Constitutive promoters and their uses
A promoter and constitutive technology, applied in the direction of application, recombinant DNA technology, using vectors to introduce foreign genetic material, etc., can solve the problem of low efficiency
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no. 1 example
[0059] The first embodiment, the synthesis of prBrTsf1 promoter sequence
[0060] Obtain the constitutive promoter prBrTsf1 of the present invention, as shown in SEQ ID NO: 1 in the sequence listing; the prBrTsf1 promoter sequence (shown in SEQ ID NO: 1 in the sequence listing) was synthesized by Nanjing GenScript Biotechnology Co., Ltd.
no. 2 example
[0061] The second embodiment, the construction of the recombinant expression vector DBN100040 containing the prBrTsf1 promoter sequence and the transformation of the recombinant expression vector into Agrobacterium
[0062] 1. Construction of the recombinant cloning vector pT-prBrTsf1 containing the prBrTsf1 promoter sequence
[0063] The prBrTsf1 promoter sequence was connected to the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector pT-prBrTsf1.
[0064] Then, the recombinant cloning vector pT-prBrTsf1 was transformed into Escherichia coli T1 competent cells (Transgen, Beijing, China, CAT: CD501) by heat shock method. The heat shock conditions were: 50 μl E. coli T1 competent cells, 10 μl plasmid DNA (recombinant Cloning vector pT-prBrTsf1), water bath at 42°C for 30 seconds; shake culture at 37°C for 1 hour...
no. 3 example
[0074] The third embodiment, the acquisition of soybean plants transferred to the prBrTsf1 promoter sequence
[0075] According to the commonly used Agrobacterium infection method, the cotyledon node tissue of the aseptically cultured soybean variety Zhonghuang 13 was co-cultured with the Agrobacterium described in 4 in the second embodiment to construct 2 and 3 in the second embodiment The T-DNA in the recombinant expression vector DBN100040 and DBN100036 (including eFMV: prBrTsf1 nucleotide sequence, eFMV: prAtTsf1 nucleotide sequence, CTP2 nucleotide sequence, EPSPS gene, E9 terminator sequence, prCaMV35S promoter sequence, PAT Gene and tCaMV35S terminator sequence) were transferred into the soybean genome, and soybean plants transferred to the prBrTsf1 promoter sequence and soybean plants transferred to the prAtTsf1 promoter sequence were obtained; at the same time, wild-type soybean plants were used as controls.
[0076] For Agrobacterium-mediated soybean transformation, ...
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