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A kind of Agrobacterium transformation vector composition for obtaining marker-free transgenic plants and its application
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A technology for transgenic plants and marker genes, applied in the field of Agrobacterium transformation vector composition, can solve the problems of inconvenient target gene replacement and application that have not been reported yet
Active Publication Date: 2016-06-08
INST OF CROP SCI CHINESE ACAD OF AGRI SCI
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[0011] In summary, the above studies only adopted a vector construction strategy and applied it to the genetic transformation of individual plants, and the application in wheat and other crops has not been reported yet.
In addition, the construction of most of the vectors is to verify the feasibility of Agrobacterium to transmit 2 independent T-DNAs to produce mark-free plants. Therefore, many vectors have only been transformed in model plants such as tobacco, etc., and have not been used in important crops such as wheat. middle application
In addition, most of the currently reported vectors used to produce marker-free plants are constructed by traditional enzyme digestion and ligation. Many vectors use the gus gene as the target gene. Due to the restriction of enzyme digestion sites, some vectors are not easy to use. Replacement of the target gene
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Embodiment 1
[0081] Example 1. Agrobacterium-specific vector composition for obtaining marker-free transgenic plants
[0082] 1. Construction of Gateway-compatible pCG1811, pCG1851, pCS1671 universal binary vectors
[0083] The structure of pCG181, pCG185, pCS167 vector is as follows figure 1 , the DNA fragment attR1-cm inserted into the ccdB reading frame in its T-DNA, respectively R -ccdB-attR2 (the nucleotide sequence of the DNA fragment is sequence 3 in the sequence table, purchased from Invitrogen Company), to obtain binary vectors pCG1811, pCG1851, pCS1671; the specific process is as follows:
[0084] 1. Construction of pCG1811 vector
[0085] 1) Obtaining the P-free blunt-end vector backbone
[0086] The pCG181 vector was digested with NotI, and the 2718bp digested product was recovered; then the above-recovered digested product was sequentially filled with Klenow fragments to blunt the protruding end, purified, and de-P reaction to obtain the P-depleted blunt-ended pCG181 vector...
Embodiment 2
[0193] Example 2. Application of Agrobacterium-Specific Vector Composition for Obtaining Marker-Free Transgenic Plants
[0194] 1. Transformation of Agrobacterium with special carrier composition for Agrobacterium
[0195]After mixing the carriers in each carrier composition obtained in Example 1 at a mass ratio of 1:1, transform Agrobacterium AGL1 into 2×YT solid medium (containing 200mgL -1 Carb, 100mg / LKan) were coated on the plate, cultured in the dark at 28°C for 48h, and the recombinant bacteria AGL1 / pG1811-UG / pS167-UB (1G7B), AGL1 / pG1851-UG / pS167-UB (5G7B), AGL1 / pG1852- UG / 154 (5BTG154), AGL1 / pG1853-UG / 154 (5LBTG154) and AGL1 / pG1854-UG / 154 (5TBTG154) ( Figure 9 ).
[0196] Use gusF / R and baF / R primers to amplify the above-mentioned recombinant vector to obtain products of 1051bp and 444bp, which are positive recombinant bacteria.
[0197] AGL1 / pG1811-UG / pS167-UB (1G7B) identified as positive was named 1G7B (pG1811-UG / pS167-UB);
[0198] AGL1 / pG1851-UG / pS167-UB (5G7...
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Abstract
The invention discloses an agrobacterium-mediated transformation vector composition for obtaining marker-free transgenic plants and application of the agrobacterium-mediated transformation vector composition, and provides a special agrobacterium vector composition for obtaining marker-free transgenic plants. The special vector composition is composed of an individually-packed recombinant vector and the pClean-Soup vector of a pClean series vector; a target gene is inserted into the T-DNA region of the pClean-Green vector of the pClean seriesvector and a marker gene is inserted into the other T-DNA region of the pClean-Green vector to obtain the recombinant vector with the expression of the target gene and the marker gene. As can be seen, with the adoption of the 5TBTG154 composition of the target gene and the marker gene in different T-DNA regions of the same vector, marker-free transgenic plants in a higher proportion can be obtained from progenies. The agrobacterium-mediated transformation vector composition for obtaining the marker-free transgenic plants and the application of the agrobacterium-mediated transformation vector composition have great significance on the transgenic plant research, especially on acquisition and utilization of wheat marker-free transgenic plants.
Description
technical field [0001] The invention relates to the field of biotechnology, in particular to an Agrobacterium transformation vector composition for obtaining marker-free transgenic plants and its application. Background technique [0002] Plant transgenic technology is an important tool for gene function research and plant genetic improvement through genetic engineering. Since the first commercial planting of genetically modified crops in 1996, countries around the world have approved a total of 23 types of genetically modified crops for commercial production, involving 13 types of target traits, including insect resistance, herbicide resistance, disease resistance, fertility change, and quality improvement Wait. Genetically modified crops such as soybeans, corn, cotton, and rapeseed, which are mainly resistant to herbicides and insects, have been planted in 22 countries and regions around the world, and the planted area reached 170 million hectares in 2012. The industrial...
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