分裂前的二倍体细胞中, 并在授粉后停止工作, 以避免杂 交制种时, 外源基因被提前删除; 同时防止 代植株外源基因删除效率的下降 (如果启动子活性发生在减数分裂, 将导致二元系统因减数分裂而分离)。 通过 大量的启动子分析筛选, 最终获得了适宜的花原基特异启动子, 用所述花原基特 异启动子控制转录激活系统, 而转录激活系统控制重组酶系统, 实现了外源基因 在?1杂交种中稳定存在, 仅在 植株的花粉和种子中被自动删除, 从而满足 杂交作物转基因安全控制的需要。
本发明中用于控制转录激活因子基因的组织特异启动子包括来自植物、 动 物、 微生物的天然启动子, 或人工改造或合成的启动子; 作为本发明概念性
(conceptual) 描述的此类启动子是烟草花发育 C基因 ntAG (tobacco AGAMOUSwwo/og^的启动子 ntAGIPl, 其核苷酸序列如 SEQ ID NO. 5所示。
根据本发明的另一方面, 为实现杂交作物转基因安全控制目的, 将上述植物 花原基特异启动子应用于基因自动删除双元系统(也称基因删除系统) 中, 所述 基因删除系统包括分别位于第一植物表达载体和第二植物表达载体且相互配合 的重组酶系统、转录激活系统和外源基因表达控制系统; 所述重组酶系统包括重 组酶和该重组酶的特异识别位点; 所述转录激活系统包括转录激活因子基因、 由 该转录激活因子控制的靶标启动子和控制该转录激活因子基因的植物花原基细 胞特异启动子;所述外源基因表达控制系统包括控制外源基因表达的启动子和导 入的外源基因。 二个植物表达载体的构成为: 第一植物表达载体 (也称 Trigger 植物表达载体, 其中所包含的各控制元件称 Tngger元件) 包括两个同向的重组 酶特异识别位点以及在所述两个重组酶特异性识别位点之间的下述基因或核苷 酸: 控制外源基因表达的启动子; 用于导入外源基因的多克隆位点; 转录激活因 子基因; 以及植物花原基细胞特异启动子, 用于控制转录激活因子基因的启动。 第二植物表达载体 (也称 Deleter植物表达载体, 其中所包含的各控制元件称 Deleter元件), 所述表达载体包括两个同向的重组酶特异识别位点以及在所述两 个重组酶特异性识别位点之间的下述基因或核苷酸: 控制外源基因表达的启动 子; 用于导入外源基因的多克隆位点; 重组酶基因; 以及靶标启动子, 所述靶标 启动子由所述转录激活因子基因控制, 当被激活时, 启动重组酶基因表达。
在上述植物表达载体中, 优选的重组酶特异性识别位点选自 ΙοχΡ, 1οχ2272, 1οχ5171和 FRT识别位点, 重组酶基因选自 FLP、 Cre和 Cremt重组酶基因, 由 靶标启动子及与之配合的转录激活因子基因组成的转录激活系统为 pOp/LhG4转 录激活系统。
根据本发明的又一个方面,本发明也提供了制备安全的转基因植物的方法, 当分别携带 Deleter元件和 Tngger元件的植物作为亲本相互杂交, 获得具有目标 性状的杂交种, 大田生产体现目标性状在根、 茎、 叶等组织中的生物学功能, 而 仅在 减数分裂发生之前的花原基细胞中高效启动系统的删除功能, 实现包 括重组酶基因在内的所有外源基因的删除,达到控制杂交作物外源基因生物安全 性的目的, 可制备安全的转基因植物。
本发明用于转基因杂交作物生产非转基因花粉和种子的基因自动删除双元 系统及其制备安全的转基因杂交作物的方法具有以下有益效果:
本发明针对 "GAEBS"基因自动删除双元系统无法维持外源基因在杂交作物 杂交制种中稳定遗传的缺点, 根据杂交作物的特点并结合三系、两系等杂交制种 技术要求,创造性地提出选取植物花原基细胞特异启动子控制" GAEBS"系统转录 激活子基因的表达, 进而实现" GAEBS"系统仅在 代植株减数分裂发生之前高 效开启, 将所有外源基因从花原基细胞中彻底删除, 产生无任何外源基因的花粉 和安全食用种子。
本发明适用于玉米、水稻、 油菜以及蔬菜等通过三系或两系法制种的杂交作 物, 将第一植物表达载体和第二植物表达载体分别导入作物的保持系 /不育系和 恢复系中, 转基因不育系和恢复系材料经传代选育和纯化, 用于杂交制种。 由于 杂交制种需要的面积远小于杂交 植株的栽种面积, 可以由种子公司在封闭的 环境中进行, 可大大降低外源基因 "飘逸"带来的生态隐患。 大面积栽种的杂交 植株的营养器官由于外源基因的存在继续发挥转基因的功效(抗虫、 抗病、 抗 除草剂等), 但其产生的花粉和种子中的外源基因则被全部删除, 通过花粉和种 子途径的 "基因逃逸"途径可以得到有效阻断。 更重要的是, 由于作物(特别是 像水稻、玉米等这类作为主食的作物)的种子或果实Frmts;)中已经没有任何外源 基因及其编码产物的存在, 转基因生物存在的食品安全隐患得以消除, 公众对转 基因食物安全性的忧虑和反对, 也会被打消。
转基因烟草实验结果表明,通过第一转基因植物与第二转基因植物的有性杂 交,在植物组织特异性启动子 ntAGIPl调控的基因自动删除系统的指导下,杂交 制种不但能高效获得杂交种,而且能有效保证大田生产中外源功能基因在根、茎、 叶等目标组织中有效表达, 实现其生物学功能; 同时, 能 100%地将来源于双亲 的所有外源基因从杂交后代的花原基细胞中彻底删除,生产无任何外源基因的花 粉和食用种子。 附图说明
图 1 : ntAGIPl启动子控制 LhG4ATO表达的 Trigger植物表达载体的结构示 意图。
ΝΡΤΠΙ,卡那霉素抗性基因; 2 X 35S,来源于花椰菜花叶病毒的植物组成性启 动子; GUS:NPTII, β-葡萄糖酸苷酶基因 (GUS)和新霉素磷酸转移酶基因(ΝΡΤΠ) 融合基因, 用于转基因植株的筛选与鉴定; nos, 冠瘿碱合成酶基因转录终止序 列; LB, T-DNA左边界; RB, T-DNA右边界; loxp, cre/loxp重组酶系统的识 别位点序列。用于构建植物表达载体的骨架载体 pL35SLhG4参见 201110179613.2 号申请。
图 2: F1代植株叶片中外源基因非特异性删除的 PCR分析。
检测的目的基因为 m ^和 基因。 杂交 代植株中应同时检测到约 1.2kb的 cre'"f和约 1.4kb的 2G ro特异条带。 M泳道: DNA分子 marker; H泳 道: 水为模板; WT泳道: 野生型烟草 DNA为模板; 1, 2, 4-11, 13-17泳道: 杂交 代根、 茎、 叶的总 DNA为模板; 3泳道: Trigger亲本 DNA为模板; 12 泳道: D198亲本 DNA为模板。
图 3: 杂交途径中 GUS和 GFP的表达分析。
A. GUS和 GFP在亲本 Deleter和 Trigger中的表达分析。在幼苗 (A-i)、茎 (A-ii;)、 叶 (A-iii;)、 花 (A-iv)和花粉 (Α-ν)中均检测到 GUS和 GFP基因的表达。 B. GUS和 GFP在亲本 Deleter和 Trigger杂交 代中的表达分析。 代花粉 (B-v)和花原基 中心(雌蕊和雄蕊) (B-iv)中没有检测到 GUS和 GFP基因的表达,而在 F1茎 (B-ii;)、 叶 (B-iii)和幼苗 (B-i)非删除组织中检测到 GUS和 GFP基因的表达; C.GUS和 GFP 在杂交 F2代中的表达分析.在来自 F2代幼苗、 茎、 叶、 花和花粉中均未检测到 GUS和 GFP基因的表达。 P: 纯合亲本; GUS: GUS组织化学染色检测; GFP: 绿色荧光显微检测; Fl : Deleter与 Trigger的杂交 F1代植株; F2: F1代自交后 代 ; 1: 幼苗; 11: 茎横切; in: 叶; ιν:2-3时期花纵切; V: 成熟花粉。
图 4: ntAGIPl指导系统删除外源基因时空特点的 GFP荧光检测
A-C、 杂交 花器官 GFP绿色荧光检测结果。 a: -7时期以前的花; b: 约 -7一 -6时期的花; c: 约 -4时期的花:; B: -2时期的花; C: -1时期的花。 D和 E、 Deleter转基因植株花器官 GFP绿色荧光检测结果。 d: -7时期以前的花; e: 约 -5时期的花; f: 约 -2时期的花; g: -1时期的花。 E: 2-3时期花。 Bar=3mm. 图 5: 用于控制杂交作物外源基因生物安全的基因自动删除双元系统的工作 原理图
利用转录激活系统的反式作用原理, 将重组酶删除系统一分为二, 产生两个 元件: Deleter和 Trigger。 Deleter元件包括 "Target promoter" ( θρ) 控制的重 组酶基因 (Cre) 和外源基因 (trait gene) ; Trigger元件包括种系细胞特异启动子 (germlme P, 如花原基组织特异启动子) 控制的杂合转录因子 (LhG4) 基因以 及外源基因。 将两个元件中的所有基因都置于重组酶识别位点 (L) 之间, 构建 植物表达载体。 将 Trigger和 Deleter分别导入植物基因组中。 在 Trigger转基因 植株中, 由于没有重组酶基因, 外源基因不会被删除; 在 Deleter转基因植株中, 由于没有杂合转录因子基因, ρθρ启动子关闭, 所控制的重组酶基因不表达, 外 源基因也不会被删除。这样就可以确保外源基因在植物有性世代中稳定遗传 (: I )o 当生产杂交种子时,通过有性杂交, Trigger和 Deleter在合子中重新组合在一起。 在 germlme P启动子控制下, Trigger在合子、 杂交种子及 F1代营养体根、 茎、 叶保持沉默, 基因删除继续关闭,保证目标性状基因(如抗虫、抗除草剂等基因) 在杂交 代营养体根、 茎、 叶等中实现其生物学功能 (11 )。 当 F1代植株进入 开花期时, germline P启动子在花原基细胞中启动 Trigger, LhG4基因表达, 特 异激活 ρθρ 启动子, 开启重组酶基因表达, 进而删除所有外源基因, 随后, 无 外源基因的花原基细胞分化产生不再具有任何外源基因的花粉和种子,控制大田 生产中花粉和种子可能带来的生物安全隐患 (111)。
图 6是按照申请号 201110179613.2所述的方法构建的 Deleter植物表达载体 的结构示意图。 具体实施方式
以下结合附图对本发明进行进一歩的详细说明,但以下说明并不意味着对本 发明进行限定。
本发明实例中的试剂药品未做具体说明的均为普通市售,材料方法未做具体 说明的均参考 《分子克隆实验指南》。
【实施例 1】 DNA的提取和目的片段序列的扩增
1.DNA的提取
选取烟草 (Nicotiana tabacum, Xanthin) 幼嫩叶 0.3-0.5g, 在液氮中迅速磨 成白色粉末, 转入 10mL离心管, 加入 3 mL 65 °C预热的 DNA提取缓冲液, 快速 振荡混匀。 65°C水浴 45 min, 期间混匀 2-3次。 然后加入 1 mL 5 mol/L KAc, 冰 浴 20 min。 用等体积 (4mL;>的氯仿: 异戊醇 (24: 1)抽提 1次 (10,000 r/min, 25 °C离 心 10 min:)。 取上清, 加入 2/3倍体积的 -20 °C预冷异丙醇, 混匀, 室温静置约 30 mm。 挑出絮状沉淀, 用 75%的乙醇反复漂洗两次, 再用无水乙醇漂洗 1次。 室温吹干, 重悬于 600 TE中。 加入 1 RNaseA (10 mg/mL), 37°C处理 1 h 去除样品中的 RNA。再用酚 (Ph8.0):氯仿:异戊醇 (25:24: 1)和氯仿:异戊醇 (24: 1) 各抽提 1次 (10,000 r/min, 离心 10 min), 取上清, 2.5倍体积无水乙醇于 -20°C沉 淀 30min以上。 13,000 r/min, 离心 lO min收集沉淀, 弃上清, 沉淀用 75%的乙 醇漂洗。 减压离心干燥, 沉淀最终溶于 50-200 TE中, -20 °C保存备用。
2. 目的片段序列的 PCR扩增
ΙΟχΕχ PCR buffer (Mg2+ free) 2.5 μ
2.5 mmol/L dNTPs 2 μ
25 mmol/L MgCl2 2 μ
上游引物 (5 μπιοΙ/L) 1 μ
下游引物 (5 μπιοΙ/L) 1 μ
Ex Taq DNA聚合酶 1 U
DNA 约 20 ng
25 μ 的扩增体系
扩增程序为: 94°C, 5 min; 94°C, 30 sec, 56°C, 30 sec, 72°C, l〜4 min, 35个循环; 72°C延伸 10 min。
3. DNA片段回收, 连接, 转化大肠杆菌。
紫外灯下, 用洁净的刀片切下含目的片段的琼脂糖凝胶块。 回收方法参照试 剂盒 (Roche公司) 的使用说明书进行。 回收片段在琼脂糖凝胶上电泳定量。
酶切体系的建立和反应条件均参考 Roche 公司限制性内切酶试剂盒的说明 书进行。酶切回收的片段, 或者扩增获得的回收片段按连接酶试剂盒说明书克隆 到 pUCm-T (上海 Sangon)或 GEM-T/pGEM-T Easy(Promega)载体上。连接反应体 系如下:
10xT4 DNA连接缓冲液 1
载体 DNA片段 l L (50ng)
外源连接产物 DNA片段 l L
T4 DNA连接酶 1 μ
用 dd¾0补足 10 μ 的连接体系
载体 DNA片段与外源连接产物 DNA片段摩尔比 =1 :3。
16°C连接 2-8h。之后将连接产物转化大肠杆菌 DH5a感受态细胞, 37°C培养。 【实施例 2】 花原基细胞特异启动子控制转录激活因子基因的 Trigger植物表 达载体的构建
1.植物花原基细胞特异启动子的获得
根据质粒 pER8 (GenBank登录号: AF309825.2)上 CaMV 35S 最小核心启 动子序列, 设计引物 (SEQ ID NO. 1和 2), 以质粒 pER8为模板, PCR扩增得到 长约 600 bp的序列。测序结果表明该序列全长 602 bp, 含有 58 bp 长的 CaMV 35S (-46 to +12) 核心启动子 (简称 minP)、 多克隆位点 MCS和豌豆核酮糖 -1, 5-二 磷酸羧化酶小亚基 rbcS-3A基因的 polyA序列 (简称 "T3A")。
根据烟草花发育 AGAMOUS(AG)同源基因 1的第二内含子序列 ( GenBank登 录号: GU143404.1 ) , 设计引物 (SEQ ID NO. 3, 4), 从烟草基因组中 PCR扩增获 得一个约 4.1 kb的片段, 将扩增 DNA片段克隆后测序分析表明为烟草 AG同源基 因 1的第二内含子调控序列。将该序列反向插入上述克隆的 CaMV 35S minP启动 子上游, 构建融合启动子, 命名为 ntAGIPl ( SEQ ID NO. 5
根据烟草花发育 AGAMOUS(AG)同源基因 2的第二内含子序列 (GenBank登 录号: GU143405.1 ) , 设计引物 (SEQ ID NO. 6, 7), 从烟草基因组中 PCR扩增获 得一个约 4.1 kb的片段, 将扩增 DNA片段克隆后测序分析表明为烟草 AG同源基 因 2的第二内含子序列。将该序列反向插入上述克隆的 CaMV 35S minP启动子上 游, 构建融合启动子, 命名为 ntAGIP2 ( SEQ ID N0.22
根据拟南芥花发育 AGAMOUS(AG)基因的第二内含子调控序列 ( GenBank 登录号: AT4G18960) , 设计引物 (SEQ ID NO.8,9), 从拟南芥基因组中 PCR扩增 获得一个约 1.7 kb的片段, 将扩增 DNA片段克隆后测序分析表明为拟南芥 AG基 因的第二内含子的核心调控序列。 同样, 将该序列反向插入上述克隆的 CaMV 35S minP启动子上游, 构建融合启动子, 命名为 AGIP EQ ID N0.23;>。
根据拟南芥花原基发育特异启动子 API的序列 (GenBank登录号:
AT1G69120), 设计引物 (SEQ ID NO.10, 11), 以拟南芥基因组 DNA为模板扩增 API启动子, 获得了一个约 1.8 kb长的片段, 将扩增 DNA片段克隆后测序分析表 明为拟南芥的 API特异启动子, 命名为 API。
根据拟南芥孢子母细胞发育特异启动子 SPL (GenBank登录号: AT4G27330) 的序列,设计引物 (SEQ ID N0.12, 13),以拟南芥基因组 DNA为模板 PCR扩增 SPL 启动子, 获得了一个约 2.7 kb长的片段, 将扩增 DNA片段克隆后测序分析表明为 拟南芥的 SPL特异启动子, 命名为 SPL。
根据拟南芥孢子母细胞特异启动子 EMS1的序列 (GenBank登录号:
AT5G07280), 设计引物 (SEQ ID N0.14, 15), 以拟南芥基因组 DNA为模板扩增 EMS1启动子, 获得了一个约 3.0 kb长的片段, 将扩增 DNA片段克隆后测序分析 表明为拟南芥的 EMS1特异启动子, 命名为 EMS1。
根据番茄果实发育早期特异启动子 Lefsml的序列 (GenBank登录号:
AJ583670.1 ), 设计引物 (SEQ ID NO.16, 17), 以番茄基因组 DNA为模板扩增 Lefsml启动子, 获得了一个约 1.0 kb长的片段, 将扩增 DNA片段克隆后测序分析 表明为番茄的 Lefsml特异启动子, 命名为 Lefsml。 2. 花原基细胞特异启动子控制转录激活子基因 Tngger植物表达载体的构建 简单地说, 用酶切连接技术将 pL35SLhG4载体 (参见申请号为
201110179613.2的专利申请所公开的相关内容)中的 35S启动子分别替换为上述 花原基细胞特异启动子, 构建不同花原基细胞特异启动子控制 LhG4ATO转录因 子基因的植物表达载体, 分别命名为 ntAGIPl ::Trigger, ntAGIP2:: Trigger,
AGIP:: Trigger, API:: Trigger, SPL:: Trigger, EMSl ::Trigger, Lefsml:: Trigger植物 表达载体。 其中, ntAGIPl ::Trigger植物表达载体的结构示意图见图 1, 其余 Trigger植物表达载体的结构与之相似。所有限制性内切酶购自 Roche公司, 按照 使用说明书操作。
3. 用电激法将构建的植物表达载体质粒导入农杆菌 EHA105中。
参考 Bio-RAD MicroPulser用户说明书, 将上述载体通过电激转化法导入农 杆菌 EHA105。
【实施 3】 农杆菌介导的烟草遗传转化
通过根癌农杆菌介导的方法进行烟草的遗传转化所用培养基见表 1 表 1: 根癌农杆菌介导的烟草遗传转化用培养基
培养基名称 成分
基本培养基 MSB (MS无机盐 +B5有机)
(MSB)
种子萌发培养 MSB+30g/L葡萄糖 +7.5 g/L琼脂, pH5.8
基 (MSB0)
; tt+立类 +立类;
口芥 口芥¾ it¾ MSB+2.0mg/L 6-BA+0.5mg/L IAA+6g/L琼月旨,, pH5.8
(MSB1 )
筛选培养基 MSB+2.0mg/L 6-BA+0.5mg/L IAA +200mg/L Cef+ 50mg/L
(MSB2) Kan+6g/L琼脂, pH5.8
诱芽培养基 MSB+200mg/L Cef+ 50mg/L Kan+6g/L琼脂, pH5.8
(MSB3 )
生根培养基 MSB+200mg/L Cef+6g/L琼脂, pH5.8
(MSB4)
MS: Murashige & Skoog, 1962
B5: Gamborg, 1986
农杆菌介导叶盘的方法导入烟草。 具体方法如下:
烟草种子用 1%的次氯酸钠消毒后, 在固体培养基 MSBQ上萌发, 培养条件 为 25°C、 16 hr光照 /8 hr黑暗的光周期。 约一个月后生长健壮的无菌苗即可用作 转化外植体。 选取健壮叶片, 超净工作台上切成约 0.5Cmx0.5cm的叶盘, 保持湿润备用。 牙签挑取 YEB平板上培养的转化用农杆菌单菌落, 液体培养活化, 再转入三角 瓶中培养至 OD6QQ = 0.5。 把切好的叶盘浸泡于 MSB液体基本培养基重悬的农杆 菌菌液中, 静止浸染 10-20 mm。 倾去菌液, 用无菌的吸水纸吸去叶盘表面多余 的菌液, 将叶盘接入共培养基 MSBi上, 24 °C暗培养 2 d。 共培养完成后, 将叶 盘接入筛选培养基 MSB2中进行分化培养 2周,培养条件为 25°C、 16 hr光照 /8 hr 黑暗的光周期。 出现再生绿色愈伤后, 转入诱芽培养基 MSB3促进芽的产生。 当 抗性苗生长到 3-4 cm长时, 切下转入生根培养基 MSB4中, 诱导生根。 当抗性苗 的根生长到 2-3 cm长时, 洗净培养基, 并水培炼苗 2-3d, 移栽到营养钵中, 于 温室生长。
【实施例 4】 Trigger植株的获得
参照实施例 3, 利用农杆菌介导法将实施例 2构建的 Trigger植物表达载体 转化烟草, GUS组织化学染色和 PCR筛选鉴定转基因植株, 并利用 GUS组织化 学染色等技术分析和筛选单拷贝 Tngger转基因植株的后代纯合株系, 用于进一 歩研究。
【实施例 5】 Deleter植物表达载体及 Deleter植株的获得
按照申请号 201110179613.2 的发明专利申请中所述的方法构建本发明所称 的第二植物表达载体 Deleter (如图 6), 并获得 Deleter转基因植物。
【实施例 6】 转基因烟草的杂交试验
为了评价不同花原基细胞特异启动子指导 GAEBS 系统通过杂交途径删除 外源基因的特点和效率, 首先, 按照申请号 201110179613.2的发明专利申请中 所述的方法筛选获得具有 100%删除效率的高效 Deleter转基因株系: D198 (即 实施例 5所制备的 Deleter植株); 然后, 以 D198后代纯合株系为父本, 分别与 Trigger激活子纯合株系杂交。 杂交方法参见刘仁祥等 (刘仁祥 et al., 2000)所述, 在烟草盛花期,每天下午采集第 2 天要裂开的花药存于实验室,待第 2 天早上, 花药已裂开, 用毛笔粘取花粉涂于要授粉的柱头 (去雄蕊)上, 即完成授粉过程。
【实施例 7】 不同花原基细胞特异启动子控制的 GAEBS系统删除外源基 因效率的统计分析
选取花原基细胞特异启动子指导 GAEBS系统从 F1代种系细胞中删除外源 基因, 进而产生无任何外源基因的种子和花粉是本发明的最终目的。 因此, 通 过该系统创制的转基因植株杂交后, 系统在 F1代植株中合拢后, 花原基细胞特 异启动子开启系统在种系细胞中删除外源基因, 产生无外源基因的种子和花粉 的频率是评价花原基细胞特异启动子控制的 GAEBS系统删除效率的关键。 为 此, 首先以 自交种子萌发产生的 F2代幼苗中 和 基因的表型为标 准分别统计系统对 Deleter和 Tngger的删除效率。根据孟德尔遗传分离规律,在 没有任何外源基因删除时, F2代植株应有四种表型: GFP+/GUS+ 、 GFP+/GUS―、 GFP7GUS+ 和 GFP7GUS—, 分离比为 9:3:3:1。 其中以 GFP为标准的 Deleter外 源基因的分离比为 GFP+: GF =3:1 ; 以 GUS为标准的 Trigger外源基因的分离 比为 GUS+: GUS— =3:1。 通过统计不同表型的植株数, 根据单基因 3:1的分离规 律, 得到了系统删除 Trigger和 Deleter转基因的效率, 统计结果见表 2所示。 从表中可以看出, 不同启动子指导系统的删除效率存在明显的差异。 其中, ntAGIPl 启动子指导系统删除 Trigger 和 Deleter 外源基因的最高效率均可达 100%, 系统的平均效率为 68.6%, 显著高于其他启动子控制的系统的删除效率。 因此, 选取来自烟草的 ntAGIPl启动子用于进一歩研究。
表 2不同花原基细胞特异启动子指导 GAEBS 系统删除外源基因效率的统计分析 启动子 a分析的独立 Trigger转 b删除 Deleter外源 e删除 Trigger外源 d平均删除 e删除效率为 100%
基因植株数 基因的效率(%) 基因的效率(%) 效率 (%) 的 Trigger植株数 ntAGIPl 25 0-100 0-100 68.6 3 ntAGIP2 15 0-80 0-100 33.0 0
AGIP 10 0-74 0-79 40.5 0
API 10 0-63 0-66 33.7 0
SPL 11 0-22 0-4 4.1 0
EMS1 17 0-92 0-100 38 0
Lefsml 9 0 0-6 1 0
a每个启动子平均选取 15株独立的 Trigger转基因植株作为父本与 D198 Deleter杂交, 获得杂交种 (F1代) , 种植 F1代植 株, 分析 F1代自交种子中 GFP和 GUS的酶活。 所有 Trigger转基因植株均为单拷贝纯合植株。
a删除 Deleter效率 (%) = (4 X GFF幼苗数-分析幼苗总数) /3 X分析幼苗总数 X 100;
b删除 Trigger效率(%) = (4 X GUS-幼苗数-分析幼苗总数) /3 X分析幼苗总数 X 100;
e平均删除效率 (%) = (删除 Deleter效率 +删除 Trigger效率) /2。
e100%删除效率是指来自双亲的所有外源基因 (包括 Trigger和 Deleter) 均 100%从 F1代自交种子中删除。
2分析 F2代幼苗中 GUS-和 GFF表型幼苗分离比对 3:1比例的适合度。 当 GUS-或 GFF幼苗分离比显著偏离 3:1时, 表明 外源基因删除发生, 并计算删除效率; 否则, 删除效率记为 0。
进一歩分析发现, 在 ntAGIPl指导的 GAEBS系统中, 以 GUS为标准统计 的系统对 Trigger的平均删除效率为 71%; 以 GFP为标准统计的系统对 Deleter 的删除效率为 66%。 在 25株独立 ntAGIPl ::Trigger与 D198杂交组合中, 系统 在 3株 ntAGIPl ::Trigger (ntAGIPl -85, -126, -137) 与 D198杂交 F1代种子中 删除效率均为 100% (平均统计约 2,000颗种子中), 能 100%产生无任何外源基 因的 F1代种子 (表 3 )。 表 3 ntAGIPl指导系统删 1除效率的统计分析
Trigger lines 分析的 GFP+/GFF苗 a删除 Deleter效 GUS+/GUS-苗数 b删除 Trigger效 e平均删除效率 总苗数 数 率 (%·) 率 (%·) (%) ntAGIPl-6 150 14/136 87 3/147 97 92 ntAGIPl-3 200 150/50 0 198/2 0 0 ntAGIPl-2 200 60/140 60 12/188 92 76 ntAGIPl-10 150 25/125 78 43/107 62 70 ntAGIPl-4 200 26/174 83 0/200 100 91 ntAGIPl-17 100 38/63 50 43/57 43 46 ntAGIPl-11 225 45/180 73 9/216 95 84 ntAGIPl-8 150 99/51 12 86/64 24 18 ntAGIPl-5 120 19/101 79 22/98 76 77 ntAGIPl-9 2000 27/1973 98 20/1980 99 98 ntAGIPl-132 192 10/182 93 1/191 99 96 ntAGIPl-113 192 79/113 45 93/99 35 40 ntAGIPl-109 193 58/135 60 0/193 100 80 ntAGIPl-134 192 10/182 93 177/15 0 47 ntAGIPl-95 192 132/60 8 29/163 80 44 ntAGIPl-93 187 42/145 70 13/174 91 80 ntAGIPl-138 361 3/358 99 1/360 100 99 ntAGIPl-137 2000 0/2000 100 0/2000 100 100 ntAGIPl-112 96 58/38 20 43/53 40 30 ntAGIPl-103 96 22/74 70 33/63 54 62 ntAGIPl-122 96 71/25 2 33/63 54 28 ntAGIPl-85 1940 0/1940 100 0/1940 100 100 ntAGIPl-125 50 4/46 89 10/40 73 81 ntAGIPl-89 96 13/83 82 26/70 64 73 ntAGIPl-126 1640 0/1640 100 0/1640 100 100 只有当 2禾卩 5fc2大于 3.84时, 才分别估测 GFP和 GUS外源基因被删除的效率。
a删除 Deleter效率 (%) = (4><GFF幼苗数-分析幼苗总数) /3><分析幼苗总数 χ100:
b删除 Trigger效率 (%) = (4><GUS-幼苗数-分析幼苗总数) /3><分析幼苗总数 χΙΟΟ;
e平均删除效率 (%■) = (删除 Deleter效率 +删除 Trigger效率) /2。
χ2分析 F2代幼苗中 GUS-和 GFF表型幼苗分离比对 3:1 比例的适合度。 当 GUS ¾ GFF幼苗分离比显著偏离 3:1时, 表明外 源基因删除发生, 并计算删除效率; 否则, 删除效率记为 0.
另外, 田间生产中, F1花粉是带来转基因生物安全隐患的主要途径。为此, 进一歩检测了 F1 代成熟花粉中 GUS 和 GFP 的表达活性, 结果表明, 在 ntAGIP-85,-126,-137 三株 Triggers分别与 D198 Deleter杂交 Fl代的花粉中, 均 未检测到 GUS和 GFP的表达活性(评价统计花粉数 20,000以上)(图 3 )。这些 结果表明, 系统能 100%将外源基因从 F1代花粉中删除, 产生无任何外源基因 的花粉。
【实施例 8】 ntAGIPl启动子控制的 GAEBS系统通过杂交途径维持外源基因 稳定传递的分析
l.ntAGIPl指导的 GAEBS系统维持外源基因在杂交种中稳定传递的分析
为了分析外源基因在杂交当代种子中的非特异性删除情况, 实验条件下, 萌发杂交种子, 利用 GFP荧光检测法和 GUS组织化学染色法检测 7d龄幼苗中 GFP和 基因的表达。根据孟德尔遗传规律,如果没有发生外源基因的删除, 应该只观察到一种表型的幼苗: GFP+/GUS+ (即同时含绿色荧光和 GUS蓝色信 号的幼苗), 如果发生删除, 有可能观察到以下三种表型的幼苗: GFP+/GUS_、 GFP7GUS+ 和 GFP7GUS―。统计分析结果见表 4所示。从表中可以看出,在 D198 与 25株 Tngger转基因植株的杂交组合中, 系统仅在 2个杂交组合中删除外源 基因, 其余杂交组合中, 没有检测到外源基因的删除, 结果表明 ntAGIPl启动 子指导的 GAEBS系统能高效维持外源功能基因在杂交种中的传递, 杂交制种 能获得杂交种。
表 4 系统在杂交种中非特异删除外源基因的统计分析
Trigger 株 分析的 GFP+苗数 / GUS+苗数 / e删除
芽总数 GFF苗数 GUS-苗数
ntAGIPl -2 347 347/0 347/0 N ntAGIPl -3 496 496/0 496/0 N ntAGIPl -4 377 377/0 377/0 N ntAGIPl -5 371 371/0 371/0 N ntAGIPl -6 406 406/0 406/0 N ntAGIPl -8 366 366/0 366/0 N ntAGIPl -9 399 399/110 399/45 Y ntAGIPl-10 390 390/0 390/0 N ntAGIPl-11 325 325/0 325/0 N ntAGIPl-17 331 331/34 331/65 Y ntAGIPl-85 251 251/0 251/0 N ntAGIPl-89 205 205/0 205/0 N ntAGIPl-93 274 274/0 274/0 N ntAGIPl-95 246 246/0 246/0 N ntAGIPl-103 345 345/0 345/0 N Trigger 株 分析的 GFP+苗数 / GUS+苗数 / e删除
芽总数 GFF苗数 GUS-苗数
ntAGIPl-109 381 381/0 381/0 N ntAGIPl-112 303 303/0 303/0 N ntAGIPl-113 334 334/0 334/0 N ntAGIPl-122 236 236/0 236/0 N ntAGIPl-125 338 338/0 338/0 N ntAGIPl-126 328 328/0 328/0 N ntAGIPl-132 238 238/0 238/0 N ntAGIPl-134 208 208/0 208/0 N ntAGIPl-137 310 310/0 310/0 N ntAGIPl-138 128 128/0 128/0 N
¾: 无外源基因删除发生; Y: 有外源基因删除发生。
2.ntAGIPl指导的 GAEBS系统维持外源功能基因在 F1代非删除组织中有效表 达的分析
系统在 代营养体组织中的稳定性是实现外源性状基因(如抗虫、抗病等) 在这些组织中生物学功能的关键。 为此, 首先用 PCR检测了系统在 F1代植株 中的稳定性, 扩增的目标基因为 cre'" n 7J2G ro基因。 提取植物叶片的基因组 DNA, 用引物对 (SEQ ID No. 18, 19和 SEQ ID No. 20, 21 ) 分别检测 cre'" f] 2(^ ^基因在这些组织基因组中的稳定性。 PCR检测结果显示, 所有 F1代植 株基因组中均同时检测到长约 1.2 kb cre'"f和长 1.3 kb 7J?G ro基因的存在,表明 这些植株同时含有 Deleter和 Trigger转基因,外源基因在这些组织中没有被删除 (图 2)。
进一歩, 利用 GFP荧光检测法和 GUS组织化学染色法分析 代根、 茎、 叶等组织中 和 报告基因的表达情况。结果表明, 在 F1代植株根、茎、 叶和花等非删除组织中均检测到 GFP和 GUS基因的高效表达 (图 3-B)。
上述结果表明,在 ntAGIPl启动子调控下, GAEBS系统在杂交后代合拢后, 依然能有效维持外源功能基因 (GFP和 GUS)在 F1代非删除组织(根、 茎、 叶 和花) 中的稳定表达, 实现外源功能基因的生物学功能。
【实施例 9】 ntAGIPl启动子控制的 GAEBS系统在 代花发育时期删除 外源基因的时空特点分析
为了分析 ntAGIPl启动子控制的 GAEBS系统在 代花发育时期删除外源基 因的时空特点,参见 Koltimow等 (1990)关于烟草花发育时期的划分,我们分析了 F1代花发育 2-3时期 (stage 2-3 ) GUS和 GFP的表达特征。 结果显示, GUS和 GFP活性均特异地从雄蕊和雌蕊中消失,表明外源基因已从这些组织中被彻底删 除 (图 3-B)。 而且, 在 F2代幼苗、 茎、 叶和花中均未检测到 GUS和 GFP基因 的表达活性,表明由外源基因已被删除的花原基细胞发育能产生无外源基因的非 转基因植株 (图 3-C)。
ntAGIPl启动子在烟草花发育 -7时期以前就开始启动靶标基因在烟草花原基 中心(包括雌蕊原基和雄蕊原基) 强烈持续表达, 直到开花前 ¾ng et al., 2010;)。 因此, 理论上, 系统在 代细胞中合拢后, ntAGIPl 启动子将特异启动系统从 ΐι代花原基发早期开始将外源基因从花原基中心删除, 据此, 进一歩利用 GFP 绿色荧光检测技术追踪了 花器官中外源基因删除的时间窗。图 4显示, GFP 绿色荧光从杂交后代 -7时期以前花原基中心特异性消失,在随后的 -6— -1时期的 雄蕊和雌蕊群中均没检测到绿色荧光信号, 而只检测到红色的荧光信号 (图 4A, B, C)。 而亲本 D198中, 荧光信号在各时期的花中均一强烈出现(图 4D, E)。 这 些结果表明, ntAGIPl启动子在 F1代 -7时期以前的早期花原基中心就开始启动 基因自动删除双元系统高效删除外源基因。
上述实例表明, 本发明利用 ntAGIPl等花原基细胞特异启动子控制 GAEBS 系统的时空删除,成功构建了用于控制杂交作物外源基因生物安全的基因自动删 除双元系统 GAEBS, 其工作原理见图 5。 目标性状基因通过该系统导入杂交作 物中, 从而实现: (1 ) 维持了目标性状基因在亲本有性世代中的稳定遗传, 便于优良转基因株系的筛选; (2)维持了目标性状基因在杂交种中的稳定传 递, 杂交制种能获得生产用转基因杂交种子; (3 )大田生产中, 能有效保证目标 性状基因在 代非删除组织 (如根、 茎和叶) 中实现其功能 (如抗虫、 抗病和 抗除草剂等); (4) 基因自动删除双元系统在 代植物减数分裂发生之前的 性细胞中启动将包括重组酶系统在内的所有外源基因 100%删除, 产生不含有任 何外源基因的花粉和种子。
本发明方法简便易行, 在需要删除外源基因时, 能 100%删除所有外源基因, 效果显著, 具有很好的应用前景。
以上对本发明的详细描述并不限制本发明, 本领域的技术人员可以根据本 发明做出各种变形和改变, 只要不脱离本发明的精神, 均应属于本发明所附权利 要求所定义的范围。 参考文献
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290-298 Koltunow AM, Truettner J, Cox KH, Wallroth M, Goldberg RB (1990) Different Temporal and Spatial Gene Expression Patterns Occur during Anther Development. Plant Cell 2: 1201-1224
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刘仁祥, 丁伟, 杨俊, 冯勇刚 (2000) 烤烟雄性不育杂交种制种技术研究 [J]. 中国烟草科 学 21 : 7

Claims

权利要求书
1.一种杂交作物转基因安全控制的方法, 其中构建包括重组酶系统、转录激 活系统以及外源基因表达控制系统的转基因植物自动删除双元系统,通过将植物 花原基细胞特异启动子作为所述转基因植物自动删除双元系统中控制转录激活 系统的启动子, 所述转录激活系统控制重组酶系统的启动, 使导入到植物中的外 源基因在 F1代杂交种中以及 F1代的根、茎、 叶等非删除组织中稳定存在, 但在 F1代植株的花粉和种子中所述外源基因被删除, 以实现杂交作物转基因安全控 制。
2.权利要求 1所述的方法,其中所述转基因植物自动删除双元系统包括分别 位于第一植物表达载体和第二植物表达载体且相互配合的重组酶系统、转录激活 系统和外源基因表达控制系统;
所述重组酶系统包括重组酶和该重组酶的特异识别位点;
所述转录激活系统包括转录激活因子基因、由该转录激活因子基因控制的靶 标启动子和控制该转录激活因子基因的植物花原基细胞特异启动子;
所述外源基因表达控制系统包括控制外源基因表达的启动子和导入的外源 基因。
3. 权利要求 2所述的方法, 其中所述转基因植物自动删除双元系统包括: 第一植物表达载体,所述表达载体包括两个同向的重组酶特异识别位点以及 位于所述两个重组酶特异性识别位点之间的下述基因或核苷酸:
控制外源基因表达的启动子;
用于导入外源基因的多克隆位点;
转录激活因子基因; 以及
植物花原基细胞特异启动子, 用于控制转录激活因子基因的启动; 第二植物表达载体,所述表达载体包括两个同向的重组酶特异识别位点以及 位于所述两个重组酶特异性识别位点之间的下述基因或核苷酸:
控制外源基因表达的启动子;
用于导入外源基因的多克隆位点;
重组酶基因; 以及
控制重组酶基因的靶标启动子,所述靶标启动子由所述转录激活因子基因控 制, 当被激活时, 启动重组酶基因表达。
4. 权利要求 1至 3任一项所述的方法, 其中所述植物花原基细胞特异启动 子为烟草花发育 C基因 MAG的启动子 ntAGIPl。
5. 植物花原基细胞特异启动子在制备安全的转基因植物中的应用, 其中通 过将所述植物花原基细胞特异启动子作为转基因植物自动删除双元系统中控制 转录激活系统的启动子, 所述转录激活系统控制重组酶系统的启动, 使导入到 植物中的外源基因在杂交种以及 Fl代的根、 茎、 叶等非删除组织中稳定存在, 但在 F1代植株的花粉和种子中所述外源基因被删除,以实现在转基因杂交作物 中生产非转基因花粉和种子。
6. 权利要求 5所述的应用, 其中所述转基因植物自动删除双元系统包括: 第一植物表达载体,所述表达载体包括两个同向的重组酶特异识别位点以及 位于所述两个重组酶特异性识别位点之间的下述基因或核苷酸:
控制外源基因表达的启动子;
用于导入外源基因的多克隆位点;
转录激活因子基因; 以及
植物花原基细胞特异启动子, 用于控制转录激活因子基因的启动; 第二植物表达载体,所述表达载体包括两个同向的重组酶特异识别位点以及 位于所述两个重组酶特异性识别位点之间的下述基因或核苷酸:
控制外源基因表达的启动子;
用于导入外源基因的多克隆位点;
重组酶基因; 以及
控制重组酶基因的靶标启动子,所述靶标启动子由所述转录激活因子基因控 制, 当被激活时, 启动重组酶基因表达。
7. 权利要求 5或 6所述的应用, 其中所述植物花原基细胞特异启动子为烟草 花发育 C基因 ntAG的启动子 ntAGIPl。
8. 一种用于杂交作物转基因安全控制的基因自动删除双元系统, 包括 第一植物表达载体,所述表达载体包括两个同向的重组酶特异识别位点以及 位于所述两个重组酶特异性识别位点之间的下述基因或核苷酸:
控制外源基因表达的启动子;
用于导入外源基因的多克隆位点;
转录激活因子基因; 以及
植物花原基细胞特异启动子, 用于控制转录激活因子基因的启动; 第二植物表达载体,所述表达载体包括两个同向的重组酶特异识别位点以及 位于所述两个重组酶特异性识别位点之间的下述基因或核苷酸:
控制外源基因表达的启动子;
用于导入外源基因的多克隆位点;
重组酶基因; 以及
控制重组酶基因的靶标启动子,所述靶标启动子由所述转录激活因子基因控 制, 当被激活时, 启动重组酶基因表达。
9. 权利要求 8所述的基因自动删除双元系统, 其中所述植物花原基细胞特异 启动子为烟草花发育 C基因 ntAG的启动子 ntAGIPl。
10. 权利要求 8或 9所述的基因自动删除双元系统, 其中所述重组酶特异性 识别位点选自 loxP, lox2272, 10x5171和 FRT识别位点, 所述重组酶基因选自 FLP、 Cre和 Cremt重组酶基因, 所述靶标启动子及与之配合的转录激活因子基因 为 pOp/LhG4转录激活系统。
11. 一种制备转基因植物的方法, 包括下述歩骤:
1 ) 构建权利要求 7所述的基因自动删除双元系统;
2) 将权利要求 7所述的第一植物表达载体导入植物基因组, 制备第一转基 因植物;
3 ) 将权利要求 7所述的第二植物表达载体导入植物基因组, 制备第二转基 因植物;
4) 将第一转基因植物和第二转基因植物作为亲本相互杂交, 获得 F1代转基 因植物。
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